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Nitric oxide in focal cerebral ischemia, an experimental study

Coert, B.A.

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Citation for published version (APA):Coert, B. A. (2008). Nitric oxide in focal cerebral ischemia, an experimental study.

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Nitric Oxide in Focal Cerebral Ischemia, an Experimental Study

Nitric O

xide in Focal Cerebral Ischemia, an Experim

ental StudyB.A

. Coert B.A. Coert


B.A. Coert

Copyright 2008 B.A. Coert

Nitric Oxide in Focal Cerebral Ischemia, an Experimental StudyB.A. Coert, proefschrift Universiteit van Amsterdam, Amsterdam, The Netherlands (met Nederlandse samenvatting / with Dutch summary)ISBN 978-90-8559-356-0Projects were funded by Grant No. RO1-25374 from the National Institute of Health, and by de Netherlandse Hartstichting, Dr. H. Muller Vaderlandsch Fonds and Genootschap “Noorthey”.Financial support has been given by:SynthesVan Leersum fonds KNAWZeiss

Printed by Optima Gra� sche Communicatie, Rotterdam


ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctoraan de Universiteit van Amsterdamop gezag van de Rector Magni� cus

prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde

commissie, in het openbaar te verdedigen in de Agnietenkapel

op donderdag 6 maart 2008, te 10.00 uur

door Bernard Arthur Coert

geboren te Tilburg

PROMOTIECOMMISSIE

Promotor: Prof. dr. W.P. Vandertop

Overige leden: Prof. dr. C.A.F. Tulleken Prof. dr. J. Stam Prof. dr. M. Vermeulen Prof. dr. C.J.F. van Noorden Prof. dr. E.T. van Bavel

Faculteit der Geneeskunde

In loving memory of my mother Jannetje Coert - de Vries

CONTENTS

List of abbreviations 8

Chapter 1 Introduction 9

Chapter 2 Reproducibility of Cerebral Cortical Infarction in the Wistar Rat After Middle Cerebral Artery Occlusion

17

Chapter 3 A Comparative Study of the E� ects of Two Nitric Oxide Syn-thase Inhibitors and Two Nitric Oxide Donors on Temporary Focal Ischemia in the Wistar Rat

33

Chapter 4 Exogenous Spermine Reduces Ischemic Damage in a Model of Focal Cerebral Ischemia

49

Chapter 5 Is the Neuroprotective E� cacy of nNOS Inhibitor 7-NI Dependent on Ischemic Intracellular pH ?

59

Chapter 6 E� ects of the Nitric Oxide Donor 3-Morpholinosydnomine (SIN-1) in Focal Cerebral Ischemia Dependent on Intracel-lular pH.

77

Chapter 7 Discussion 93

Chapter 8 Summary 103

Samenvatting 113

Chapter 9 Acknowledgements 121

Chapter 10 Curriculum Vitae 125

LIST OF ABBREVIATIONS

7-NI 7-nitroindazole

AT-1 angiotensin II type I

CBF cerebral blood � ow

CCA common carotid artery

CCAo common carotid artery occlussion

cNOS constitutive nitric oxide synthase

CV coe� cient of variation

DETA NO (Z)-1-[2(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate

DMSO dimethyl sulfoxide

EDRF endothelium derived relaxing factor

eNOS endothelial nitric oxide synthase

H&E Hematoxylin and eosin

HMG-coA 3-hydroxy-3-methyl-glutaryl-coA

iNOS inducble nitric oxide synthase

LCBF local cerebral blood � ow

L-NAME NG-nitro-L-Arginine-methyl-esther

MABP mean arterial blood pressure

MCA middle cerebral artery

MCAo middle cerebral artery occlussion

NaCl sodium chloride

NADH nicotinamide adenine dinucleotide

NMDA N-methyl-D-aspartate

nNOS neuronal nitric oxide synthase

NO nitric oxide

NOS nitric oxide synthase

PA polyamine

PaCO2 arterial bloodgas carbon dioxide pressure

PaO2 arterial bloodgas oxygen pressure

pHi intracellulaire pH

rCBF regional cerebral blood � ow

SAMD S-adenosyl methionine decarboxylase

SD standard deviation

SE standard error

SIN-1 3-morpholinosydnomine

SSAT spermidine/spermine acetyl transferase

TBARS thiobarbituric acid-reactive substance

TTC 2,3,5,-triphenyltetrazolium chloride

Chapter 1

Introduction

Introduction 11

INTRODUCTION

Cerebral ischemia is an important cause of brain damage and can lead to temporary or permanent loss of function in various disease entities. De� cits in essential substrate delivery and insu� cient clearance of toxic metabolic waste products both create a harm-ful environment for neural structures and their surroundings. In-depth knowledge of the pathophysiological mechanisms and cascades activated during this process will help to develop therapeutic strategies. An array of di� erent experimental in vivo and in vitro models is available to study the pathophysiology of cerebral ischemia, all mimicking vari-ous types of ischemia (global versus focal). Although in vitro models provide more con-trolled environments, they cannot incorporate important outcome determining factors such as auto regulation and microvasculary changes. For this purpose, in vivo models are far more suitable. Considerations that should be made before using existing in vivo models include the type of ischemia studied and the reproducibility of the ischemia. This reproducibility, which has been found to be a� ected by many factors like animal strain18, vendor18, location and duration of arterial occlusion and anesthesia technique25, is the � rst topic of this thesis.

NO production is a cardinal step in the pathophysiology of cerebral ischemia5,7. The discovery of NO as an endothelium-derived relaxing factor (EDRF) revolutionized think-ing about cell to cell communication. Its importance was con� rmed with a Nobel price for Robert Furchgott, Louis Ignarro and Ferid Murad in 1998. NO production was found to be an important mediator of ischemic damage21. On the other hand NO’s powerful vasodilator action and inhibition of platelet aggregation contributes to collateral � ow, thereby reducing ischemic damage. Di� erent sources of NO production have been iden-ti� ed in the di� erent isoforms of the enzyme: neuronal NO-Synthase (nNOS), endothelial NO-Synthase (eNOS) and inducible NO-Synthase (iNOS). In cell cultures, NO was found to be toxic in a dose dependent way8, but in vivo the e� ect varies from detrimental to neuroprotective12. Since direct measurements of NO, which in an oxygen-containing environment has a very short half-life in the order of seconds13, are complicated, NOS inhibitors and NO donors have been used to study the role of NO in the pathophysiology of cerebral ischemia. Information on the mechanisms and actions of NO has been largely derived from studies in which NOS inhibitors have been used in vivo, but published re-ports have shown contradictory results, with both cerebroprotective and detrimental e� ects of NOS inhibition3. A comparison of the e� ects of NO modulators in vivo between di� erent investigations is complicated by di� erences in methodology, including animal model, anesthetic agent, occlusion technique, duration of ischemia, and drug dosing. To reduce the risk of alternative, a-speci� c e� ects of the inhibitors and donors, we set out to

12 Chapter 1

compare the e� ects of two di� erent NOS inhibitors and NO donors in a focal temporary ischemia model.

Polyamines with NO donor capacity reduce ischemic injury20; while in general they are important for cell growth, di� erentiation23 and numerous cellular functions including protein phosphorylation and protein synthesis16, regulation of gene expression, pro-grammed cell death, inhibition of the mitochondrial inner membrane permeability15, regulation of mitochondria Ca2+ transport17, free radical scavenging24 and the regulation of nitric oxide synthase (NOS)11. The role of the polyamine part itself in the pathophysiol-ogy of cerebral ischemia has not been elucidated. Following either transient global, focal, or focal permanent cerebral ischemia, there is a signi� cant increase in polyamine me-tabolism with increased ornithine decarboxylase (ODC) activity and increased putrescine levels19, which is subsequently metabolized to spermidine and spermine22. It has been demonstrated that spermine concentrations in brain are either slightly reduced14 or moderately increased14 following global cerebral ischemia. In focal cerebral ischemia, signi� cant reductions or no alterations in spermine concentrations have been dem-onstrated4,19. Since the polyamine may contribute to the protection against ischemic damage, we tested the e� ects of exogenous spermine on stroke size in a focal cerebral ischemia model.

Although variable results of NO modulations in experimental focal cerebral ischemia1 can be partly explained by previously discussed variables like strain, dosing and model, we investigated the e� ect of intra-ischemic intracellular pH (pHi). The activities of endothe-lial and neuronal NOS enzymes have been shown to be dependent on brain intracellular pH (pHi), with an optimal enzyme activity at a pH of 7.6 for (microsomal) eNOS10 and at a pH of 6.7 for nNOS9. The hypothesis is tested that the severity of ischemic brain acidosis a� ects the activity of the NOS enzyme and with this its contribution to ischemic dam-age. As it is not possible to measure brain pH and NO simultaneously in vivo, given the current technology, the neuroprotective e� ects of NOS inhibitors and NO donors were used to study the e� ect of pHi on the production of NO. The degree of brain acidosis was manipulated by altering serum glucose concentrations2,6.

Introduction 13

In this thesis the following questions are addressed:

Does the severity of ischemia a� ect the reproducibility of cortical infarction • in a temporary focal cerebral ischemia model in the Wistar rat (chapter 2)?

How do the e� ects of NOS inhibition compare to NO donor treatment in this • focal cerebral ischemia model (chapter 3)?

Can spermine contribute to the protective e� ects of the NO donor spermine • NONOate (chapter 4)?

Can intracellular pH explain the reduced e� ectivity of NOS inhibition in more • severe cerebral ischemia (chapter 5)?

Do the observed e� ects of NO donor treatment in more severe cerebral isch-• emia comply with the e� ect of pHi on the NOS enzyme (chapter 6)?

To discuss subsequent developments chapter 7 addresses the transfer of knowledge from laboratory to clinic and the possibles roles for NO in future therapies. Chapter 8 contains the summary.

14 Chapter 1

REFERENCES

1. Adachi N, Lei B, Soutani M, Arai T: Di� erent roles of neuronal and endothelial nitric oxide synthases on ischemic nitric oxide production in gerbil striatum. Neurosci Lett 288:151-154, 2000

2. Anderson RE, Tan WK, Martin HS, Meyer FB: E� ects of glucose and PaO2 modulation on cortical in-tracellular acidosis, NADH redox state, and infarction in the ischemic penumbra. Stroke 30:160-170, 1999

3. Ashwal S, Cole DJ, Osborne TN, Pearce WJ: Dual e� ects of L-NAME during transient focal cerebral ischemia in spontaneously hypertensive rats. Am J Physiol 267:H276-284, 1994

4. Baskaya MK, Rao AM, Dogan A, Donaldson D, Gellin G, Dempsey RJ: Regional brain polyamine levels in permanent focal cerebral ischemia. Brain Res 744:302-308, 1997

5. Choi DW: Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623-634, 1988

6. Chopp M, Welch KM, Tidwell CD, Helpern JA: Global cerebral ischemia and intracellular pH during hyperglycemia and hypoglycemia in cats. Stroke 19:1383-1387, 1988

7. Dawson VL, Dawson TM, Bartley DA, Uhl GR, Snyder SH: Mechanisms of nitric oxide-mediated neu-rotoxicity in primary brain cultures. J Neurosci 13:2651-2661, 1993

8. Dawson VL, Dawson TM, London ED, Bredt DS, Snyder SH: Nitric oxide mediates glutamate neuro-toxicity in primary cortical cultures. Proc Natl Acad Sci U S A 88:6368-6371, 1991

9. Gorren AC, Schrammel A, Schmidt K, Mayer B: E� ects of pH on the structure and function of neu-ronal nitric oxide synthase. Biochem J 331 ( Pt 3):801-807, 1998

10. Hecker M, Mulsch A, Busse R: Subcellular localization and characterization of neuronal nitric oxide synthase. J Neurochem 62:1524-1529, 1994

11. Hu J, Mahmoud MI, el-Fakahany EE: Polyamines inhibit nitric oxide synthase in rat cerebellum. Neurosci Lett 175:41-45, 1994

12. Iadecola C: Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci 20:132-139, 1997

13. Kelm M, Feelisch M, Spahr R, Piper HM, Noack E, Schrader J: Quantitative and kinetic character-ization of nitric oxide and EDRF released from cultured endothelial cells. Biochem Biophys Res Commun 154:236-244, 1988

14. Koenig H, Goldstone AD, Lu CY, Trout JJ: Brain polyamines are controlled by N-methyl-D-aspartate receptors during ischemia and recirculation. Stroke 21:III98-102, 1990

15. Lapidus RG, Sokolove PM: Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria. FEBS Lett 313:314-318, 1992

16. Lenzen S, Hickethier R, Panten U: Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport. J Biol Chem 261:16478-16483, 1986

17. Lenzen S, Munster W, Rustenbeck I: Dual e� ect of spermine on mitochondrial Ca2+ transport. Biochem J 286 ( Pt 2):597-602, 1992

18. Oli� HS, Weber E, Eilon G, Marek P: The role of strain/vendor di� erences on the outcome of focal ischemia induced by intraluminal middle cerebral artery occlusion in the rat. Brain Res 675:20-26, 1995

19. Paschen W, Csiba L, Rohn G, Bereczki D: Polyamine metabolism in transient focal ischemia of rat brain. Brain Res 566:354-357, 1991

Introduction 15

20. Salom JB, Orti M, Centeno JM, Torregrosa G, Alborch E: Reduction of infarct size by the NO donors sodium nitroprusside and spermine/NO after transient focal cerebral ischemia in rats. Brain Res 865:149-156, 2000

21. Samdani AF, Dawson TM, Dawson VL: Nitric oxide synthase in models of focal ischemia. Stroke 28:1283-1288, 1997

22. Seiler N: Polyamine oxidase, properties and functions. Prog Brain Res 106:333-344, 1995

23. Tabor CW, Tabor H: Polyamines. Annu Rev Biochem 53:749-790, 1984

24. Tadolini B: The in� uence of polyamine-nucleic acid complexes on Fe2+ autoxidation. Mol Cell Biochem 83:179-185, 1988

25. Warner DS, Ludwig PS, Pearlstein R, Brinkhous AD: Halothane reduces focal ischemic injury in the rat when brain temperature is controlled. Anesthesiology 82:1237-1245; discussion 1227A, 1995

Chapter 2

Reproducibility of Cerebral Cortical Infarction in the Wistar Rat After Middle

Cerebral Artery Occlusion

Bert A. Coert MD

Robert E. Anderson BS

Fredric B. Meyer MD

Journal of Stroke and Cerebrovascular Diseases vol.8, No.6 (November-December), 1999 pp380-387

18 Chapter 2

ABSTRACT

Although middle cerebral artery (MCA) occlusion in the rat is often used to study focal cerebral ischemia, the model of ischemia a� ects the size and reproducibility of infarction. The purpose of this experiment was to methodically examine di� erent preparations to determine the optimum focal cerebral ischemia model to produce a reproducible severe ischemic injury. Eighty-two Wistar rats underwent 1 hour, 3 hour, or permanent MCA oc-clusion combined with no, unilateral, or bilateral common carotid artery (CCA) occlusion. Three days after ischemia, the animals were prepared for tetrazolium chloride assess-ment of infarction size. One-hour MCA occlusion produced a coe� cient of variation (CV) of 200% with an infarction volume of 20.3 ± 10.5 mm3. Adding unilateral or bilateral CCA occlusion resulted in a CV of 134% and 101%, respectively. Three-hour MCA occlusion combined with bilateral CCA occlusion decreased the CV to 58% with a cortical infarction volume of 82.6 ± 12.1 mm3, P < 0.05, compared with 1-hour MCA occlusion with or with-out CCA occlusion. Permanent MCA occlusion combined with 3 hours of bilateral CCA occlusion resulted in a CV of 47% with a cortical infarction volume of 89.6 ± 16.0 mm3. These results indicate that 3-hour MCA occlusion combined with bilateral CCA occlusion consistently provides a large infarction volume after temporary focal cerebral ischemia.

Reproducibility of infarction after MCAo 19

INTRODUCTION

Numerous experimental animal models have been developed to study the pathophysiol-ogy and to test therapeutic interventions in focal cerebral ischemia. Rodent models have gained considerable interest for a variety of reasons including their low cost and wide availability, and new possibilities with genetically altered animals. Because of Tamura et al’s1 description of a middle cerebral artery occlusion (MCAO) technique, MCAO in rats has been widely used as a model of focal cerebral ischemia. Unfortunately, there is signi� cant variability in the reproducibility and size of cortical infarction using the MCAO model in Wistar rats2,3. This makes it problematic to investigate the pathophysiology and to compare the e� cacy of therapeutic interventions. This variability or inconsistency can be attributed to the model (location and duration of occlusion), strain/vendor4,5, age/weight6,7, anesthesia8, brain and body temperature9, systemic parameters (PaO2, PaCO2, and pH); blood pressure10,11 and serum glucose levels (Table 1) 12-14. The purpose of this experiment was to rigorously examine various models of temporary focal cerebral is-chemia in the Wistar rat to improve consistency and reproducibility of cortical infarc-tion.

MATERIAL AND METHODS

After review and approval by the Institutional Animal Care and Use Committee, 82 adult male Wistar rats weighing between 350 and 450 g were anesthetized with halothane at 1.5% during the surgical exposure and at 1.0% during the occlusion period. The animals were spontaneously breathing with a mixture of air and oxygen through a face mask. Atropine was administered preoperatively at 0.1 mg/kg subcutaneously to reduce res-piratory secretions. Core body temperature was monitored with a rectal probe and was maintained at 37 ± 0.5°C by using an infrared heating lamp which warmed both the head and body simultaneously. Head and brain temperature, therefore, was maintained at 37 ± 0.5°C throughout the entire experiment from the beginning of the surgical prepara-tion. A polyethylene catheter (PE-50; IntraMedic AB, Balsta, Sweden) was inserted into the right femoral artery to monitor arterial blood pressure and to sample blood for meas-urements of PaO2, PaCO2, pH, and serum glucose. All animals had free access to water and food before and after surgery. Body weight of the rats was recorded preoperatively and before they were killed to examine weight loss.

Temporary Left MCAO

Temporary MCAO was obtained through a modi� cation of the technique originally de-scribed by Tamura et al1. Brie� y, a 2-cm skin incision was made between the left outer

20 Chapter 2

Table

1. Re

view

of te

mpo

rary

clip

and l

igatu

re M

CA oc

clusio

ns in

rats

(mea

n ± SE

)

(Abb

revia

tions

: MCA

, midd

le ce

rebr

al ar

tery

; SE,

stand

ard e

rror;

n.a.,

not a

vaila

ble; s

b, sp

onta

neou

s bre

athin

g; M

ABP,

mea

n arte

rial b

lood p

ressu

re; M

CAO,

midd

le ce

rebr

al ar

tery

occlu

sion;

perm

, per

man

ent;

tem

p, te

mpo

rary

; coa

g, co

agula

tion;

olf, o

lfacto

ry tr

act;

bilat

, bila

tera

l; CCA

O, co

mm

on ca

rotid

arte

ry oc

clusio

n; H

& E,

hem

atox

ylin a

nd eo

sin; T

TC, 2

,3,5-

triph

enyl

tetra

zoliu

m.)


canthus and the tragus. After de� ecting the temporal muscle anteriorly, the middle part of the left zygomatic arch was removed without damaging the facial nerve. Muscles were retracted both downward and anteriorly, after which the mandibular nerve was identi-� ed and followed back to the foramen ovale. A 4 mm craniectomy was made just anterior and superior to the foramen ovale with the use of a high speed air drill. After opening the dura with a sharp needle, the left MCA was dissected free of the arachnoid. A Sundt #2 AVM microclip (Codman and Shurtle� , Inc, Raynham, MA) was applied to the MCA crossing the olfactory tract.

Bilateral Common Carotid Artery Occlusion (CCAO)

A ventral midline incision was made and both CCAs were exposed and isolated from the surrounding tissue. The contralateral CCA was permanently ligated using a 3.0 silk suture, the ipsilateral CCAO with a miniature May� eld aneurysm clip was temporary (1 hour or 3 hours).

Permanent Left MCAO

Surgical exposure was previously described for the temporary occlusion of the MCA. Af-ter freeing the MCA from the arachnoid, a bipolar forceps was used to carefully coagulate the MCA from the olfactory tract to the inferior cerebral vein. To prevent recanalization, the MCA was transected using microscissors.

Experimental Study Groups

The animals were divided into 7 groups. The � rst 3 groups underwent 1-hour MCAO with either 1 day (n = 13), 3 days (n =15), or 7 days (n = 6) of reperfusion to examine for pos-sible e� ects of reperfusion time on infarction volume. Groups 4 through 7 had 3 days of reperfusion after the ischemic experiment before histological preparation. Group 4 (n = 10) underwent 1-hour MCAO with ipsilateral CCA occlusion. Group 5 (n = 16) had 1-hour MCAO and bilateral CCAO (MCAO + 2 CCAO). In group 6 (n = 16), the duration of occlusion was 3 hours for both MCAO and bilateral CCAO. In group 7 (n = 6), animals underwent permanent MCAO with 3-hour-bilateral CCAO. Ipsilateral CCAO was always temporary, whereas the contralateral CCAO (only in the bilateral CCAO groups) was permanent.

Histopathology

After the designated reperfusion period, the animals were reanesthetized, weighed, and perfused intracardially with a warm (37˚C) 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution. The brains were quickly removed and then immersed in 37˚C, 2% TTC solution for 15 minutes to enhance staining and then placed in a 10% bu� ered formaldehyde solution for 5 days. Twelve serial coronal sections were cut at 1-mm intervals, beginning at the frontal pole, by using a rodent brain matrix (ASI Instruments Inc, Warren, MI), and photographed (Fig 1). Areas of unstained tissue (infarcted) were easily disseminated

22 Chapter 2

from areas of viable tissue, which stained red or pink. The infarcted area of each tis-sue section was traced by a computer-assisted image analyzer (JAVA; Jandel Scienti� c Software, SPSS, Inc, Chicago, IL). This analyzer was previously calibrated to express the measurement in square millimeters. Total cortical infarction volume was calculated by integrating the infarcted area in each slide (area of infarction in square millimeters times thickness of the slice, 1 mm). The percentage of animals with infarction was determined by dividing the number of infarcted animals by the total number of animals within each study group. Edema ratio was determined by dividing the left hemispheric area by the right hemispheric area.

Figure 1. TTC histology of adjacent serial coronal brain sections that were cut at l-mm intervals after 3 hours MCA and bilateral CCA occlusion, followed by a 3-day reperfusion period. For colour image see front cover.


Statistical Analysis

Analysis of variance (ANOVA) with Tukey’s posthoc test for multiple comparisons was used for statistical analysis of infarction volume, edema ratio, and percentage of weight loss. The coe� cient of variation (CV) was computed to provide an index of experimental variation for each group. Two-sample F test was used for statistical analysis of rate of infarction and CV. Statistical analysis of systemic parameters were performed using the unpaired t-test. Data were expressed as mean and standard error. P values less than 0.05 were considered to be signi� cant.

RESULTS

Systemic Parameters

The physiologic parameters, which were mean arterial blood pressure (MABP), PaO2, PaCO2, pH, serum glucose, and core body temperature, are shown in Table 2. There were no signi� cant di� erences between the groups studied.

Table 2. Systemic parameters (mean ± SE)

(Abbreviations: SE, standard error; MABP mean arterial blood pressure; MCAO, middle cerebral artery occlusion; n.a., not available; CCAO, common carotid artery occlusion). NOTE: Core body and head temperature were maintained at 37 ± 0.5ºC. These systemic parameters were measured at the end of the ischemic period.

Table 3. Measured parameters (mean ± SE)

(Abbreviations: SE, standard error; CV, coe� cient of variation; MCAO, middle cerebral artery occlusion; CCAO, common carotid artery occlusion.)*Signi� cantly di� erent from 1 hr MCAO 1-, 3-, and 7-day sacri� ce groups and 1 hr MCAO + 1 CCAO group, P < 0.05.† Signi� cantly di� erent from 1 hr MCAO + 2 CCAO group, P < 0.05.‡ Signi� cantly di� erent from 1 hr MCAO 1-day sacri� ce group, P < 0.05.§ Signi� cantly di� erent from 1 hr MCAO 3-day sacri� ce group, P < 0.05.

24 Chapter 2

Experimental Groups

E� ect of Reperfusion on Infarction Rate and Volume (groups 1, 2, and 3)

The infarction rates and infarction volumes for the 3 1-hour MCAO without CCAO groups that were killed 1, 3, or 7 days after ischemia are described in Table 3. There were no signi� cant di� erences in infarction rate, CV, or infarction volume among these groups.

72-Hour Reperfusion (groups 4, 5, 6, and 7)

The 1-hour MCAO group without CCAO resulted in infarction in 7 of 15 animals with a CV of 200%. Adding ipsilateral and bilateral CCAO resulted in infarction in 6 of 10 animals with a CV of 134% and in 12 of 16 animals with a CV of 101%. The group with 3-hour MCAO and 2 CCAOs increased the infarction rate to 88% (14/16) with a signi� cant decrease in the CV to 58%, compared with the 1-hour MCAO group. Permanent MCAO combined with 3-hour bilateral CCAO resulted in cortical infarction in all animals (6/6) with a CV of 47%, signi� cantly di� erent from the 1-hour MCAO group (P < 0.05) (Table 3).

The cortical infarction volume in the 1-hour MCAO measured 20.3 ± 10.5 mm3. Ipsilat-eral CCAO added to the 1-hour MCAO did not signi� cantly alter the average infarction volume (21.3 ± 9.0 mm3), whereas bilateral CCAO did signi� cantly (P < 0.05) increase infarction volume to 51.9 ± 13.1 mm3 (Table 3). When the occlusion time for MCA and bilateral CCA was extended to 3 hours, there was a signi� cant (P < 0.05) increase in the volume of infarction to 82.6 ± 12.1 mm3, when compared with the group with 1-hour MCAO and 2 CCAOs. In the group with permanent MCAO and 3-hour bilateral CCAO, the infarction volume was 89.6 ± 16.0 mm3. The di� erence in infarction volume between the group with 3-hour MCAO and bilateral CCAO and the group with 1-hour MCAO and CCAO was signi� cant (P < 0.05). There was no signi� cant di� erence between the 3-hour MCAO with bilateral CCAO group and the group with permanent MCAO and 2 CCAOs.

Edema

Infarction volumes of both hemispheres were measured and compared to identify pos-sible edema. Edema in the ischemic hemisphere would increase the overall measured infarction volume. Because edema has been described to peak at 24 hours after occlu-sion15, as the di� erent reperfusion intervals (1, 3, and 7 days) were considered separately (Table 3). Overall, the average ischemic hemispheric volumes were 580.3 ± 6.1 mm3 and 574.4 ± 6.6 mm3 for the nonischemic (right) hemisphere and ischemic (left) hemisphere, respectively. There were no di� erences among any of these groups.


Weight Loss

Average weight loss in the total group of 82 animals measured was 52.1 g (19.1% of the preoperative body weight). Weight loss correlated well with the time between surgery and death when comparing the 3 1-hour MCAO (1, 3, and 7 day) groups that were killed (Table 3). Increasing the severity of the ischemic insult from 1-hour MCAO with 2 CCAOs, to 3-hour MCAO with 2 CCAOs, to permanent MCAO with 2 CCAOs signi� cantly increased weight loss in these groups (P < 0.05) (Table 3).

DISCUSSION

Volume and reproducibility of cortical infarction in rat MCAO models depend on many factors including exact location of occlusion16, duration of occlusion17, anesthetic8, brain and body temperature9, blood pressure18, glucose12, and histological techniques. Rat strain and vendor have also been identi� ed in several studies to e� ect infarction vol-ume and reproducibility in rat MCAO models17,19. Di� erent start-up stocks and breed-ing strategies make outbred strains like Sprague Dawley and Wistar more susceptible to vendor-dependent genetic divergence5. The incidence of abnormalities in the rat’s circle of Willis could explain the reported higher variability of infarction volume in these strains when compared with F344 rats19. Anatomical studies have shown that the presence of more proximal MCA side branches may be responsible for the di� erences in variability in the neuropathological outcome of focal cerebral ischemia between F344 and Wistar rats4. Age-related di� erences in ischemic susceptibility in rats have been proposed by Sutherland et al.7, who found signi� cantly larger infarctions in older Wistar rats (26 to 28 months, 595 g) as compared with younger Wistar rats (2 to 3 months, 353 g). However, Duverger and MacKenzie19, in a study with F344 rats, did not � nd signi� cant di� erences in the 3 di� erent age groups.

The staining technique with TTC in this study is an accurate, rapid, and inexpensive way to delineate cerebral infarction20. Tissue changes caused by ischemia have been reported to parallel irreversible changes seen in hematoxylin and eosin (H&E) staining at 1 day and 3 days15,16. The accuracy of TTC at 7 days is unknown because in� ammatory response may result in unreliable TTC staining21. This is the rationale of choosing the 3-day time for comparing the models in this experiment. Isayama et al. 20, compared the TTC staining technique with that of H&E staining in a permanent MCAO model. The TTC staining tech-nique tended to show larger infarctions than H&E staining, for which 2 possible explana-tions were given: compromised accessibility of TTC to the tissue in permanent MCAO and tissue shrinkage in conventional histologic techniques20. Random assessment by H&E staining did not show any di� erence when compared with TTC staining.

26 Chapter 2

Brain edema in rats can cause overestimation of infarction volume by up to 22%2. Some investigators have found that maximal infarction volume was reached at 24 hours after occlusion22,23. Other investigators have suggested that the duration of occlusion deter-mines the incidence and time course of cerebral edema24. Indirect measurements have been proposed to minimize this error25. Lin et al.15, reported smaller infarction volumes at 7 days of reperfusion when compared with 1 day and 3 days, even with indirect measure-ment23. Anesthetics, such as halothane, in higher doses can reduce ischemic brain edema by counteracting superimposed vasogenic edema during re� ow by reducing hyperemia through a decrease in MABP and cerebral perfusion pressure26. Although reperfusion has been reported to aggravate edema formation in other species23, Kaplan et al.27 found no di� erence in edema volume between the 3 and 4 hours of temporary MCAO and perma-nent occlusion groups. Speci� c gravity measurements in Shigeno’s study showed edema in permanent occlusion groups and not in any of the reperfusion groups28. A close correlation was found between the size of infarction and edema volume27. This could indicate that the origin of the edema was more likely cytotoxic than vasogenic. Left and right hemispheric volumes were not signi� cantly di� erent in any group in our study.

One-hour MCAO in our experience did not consistently result in infarction. Hiramatsu et al. 3, showed that none of the 1-hour and 2-hour MCA-occluded rats had infarctions. Focal permanent occlusion in Bederson’s16 study resulted in infarction in 67% (site of occlusion proximal to the olfactory tract), 13% (MCA origin from internal carotid artery), or 0% (distal of the inferior cerebral vein). Coyle, in his study on the collateral circulation in rats, conclud-ed that the location of MCAO determined the collateral � eld29. Reduction of collateral � ow after MCAO will increase the severity of cerebral ischemia and MCA territory infarction30. Divergent genetic coding for vascular collateral � ow could explain variability in infarctions after MCAO in rats31. In our study, adding ipsilateral CCAO to reduce collateral � ow resulted in a 60% infarction rate, whereas, in the bilateral CCAO group, 75% of rats had infarctions. Relative blood � ow in the cortex was reduced from 62% with distal MCAO without CCAO, to 48% with ipsilateral CCAO, and to 18% with bilateral CCAO in laser Doppler � ow studies32. Addition of the bilateral CCAO with 3 hours of MCAO resulted in an increase in the infarction rate from 25% to 100% in Hiramatsu’s study3. Extending occlusion time from 1 hour to 3 hours in his study resulted in a more reproducible infarction volume; this suggests that the severity of ischemia may a� ect variability of outcome. When occlusion time is prolonged, infarction volume enlarges progressively until it approximates permanent occlusion27,33,34. In our study, the duration of occlusion was extended from 1 hour to 3 hours to improve reproducibility. In the group with 3-hour MCA and 2 CCAO, the CV was 58%. To con� rm that 3-hour MCAO and 2 CCAOs could equal permanent occlusion, a group of rats was permanently occluded with 3 hours of 2 CCAOs. This resulted in a CV of 47% with an aver-age infarction volume of 89.6 ± 16.0 mm3, whereas 3 hours of MCAO and 2 CCAOs resulted in an average infarction volume of 82.6 ± 12.1 mm3. The di� erence between these groups


was not signi� cant. By contrast, Hiramatsu’s study3 showed that 3 hours of MCAO with 2 CCAOs in Sprague Dawley rats resulted in an infarction volume of 177.4 ± 6.3 mm3 with a CV of 11%. Herz et al.4, using permanent MCAO in Wistar rats, showed an infarction volume of 44.2 ± 11.3 mm3, and the CV was 26%. Xue et al.35 in Wistar rats, showed an infarction volume of 211 ± 14 mm3, with a CV of 17% as a result of 3 hours of MCAO. Xue, using per-manent MCAO, however, showed an infarction volume of 142 ± 18 mm3 with a CV of 31%. These results depict a wide variation in infarction volume and reproducibility among inves-tigators and also within an investigation (permanent v temporary occlusion). In a review of the literature, it is not possible to ascertain the reasons for the discrepancies in the same species (e.g. the Wistar rat), because of the extreme variability in experimental protocols. Brain temperature during and after ischemia has been reported to a� ect distribution and extent of ischemic injury8,9,36. Anesthetics, such as halothane, iso� urane, and thiopental, are cerebroprotective during ischemic injury37. Hypothermia induced by anesthesia could be partly responsible for this e� ect. The e� ect of halothane on MABP should also be taken into consideration. Oli� et al.5 reported that raising the halothane concentration from 2% to 2.5% reduced MABP from 102 mmHg to 74 mmHg, which resulted in an 8-fold increase in cortical infarct volumes from 14.2 mm3 (2% halothane) to 113.6 mm3 (2.5% halothane). Lowering MABP increased the severity of the ischemic event18 by decreasing cerebral perfusion pressure, which reduced collateral � ow. Zhu and Auer11 reported mortality rates of 50% for rats undergoing 2-hour MCAO at a MABP of 40 mmHg, which resulted in an average infarction volume of 174 mm3. In our study, core body and head temperature was controlled at 37 ± 0.5˚C with an average MABP of 91.4 ± 1.0 mmHg under 1% halothane anesthesia. Data from the study by Oli� et al.5, including average MABPs for di� erent rat strains and average infarction volumes with MCAO and CCAO, showed an obvious decrease in infarction volume with increasing MABP. Phenylephrine-induced hypertension during MCAO attenuated size of infarction zones of severely decreased local cerebral blood � ow (LCBF) (0 to 15 mL/100 g/min)10. Increased cerebral perfusion pressure-dependent collat-eral CBF and inverse steal caused by phenylephrine-induced vasoconstriction in normal brain regions were identi� ed as possible mechanisms for these changes in CBF10.

