Hydraulic Linkage Between the Cerebrospinal Fluid Cornpartment and Cervical Lymphatics

lm M. F. Silver

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Labontory Medicine and Pathobiology University of Toronto

O Copyright by Ian Michael Francis Silver 1999

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Hydraulic Linkage Between the Cerebrospinal Fiuid Cornpartment and Cervical Lymphatics

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Laboratory Medicine and Pathobiology University of Toronto. 1999

Ian 'ulichaef Francis Silver

Abstract

Previous reports have dernonstrated thac about one-half of the total volume of cerebrospind

fluid (CSF) removed from the cranial vault in sheep is transported into extracranial lymphatics,

especially cervical lymphatic vessels in the neck (Boulton et al., 1996; Boulton et al., 1997;

Boulton et al., 1998a; Boulton et al., 1998: Boulton et al., 1999). It has aiso been noted that

increasing intracraniai pressure resuits in elevated recovery of a CSF protein tracer in ceMcal

lymph (Boulton et al., 1998b). In this study we tested the hypotheses that an elevation of

intracraniai pressure (ICP) would increase cenicd lymphatic pressure and lyrnph Bow rates in

anesthetized sheep, and that blockage of CSF access to ceMcal lyrnphatics would increase

resistance to CSF outflow (%,,). To test the relationship between ICP and cervical lymphatic

pressure and flow, catheters were inserted into both Iaterai ventricles, the cistema magna,

cervical lymphatics and jugula vein. A ventricule-cistemal perfùsion system was employed to

regulate ICP. Mean @=0.008), peak @=0.007) and diastolic (p=0.0 1 3) cervicai rymphatic - pressures increased as ICP was eievated f?om 10 to 70 cm H20 in 20 cm Hz0 increments.

Similarly, cervical lymph flow rates increased @<0.001) with flows at 70 cm Hfi ICP observed

to be approxhately 4 fold higher than those at IO cm H20 ICP. No changes were observed in

mesentenc lymph flow rates (vessels not e-xpected to drain CSF). To test the effect of blocking

access of CSF to the cervical lymphatics, a catheter was inserted into one lateral venricle and the

cistema magna. A constant-flow syringe pump was used to adrninister bolus injections of

artificial CSF, and the extracranial aspect of the minform plate was exposed and sealed. Mean L

R,,,, values increased almost 300% (p=û.003) &er the cribrifom plate was seaied, fkom 71.9 +

1 7.5 to 1 95. I ,; 34.3 cm H-O/rnYmin. In contrast, there was no s i s i ficant change in mem bu,

values in sham operated animais. These results indicare that blocking access of CSF to

extracranial lymphatics ~i~gpificanrly increases the resistance to drainage of CSF fiom the clanid

cavity.

Acknowledgements

1 would like to thank my thesis supervisor, Dr. Miles Johnston, for the wondefil

guidance and support he gave me throughout my graduate studies. I will always owe him

a tremendous debt of gratitude for what he has taught me about good science. 1 would

also like to thank Mrs. Dianna Armstrong, Dr. Melfort Boulton and Dr. Baohua Li for

giving me the benefit of their skiil and insight in laboratory experimentation. In addition,

1 would like to thank a highly talented swnmer student, Ms. Catherine Kim, for the

precious assistance she gave me in carrying out some of the expenments on which this

thesis is based.

My acknowledgements would not be complete without mentioning three other

very important people: Drs. Marie-France and Arthur Silver (my parents), and my wife,

Kirsten Meyer who were always willing to discuss my conclusions with me.

Statement

Widi the exception of the insertion of vennicular catheters in the sheep (whkh 1

pe~ormed), al1 surgical procedures in the lymphatic pressure and flow experbents were

performed by Dr. Baohua Li tvith my assisrance. The cribnform plate blockade experiments

were performed by Dr. Li, Ms. Catherine Kim and me. -411 data analyses contained within this

thesis were performed by me with the assistance of Dr. Li and Ms. Kim.

Table of Contents

7 1.1 Overview ........,.,........-....-.-.-o...--.-....... ..,.... ...... .......-"...... .- .. .- .- .......- ................. ... .. 1.2 Anatomical considerations ..,.. ...................... .".- ..... .-.. ..... - ... ........... ....... - 3 ....w.......Hm..H..nnC-.-

1 3 Ertnce1lul;ir fluid formation ......-...-.------.I)I .- .- ....O.- ......-....... .................. ....-.-.-3

1.4 CSF drainage by arachnoid villi -, ......... - ......... - ............................ ..... ....-.-..- 7

15 Surnmnry of classical view ofECF dynamics in the CNS .,., .... - ..... - 8 ..,,.....-....n....C....~wt.

1.6 Problems with the classical view of CSF absorption -,.....,.......-.-.-...-..rr....t... "- ...U.u.U....-..- 9

1.7 Ly mp hatic drainage of CSF ..---.. "ne-."........ .... .- ....-.--........- 10

1.8 Quantitation of lymphatic drainage of CSF using protein tracers ..lul-.-..............H....n.tUI...- 11

1.9 Response of cervical lyrnphatics to changes in CSF prrssure 15

1.1 0 Effect of iymphatic O bsbuc tion on ICP --- ..--..I........,...w-...ll.-.-...--........ ..... .....w..-- 12

... 1.1 1 Generai hypothesis , ... ........ - ..,,-.....-..- ..,.,,,.. -., ..,...,...-..H.M...M.rm.mr.. 14

1.12 Objectives of the esperimental studies outlined in this thais , .--.-........---... ".-.--A4

MATERIALS AND METHODS 15

. 2.1 Surgicnl preparation .... ...-..... -.-.. ..............- ...-..-....... .- ........................... 16

2.1 .M easurement of lymph flow rat es....-.. .... .....-...-..... .... ..--.........----.. ... 2.3 Measurement of lymphatic pressure, ...-. ........ ........-. ..... ...........-..-.-................-- ..19

2.1 Experimental design .,. -.-... ..--.-....- ...-..............-*W... .. .-.-.- .....-...........-..-- 20

2 5 Data rnalysis ,,.. ........., _, ...................... ....... - ,. ....... .21

RESULTS 23

3.1 Rehtionship between ICP and cervical lymphatic pressure .....m..-.. ..-....m....-.. ..24

3.2 Relationsbip between ICP and cervical lymph flow rates ...... ..-.............-.. 26

3 3 Changes in lymphatic contractile patterns wiib elevation of ICP -...- .......-.......A 1