Temporomandibular joint dysfunction has been described as interfering with the animal’s ability to eat38. Weight loss did appear after surgery in our study. Neurologi-cal de� cit could also account for the reduced body weight when the animal was killed. Although we did not perform an extensive neurological evaluation in our study, no focal neurological signs were observed except for some limping, which can be attributed to femoral artery occlusion after removal of the femoral arterial catheter. In general, agility appeared to be reduced in most animals. Duverger and MacKenzie 19 described hyperac-tive and aggressive behaviour postoperatively that we did not observe. In comparing our 1-, 3-, and 7-day reperfusion groups, we observed an almost linear weight loss over time.

28 Chapter 2

Weight loss has also been described in the intraluminal cerebral ischemia model in which temporomandibular dysfunction does not occur. A correlation was found in this study between duration of MCAO, MABP, and weight loss11. Yamamoto et al., in their study, found a 12.5% weight loss after 1-hour MCAO and 7 days of reperfusion38.

In conclusion, 1-hour proximal MCAO in Wistar rats did not consistently result in infarction. Because edema has been reported to maximize at approximately 24 hours22,23, and post-22,23, and post-22,23

operative weight loss in our study averaged 24.2% of total body weight at 7 days, a 3-day reperfusion period was chosen. Reducing collateral � ow through unilateral or bilateral CCAO augmented infarction rate to 60% and 75% and reduced variability to 134% and 101%, re-spectively. Because increased severity of ischemia can reduce variability3spectively. Because increased severity of ischemia can reduce variability3spectively. Because increased severity of ischemia can reduce variability , occlusion time was 3, occlusion time was 3

extended to 3 hours, resulting in an infarction rate of 88% and a CV of 58%, with an infarction volume of 82.6 ± 12.1 mm3, which was signi� cantly greater when compared with the 1-hour 3, which was signi� cantly greater when compared with the 1-hour 3

MCAO groups with or without CCAO. Reproducibility is a major concern in rat models of tem-porary focal cerebral ischemia. This study shows that 3-hour MCAO combined with bilateral CCAO produces a reliable infarction after temporary focal ischemia.


REFERENCES

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2. Brint S, Jacewicz M, Kiessling M, et al. Focal brain ischemia in the rat: Methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral com-mon carotid arteries. J Cereb Blood Flow Metab 1988;8:474-485.

3. Hiramatsu K, KasseU NF, Goto Y, et al. A reproducible model of reversible, focal, neocortical isch-emia in Sprague- Dawley rat. Acta Neurochir 1993;120:66-71.

4. Herz RC, Jonker M, Verheul HB, et al. Middle cerebral artery occlusion in Wistar and Fischer-344 rats: Functional and morphological assessment of the model. J Cereb Blood Flow Metab 1996;16:296-302.

5. Oli� HS, Weber E, Eilon G, et al. The role of strain/ vendor di� erences on the outcome of fo-cal ischemia induced by intraluminal middle cerebral artery occlusion in the rat. Brain Res 1995;675:20-26.

6. Coyle P. Middle cerebral artery occlusion in the young rat. Stroke 1982;13:855-859.

7. Sutherland GR, Dix GA, Auer RN. E� ect of age in rodent models of focal and forebrain ischemia. Stroke 1996;27: 1663-1668.

8. Warner DS, Ludwig PS, Pearlstein R, et al. Halothane reduces focal ischemic injury in the rat when brain temperature is controlled. Anesthesiology 1995;82:1237-1245.

9. Goto Y, Kassell NF, Hiramatsu K, et al. E� ects of intraischemic hypothermia on cerebral damage in a model of reversible focal ischemia. Neurosurgery 1993;32:980-985.

10. Drummond JC, Oh YS, Cole DJ, et al. Phenylephrineinduced hypertension reduces ischemia fol-lowing middle cerebral artery occlusion in rats. Stroke 1989;20:1538-1544.

11. Zhu CZ, Auer RN. Graded hypotension and MCA occlusion duration: E� ect in transient focal isch-emia. J Cereb Blood Flow Metab 1995;15:980-988.

12. Nedergaard M. Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction. Brain Res 1987;408:79-85.

13. Tan WK, Anderson RE, Meyer FB. Glucose and Pao2 modulation of cerebral metabolic responses following cerebral ischemia. J Cereb Blood Flow Metab 1997;17: $303, (suppl 1, abstr).

14. Warner DS, Gionet TX, Todd MM, et al. Insulin-induced normoglycemia improves ischemic out-come in hyperglycaemic rats. Stroke 1992;23:1775-1781.

15. Lin TN, He YY, Wu G, et al. E� ect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 1993;24:117-121.

16. Bederson JB, Pitts LH, Tsuji M, et al. Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination. Stroke 1986;17:472- 476.

17. Buchan AM, Xue D, Slivka A. A new model of temporary focal neocortical ischemia in the rat. Stroke 1992;23:273- 279.

18. Gionet TX, Warner DS, Verhaegen M, et al. E� ects of intra-ischemic blood pressure on outcome from 2-vessel occlusion forebrain ischemia in the rat. Brain Res 1992;586: 188-194.

30 Chapter 2

19. Duverger D, MacKenzie ET. The quanti� cation of cerebral infarction following focal ischemia in the rat: In� uence of strain, arterial pressure, blood glucose concentration, and age. J Cereb Blood Flow Metab 1988;8:449-461.

20. Isayama K, Pitts LH, Nishimura MC. Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. Stroke 1991;22:1394-1398.

21. Clark RK, Lee EV, Fish CJ, et al. Development of tissue damage, in� ammation and resolution following stroke: An immunohistochemical and quantitative planimetric study. Brain Res Bull 1993;31:565-572.

22. Jacewicz M, Tanabe J, Pulsinelli WA. The CBF threshold and dynamics for focal cerebral infarction in spontaneously hypertensive rats. J Cereb Blood Flow Metab 1992;12:359-370.

23. Sundt TM Jr, Grant WC, Garcia JH. Restoration of middle cerebral artery � ow in experimental infarc-tion. J Neurosurg 1969;31:311-321.

24. Ito U, Go KG, Walker JT Jr, et al. Experimental cerebral ischemia in Mongolian gerbils III. Behaviour of the blood-brain barrier. Acta Neuropatho11976;34:1-6.

25. Swanson RA, Morton MT, Tsao-Wu G, et al. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 1990;10:290-293.

26. Cahn R, Dupont JM, Borzeix MG, et al. E� ect of halothane on ischemic brain edema. Adv Neurol 1990;52: 93-96.

27. Kaplan B, Brint S, Tanabe J, et al. Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia. Stroke 1991;22:1032-1039.

28. Shigeno T, Teasdale GM, McCulloch J, et al. Recirculation model following MCA occlusion in rats. Ce-rebral blood � ow, cerebrovascular permeability, and brain edema. J Neurosurg 1985;63:272-277.

29. Coyle P, Jokelainen PT. Dorsal cerebral arterial collaterals of the rat. Anat Rec 1982;203:397-404.

30. Chen ST, Hsu CY, Hogan EL, et al. A model of focal ischemic stroke in the rat: Reproducible exten-sive cortical infarction. Stroke 1986;17:738-743.

31. Coyle P. Di� erent susceptibilities to cerebral infarction in spontaneously hypertensive (SHR) and normotensive Sprague-Dawley rats. Stroke 1986;17:520-525.

32. Chen Q, Chopp M, Bodzin G, et al. Temperature modulation of cerebral depolarization dur-ing focal cerebral ischemia in rats: Correlation with ischemic injury. J Cereb Blood Flow Metab 1993;13:389-394.

33. Aronowski J, Ostrow P, Samways E, et al. Graded bioassay for demonstration of brain rescue from experimental acute ischemia in rats. Stroke 1994;25:2235-2240.

34. Margaill I, Parmentier S, Callebert J, et al. Short therapeutic window for MK-801 in transient focal cerebral ischemia in normotensive rats. J Cereb Blood Flow Metab 1996;16:107-113.

35. Xue D, Huang ZG, Smith KE, et al. Immediate or delayed mild hypothermia prevents focal cerebral infarction. Brain Res 1992;587:66-72.

36. Markarian GZ, Lee JH, Stein DJ, et al. Mild hypothermia: Therapeutic window after experimental cerebral ischemia. Neurosurgery 1996;38:542-551.

37. Warner DS, McFa� ane C, Todd MM, et al. Sevo� urane and halothane reduce focal ischemic brain damage in the rat. Possible in� uence on thermoregulation. Anesthesiology 1993;79:985-992.

38. Yamamoto M, Tamura A, Kirino T, et al. Behavioral changes after focal cerebral ischemia by left middle cerebral artery occlusion in rats. Brain Res 1988;452:323- 328


39. Morikawa E, Ginsberg MD, Dietrich WD, et al. The signi� cance of brain temperature in focal cere-bral ischemia: Histopathological consequences of middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 1992;12:380-389.

40. Selman W, Bhatti SU, Rosenstein CC, et al. Temporary vessel occlusion in spontaneously hyper-tensive and normotensive rats. E� ect of single and multiple episodes on tissue metabolism and volume of infarction. J Neurosurg 1994;80:1085-1090.

41. David CA, Prado R, Dietrich WD. Cerebral protection by intermittent reperfusion during temporary focal ischemia in the rat. J Neurosurg 1996;85:923-928.

42. Drummond JC, Cole DJ, Patel PM, et al. Focal cerebral ischemia during anesthesia with etomi-date, iso� urane, or thiopental: A comparison of the extent of cerebral injury. Neurosurgery 1995;37:742-749.

Chapter 3

A Comparative Study of the E� ects of Two Nitric Oxide Synthase Inhibitors and Two Nitric Oxide Donors on Temporary Focal

Ischemia in the Wistar Rat

Bert A. Coert MD


Fredric B. Meyer MD

Journal of Neurosurgery Vol.90 February 1999 pp 332-338

34 Chapter 3

ABSTRACT

A critical review of the literature indicates that the e� ects of nitric oxide synthase (NOS) inhibitors on focal cerebral ischemia are contradictory. In this experiment the authors methodically examined the dose-dependent e� ects of two NOS inhibitors and two NO donors on cortical infarction volume in an animal model of temporary focal cerebral ischemia simulating potential ischemia during neurovascular interventions. Ninety-two Wistar rats underwent 3 hours of combined left middle cerebral artery and bilateral common carotid artery occlusion after having been anesthetized with 1% halo thane. A nonselective NOS inhibitor, NG-nitro- L-arginine-methyl-ester (L-NAME), and two NO donors, 3-morpholinosydnonimine hydrochloride and NOC-18, DETA/NO, (Z)-1-[2(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, were administered in-travenously 30 minutes before ischemia was induced. A selective neuronal NOS inhibitor, 7-nitroindazole (7-NI), was administered intraperitoneally in dimethyl sulfoxide (DMSO) 60 minutes before ischemia was induced. Two ischemic control groups, to which either saline or DMSO was administered, were also included in this study. Seventy-two hours after � ow restoration, the animals were perfused with tetrazolium chloride for histologi-cal evaluation. Cortical infarction volume was signi� cantly reduced by 71% in the group treated with 1 mg/kg L-NAME when compared with the saline-treated ischemic control group (27.1 ± 37 mm3 compared with 92.5 ± 26 mm3, p < 0.05). The NOS inhibitor 7-NI signi� cantly reduced cortical infarction volume by 70% and by 92% at doses of 10 and 100 mg/kg: 35.2 ± 32 mm3 (p < 0.05) and 9 ± 13 mm3 (p < 0.005), respectively, when compared with the DMSO-treated ischemic control group (119 ± 43 mm3). There was no signi� cant di� erence between the saline-treated and DMSO-treated ischemic control groups. Treatment with NO donors did not signi� cantly alter cortical infarction volume. These results support an important role for NO in ischemic neurotoxicity and indicate that neuronal NOS inhibition may be valuable in reducing cortical injury in patients suf-fering temporary focal cerebral ischemia during neurovascular procedures.

Comparative study of two NOS inhibitors and two NO donors 35

INTRODUCTION

During cerebral ischemia, excitatory amino acid release activates the N-methyl-D-aspartate (NMDA) receptor, which mediates a transmembrane in� ux of calcium with subsequent deleterious e� ects7,43,44. This rise in intracellular calcium activates calcium-dependent enzymes including neuronal (n) and endothelial (e) nitric oxide synthase (NOS) 45. Inducible NOS, which is calcium independent, requires protein synthesis to be expressed and, thereby, slowly produces large amounts of nitric oxide (NO) over a long period of time during the postischemic phase10. Excessive concentration of NO may mediate the majority of NMDA toxicity45. The half-life of NO in an oxygen-containing environment is short, in the order of seconds29, which complicates direct measurements of NO levels in ischemic tissue. Accordingly, information on the mechanisms and actions of NO has been largely derived from studies in which NOS inhibitors have been used in vivo. Published reports have shown contradictory results, with both cerebroprotec-tive2,50 and detrimental30 e� ects of NOS inhibition. Combining these results has led to a proposed dose-dependent dual role for NO1,10. Selective inhibitors of nNOS have dem-onstrated more consistent protective results24, suggesting that this dualism is related to the di� erent isoforms of NOS. However, even the selectivity and mechanisms of action of NOS antagonists have been questioned40,51. With the availability of genetically altered animals, it has been possible to by-pass possible nonspeci� c side e� ects of pharmaceu-tical intervention. nNOS “knockout” animals developed smaller infarctions after middle cerebral artery (MCA) occlusion when compared with the wild type13,20. The successful use of NO donors in acute myocardial ischemia has raised questions about its similar e� -cacy in focal cerebral ischemia. In a rat model of focal cerebral ischemia, NO donors were protective if hypotension was avoided53. Alternately, in cortical cultures, NO donors were neurotoxic12. A comparison of the e� ects of di� erent NO modulators in vivo between di� erent investigations is complicated by di� erences in methodology, including animal model, anesthetic agent, occlusion technique, duration of ischemia, and drug dosing. The objective of this study was to examine rigorously the dose-dependent e� ects of two NOS inhibitors and two NO donors on cortical infarction volume in a model of temporary focal cerebral ischemia that simulates possible ischemia during neurovascular procedures.

MATERIALS AND METHODS

Following review and approval of the protocol by our institutional Animal Care and Use Committee, 92 adult male Wistar rats were administered halothane in a mixture of oxy-gen and air through a face mask at 1.5% during the surgical procedure and 1% during the occlusion period. Subcutaneous glycopyrrolate was administered preoperatively

36 Chapter 3

at 4 mg/kg to reduce respiratory secretions. Core body temperature was continuously monitored at the beginning of the surgical preparation and throughout the experiment by using a rectal probe. This rectal probe was connected to an infrared heating lamp that maintained both body and head temperature at 37 ± 0.5°C throughout the experiment. Polyethylene catheters (PE- 50) were inserted into the right femoral artery and vein to monitor mean arterial blood pressure (MABP) and arterial blood sampling (pH, PaCO2, PaO2, and serum glucose). These physiological parameters were measured at the begin-ning of the surgical preparation, 15 minutes before ischemia was induced, and at the completion of the ischemia experiment.

Model of Focal Cerebral Ischemia

The original technique, as described by Tamura, et al. 47, was modi� ed for our experi-ments to increase the severity and reliability of the ischemic model8. A ventral midline incision was made for exposure of both common carotid arteries (CCAs). The contralat-eral (right) CCA was permanently ligated using a No. 3.0 silk suture. The ipsilateral (left) CCA was temporarily occluded (for 3 hours) using a May� eld microaneurysm clip. A skin incision was made between the left outer canthus and the tragus. The temporal muscle was de� ected anteriorly and a portion of the left zygomatic arch was removed. Care was taken to avoid damaging the facial nerve. After anterior and downward retraction of the musculature, the mandibular nerve was identi� ed and followed back to the foramen ovale. Using a high-speed air drill, a 3- to 4-mm craniectomy was made just anterior and superior to the foramen ovale. The dura was opened with a sharp needle and the MCA was freed from arachnoid. The portion of the left MCA that crosses the olfactory tract was temporarily occluded (for 3 hours) using a No. 3 Sundt arteriovenous malformation microclip.

Experimental Groups

The animals were divided into 16 groups as follows. The nonspeci� c NOS inhibitor, NG-nitro-L-arginine-methyl-ester (L-NAME), was administered intravenously 30 minutes before MCA occlusion at doses of 0.1 (six rats), 1 (� ve rats), 10 (� ve rats), and 30 (six rats) mg/kg. A selective nNOS inhibitor, 7-nitroindazole (7- NI), was administered intraperi-toneally 60 minutes before MCA occlusion at doses of 0.1 (� ve rats), 1 (six rats), 10 (six rats), and 100 (six rats) mg/kg. Both NO donors, 3-morpholinosydnonimine (SIN- 1) and NOC-18, DETA/NO, (Z)-1-[2(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA NONOate), were administered intravenously 30 minutes before MCA occlusion. The SIN-1 was given at doses of 0.1 (� ve rats), 1 (six rats), and 10 (six rats) mg/kg, and the DETA NONOate at doses of 0.1, 1, and 10 mg/kg (six rats in each group). The L-NAME, DETA NONOate, and SIN-1 were dissolved in 0.35 ml of 0.9% NaCl. Because of its poor solubility in aqueous solution, the 7-NI was dissolved in 0.35 ml dimethyl sulfoxide


(DMSO). Two groups of ischemic control animals (each group consisting of six animals) were administered 0.35 ml of either 0.9% NaCl (intravenously) or DMSO (intraperitone-ally) at 30 or 60 minutes before MCA occlusion, respectively. Each solution was prepared directly before administration. Changes in the animals’ cardiovascular status (heart rate and arterial blood pressure) were measured and recorded.

Histological Study

Three days (72 hours) after removal of the left MCA and CCA clips, anesthesia was again induced in the animals by using pentobarbital and the rats were intracardially perfused with a warm (37°C) 2% 2,3,5,-triphenyltetrazolium chloride solution. The rat brains were quickly removed, immersed in the 37°C 2,3,5,-triphenyltetrazolium chloride solution for 15 minutes to enhance staining, and placed in 10% bu� ered formaldehyde for 5 days. Twelve serial sections from each brain were cut at 1-mm intervals from the frontal pole and photographed. Photographic slides were analyzed using a computer-assisted image analyzer. Total cortical infarction volume was calculated by integrating the infarcted ar-eas of all twelve sections (area of infarction in square millimeters x thickness of section).


Statistical analysis was performed using analysis of variance with Sche� é’s post hoc test for multiple comparisons. Di� erences were considered signi� cant if the probability value was less than 0.05. The data are depicted as the mean ± standard deviation.

Sources of Supplies and Equipment

The L-NAME was purchased from Sigma Chemical Co. (St. Louis, MO) and the 7-NI, SIN-1, and DETA NONOate from Alexis Biochemicals Corp. (San Diego, CA). The JAVA image analyzer was obtained from SPSS Inc. (Chicago, IL) and the Sundt No. 3 arteriovenous malformation microclip from Johnson & Johnson Professional, Inc. (Raynham, MA).

RESULTS

Physiological Measurements

Physiological parameters measured just before completion of the ischemia experiment for each of the 16 groups are listed in Table 1. These physiological measurements were similar to those obtained at the beginning of the experiment and just prior to induction of ischemia. The MABP decreased momentarily after the intravenous bolus administra-tion of SIN-1 but normalized before left MCA and bilateral CCA occlusion. A slight, non-signi� cant reduction in MABP was noted in the DETA NONOate- treated groups. Mean ar-terial blood pressure was signi� cantly higher in the L-NAME groups when compared with

38 Chapter 3

the saline-treated ischemic control group. In the group treated with the highest dose of 7-NI (100 mg/kg administered intraperitoneally), bradycardia occurred with heart rates decreasing to 150 to 200/minute (normal approximately 300/minute).

Ischemic Control Groups

Three hours of left MCA and bilateral CCA occlusion resulted in mean cortical infarction volumes of 92.5 ± 26 mm3 and 119 ± 43 mm3 in the saline- and DMSO-treated ischemic control groups, respectively (Fig. 1). There was no signi� cant di� erence (Student’s un-paired t-test) between these two groups. Because the infarcted area was not directly located at the craniotomy site, it was easily distinguishable from possible direct damage caused by the surgical procedure. To assess the presence and extent of edema, the ische-mic hemisphere volume was compared with the hemisphere volume on the contralateral side. In the saline treated group, the mean hemisphere volume was 648.1 ± 33 mm3 for the ischemic (left) side and 621.2 ± 29 mm3 for the nonischemic (right) side. The right/left ratio (the right hemispheric volume divided by the left hemispheric volume) was 0.96 for this group. The range of the right/ left ratio was 0.96 to 1.01 in all groups studied, with no signi� cant di� erences between groups.

Table 1 Physiological parameters in rats subjected to temporary focal cerebral ischemia.

no significant difference (Student’s unpaired t-test) be-tween these two groups. Because the infarcted area wasnot directly located at the craniotomy site, it was easilydistinguishable from possible direct damage caused by thesurgical procedure.

To assess the presence and extent of edema, the isch-emic hemisphere volume was compared with the hemi-sphere volume on the contralateral side. In the saline-treated group, the mean hemisphere volume was 648.1 �33 mm3 for the ischemic (left) side and 621.2 � 29 mm3

for the nonischemic (right) side. The right/left ratio (theright hemispheric volume divided by the left hemisphericvolume) was 0.96 for this group. The range of the right/left ratio was 0.96 to 1.01 in all groups studied, with nosignificant differences between groups.

Drug Treatment Groups

The L-NAME–Treated Groups. The mean cortical infarc-tion volume was reduced by 50 to 71% in all four L-NAME–treated groups studied when compared with thesaline-treated ischemic control group (Fig. 1A). Only inthe 1 mg/kg group was this reduction significant (27.1 �37 mm3 compared with 92.5 � 26 mm3; p � 0.05).

The 7-NI–Treated Groups. The mean cortical infarctionvolume was significantly reduced in the 10 mg/kg–treatedgroup (35.2 � 32 mm3, p � 0.05) and the 100 mg/kg–treated group (9 � 13 mm3; p � 0.005) when comparedwith the DMSO-treated ischemic control group (119 � 43mm3 Fig. 1B). The high-dose of 7-NI (100 mg/kg) tendedto potentiate anesthesia and delay recovery.

The DETA NONOate–Treated Groups. No significant

difference was found between the mean cortical infarctionvolumes in the DETA NONOate–treated groups and thesaline-treated ischemic control group (Fig. 1C).

The SIN-1–Treated Groups. The mean infarction vol-umes in the SIN-1–treated groups were not significantlydifferent from those in the saline-treated ischemic controlgroup (Fig. 1D).

DiscussionNitric oxide has been recognized as an important medi-

ator of NMDA and hypoxic neurotoxicity.13 Malinski, etal.,35 conducted the first direct NO measurement study inwhich a NO-sensitive microsensor was used, and theirresults confirmed increased levels of NO in focal cerebralischemia and provided valuable information about thetime course of NO production. In that study, a rapid in-crease in the NO signal was found in the parietal cortexafter the onset of focal cerebral ischemia; this increasereached a semiplateau at 6 minutes and the signal de-creased to below detectable levels by 60 minutes.35 Usinga nitrite assay technique as an indirect method of measur-ing NO levels, Kader, et al.,27 found a similar pattern witha rise in nitrite levels maximizing at 5 to 10 minutes andnormalizing within 60 minutes after onset of ischemia.Although of great importance, absolute values of NO pro-duction are very dependent on the local microenvironmentand measurement methodology.35

Nitric Oxide Synthase Inhibitors

Nitric oxide synthase inhibitors have been widely used

B. A. Coert, R. E. Anderson, and F. B. Meyer

334 J. Neurosurg. / Volume 90 / February, 1999

TABLE 1Physiological parameters in rats subjected to temporary focal cerebral ischemia*

PaCO2 PaO2 Glucose MABPGroups (no. of rats) Weight (g) pH (mm Hg) (mm Hg) (mg/dl) (mm Hg)

L-NAME0.1 mg/kg (6) 413 � 42 7.35 � 0.04 48.1 � 2.3 251 � 46 194 � 28 104 � 8†1 mg/kg (5) 362 � 56 7.36 � 0.02 51.3 � 4.5 218 � 54 175 � 33 103 � 6†10 mg/kg (5) 390 � 60 7.35 � 0.03 47.0 � 3.4 192 � 54 174 � 24 104 � 8†30 mg/kg (6) 419 � 8 7.43 � 0.05 38.4 � 8.6 241 � 18 179 � 34 104 � 7†

7-NI0.1 mg/kg (5) 349 � 8 7.40 � 0.02 40.7 � 4.0 196 � 18 159 � 11 94 � 61 mg/kg (6) 338 � 21 7.42 � 0.05 37.8 � 6.2 222 � 35 168 � 59 94 � 510 mg/kg (6) 368 � 31 7.42 � 0.05 44.9 � 9.6 275 � 54 173 � 16 95 � 5100 mg/kg (6) 352 � 12 7.43 � 0.06 38.2 � 6.4 247 � 45 219 � 64 95 � 6

SIN-10.1 mg/kg (5) 371 � 11 7.40 � 0.08 47.1 � 10 265 � 84 174 � 22 103 � 61 mg/kg (6) 348 � 21 7.41 � 0.08 41.5 � 9.2 243 � 59 161 � 36 93 � 910 mg/kg (6) 379 � 48 7.45 � 0.09 42.1 � 11 224 � 25 172 � 22 94 � 6

DETA NONOate0.1 mg/kg (6) 360 � 24 7.47 � 0.04 38.6 � 5.4 239 � 31 186 � 32 93 � 61 mg/kg (6) 334 � 42 7.46 � 0.06 38.1 � 6.5 218 � 45 166 � 30 96 � 710 mg/kg (6) 419 � 18 7.41 � 0.04 42.9 � 4.3 246 � 64 170 � 22 90 � 8

ischemic control0.9% NaCl (6) 372 � 45 7.44 � 0.07 38.9 � 6.8 240 � 60 192 � 36 91 � 6DMSO (6) 342 � 16 7.46 � 0.04 36.6 � 4.9 224 � 24 158 � 19 90 � 6

* Head and body temperature was maintained at 37 � 0.5˚C throughout the experiment. The parameters listed in this table are thoseobtained at the third measurement just before completion of the ischemia experiment. Physiological parameters obtained in the firsttwo measurement periods at the beginning of the experimental preparation and just prior to ischemia were similar to these values andare not listed. Values are expressed as the mean � standard deviation.

† p � 0.05 when compared with the ischemic control group (0.9% NaCl).

Head and body temperature was maintained at 37 ± 0.5°C throughout the experiment. The parameters listed in this table are those obtained at the third measurement just before completion of the ischemia experiment. Physiological parameters obtained in the � rst two measurement periods at the beginning of the experimental preparation and just prior to ischemia were similar to these values and are not listed. Values are expressed as the mean ± standard deviation. † p < 0.05 when compared with the ischemic control group (0.9% NaCl).


Drug Treatment Groups

The L-NAME-Treated Groups

The mean cortical infarction volume was reduced by 50 to 71% in all four L-NAME- treated groups studied when compared with the saline-treated ischemic control group (Fig. 1A). Only in the 1 mg/kg group was this reduction signi� cant (27.1 ± 37 mm3 compared with 92.5 ± 26 mm3; p < 0.05).

to determine the overall effect of NO on ischemic damage.Selective inhibitors and genetically altered animals havemade it possible to estimate contributions of the differentisoforms of NOS to neurotoxicity and neuroprotection.Results with nonselective inhibitors such as L-NAMEhave shown a wide spectrum of results varying from ce-rebroprotection2 to augmentation of ischemic damage.19

Selective nNOS inhibitors and nNOS knockout animalstudies have shown consistent protective results.24

Inhibition of NO’s physiological activity may accountfor some side effects. Because the head and core bodytemperature was maintained throughout the experiment at36.5 to 37.5˚C, the effects of the NOS antagonists oninfarction size were not related to a hypothermic effect.Vasoconstriction resulting in a rise in MABP was found in

studies in which nonspecific NOS inhibitors were used,such as L-NAME33 administered intravenously at highdosages, whereas reductions in cerebral blood flow (CBF)were observed in both nNOS-specific (7-NI) and non-spe-cific (for example, L-NAME) inhibitors.28,33 Bradycardiaand sedation have been reported with the use of L-NAMEat higher doses.33 The 7-NI has potent antinociceptiveproperties38 and induces central nervous system depres-sion similar to high doses of narcotic/sedative/hypnoticagents.15,40 It may also inhibit eNOS and, therefore, maynot be a completely selective nNOS inhibitor.51 In ourpresent study, bradycardia and sedation were seen in thegroup treated with an intraperitoneal injection of 100 mg/kg of 7-NI. In the L-NAME groups studied, the MABP in-creased after intravenous administration of L-NAME.

J. Neurosurg. / Volume 90 / February, 1999

Nitric oxide in focal cerebral ischemia

335

FIG. 1. Bar graphs showing infarction volumes in the four drug groups (cross-hatched bars) and their respectivesaline- or DMSO-treated ischemic control group (slashed bars). A: Groups treated with L-NAME, a nonselective NOSinhibitor, at four different doses. Note a subtle bell-shaped dose response with a significant decrease in infarction volumeobserved only at the 1-mg/kg dose. B: Groups treated with 7-NI, a selective NOS inhibitor, at four different doses. Notethe uniphase dose response, with the two highest doses resulting in significant reduction in infarction volume, mostnotably at the 100-mg/kg dose. C: Groups treated with DETA NONOate, a NO donor, at three different doses. Therewas no significant alteration in infarction volume associated with any dose. D: Groups treated with SIN-1, an NOdonor, at three different doses. There was no significant alteration in infarction volume in this group associated with anydose. Values are expressed as the mean � standard deviation for each group. *p � 0.05; **p � 0.005.

Figure 1. Bar graphs showing infarction volumes in the four drug groups (cross-hatched bars) and their respective saline- or DMSO-treated ischemic control group (slashed bars). A: Groups treated with L-NAME, a nonselective NOS inhibitor, at four di� erent doses. Note a subtle bell-shaped dose response with a signi� cant decrease in infarction volume observed only at the 1-mg/kg dose. B: Groups treated with 7-NI, a selective NOS inhibitor, at four di� erent doses. Note the uniphase dose response, with the two highest doses resulting in signi� cant reduction in infarction volume, most notably at the 100-mg/kg dose. C: Groups treated with DETA NONOate, a NO donor, at three di� erent doses. There was no signi� cant alteration in infarction volume associated with any dose. D: Groups treated with SIN-1, an NO donor, at three di� erent doses. There was no signi� cant alteration in infarction volume in this group associated with any dose. Values are expressed as the mean ± standard deviation for each group. *p < 0.05; **p < 0.005.

40 Chapter 3

The 7-NI-Treated Groups

The mean cortical infarction volume was signi� cantly reduced in the 10 mg/kg-treated group (35.2 ± 32 mm3, p < 0.05) and the 100 mg/kg- treated group (9 ± 13 mm3; p < 0.005) when compared with the DMSO-treated ischemic control group (119 ± 43 mm3

Fig. 1B). The high-dose of 7-NI (100 mg/kg) tended to potentiate anesthesia and delay recovery.

The DETA NONOate-Treated Groups

No signi� cant di� erence was found between the mean cortical infarction volumes in the DETA NONOate-treated groups and the saline-treated ischemic control group (Fig. 1C).

The SIN-1-Treated Groups

The mean infarction volumes in the SIN-1–treated groups were not signi� cantly di� erent from those in the saline-treated ischemic control group (Fig. 1D).

DISCUSSION

Nitric oxide has been recognized as an important mediator of NMDA and hypoxic neu-rotoxicity13. Malinski, et al.35, conducted the � rst direct NO measurement study in which a NO-sensitive microsensor was used, and their results con� rmed increased levels of NO in focal cerebral ischemia and provided valuable information about the time course of NO production. In that study, a rapid increase in the NO signal was found in the parietal cortex after the onset of focal cerebral ischemia; this increase reached a semiplateau at 6 minutes and the signal decreased to below detectable levels by 60 minutes35. Using a nitrite assay technique as an indirect method of measuring NO levels, Kader, et al.27, found a similar pattern with a rise in nitrite levels maximizing at 5 to 10 minutes and normalizing within 60 minutes after onset of ischemia. Although of great importance, absolute values of NO production are very dependent on the local microenvironment and measurement methodology35.

Nitric Oxide Synthase Inhibitors

Nitric oxide synthase inhibitors have been widely used to determine the overall e� ect of NO on ischemic damage. Selective inhibitors and genetically altered animals have made it possible to estimate contributions of the di� erent isoforms of NOS to neurotoxicity and neuroprotection. Results with nonselective inhibitors such as L-NAME have shown a wide spectrum of results varying from cerebroprotection2 to augmentation of ischemic dam-age19. Selective nNOS inhibitors and nNOS knockout animal studies have shown consis-tent protective results24. Inhibition of NO’s physiological activity may account for some


side e� ects. Because the head and core body temperature was maintained throughout the experiment at 36.5 to 37.5°C, the e� ects of the NOS antagonists on infarction size were not related to hypothermia. Vasoconstriction resulting in a rise in MABP was found in studies in which nonspeci� c NOS inhibitors were used, such as L-NAME33 administered intravenously at high dosages, whereas reductions in cerebral blood � ow (CBF) were observed in both nNOS-speci� c (7-NI) and non-speci� c (for example, L-NAME) inhibi-tors28,33. Bradycardia and sedation have been reported with the use of L-NAME at higher doses33. The 7-NI has potent antinociceptive properties38 and induces central nervous system depression similar to high doses of narcotic/sedative/hypnotic agents15,40. It may also inhibit eNOS and, therefore, may not be a completely selective nNOS inhibitor51. In our present study, bradycardia and sedation were seen in the group treated with an intraperitoneal injection of 100 mg/ kg of 7-NI. In the L-NAME groups studied, the MABP increased after intravenous administration of L-NAME. Brain NOS activity has been reported to diminish for at least 6 hours48 and up to 96 hours after a single dose of L-NAME is given intravenously26. The inhibitor L-NAME has been shown to be e� ective in reducing infarction size mainly at lower dosages and in accentuating ischemia at high dosages25. This suggests that partial inhibition of nNOS from lower dosages is su� cient to acquire an optimum neuroprotective e� ect, but that higher doses can result in inhibi-tion of eNOS, thereby exacerbating cerebral ischemia via vasoconstriction. Intraperito-neal 7-NI administration in rats has resulted in maximum brain nNOS inhibition at 30 minutes and in complete recovery at 24 hours32. Signi� cant reductions in mean cortical infarction volumes were seen in two of the 7-NI groups (10 and 100 mg/ kg administered intraperitoneally in DMSO). A DMSO treated ischemic control group was added to the 7-NI–treated groups because, as an HO• scavenger, DMSO can reduce oxidant injury3. The mean cortical infarction volume in this DMSO-treated group was 119 ± 43 mm3, which was not signi� cantly di� erent (Student’s unpaired t-test) from that found in the saline-treated ischemic control group (92.5 ± 26 mm3). Overall, the maximum protective e� ect was seen in the 100-mg/kg 7-NI group, in which a mean cortical infarction volume reduction of 92% was seen. The enhancement and prolongation of the anesthetic e� ect observed in this group could have contributed to its protective e� ects.

Nitric Oxide Donors

The anti-schemic e� ects of nitrovasodilators in coronary artery disease have been rec-ognized for more than 100 years6 and are still used to treat acute coronary syndromes. In focal cerebral ischemia NO donors appeared to reduce ischemic brain damage if hy-potension was pharmacologically avoided by coadministration of an ionotrope25. Cyclic guanosine monophosphate-mediated vasodilation and reduction in platelet aggrega-tion37, as well as direct downregulation of the NMDA receptor by reacting with the redox modulary site31, have been proposed to contribute to the cerebroprotective e� ect of NO.

42 Chapter 3

No protective e� ect was found in vitro, however. The NO donor sodium nitroprusside showed concentration-dependent cell death curves similar to those of NMDA12. A dual role for NO was proposed in which neuronal NO overproduction played an important role in the development of ischemic damage, whereas endothelial and perivascular NO could protect against ischemia by increasing regional CBF and preventing platelet ag-gregation1,10. One of the two NO donors used in this study, SIN-1, is routinely used in interventional cardiology as a coronary artery bolus injection18. In a study by Shukla, et al. 46, SIN- 1 was shown to cross the blood-brain barrier in rats. Nitric oxide formation from SIN-1 occurs spontaneously and does not require the presence of cysteine17. Two factors were identi� ed as in� uencing NO release: PO2 and pH5. During cerebral ischemia, oxida-tive capacity appeared to be high enough to guarantee NO release5. With the exception of NO, superoxide was shown to be a product of the SIN-1 oxidation22. Superoxide gen-eration was also described with puri� ed nNOS at suboptimal L-arginine concentrations21. By preventing superoxide formation, L-arginine infusion could provide additional pro-tection to the previously discovered bene� cial e� ects on regional CBF39. Together super-oxide and NO form peroxynitrite, which is the major contributor to NO and superoxide toxicity. Care should be taken in handling sydnonimines such as SIN-1 because they are highly susceptible to oxygen and light16. For this reason all our SIN-1 solutions were pre-pared directly before use and administered as an intravenous bolus. The half-life of NO in air-saturated bu� er was calculated to be 6 seconds29, indicating that NO formation is the main determinant of NO levels. Noack and Feelisch41 studied time-dependent formation of various metabolites of SIN-1 and their velocity of NO liberation, revealing the half-life of SIN-1 to be approximately 150 minutes. The SIN-1 metabolite SIN-1A reached a peak concentration at approximately 75 minutes with a half-life of approximately 300 minutes. The initial NO liberation velocity for SIN-1A was measured to be four times higher than that for SIN-1. Accounting for each relative NO liberating capacity, the overall half-life for NO donation is approximately 230 minutes and the maximum NO liberation is at ap-proximately 50 minutes41. In our study a temporary reduction in MABP was seen after intravenous administration. Before MCA and bilateral CCA occlusion, the MABP had re-turned to baseline levels without the use of vasopressor agents. The protective e� ect of a NO donor may be di� cult to determine when a vasopressor is used simultaneously to maintain blood pressure by some investigators52,53. Using a permanent MCA occlusion model with phenylephrine-induced hypertension, Drummond and associates14 showed a reduction in brain regions in which local CBF is equal to or lower than levels that may result in neuronal death. Using permanent MCA occlusion in spontaneously hypertensive rats, Maiese, et al. 34, found no signi� cant di� erence in infarction volume when the MABP was elevated using phenylephrine. One possible explanation for this discrepancy may be that the protective e� ect of phenylephrine depends on improvement in collateral � ow. Collateral circulation is less developed in spontaneously hypertensive rats9, which may


explain the lack of e� cacy of phenylephrine. In this present study, SIN-1 without vaso-pressor did not signi� cantly alter mean cortical infarction volume. The other NO donor used in this study, DETA NONOate, is a zwitterionic polyamine/NO adduct23 that releases two molecules of NO per molecule of DETA NONOate49. The half-life for DETA NONOate was found to be 3400 minutes23; therefore, it is more stable and longer acting than SIN-1. Although it is longer acting and releases higher concentrations of NO than SIN-1, DETA NONOate also did not signi� cantly a� ect mean cortical infarction volume in our study. In our model of focal cerebral ischemia in the Wistar rat, NO donors resulted in unchanged mean cortical infarction volumes throughout the concentration range used. However, both a nonspeci� c (L-NAME) and an nNOS speci� c (7-NI) inhibitor reduced infarction vol-ume, which suggests that excessive NO production is detrimental. However, the e� ect of NOS inhibitors on other physiological roles of NO should not be overlooked11,25. Pajewski and associates42 studied the e� ect of L-NAME and 7-NI on anesthesia, discovering that inhibition of the NO pathway decreased levels of consciousness, augmenting sedation, analgesia, and anesthesia. Potencies for 7-NI were found to vary between mice and rats in a study by Moore, et al.38 In that study, the authors showed that there was inhibition of nNOS without alterations in MABP in a dose range of 10 to 80 mg/kg. The lack of e� ect on MABP would indicate that there would be minimal or no e� ect on eNOS. Traystman and colleagues 48 showed that a 20-mg/kg intravenous injection of L-NAME produced the same amount of NOS enzyme inhibition (> 70%) but the half-lives varied widely among cats, dogs, and pigs. These observations suggest that extrapolations between species can lead to inaccurate dosing. The most e� ective dose for 7-NI found in our study was 100 mg/kg administered intraperitoneally, although a signi� cant reduction in infarction volume was also demonstrated at 10 mg/kg. Dalkara, et al.10, found 25- and 50-mg/kg doses to be e� ective in reducing infarction volume by 25 to 27%. The e� cacy of di� erent therapies appears to depend on the severity of the ischemic insult, time of onset, dosing of therapy, and the patient’s general vascular condition. Mar-gaill, et al.36, described the time course of glutamate concentrations during temporary MCA and bilateral CCA occlusion. Because the glutamate surge occurred minutes after occlusion and lasted for only approximately 80 minutes, the therapeutic window for in-tervention was found to be very short36. Direct measurement of NO production showed a similar pattern35. Atherosclerotic lesions tended to modify the response of human (coronary) arteries to vasoactive substances4 that curtailed the e� cacy of the therapy. Dosing appeared to be a major determinant of the overall e� ect, especially in the case of L-NAME, a low dose of which (0.1 mg/kg) has been shown to be protective2 with a minimum e� ect on MABP and a high dose (10 mg/kg) was more often detrimental52. Be-fore extrapolations can be made to human pathophysiological states, more information should be obtained about the activation of pathophysiological events in humans. The therapeutic window for NOS inhibition appears to be short35, limiting its e� cacy in cases

44 Chapter 3

of stroke. In temporary arterial occlusion during vascular surgery, drug therapy could be instituted before occlusion to prevent ischemic brain damage. The results of this ex-periment indicate that NOS inhibition may be a valuable neuroprotective intervention during neurovascular or endovascular procedures.


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46 Chapter 3

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Chapter 4

Exogenous Spermine Reduces Ischemic Damage in a Model of Focal Cerebral

Ischemia

Bert A. Coert MD


Fredric B. Meyer MD

Neuroscience Letters 282(2000) pp 5-8

50 Chapter 4

ABSTRACT

Alterations in polyamine metabolism during and after global or focal cerebral ischemia can produce a multiplicity of e� ects on the brain, such as modi� cation in the mitochon-drial calcium bu� ering capacity, exacerbating glutamate-mediated neurotoxicity, and impairment of the blood-brain barrier. In this study, the endogenous polyamine spermine was administered intravenously 30 minutes prior to temporary focal cerebral ischemia in rats induced by clipping of the left middle cerebral and bilateral common carotid arter-ies for 3 hours. Three days after removal of the microclips, intracardiac perfusion with 2% 2,3,5-triphenyl tetrazolium chloride was performed. Coronal slices were cut, photo-graphed, and examined for cortical infarct volume. Spermine reduced infarct volume in a dose-dependent fashion. This study demonstrates that the use of polyamines may be considered as a powerful tool in the prevention of ischemic tissue damage following focal cerebral ischemia.

Exogenous spermine reduces ischemic damage 51

INTRODUCTION

The polyamines (PA) putrescine, spermine, and spermidine are important for cell growth and di� erentiation29. They are involved with numerous cellular functions including protein phosphorylation and protein synthesis17, regulation of gene expression4, pro-grammed cell death9 , inhibition of membrane permeability transition of mitochondria16, regulation of mitochondria Ca2+ transport13,18, regulation of nitric oxide synthase (NOS)12, and free radical scavenging10,30. Spermine in particular, found in millimolar concentra-tions in the nucleus of the cell25 was shown to prevent endonuclease-mediated DNA fragmentation3. The role of polyamines in the pathophysiology of cerebral ischemia has not been very well elucidated. Following either transient global, focal, or focal permanent cerebral ischemia there is a signi� cant increase in polyamine metabolism which is char-acterized by increased ornithine decarboxylase (ODC) activity with increased putrescine levels21,23 which is subsequently metabolized to spermidine and spermine29. It has been demonstrated that spermine concentrations in brain is either slightly reduced 20,22,23 or moderately increased15 following global cerebral ischemia. However, in models of focal cerebral ischemia, signi� cant reductions21,26 or no alterations2 in Spermine concentra-tions have been demonstrated. It has been shown that in di� erent models of ischemia, polyamine metabolism is dependent on changes in ATP and acetyl CoA27. Spermine has been reported to be neuroprotective in a model of forebrain ischemia. Gilad and Gilad8, demonstrated in gerbils that intra-peritoneal administration of Spermine signi� cantly decreased hippocampus and striatal cell loss. In a rat model of forebrain ischemia, Far-biszewski et al.7, showed that spermine signi� cantly reversed the decrease in superoxide dismutase (SOD) activity in the cortex. To date there have been no studies performed to ascertain the e� ects of endogenous spermine in a rat model of reversible focal cerebral ischemia. In this study we demonstrate that spermine can be signi� cantly neuroprotec-tive in this model.


Following review and approval by the Institutional Animal Care and Use Committee, 24 adult male Wistar rats were administered halothane anesthesia in a mixture of oxygen and air through a face mask at 1.5% during the surgical procedure and 1.0% during the occlusion period. Subcutaneous glycopyrrolate (Robinul-V) was administered pre-operatively at 4 mg/kg to reduce respiratory secretions. Core body temperature was con-tinuously monitored at the beginning of the surgical preparation and throughout the experiment with a rectal probe. This rectal probe was connected to an infrared heating lamp, which maintained both the body and head temperature at 37.0 ± 0.5ºC through-

52 Chapter 4

out the experiment. Polyethylene catheters (PE-50) were inserted into the right femoral artery and vein for monitoring of mean arterial blood pressure (MABP) and arterial blood sampling (pHa, PaCO2, PaO2, hematocrit, serum glucose, and lactate). These physiological parameters were measured at the beginning of the surgical preparation, 15 min prior to ischemia, and at the completion of the ischemia experiment. The original technique as described by Tamura et al. 31, was modi� ed for our experiments to increase the severity and reliability of the ischemic model5. A ventral midline incision was made for exposure of both common carotid arteries (CCA). The contralateral right CCA was permanently ligated using a 3/0 silk suture. The ipsilateral (left) CCA was temporarily occluded for 3 hours using a May� eld micro-aneurysm clip. A skin incision was made between the left outer canthus and the tragus. The temporal muscle was de� ected anteriorly and part of the left zygomatic arch was removed. Care was taken to avoid damaging the facial nerve. After anterior and downward retraction of the musculature, the mandibular nerve was identi� ed and followed back to the foramen ovale. Using a high-speed air drill with irriga-tion, a 3-4 mm craniectomy was made just anterior and superior to the foramen ovale. The dura was opened with a sharp needle and the middle cerebral artery (MCA) was freed of arachnoid. The left MCA crossing the olfactory tract was temporarily occluded (3 h) using a Sundt #3 AVM microclip (Codman and Shurtle� Inc.).

Experimental groups

The 24 animals were divided into four groups as follows: spermine (Sigma) dissolved in NaCl 0.9 % was administered intravenously (i.v.) 30 min prior to middle cerebral artery occlusion (MCAo) at doses of 0.1 (n = 6), 1.0 (n = 6), and 10.0 (n = 6) mg/kg. An ischemic control group was included in which 0.35 ml of NaCl 0.9% i.v. (n = 6) was administered 30 min prior to MCA occlusion. Each solution was prepared directly before administration.

Histopathology

Three days (72 h) after removal of the left MCA and CCA clips, the animals were reanes-thetized with pentobarbital and intracardially perfused with a warm (378 C) 2% TTC (2,3,5,-triphenyltetrazolium chloride) solution. Their brains were quickly removed, im-mersed in the 370C TTC solution for 15 min to enhance staining and then placed in 10% bu� ered formaldehyde for 5 days. Twelve serial coronal sections from each brain were cut at 1 mm intervals beginning at 3.7 mm from the bregma using a rodent brain matrix (ASI Instruments, Inc.) and the anterior side of each section photographed. Photographic slides were analyzed in a blinded fashion using a computer-assisted image analyzer (JAVA, Jandel Scienti� c Software). Total cortical infarct volume was calculated by integrating the infarcted area of all 12 sections (area of infarct in mm2 x thickness of section).



Statistical analysis was carried out using ANOVA with Tukey’s post hoc test for multiple comparisons. Di� erences were considered signi� cant if P < 0.05. The data is depicted as mean and standard error.

RESULTS

There were no signi� cant di� erences in the measurement of systemic parameters includ-ing PaCO2 (40 ± 1 mmHg), PaO2 (214 ± 9 mmHg), pHa (7.434 ± 0.016), mean arterial blood pressure (96 ± 2 mmHg), and hematocrit (38 ± 1%) between the measurements taken pri-or to onset of ischemia and at the completion of the experiment and between all groups studied. Serum glucose decreased signi� cantly (P < 0.05), but not in a dose-dependent manner, by an average of 30% after administration of spermine. Serum lactate decreased in a dose-dependent manner by 4, 27 and 48% after administration of 0.1, 1.0 and 10.0 mg/kg spermine, respectively. These values, however, were not signi� cantly di� erent from one to the other by ANOVA. Three hours of left MCA and bilateral CCA occlusion resulted in mean cortical infarct volume of 95.0 ± 7.4 mm3 (Fig. 1). Intravenous admin-istration of spermine 30 min prior to ischemia resulted in a dose-dependent response in the reduction of infarct volume (Fig. 1). A signi� cant reduction (P < 0.05) of infarct volume was seen only in the 10 mg/kg spermine treated group compared to the control group (31.1 ± 17.4 vs. 95.0 ± 7.4 mm3).

DISCUSSION

For the � rst time in a rat model of reversible focal cerebral ischemia, the � ndings of this study demonstrate an impressive and signi� cant reduction of cortical infarct volume when spermine, an aliphatic polyamine, was administered intravenously 30 min prior to middle cerebral and common carotid artery cerebral occlusion. It also demonstrated no adverse e� ects on blood pressure, temperature, or blood glucose levels during the experimental period. Spermine is fully protonated at physiological pH and is readily solu-blized in organic and aqueous media. It has been shown that spermine can easily pass the blood-brain barrier 8,11,24. Many early studies have shown increases, decreases, or no change in spermine concentrations in brain during and/or after reperfusion. It has shown that spermine is slightly reduced following global cerebral ischemia 20,22,23, while Koenig et al.15, reported modest increase in tissue levels of spermine. In a rat model of focal cerebral ischemia by reversible middle cerebral artery embolization, Paschen et al.21, reported a signi� cant (≈23%) reduction in cortical spermine concentration during

54 Chapter 4

ischemia. Most notably, spermine levels in both the contralateral and ipsilateral cortex after 24 h of re� ow were less than the level seen in the ipsilateral cortex during ischemia. Sauer et al.26, in a model of permanent focal brain ischemia, demonstrated a signi� cant <50% reduction in spermine levels.

Baskaya et al.2, using a model of focal cerebral ischemia in the cat, found no di� erences in spermine levels between ipsilateral ischemic cortex and penumbra compared to that of the contralateral side. Spermine synthesis involves S-adenosylmethioine derived from ATP and interconversion via acetylation steps involving acetyl-CoA. Therefore, in di� er-ent models of ischemia, polyamine metabolism is dependent on changes in ATP and acetyl-CoA 27. Many di� erent mechanisms have been proposed to explain the protective and neurotoxic e� ects of polyamines. Blood-brain barrier (BBB) disruption was reported with the suggestion that the hyperosmolar (mannitol) BBB disruption could be medi-ated by increased ODC activity with signi� cantly increased putrescine levels and slightly increased levels of spermine and spermidine14. N-methyl-D-aspartate (NMDA) receptor modulation at a speci� c site has also been proposed with a biphasic response with lower

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Figure. 1. Graph depicting cortical infarction volume in the ischemic control group (grey bar) and the spermine-treated groups (black bars) at 0.1, 1.0 and 10.0 mg/kg. Note that spermine was signi� cantly (*P < 0:05, ANOVA) e� ective at the 10.0 mg/kg dosage.


doses of spermine (<50 nmol) attenuating NMDA damage while higher dosages increased excitotoxic damage19. Separate recognition sites were identi� ed for polyamines on the NMDA receptor, the presence of both positive and negative modulation implying more than one site19. Spermine and, to a lesser extent, spermidine were found to increase rate and a� nity of Ca2+ uptake by mitochondria. This e� ect was progressively less potent with increasing Ca2+ concentration. Polyamines may play a critical role in the regulation of the intracellular Ca2+ concentration especially during activation18. Hu et al.12 demon-strated an interaction between the positively charged amine groups in PA and the NADPH electron transfer of nNOS resulting in an inhibition of activity. Farbiszewski et al.7, dem-onstrated a protective e� ect of exogenous spermine as an antioxidant enzyme defense in an in vivo rat model. Thiobarbituric acid-reactive substance (TBARS), an indicator of lipid peroxidase was diminished in this study by the addition of spermine (5 mg/kg i.v.) 7. Interconversion enzyme activity SAMD (S-adenosyl methionine decarboxylase) and SSAT (spermidine/spermine acetyl transferase) regulate the interconversion from putrescine

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Figure. 2. Typical photographs of histological assessment using TTC staining after 3 h of focal cerebral ischemia followed by 72 h of reperfusion: (A) ischemic control animal; (B) 0.1 mg/kg spermine treated animal; (C) 1.0 mg/kg spermine treated animal; (D) 10.0 mg/kg spermine treated animal. Sections are at 2 mm intervals in descending order (anterior to posterior) beginning at the top row 3.7 mm from the bregma.

56 Chapter 4

to spermidine to spermine and vice versa. Spermine has been shown to be protective in neurotoxic insult of brain neurons in culture1. Spermine, given intra-peritoneally, signi� -cantly reduced hippocampal and striatal cell loss after an ischemic insult8. Many of the protective e� ects of PA attributed to spermine, while putrescine levels were reported to correlate well with the extent of tissue damage20. By changing the ratio of production of the di� erent polyamines, SSAT activity at critical post-lesion periods may be relevant in determining the e� ect of PA6. Recently the polyamine oxidase activity in a traumatic brain model was found to contribute to edema formation and necrotic cavitation6. The harmful e� ects were found to be the production of putrescine, hydrogen peroxide and a toxic aldehyde 28. Intravenously administered exogenous spermine in our model resulted in a signi� cant reduction of infarct size. Further investigations are required to con� rm this protective e� ect and to further ascertain the mechanism of action.


REFERENCES

1. Abe, K., Nishiyama, N. and Saito, H., Spermine promotes the survival of primary cultured brain neurons. Brain Res., 605 (1993) 322-326.

2. Baskaya, M.F., Rao, A.M., Dogan, A., Donaldson, D., Gellin, G. and Dempsey, R.J., Regional brain polyamine levels in permanent focal cerebral ischemia. Brain Res., 744 (1997) 302-308.

3. Brüne, B., Hartzell, P., Nicotera, P. and Orrenius, S., Spermine prevents endonuclease activation and apoptosis in thymocytes. Exp. Cell. Res., 195 (1991) 323-329.

4. Celano, P., Baylin, S.B. and Casero Jr., R.A., Polyamines di� erentially modulate the transcription of growth-associated genes in human colon carcinoma cells. J. Biol.Chem., 264 (1989) 8922-8927.

5. Coert, B.A., Anderson, R.E. and Meyer, F.B., Reproducibility of cerebral cortical infarction in the Wistar rat following middle cerebral artery occlusion. J. Stroke Cerebrovasc.Dis., Vol.8, No 6 (1999): pp380-287.

6. Dogan, A., Rao, A.M., Baskaya, M.K., Hatcher, J., Temiz, C.,Rao, V.L.R. and Dempsey, R.J., Contribution of polyamine oxidase to brain injury after trauma. J. Neurosurg., 90(1999) 1078-1082.

7. Farbiszewski, R., Bielawski, K., Bielawska, A. and Sobaniec,W., Spermine protects in vivo the antioxi-dant enzymes in transiently hypoperfused rat brain. Acta Neurobiol. Exp., 55(1995) 253-258.

8. Gilad, G.M. and Gilad, V.H., Polyamines can protect against ischemia-induced nerve cell death in gerbil forebrain. Exp.Neurol., 111 (1991) 349-355.

9. Ha, H.C., Woster, P.M., Yager, J.D. and Casero Jr., R.A., The role polyamines catabolism in polyamine analogue induced programmed cell death. Proc. Natl. Acad. Sci.USA, 94 (1997) 11557-11562.

10. Ha, H.C., Sirisoma, N.S., Kuppisamy, P., Zweier, J.L.,Woster, P.M. and Casero Jr., R.A., The natural polyamine spermine functions directly as a free radical scavenger.Proc. Natl. Acad. Sci. USA, 95 (1998) 11140-11145.

11. Halliday, C.A. and Shaw, G.G., Clearance of the polyamines from the perfused cerebroventricular system of the rabbit. J. Neurochem., 30 (1978) 807-812.

12. Hu, J., Mahmoud, M.I. and El-Fakahany, E.E., Polyamines inhibit nitric oxide synthase in rat cerebel-lum. Neurosci.Lett., 175 (1994) 41-45.

13. Jensen, J.R., Lynch, G. and Baudry, M., Polyamines stimulate mitochondrial calcium transport in rat brain. J. Neurochem.,48 (1987) 765-772.

14. Koenig, H., Goldstone, A.D. and Lu, C.Y., Polyamines mediate the reversible opening of the blood-brain barrier by the intracarotid infusion of hyperosmolal mannitol. Brain Res., 483 (1989) 110-116.

15. Koenig, H., Goldstone, A.D., Lu, C.Y. and Trout, J.J., Brain polyamines are controlled by N-methyl-D-aspartate receptors during ischaemia and recirculation. Stroke, 21 (1992)98-102.

16. Lapidus, R.G. and Sokolove, P.M., Inhibition by spermine of the inner membrane permeability tran-sition of isolated rat heart mitochondria. FEBS Lett., 313 (1992) 314-318.

17. Lenzen, S., Hickethier, G. and Paten, U., Interactions between spermine and Mg21 on mitochon-drial Ca21 transport. J. Biol. Chem., 261 (1986) 16478-16483.

18. Lenzen, S., MuÈ nster, W. and Rustenbeck, I., Dual e� ect of spermine on mitochondrial Ca21 trans-port. Biochem. J., 286 (1992) 597-602.

19. Munir, M., Subramamiam, S. and McGonigle, P., Polyamines modulate the neurotoxic e� ects of NMDA in vivo. Brain Res., 616 (1993) 163-170.

58 Chapter 4

20. Paschen, W., Schmidt-Kastner, R., Hallmayer, J. and Djuricic, B., Polyamines in cerebral ischemia. Neurochem.Pathol., 9 (1988) 1-20.

21. Paschen, W., Csiba, L., Röhn, G. and Bereczki, D., Polyamine metabolism in transient focal ischemia of rat brain. BrainRes., 566 (1991) 354-357.

22. Paschen, W., Widmann, R. and Weber, C., Changes in regionalpolyamine pro� les in rat brains after transient cerebral ischemia (single versus repetitive ischemia): evidence for release of polyamines from injured neurons. Neurosci.Lett., 135 (1992) 121-124.

23. Paschen, W., Polyamine metabolism in reversible cerebral ischaemia. Cerebrovasc. Brain Metab. Rev., 4 (1992) 59-88.

24. Pateman, A.J. and Shaw, G.G., The uptake of Spermidine and spermine by slices of mouse cerebral hemispheres. J.Neurochem., 25 (1975) 341-345.

25. Sarhan, S. and Seiler, N., On the subcellular localization of the polyamines. Biol. Chem. Hoppe-Seyler, 370 (1989)1279-1284.

26. Sauer, D., Martin, P., Allegrini, P.R., Bernasconi, R., Amacker, H. and Fagg, G.E., Di� ering e� ects of α-di� uoromethylornithine and CGP 40116 on polyamine levels and infarct volume in a rat model focal cerebral ischaemia. Neurosci. Lett., 141 (1992) 131-135.

27. Seiler, N., Pharmacological properties of the natural polyamines and their depletion by biosynthe-sis inhibitors as a therapeutic approach. Prog. Drug Res., 37 (1991) 107-159.

28. Seiler, N., Polyamine oxidase, properties, and functions. Prog. Brain Res., 106 (1995) 333-344.

29. Tabor, C.W. and Tabor, H., Polyamines. Annu. Rev.Biochem., 53 (1984) 749-790.

30. Tadolini, B., Polyamine inhibition of lipoperoxidation: the in� uence of polyamines on iron oxida-tion in the presence of compounds mimicking phospholipid heads. Biochem. J.,249 (1998) 33-36.

31. Tamura, A., Graham, D.I., McCulloch, J. and Teasdale, G.M., Focal cerebral ischaemia in the rat: I. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab., 1 (1981) 53-60.

Chapter 5

Is the Neuroprotective E� cacy of nNOS Inhibitor 7-NI Dependent on Ischemic

Intracellular pH?

Bernard A. Coert MD


Fredric B. Meyer MD

American Journal of Physiology Heart and Circulatory Physiology 284:151-159, 2003

60 Chapter 5

ABSTRACT

The purpose of this study was to test the hypothesis that the e� cacy of 7-nitroindazole (7-NI), a selective neuronal nitric oxide (NO) synthase (NOS) inhibitor, is pH dependent in vivo during focal cerebral ischemia. Wistar rats underwent 2 hours of focal cerebral ischemia under 1% halothane anesthesia. 7-NI, 10 and 100 mg/kg in 0.1 ml/kg DMSO, was administered 30 minutes before occlusion. Ischemic brain acidosis was manipulated by altering serum glucose concentrations. Con� rmation of the e� ects of these serum glucose manipulations on brain intracellular pH (pHi) was obtained in a group of acute experiments utilizing umbelliferone � uorescence. The animals were euthanized at 72 hours for histology. 7-NI signi� cantly (P < 0.05) reduced infarction volume in both the normoglycemic by 93.3% and hyperglycemic animals by 27.5%. In the moderate hypo-glycemic animals, the reduction in infarction volume did not reach signi� cance because moderate hypoglycaemia in itself dramatically reduced infarction volume. We hypoth-esize that a mechanism to explain the published discrepancies of the results with neu-ronal NOS inhibitors in vivo may be e� ects of di� erent levels of ischemic brain acidosis on the production of NO.

Is the neuroprotective e� ect of selective nNOS inhibition pHi dependent? 61

INTRODUCTION

Elaborate studies on the role of nitric oxide (NO) in the pathophysiology of stroke have revealed complex and contradictory results34,55. The e� ects of NO synthase (NOS) inhibi-tors such as NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), and 7-nitroindazole (7-NI) for treatment of cerebral ischemia vary from protective to cytotoxic1,2,3,7,10,17,19,22,26,27,30,35,41,44,38,49,53,59,66,67,70 depending on the ischemia model, isoenzyme speci� city, the timing, and dosage administered. In the early phase of cere-bral ischemia, NO has been shown to be bene� cial69 by increasing collateral circulation and inhibiting platelet aggregation. Treatment with NO donors69 and with L-arginine 47during this phase has been shown to be e� ective in reducing ischemic brain damage. Neuronal NOS (nNOS) activation appears to increase ischemic damage, whereas selec-tive inhibition reduces cerebral infarct volume17,19,22,26,30,35,49,67. It has been reported that nNOS-de� cient mice develop smaller infarcts after middle cerebral artery (MCA) occlu-sion33. However, the severity of the ischemic insult may determine the neuroprotective e� cacy of NOS inhibitors3,30. For example, severe ischemia resulted in loss of protective e� ects by the nonselective NOS inhibitor L-NAME3 and nNOS inhibitor AR-R1747730. It has been hypothesized that increasing severity of ischemia results in more pronounced intracellular acidosis, which subsequently attenuates NOS enzyme activity3. Three in vitro studies25,31,54 have demonstrated a biphasic pH sensitivity for NOS enzymes with a pH optimum for nNOS of 6.7-7.025,31,54. Accordingly, the variable published e� ects of NOS inhibition might not only be related to the ischemia model, dosage, and timing but also to the e� ects of intra- and peri-ischemic intracellular pH (pHi) on NOS activity. To investigate the possible in� uence of brain pHi on nNOS inhibition in vivo, we utilized hyperglycemic and moderate hypoglycemic conditions during cerebral ischemia to exacerbate and attenuate intracellular acidosis and compared the protective e� ects of selective nNOS inhibition on ischemic brain damage. Given the current technology, it is not possible to simultaneously measure intracellular brain pH and NO in vivo. Therefore, a comparative analysis of the literature is made to elucidate the possible e� ects of pHi on NOS activity.