3.4 Effect of seding cribriform plate on outflow resistance ..- ...CMIIIII....m.......-.- A 3

DISCUSSION 36

4.1 Relationship benveen ICP and cervical lymphatic pressure and flow .. ...-,....,....,...-.-. 37

4.1 Elevated cervical Iymphotic pressure and flow rate in response to increased CCP is due to enhanced delive- of CSF to the cemicd vessels ..-.- ..--.........-......n..n....HH.-..C.............. .38

........ 4 3 Cervical l~mphatic pressure and flow responses at high leveis of ICP ...NU.....W...--.-.......... 40

4.4 Elevation of F&,,,. after sealing the cribriform piste .--.-.-.-... ....................-.....-.. 4 3

REFERENCE LIST 46

Chapter 1

Introduction

Extracranial lymphatic vessels play a si-nificant roIe in the drainage of cerebrospinal

fluid (CSF). Although evidence to support this concept has been in existence since the

nineteenth century, it wasn't mtil recently that the research community started embracing the

idea. This is probably due to nvo principal reasons: (a) an aiternative pathway for draining CSF

had already been described and (b) there are no lymphatic vessels in the central nervous system.

Newer studies have removed these barriers to research in lymphatic drainage of CSF by bringing

to light Iimitations in the old theory of CSF drainage and b y eiucidating routes b y which CSF can

eain access to extracranial lymphatic vessels. - The fact that lyrnphatics play a role in the drainage of CSF may have important

consequences for the pathogenesis of a variety of disease States where intracranial pressure (ICP)

is elevated. Pediatric hydrocephalus and subarachnoid hemorrhage are just two examples of

conditions associated with elevated ICP where impairment of lympbahc drainage of CSF may

play an important role. In this light, the goal of our experimental studies was to provide a better

understanding of the dynamics of extrace1Iular fluid drainage via extracranial lymp hatics. Using

sheep in our in vivo experùnental preparations, we focused on the response of lymphatics to

- aIterations in ICP. From these studies, we conclude that there is a direct reiationship between

ICP and cervical lymphatic pressure and flow, that this reiationship is due to an hydrauiic

linkage between the two compartments, and that this reiationship holds true over a wide range of

ICPs.

1.2 Anatornical considerations

The human brain consists of an e.wemeiy complex array of approximately 100 billion

neurons, each fonning anywhere kom LOO0 to 10,000 synaptic connections with oîher nerve

cells (Carola et al., 1992). This complex arrangement suggests a requirement for strict regulation

of volume within the cranial cavity (Fenstermacher, 1984). In the late 1 8<h and early 19" century,

Alexander Munro and George Kellie noted that within the craniai vault there were three

relatively incompressible components: brai% blood and CSF. The ~Munro-Kellie hypothesis

evolved to allow for changes in blood volume with reciprocal changes in brain ancilor CSF

volumes, as long as the total volume of al1 three components remained unchanged.

The C N S differs fkom other tissues in the body in terms of generation and removal of

extracellular fluid, perhaps because of its requirernent for accurate volume regdation.

DiRerences fiom other tissues include the presence of the blood-brain bamer (BBB), the absence

of lymphatic vessels and the presence of arachnoid villi.

1.3 E ~ c e ï Z u f a r j W d formation

There are two types of extracellular fluid in the CNS: cerebrospinal fluid (CSF) and

parenchymal interstitial fluid (PIF). PIF is found in the interstitium of the brain parenchyma, and

accounts for approximately 20% of parenc hymal volume (Bradbury, 1979; Levine et al., 1970).

It is formed f?om the cerebral capillary, but unlike interstitial fluid in penpheral tissues its

formation is not due to hydrostatic pressure-driven ultrafiltration. Rather, regulation of

molecular movement across the BBB allows the CNS to closely control the composition and

therefore osmotic pressure of its extracellular environment. As the drivhg force of capillaq

hydrostatic pressure is minimized by the BBB, movement of water across the capillary is L

determined largely by osmotic gradients (Fenstermacher. 1984). The interstitiai fluid thus

produced must be rernoved in order to maintain a physiological steady state. As the CNS

contains no lynphatic vessels, the fluid exits the interstitium via the perivascular or Virchow-

Robin spaces and korn there flows into the subarachnoid space. Blood vessels penetrate the

pia/glial surface of the CNS at numerous points and branch extensively throughout the

parenchyma (Hutchings, Weller, 1986; Ichimura et al., 199 1; Zhang et al.. 1990). Benveen these

vessels and the brain parenchyma is a fluid fiiled space that is a n a t o m i d y continuous with the

subpial space. The pia does not bar passage of solutes and fluid, thus the Virchow-Robin spaces

are functionally c o n ~ u o u s with the subarachnoid space (Fi-me 1- 1). PIF flow into the

subarachnoid space accounts fcr 11% of total CSF production in rat and 6% in the rabbit

(Szentistvanyi et al., 1984).

CC

and

Figure 1.1

Schematic illustrating the relationship berneen the perivascnlar and subarac

(Modified fiom Ichimura et al., 1991)

Glia

:hnoid space.

Unlike PIF, cerebrospinal fluid is located within the cerebral ventricles and the

subarachnoid space surrounding the brah and spinal cord. W i W each of the four venmcles are

- tufis of specialized capillaries covered by a simple cuboidal epithelium denved £kom ependyma

These projections are known as choroid plexuses, which are responsible for the formation and

secretion of CSF (Ames et al., 1964; Ames et al., 1965; Fishrnan, 1992). The choroid plexus

consists of numerous villi (Figure 1.2), each with a single Iayer of choroidal epithelia on the

outer surface and a fenestrated capillary in the centre. A blood-CSF barrïer is produced by tight

junctions at the apical end of the epithelia, thereby preventing filtration of plasma components

directly into the veniricular caOity (Becker et al., 1967). The production of CSF is a two step

process. where first an ultrafiltrate is produced by passage of plasma through the capillary

fenestrae into the connective tissue stroma underlying the choroidal epithelium (Davson, Sesai,

1995), followed by movement of fluid across the choroidal epithelium dong an actively

generated osmotic gradient (Pollay et al., 1973; Pollay et al., 1985; Sesal. 1993; Wright, 1972;

Wright, 1977).

1

Figure 1.2

Choroidal villus (modified nom Millen and Woollam, 1958)

The flow of CSF is fiom each of the m o laterai ventricles, through the intervenhicular

foramen of Munro, into the third ventricle, through the cerebral aqueduct of Sylvius and into the

fourth ventricle (Ingraham et al., 1948). From here, CSF flows into the subarachnoid space of

the cistema magna via the foramen of Magendie and the bilateral foramina of Luschka m i n g

of the venmcuiar CSF with parenchpal interstitial fluid occurs within the subarachnoid space

(Ichimura et al., 1991).

1.4 CSF droirzage by arachnoid

in generai, eady inquiries into

vil&

CSF drainage were limited to gross descriptions of

anatornicai structures that came into contact with the subarachnoid fluid. Detailed accounts of

amchnoid Yoranuiarions were presenr in the ~ite~iiture of Willis, -Magendie, Pacchioni and Lushka

(reviewed in Pollay, 1989). These structures were noted to be yellow-gay& rounded

o~mulations, which occurred in isolation or groupings dong the arachnoid membrane. Clark Y

(1920) later reponed that the arachnoid granulations were enlarged versions of the normal

arachnoid villi, and that the macroscopic structures were conspicuously absent in animais and

children under trvo years of age (Clark 1920). Theu relationship to CSF drainage remained

unknown until 1875, when Key and Retzius injected a blue gelatin solution, at a pressure of 80

mm Hg, into the subarachnoid space of human cadavers. GeIatin movement was tracked dong

the subarachnoid space and it penetrated the CpnuIations to enter the dural venous sinuses. CSF

was therefore proposed to drain fkom the subarachnoid space into the dural sinuses via the

arachnoid granulations. Much of this work was later confirmed by Weed, and extended to

propose hydrostatic pressure as the driving force for CSF absorption (Weed, 1914; Weed, 1935).

Spinal arachnoid villi have also been descnied and are believed to be responsible for the

removal of CSF fiom the spinal subarachnoid space into adjacent spinal veins (WeIch, PoUay,

1963). By perfusing an isolated segment of venous sinus wall, Pollay and Welch (Pollay, Welch,

1962) were able to demonstrate that transvillus flow was unidirectional, began at 3-5 cm H20,

and increased in a non-iinear fashion. Osmotic gradients did not affect flow, and particles as L

I q e as canine red blood cells (5.3-7.2 pm) were able to traverse the villi unhindered-

Polystyrene spheres larger than 8.0 p were excluded f5om enterinz the sinus. CNS perfusion

studies demonsmed many parallei observations for total CSF removal (Cutler et al., 1968;

Davson et al., 1970; Lorenzo et ai., 1970) and entrenched the idea that arachnoid villi are largely

responsible for the rernovd of extracellular fluid fiom the Ois.

1.5 Stimmay of cCussicul virw of ECF dynamics in the CNS

Having esrablished the choroid plexuses and arachnoid villi as the primary sites of CSF

production and drainage, respective1 y, a general understanding of CSF d yarnics evolved. CSF

produced by the choroid plexuses flows firom the ventricles into the cistema magna From this

subarachnoid pool, the main CSF current moves around to the venuai aspect of the meduila

oblon_eata, fonvard into the basal cistems, and over the cerebral hemispheres. As the CSF travels

along the cerebral sulci, it mires with PIF that is c o n v e c ~ g ouhvard fkom the perivascular

spaces (McComb et al., 1977). The arachnoid vilfi and granulations located predomuiately along

the parasagittal region provide a direct drainage route for CSF by projecting into the dural

venous sinuses (Figure 1.3). Hydrostatic pressure gradients behveen CSF and blood provide the

dnving force necessary for drainage, where the overall CSF pools is renewed 3-4 times daily.

Arachnoid Arachnoid ElidotbIium Membrane

\ pia r o t e r Sumior ,Sinus

Oum Mater

Cerebca c e -

Blood Vessel and Perivascu lar S m Cerebri Space - -

Figure 1.3

Projection of an arachnoid villus into the superior sagittal sinus.

(modified fiom Davson and Segal, 1995)

1.6 Problems wiîh the classicd view of CSF absorption

Various clinical and experimental observations have pointed to hadequacies in the

classical view of CSF absorption. Perhaps the most problematic is the possibility that arachnoid

villi and arachnoid granulations may not exist or exist in very mal1 numbers in the prenatal

penod (Raimondi, 1986). Arachnoid granulations have been difficult to identify in neonates, but

smctures with morphological characteristics similar to those of the adult have been descnied in

newborn babies (Gomez et al., 1983). Granulations appear in greater numbers as the child ages,

with structures clearly visible by about two years of age (Clark, 1920). As the macroscopic

granulations are simply exaggerated foms of the microscopic villi, it is possible that CSF L

drainage occurs through villi that are not visible to the naked eye. However, in two rnicroscopic

studies of autopsy specimens fÏom individuals up to 56 days old (Osaka et al., 1980) and h m 18

weeks gestation to 80 years (Fox et al., 1996), no arachnoid villi or granulations were observed -

before birth. in newborn infants. few granulations were noted but in al1 specimens fÏom adults,

nurnerous arachnoid granulations were found and these increased in size and number with

increasing a p . One mi&t argue that the feus has less need to drain CSF. However, the choroid

plesus begns produchg CSF re1ativeIy early in deveIoprnent (Portugal. Brock, 19621, so it

would appear that the fetus has need of effecrive CSF ctearance.

To add to this puzzle, there is no apparent correlation between children with congenital

hydrocephalus (documented CSF outflow deficit) and the presence or absence of arachnoid villi

(Gutierrez et ai., 1975; Winkelman, Fay, 1930)- Additionally, ligation of intracranial venous

sinuses in experïmental animais, a procedure expected to impair drainage via arachnoid villi,

fails to induce hydrocephalus (McComb, 1983). Al1 of this evidence seems to indicate the

presence of another pathway capable of draining CSF.

1.7 Lyinphutic drainage of CSF ,

Despite numerous citations attnbuting CSF drainage to the arachnoid villi, there is a

substantial volume of evidence revealing an anatornical and physiological cornation between

CSF and extracranial lymphatic vessels. Studies that are ofien cited as evidence for drainage of

CSF via arachnoid vi1Ii often contain equally compelhg data of CSF drainage by lymphatic

pathways. In their original publication pmposing the arachnoid villi as the major site of CSF

absorption, Key and Reîzius dso noted coloured gelatin extendhg dong the cranial nerves and

entering the lymphatic system (Key, Retzius, 1875). Weed aiso went to great lengths to describe I

the presence of precipitated -granules of Pnxssian blue within the craniaI nenre sheaths, nasal

submucosa, paranasal sinuses. orbital sclera, lumen of lymphatic vessels and cervicd lymph

nodes (Weed. 19 14). Howeve. Weed dismissed these findings as insiyenificant. Despite such a

negative forecasr several subsequent studies introduced various tracers into the CSF in

attempt to unveil the anatomical connections between the subarachnoid space and extracranial

ldmphatic vesseis (reviewed in Faber. 1937). The repeated conclusion fiom snidies upon dead

anïmals. induding human cadavers, as weil as living rats, rabbits, cats, dop. and primates is that

despite the lack of Iynphatic vessels within the C N S , there clearly exists a communication

between the subarachnoid space and peripheml lymph nodes.