After we received approval by the Institutional Animal Care and Use Committee, 41 adult male Wistar rats weighing 300–450 g were fasted overnight and allowed free access to water. The animals were induced with halothane in a mixture of oxygen and air through a face mask at 2.0% during the surgical procedure and 1.0% during the occlusion pe-riod. Atropine was administered subcutaneously (0.08 mg/kg) preoperatively to reduce

62 Chapter 5

respiratory secretions. Core body temperature was maintained at 36.5-37.5°C by using a heating pad and monitored continuously with a rectal temperature probe. Polyethylene catheters (PE-50) were inserted into the femoral artery to monitor arterial blood pressure and for arterial blood sampling [arterial PCO2 (PaCO2), arterial PO2 (PaO2), pH, hematocrit, and serum glucose] and inserted into a vein for administration of drugs. These physi-ological parameters were recorded 30 and 60 min before, during the 2-h ischemic period, and 30 min after � ow restoration. Antibiotics (Durapen, 30,000 units) were administered intramuscularly before wound closure.

Model of focal cerebral ischemia

A modi� cation by Coert et al. 16 of the technique described by Tamura et al. 60 was used. A 2-cm ventral coronal incision was made in the neck to expose and place snares (silk no. 2) around both common carotid arteries. A 2-cm skin incision was then made between the right outer canthus and the tragus. The temporal muscle was de� ected anteriorly, and the zygomatic arch was partially removed. After retraction of the musculature, the mandibular nerve was identi� ed and followed to the foramen ovale. With the use of a high-speed drill (Hall Surgical), a small 3- to 4-mm craniectomy was made just anterior and superior to the foramen ovale. The dura was then opened with a sharp needle and the MCA freed of arachnoid. The MCA was temporarily occluded for 2 h at its crossing with the olfactory tract with the use of a no. 3 Sundt arteriovenous malformation micro-clip (Johnson and Johnson Professional). Simultaneously with occlusion of MCA, both common carotid arteries (CCAs) were temporarily occluded with the use of the snares. After restoration of � ow, the MCA was observed for patency. Criteria for a patent vessel was the return to its normal size and bright red colorization. A nonpatent vessel would exhibit a dark blue colorization. In three animals used for � uorescent imaging, an extra 4 x 5-mm craniectomy was made over the frontal and parietal area of cortex. The dura was then removed, and edges were carefully cauterized at the margins of the craniec-tomy and then covered with Saran Wrap to prevent surface oxygenation and to keep the brain moist.

Experimental groups

Animals were divided into seven groups. Three control groups: 1) normoglycemic (n = 7), 2) moderate hypoglycemic (n = 7), and 3) hyperglycemic (n = 7), and three treatment groups that were given 7-NI intraperitoneally at 100 mg/kg in 0.1 ml/kg DMSO 1 h before ischemia for the normoglycemic (n = 6) and hyperglycemic (n = 7) groups and 10 mg/kg for the moderate hypoglycemic group (n = 7). A high mortality rate (86%) was found in the moderate hypoglycaemic 7-NI-treated group (100 mg/kg), which then prompted a reduction in the 7-NI dose to 10 mg/kg. Because of its lipid solubility, MacKenzie et al. 43

and Bush and Pollack 11 demonstrated that the preferred route of administration of 7-NI


is by intraperitoneal (i.p.) injection. DMSO was the preferred carrier as opposed to peanut oil or arachis oil because of its higher rate of absorption. The dosages used in this study were based on our previous study17 and others35,67. Ishida et al. 35 demonstrated that 7-NI at 50 mg/kg transiently inhibited nNOS activity by 40% at 1 h, and 100 mg/kg inhibited nNOS activity by 56% at 1 h with signi� cantly sustained reduction. Although 7-NI has been demonstrated to inhibit bovine endothelial NOS in vitro8, 7-NI has not been shown to alter blood pressure at the dosages used in this study1,45,67. Therefore, 7-NI is a selec-tive nNOS inhibitor at these dosages. In the seventh group, three animals were studied to con� rm the relationship between pHi and moderate hypoglycemia, normoglycemia, and hyperglycemia using in vivo � uorescent imaging. This group was done to demon-strate the expected levels of regional cerebral blood � ow (rCBF), pHi, and NADH redox state under moderate hypoglycemic, normoglycemic, and hyperglycemic conditions in the six chronic study groups as described above. Moderate hypoglycemia (3-5 mmol/l) was obtained by using a single dose of Humilin-N U-100 (Lilly) 1.0 U/kg administered subcutaneously 1 h before ischemia. Hyperglycemia (17- 27 mmol/l) was achieved by intravenous infusion of dextrose 0.5 g/ml (50%) at 6 g/kg/h over 20 min followed by a 1.2 g/kg/h infusion for maintenance.

Serum glucose manipulations of pHi

Con� rmation of the e� ects of serum glucose manipulations on brain pHi was con� rmed in a group of three acute experiments using � uorescent imaging before, during, and af-ter focal cerebral ischemia (see discussion). As published previously, pHi as well as rCBF and NADH redox state can be measured in vivo by using umbelliferone, a � uorescent indicator 5. A PE-10 catheter was placed in the right external carotid artery with the tip at the carotid bifurcation for retrograde injection of umbelliferone. A video-� uorescent microscope was focused on the parietal cortex for brain pHi, rCBF, and NADH redox state measurements. Umbelliferone solution (0.2 g in 200 ml of 5% glucose) was injected into the external carotid line at 30- to 60-min intervals before, during, and after MCA and bilateral CCA occlusion. The pH indicator umbelliferone has two � uorophors, anionic and isobestic, which are excited at 370 and 340 nm, respectively, and have a common emission wavelength of 450 nm. The � uorescence of the anion varies directly with pH, whereas the isobestic � uorescence varies with concentration. Brain pHi can be then cal-culated from the 340-to-370 nm ratio. NADH � uorescent images excited at 370 nm are acquired before umbelliferone injection for correction of background � uorescence and for analysis of mitochondrial function61. The scale factor for the percent change in NADH � uorescence from baseline is set so that 100% represents the level of NADH � uorescence in the normal brain, whereas an increase to 300% represents brain death61. The images from the 340-nm excitation were processed to compute rCBF by using the 1-min initial slope index using a partition coe� cient of unity for umbelliferone5. All images of pHi,

64 Chapter 5

rCBF, and NADH redox states are stored on tape for analysis. rCBF as measured by um-belliferone is de� ned as those areas that are relatively avascular and contain primarily arterioles and capillary beds5. The imaging system allows the measurement of rCBF by allowing the investigator to outline cortical areas of interest, which are devoid of major surface conducting vessels.

Histopathology

Seventy-two hours after � ow restoration, the animals were reanesthetized with pento-barbital and then intracardially perfused with warm (37°C) 2% 2,3,5,-triphenyltetrazolium chloride (TTC) solution. The brains were quickly removed and immersed in the TTC solu-tion for 15 min to enhance staining after which they were then placed in a 10% bu� ered formaldehyde solution for 5 days. During this time period there was no diminution of TTC staining. Eleven serial coronal sections were cut from each brain at 1-mm intervals and photographed. Total cortical infarction volume was measured and calculated in a blinded fashion by integrating the infarct areas of all 11 sections (area of infarction in square millimeter x thickness of section). The total infarction volume was multiplied by the ratio of the total left hemisphere volume to that of the total right hemisphere volume to correct for cerebral edema24.

Statistical analysis

ANOVA followed by Fisher PLSD post hoc test was used to test the statistical signi� cance of di� erences between groups. A P value of < 0.05 was considered signi� cant. Polynomial regression was performed by using the individual data points from each group. Data are presented as means ± SE for all groups, with exception of region-of-interest data (brain pHi, rCBF, and NADH redox state), which from the video images are presented as means ± SD. The region-of-interest data are intended to show the expected alterations in rCBF, pHi, and NADH state under di� erent glycemic states used in the six chronic groups in this study. All analysis was conducted using SATVIEW statistical software.

RESULTS

In vivo fl uorescence imaging of brain pHi, rCBF, and NADH redox state

In three typical animals, brain pHi, rCBF, and NADH redox state were determined (Figs. 1 (for colour version see back cover) and 2). In normoglycemic animals [serum glucose, 10.7 mmol/l; PaCO2, 45 mmHg; arterial pH (pHa), 7.329; and mean arterial blood pressure (MABP) 86 mmHg] baseline brain pHi was 7.01 ± 0.03, NADH redox state measured 100%, and rCBF was 66.0 ± 15.5 ml /100 g / min. Brain pHi declined signi� cantly in the 2 h of ischemia to 6.58 ± 0.07, whereas NADH redox state markedly increased by 44%. rCBF


signi� cantly declined to 23.7 ± 13.4 ml / 100 g / min. Thirty minutes after restoration of � ow, brain pHi was 6.69 ± 0.03, NADH redox state decreased to near baseline levels, and rCBF increased to 85.7 ± 13.8 ml /100 g / min. In moderate hypoglycemic rats (serum glucose, 5.2 mmol; PaCO2,43 mmHg; pHa, 7.397; and MABP, 86 mmHg) baseline brain pHi was 7.01 ± 0.08 and then decreased to 6.79 ± 0.06 after 2 h of ischemia, whereas NADH redox state increased by 14% and rCBF declined from 79.8 ± 14.4 to 32.8 ± 10.3 ml / 100 g / min. Therefore, although CBF declined signi� cantly from baseline, there was no signi� cant brain acidosis during ischemia. Thirty minutes after restoration of � ow, brain pHi was 6.89 ± 0.05, NADH redox state decreased by 17% but still remained elevated, and rCBF increased to 89.1 ± 20.1 ml / 100 g/ min. In hyperglycemic animals (serum glucose, 19 mmol/l; PaCO2, 48 mmHg; pHa, 7.436; and MABP, 89 mmHg) after 2 h of ischemia, brain pHi decreased from 7.01 ± 0.07 to 6.12 ± 0.05, NADH redox state increased by 75%, and rCBF declined from 75.3 ± 24.4 to 16.44 ± 10.7 ml / 100 g / min. Thirty minutes after � ow restoration, brain pHi was 6.45 ± 0.10, NADH redox state decreased by 60%, and rCBF increased to 46.4 ± 17.4 ml / 100 g / min. The di� erence in brain pHi during ischemia between the normoglycemic and hyperglycaemic groups was signi� cant (P < 0.005).

Physiological parameters

There were no signi� cant di� erences in PaCO2, PaO2, pHa, body temperature, hematocrit, and MABP (Table 1). Glucose levels in moderate hypoglycemic and hyperglycemic treat-ment and control groups were signi� cantly (P < 0.05) di� erent from the normoglycemic control and treatment groups. Weight loss was signi� cantly (P < 0.05) increased only in the moderate hypoglycemic 7-NI-treated group compared with the control group.

sented as means � SE for all groups, with exception ofregion-of-interest data (brain pHi, rCBF, and NADH redoxstate), which from the video images are presented asmeans � SD. The region-of-interest data are intended toshow the expected alterations in rCBF, pHi, and NADH stateunder different glycemic states used in the six chronic groupsin this study. All analysis was conducted using SATVIEWstatistical software.

RESULTS

In vivo fluorescence imaging of brain pHi, rCBF, andNADH redox state. In three typical animals, brain pHi,rCBF, and NADH redox state were determined (Figs. 1and 2). In normoglycemic animals [serum glucose, 10.7mmol/l; PaCO2

, 45 mmHg; arterial pH (pHa), 7.329; andmean arterial blood pressure (MABP) 86 mmHg] base-line brain pHi was 7.01 � 0.03, NADH redox statemeasured 100%, and rCBF was 66.0 � 15.5 ml �100g�1 �min�1. Brain pHi declined significantly in the 2 hof ischemia to 6.58 � 0.07, whereas NADH redox statemarkedly increased by 44%. rCBF significantly de-clined to 23.7 � 13.4 ml �100 g�1 �min�1. Thirty min-utes after restoration of flow, brain pHi was 6.69 �0.03, NADH redox state decreased to near baselinelevels, and rCBF increased to 85.7 � 13.8 ml �100g�1 �min�1.

In moderate hypoglycemic rats (serum glucose, 5.2mmol; PaCO2

,43 mmHg; pHa, 7.397; and MABP, 86mmHg) baseline brain pHi was 7.01 � 0.08 and thendecreased to 6.79 � 0.06 after 2 h of ischemia, whereasNADH redox state increased by 14% and rCBF de-clined from 79.8 � 14.4 to 32.8 � 10.3 ml �100g�1 �min�1. Therefore, although CBF declined signifi-cantly from baseline, there was no significant brainacidosis during ischemia. Thirty minutes after restora-

tion of flow, brain pHi was 6.89 � 0.05, NADH redoxstate decreased by 17% but still remained elevated,and rCBF increased to 89.1 � 20.1 ml �100 g�1 �min�1.

In hyperglycemic animals (serum glucose, 19 mmol/l;PaCO2

, 48 mmHg; pHa,7.436; and MABP, 89 mmHg)after 2 h of ischemia, brain pHi decreased from 7.01 �0.07 to 6.12 � 0.05, NADH redox state increased by75%, and rCBF declined from 75.3 � 24.4 to 16.44 �10.7 ml �100 g�1 �min�1. Thirty minutes after flow res-toration, brain pHi was 6.45 � 0.10, NADH redox statedecreased by 60%, and rCBF increased to 46.4 � 17.4ml �100 g�1 �min�1. The difference in brain pHi duringischemia between the normoglycemic and hyperglyce-mic groups was significant (P � 0.005).

Physiological parameters. There were no significantdifferences in PaCO2

, PaO2, pHa, body temperature, he-

matocrit, and MABP (Table 1). Glucose levels in mod-erate hypoglycemic and hyperglycemic treatment andcontrol groups were significantly (P � 0.05) differentfrom the normoglycemic control and treatment groups.Weight loss was significantly (P � 0.05) increased onlyin the moderate hypoglycemic 7-NI-treated group com-pared with the control group.

Infarction volume. A significant (P � 0.0009) reduc-tion in cortical infarction volume (95.8 � 11.7 to 19.1 �10.4 mm3) was achieved by lowering serum glucoselevels from normoglycemia (�10 mmol/l) to moderatehypoglycemia (�3.3 mmol/l) (Fig. 3). Raising serumglucose levels to 22.4 � 32 mmol/l resulted in anexacerbation of cortical ischemic damage by 177.8% to170.3 � 13.8 mm3 (P � 0.0014 compared with normo-glycemia animals).

Treatment with 7-NI in the normoglycemic groupresulted in a significant (P � 0.0001) 93.5% reduction

Fig. 1. Video pictures of brain intracellular pH (pHi), NADH redox state, and regional cerebral blood flow (rCBF)in three typical animals during moderate hypoglycemia (serum glucose 5.2 mmol/l) (A), normoglycemia (serumglucose 10.7 mmol/l) (B), and hyperglycemia (serum glucose 19 mmol/l) (C). Calibration bars for pHi and rCBF areto the right of the three sets of images. Regions of interest (�13,600 �m2) for comparison of brain pHi, NADH redoxstate, and rCBF in these experiments are outlined in yellow. Each video frame is �0.5 � 0.5 cm.

H153IS NNOS ACTIVITY DEPENDENT ON ISCHEMIC BRAIN PHI?

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Fig. 1. Video pictures of brain intracellular pH (pHi), NADH redox state, and regional cerebral blood � ow (rCBF) in three typical animals during moderate hypoglycemia (serum glucose 5.2 mmol/l) (A), normoglycemia (serum glucose 10.7 mmol/l) (B), and hyperglycemia (serum glucose 19 mmol/l) (C). (For colour version see back cover) Calibration bars for pHi and rCBF are to the right of the three sets of images. Regions of interest (≈13,600 µm2) for comparison of brain pHi, NADH redox state, and rCBF in these experiments are outlined in yellow. Each video frame is ≈ 0.5 x 0.5 cm.

66 Chapter 5

Infarction volume

A signi� cant (P = 0.0009) reduction in cortical infarction volume (95.8 ± 11.7 to 19.1± 10.4 mm3) was achieved by lowering serum glucose levels from normoglycemia (≈10 mmol/l) to moderate hypoglycemia (≈3.3 mmol/l) (Fig. 3). Raising serum glucose levels to 22.4 ± 32 mmol/l resulted in an exacerbation of cortical ischemic damage to 177.8% to 170.3 ± 13.8 mm3 (P = 0.0014 compared with normoglycemia animals). Treatment with 7-NI in the normoglycemic group resulted in a signi� cant (P = 0.0001) 93.5% reduction in a corti-cal infarct volume compared with normoglycemic controls (6.4 ± 4.4 vs. 95.8 ± 11.7 mm3).

in a cortical infarct volume compared with normogly-cemic controls (6.4 � 4.4 vs. 95.8 � 11.7 mm3). Hyper-glycemic 7-NI-treated animals also demonstrated asignificant (P � 0.0216) 27.5% reduction in corticalinfarct volume compared with their hyperglycemic con-trols (123.6 � 11.1 vs. 170.3 � 13.8 mm3). Undermoderate hypoglycemic conditions treatment with 10mg/kg ip 7-NI resulted in a 72.6% reduction in corticalischemic damage compared with their moderate hypo-glycemic controls (5.3 � 4.1 vs. 19.1 � 10.4 mm3). Thiswas not statistically significant (P � 0.277).

DISCUSSION

This study provides evidence that one of the effects ofnNOS may be dependent on brain pHi in a model offocal cerebral ischemia. The protective effect of 7-NIexpressed as percent reduction of cortical infarct vol-ume was 93.3% in normoglycemia and 27.5% in hyper-glycemia. In the moderate hypoglycemic animals, thereduction in infarction volume did not reach signifi-cance because moderate hypoglycemia in itself dramat-ically reduced infarction volume. Therefore, 7-NI was

Fig. 2. Bar graph of rCBF, pHi, andNADH redox state from a moderatehypoglycemic (solid bar), normoglyce-mic (front slashed bar), and hypergly-cemic (back slashed bar) animal. Datadepicted are from the region of interestfrom each video image at their respec-tive time points from each animal.Graph demonstrates the findings ofrCBF, pHi, and NADH redox state un-der different glycemic conditions. Notethat before ischemia, the values ofrCBF, pHi, and NADH redox state arequite similar among the different gly-cemic states. During the ischemic pe-riod there are distinct gradated levelsof pHi and NADH redox state accord-ing to the level of glycemia. The wors-ening of rCBF at 2 h of ischemia and itsslow recovery after restoration of floware due mainly to edema. Data areexpressed as means � SD.

H154 IS NNOS ACTIVITY DEPENDENT ON ISCHEMIC BRAIN PHI?



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Fig. 2. Bar graph of rCBF, pHi, and NADH redox state from a moderate hypoglycemic (solid bar), normoglycemic (front slashed bar), and hyperglycaemic (back slashed bar) animal. Data depicted are from the region of interest from each video image at their respective time points from each animal. Graph demonstrates the � ndings of rCBF, pHi, and NADH redox state under di� erent glycemic conditions. Note that before ischemia, the values of rCBF, pHi, and NADH redox state are quite similar among the di� erent glycemic states. During the ischemic period there are distinct gradated levels of pHi and NADH redox state according to the level of glycemia. The worsening of rCBF at 2 h of ischemia and its slow recovery after restoration of � ow are due mainly to edema. Data are expressed as means ± SD.


Hyperglycemic 7-NI-treated animals also demonstrated a signi� cant (P = 0.0216) 27.5% reduction in cortical infarct volume compared with their hyperglycemic controls (123.6 ± 11.1 vs. 170.3 ± 13.8 mm3). Under moderate hypoglycemic conditions treatment with 10 mg/kg i.p. 7-NI resulted in a 72.6% reduction in cortical ischemic damage compared

Table 1. Physiological parameters

less effective at lower brain pHi conditions. The resultsof this study mirror that of the in vitro study by Heckeret al. (31) in which they demonstrated that the activityof microsomal constitutive NOS obtained from rat cer-ebellum was pH dependent in a biphasic fashion,where pH sensitivity was lost 1 unit above or below theoptimum pH of 6.7. Two other in vitro studies, Riveros-Moreno et al. (54) and Gorren et al. (25), also demon-strated a biphasic response between nNOS activity andpH. Combining the data with those of Hecker et al.(31), Riveros-Moreno et al. (54), Gorren et al. (25), andours, we can conclude that within the encountered pHrange (6.12–6.82), enzyme activity would vary consid-erably. Support for this includes the following: 1) in-creased intracellular acidosis achieved by augmentingthe severity of ischemia resulted in loss of the neuro-protective effect by the nonselective NOS inhibitor,L-NAME (3); and 2) in a separate study (18) adminis-tration of 3-morpholinosydnonimine hydrochloride(SIN-1), a NO donor, was far more effective duringhyperglycemia (�48% reduction vs. 27.5% in thisstudy), and less effective during normoglycemia (�71%reduction vs. 93.5% in this study) and moderate hypo-glycemia (�61% reduction vs. 72.6% in this study).Consideration of the binding properties of 7-NI tonNOS under different pH conditions must be takeninto account. To our knowledge, the pKa of 7-NI and its


PaCO2,mmHg

PaO2,mmHg pHa

MABP,mmHg

Glucose,mmol/l Hct, %

Temperature,°C

WeightLoss, %

HypoglycemiaIschemic control group

Before occlusion 37�1 210�8 7.437�0.012 80�3 4.0�0.6* 39.5�1.0 37.0�0.11 h ischemia 41�2 190�7 7.408�0.015 81�1 3.0�0.6* 38.9�0.5 37.0�0.12 h ischemia 39�2 181�7 7.435�0.017 83�3 3.0�0.5* 38.4�0.6 36.9�0.1 0.6�1.9

Treatment group(7-NI 10.0 mg/kg)

Before occlusion 38�2 234�11 7.448�0.010 87�5 4.0�0.2* 42.7�0.9 36.9�0.11 h ischemia 42�1 208�9 7.364�0.010 92�4 3.4�0.3* 43.0�1.2 37.0�0.02 h ischemia 40�1 198�9 7.404�0.015 95�6 3.1�0.0* 42.4�1.6 37.0�0.1 7.3�1.3†

NormoglycemiaIschemic control group

Before occlusion 38�3 185�10 7.442�0.047 82�2 9.8�1.0 37.7�1.0 36.9�0.11 h ischemia 38�4 163�11 7.463�0.041 81�2 9.5�1.1 36.9�1.1 36.9�0.12 h ischemia 36�3 158�13 7.479�0.040 87�4 9.7�0.8 35.1�1.7 37.0�0.2 15.2�2.3



HyperglycemiaIschemic control group




Values are means � SE. PaCO2, arterial PCO2; PaO2, arterial PO2; pHa, arterial pH; MABP, mean arterial blood pressure; Hct, hematocrit;7-NI, 7-nitroindazole. *Statistically different from normoglycemic control values (P � 0.05); † Statistically different from respectiveischemic control groups (P � 0.05).

Fig. 3. Bar graph of infarction volume (in mm3) comparing 7-nitro-indazole (7-NI)-treated animals (crosshatched bars) with ischemiccontrols (solid bars) at moderate hypoglycemia, normoglycemia, andhyperglycemia. Percent difference in infarction volumes betweentreated and nontreated animals in the hyperglycemic group was27.5% (P � 0.0216) and in the normoglycemic group it was 93.3%(P � 0.0001). Therefore, 7-NI was less effective in reducing infarctionvolume in the hyperglycemic group. Although there was a differenceof 72.6% during moderate hypoglycemia between the treated andnontreated groups, it was not statistically significant (P � 0.277).Data are expressed as means � SE. †Statistically different fromrespective ischemic control values, P � 0.05. *Statistically differentfrom normoglycemic ischemic control values, P � 0.005.




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Values are means ± SE. (PaCO2, arterial PCO2; PaO2, arterial PO2; pHa, arterial pH; MABP, mean arterial blood pressure; Hct, hematocrit; 7-NI, 7-nitroindazole.) *Statistically di� erent from normoglycemic control values (P < 0.05); † Statistically di� erent from respective ischemic control groups (P < 0.05).

less effective at lower brain pHi conditions. The resultsof this study mirror that of the in vitro study by Heckeret al. (31) in which they demonstrated that the activityof microsomal constitutive NOS obtained from rat cer-ebellum was pH dependent in a biphasic fashion,where pH sensitivity was lost 1 unit above or below theoptimum pH of 6.7. Two other in vitro studies, Riveros-Moreno et al. (54) and Gorren et al. (25), also demon-strated a biphasic response between nNOS activity andpH. Combining the data with those of Hecker et al.(31), Riveros-Moreno et al. (54), Gorren et al. (25), andours, we can conclude that within the encountered pHrange (6.12–6.82), enzyme activity would vary consid-erably. Support for this includes the following: 1) in-creased intracellular acidosis achieved by augmentingthe severity of ischemia resulted in loss of the neuro-protective effect by the nonselective NOS inhibitor,L-NAME (3); and 2) in a separate study (18) adminis-tration of 3-morpholinosydnonimine hydrochloride(SIN-1), a NO donor, was far more effective duringhyperglycemia (�48% reduction vs. 27.5% in thisstudy), and less effective during normoglycemia (�71%reduction vs. 93.5% in this study) and moderate hypo-glycemia (�61% reduction vs. 72.6% in this study).Consideration of the binding properties of 7-NI tonNOS under different pH conditions must be takeninto account. To our knowledge, the pKa of 7-NI and its


PaCO2,mmHg

PaO2,mmHg pHa

MABP,mmHg

Glucose,mmol/l Hct, %

Temperature,°C

WeightLoss, %

HypoglycemiaIschemic control group



Before occlusion 38�2 234�11 7.448�0.010 87�5 4.0�0.2* 42.7�0.9 36.9�0.11 h ischemia 42�1 208�9 7.364�0.010 92�4 3.4�0.3* 43.0�1.2 37.0�0.02 h ischemia 40�1 198�9 7.404�0.015 95�6 3.1�0.0* 42.4�1.6 37.0�0.1 7.3�1.3†

NormoglycemiaIschemic control group




HyperglycemiaIschemic control group




Values are means � SE. PaCO2, arterial PCO2; PaO2, arterial PO2; pHa, arterial pH; MABP, mean arterial blood pressure; Hct, hematocrit;7-NI, 7-nitroindazole. *Statistically different from normoglycemic control values (P � 0.05); † Statistically different from respectiveischemic control groups (P � 0.05).

Fig. 3. Bar graph of infarction volume (in mm3) comparing 7-nitro-indazole (7-NI)-treated animals (crosshatched bars) with ischemiccontrols (solid bars) at moderate hypoglycemia, normoglycemia, andhyperglycemia. Percent difference in infarction volumes betweentreated and nontreated animals in the hyperglycemic group was27.5% (P � 0.0216) and in the normoglycemic group it was 93.3%(P � 0.0001). Therefore, 7-NI was less effective in reducing infarctionvolume in the hyperglycemic group. Although there was a differenceof 72.6% during moderate hypoglycemia between the treated andnontreated groups, it was not statistically significant (P � 0.277).Data are expressed as means � SE. †Statistically different fromrespective ischemic control values, P � 0.05. *Statistically differentfrom normoglycemic ischemic control values, P � 0.005.




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Fig. 3. Bar graph of infarction volume (in mm3) comparing 7-nitroindazole (7-NI)-treated animals (crosshatched bars) with ischemic controls (solid bars) at moderate hypoglycemia, normoglycemia, and hyperglycemia. Percent di� erence in infarction volumes between treated and nontreated animals in the hyperglycemic group was 27.5% (P = 0.0216) and in the normoglycemic group it was 93.3% (P = 0.0001). Therefore, 7-NI was less e� ective in reducing infarction volume in the hyperglycemic group. Although there was a di� erence of 72.6% during moderate hypoglycemia between the treated and nontreated groups, it was not statistically signi� cant (P = 0.277). Data are expressed as means ± SE. †Statistically di� erent from respective ischemic control values, P < 0.05. *Statistically di� erent from normoglycemic ischemic control values, P < 0.005.

68 Chapter 5

with their moderate hypoglycaemic controls (5.3 ± 4.1 vs. 19.1 ± 10.4 mm3). This was not statistically signi� cant (P = 0.277).

DISCUSSION

This study provides evidence that one of the e� ects of nNOS may be dependent on brain pHi in a model of focal cerebral ischemia. The protective e� ect of 7-NI expressed as per-cent reduction of cortical infarct volume was 93.3% in normoglycemia and 27.5% in hy-perglycemia. In the moderate hypoglycemic animals, the reduction in infarction volume did not reach signi� cance because moderate hypoglycemia in itself dramatically reduced infarction volume. Therefore, 7-NI was less e� ective at lower brain pHi. The results of this study mirror that of the in vitro study by Hecker et al.31 in which they demonstrated that the activity of microsomal constitutive NOS obtained from rat cerebellum was pH dependent in a biphasic fashion, where pH sensitivity was lost 1 unit above or below the optimum pH of 6.7. Two other in vitro studies, Riveros-Moreno et al. 54 and Gorren et al.25, also demonstrated a biphasic response between nNOS activity and pH. Combining our data with those of Hecker et al.31, Riveros-Moreno et al.54, and Gorren et al.25, we can conclude that within the encountered pH range (6.12- 6.82), enzyme activity would vary considerably. Support for this includes the following: 1) increased intracellular acidosis achieved by augmenting the severity of ischemia resulted in loss of the neuroprotective e� ect by the non-selective NOS inhibitor, L-NAME3; and 2) in a separate study18 admin-istration of 3-morpholinosydnonimine hydrochloride (SIN-1), a NO donor, was far more e� ective during hyperglycemia (≈ 48% reduction vs. 27.5% in this study), and less ef-fective during normoglycemia (≈ 71% reduction vs. 93.5% in this study) and moderate hypoglycaemia (≈ 61% reduction vs. 72.6% in this study). Consideration of the binding properties of 7-NI to nNOS under di� erent pH conditions must be taken into account. To our knowledge, the pKa of 7-NI and its sensitivity to pH in neurons are unknown. However, because SIN-1, an NO donor, was far more e� ective than 7-NI during hypergly-cemia18, it is more than likely that if 7-NI had greater binding properties at more acidotic levels, it would have been less e� ective in reducing infarction volume. On the basis of this study, the following suggestion can be made. In the normal brain, NO is produced to maintain basal tone. In cerebral ischemia, NO production increases as the brain becomes ischemic to a pH optimum (≈ 6.7- 6.8). As the brain becomes more acidotic, NO produc-tion decreases. Therefore, it follows that with worsening acidosis below pH ≈ 6.7- 6.8, NO donors become more e� ective compared with NOS inhibitors and that decreasing acidosis above pH ≈ 6.7- 6.8, inhibition of nNOS becomes more e� ective.