I.8 Quantitation of lynpharic drainage MCSF using protein trucers

Recent siudies have demonsûared the quantitative si-pificame of the lymphatic route in

resting States. Approximatek one half of the total CSF to plasma nawport of a protein tracer

occurs through extracranial lymphatics in adult sheep (Boulton et al., 1997) and rats (Boulton et

ai., I 999). Additionail y, tracer recovery data in a three cornpartment mathematical mode1 have

been used to estimate the volurnetric CSF clearance into lymphatics. Remarkably, this study

demonstrated that about one half of the total volume of CSF absorbed kom the cranid vault was

removed by lymphaîic vessels (Boulton et al., 1998). These studies herald a major change in our

understanding of the mechanisms of drainage of CSF, and portend a shift in the direction of

future research into diseases uivolving impaired CSF drainage.

1.9 Response of cervical lynpkatics tu changes in CSFpressure

The cervical lymphatic vessels in die neck provide the most important lpphanc pathway

for CSF clearance. h this regard, raised K P efevates the concentrations of CSF protein tracers -

in cervical lymph nodes (McComb et al., 1982) and in cervical lymph (Boulton et al., 1998b).

hdeed. there are reports in the Iiterature suggesting that total cervical lyrnph flow is associated

wirh elevation of ICP in cats (Love, Leslie. 1981), dogs (Hasuo et al.. 1983), rabbits (Tsay, Lin,

1997) and sheep (Boulton et al., 1998b)- However. the retationship benveen ICP and cervical

lymphatic parameters has not been assessed systematically. Cervical lymphatics collect

interstitial liquid and solutes kom a wide variety of tissues in the head and neck region, not just

fluid originating as CSF. One could argue that the increased lymph flow observed with elevated

ICP was secondary to some KP-induced systemic aiteration. In addition, the ability of the

cervical vessels to transport CSF may be restricted by the nature of the anatomicai pathways that

deliver CSF to lymph accessible sites, Bradbury and Westrop (Bradbury, Westrop, 1983) have

speculated that the greatest resisiance to CSF transport to extracranial lymphatics rnay occur as

the CSF moves through the perineural spaces within the ngid bone of the cnbnfonn plate. Ifthis

is the case, the ability of c e ~ c a l vessets to transport CSF in response to raised 1CP may be

limi t ed.

1.10 Eflect of ZympIzatic obstruction on ICP

The fact that cervical lymphatic vessels participate in the clearance of CSF suggests a

possible roIe for extracranial lymphatics in pathophysiologicai processes invoiving intracranial

fluid accumulation and subsequent elevation of ICP. If cervical lymphatics play such a mie, it

stands to reason that obstmcting them should have an effect on ICP- Indeed, in some species,

cervical lymphatic blockade results in marked anatomical, histological and functional defects

(Foldi et al..

characteristic

'lympbostatic

1968). In dog, for example, ligation of the cervical lyrnph duc= causes

changes - in behaviùur and altered EEG patterns that were attributed to

encephalopathy" (Csanda et al., 1983). Cerebral edema is a characteristic f e a k

of these experïments. Other investigators have nored more subtle changes in br- morphology

and there is some disagreement as to the functional si-pificance of these alterations (Csanda et

ai.. 1983). On refiection, it is not surpnsing to leam that lymphatic ligation produces mixed

results. First. it is well accepted in the field that complete long-term lymphatic obstruction is

very difficult to achieve experimentally due to the enormous regenerative capacity of the vessels,

the npid development of collateral pathways and the potential for the formation of new lymph-

venous anastarnoses (PiIler, Clodius, 1985). Second, the cornpliance-volume characteristics of

the lymphatics and any possible intermediate fluid cornpartment (such as the nasal submucosa)

may have to be saîurated before intracranial pressure rises. Third, acute perturbations likely to

increase ICP can be dealt with over the short tenn by the animal. Distention of the meninges,

compression of vascular elements and venting of CSF through arachnoid villi all serve to protect

the br in &om increases in ICP. Indeed, early attempts on our part to f i ~ d an effect of cervical

lympharic blockade on intracranial pressure and CSF outfiow resistance led to inconsistent

results, probably due to some of the reasons mentioned above. If one wishes to detemiine if the

perineural/lymphatic pathway is capable of affecthg ICP, it becornes necessary to minimize the

effects of these factors as far as possible.

1. I l Geiieral Izypotlresis

Extracranial lymphatic vessels are capable of responding to changes in intracranial

pressure, and biockina access of cerebrospinai fluid to 1-ymphatics increases the resistance to its

outflow.

1-12 Objectives of the qperiin e~ztui sîudies oirtlin ed in this th esis

The purpose of the experirnents outlined this report was to test the hypotheses that (1)

cervical lymphatic pressure and flow are reiated directly to ICP (2) the increaçed lyrnph flow

associated with raised ICP is due primarily to CSF transport to the vesseis (3) the etevated CSF

clearance by cervical lymphatics occurs over a wide range of ICPs and (4) blocking flow of CSF

alon% the perineurai sheath of the olfactory nerve increases the outflow resistance to CSF

drainage.

Chapter 2

mate rials and Methods

Randomly bred female sheep weighing 10-40 kg were purchased fkom LeDo farms

(Ontario) for this investigation. They were fed hay, pellets and water ad iibidum, but were fasted

24 hours before surgery. Experiments were approved by the ethics cornmittee at Sunnybrook

HeaIth Science Centre. and confonned to the guidelines set by the Canadian Council on Animal

Care and the Animals for Research Act of Ontario-

The sheep were anesthetized initially by intravenous infusion of 5% sodium pentothai

solution. FoIlowing this, the animals were intubated and surgical anesthesia was rnaintained

using haIothme administered through a Narkomed 2 respirator. A nid-sagittal incision was

made in the sheep's scalp to reveal the junction of the sagittal and lambdoid sutures. Two 1/8

inch burr holes were made bilateraily 1.5 cm anterior and 1.5 cm lateral to the lambda, at an

angle of 10" Çom the sagittal plane. A single catheter ,ouide screw was insened in each hole. A

16 gauge Novalon i.v. catheter (Becton Dickinson, Sandy, Utah, USA) was then attached to a

column of fiIter-stenlized artificid CSF (as descnied in Chodobski et al., 1992) and fed through

the guide screw. Entry of the catbeters into the laterai ventricles was confirmed by a sudden

drop in artificial CSF volume in the column.

2.1.1 Pressure and flow experiments

One of the catheters was connected to a raised reservoir filled with artificial CSF, while the other

was coupled to a pressure transducer (CDXpress, COBE, Lakewood, Colorado, USA). A

laminectomy was perfaxmed on C l in order to expose the cistema magna, which was cannulated

with a 140 cm long vinyl catheter filled with argficial CSF (Dural Clear Vinyi Tube; i.d. 1.00 L

mm; O-d. 1.50 mm). The catheter was secured to the dura and exteriorized. ICP was controlled

by adjusting the height of the inflow reservoir and the outflow cartieter appropriately. An

incision was made in the neck so that the jugular vein and cervical lymphatic vessels could be

exposed- A vinyl catheter (i-d. 1 .O0 mm, O-d. 1.50 mm) was inserted into the jugular v e h The

outflow end of this catheter was connected to a stopcock, one arm of which \vas in continuity

with a pressure transducer (CDXpress. Cobe) for monitoring of centrai venous pressure (CVP).

In the majorïty ofexperirnents, ICP and CVP were recorded on w o channels of a physiologicd

recorder (Hewlett Packard 7758A). In severai experirnents, data were recorded on a computer

based data acquisition system (A-Tech Instruments, Visuai Designer Software).

2.1.2 Ourflo IV resistame experiments

One of the ventricular catheters was connected to a 20 C.C. syringe (Becton Dickinson,

Sandy, Utah, USA) filled with artificid CSF via a 4 foot i.v. iine attached to a stenle syringe

fiiter (0.45 pm, Coming, USA) and mounted on a syringe pump (Mode1 260, KD Scientific,

USA). A Iaminectomy was performed on Cl in order to expose the cistema magna, which was

cannulated with a 70 cm long vinyl catheter filled with artificial CSF (Dural Clear Vinyl Tube;

i.d. 1.00 mm; 0.d. 1.50 mm). The catheter was secured to the dura and exteriorized. The

catheter was then connected to a pressure transducer (CDXpress, Cobe) for monitoring of CSF

pressure. Data were recorded on a computer based data acquisition system (A-Tech Instruments,

Visual Designer Software). Parallel sagittal incisions approximately 5cm in length were made

Iongitudinally throua the skin and nasal bone. The caudal border of the incisions would began

at the level of a line bisecting the nasal canthi. The caudal and rostral borders of the two

incisions were joined by two transverse incisions. The skin was then replaced over the wound. In L

experimental animals. the nasal mucosa, olfactory nerves and al1 soft tissue on the extracranial

surface of the cribriform plate were later scraped away with a curette and sealed with bone wax

(Johnson & Johnson, Canada), while the sham-operated animals undenvent no m e r treatment.

2.2 Measurement of lynph flow rates

A cervical lymphatic vessel was cannulated using an LS gauge Novaion i.v. catheter

artached to a 3-way stopcock. This in turn was connected to a vinyl catheter (i.d. 1-00 mm, 0.d.

1.50 mm). In the event of lymph clotting, the catheter could be nushed with a heparin-saiine

solution. Al1 side branches, tributaries and other cenical lyrnphatics were tied off. The c e ~ c a i

lymphatics empty into the venous system at the base of the neck and therefore ceMcal lymph

flow is opposed by the central venous pressure (CVP). Since the cervical duct was cannulated

and the lymph diverted, the normal outflow pressure into which this vessel transports lymph

would be altered. To maintain the physiological relationships as close as possible to the n a d

state, we simulated the outfIow pressure encountered by the cervical lymphatic at its normal

lymphatic-venous anastomosis by adjusting the height of the lymphatic outflow catheter to create

a total outflow resistance equivalent to the CVP, which was monitored continuously throughout

the experirnent (described in Boulton et al., l998b). However, in two of the six animals used for

data analysis in this part of the study, this procedure reduced the cervical lymph flow to zero. To

re-establish flow in these huo preparations, the outflow end of the catheter was lowered 2.5 cm

below the measured CVP. Next, an incision was made in the nght abdominal wall and the

mesentery was exteriorized The main mesentenc aunk draining the ileo-cecal junction was

cannulated using a 72 cm long vinyl catheter (i-d. 1.00 mm, o.d, 1.50 mm) and the mesentery

was returned to the abdominal cavity. The outflow end was set at mid-thoracic height. t.

The openings of the cervical and mesentenc outflow catheters were placed imniediately

adjacent to tsvo lever-arm isometric transducers (GouId Statham Model UC3), wbich were

connected to one of the channels of a second physiolo~cal recorder (R511A; Bechan

Insmunenrs). The outflow catheters were positioned such that lymph flowed onto the arm of thé

transducers As the drop of l p p h formed on the transducer arm, an increase in tension was

recorded. When the drop fell off the lever, the transducer was automatically reset (Hayashi et

ai.- 1987). Ttie drop counter was callbrated fiom the cenical and mesenteric lymph that was

coIIected at 15 minute intewals throughout the expenment.

2.3 Measurement of lymphatic pressure

Two vinyl catheters (15 cm in len@l, i.d. 1.50 mm, 0.d. 2.50 mm) were inserted into the

cervical lymphatic, one against the direction of flow and one downstream in the direction of

flow. The outflow end of the upstream catheter and the inflow end of the downstream catheter

were attached to a plastic t-piece such that cervical lymph continued to flow hto the venous

system. A Millar soiid state pressure transducer catheter (SPR-307, Houston, Texas, USA) was

placed through a Cobe sarnpling plug into the side-am of the t-piece. in two of the five

expenments designed to investigate the relatiouship between ICP and ceMcal lymphatic

pressure, ICP, CVP and lymphatic pressures were recorded on the physiological recorder. In

three experiments, the outputs f Ï m the transducers was fed directly to the cornputer based data

acquisition system.

Figure 2.1

Sc hematic illustrating setup for measuring intral ymp hatic pressure.

2.4 Experimentai design

Lyrnphatic pressures and flows were assessed at four different ICPs in each animal. The

ICP was set originally at 10 cm Hfl (- resting ICP in sheep) and raised incrementally to 30, 50

and 70 cm H20. Lymph flow rates and lymphatic and central venous pressures were monitored

continuously for 45 minutes at each pressure.

In the outflow resistance experiments, each animal received one or two priming

injections of 2.5 mi artificial CSF at a rate of 5 ml/min. The animal was considered primed

when on the next injection the post-bolus L ICP retunied to the pre-bolus pressure (-30 minutes).