Relationship among systemic glucose and pHi, rCBF, and NO

To determine the relationship between intracellular brain pH and the e� cacy of nNOS inhibition by 7-NI, animals were made moderately hypoglycemic, normoglycemic, and hyperglycemic to provide three graded levels of brain acidosis. In the last several dec-ades numerous investigations have been performed to study the e� ects of intracellular acidosis on cerebral ischemia by modi� cation of serum glucose levels via addition of glucose and/or insulin. Manipulation of serum glucose is a well-documented method to alter brain pHi during cerebral ischemia. For example, there have been several published studies4,6,13,14,21,29,32,39,40,57,58,62,64 in which brain pHi has been altered in both models of global and focal cerebral ischemia under hypoglycemic, normoglycemic, and hyperg-lycemic conditions. Studies37,39 have demonstrated that acidosis is one of the primary contributing factors of ischemic damage. Intracellular brain pHi becomes increasingly acidotic during ischemia declining from ≈ 6.7 to < 6.0 concomitant with serum glucose levels (≈ 6.5 to > 28 mmol/l). Conversely, brain pHi becomes less acidotic (6.7 to 7.0) as se-rum glucose levels decline toward moderately hypoglycemic levels (≈6.7 to ≈7.0 mmol/l)4,58. Cerebral infarction is reduced under moderate hypoglycemic conditions, whereas it becomes exacerbated under hyperglycemic conditions4,28. In our present study, moder-ate hypoglycemia (serum glucose ≈ 3 - 4 mmol/l) reduced cortical infarct volume by ≈ 80% to 19.1 ± 10.4 mm3 from 95.8 ± 11.7 mm3 in the normoglycemic group, whereas in the hyperglycemic animals this resulted in a 178% increase in infarction volume to 170 ± 14 mm3. To con� rm the e� ects of serum glucose concentrations on brain pHi in our rat model of focal cerebral ischemia used in this study, animals in the acute setting were studied and found to produce acidosis comparable to the di� erent glycemic levels as described above. rCBF is not altered during moderate hypoglycaemia in serum glucose concentrations of ≈ 3.0 mmol/l and at lower concentrations, rCBF increases signi� cant-ly9,15. Under hyperglycemic steady-state conditions, if the serum glucose concentration is ≈ 25 mmol/l or less, there is no signi� cant change in rCBF50,51. In concentrations greater than ≈ 25 mmol/l, rCBF is signi� cantly decreased. However, during ischemia, rCBF has been shown to be signi� cantly lower compared with normoglycemic conditions mainly due to edema formation38. These � ndings support the expected rCBF results in the ani-mals studied in the acute setting used in this study. Wei and Quast63, using microdialysis in rats, showed that hyperglycemia did not alter citruline levels compared with normo-glycemic rats before focal cerebral ischemia. Citruline is a by-product in the transforma-tion of arginine to NO. Citruline levels increased during focal cerebral ischemia, however, not signi� cantly at the majority of time points compared with normoglycemic rats. After restoration of � ow, citruline increased signi� cantly compared with normoglycemic rats. This follows that after restoration of � ow, brain (pHi) becomes less acidotic, and then, according to the in vitro NOS data25,31,54, NO production would increase. In nonischemic animals, Yu et al. 68 found that there were no di� erences in the activity and gene expres-

70 Chapter 5

sion of nNOS between hyperglycemic and normoglycemic nonischemic rats. It should be noted that hyperglycemia may have additional e� ects on extracellular excitatory amino acids23,42 and free fatty acids during ischemia52. With the use of in vivo microdialysis in rabbits, the e� ect of hyperglycemia on peri-ischemic extracellular glutamate concentra-tion in global ischemia revealed decreased glutamate concentrations compared with normoglycemic ischemic controls12. A reduction in glutamate e� ux under hyperglycemic conditions was also reported by Phyllis et al. 52. Raising blood glucose levels from 90 to 373 mg/dl (= 5 to 21 mmol/l) during severe focal ischemia in the rat increased glutamate levels in the neocortex with twofold higher peak values42. Di� erences between stud-ies were explained with the observation that glutamate release during ischemia varies with the experimental conditions and areas chosen for sampling42. However, in contrast, Yamamoto et al. 65 demonstrated in gerbils during hypothermia that ischemic damage was still reduced when given an intracerebral CA1 injection of glutamate. Nonetheless, it is conceivable that the measured e� ects of variable serum glucose on NO production in this study were multifactorial in addition to the alterations in brain pHi.

NOS inhibition in cerebral ischemia

7-NI is found to be a potent inhibitor of the neuronal constitutive isoforms of NOS and causes a dose-related antinociception45,46. MacKenzie et al. 43 studied the time course of brain NOS inhibition after administration of 7-NI intraperitoneally. At 30 mg/kg i.p., maxi-mal inhibition (85%) was reached 30 min after 7-NI administration. After 3 h, NOS activity was reduced to 29% of baseline and returned to baseline level at 24 h43. When 7-NI in arachis oil was administered at 10 mg/kg i.p. in young male Wistar rats, NOS activity was reduced maximally (85%) at 1 hour, whereas after 4 hours it was 60%, and at 24 hours it was 20%59. Studying the pharmacokinetics of 7-NI, Bush and Pollack11 discovered a marked nonlinearity consistent with saturable elimination after intraperitoneal adminis-tration of 7-NI dissolved in peanut oil. At a higher dose this resulted in a disproportion-ally increased exposure to 7-NI. Clearly, a critical review of the literature demonstrates contradictory results regarding the protective e� ects of NOS inhibitors. A study on time-dependent e� ects of NOS inhibition on ischemic cerebral damage using the nonselec-tive NOS inhibitor L-NAME at 3 mg/kg iv revealed that in a permanent focal ischemia model in the rat, early treatment (< 5 min of MCA occlusion) increased infarct volume, whereas given 3 h later, it slightly reduced infarct volume70. In contrast early treatment with L-NAME at 3 mg/kg iv repeated at 3, 6, 24, and 36 h in a similar MCA occlusion model reduced cortical infarct volume by 43%10. Margaill et al. 44 reported protective e� ects of treatment with L-NAME (1 mg/kg) up to 8 h after transient focal ischemia. Adachi et al. 1, using a gerbil model of global ischemia, demonstrated that both L-NAME and 7-NI exacerbated ischemic damage in the striatum after 5 min of occlusion compared with that of the saline control group. At 10 min of occlusion, L-NAME and 7-NI had no e� ect


on ischemic damage. This suggests that, at least in part, as ischemia worsens, these NOS inhibitors have reduced e� cacy in reducing infarction. In a model of neonatal hypoxia, Muramatsu et al. 48 showed a trend toward neuroprotection using 7-NI only at high dos-ages of 50 mg/kg i.p. In contrast, using 7-NI, Yoshida et al. 67 revealed a reduction of corti-cal damage of 24% and 25% for doses of 25 and 50 mg/kg i.p. in peanut oil, respectively. Signi� cant reductions in cortical infarct volume were previously reported by our group for doses of 10 and 100 mg/kg i.p. in 0.1 ml/kg DMSO17. The protective e� ects have been described for DMSO20,56; these studies used dosages (> 0.1 ml) much greater than the dosage used in this present study. DMSO at 0.1 ml/kg did not alter cortical infarct volume in the present study as well as in our previous study17 and in another study by Jiang et al. 36. At a dose of 100 mg/kg 7-NI, 60 min before occlusion, cortical infarct volume was reduced by 92% (from 92.5 to 9.0 mm3) 17. In the present study in which the occlusion time was reduced to 2 h, mean cortical infarct was reduced by 90% in the normoglycemic 7-NI-treated group when compared with the normoglycemic controls (from 102.3 to 9.9 mm3). Comparison of the e� ects of 7-NI (50 mg/kg i.p. in peanut oil) and NG-nitro-L-arginine (L-NNA, 1 mg/kg i.p.) on focal ischemic damage in the mouse revealed that L-NNA still reduced ischemic damage when given just before reperfusion, whereas 7-NI did not provide protection at this point26. Ninety minutes into ischemia, 7-NI (60 mg/kg i.p.) was also shown not to be neuroprotective, whereas the same dose was neuroprotective when given 5 min after MCA occlusion22. Although 7-NI did not reduce ischemic damage, it did reduce nitrotyrosine formation (a measure of peroxynitrite formation) to an extent comparable to L-NNA26. It was proposed that the partial inhibition of endothelial NOS provided the optimum bene� t by inhibiting peroxynitrite formation without signi� cantly increasing intravascular clogging26.

In conclusion, in this experiment the protective e� ect of selective nNOS inhibition by 7-NI during focal cerebral ischemia was altered by serum glucose concentrations, which in e� ect was manipulation of brain pHi. This apparent e� ect of pHi on NO is consistent with in vitro data25,31,54. We propose that brain pHi may be an important factor for determining NOS activity and that the observed variability in e� ects of NOS inhibition in di� erent models of cerebral ischemia is partly due to di� erences in brain pHi during ischemia. The e� ect of pH on nNOS activity provides an additional mechanism by which acidosis contributes to ischemic brain damage. Further investigations will be needed to elucidate the mechanism by which hydrogen ion concentration a� ects nNOS enzyme activity.

72 Chapter 5

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30. Harunki I, Traystman RJ, and Kirsch JR. E� ects of AR-R 17477, a potent neuronal nitric oxide syn-thase inhibitor on infarction volume resulting from permanent focal ischemia in rats. Crit Care Med 27: 2508–2511, 1999.

31. Hecker M, Mulsch A, and Busse R. Subcellular localization and characterization of neuronal nitric oxide synthase. J Neurochem 62: 1524–1529, 1994.

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33. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, and Moskowitz MA. E� ects of cerebral ischemia in mice de� cient in neuronal nitric oxide synthase. Science 265: 1883–1886,1994.

34. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci 20: 132–139, 1997.

35. Ishida A, Trescher WH, Lange MS, and Johnson MV. Prolonged suppression of brain nitric oxide synthase activity by 7-nitroindazole protects against cerebral hypoxic-ischemic injury in neonatal rat. Brain Dev 23: 349–354, 2001.

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74 Chapter 5

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41. Kuluz JW, Prado RJ, Dietrich WD, Schleien CL, and Watson BD. The e� ect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats. Stroke 24: 2023–2029, 1993.

42. Li PA, Shuaib A, Miyashita H, He QP, and Siesjo BK. Hyperglycemia enhances extracellular gluta-mate accumulation in rats subjected to forebrain ischemia. Stroke 31: 183–192, 2000.

43. MacKenzie GM, Rose S, Bland-Ward PA, Moore PK, Jenner P, and Marsden D. Time course of brain nitric oxide synthase by 7-nitroindazole. Neuroreport 5: 1993–1996, 1994.

44. Margaill I, Allix M, Boulu RG, and Plotkine M. Dose- and time dependence of L-NAME neuroprotec-tion in transient focal cerebral ischemia in rats. Br J Pharmacol 120: 160–163, 1997.

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46. Moore PK, Wallace P, Ga� en Z, Hart SL, and Babbedge RC. Characterization of the novel nitric oxide synthase inhibitor 7-nitroindazole and related indazoles: antinociceptive and cardiovascular ef-fects. Br J Pharmacol 110: 219–224, 1993.

47. Morikawa E, Moskowitz MA, Huang Z, Yoshida T, Irikura K, and Dalkara T. L-Arginine infusion promotes nitric oxidedependent vasodilation, increases regional blood � ow and reduces infarct volume in the rat. Stroke 25: 429–435, 1994.

48. Muramatsu K, Sheldon RA, Black SM, Tauber M, and Ferriero DM. Nitric oxide synthase activity and inhibition after neonatal hypoxia ischemia in the mouse brain. Brain Res Dev Brain Res 123: 119–127, 2000.

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50. Nedergaard M, Jakobesen J, and Diemer NH. Autoradiographic determination of cerebral glucose content, blood � ow, and glucose utilization in focal ischemia of the rat brain: in� uence of the plasma glucose concentration. J Cereb Blood Flow Metab 8: 100–108, 1988.

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53. Regli L, Held M, Anderson RE, and Meyer FB. Nitric oxide synthase inhibition by L-NAME prevents brain acidosis during focal cerebral ischemia in rabbits. J Cereb Blood Flow Metab 16: 988–995, 1996.

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58. Smith ML, von Hanwehr R, and Siesjo BK. Changes in extra- and intracellular pH in the brain during and following ischemia in hyperglycemic and in moderately hypoglycemic rats. J Cereb Blood Flow Metab 6: 574–583, 1986.

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60. Tamura A, Graham DI, McCulloch J, and Teasdale GM. Focal cerebral ischaemia in the rat: descrip-tion of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1: 53–60, 1981.

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62. Tomlinson FH, Anderson RE, and Meyer FB. E� ect of arterial blood pressure and serum glucose on brain intracellular pH, cerebral and cortical blood � ow during status epilepticus in the white New Zealand rabbit. Epilepsy Res 14: 123–137, 1993.

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65. Yamamoto H, Mitani A, Cui Y, Takechi S, Irita J, Suga T, Arai T, and Kataoka K. Neuroprotective ef-fects of mild hypothermia cannot be explained in terms of a reduction of glutamate release during ischemia. Neuroscience 91: 501–509, 1999.

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Chapter 6

E� ects of the Nitric Oxide Donor 3-Morpholinosydnomine (SIN-1) in

Focal Cerebral Ischemia Dependent on Intracellular pH.

Bernard A. Coert MD


Fredric B. Meyer MD

Journal of Neurosurgery, Vol. 97 October 2002 pp 914-921

78 Chapter 6

ABSTRACT

A nitric oxide (NO) donor that has been successfully used in the treatment of myocardial infarction, 3-morpholinosydnonimine (SIN-1), may be a potential neuroprotective agent. Production of NO in brain microsomes is dependent on the pH. The purpose of this study was to determine the e� cacy of SIN-1 and its dependence on pH in vivo during periods of focal cerebral ischemia. At 0.1 or 1 mg/kg, SIN-1 was administered to 54 Wistar rats 30 minutes before a 2-hour period of focal cerebral ischemia under moderate hypo-, normo-, and hyperglycemic conditions. Measurements of brain intracellular pH (pHi); regional cortical blood � ow, and the redox state of nicotinamide adenine dinucleotide were obtained in three additional animals to con� rm the e� ects of the serum glucose manipulations. The animals were killed at 72 hours after the ischemic period to obtain infarction volumes. Administration of SIN-1 signi� cantly reduced infarction in normogly-cemic animals and, to a lesser extent, in hyperglycemic animals, indicating that SIN-1 was less e� ective under hyperglycemic conditions. At either dose SIN-1 had no signi� cant ef-fect on infarction volume in moderately hypoglycemic animals because moderate hypo-glycemia in itself signi� cantly (p < 0.005) reduced infarction volume. The NO donor SIN-1 may be a useful intraoperative cerebral protective agent. Furthermore, it is hypothesized that a mechanism that could explain the published discrepancies regarding the e� ects of NO donors in vivo may be a� ected by di� erences in ischemic brain acidosis.

Are the e� ects of NO donor treatment dependent on pHi? 79

INTRODUCTION

The role of NO in the development of ischemic brain damage is complex. Endothelial cells, which produce endothelium-derived relaxing factor NO, regulate the basal tone of cerebral vessels37, platelet aggregation36, neutrophil in� ltration7, and neuronal function44. The NO donor SIN-1 is currently being used in interventional cardiology to minimize myocardial infarction17. Treatment by NO donors in di� erent animal studies has shown a trend toward neuroprotection11,13,30,39,50-52; however, isoenzyme- and con-centration-dependent dual mechanisms have been proposed to explain both protective and detrimental e� ects of nonselective inhibitors of NOS at di� erent dosages15. Hecker, et al.22,23, demonstrated that the activities of endothelial and neuronal NOS enzymes23

were dependent on pH. The “enzyme activity pH curve” was shown to be a narrow bell-shaped curve with an optimal enzyme activity at a pH of 7.6 for eNOS22 and at a pH of 6.7 for nNOS23. Therefore, the variable e� ects of endogenous NO donors described in dif-ferent published studies not only might be related to the ischemia model, dosage of the drug treatment, and timing, but also to the e� ects of intra- and periischemic pHi on NO activity. Supporting this concept is the observation that the nonselective NOS inhibitor, L-NAME, was not as e� cacious in situations of severe focal cerebral ischemia compared with those of moderate ischemia1.

In this study we tested the e� cacy of SIN-1, an NO donor, as a neuroprotectant, as well as the hypothesis that the severity of ischemic brain acidosis in� uences the neuroprotec-tive e� ects of the NO donor. The degree of brain acidosis was manipulated by altering serum glucose concentrations. A sydnonimine, SIN-1 is considered to be an endogenous endothelium-derived relaxing factor that is produced by the endothelium and is used to mimic the intravascular actions of NO32. Given the current technology, it is not possible to measure brain pH and NO simultaneously in vivo. We therefore conducted a compara-tive analysis of the literature to elucidate the possible e� ects of pHi on the production of NO.


Following approval by the Institutional Animal Care and Use Committee, anesthesia was induced in 60 adult male Wistar rats, each weighing between 300 and 450 g. Anesthesia was achieved by administration of halothane in a mixture of O2 and air, which was given through a face mask at a concentration of 2% during the surgical procedure and 1% during the occlusion period. Atropine was administered subcutaneously (0.08 mg/kg) preoperatively to reduce respiratory secretions. Core body temperature was maintained at 36.5 to 37.5°C by using a heating pad, and the temperature was monitored continu-

80 Chapter 6

ously by using a rectal temperature probe. Polyethylene catheters (type PE-50) were in-serted into the femoral artery to monitor arterial blood pressure and to facilitate arterial blood sampling (PaCO2, PaO2, pH, hematocrit, and serum glucose); similar catheters were inserted into a vein for administration of drugs. These animals’ physiological parameters were recorded 30 and 60 minutes before experimental ischemia was initiated, during the 2- hour ischemic period, and 30 minutes after restoration of blood � ow. Antibiotic medi-cation (Durapen, 30,000 U) was administered intramuscularly before wound closure.

Model of Focal Cerebral Ischemia

A modi� cation by Coert, et al. 14, of the technique described by Tamura, et al. 45, was used. A 2-cm ventral coronal incision was made in the neck to expose and place snares (No. 2 silk suture) around both CCAs. A 2-cm skin incision was made between the right outer canthus and the tragus of the right ear. The temporal muscle was de� ected anteriorly and the zygomatic arch was partially removed. After retraction of the musculature, the man-dibular nerve was identi� ed and followed back to the foramen ovale. Using a high-speed drill (Hall Surgical, Largo, FL) a small 3- to 4-mm craniectomy was made just anterior and superior to the foramen ovale. The dura mater was then opened with a sharp needle and the MCA was freed from the arachnoid. The MCA was temporarily occluded for 2 hours at the point where it crosses with the olfactory tract, by using a No. 3 Sundt arteriovenous malformation microclip (Johnson and Johnson Professional, Inc., Raynham, MA). Both CCAs were simultaneously temporarily occluded using the snares. In three animals used for � uorescent imaging, an extra 4 x 5 mm craniectomy was made over the frontal and parietal area of the cortex. The dura was removed and the edges at the margins of the craniectomy were carefully cauterized; the cortex was then covered with plastic wrap (Saran Wrap) to prevent surface oxygenation and keep the brain moist.

Experimental Groups

The animals were randomly divided into nine groups in the following arrangement: 1) there were three control groups: moderate hypoglycemia, normoglycemia, and hyper-glycemia (seven rats each); 2) three treatment groups in which SIN-1 (0.1 mg/kg) was administered intravenously to animals with moderate hypoglycemia (seven rats), normo-glycemia (six rats), and hyperglycemia (six rats); and 3) three treatment groups in which SIN-1 (1 mg/kg) was administered intravenously to rats with moderate hypoglycemia, normoglycemia, and hyperglycemia (seven rats each). Three additional animals, one with moderate hypoglycemia, one with normoglycemia, and one with hyperglycemia were studied before, during, and after induction of focal cerebral ischemia for imaging of brain pHi, regional cortical blood � ow, and the NADH redox state. Moderate hypoglycemia (1.7-4.4 mmol/l) was obtained using a single dose of Humilin-N U-100 (1 U/kg; Eli Lilly and Co., Indianapolis, IN), which was administered subcutaneously 1 hour before ischemia


was induced. Hyperglycemia (16.6-27.8 mmol/l) was achieved by an intravenous infusion of 0.5 g/ml dextrose (50% concentration) at 6 g/kg/hr over a 20-minute period, followed by 1.2 g/kg/hr for maintenance. Fresh solutions of SIN-1 were administered intravenously at 0.1 or 1 mg/kg, dissolved in saline (1 ml/kg) as a bolus injection, 30 minutes before MCA and bilateral CCA occlusion.

In Vivo Fluorescent Instrumentation

To determine changes in brain pHi in the cortex, � uorescent imaging was used before, during, and after focal cerebral ischemia. As has been described previously, pHi as well as regional cortical blood � ow, and the NADH redox state, can be measured in vivo by using umbelliferone, a � uorescent indicator3. A PE-10 catheter was placed in the right external CA, with the tip at the carotid bifurcation for retrograde injection of umbelliferone. A video-� uorescent microscope was focused on the parietal cortex to measure brain pHi, regional cortical blood � ow, and the NADH redox state. The umbelliferone solution (0.2 g in 200 ml of 5% glucose) was injected into the external CA catheter at 30- to 60-minute intervals before, during, and after MCA and bilateral CCA occlusion. The pH indicator, um-belliferone, has two � uorophors, anionic and isobestic, which are excited at 370 and 340 nm, respectively, and have a common emission wavelength of 450 nm. The � uorescence of the anion varies directly with the pH, whereas the isobestic � uorescence varies with the drug concentration. Brain pHi can then be calculated from the 340/370–nm ratio. The NADH � uorescent images excited at 370 nm are acquired before umbelliferone is injected to correct for background � uorescence and for analysis of mitochondrial function46. The scale factor for the percentage of change in NADH � uorescence from baseline is set so that 100% represents the level of NADH � uorescence in the healthy brain, whereas an increase to 300% represents brain death. The images obtained from the 340-nm excita-tion were processed to compute the regional cortical blood � ow by using the 1-minute initial slope index with a partition coe� cient of unity for umbelliferone3. All images of pHi, regional cortical blood � ow, and NADH redox states are stored on tape for analy-sis. Regional cortical blood � ow as measured using umbelliferone is de� ned as those areas that are relatively avascular and primarily contain arterioles and capillary beds3. The imaging system allows the measurement of regional cortical blood � ow by allowing the investigator to outline cortical areas of interest, which are devoid of major surface conducting vessels.

Histopathological Study

Seventy-two hours after � ow restoration, the animals were again placed in a state of an-esthesia, induced by pentobarbital, and then intracardially perfused using a warm (37°C) 2% TTC solution. The animals’ brains were quickly removed and immersed in the TTC solution for 15 minutes to enhance staining, after which the brains were placed in a 10%

82 Chapter 6

bu� ered formaldehyde solution for 5 days. Eleven serial coronal sections were cut from each brain at 1-mm intervals and photographed. Total cortical infarction volume was calculated by integrating the infarct areas of all 11 sections (area of infarction in square millimeters x thickness of section). The total infarction volume was multiplied by the ratio between total left hemisphere volume and total right hemisphere volume to correct for cerebral edema19.


Analysis of variance was followed by the Fisher post hoc test for multiple comparisons to test the statistical signi� cance of di� erences between groups. The Student unpaired t-test was used to compare measurements between di� erent time points within a group. A probability value lower than 0.05 was considered signi� cant. Data are presented as the means ± standard errors of the means for all groups, with the exception of data obtained from the video images (brain pHi and regional cortical blood � ow), which are presented as the means ± standard deviations. All analysis was conducted using STATVIEW statisti-cal software (Abacus Concepts, Inc., Berkeley, CA).

RESULTS

In Vivo Fluorescence Imaging of Brain pHi, regional cortical blood fl ow, and the NADH Redox State

Brain pHi, the NADH redox state, and regional cortical blood � ow were measured in three typical animals (Fig.1 for colour version see backcover). Baseline values measured before initiation of ischemia in the normoglycemic animal (serum glucose 10.7 mmol/L, PaCO2

45.1 mm Hg, pHa 7.329, and MABP 86 mm Hg) were as follows: brain pHi 7.01 ± 0.03, the NADH redox state 100%, and regional cortical blood � ow 66 ± 15.5 ml/100 g/min. After 2 hours of ischemia the brain pHi had declined to 6.58 ± 0.07, the NADH redox state had increased by 44% of baseline, and the regional cortical blood � ow had signi� cantly declined to 23.7 ± 13.4 ml/100 g/min. Thirty minutes after restoration of blood � ow, the brain pHi was 6.69 ± 0.03, the NADH redox state had decreased to near baseline levels, and the regional cortical blood � ow had increased to 85.7 ± 13.8 ml/100 g/min. In the moderately hypoglycemic animal (serum glucose 5.2 mmol/L, PaCO2 43 mm Hg, pHa 7.397, and MABP 86 mm Hg) the baseline brain pHi, which had been 7.01 ± 0.08, had decreased to 6.79 ± 0.06 after 2 hours of ischemia, the NADH redox state had increased by 14% of baseline, and the regional cortical blood � ow had declined from 79.8 ± 14.4 to 32.8 ± 10.3 ml/100 g/min. Thirty minutes after restoration of blood � ow, the brain pHi was 6.89 ± 0.05, the NADH redox state had decreased by 17%, and the regional cortical blood � ow had increased to 89.1 ± 20.1 ml/100 g/min. Baseline values in the hypergly-cemic animal (serum glucose 19 mmol/L, PaCO2 48 mm Hg, pHa 7.436, and MABP 89 mm


Hg) before initiation of ischemia were the following: brain pHi 7.01 ± 0.07, the NADH redox state 100%, and regional cortical blood � ow 75.3 ± 24.4 ml/100 g/min. After 2 hours of ischemia, the brain pHi had decreased to 6.12 ± 0.05, the NADH redox state had increased by 75% of baseline, and the regional cortical blood � ow declined to 16.4 ± 10.7 ml/100 g/min. Thirty minutes after restoration of blood � ow, the brain pHi was 6.45 ± 0.1, NADH redox state had decreased by 60%, and the regional cortical blood � ow had increased to 46.4 ± 17.4 ml/100 g/min. The di� erence in brain pHi during periods of focal cerebral ischemia between the normoglycemic and hyperglycaemic groups was signi� cant (p < 0.005).

Physiological Parameters

Treatment with SIN-1 did result in a temporary reduction in MABP directly after intrave-nous injection, but MABP recovered during the 30 minutes between injection of SIN-1 and occlusion of the MCA and CCAs. Weight loss was reduced signi� cantly (p < 0.05) in both normoglycemic and hyperglycemic SIN-1–treated animals in response to both low and high doses. The SIN-1 did not signi� cantly a� ect the glucose response to insulin in the hypoglycemic groups (Table 1).

Infarction Volume

The decrease in serum glucose levels from 10 to 3.3 mmol/L resulted in a signi� cant (p < 0.001) reduction in cortical infarction volume by 80%, from 95.8 ± 12 to 19.1 ± 11 mm3, compared with the normoglycemic group (Fig. 2). Hyperglycemia (glucose level 22.4 mmol/L) resulted in exacerbation of cortical ischemic damage by 178%, to 170.3 ± 14 mm3 (p < 0.005 compared with normoglycemic animals). When compared with the normoglycemic controls, SIN-1 at 0.1 mg/kg signi� cantly decreased infarction volume

1). Baseline values measured before initiation of ischemiain the normoglycemic animal (serum glucose 10.7mmol/L, PaCO2 45.1 mm Hg, pHa 7.329, and MABP 86mm Hg) were as follows: brain pHi 7.01 � 0.03, theNADH redox state 100%, and regional cortical blood flow66 � 15.5 ml/100 g/min. After 2 hours of ischemia thebrain pHi had declined to 6.58 � 0.07, the NADH redoxstate had increased to 44% of baseline, and the regionalcortical blood flow had significantly declined to 23.7 �13.4 ml/100 g/min. Thirty minutes after restoration ofblood flow, the brain pHi was 6.69 � 0.03, the NADHredox state had decreased to near baseline levels, and theregional cortical blood flow had increased to 85.7 � 13.8ml/100 g/min.

In the moderately hypoglycemic animal (serum glucose5.2 mmol/L, PaCO2 43 mm Hg, pHa 7.397, and MABP 86mm Hg) the baseline brain pHi, which had been 7.01 �0.08, had decreased to 6.79 � 0.06 after 2 hours ofischemia, the NADH redox state had increased by 14% ofbaseline, and the regional cortical blood flow had declinedfrom 79.8 � 14.4 to 32.8 � 10.3 ml/100 g/min. Thirtyminutes after restoration of blood flow, the brain pHi was6.89 � 0.05, the NADH redox state had decreased by17%, and the regional cortical blood flow had increased to89.1 � 20.1 ml/100 g/min.

Baseline values in the hyperglycemic animal (serum glu-cose 19 mmol/L, PaCO2 48 mm Hg, pHa 7.436, and MABP89 mm Hg) before initiation of ischemia were the follow-ing: brain pHi 7.01 � 0.07, the NADH redox state 100%,and regional cortical blood flow 75.3 � 24.4 ml/100 g/min.After 2 hours of ischemia, the brain pHi had decreased to6.12 � 0.05, the NADH redox state had increased to 75%of baseline, and the regional cortical blood flow declined to16.4 � 10.7 ml/100 g/min. Thirty minutes after restorationof blood flow, the brain pHi was 6.45 � 0.1, NADH redoxstate had decreased by 60%, and the regional cortical bloodflow had increased to 46.4 � 17.4 ml/100 g/min. The dif-ference in brain pHi during periods of focal cerebralischemia between the normoglycemic and hyperglycemicgroups was significant (p � 0.005).

Physiological ParametersTreatment with SIN-1 did result in a temporary reduc-

tion in MABP directly after intravenous injection, butrecovered during the 30 minutes between injection ofSIN-1 and occlusion of the MCA and CCAs. Weight losswas reduced significantly (p � 0.05) in both normogly-cemic and hyperglycemic SIN-1–treated animals in re-sponse to both low and high doses. The SIN-1 did not sig-nificantly affect the glucose response to insulin in thehypoglycemic groups (Table 1).

Infarction VolumeThe decrease in serum glucose levels from 10 to 3.3

mmol/L resulted in a significant (p � 0.001) reduction incortical infarction volume by 80%, from 95.8 � 12 to 19.1 � 11 mm3, compared with the normoglycemic group(Fig. 2). Hyperglycemia (glucose level 22.4 mmol/L)resulted in exacerbation of cortical ischemic damage by178%, to 170.3 � 14 mm3 (p � 0.005 compared withnormoglycemic animals).

When compared with the normoglycemic controls, SIN-1 at 0.1 mg/kg significantly decreased infarction volume by71% (p � 0.003), from 95.8 � 12 to 27.9 � 12 mm3. In-creasing the dose of SIN-1 to 1 mg/kg also caused a signif-icant reduction in ischemic damage by 69% (p � 0.018), to33.4 � 10 mm3, compared with the normoglycemic controlgroup. Infarction volumes between the two normoglycemicSIN-1–treated groups were not significantly different.

In hyperglycemic animals SIN-1 treatment at 0.1 and 1mg/kg significantly reduced cortical infarction volume by45% (p � 0.025) and 51% (p � 0.023), from 170.3 � 14mm3 to 87.3 � 18.7 and 83.5 � 17.3 mm3, respectively.Increasing the SIN-1 treatment dose in hyperglycemic ani-mals from 0.1 to 1 mg/kg did not significantly decreasecortical infarction volume.

The differences in infarction volumes between the SIN-1–treated normoglycemic groups (69–71%) and SIN–treatedhyperglycemic groups (45–51%) was statistically signifi-cant (p � 0.05).


916 J. Neurosurg. / Volume 97 / October, 2002

FIG. 1. Still pictures from videotapes of brain pHi, NADH redox state, and regional cortical blood flow (rCBF) in threetypical animals during the following periods: moderate hypoglycemia (serum glucose 5.2 mmol/L; left), normoglycemia(serum glucose 10.7 mmol/L; center), and hyperglycemia (serum glucose 19 mmol/L; right). Calibration bars for pHi andregional cortical blood flow are placed at the far right of the three sets of images. The regions of interest (approximate-ly 14,000 �m2) for a comparison of brain pHi, the NADH redox state, and regional cortical blood flow in these experi-ments are outlined in white. Each video frame originally was approximately 0.5 � 0.5 cm.

Figure. 1. Still pictures from videotapes of brain pHi, NADH redox state, and regional cortical blood � ow (rCBF) in three typical animals during the following periods: moderate hypoglycemia (serum glucose 5.2 mmol/L; left), normoglycemia (serum glucose 10.7 mmol/L; center), and hyperglycemia (serum glucose 19 mmol/L; right). Calibration bars for pHi and regional cortical blood � ow are placed at the far right of the three sets of images. The regions of interest (approximately 14,000 µm2) for a comparison of brain pHi, the NADH redox state, and regional cortical blood � ow in these experiments are outlined in white. Each video frame originally was approximately 0.5 x 0.5 cm. (For the colour version see the backcover.)

84 Chapter 6

by 71% (p < 0.003), from 95.8 ± 12 to 27.9 ± 12 mm3. Increasing the dose of SIN-1 to 1 mg/kg also caused a signi� cant reduction in ischemic damage by 69% (p < 0.018), to 33.4 ± 10 mm3, compared with the normoglycemic control group. Infarction volumes between the two normoglycemic SIN-1–treated groups were not signi� cantly di� erent. In hyperglycemic animals SIN-1 treatment at 0.1 and 1 mg/kg signi� cantly reduced corti-cal infarction volume by 45% (p < 0.025) and 51% (p < 0.023), from 170.3 ± 14 mm3 to 87.3 ± 18.7 and 83.5 ± 17.3 mm3, respectively. Increasing the SIN-1 treatment dose in hyperglycemic animals from 0.1 to 1 mg/kg did not signi� cantly decrease cortical infarc-tion volume. The di� erences in infarction volumes between the SIN-1- treated normogly-cemic groups (69-71%) and SIN–treated hyperglycemic groups (45–51%) was statistically signi� cant (p < 0.05). In the insulin-induced moderately hypoglycemic animals, SIN-1 tended to reduce infarction volume at both dosages of 0.1 mg/kg (8.4 ± 4.9 mm3) and 1 mg/kg (6.6 ± 3.4 mm3), but this reduction did not reach statistical signi� cance (p < 0.22 and p < 0.29, respectively).