Each animal then received another bolus injection, and when the ICP retumed to the pre-bolus

pressure, the animal had its cnbrifonn plate scnped and sealed, followed by four more identicai

injections (2.5 ml at a rate of 5 mumin. with each injection given when the ICP retumed to its %

pre-bolus level). Shams undenvent the same procedure, with the exception of having their

cribriform plates scraped and sealed.

We assessed the ICP vs. lymph 80w relationships in eight animals. Mesenteric flow data

were obtained in ali sheep, but cervical Iymph flow data were obtained in six anùnals due to

lymph cIotting in two sheep. ICP vs. cervical lymphatic pressure \vas assessed in six sheep but

the data fYom one animal had to be omirted due to the deposition of fibrin on the Millar

transducer tip .

The baseline, peak and rnean lyrnphatic pressures and normalized lymph flow rates were

plotted over tirne. Norrnaiization of the Idmph fiow data was achieved by dividing each value by

the maximum value obtained in that vesse1 for that animal. This permitted meaninfil

cornparisons because of the variability in flow rates measured from vessels of different sizes.

Mean changes in lymphatic pressure or lymphatic flow rates as a firnction o f ICP were analysed

through repeated measures ANOVA.

The effect of blocking the cribriform plate on outflow resistance was assessed in ten

animais, five shams and five experimental sheep. Outflow resistance was calculated using

equations 1 and 2 for pressure-volume index (PVQ and respectively, as described by

Marmarou (Marmarou et al., 1975; Mannarou et al., 1978). Mean changes of were analysed

through two-way repeated measures ANOVA. L

Equation 1

Equation 2

PVI = A V PP log 10 - Po

A volume AV is injected into the CSF space, raising pressure fiom an initial levei of Po to

a peak pressure of P,. The PVI is then calculated according to equation 1. Following diis, kUt

cm be calculated according to equation 2. where P2 = the instantaneous pressure at an elapsed

time (t2) on the recovery dope of the pressure recording and tz = the elapsed tirne Eom the

instant of volume injection to the point at which P2 is determined. For each injection, five %

values were calculated using five diEerent t tirnepoints (100, 150, 200, 250 and 300 seconds

post-injection). The mean of the five values was then used as the calculated but value for that

in. ection.

Chapter 3

Results

3. I Relationship between ICP and cervical lympliatic pressure

In dl anirnals. a rise in ICP was associated with an increase in cervical Lyrnphatic

pressure (example illustrated in Figure 3.1 ). In these experiments, the cervical vessels continued

to empty into the venous system at the base of rhe neck. Therefo- a change in CVP could

affect cervical lymphatic pressure by altering the outflow pressure experienced by these vessels.

However, we did not observe changes in CVP as ICP was elevated in any of the experiments

performed. The mean data from five anirnals are proned in Figure 3.2. Taking the average of

the last 15 minutes of each monitoring penod. we observed a significant increase in the baseline

(p=0.013), mean (p=0.008) and peak Iwiphatic pressure (p4.007) as K P was elevated.

Behveen 10 and 30 cm HzO ICP, mean iymphatic pressure rose only slightly (from 2.58 to 3.60

cm HzO). However, as ICP was raised fiom 30 to 50 and 50 to 70 cm HzO, cervical lymphatic

pressure increased to 5.79 and 8.06 cm H 2 0 respectively.

100 - lntracranial Pressure

25 - Cervical Lymphatic Pressure

14 t Central Venous Pressure

12 - I

Time (sec)

Figure 3.1

Relationship between intracranial pressure (ICP), cervical lymph pressure and central venous

pressure (CVP) in one sheep. L

i Baseline / i n ~ e a n 1 !.Peak !

10 30 50 70

ICP (cm H G )

Figure 3.2

Relationship between intracranial pressure (ICP) and cervical lymph pressure. A one-way

repeated measures ANOVA revealed a significant effect of ICP on baseline (p=O.013), mean

(~4.008) and peak cervical lymphatic pressure (p=û.007), benueen ICPs of 10 and 70 cm H20.

3.2 Relationship befween ICP and cervical lynph flow rates

As was the case with cervical lymphatic pressure, a rise in ICP produced an increase in

ceMcal lymphatic flow rates (example illusmted in Figure 3.3). Between 10 and 30 cm Hz0

ICP, the change in lymph flow was very small. However, as ICP was elevated M e r , cervical

lymphatic now rates increased coasiderably. Similarly, lowering of ICP resulted in a reduction

of cervical lymph flow rates (see Figure 3.4). Mesenteric Lymph Dow rates monitored in the

same animal did not change when ICP was elevated or lowered (also in Figure 3.4). Changes in

ICP resulted in fairly rapid c e ~ c a i flow responses. Furthemore, it appeared as though the

magnitude of the delay in c e ~ k a l response becarne shorter as ICP was elevated In the example

illusu-ated in Fiapre 3.3, elevation of ICP from 10 to 30 cm HzO produced litîle discernaHe

increase in cervical lyrnph flow rate, but an elevatïon of ICP to 50 cm Hz0 produced a change

within - 5 minutes. The chanse in ICP firom 50 to 70 cm H 2 0 produced an ahost immediate

increase in l p p h flow rate- In the exampIe illustrated in Fi,oure 3-4, ICP \vas reduced ftom 70

to 10 cm HzO and cervical lymph flow decreased within a few minutes. In this same exampie,

raising ICP back to 70 cm HiO had an aimost immediate effect on cervical flows.

5 20 35 50 65 80 95 110 125 140 155 170

Time (min)

Figure 3.3

Relationship between intracranial pressure (ICP) and cervical lymph flow rate in one animai.

t Cervical -..

Figure 3.4

Example of concurrent lymph flow recordings in one animal as ICP was altered. Note that

mesenteric lymph flow is unaffected by changes in ICP.

Andysis of the mean normalized data (Figure 3.5A) demonstrated a significant increase

in ceMcal lyrnph flow associated with elevation of ICP (p4.001). Flow rates increased - four fold as ICP was eIevated fkom 10 to 70 cm HtO. Absolute flow rates averaged 0.82 + 0.02, 1.05 + 0.03, 1.75 t 0.04 and 3.15 f 2.32 mUhr at 10, 30, 50 and 70 cm Hz0 ICP respectively. No

si-gnificant changes in mesenteric lyrnphatic flow rates measured concurrently were observed

(Figure 3.5B).

ICP (cm H20)

10 30 50 70

ICP (CM H20)

Relationship between intracranial pressure (ICP) and ceMcal (A) and mesenteric (B) lymph

flow rates. Data is presented as mean L SEM. A repeated mesures ANOVA revealed a

si-gnificant effect of ICP on ceMcal flow rates (pc0.001) but not on mesenteric vessels.

* represents a significant diifference compared to values at 10 cm H20 ICP as determined by

post hoc analysis with the Newman Keuls test. In (A), the dope or change of the ICP-cervical

lymph flow between 50 and 70 cm EU0 was significantly greater tha. that between 10 and 30

cm H20 as determined by the presence of an interaction effect detected by ANOVA- L

In attempting to determine the rate of change of ceMcal lympharic flow rates for a given

change in ICP, it appeared as though there were hvo distinct slopes for the ICP vs. lymph flow

relationships. Betsveen 10 and 30 cm H20 ICP the dope was relativety shailow, but it increased

benveen 30 and 70 cm HzO. To test the change in the nvo slopes directly, another rtVOVA was

performed. The four pressure levels were now d e h e d by two within-subject factors: TIME

(early vs. iate) and PRESSUEE (low vs. hi&) ~virhin each 2evei o f TIME, As the dependent

rneasure represented a proportion, it was subjected to an arcsine transformation prior to analysis

(Cohen, 1983). The interaction tenn (TIME by PRESSURE) which assesses the difference

berween the two slopes directly was statistically s i - d c m t (~4.0246) .

3.3 Changes in Cynphatic contractt'Iepaîîmns with elmation of ICP

When analysins the flow data from the chart recorder, it was noted that the pattern of

flow and not just the rate of flow was altered in ceMcal lymphatics upon elevation of ICP.

Whenever the lymphatic would contract, an increase in strain was monitored on the data recorder

due to a build-up of fluid on the transducer tip. If al1 of the lymphangioos in the vesse1 were to

contract at the same the, a relatively large amount of fluid would be displaced onto the

transducer tip, and this would be recorded as a pulse. Typically, in the case of the cervical

lymphatic vessels, this would occur several times before the drop of fluid on the txansducer tip

was big enough to fa11 off and reset the transducer. However, at the lower ICP leveb, the

buildup of lymph on the transducer tip appeared srnooth rather dian pulsatile, either because the

contractions were too weak to propel a signincant amount of fluid at one tirne, or because the

lymphangions were contracting in a peristaitic pattern, thus pmpeiing the fluid at a more ~ o r m L

flow rate. As it was difficult to quanti@ the change in contractile pattern we venfied whether or

not the effect was consistent across animals. ln every sheep. an increase in the pulsatility o f flow

was observed as ICP was raised £kom 10 to 70 cm HzO.

F~M? 1996). this was a stahsucally significant change

pattern.

According to the s i 9 test (Anderson,

(p4.04) in the lymphatic contractil=

SMOOTH FLOW, NO PULSES EVIDENT

B PLTLSATILE now, PULSES CLEARLY VISIBLE

I Zr. 1 , t I a , 3 3

Figure 3.6

Example of the change in cervical lymphatic puisatility in one animal &ter intracraniai

pressure is raised from 10 cm HzO (A) to 70 cm HtO (B).

3.4 Effect of seding cribr#îonn plate on outjlow resisrance

In al1 animals tested, sealing the cribriform plate increased the &,,. When the

data were anaiysed by means of a two-way, repeated mesures ANl3V-A. a significant

interaction effect was found between the terms GROUP (expenmental vs sham operated

animals) and PHASE (pre vs post-treamient) on the su, values (p=0.006). This effect

\vas due to the observation that there was a si-gnificant increase in experimental animals

after sealing the cribriform plate, but there was no such increase observed in the shams.

The bu, values used were calculated fiom the Iast bolus injection pnor to treatment, as

well as f?om the last post-treatment injection. Post-hoc analysis by paired t-test revealed

a si-gificant difference between experimental mean K., values before vs d e r sealing the

cribrifom plate @=0.003), but there was no si-glificant difference in sham animais

benveen the pre and post-treatment values. Unpaired t-test mdysis revealed a significant

difference between post-treatment (p=0.02) but not pre-treatment expenmental vs sham

Ku, values. Fi,aure 3.7 shows the mean values for al1 animds.

Figure 3.7

Mean values fkom al1 animals. The h t point in each line represents the pre-

treatment Rut value, while the subsequent points represent the values for each

subsequent post-treamient injection. In the experimental group, Rut values increased

approximately three fold.

In addition to increases in outflow resistance, sealing the cnbriform plate

increased the peak pressure obtained when giving the bolus injections, as illustrated in

Figure 3.8, and also siowed the rate at which ICP retumed to the pre-bolus level.

SrniVI TREATiMENT SHAM OPERATED

70 - SHEEP

I

SEALING OF CRIBRIFORM PLATE EXPERIMENT-LU, I SEIEEP

Figure 3.8

Example ICP traces in a sham operated sheep (top panel) and a sheep that had its

. cnbriform plate sealed (lower panel). The broken line represents the peak pressure of the

pre-treatment injection.

Chapter 4

Discussion

4. I Relationship between ïCP and cetvieal lympIhztic pressure and flow

The data outlined in this thesis support the concept of hydraulic couphg benveen CSF

and cervical lymph in sheep. Incremental changes in ICP were reflected by significant increases

in cervical lyrnphatic pressures and flow rates. In anesthetized cats. cenical lymph flow rates

increased as CSF pressures were raised following infusion of artificiai CSF h t o the cistema

magna (Love, Leslie. 1984), but the increase was not rnaintained as the i n h i o n continued. In

Our studies, cervical flow- rates were retativeiy stable once eqniiibnum had been reached- Th+ may be due to the better control of ICP af5orded by the venmculo-ciste perfusion method

employed in our expenments.

The response t h e of CSF-lyrnph transport to perturùations in ICP undoubtedly relates to

the nature and lena& of the matomicai connections that fink CSF with the cervical vessels. In

rats, lymphatic channels fkom the nasal submucosa approach the cribriform plate and appear to

be in direct continuity with the subarachnoid space associated with perineural olfactory conduits

(Kida et al., 1 993). Altematively, CSF may exit the perineural space to enter the interstitium of

the nasal submucosa Lymphatic vessels present in this tissue collect and drain away the CSF

which has become mixed with nasal interstitiai fluid Clearly, anatomical studies are needed in

the sheep before this issue c m be resolved satisfactorily, In any case, the tirne required to

saturate the pathways leading directly to the cervical ducts or to the nasal submucosal

intermediate cornpartment probably accounts for the initial delay in reflection of ICP changes in

the c e ~ c a l flow and pressure responses. Once saturated the response of the cervicai vessels to

changes in ICP would be expected to occur more rapidly. In support of this, once a new steady-

state flow had been established at high ICP levels, lowering 1CP resulted in a faster cervical

lyrnph flow response. In the experiment illustrated in Figure 3.4, decreasing ICP b m 70 to 10 L

cm H 2 0 caused a cervical flow change in approximately nvo to three minutes and abruptly

increasing ICP back to 70 cm HzO produced an almost immediate increase in cervicai flow.