In the insulin-induced moderately hypoglycemic ani-mals, SIN-1 tended to reduce infarction volume at bothdosages of 0.1 mg/kg (8.4 � 4.9 mm3) and 1 mg/kg (6.6 �3.4 mm3), but this reduction did not reach statistical sig-nificance (p � 0.22 and p � 0.29, respectively).

DiscussionThree hypotheses can be supported by this study. First,

SIN-1, an NO donor that has been used in the treatment ofmyocardial infarction may also be useful as an intraoper-ative neuroprotectant and as a treatment for stroke. Sec-ond, the efficacy of SIN-1 is dependent on brain pHi, andmay be more effective in cases of moderate ischemia thanin those of severe ischemia. Third, when evaluating thepotential effects of pharmaceutical agents on the treatmentof central nervous system disorders, such as cerebrovas-cular disease and brain tumors, brain pHi must be consid-ered to be a possible influencing factor.

The NO Donor SIN-1

The NO donor SIN-1 is currently used in interventionalcardiology because it produces antispastic and vasodilatoryeffects without inducing tolerance.17 In human acute coro-nary syndromes, titration of SIN-1 to the desired antiisch-

emic effect was suggested to lie within the flow rate of 0.2and 1.6 mg/hour, and titration to the desired vasodilatoryeffect, without untoward action on filling pressures, cardiacindex, or heart rate was said to lie with doses of approxi-mately 1 mg/hour. Comparing these suggested dosageswith the dosages used in the present study and in other ani-mal studies39,50,51,52 we have to conclude that the amountsused in this study—0.1 and 1 mg/kg in rats weighing be-tween 300 and 450 g—are relatively high doses.

In this analysis of SIN-1, Feelisch, et al.,16 reported thatsimultaneous with NO release, SIN-1 generates superox-ide. Bohn and Schönafinger8 identified two factors that in-fluence NO release, PO2 and pH, and found that the oxida-tive capacity in vivo was high enough to guarantee NOrelease under ischemic conditions. Singh, et al.,41 reportedthat the pure NO donor behavior exhibited by SIN-1 invivo, without superoxide production, occurred because ofthe presence of biological electron acceptors that outcom-pete O2.

Combining the data from Noack and Feelisch32 on time-dependent formation of various metabolites and theirvelocity of initial NO liberation, the overall half-life forthe NO donation of SIN-1 was estimated to be 230 min-utes. As we reported earlier13 and in the present study, weobserved a temporary reduction in MABP after intra-venous administration of SIN-1, but MABP returned tobaseline within 30 minutes between injection of the drugand onset of ischemia.

Although reports of the protective effect of NO donorssuch as SIN-1 have been limited primarily to permanentischemia models, NO donors have been demonstrated tobe efficacious in the present study and in the work ofSalom, et al.,39 in which a model of temporary focal cere-bral ischemia was used. Proposed mechanisms of protec-tion include the following: the reduction of activation andrecruitment of neutrophils that produce cytokines and pro-teases, amplifying endothelial dysfunction and promotingtissue damage, and the loss of vasomotor control con-tributing to the “no reflow” phenomenon.24

The agent SIN-1 also has a negative insulinotropic ac-tion,5,42 whereas L-arginine was found to induce insulinsecretion from pancreatic � cells.40 In their study, Zhang,et al.,52 reported an approximately 20% increase in glucoselevels in SIN-1– and phenylephrine-treated groups, butfound in a separate group that SIN-1 (3 mg/kg) withoutphenylephrine did not significantly alter serum glucoselevels, thus attributing that effect to phenylephrine. In ourexperiment in normoglycemic, moderately hypoglycemic,and hyperglycemic rats, SIN-1 treatment did not result inglucose levels that were different from those measured inthe moderately hypo-, normo-, and hyperglycemic controlgroups.

In this study, the efficacy of SIN-1, expressed as a per-centage of reduction in cortical infarction volume, wasapproximately 70% in rats with normoglycemia, approxi-mately 61% in rats with hypoglycemia, and 48% in ratswith hyperglycemia. Therefore, SIN-1 was less effectiveunder more acidic brain conditions. These data closelymirror the endothelial cNOS response curve that wasdemonstrated in vitro by Hecker, et al.22 In their study, theconcentration of hydrogen ions was found to be an impor-tant determinant of endothelial cNOS activity, whereas pHsensitivity was lost one unit above or below the optimum

J. Neurosurg. / Volume 97 / October, 2002

Intracellular brain pHi and SIN-1

917

FIG. 2. Bar graph of infarction volumes shown in cubic mil-limeters comparing animals treated with SIN-1 at 0.1 and 1 mg/kgwith ischemic controls during moderate hypoglycemia, normo-glycemia, and hyperglycemia. The percentage differences ininfarction volumes between treated and nontreated animals were45 � 11% and 51 � 10% in the hyperglycemic group and 71 �13% and 69 � 10% in the normoglycemic group for 0.1 mg/kg and1 mg/kg SIN-1, respectively. Therefore, SIN-1 was less effectivein reducing infarction volume as brain pHi became more acidic.During moderate hypoglycemia, differences between the treatedand nontreated groups were 56 � 27% and 66 � 19% for 0.1 and1 mg/kg SIN-1, respectively; this was not statistically significant.Data are expressed as the means � standard errors of the means.*Statistically different from normoglycemic ischemic control val-ues (p � 0.005). †Statistically different from respective ischemiccontrol values (p � 0.025, analysis of variance).

Figure. 2. Bar graph of infarction volumes shown in cubic millimeters comparing animals treated with SIN-1 at 0.1 and 1 mg/kg with ischemic controls during moderate hypoglycemia, normoglycemia, and hyperglycemia. The percentage di� erences in infarction volumes between treated and nontreated animals were 45 ± 11% and 51 ± 10% in the hyperglycemic group and 71 ± 13% and 69 ± 10% in the normoglycemic group for 0.1 mg/kg and 1 mg/kg SIN-1, respectively. Therefore, SIN-1 was less e� ective in reducing infarction volume as brain pHi became more acidic. During moderate hypoglycemia, di� erences between the treated and nontreated groups were 56 ± 27% and 66 ± 19% for 0.1 and 1 mg/kg SIN-1, respectively; this was not statistically signi� cant. Data are expressed as the means ± standard errors of the means. *Statistically di� erent from normoglycemic ischemic control values (p < 0.005). †Statistically di� erent from respective ischemic control values (p < 0.025, analysis of variance).


DISCUSSION

Three hypotheses can be supported by this study. First, SIN-1, an NO donor that has been used in the treatment of myocardial infarction may also be useful as an intraoperative neuroprotectant and as a treatment for stroke. Second, the e� cacy of SIN-1 is dependent on brain pHi, and may be more e� ective in cases of moderate ischemia than in those of severe ischemia. Third, when evaluating the potential e� ects of pharmaceutical agents on the treatment of central nervous system disorders, such as cerebrovascular disease and brain tumors, brain pHi must be considered to be a possible in� uencing factor.

The NO Donor SIN-1

The NO donor SIN-1 is currently used in interventional cardiology because it produces antispastic and vasodilatory e� ects without inducing tolerance17. In human acute coro-nary syndromes, titration of SIN-1 to the desired antiischemic e� ect was suggested to lie within the � ow rate of 0.2 and 1.6 mg/hour, and titration to the desired vasodilatory

Table 1 The mean systemic parameters in the study groups

(pH 7.6). Similar studies on pH sensitivity of the neuronalisoform of cNOS revealed an optimal pH of 6.7 within abell-shaped curve similar to endothelial cNOS.23 Com-bining their data on both cNOS enzymes, we can concludethat, within the pH range encountered (6.12–6.82), en-zyme activity would vary considerably. The followingsupports this hypothesis: 1) increased intracellular acido-sis achieved by augmenting the severity of ischemiacaused a loss of the neuroprotective effect by the nonse-lective NOS inhibitor, L-NAME, suggesting that NOSinhibition was less effective because of inhibition of NOSactivity in acidosis;1 2) in a separate study (Coert, et al., inpress), administration of 7-NI, a selective nNOS inhibitor,was far less effective during periods of hyperglycemia(27.5% reduction compared with approximately 48% inthis study) and more effective during periods of both nor-moglycemia (93.5% reduction compared with approxi-mately 70% in this study) and hypoglycemia (72.6% re-duction compared with approximately 61% in this study).

Based on the data the following hypothesis can be sup-ported. In the healthy brain, NO is produced to maintainbasal tone. In the brain with cerebral ischemia, NO pro-duction increases as the brain becomes ischemic, to a pHoptimum of NO (approximately 6.7–6.8). Thereafter, pro-duction decreases as the brain becomes more acidotic.Therefore, it follows that, with worsening acidosis—a pHbelow approximately 6.7—NO donors become more ef-fective compared with NOS inhibitors, and with improv-ing acidosis—a pH above approximately 6.7—inhibitionof nNOS becomes more effective.

Role of NO in Cerebral Ischemia

Direct measurements of NO production in vivo haverevealed both increased28 and decreased NO concentrationduring cerebral ischemia.29 Using a porphyrinic microsen-sor, Malinski, et al.,28 reported an increase from a baselinelevel of lower than 10�8 M to an approximate level of


918 J. Neurosurg. / Volume 97 / October, 2002

TABLE 1The mean systemic parameters in the study groups*

PaCO2 PaO2 MABP Glucose Hematocrit Temperature Weight Group (mm Hg) (mm Hg) pHa (mm Hg) (mmol/L) (%) (°C) Loss (%)

hypoglycemiaischemic control group

before occlusion 36.7 � 0.8 210.3 � 7.6 7.437 � 0.012 79.8 � 2.7 4.0 � 0.6† 39.5 � 1.0 37.0 � 0.1at 1 hr ischemia 40.7 � 2.2 190.4 � 7.3 7.408 � 1.015 80.7 � 1.0 3.0 � 0.6† 38.9 � 0.5 37.0 � 0.1at 2 hrs ischemia 39.1 � 1.9 180.7 � 7.0 7.435 � 0.017 83.2 � 2.9 3.0 � 0.5† 38.4 � 0.6 36.9 � 0.1 0.6 � 1.9

SIN-1 0.1 mg/kg treatment groupbefore occlusion 34.8 � 1.7 194.7 � 19.0 7.454 � 0.020 84.0 � 3.9 4.2 � 0.4† 36.2 � 1.1 38.3 � 0.4at 1 hr ischemia 42.4 � 1.5 193.3 � 9.0 7.416 � 0.016 89.1 � 2.7 2.7 � 0.6† 36.0 � 1.5 38.2 � 0.4at 2 hrs ischemia 41.0 � 2.3 181.3 � 7.0 7.412 � 0.021 89.5 � 3.0 2.4 � 0.3† 36.2 � 1.2 38.2 � 0.4 5.6 � 2.7

SIN-1 1 mg/kg treatment groupbefore occlusion 39.7 � 1.5 212.9 � 3.7 7.442 � 0.013 81.4 � 2.2 3.5 � 0.6† 39.2 � 0.6 37.0 � 0.0at 1 hr ischemia 36.4 � 1.6 207.1 � 5.9 7.450 � 0.015 84.4 � 2.9 2.3 � 0.2† 38.1 � 1.1 37.0 � 0.1at 2 hrs ischemia 38.9 � 1.5 195.1 � 6.7 7.431 � 0.016 84.9 � 2.8 1.9 � 0.3† 39.2 � 0.5 37.0 � 1.0 6.4 � 2.3

normoglycemiaischemic control group

before occlusion 37.8 � 2.6 185.0 � 10.2 7.442 � 0.047 81.9 � 1.9 9.8 � 1.0 37.7 � 1.0 36.9 � 0.1at 1 hr ischemia 37.7 � 4.1 163.4 � 10.7 7.463 � 0.041 80.8 � 2.1 9.5 � 1.1 36.9 � 1.1 36.9 � 0.1at 2 hrs ischemia 36.1 � 2.5 158.1 � 13.3 7.479 � 0.040 87.4 � 3.6 9.7 � 0.8 35.1 � 1.7 37.0 � 0.2 15.2 � 2.3

SIN-1 0.1 mg/kg treatment groupbefore occlusion 36.3 � 1.4 191.3 � 8.8 7.429 � 0.013 82.0 � 2.9 8.2 � 0.5 38.5 � 0.9 37.0 � 1.0at 1 hr ischemia 41.8 � 0.6 190.5 � 11.1 7.401 � 0.013 86.3 � 2.9 7.6 � 0.4 38.4 � 1.7 37.0 � 1.0at 2 hrs ischemia 40.4 � 0.7 178.3 � 17.0 7.404 � 0.008 77.7 � 6.0 7.3 � 0.5 37.2 � 1.4 37.0 � 1.0 2.5 � 1.6‡

SIN-1 1 mg/kg treatment groupbefore occlusion 34.0 � 1.1 163.0 � 12.6 7.424 � 0.018 82.6 � 2.2 8.4 � 0.5 37.6 � 0.4 37.0 � 1.0at 1 hr ischemia 38.3 � 1.6 161.9 � 10.9 7.418 � 0.016 84.3 � 1.5 8.1 � 0.7 37.6 � 0.8 37.0 � 1.0at 2 hrs ischemia 39.5 � 1.9 162.4 � 10.9 7.421 � 0.012 90.8 � 1.5 8.4 � 0.9 37.4 � 1.2 37.0 � 1.0 6.4 � 1.8‡

hyperglycemiaischemic control group

before occlusion 42.5 � 3.1 166.0 � 21.0 7.372 � 0.017 86.0 � 1.0 15.9 � 3.8† 37.5 � 0.9 36.9 � 0.1at 1 hr ischemia 41.1 � 1.1 160.2 � 20.2 7.362 � 0.024 87.0 � 1.0 26.5 � 0.7† 37.1 � 0.7 37.0 � 0.1at 2 hrs ischemia 39.8 � 1.1 155.5 � 18.3 7.400 � 0.018 86.0 � 1.0 18.5 � 2.2† 36.3 � 0.5 37.0 � 0.1 15.7 � 2.2

SIN-1 0.1 mg/kg treatment groupbefore occlusion 40.7 � 2.2 168.3 � 7.9 7.377 � 0.020 86.8 � 1.5 22.9 � 2.2† 38.5 � 1.0 37.0 � 1.0at 1 hr ischemia 39.7 � 1.7 154.7 � 15.8 7.384 � 0.019 84.6 � 2.9 21.4 � 1.7† 37.0 � 1.6 37.0 � 1.0at 2 hrs ischemia 43.2 � 1.8 145.8 � 11.4 7.372 � 0.016 87.2 � 1.7 18.3 � 0.3† 38.8 � 1.6 37.0 � 1.0 6.4 � 1.6‡

SIN-1 1 mg/kg treatment groupbefore occlusion 35.0 � 2.1 190.1 � 4.0 7.403 � 0.013 82.2 � 1.4 22.4 � 2.1† 35.1 � 1.3 37.0 � 1.0at 1 hr ischemia 41.0 � 1.9 181.7 � 9.8 7.390 � 0.014 87.6 � 2.1 22.7 � 1.1† 35.4 � 1.2 37.0 � 1.0at 2 hrs ischemia 39.5 � 3.0 188.7 � 5.4 7.410 � 0.022 87.0 � 0.9 20.9 � 1.2† 36.0 � 1.1 37.0 � 1.0 8.0 � 1.9‡

* Values are expressed as the means � standard errors of the means.† Statistically different from normoglycemic control values (p � 0.05).‡ Statistically different from respective ischemic control values (p � 0.05).

* Values are expressed as the means ± standard errors of the means.† Statistically di� erent from normoglycemic control values (p < 0.05).‡ Statistically di� erent from respective ischemic control values (p < 0.05).

86 Chapter 6

e� ect, without untoward action on � lling pressures, cardiac index, or heart rate was said to lie with doses of approximately 1 mg/hour. Comparing these suggested dosages with the dosages used in the present study and in other animal studies39,50,51,52 we have to conclude that the amounts used in this study, 0.1 and 1 mg/kg in rats weighing between 300 and 450 g, are relatively high. In this analysis of SIN-1, Feelisch, et al. 16, reported that simultaneous with NO release, SIN-1 generates superoxide. Bohn and Schöna� nger8

identi� ed two factors that in� uence NO release, PO2 and pH, and found that the oxidative capacity in vivo was high enough to guarantee NO release under ischemic conditions. Singh, et al. 41, reported that the pure NO donor behaviour exhibited by SIN-1 in vivo, without superoxide production, occurred because of the presence of biological electron acceptors that outcompete O2. Combining the data from Noack and Feelisch32 on time dependent formation of various metabolites and their velocity of initial NO liberation, the overall half-life for the NO donation of SIN-1 was estimated to be 230 minutes. As we reported earlier13 and in the present study, we observed a temporary reduction in MABP after intravenous administration of SIN-1, but MABP returned to baseline within 30 minutes between injection of the drug and onset of ischemia. Although reports of the protective e� ect of NO donors such as SIN-1 have been limited primarily to permanent ischemia models, NO donors have been demonstrated to be e� cacious in the present study and in the work of Salom, et al. 39, in which a model of temporary focal cerebral ischemia was used. Proposed mechanisms of protection include the following: the reduc-tion of activation and recruitment of neutrophils that produce cytokines and proteases, amplifying endothelial dysfunction and promoting tissue damage, and the loss of vaso-motor control contributing to the “no re� ow” phenomenon24. The agent SIN-1 also has a negative insulinotropic action5,42, whereas L-arginine was found to induce insulin secre-tion from pancreatic ß cells40. In their study, Zhang, et al. 52, reported an approximately 20% increase in glucose levels in SIN-1- and phenylephrine-treated groups, but found in a separate group that SIN-1 (3 mg/kg) without phenylephrine did not signi� cantly alter serum glucose levels, thus attributing that e� ect to phenylephrine. In our experiment in normoglycemic, moderately hypoglycemic, and hyperglycemic rats, SIN-1 treatment did not result in glucose levels that were di� erent from those measured in the moderately hypo-, normo-, and hyperglycemic control groups. In this study, the e� cacy of SIN-1, expressed as a percentage of reduction in cortical infarction volume, was approximately 70% in rats with normoglycemia, approximately 61% in rats with hypoglycemia, and 48% in rats with hyperglycemia. Therefore, SIN-1 was less e� ective under more acidic brain conditions. These data closely mirror the endothelial cNOS response curve that was demonstrated in vitro by Hecker, et al.22. In their study, the concentration of hydrogen ions was found to be an important determinant of endothelial cNOS activity, whereas pH sensitivity was lost one unit above or below the optimum pH 7.6). Similar studies on pH sensitivity of the neuronal isoform of cNOS revealed an optimal pH of 6.7 within


a bell-shaped curve similar to endothelial cNOS23. Combining their data on both cNOS enzymes, we can conclude that, within the pH range encountered (6.12- 6.82), enzyme activity would vary considerably. The following supports this hypothesis: 1) increased intracellular acidosis achieved by augmenting the severity of ischemia caused a loss of the neuroprotective e� ect by the nonselective NOS inhibitor, L-NAME, suggesting that NOS inhibition was less e� ective because of inhibition of NOS activity in acidosis1; 2) in a separate study (Chapter 5), administration of 7-NI, a selective nNOS inhibitor, was far less e� ective during periods of hyperglycemia (27.5% reduction compared with ap-proximately 48% in this study) and more e� ective during periods of both normoglycemia (93.5% reduction compared with approximately 70% in this study) and hypoglycemia (72.6% reduction compared with approximately 61% in this study). Based on the data the following hypothesis can be supported. In the healthy brain, NO is produced to maintain basal tone. In the brain with cerebral ischemia, NO production increases as the brain be-comes ischemic, to a pH optimum of NO (approximately 6.7- 6.8). Thereafter, production decreases as the brain becomes more acidotic. Therefore, it follows that, with worsening acidosis, a pH below approximately 6.7, NO donors become more e� ective compared with NOS inhibitors, and with improving acidosis, a pH above approximately 6.7, inhibi-tion of nNOS becomes more e� ective.

Role of NO in Cerebral Ischemia

Direct measurements of NO production in vivo have revealed both increased28 and de-creased NO concentration during cerebral ischemia29. Using a porphyrinic microsensor, Malinski, et al. 28, reported an increase from a baseline level of lower than 10-8 M to an ap-proximate level of 10-6 M in cases of focal ischemia. Using Na� on and porphyrine-coated carbon � ber electrodes, Lin, et al. 26, recorded NO production in rats during 40 minutes of combined MCA and bilateral CCA occlusion. Basal extracellular NO concentration in-creased to a mean of 18.8 ± 3.4 nmol/L. The di� erences between these values and those reported by Malinski, et al.28, were explained by the smaller tip of the probe, a di� erent occlusion technique, more super� cial cortical measurements, and the higher selectivity of the probe26. In a cat model of focal cerebral ischemia, NO concentrations were shown to increase during the � rst 10 minutes in regions exhibiting depolarization. The course of NO production after this was found to be variable34 and heterogeneous, ranging from a continuous reduction to a sustained overproduction35. It was suggested that NO produc-tion could be pH dependent35. Altogether, these data suggest that outcome is determined by the individual contributions of eNOS and nNOS through their speci� c and local e� ects rather than by the absolute concentration of NO during ischemia. A limited number of in vivo studies have been performed in which the e� ect of NO donors in focal cerebral ischemia was shown to be primarily protective 30,39,50-52. Di� erences in methodology, in-cluding animal models, anesthetic agents, occlusion techniques, duration of ischemia, and

88 Chapter 6

drug delivery and dosing complicate a comparison of e� ects in these di� erent studies. In the study by Morikawa, et al. 30, in which 300 mg/kg L-arginine was used as a treatment dose, a 28 to 35% reduction in ischemic damage was reported. Sodium nitroprusside was used in three studies39,50,52 in dosages ranging from 0.11 mg/kg/hr (total dose 0.22 mg/kg/hr) to 3 mg/kg/hr (total dose 3 mg/kg/hr), resulting in reductions in infarction volume of 67 and 27%, respectively. In a study by Salom, et al. 39, a high dose of SNP (1.1 mg/kg/hr administered intravenously for 2 hours), which did not signi� cantly reduce regional corti-cal blood � ow but signi� cantly reduced MABP, did not attenuate ischemic damage com-pared with a lower dose (0.11 mg/kg/hr), which improved treatment outcome. In contrast, a high dose of SNP with addition of phenylephrine (10–100 µg/hour intracarotid infusion) to prevent hypotension was protective in the study by Zhang, et al. 52 At 3 mg/kg/hr SIN-1 was shown to attenuate ischemic damage in a permanent model of focal ischemia when administered up to 60 minutes after onset of ischemia51. In a previous study by Coert, et al. 13, intravenous administration of SIN-1 at 1 mg/kg reduced the mean cortical infarction volume, but this reduction was not statistically signi� cant. Reducing occlusion times from 3 to 2 hours in the present study did not signi� cantly change cortical infarction volume or variability, although a reduction in the occlusion time from 3 hours to 1 hour of ischemia in this same model did just that14. Initiating intravenous SNP (0.19 µg/kg/min) in patients with white-matter lacunar (four patients) or cortical infarcts (18 patients) at a mean of 21.3 hours (range 9.3–27 hours) after onset of stroke, Butterworth, et al. 11, were able to im-prove cerebral blood � ow, although MABP was reduced. A reduction in platelet function was also found in patients who were not on a regimen of aspirin prior to their ischemic strokes. Overall outcome in the SNP-treated group, however, was not di� erent from that of the control group11.The neuroprotectiveness of NO donors can be attributed to either a parenchymal or vascular e� ect. It has been demonstrated that NO donors enhance re-gional cortical blood � ow by vasodilation, which reduces neuronal damage in the area surrounding the ischemic core50-52. These studies used either SNP or SIN-1, which was given by intracarotid infusion, whereas in three other studies12,30,39 these donors were given intravenously and demonstrated no signi� cant changes in regional cortical blood � ow. This may suggest that intracarotid infusion may result in a higher concentration of NO donor in the brain, thereby exerting a profound dilatory e� ect, or there could be a loss of vascular reactivity because of cerebral ischemia. In this study we chose the intravenous route for administration of SIN-1 because clinically it is routinely given intravenously. On the other hand, a parenchymal e� ect of NO could, for example, decrease neuronal death by attenuating the rise in intracellular calcium27 or by reducing free radical formation47. Inhibition of NOS also has been demonstrated in a number of published reports to be either neuroprotective2,6,9,10,13,31,38 or neurotoxic20,25,33,49. This suggests in part that NO can be neuroprotective or neurotoxic, depending on the ischemic environment, which in part may be pH dependent.


Model of Graded Focal Cerebral Ischemia

To ascertain the relationship between intracellular brain acidosis and NO, animals were subjected to moderate hypo-, normo-, or hyperglycemia to provide models of three graded levels of ischemia. There have been several studies4,43 in which brain pHi has been measured before, during, and after global and focal cerebral ischemia. Brain pHi becomes more acidotic during ongoing ischemia, declining from approximately 6.7 to less than 6 in response to increasing serum glucose levels (approximately 6.5 mmol/L to > 28 mmol/L). Conversely, as serum glucose levels become more hypoglycemic (ap-proximately 6.7- 7 mmol/L to approximately 7 mmol/L), brain pHi becomes less acidotic (6.7–7.0) 4,43. Cerebral infarction is reduced under moderate hypoglycemic conditions, whereas it becomes exacerbated under hyperglycemic conditions4,21. In our present study, hypoglycemia (serum glucose level of approximately 3 mmol/L) reduced the corti-cal infarction volume by approximately 80%, from 95.8 ± 12 to 19.1 ± 10 mm3 in the normoglycemic control group, whereas in the hyperglycemic animals there was a 178% increase in infarction volume (to 170.3 ± 14 mm3).

CONCLUSIONS

In this experiment the protective e� ect of NO enhancement by SIN-1 during focal ce-rebral ischemia was altered by serum glucose concentrations, which in e� ect re� ected a manipulation of brain pHi. As a neuroprotectant in this study SIN-1 was signi� cantly e� ective in reducing infarction volume in animals with hyperglycemia and, to a greater extent, in animals with normoglycemia. The successful use of NO donors in the treatment of myocardial ischemia makes them attractive candidates for use as neuroprotective agents. The apparent e� ect of pHi on NO is consistent with in vitro data22,23. We propose that brain pHi is an important factor for determining NO activity and that the observed variability in e� ects of NO enhancement indi� erent models of cerebral ischemia is partly due to di� erences in brain pHi during ischemia. The e� ect of pH on NO enhancement provides an additional mechanism by which acidosis contributes to ischemic brain dam-age. We also propose that, depending on the environment, brain pHi might in� uence how pharmaceutical agents perform as a treatment modality. For example, brain tumors have been shown to alter pHi18,28. Further investigations will need to be undertaken to elucidate the mechanism by which the concentration of H+ a� ects NO activity.

90 Chapter 6

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3. Anderson RE, Meyer FB, Tomlinson FH: Focal cortical distribution of blood � ow and brain pHi de-termined by in vivo � uorescent imaging. Am J Physiol 263:H565–H575, 1992

4. Anderson RE, Tan WK, Martin HS, et al: E� ects of glucose and PaO2 modulation on cortical intracel-lular acidosis, NADH redox state, and infarction in the ischemic penumbra. Stroke 30: 160–170, 1999

5. Antoine MH, Ouedraogo R, Hermann M, et al: 3-Morpholinosydnonimine as instigator of a glib-enclamide-sensitive reduction in the insulin secretory rate. Biochem Pharmacol 53: 1211–1213, 1997

6. Ashwal S, Cole DJ, Osborne TN, et al: Dual e� ects of LNAME during transient focal cerebral isch-emia in spontaneously hypertensive rats. Am J Physiol 267:H276–H284, 1994

7. Batteur-Parmentier S, Margaill I, Plotkine M: Modulation by nitric oxide of cerebral neutrophil ac-cumulation after transient focal ischemia in rats. J Cereb Blood Flow Metab 20: 812–819, 2000

8. Bohn H, Schöna� nger K: Oxygen and oxidation promote the release of nitric oxide from syd-nonimines. J Cardiovasc Pharmacol 14 (Suppl 11):S6–S12, 1989

9. Buisson A, Margaill I, Callebert J, et al: Mechanisms involved in the neuroprotective activity of a nitric oxide synthase inhibitor during focal cerebral ischemia. J Neurochem 61:690–696, 1993

10. Buisson A, Plotkine M, Boulu RG: The neuroprotective e� ect of a nitric oxide inhibitor in a rat model of focal cerebral ischemia. Br J Pharmacol 106:766–767, 1992

11. Butterworth RJ, Cluckie A, Jackson SHD, et al: Pathophysiological assessment of nitric oxide (given as sodium nitroprusside) in acute ischaemic stroke. Cerebrovasc Dis 8:158–165, 1998

12. Chi OZ, Wei HM, Weiss HR: E� ects of CAS 754, new nitric oxide donor, on regional cerebral blood � ow in focal cerebral ischemia. Anesth Analg 80:703–708, 1995

13. Coert BA, Anderson RE, Meyer FB: A comparative study of the e� ect of two nitric oxide synthase inhibitors and two nitric oxide donors on temporary focal ischemia in the Wistar rat. J Neurosurg 90:332–338, 1999

14. Coert BA, Anderson RE, Meyer FB: Reproducibility of cerebral cortical infarction in the Wistar rat after middle cerebral artery occlusion. J Stroke Cerebrovasc Dis 8:380–387, 1999

15. Dalkara T, Yoshida K, Irikura K, et al: Dual role of nitric oxide in focal cerebral ischemia. Neurophar-macology 33: 1447–1452, 1994

16. Feelisch M, Ostrowski J, Noack E: On the mechanism of NO release from sydnonimines. J Cardio-vasc Pharmacol 14 (Suppl 11):S13–S22, 1989

17. Foucher-Lavergne A, Kolsky H, Spreux-Varoquaux O, et al: Hemodynamics, tolerability, and phar-macokinetics of linsidomine (SIN-1) infusion during the acute phase of uncomplicated myocardial infarction. J Cardiovasc Pharmacol 22: 779–784, 1993

18. Gerweck LE, Seetharaman K: Cellular pH gradient in tumor versus normal tissue: potential exploi-tation for the treatment of cancer. Cancer Res 56:1194–1198, 1996


19. Golanov EV, Reis DJ: Contribution of cerebral edema to the neuronal salvage elicited by stimula-tion of cerebellar fastigial nucleus after occlusion of the middle cerebral artery in rat. J Cereb Blood Flow Metab 15:172–174, 1995

20. Hamada J, Greenberg JH, Croul S, et al: E� ects of central inhibition of nitric oxide synthase on focal cerebral ischemia in rats. J Cereb Blood Flow Metab 15:779–786, 1995

21. Hamilton HG, Tranmer BI, Auer RN: Insulin reduction of cerebral infarction due to transient focal ischemia. J Neurosurg 82:262–268, 1995

22. Hecker M, Mülsch A, Bassenge E, et al: Subcellular localization and characterization of nitric oxide synthase(s) in endothelial cells: physiologic implications. Biochem J 299:247–252, 1994

23. Hecker M, Mülsch A, Busse R: Subcellular localization and characterization of neuronal nitric oxide synthase. J Neurochem 62:1524–1529, 1994

24. Johnson D, Freischlag JA, Lesniak R, et al: Endothelial damage due to ischemia and reperfusion is prevented with SIN-1. Cardiovasc Surg 6:367–372, 1998

25. Kuluz JW, Prado RJ, Dietrich WD, et al: The e� ect of nitric oxide synthase inhibition on infarct vol-ume after reversible focal cerebral ischemia in conscious rats. Stroke 24: 2023–2029, 1993

26. Lin SZ, Chiou AL, Wang Y: Ketamine antagonizes nitric oxide release from cerebral cortex after middle cerebral artery ligation in rats. Stroke 27:747–752, 1996

27. Lipton SA, Choi YB, Pan ZH, et al: A redox-based mechanism for the neuroprotective and neurode-structive e� ects of nitric oxide and related nitroso-compounds. Nature 364:626–632, 1993

28. Malinski T, Bailey F, Zhang ZG, et al: Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 13: 355–358, 1993

29. Mason RB, Pluta RM, Walbridge S, et al: Production of reactive oxygen species after reperfusion in vitro and in vivo: protective e� ect of nitric oxide. J Neurosurg 93:99–107, 2000

30. Morikawa E, Moskowitz MA, Huang Z, et al: L-arginine infusion promotes nitric oxide-depen-dent vasodilation, increases regional blood � ow, and reduces infarct volume in the rat. Stroke 25:429–435, 1994

31. Nishikawa T, Kirsch JR, Koehler RC, et al: Nitric oxide synthase inhibition reduces caudate injury following transient focal ischemia in cats. Stroke 25:877–885, 1994

32. Noack E, Feelisch M: Molecular aspects underlying the vasodilator action of molsidomine. J Car-diovasc Pharmacol 14 (Suppl 11):S1–S5, 1989

33. Nowicki JP, Duval D, Poignet H, et al: Nitric oxide mediates neuronal death after focal cerebral ischemia in the mouse. Eur J Pharmacol 204:339–340, 1991

34. Ohta K, Graf R, Rosner G, et al: Early nitric oxide increase in depolarized tissue of cat focal cerebral ischemia. Neuroreport 8:143–148, 1996

35. Ohta K, Graf R, Rosner G, et al: Pro� les of cortical tissue depolarization in cat focal cerebral isch-emia in relation to calcium ion homeostasis and nitric oxide production. J Cereb Blood Flow Metab 17:1170–1181, 1997

36. Radomski MW, Palmer RMJ, Moncada S: An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci USA 87:5193–5197, 1990

37. Rees DD, Palmer RMJ, Moncada S: Role of the endotheliumderived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci USA 86:3375–3378, 1989

38. Regli L, Held MC, Anderson RE, et al: Nitric oxide synthase inhibition by L-NAME prevents brain acidosis during focal cerebral ischemia in rabbits. J Cereb Blood Flow Metab 16: 988–995, 1996

92 Chapter 7

39. Salom JB, Orti JM, Centeno JM, et al: Reduction of infarct size by the NO donors sodium nitrop-russide and spermine/NO after transient focal cerebral ischemia in rats. Brain Res 865: 149–156, 2000

40. Schmidt HH, Warner TD, Ishii K, et al: Insulin secretion from pancreatic B cells caused by L-arginine-derived nitrogen oxides. Science 255:721–723, 1992

41. Singh RJ, Hogg H, Joseph J, et al: The peroxynitrite generator, SIN-1, becomes a nitric oxide donor in the presence of electron acceptors. Arch Biochem Biophys 361:331–339, 1999

42. Sjöholm Å: Nitric oxide donor SIN-1 inhibits insulin release. Am J Physiol 271:C1098–C1102, 1996

43. Smith ML, von Hanwehr R, Siesjö BK: Changes in extra- and intracellular pH in the brain during and following ischemia in hyperglycemic and in moderately hypoglycemic rats. J Cereb Blood Flow Metab 6:574–583, 1986

44. Snyder SH: Nitric oxide: � rst in a new class of neurotransmitters. Science 257:494–496, 1992

45. Tamura A, Graham DI, McCulloch J, et al: Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1:53–60, 1981

46. Tomlinson FH, Anderson RE, Meyer FB: Acidic foci within the ischemic penumbra of the New Zea-land white rabbit. Stroke 24:2030–2040, 1993

47. Wink DA, Hanbauer I, Krishna MC, et al: Nitric oxide protects against cellular damage and cytotox-icity from reactive oxygen species. Proc Natl Acad Sci USA 90:9813–9817, 1993

48. Winter PM, Poptani H, Bansal N: E� ects of chemotherapy by 1,3-bis(2-chloroethyl)-1-nitrosourea on single-quantum- and triple-quantum-� ltered 23Na and 31P nuclear magnetic resonance of the subcutaneously implanted 9L glioma. Cancer Res 61:2002–2007, 2001

49. Yamamoto S, Golanov EV, Berger SB, et al: Inhibition of nitric oxide synthesis increases focal isch-emic infarction in rat. J Cereb Blood Flow Metab 12:717–726, 1992

50. Zhang F, Iadecola C: Nitroprusside improves blood � ow and reduces brain damage after focal ischemia. Neuroreport 4: 559–562, 1993

51. Zhang F, Iadecola C: Reduction of focal cerebral ischemia by delayed treatment with nitric oxide donors. J Cereb Blood Flow Metab 14:574–580, 1994

52. Zhang F, White JG, Iadecola C: Nitric oxide donors increase blood � ow and reduce brain damage in focal ischemia: evidence that nitric oxide is bene� cial in the early stages of cerebral ischemia. J Cereb Blood Flow Metab 14:217–226, 1994

Chapter 7

Discussion

Discussion 95

DISCUSSION

Model to clinic

The development of experimental models of focal cerebral ischemia has allowed for a better knowledge of its pathophysiology and for testing of therapeutic strategies. Nu-merous studies of neuroprotective compounds have shown reduction of infarct volumes in animal stroke models and in some cases promising phase II results while none have been proven e� cacious on the basis of a positive phase III trial26,30,45. Many factors may have contributed to this phenomenon including: inadequate interpretation of pre-clincal data; underpowered phase III trials; patients are included that are unlikely to respond to the drugs being tested; the chosen primary endpoint may be inadequate to fully evaluate the drugs e� ect24. Phase III studies continue to be conducted despite limited pharmacokinetic data from animal and preliminary human studies3,25. Animal model de� ciencies as well as inappropriate dosing and timing of therapy are also potential rea-sons for the failure of neuroprotective drugs in clinical e� cacy trials24. Other important reasons for the negative neuroprotective trials probably are the lack of preclinical data to support the time window chosen for e� cacy trials and the rapid movement to pivotal trials without in-depth understanding of side e� ect pro� les and the subtypes of stroke patients that are most likely to bene� t24. Stroke Therapy Academic Industry Roundtable (STAIR) meetings have focused on di� erent aspects of the development and assessment of new neuroprotective stroke therapies. Robustness of the neuroprotective e� ects was found to be an important factor in determining which therapy should advance from pre-clinical to clinical development1. It was stated that the neuroprotective e� ects should be con� rmed in at least two independent laboratories of which at least one should be independent of the sponsoring company1. Rather than moving directly to interventional studies in humans, the use of several appropriate animal models is encouraged20. The usage of at least two outcome measures was recommended to evaluate both functional and histological response1,16. Neurological de� cits after experimental cerebral ischemia is sometimes di� cult to detect in rodents7. Reports on the correlation between neuro-logical de� cits and stroke volumes have varied from no correlation60 to signi� cant cor-relations51. In clinical trials, however, functional recovery is the major end point2.

No animal model can exactly mimic stroke in humans. The relevance to the human situation is essential before pharmacokinetic and time window issues can be resolved67. Comparison of positron emission tomography (PET) studies of stroke patients to a cat MCA occlusion model revealed great resemblance in the development of “misery per-fused” penumbral tissue and its centrifugal conversion into necrosis,31 which is impor-tant proof of relevance of this model. When embolic MCA occlusion in Sprague Dawley rats was directly compared to a suture MCA occlusion it was noted that there are im-portant di� erences in acute ischemic lesion evolution33, mimicking two di� erent clinical

96 Chapter 7

problems. The validity, complications and side e� ects were studied for three techniques of MCA occlusion by Gerriet et al. 29. Subarachnoid hemorrhage was noted in some ani-mals while in others the occlusion was inadequate with patent � ow on MRA. In 7 of 37 cases, model failure was noted using MRA. This study provides us with important data on model failure. The age, gender species and strain of the laboratory animal used prob-ably in� uence its relevance to stroke in humans1. Elderly animals had di� erent response mechanisms, ischemic consequences and histological changes52,53. Frequently used young animal models may have limited e� cacy in predicting clinical neuroprotective ef-� cacy in a disease primarily a� ecting the elderly. Between di� erent rat strains substantial di� erences in acute ischemic lesion evolution was demonstrated6,48,61. Gender speci� c di� erences in outcome were noted in di� erent models,11,62,63,65 raising the possibility that therapeutic interventions should be gender speci� c. In 1999 STAIR, rodent models of focal cerebral ischemia like the Tamura model57 were recommended for the evalua-tion of putative neuroprotective drugs with careful dos-response and toxicology studies to enable future clinical trials1. To date new animal stroke models are being developed 32,36,63. The development of models of focal cerebral ischemia must take into account known species di� erences and idiosyncrasies, underlying vascular disease processes, the nature of thrombotic processes, cellular reactivities, the presence of co-stimulation (e.g. in� ammation), the characteristics of immunological and reporter molecules, the coinci-dent use of other pharmacologic modi� ers (e.g. anesthesia), and stress20. These elements are potential contributors to cerebral tissue injury and its assessment and may a� ect species di� erentially20.

Stroke models have been a valuable instrument to study many facets of the pathophysiology of stroke. The transfer of these data to the clinical setting, however, has been mainly unsuc-cessful. This has made us realize that stroke models are a powerful tool in the quest to unravel the complex pathophysiology of stroke but care should be taken to extrapolate data to the clinical situation in humans. Stricter adherence to the recommendations based on previous experiences will hopefully prevent disappointing results from premature advancement to clinical trials.

Nitric Oxide in focal cerebral ischemia

An important and complex role for NO has been proposed in the pathophysiology of cerebral ischemia17. Whether overall NO is bene� cial or detrimental seems to depend upon its dose timing and location44. NO is a short-lived, di� usible, reactive free radical gas that is synthesized from l-arginine through the NO synthase enzyme. Three isoforms were identi� ed for this enzyme: type I neuronal or nNOS, type II inducible or iNOS and type III endothial or eNOS. While neuronal and endothial NOS were found in neurons en endothial cells respectively, inducible NOS was found in astrocytes. NO was found to

Discussion 97

regulate vascular tone, platelet aggregation49 and neurotransmission55,18. Under patho-logical circumstances it was also found to be an important mediator of N-methyl-d-as-partate (NMDA) mediated toxicity19. Direct measurements of NO in vivo are hindered by NO’s short half life4,59. Uncoupling of constitutive NOS (endothelial and neuronal) leads to overproduction of superoxide (O2

-) and peroxynitrite (ONOO-), 2 potent oxidants and O2

- and ONOO- which triggers the release of aggressive radicals40. Arginine analogue NO synthase inhibitors with selectivity for a speci� c iso-enzymes have been used to study the role of di� erent sources of NO in cerebral ischemia50,64. Targeted gene disruptions of e- or nNOS isoforms are an alternative approach. Neuronal NOS knockout mice were found to be resistant to brain injury after focal cerebral ischemia35 while e-NOS knockout mice developed larger infarcts34. This was consistent with studies using selective nNOS inhibitors64,14 and data using nonselective NOS inhibitors that a� ect eNOS14. NO donor treatment mimicking endothelial NO54 was found to protect brain tissue15,66. The en-dothelium plays a critical role in maintaining vascular tone by releasing nitric oxide (NO). Endothelium derived NO di� uses to smooth muscles, triggering their relaxation5. Modali-ties that upregulate eNOS expression and/or activity like HMG Co-A reductase inhibitors, steroid hormones, nutrients and physical activity were found to enhance cerebral blood � ow and protect from ischemic stroke23. In animal models the protective e� ect of HMG Co-A reductase inhibitor simvastatin through eNOS activation was con� rmed for adult animals if the statin was administered within 3-6 hours after ischemia13. Nitric oxide re-lease from the endothelium of spontaneously hypertensive rats was found to be reduced when compared to controls8. The de� ciency in NO concentration correlated positively with the increase of cerebral ischemia/reperfusion injury21,38. Racial di� erences in the predisposition to vascular diseases were explained by predispositions to endothelial dys-function during ongoing vascular disturbances37. The clinical e� cacy of third generation beta-adrenolytics like nebivolol was found to be through inhibition of endothelial dys-function42 and thus indirectly through release of NO56. Experimental evidence indicated that adventitial NO has an important role in the pathogenesis of cerebral vasospasm after SAH47. Markers of endothelial damage like von Willebrand factor were found to predict vasospasm43,28,27. A strong, graded and independent association was observed between blood concentrations of markers of endothelial activation (E selectin and Von Willebrand factor) and experimental ischemic stroke12. Nitric oxide synthase dysfunction is a therapeutic target in the treatment for delayed cerebral vasospasm after SAH46,39. A predisposition toward cerebral vasospasm may be related partially to genetic factors. In a study on 28 consecutive Fisher grade 3 patients with an aneurysmal subarachnoid hemorrhage DNA analysis revealed eNOS polymorphisms of the eNOS T 786C single nucleotide. Polymorphism correlated with the presence and severity of vasospasm38.

98 Chapter 7

In chapter 3 selective nNOS inhibition and eNOS augmentation reduced ischemic dam-age in experimental focal cerebral ischemia. Although very e� ective, selective nNOS inhibitors like 7-NI used in chapters 3 and 5 caused CNS depression22, probably related to nNOS’ physiological role. These side e� ects, but also the complex pharmacokinetics10 and non water solubility make selective nNOS inhibitors like 7- NI less attractive. In contrast, NO donors have been used widely9 since discovery of their vasodilatory e� ects. Currently focus is directed more towards the endothelium and eNOS. Better understanding of the role of NO from its various sources under physiologic and pathologic circumstances will hopefully lead to new therapeutic options.

The discovery of NO has led to a whole new � eld of research. The absence of an easy way to directly measure NO 41 has led to more indirect approaches yielding results which are inher-ently more di� cult to interpret. Convincingly it has been demonstrated that inhibition of the neuronal isoform of NOS leads to smaller cerebral infarcts. The use of neuronal NOS inhibitors however, is limited by serious side e� ects that appear to be a direct result its physiological function. Exogenous NO (NO donor treatement), mimicking enhancement of endothelial NO, and l-arginine, the substrate of nitric oxide synthase and the main precursor of nitric oxide have similar e� ects58. Attention has shifted to eNOS which seem to play a pivotal role in the protective e� ects of statins and exercise in stroke and cardiovascular disease. The observed variability in ischemic complications after subarachnoid hemorrhage has been found to cor-relate with polymorphisms in e-NOS, con� rming an important role for eNOS. Whether this will lead to therapeutic options remains to be seen. Further studies will have to clarify the e� ects of NO on platelet and endothelial function, but in this process, we need better techniques to measure or visualize NO. Nevertheless, NO will remain an important subject of study in future cerebrovascular research.

Discussion 99

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9. Brunton T: Use of nitrite of amyl in angina pectoris. Lancet 2:97-98, 1867

10. Bush MA, Pollack GM: Pharmacokinetics and protein binding of the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole. Biopharm Drug Dispos 21:221-228, 2000

11. Chen CH, Toung TJ, Hurn PD, Koehler RC, Bhardwaj A: Ischemic neuroprotection with selective kappa-opioid receptor agonist is gender speci� c. Stroke 36:1557-1561, 2005

12. Cherian P, Hankey GJ, Eikelboom JW, Thom J, Baker RI, McQuillan A, et al: Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes. Stroke 34:2132-2137, 2003

13. Cimino M, Balduini W, Carloni S, Gelosa P, Guerrini U, Tremoli E, et al: Neuroprotective e� ect of simvastatin in stroke: a comparison between adult and neonatal rat models of cerebral ischemia. Neurotoxicology 26:929-933, 2005

14. Coert BA, Anderson RE, Meyer FB: A comparative study of the e� ects of two nitric oxide synthase inhibitors and two nitric oxide donors on temporary focal cerebral ischemia in the Wistar rat. J Neurosurg 90:332-338, 1999

15. Coert BA, Anderson RE, Meyer FB: E� ects of the nitric oxide donor 3-morpholinosydnonimine (SIN-1) in focal cerebral ischemia dependent on intracellular brain pH. J Neurosurg 97:914-921, 2002

16. Corbett D, Nurse S: The problem of assessing e� ective neuroprotection in experimental cerebral ischemia. Prog Neurobiol 54:531-548, 1998

17. Dalkara T, Moskowitz MA: The complex role of nitric oxide in the pathophysiology of focal cerebral ischemia. Brain Pathol 4:49-57, 1994

18. Dawson TM, Dawson VL, Snyder SH: A novel neuronal messenger molecule in brain: the free radi-cal, nitric oxide. Ann Neurol 32:297-311, 1992

19. Dawson VL, Dawson TM, London ED, Bredt DS, Snyder SH: Nitric oxide mediates glutamate neuro-toxicity in primary cortical cultures. Proc Natl Acad Sci U S A 88:6368-6371, 1991

100 Chapter 7

20. del Zoppo GJ: Clinical trials in acute stroke: why have they not been successful? Neurology 51:S59-61, 1998

21. Dobrucki LW, Kalinowski L, Uracz W, Malinski T: The protective role of nitric oxide in the brain ischemia. J Physiol Pharmacol 51:695-703, 2000

22. Dzoljic MR, de Vries R, van Leeuwen R: Sleep and nitric oxide: e� ects of 7-nitro indazole, inhibitor of brain nitric oxide synthase. Brain Res 718:145-150, 1996

23. Endres M, Laufs U, Liao JK, Moskowitz MA: Targeting eNOS for stroke protection. Trends Neurosci 27:283-289, 2004

24. Fisher M: Recommendations for advancing development of acute stroke therapies: Stroke Therapy Academic Industry Roundtable 3. Stroke 34:1539-1546, 2003

25. Fisher M, Albers GW, Donnan GA, Furlan AJ, Grotta JC, Kidwell CS, et al: Enhancing the devel-opment and approval of acute stroke therapies: Stroke Therapy Academic Industry roundtable. Stroke 36:1808-1813, 2005

26. Fisher M, Schaebitz W: An overview of acute stroke therapy: past, present, and future. Arch Intern Med 160:3196-3206, 2000

27. Frijns CJ, Kasius KM, Algra A, Fijnheer R, Rinkel GJ: Endothelial cell activation markers and delayed cerebral ischaemia in patients with subarachnoid haemorrhage. J Neurol Neurosurg Psychiatry 77:863-867, 2006

28. Frijns CJ, Rinkel GJ, Castigliego D, Van Gijn J, Sixma JJ, Fijnheer R: Endothelial cell activation after subarachnoid hemorrhage. Neurosurgery 50:1223-1229; discussion 1229-1230, 2002

29. Gerriets T, Stolz E, Walberer M, Muller C, Rottger C, Kluge A, et al: Complications and pitfalls in rat stroke models for middle cerebral artery occlusion: a comparison between the suture and the macrosphere model using magnetic resonance angiography. Stroke 35:2372-2377, 2004

30. Gorelick PB: Neuroprotection in acute ischaemic stroke: a tale of for whom the bell tolls? Lancet 355:1925-1926, 2000

31. Heiss WD, Graf R, Wienhard K: Relevance of experimental ischemia in cats for stroke management: a comparative reevaluation. Cerebrovasc Dis 11:73-81, 2001

32. Henninger N, Eberius KH, Sicard KM, Kollmar R, Sommer C, Schwab S, et al: A new model of throm-boembolic stroke in the posterior circulation of the rat. J Neurosci Methods 156:1-9, 2006

33. Henninger N, Sicard KM, Schmidt KF, Bardutzky J, Fisher M: Comparison of ischemic lesion evolu-tion in embolic versus mechanical middle cerebral artery occlusion in Sprague Dawley rats using di� usion and perfusion imaging. Stroke 37:1283-1287, 2006

34. Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, et al: Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab 16:981-987, 1996

35. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA: E� ects of cerebral isch-emia in mice de� cient in neuronal nitric oxide synthase. Science 265:1883-1885, 1994

36. Imai H, Konno K, Nakamura M, Shimizu T, Kubota C, Seki K, et al: A new model of focal cerebral ischemia in the miniature pig. J Neurosurg 104:123-132, 2006

37. Kalinowski L, Dobrucki IT, Malinski T: Race-speci� c di� erences in endothelial function: predisposi-tion of African Americans to vascular diseases. Circulation 109:2511-2517, 2004

38. Khurana VG, Fox DJ, Meissner I, Meyer FB, Spetzler RF: Update on evidence for a genetic predispo-sition to cerebral vasospasm. Neurosurg Focus 21:E3, 2006

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39. Khurana VG, Smith LA, Weiler DA, Springett MJ, Parisi JE, Meyer FB, et al: Adenovirus-mediated gene transfer to human cerebral arteries. J Cereb Blood Flow Metab 20:1360-1371, 2000

40. Malinski T: Understanding nitric oxide physiology in the heart: a nanomedical approach. Am J Cardiol 96:13i-24i, 2005

41. Malinski T, Bailey F, Zhang ZG, Chopp M: Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 13:355-358, 1993

42. Mason RP, Kubant R, Jacob RF, Walter MF, Boychuk B, Malinski T: E� ect of nebivolol on endothelial nitric oxide and peroxynitrite release in hypertensive animals: Role of antioxidant activity. J Car-diovasc Pharmacol 48:862-869, 2006

43. McGirt MJ, Lynch JR, Blessing R, Warner DS, Friedman AH, Laskowitz DT: Serum von Willebrand factor, matrix metalloproteinase-9, and vascular endothelial growth factor levels predict the onset of cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Neurosurgery 51:1128-1134; discussion 1134-1125, 2002

44. Moncada S, Palmer RM, Higgs EA: Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43:109-142, 1991

45. Muir KW: Heterogeneity of stroke pathophysiology and neuroprotective clinical trial design. Stroke 33:1545-1550, 2002

46. Pluta RM: Dysfunction of nitric oxide synthases as a cause and therapeutic target in delayed cere-bral vasospasm after SAH. Neurol Res 28:730-737, 2006

47. Pluta RM, Thompson BG, Dawson TM, Snyder SH, Boock RJ, Old� eld EH: Loss of nitric oxide syn-thase immunoreactivity in cerebral vasospasm. J Neurosurg 84:648-654, 1996

48. Prieto R, Carceller F, Roda JM, Avendano C: The intraluminal thread model revisited: rat strain dif-ferences in local cerebral blood � ow. Neurol Res 27:47-52, 2005

49. Radomski MW, Palmer RM, Moncada S: An L-arginine/nitric oxide pathway present in human plate-lets regulates aggregation. Proc Natl Acad Sci U S A 87:5193-5197, 1990

50. Rees DD, Palmer RM, Schulz R, Hodson HF, Moncada S: Characterization of three inhibitors of en-dothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 101:746-752, 1990

51. Rogers DC, Campbell CA, Stretton JL, Mackay KB: Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. Stroke 28:2060-2065; discussion 2066, 1997

52. Rosen CL, Dinapoli VA, Nagamine T, Crocco T: In� uence of age on stroke outcome following tran-sient focal ischemia. J Neurosurg 103:687-694, 2005

53. Shapira S, Sapir M, Wengier A, Grauer E, Kadar T: Aging has a complex e� ect on a rat model of ischemic stroke. Brain Res 925:148-158, 2002

54. Snyder SH: Nitric oxide. No endothelial NO. Nature 377:196-197, 1995

55. Snyder SH, Bredt DS: Nitric oxide as a neuronal messenger. Trends Pharmacol Sci 12:125-128, 1991

56. Szajerski P, Zielonka J, Sikora A, Adamus J, Marcinek A, Gebicki J, et al: Radical scavenging and NO-releasing properties of selected beta-adrenoreceptor antagonists. Free Radic Res 40:741-752, 2006

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57. Tamura A, Graham DI, McCulloch J, Teasdale GM: Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlu-sion. J Cereb Blood Flow Metab 1:53-60, 1981

58. Tousoulis D, Boger RH, Antoniades C, Siasos G, Stefanadi E, Stefanadis C: Mechanisms of disease: L-arginine in coronary atherosclerosis--a clinical perspective. Nat Clin Pract Cardiovasc Med 4:274-283, 2007

59. Vallance P, Patton S, Bhagat K, MacAllister R, Radomski M, Moncada S, et al: Direct measurement of nitric oxide in human beings. Lancet 346:153-154, 1995

60. Wahl F, Allix M, Plotkine M, Boulu RG: Neurological and behavioral outcomes of focal cerebral ischemia in rats. Stroke 23:267-272, 1992

61. Walberer M, Stolz E, Muller C, Friedrich C, Rottger C, Blaes F, et al: Experimental stroke: ischaemic lesion volume and oedema formation di� er among rat strains (a comparison between Wistar and Sprague-Dawley rats using MRI). Lab Anim 40:1-8, 2006

62. Wen TC, Rogido M, Peng H, Genetta T, Moore J, Sola A: Gender di� erences in long-term bene� -cial e� ects of erythropoietin given after neonatal stroke in postnatal day-7 rats. Neuroscience 139:803-811, 2006

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65. Zeynalov E, Nemoto M, Hurn PD, Koehler RC, Bhardwaj A: Neuroprotective e� ect of selective kappa opioid receptor agonist is gender speci� c and linked to reduced neuronal nitric oxide. J Cereb Blood Flow Metab 26:414-420, 2006

66. Zhang F, Iadecola C: Nitroprusside improves blood � ow and reduces brain damage after focal ischemia. Neuroreport 4:559-562, 1993

67. Zhang Z, Zhang RL, Jiang Q, Raman SB, Cantwell L, Chopp M: A new rat model of thrombotic focal cerebral ischemia. J Cereb Blood Flow Metab 17:123-135, 1997

Chapter 8

SummarySamenvatting

Summary 105

SUMMARY

In vivo models have been an important tool to study the pathophysiology of focal ce-rebral ischemia and test possible protective strategies. The rat MCA occlusion model is a frequently used model to study focal cerebral ischemia; it simulates ischemia during temporary arterial occlusions in neurovascular procedures. In rat MCA occlusion models volume and reproducibility of infarction were found to depend on duration of occlu-sion9, anesthetic technique39, temperature15, bloodpressure46, serum glucose levels38, strain19,29, and histologic technique. The interactions between these factors can be illus-trated by the e� ects of the anesthetics halothane and iso� urane on cerebral ischemia39. The protective e� ects of these anesthetics are attributed to e� ects on metabolism. In higher doses anesthetics can increase infarct volume by reducing blood pressure and consequently collateral � ow. Detailed appraisal of physiologic parameters like brain temperature and blood pressure combined with information on anesthetic technique can help interpret results in experimental stroke. Overall the severity of ischemia can be increased by extending the duration of occlusion and by reducing collateral � ow. Using the Tamura model, a focal cerebral ischemia model in the rat, after 1 hour MCA occlu-sion 60% of animals did not develop any cortical infarction (chapter 2)9. The addition of bilateral common carotid occlusion reduced this to 25 %. Extending the occlusion time to three hours resulted in an improvement (chapter 2)9. Cortical infarct volumes with 3 hours of MCA and bilateral CCA occlusion equaled values found in permanent MCA oc-clusion (chapter 2)9. The MCA stroke did not cause severe neurologic de� cits41, but this MCAo technique caused temporo-mandibular dysfunction interfering with the ability of the animal to eat. Reperfusion was limited to three days to limit weight loss, which aver-aged 24% after 7 days. Reproducibility is a major concern in models of cerebral ischemia. E� orts to improve reproducibility will reduce the number of animals required. Increasing the severity of cerebral ischemia increased average infarct volume and reduced variability of results. In our experiments 3 hours of MCA occlusion with bilateral CCA occlusion pro-duced cortical infarcts of 83 mm3 (average) in the male Wistar rat under (1%) Halothane anesthesia and normocapnic, normotensive, normothermic and normoxic conditions.

Studies on the cascade of events during cerebral ischemia have resulted in the identi-� cation of the glutamate activated NMDA receptor mediated transmembrane calcium in� ux8. The activation of calcium dependent enzymes mediates the deleterious e� ects down stream. The Nitric Oxide Synthase (NOS) iso-enzymes were identi� ed as impor-tant calcium dependent enzymes34 located in neurons (nNOS), endothelium (eNOS) and glial cells and macrophages (iNOS). The importance of the discovery of nitric oxide as a signaling molecule in the cardiovascular system was con� rmed in 1998 when the Nobel Price was awarded. Under pathological circumstances, like ischemia, the NOS system is

106 Chapter 8

overly activated and the excessive concentrations of NO are an important mediator of NMDA toxicity34. Because the half-life of NO is short, direct measurements are di� cult. Direct and indirect measurements of NO using di� erent methods have identi� ed peak levels of NO early after the onset of ischemia23,26. Indirect indications on the role of NO in cerebral ischemia were derived from studies with NOS inhibitors and NO donors. The e� ects of non iso-enzyme speci� c NOS inhibitors on ischemic cerebral injury have been contradictory and a dose-dependent dual role was proposed5. More consistent results were found using iso-enzyme selective NOS inhibitors. Nonspeci� c side e� ects of these drugs were excluded using knock out animals for NOS iso-enzymes. The nNOS knock-out animals, developed smaller infarcts which con� rms the deleterious e� ects of NO found in in vitro studies10. On the other hand NO donors were found to reduce ischemic damage in vivo if hypotension was avoided44 . Direct comparison of e� ects of NOS inhibition and NO donors however was complicated by important di� erences in methodology between investigators. In a model that simulates ischemia during temporary arterial occlusions in neurovascular procedures two NOS inhibitors and two NO donors were tested for dose-dependent neuroprotective e� ects. Non-selective NOS inhibitor L-NAME reduced the cortical infarct volume signi� cantly in the medium (1.0 mg/kg) dose group while the selective nNOS inhibitor 7-NI was signi� cantly protective in the higher (10 and 100 mg/kg) dose groups. Detrimental e� ects of higher dose L-NAME treatment were attributed to the e� ect on eNOS causing a reduction in collateral � ow. When compared to the ap-propriate control group 7-NI reduced infarct volume up to 92% (chapter 3, � g 1) to which the anesthetic e� ect of 7-NI may have contributed.

The anti-ischemic e� ects of NO donors were recognized for coronary artery disease for more than a century7 and are used to date in acute coronary syndromes. In our model of focal cerebral ischemia with 3 hours of MCA and bilateral CCA occlusion treatment with both NO donors reduced the average cortical infarct volume but this reduction did not reach signi� cance (chapter 3, � g 1).

Polyamines with NO donor capacity were previously reported to reduce ischemic injury in experimental focal cerebral ischemia33. Under physiologic conditions Polyamines like spermine, putrescine and spermidine were found to regulate important cellular func-tions like Ca2+ transport21,25 and nitric oxide synthase20 and free-radical scavenging21. The exact role of polyamines in the pathophysiology of stroke is unclear. Reductions of spermine levels were reported in experimental focal cerebral ischemia31,35. Treatment with spermine resulted in reduction of ischemic damage in experimental forebrain isch-emia13. To evaluate protective e� cacy of the spermine part and to enable comparison to previous experiments using NOS inhibitors and NO donors, the exact same 3 hours tandem MCA and CCA occlusion model was used. In the 10 mg/kg i.v. spermine treat-ment group infarct volume was signi� cantly reduced when compared to controls. In the

Summary 107

lower dose treatment groups the reduction in infarct volume was not signi� cant. This e� ect was stronger than the e� ect of the NO donors used in previous studies (chapter 3). Treatment with spermine resulted in a signi� cant decrease in serum glucose levels of about 30 % and a dose-dependent decrease in serum lactate. Mechanisms proposed to explain the protective e� ects of spermine include hypoglycemia and direct e� ects on NMDA receptor28 and nNOS inhibition20. The protective e� ects of spermine (10 mg/kg) approximate the e� ects of selective nNOS inhibition (7-NI, 10 mg/kg). Studies with combinations of spermine and NOS inhibitors and/or NO donors may help to determine the exact mechanisms.

Selective de� ciency or inhibition of the neuronal NOS enzyme was found to reduce cere-bral infarct volume while for non iso-enzyme speci� c NOS inhibitors more complex dos-age and time dependent e� ects have been described27,45. The � nding that the severity of the ischemic insult determined the neuroprotective e� cacy of NOS inhibitors2,16 and reports of biphasic pH sensitivity of the NOS enzyme14,24,32 led to the hypothesis that the neuroprotective e� ects of (n)NOS inhibition are dependent on ischemic intracellular pH. Manipulation of serum glucose is a well-documented method to alter brain pHi during cerebral ischemia4. Hyperglycemia may have additional e� ects on the extent of ischemic damage not mediated through the intracellular pHi however. Hyperglycemia (serum glucose levels of 20 mmol/l) was used during experimental focal cerebral ischemia to exacerbate and moderate hypoglycemia (serum glucose level 3 - 5 mmol/l) to attenuate intracellular acidosis. This resulted in an average infarct volume of 170 mm3 for hypergly-cemia (77% increase) and 19 mm3 for moderately hypoglycemia (80% decrease). In vivo � uorescence imaging techniques were used to study pHi with rCBF (regional cerebral blood � ow) and the NAD+/NADH ratios3. For the in vivo � uorescent imaging the occlu-sion time was limited to two hours, which resulted in an adjustment of the experimental protocol. This resulted in an average cortical infarct volume that was not signi� cantly di� erent from the 3 hour MCA and CCA occlusion protocol. During normoglycemic (av-erage serum glucose 9 mmol/l) ischemia pHi declined from 7.0 to 6.6. After release of the MCA and CCA occlusions, rCBF and pHi recovered. Hyperglycemia (serum glucose of 20 mmol/l resulted in a decline of pHi from 7.0 to 6.1. Under moderate hypoglycemic (serum glucose 5 mmol/l) conditions pHi reached 6.8. Under hyperglycemic conditions selective nNOS inhibition with 7-NI 100mg/kg resulted in a 28% reduction of the infarct volume, while under normoglycemic conditions this reduction was 93%. Moderate hy-poglycemia resulted in a 73% reduction. Based on in vitro studies of pH sensitivity of the NOS enzyme14,17,32 within the observed pHi range (6.1 to 6.8) enzyme activity would vary considerably. With Citruline measurements the expected subsequent increase in NOS activity on restoration of pHi was observed by Wei and Quast 40. The results of our study

108 Chapter 8

support the hypothesis that the neuroprotective e� cacy of selective nNOS inhibition is dependent of the intracellular pHi.

The bene� cial e� ects of acute treatment with NO donors has previously been report-ed42-44. NO donors like SIN-1 are used in interventional cardiology for their antispastic and vasodilatory e� ects36. The NO release capacity is a� ected by pO2 and pH but ad-equate under ischemic conditions6. The use of the single shot NO donor resulted in a temporary blood pressure reduction with spontaneous recovery within 30 min (which is before occlusion). To investigate the relationship between intracellular pH and the role of NO in focal cerebral ischemia, moderate hypoglycemia, normoglycemia or hypergly-cemia was created. The e� ect of treatment with an NO donor was evaluated under these conditions. As in the previous chapter in vivo � uorescence imaging data was used for pHi, rCBF and the NAD+/NADH ratios3. For the in vivo � uorescent imaging the occlusion time was limited to two hours (as explained in chapter 6). As presented in chapter 6 during normoglycemic ischemia pHi declined from 7.0 to 6.6. After release of the MCA and CCA occlusions the pHi recovered to 6.7. Hyperglycemia (serum glucose levels 20 mmol/l resulted in a decline of pHi from 7.0 to 6.1. Under moderate hypoglycemic (serum glucose 5 mmol/l) conditions pHi reached 6.8. In our study SIN-1 did not a� ect serum glucose levels (chapter 7, table 1). Under normoglycemic and hyperglycemic conditions the NO donor SIN-1 signi� cantly reduced cortical infarct volume, this in contrast with previous results with the 3 hour schedule when the reduction did not reach signi� cance (chapter 3, � g 1D). The reduction of infarct volume for the SIN-1 treated animals under moderately hypoglycemic condition was not signi� cant (chapter 6, � g 2). When these results are combined with data on pH sensitivity of the NOS enzyme our results indicate that under acidotic circumstances in which the NOS enzyme is less active the protective e� ects of exogenous NO is retained.

Detailed appraisal of experimental settings and their e� ects on outcome can increase the reproducibility of experimental models. Changes in variables like occlusion time, col-lateral � ow and serum glucose levels lead to important di� erences in infarct size. The translation of evidence from the basic science lab into the clinic has proven di� cult37. Attention was demanded for pharmacokinetics, dosing and choices of experimental models and outcome parameters1. This will hopefully lead to more success in the future. The complex role of NOS under physiologic circumstances and in the development of stroke and its limited therapeutic time window make it less suitable as a direct target for therapy. The evidence for an important contribution of NOS activation in the pathophysi-ology of stroke is convincing. To date the direct quanti� cation of NO is di� cult. NO donors and NOS inhibitors have made it possible to study the role of NO indirectly; bypassing direct measurements. The results of these indirect studies should be carefully interpreted

Summary 109

because e� ects of the used compounds may not only be NO related; as was illustrated by our spermine experiments. The reduced e� ectivity of nNOS inhibition in more severe ischemia was explained by the pH sensitivity of the NOS enzyme. More severe ischemia will result in more pronounced acidosis. Under these conditions the NOS enzyme is in-hibited, explaining the limited e� ects of further inhibition. Neuronal NOS inhibition did not a� ect the marked increases in lactate in the post-ischemic brain or the recovery of other energy-related metabolites18. As expected, the protective e� ects of exogenous NO was preserved under acidotic conditions. This e� ect was recently con� rmed for the NO donor sodium nitrite22. Focus has shifted from nNOS to the other iso-enzymes. In a study on gender di� erences in ischemic brain injury, estrogens’ neuroprotective e� ects were related to the attenuation of iNOS expression30. Stroke and cardiovascular event protec-tion associated with regular physical activity was found to be mediated by endothelial NOS upregulation12. The protective e� ects for Angiotensine II type 1 (AT-1) receptor in-hibitor candesartan and HMG coA (3-hydroxy-3-methyl-glutaryl-coA) reductase inhibi-tor rosuvastatin were also found to be at least partly mediated by eNOS upregulation11. Increasing knowledge on this mechanism and the role NO in physiology and pathology will be a strong basis for the development of therapies in the future.

110 Chapter 8

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40. Wei J, Quast MJ: E� ect of nitric oxide synthase inhibitor on a hyperglycemic rat model of reversible focal ischemia: detection of excitatory amino acids release and hydroxyl radical formation. Brain Res 791:146-156, 1998

41. Yamamoto M, Tamura A, Kirino T, Shimizu M, Sano K: Behavioral changes after focal cerebral isch-emia by left middle cerebral artery occlusion in rats. Brain Res 452:323-328, 1988


43. Zhang F, Iadecola C: Reduction of focal cerebral ischemic damage by delayed treatment with nitric oxide donors. J Cereb Blood Flow Metab 14:574-580, 1994

44. Zhang F, White JG, Iadecola C: Nitric oxide donors increase blood � ow and reduce brain damage in focal ischemia: evidence that nitric oxide is bene� cial in the early stages of cerebral ischemia. J Cereb Blood Flow Metab 14:217-226, 1994

45. Zhang F, Xu S, Iadecola C: Time dependence of e� ect of nitric oxide synthase inhibition on cerebral ischemic damage. J Cereb Blood Flow Metab 15:595-601, 1995

46. Zhu CZ, Auer RN: Graded hypotension and MCA occlusion duration: e� ect in transient focal isch-emia. J Cereb Blood Flow Metab 15:980-988, 1995

Samenvatting 113

SAMENVATTING

In vivo modellen zijn een belangrijk instrument bij het onderzoeken van de pathofysiolo-gie van focale cerebrale ischemie en het testen van mogelijke behandelingen. Het ratten arteria cerebri media occlusie (MCAo) model is een veel gebruikt model dat tijdelijke arteriële occlusie tijdens vasculaire operaties simuleert. Reproduceerbaarheid van het infarct in dit MCAo model in de rat bleek afhankelijk te zijn van de duur van afsluiting9, anesthesie techniek39, temperatuur15, bloeddruk46, serum bloedglucose waarde38, ras19,29 en histologische techniek. De interactie tussen deze factoren wordt geïllustreerd door e� ecten van de anesthesie middelen halothaan en iso� uraan39. Terwijl enerzijds het beschermende e� ect van narcose middelen wordt toegeschreven aan een verlaging van het metabolisme, veroorzaken deze middelen in hogere doseringen een toename van het infarct door een verlaging van de bloeddruk en dientengevolge de collaterale bloedstroom. Aandacht voor fysiologische parameters als hersentemperatuur, en bloed-druk in combinatie met informatie over anesthesie techniek kan helpen de resultaten van experimentele hersen ischemie studies beter te interpreteren.Experimenteel kan de ernst van hersen ischemie worden vergroot door het verlengen van de duur van afsluiting of door reductie van de collaterale bloedstroom. Met het Tamura model, een model voor focale cerebrale ischemie in de rat ontwikkelde 60% van de dieren na 1 uur occlusie geen corticaal infarct (hoofdstuk 2)9. Toevoeging van bilaterale carotis communis occlusie resulteerde in een reductie tot 25%. Verlenging van de occlusie tijd tot 3 uur resulteerde in een infarct in 88% van de dieren (hoofdstuk 2)9.Uiteindelijk bleek de omvang van het corticale infarct na 3 uur arteria cerebri media met bilaterale arteria carotis communis occlusie gelijk te zijn aan de waarde gevonden voor permanente media afsluiting (hoofdstuk 2)9. Het infarct in het media stroomgebied ver-oorzaakte geen ernstige neurologische beperkingen41, maar bij deze techniek wordt ten behoeve van de craniotomie de kaak geluxeerd waardoor het proefdier na de operatie moeilijker kan eten. De reperfusie periode werd beperkt tot 3 dagen omdat er na 7 dagen 24 % gewichtsverlies was. Reproduceerbaarheid is een groot probleem bij herseninfarct modellen. Pogingen om deze reproduceerbaarheid te verbeteren zullen resulteren in een reductie van het vereiste aantal proefdieren. In onze experimenten produceerde 3 uur arteria cerebri media en bilaterale arteria carotis communis occlusie een corticaal in-farct van 83 mm3 (gemiddeld) in mannelijke Wistar ratten onder (1%) halothaan narcose en normocapnische, normotensieve, normoterme en normoxische omstandigheden.

Studies over de cascade van gebeurtenissen bij cerebrale ischemie hebben geresul-teerd in de identi� catie van een glutamaat geactiveerde NMDA receptor gemedieerde transmembraan calcium stroom de cel in8. De activatie van calcium afhankelijke enzymen mediëert de verdere schadelijke e� ecten. De stikstofoxide synthase (NOS) iso-enzymen

114 Chapter 8

zijn belangrijke calcium afhankelijke enzymen34 gelokaliseerd in respectievelijk in neu-ronen (nNOS), endotheel (eNOS) en glia cellen en macrofagen (iNOS). Het belang van de ontdekking van stikstofoxide als signaal overbrengend molecuul in het cardiovasculaire systeem werd bevestigd met een Nobelprijs in 1998. Onder pathologische omstandig-heden zoals bij hersen ischemie wordt het NOS systeem overgeactiveerd en mediëert stikstofoxide NMDA toxiciteit34. Omdat de halfwaarde tijd van stikstofoxide kort is zijn directe metingen technisch moeilijk. Directe en indirecte metingen hebben piekconcen-traties geconstateerd kort na het ontstaan van ischemie23,26. Meer indirecte aanwijzingen over de rol van stikstofoxide in cerebrale ischemie komen van studies met NOS remmers en stikstofoxide donors. Het e� ect van niet iso-enzym speci� eke NOS remmers wisselt van beschermend tot schadelijk, waarbij het e� ect dosis afhankelijk is5. Meer consistente resultaten werden gevonden met iso-enzym speci� eke remmers. Aspeci� eke bije� ecten van deze remmers konden worden uitgesloten als mogelijke verklaring voor de werking door middel van studies met proefdieren waarbij dit enzym speci� ek door genetische manipulatie was uitgeschakeld; een zogenaamde “knock-out”. Proefdieren die het nNOS enzym misten ontwikkelden kleinere infarcten, wat indirect de schadelijkheid van stiksto-foxide uit in vitro studies bevestigt10. Behandeling met stikstofoxide donoren anderzijds kan ischemische schade reduceren in vivo als hypotensie wordt vermeden44. Directe ver-gelijking van de e� ecten tussen NOS remmers en stikstofoxide donoren werd bemoei-lijkt door belangrijke verschillen in de gebruikte modellen en gekozen experimentele parameters. In een model dat ischemie simuleert gedurende tijdelijke arteriële occlusies bij neurovasculaire operaties werd het dosis-afhankelijke e� ect getest van twee verschil-lende NOS remmers en twee stikstofoxide donoren. Niet selectieve NOS remmer L-NAME reduceerde het corticale infarct volume signi� cant in de midden dosis (1.0 mg/kg) groep, terwijl selectieve nNOS remmer 7-NI beschermend was bij hogere dosis (10 en 100 mg/kg). In hogere dosis verloor L-NAME zijn beschermende e� ect wat wordt toegeschreven aan eNOS gemediëerde reductie van de collaterale bloedstroom. In vergelijking met de controle groep reduceerde 7-NI het infarct volume met 92% (hoofdstuk 3, � g 1) waaraan het sederende e� ect van 7-NI kan hebben bijgedragen. De anti-ischemische vaatver-wijdende werking van stikstofoxide donoren werd al een eeuw geleden voor het eerst gerapporteerd7 voor acute coronaire syndromen en wordt tot op de dag van vandaag gebruikt. In ons model van focale cerebrale ischemie werd een trend gezien in de rich-ting van reductie van het infarct volume bij behandeling met stikstofoxide donoren maar deze reductie was niet signi� cant (hoofdstuk 3, � g 1)

Polyamines met een stikstofoxide donerende werking reduceren ischemische schade in experimentele focale cerebrale ischemie33. Onder normale (fysiologische) omstandighe-den reguleren polyamines zoals spermine, putrescine en spermidine belangrijke celfunc-ties als Ca2+ transport 21,25 en stikstofoxide productie via NOS20 en het onschadelijk maken

Samenvatting 115

van vrije radicalen21. De rol van polyamines in de pathofysiologie van het herseninfarct is onduidelijk. Gereduceerde spermine concentraties werden gevonden bij experimentele focale cerebrale ischemie31,35. Behandeling met spermine veroorzaakte een reductie van ischemische schade in experimentele, meer globale hersen ischemie13. Om de bescher-mende werking van het spermine deel te testen op een wijze die vergelijking met eerdere experimenten met NOS remmers en stikstofoxide donoren mogelijk maakt werd hetzelfde model van 3 uur arteria cerebri media met bilaterale arteria carotis communis occlusie ge-bruikt. Behandeling met spermine in de dosering van 10 mg/kg intraveneus resulteerde in een signi� cante reductie van het corticale infarct. Dit e� ect was meer uitgesproken dan dat van de behandeling met beide stikstofoxide donoren in hoofdstuk 3. De behandeling met spermine veroorzaakte een signi� cante reductie in de serum glucose waarden van ongeveer 30% en een reductie in het lactaat gehalte. Mogelijke verklaringen voor het beschermende e� ect van spermine zijn dan ook: hypoglycemie, directe werking op de NMDA receptor28 en remming van het nNOS enzym20. De beschermende werking van 10 mg/kg spermine benadert het e� ect van selectieve nNOS remming met 7-NI (hoofstuk 3). Met behulp van studies met combinaties van spermine en NOS remmers en of stikstofoxi-de donoren kan worden getracht het mechanisme van dit e� ect verder vast te stellen.

Selectieve depletie of remming van het neuronale NOS enzym resulteert in een reductie van het corticale infarct volume, terwijl niet iso-enzym speci� ek remmen van het NOS enzym in een meer complex, dosis en tijdsafhankelijk e� ect resulteert27,45. De bevinding dat de ernst van het ischemische incident de e� ectiviteit van neuroprotectie lijkt te be-palen van NOS remmers2,16, gecombineerd met gegevens over de bifasische pH gevoe-ligheid van het NOS enzym14,24,32 heeft geleid tot de hypothese dat de neuroprotectieve werking van nNOS remming afhankelijk is van de intracellulaire pH 4. Door middel van de serum glucose waarden kan de intracellulaire pH gedurende ischemie worden beïn-vloed 4. Het is mogelijk dat hyperglycemie op andere wijze dan middels de versterkte acidose de ischemische schade beïnvloedt. Hyperglycemie met serum glucose waarden van 20 mmol/l en milde hypoglycemie, met serum glucose waarden van 5 mmol/l, wer-den gebruikt om de intracellulaire acidose tijdens ischemie te beïnvloeden. Het resultaat hiervan was dat onder hyperglycemische omstandigheden het infarct toenam (met ge-middeld 77%) terwijl milde hypoglycemie resulteerde in een reductie (van gemiddeld 80%) van het infarct volume. In vivo � uorescentie beeldvormende technieken werden gebruikt om de intracellulaire pH, rCBF (regionale cerebrale bloedstroom) en de NAD+/NADH ratio te bestuderen3. In verband met deze in vivo � uorescentie techniek werd de occlusie tijd aangepast naar 2 uur. Het gemiddelde corticale infarct volume veranderde hierop niet signi� cant in vergelijking met het 3 uurs schema . In de normoglycemische groep (gemiddelde serum glucose waarde van 9 mmol/l) daalde de intracellulaire pH gedurende de 2 uur occlusie van 7.0 naar 6.6. In de hyperglycemische groep werd de pH

116 Chapter 8

6.1 en in de mild hypoglycemische groep 6.8. Onder hyperglycemische omstandigheden werd een reductie van het infarct volume van 28 % bereikt met selectieve nNOS rem-ming met 7-NI (100 mg/kg), terwijl dat onder normo- en hypoglycemische omstandighe-den respectievelijk 93 en 73% was. Uit in vitro studies over de pH gevoeligheid van het NOS enzym14,17,32 blijkt dat binnen het geobserveerde pH bereik de enzym activiteit (van nNOS) aanzienlijk varieert. Bij herstel van de pH werd door Wei en Quast de verwachte toename in NOS activiteit gemeten40. De resultaten van deze studie ondersteunen de hypothese dat het neuroprotectieve e� ect van selectieve nNOS remming afhankelijk is van de intracellulaire pH.

Het gunstige e� ect van behandeling met stikstofoxide donoren bij experimentele hersen-infarcten is eerder beschreven42-44. Stikstofoxide donoren zoals SIN-1 worden gebruikt in de interventie cardiologie in verband met het vaatverwijdende e� ect36. De stikstofoxide afgifte van deze stof wordt beïnvloed door het zuurstof gehalte en de zuurgraad, maar is intact zelfs onder ischemische omstandigheden6. Het gebruik van een stikstofoxide donor toegediend in een enkele gift resulteerde in een tijdelijke bloeddruk daling die spontaan herstelde binnen 30 minuten (voor de aanvang van de occlusie). Om de relatie tussen de rol van stikstofoxide en de intracellulaire pH in focale cerebrale ischemie verder te onderzoeken werd opnieuw naast normoglycemische omstandigheden gewerkt met hyperglycemie en milde hypoglycemie (als in hoofdstuk 5). Het e� ect van behandeling met een stikstofoxide donor werd bestudeerd onder de verschillende omstandigheden. Zoals in het vorige hoofdstuk (5) werd opnieuw in vivo � uorescentie3 gebruikt voor be-rekening van de pHi, rCBF, en NAD+/NADH ratio met aanpassing van het experimen-tele schema naar 2 uur arteria cerebri media en bilaterale carotis communis occlusie. De zuurgraad die werd bereikt variëerde van 6.6 onder normoglycemische omstandigheden tot 6.1 bij hyperglycemie (serumglucose 20 mmol/l) en 6.6 onder mild hypoglycemische omstandigheden (serumglucose 5 mmol/l). Behandeling met SIN-1 beïnvloedde het serum glucose gehalte niet (hoofdstuk 6, tabel 1). Onder normo- en hyperglycemische omstandigheden reduceerde stikstofoxide donor SIN-1 het corticale infarct volume signi-� cant, in tegenstelling tot eerdere experimenten in hoofdstuk 3, waarin de reductie niet signi� cant was bij 3 uur MCA en bilaterale CCA occlusie (hoofdstuk 3, � g 1D). De reductie van het infarct door SIN-1 was het meest uitgesproken in de normoglycemische groep. In de mild hypoglycemische groep was de reductie niet signi� cant (hoofdstuk 6, � g 2). Deze resultaten passen bij de gegevens over de pH gevoeligheid van het NOS enzym14,17,32. We kunnen concluderen dat onder acidotische omstandigheden als het eigen NOS minder actief is het e� ect van exogeen stikstofoxide blijft bestaan.

Aandacht voor details in het kiezen van experimentele parameters kan helpen de repro-duceerbaarheid van experimentele modellen te vergroten. De variabelen als occlusie tijd,

Samenvatting 117

collaterale bloedstroom en serum glucose waarden kunnen belangrijke veranderingen veroorzaken in het infarct volume. De vertaling van experimentele resultaten uit het lab naar de kliniek is moeilijk gebleken37. Er wordt meer aandacht gevraagd voor de farma-kokinetiek, bepalen van doseringen, keuze van het experimentele model en uitkomst parameters1. Verbeteringen hierin zullen hopelijk leiden tot meer klinisch bruikbare ken-nis vanuit het laboratorium.

De complexe rol van stikstofoxide onder fysiologische omstandigheden en in de ont-wikkeling van het herseninfarct in combinatie met de zeer beperkte periode van mogelijk ingrijpen maakt stikstofoxide een onaantrekkelijk doelwit voor behandeling. Het bewijs voor een belangrijke rol van de activatie van het stikstofoxide synthase (NOS) enzym in de pathofysiologie van het herseninfarct is overtuigend. Tot de dag van vandaag is direct kwanti� ceren van stikstofoxide technisch moeilijk. Met behulp van stikstofoxide dono-ren en NOS remmers kan de rol stikstofoxide worden bestudeerd op een meer indirecte wijze. De resultaten van deze indirecte studies moeten voorzichtig worden geïnterpre-teerd omdat een deel van het e� ect niet gemediëerd hoeft te zijn via stikstofoxide zoals uit de spermine experimenten uit hoofdstuk 4 bleek. De verminderde e� ectiviteit van selectieve neuronale NOS remmers bij ernstigere ischemie kan goed worden verklaard met de pH gevoeligheid van het NOS enzym. Bij ernstigere ischemie zal meer uitgespro-ken acidose ontstaan, waaronder het NOS enzym in activiteit is geremd. Neuronale NOS remming bleek geen invloed te hebben op de postischemische lactaat productie en het herstel van andere energie gerelateerde metabolieten18. Het beschermende e� ect van exogeen stikstofoxide bleef ook bij lagere pH waardes aanwezig. Dit beschermende ef-fect werd in 2006 opnieuw bevestigd in een studie met de stikstofoxide donor natrium nitriet 22. De aandacht is verschoven van het neuronale iso-enzym naar de andere iso-enzymen. Zo werd in een studie het beschermende e� ect van oestrogenen op hersen ischemie bij vrouwen gerelateerd aan verminderde iNOS expressie30. Anderzijds lijkt de beschermende werking van regelmatige lichaamsbeweging op cardiovasculaire gebeur-tenissen en het herseninfarct gemediëerd te zijn door versterkte eNOS activiteit12. Het beschermende e� ect van de angiotensine II type 1 (AT-1) receptor remmer candesartan en HMG coA (3-hydroxy-3-methyl-glutaryl-coA) reductase remmer rosuvastatine lijkt ook tenminste deels te zijn gemediëerd door versterkte eNOS activiteit11. Stikstofoxide heeft een centrale rol in de pathofysiologie van het herseninfarct maar mediëert ook de be-schermende e� ecten van bijvoorbeeld oestrogenen, statines en regelmatige lichaams-beweging. De pH gevoeligheid van het NOS enzym levert een goede verklaring voor de geobserveerd gereduceerde cerebroprotectiviteit van nNOS remming bij ernstigere ischemie en hyperglycemie. Verdere toenames van kennis over dit mechanisme en de rol van stikstofoxide onder fysiologische en pathologische omstandigheden kunnen een be-langrijk startpunt zijn voor de ontwikkeling van nieuwe behandelingen in de toekomst.

118 Chapter 8

REFERENTIES


2. Anderson RE, Meyer FB: Is intracellular brain pH a dependent factor in NOS inhibition during focal cerebral ischemia? Brain Res 856:220-226, 2000

3. Anderson RE, Meyer FB, Tomlinson FH: Focal cortical distribution of blood � ow and brain pHi de-termined by in vivo � uorescent imaging. Am J Physiol 263:H565-575, 1992



6. Bohn H, Schona� nger K: Oxygen and oxidation promote the release of nitric oxide from syd-nonimines. J Cardiovasc Pharmacol 14 Suppl 11:S6-12, 1989



9. Coert BA, Anderson, R.E., Meyer, F.B.: Reproducibility of Cerebral Cortical Infarction in the Wistar Rat After Middle Cerebral Artery Occlusion. J Stroke and Cerebrovasc Dis 8:380-387, 1999


11. Engelhorn T, Doer� er A, Heusch G, Schulz R: Reduction of cerebral infarct size by the AT1-receptor blocker candesartan, the HMG-CoA reductase inhibitor rosuvastatin and their combination. An experimental study in rats. Neurosci Lett 406:92-96, 2006

12. Gertz K, Priller J, Kronenberg G, Fink KB, Winter B, Schrock H, et al: Physical Activity Improves Long-Term Stroke Outcome via Endothelial Nitric Oxide Synthase-Dependent Augmentation of Neovascularization and Cerebral Blood Flow. Circ Res, 2006

13. Gilad GM, Gilad VH: Polyamines can protect against ischemia-induced nerve cell death in gerbil forebrain. Exp Neurol 111:349-355, 1991


15. Goto Y, Kassell NF, Hiramatsu K, Soleau SW, Lee KS: E� ects of intraischemic hypothermia on cere-bral damage in a model of reversible focal ischemia. Neurosurgery 32:980-984; discussion 984-985, 1993

16. Harukuni I, Traystman RJ, Kirsch JR: E� ect of AR-R 17477, a potent neuronal nitric oxide synthase inhibitor, on infarction volume resulting from permanent focal ischemia in rats. Crit Care Med 27:2508-2511, 1999


18. Helps SC, Sims NR: Inhibition of Nitric Oxide Synthase with 7-Nitroindazole does not Modify Early Metabolic Recovery Following Focal Cerebral Ischemia in Rats. Neurochem Res, 2006

Samenvatting 119

19. Herz RC, Jonker M, Verheul HB, Hillen B, Versteeg DH, de Wildt DJ: Middle cerebral artery occlusion in Wistar and Fischer-344 rats: functional and morphological assessment of the model. J Cereb Blood Flow Metab 16:296-302, 1996


21. Jensen JR, Lynch G, Baudry M: Polyamines stimulate mitochondrial calcium transport in rat brain. J Neurochem 48:765-772, 1987

22. Jung KH, Chu K, Ko SY, Lee ST, Sinn DI, Park DK, et al: Early intravenous infusion of sodium nitrite protects brain against in vivo ischemia-reperfusion injury. Stroke 37:2744-2750, 2006

23. Kader A, Frazzini VI, Solomon RA, Tri� letti RR: Nitric oxide production during focal cerebral isch-emia in rats. Stroke 24:1709-1716, 1993

24. Kuluz JW, Prado RJ, Dietrich WD, Schleien CL, Watson BD: The e� ect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats. Stroke 24:2023-2029, 1993



27. Margaill I, Allix M, Boulu RG, Plotkine M: Dose- and time-dependence of L-NAME neuroprotection in transient focal cerebral ischaemia in rats. Br J Pharmacol 120:160-163, 1997

28. Munir M, Subramaniam S, McGonigle P: Polyamines modulate the neurotoxic e� ects of NMDA in vivo. Brain Res 616:163-170, 1993


30. Park EM, Cho S, Frys KA, Glickstein SB, Zhou P, Anrather J, et al: Inducible nitric oxide synthase contributes to gender di� erences in ischemic brain injury. J Cereb Blood Flow Metab 26:392-401, 2006


32. Riveros-Moreno V, He� ernan B, Torres B, Chubb A, Charles I, Moncada S: Puri� cation to homogene-ity and characterisation of rat brain recombinant nitric oxide synthase. Eur J Biochem 230:52-57, 1995



35. Sauer D, Martin P, Allegrini PR, Bernasconi R, Amacker H, Fagg GE: Di� ering e� ects of alpha-di� uo-romethylornithine and CGP 40116 on polyamine levels and infarct volume in a rat model of focal cerebral ischaemia. Neurosci Lett 141:131-135, 1992

120 Chapter 8

36. Serruys PW, Deckers JW, Luijten HE, Reiber JH, Tijssen JG, Chadha D, et al: Long-acting coronary vasodilatory action of the molsidomine metabolite Sin 1: a quantitative angiographic study. Eur Heart J 8:263-270, 1987

37. Shuaib A: Neuroprotection - STAIR-Way to the Future? Cerebrovasc Dis 22 Suppl 1:10-17, 2006

38. Tan WK, Anderson, R.E., Meyer, F.B.: Glucose and PaO2 modulation of cerebral metabolic responses following cerebral ischemia. J Cereb Blood Flow Metab 17:S303, 1997


40. Wei J, Quast MJ: E� ect of nitric oxide synthase inhibitor on a hyperglycemic rat model of reversible focal ischemia: detection of excitatory amino acids release and hydroxyl radical formation. Brain Res 791:146-156, 1998

41. Yamamoto M, Tamura A, Kirino T, Shimizu M, Sano K: Behavioral changes after focal cerebral isch-emia by left middle cerebral artery occlusion in rats. Brain Res 452:323-328, 1988


43. Zhang F, Iadecola C: Reduction of focal cerebral ischemic damage by delayed treatment with nitric oxide donors. J Cereb Blood Flow Metab 14:574-580, 1994

44. Zhang F, White JG, Iadecola C: Nitric oxide donors increase blood � ow and reduce brain damage in focal ischemia: evidence that nitric oxide is bene� cial in the early stages of cerebral ischemia. J Cereb Blood Flow Metab 14:217-226, 1994

45. Zhang F, Xu S, Iadecola C: Time dependence of e� ect of nitric oxide synthase inhibition on cerebral ischemic damage. J Cereb Blood Flow Metab 15:595-601, 1995

46. Zhu CZ, Auer RN: Graded hypotension and MCA occlusion duration: e� ect in transient focal isch-emia. J Cereb Blood Flow Metab 15:980-988, 1995

Chapter 9

Acknowledgements

Acknowledgements 123

ACKNOWLEDGEMENTS

First I would like to thank Fred Meyer for giving me the chance to work in the Sundt jr. research lab in Rochester Minnesota � rst as a research trainee and special project associ-ate and later as the Sundt fellow. The experience has made a huge di� erence in my life and my career. Your help also later in my career has been priceless.

To Bob Anderson, I would like to thank you for all your mentoring. Your research skills and the incredible ability to � x anything have made a huge impression. I am proud to have worked with you.

To Heidi Martin, in the many hours we spent together in the lab working on our projects we became good friends. You are a wonderful person with incredible technical skills. I am happy for you that you have moved back to working with patients.

To Raph Thomeer, dear Raph thank you for welcoming me into the Leiden training program and mentoring me for 6 years. With your clear principles you have given me a strong base to do this di� cult job.

To Gary Steinberg, dear Gary, as a cerebrovascular expert you are one of my examples. My year at Stanford has been an incredible experience. I have learned a lot and am to this day grateful for your advice on di� cult cases.

To Peter Vandertop, dear Peter, my move to Amsterdam meant a new start. Your help and coaching in general and especially in getting this manuscript � nished has been very pleasant. We have only just begun.

To professor Tulleken, professor Stam, professor Vermeulen, professor Van Bavel and pro-fessor Van Noorden; thank you for reviewing the manuscript and accepting to be on my “promotie commissie”.

To my father, the loss of mum last year, who was the center of our small but loving family has been a huge blow. As we promised mum we will stick together and I hope to make you proud.

To Henk, you are my big brother, the one that will look after me no matter what. In the good and bad times you have been there. Your talent and ambition has inspired me.

124 Chapter 9

To Peter, I realize that living with me is not always easy. In the 7 1/2 years we have been together a lot has changed but life is still getting better. The best part is spending it with you.

Chapter 10

Curriculum Vitae

Curriculum Vitae 127

CURRICULUM VITAE

Bert was born on April 6, 1969 in Tilburg. He went to high school “ Het Chistelijk Lyceum” in Dordrecht from 1981-1987. He started college and medical school at the Erasmus University, School of Medicine, Rotterdam in September of 1987. He graduated with honors in 1994. When plans to start a research job at the Mayo Clinic were delayed, Bert worked as a resident not in training at the department of Neurosurgery of the Sloter-vaart hospital in Amsterdam. The original plan was resumed in 1996 when he moved to Rochester, Minnesota, USA. He worked at the Thoralf Sundt Jr. Neurosurgical research lab of the Mayo Clinic under supervision of F.B. Meyer M.D. and R.B. Anderson B.S. on neurovascular projects, � rst as a research trainee and later as a special project associate from 1996 to 1998. After returning to Holland he was accepted into the Neurosurgery residency program in Leiden, the Netherlands (chairman: R.T.W.M. Thomeer M.D. Ph.D.) on October 1, 1998. In his third year of residency he returned to the Mayo Clinic research lab in Rochester, Minnesota for a research fellowship (Sundt fellowship). After graduating the Neurosurgery program in Leiden, in 2004 Bert moved to Palo Alto, CA, USA to work as a clinical instructor and cerebrovascular fellow at the department of Neurosurgery of Stanford University under G.K. Steinberg M.D. Ph.D. After his return to the Netherlands he was an attending Neurosurgeon at the department of Neurosurgery at the Leiden University Medical Center (LUMC). He joined the department of Neurosurgery of the Aca-demic Medical Center (AMC) in Amsterdam (chairman: W.P. Vandertop M.D. Ph.D.) in May of 2006 and has a special interest in cerebrovascular neurosurgery.

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