4.2 Eievated cervical lymphatic pressure and flow rate in response to iiicreased ZCP is due to enhanced delivery of CSF to the cewical vessels

Srveral lines of evidence suggest that the increase in cervical flow was due to enhanced

delivery of CSF to the cervical vessels radier than to ICP-induced systemic perturbations that

could affect lymph transport uidirectly. We measured ceMcal and mesenteric flow rates

simultaneously under conditions in which ICP was varied nom low to high levels. Lymphatics

draining the intestines are not believed to have an important role in CSF clearance and elevations

of ICP had no siginincant effect on mesenteric lymph flow rates (Fi-me 3.4 and Fi-me 3.5B) but

affected cervical flows markedly. If a systemic effect of elevated ICP (such as artenal

hypertension - one of Cushing's si=) had been responsible for the increase in cervical

lymphatic flow rates, mesenteric rates would have nsen as well. The fact that elevated ICP has

an effect on cervical lymphatic but not mesentenc lymphatic flow rates over the ICP range tested

suggests that ar ICP-induced systemic perturbation was not responsible for the increases in

c e ~ c a l flow rates. This leaves an hydraulic linkage between CSF and ceMcal lyrnph as the

most plausible explanation for the effect. In addition, CVP represents an outfiow pressure

against which the cervical lymphatics are forced to flow. A change in CVP could affect cenical

lymphatic pressure and flow independent of, or in conjunction with, augxnented delivery of CSF

to the cervical vessels. Since the cervical vessels were cannulated in the flow experiments and

lymph diverted fiom the animal, any in vivo changes in CVP would not affect lymph flow rates

in our study. In the case of the pressure experiments, ceMcal l p p h continued to flow into the L

venous systern and an increase in C W could have affected lymphatic pressure. However, no

changes were observed in C W over the course of the experhents. Therefore, the mesenteric

and CVP data suggest that the increase of cervical lymphatic pressure and lymph flow rates

observed when ICP was elevated was due to enhanced CSF transport into the extracraniaï

cervical vessels.

Studies with CSF protein tracers aIso support the CSF compamnent as the source of fluid

when cervical l p p h flow rates increase following eievation of ICP- McComb et al, (McComb

et al., 1982) raised ICP in rabbits and demonstrated that the recovery of tracer infused into ihe

lateral ventricles was increased in the draining c e ~ c a i lymph nodes compared to recoveries in

control animals. Similady, in sheep we observed that the recovery of a CSF protein tracer in

cervical lyrnph increased when ICP was elevated corn IO to 30 cm H20, providing more direct

evidence that the augrnented portion of cervical lymph transport was CSF-derived (Boulton et

al., 1998b). In this laner study, lymph tracer recovery data in conjunction with mass balance

equations based on a three cornpartment mathematical mode1 were used to estimate the

volumeûic transport of CSF into the vessels. These calculahons suggested that elevations of ICP

resulted in enhanced volumetric transport of CSF into the cervical lymphatic vessels.

In a previous study conducted in our laboratory, cervical lymph fiow rates averaged 9.1

mYhr in conscious sheep (Le. CSF-denved fluid plus fluid fiom other tissues) (Boult~o et al.,

1998). The tracer-derived estimates of CSF volumetric flows into these vessels averaged

between 0.86 and 1.37 m k . This suggested that fluid orîginating as CSF represented behveen

9.5 and 15% of normal voIume tlow in these vessels. Assuming that the proportion of CSF in

cervical Iymph is similar in ane&thetized animals (this study), we can make estimates of the

proportion of CSF-derived lymph in cervical vessels at various levels of ICP. If we presume that L

IO% of the 0.82 mVhr observed at 10 cm if@ ICP in the study reported here had its origins as

CSF (-resting conditions), then the baseline Iymph flow from non-CSF-reIated sources wouid be

90% of the 0.82 r n b , or 0.74 mik. Açsuming that al1 increases in cervical lymph flow rates

relate only to the transport of CSF into the lymph. we can subnacr 0.74 mVhr h m the observed

fi ow rates at each O f the ICP levels and express the result as a percentage of the total flow rate.

In this way, we estimate that the proportion of lymph that was CSF-derived represented 10, 30,

58 and 77% of the total cenkal ljmph flow ôt ICPs of 10,30,50 and 70 cm HzO respectively.

4.3 Cewical Cynphatic pressure andflow responses at high levels of lCP

In the rabbit studies of Bradbury and Westmp (Bradbury, Westrop, 1983), infusion of

artificial CSF at increasing rates into a laterd ventricie reduced the &action of the CSF protein

tracer in cervical lymph, This is in contrast to several other published reports that demonstrated

increased transport of a CSF tracer inro cervical lyrnph nodes (McCornb et ai., 1982) or cervical

lymph (Elouiton et al-, I998b) when ICP was raised, The highest ICP achieved in the studies of

Bradbury and Westrop was 9 mm Hg and it is possible that more CSF tracer wodd have entered

cervical lymph if greater ICPs had been investigated. We could find no evidence for a plateau in

the ICP-lymph pressure or -Bow rate relationships in sheep at least up to an ICP of 70 cm HzO.

From the lowest to the highest ICP tested in this study, cervical lymph flow rates increased on

average four fold. This suggests that the pathways leading to cervical lymphatic vessels in sheep

play an important roie in the venting of CSF Eom the cranial vault at high ICP levels.

Furthemore, we observed a siwficant change in the sbpe of the ICP vs. cervical lymph flow

relationship (Figure 3SA). Between 10 and 30 cm HzO ICP, lymph flow increased 0.12 mVhr

for every 10 cm H1O increment in ICP- Between 50 and 70 cm H 2 0 ICP, this increased to 0.70

mVhr for each IO cm H 2 0 increment in ICP.

There are severd mechanisms that could contribute to enhanced CSF transport through

cervical lymphatic vessels at hi& levels of ICP. Bradbury and Westrop (Bradbury, Westrop,

1983) speculated that the highest resistance to CSF transport would occur as the CSF passed

through the channels of the cribriform plate and that other drainage parhways mi&t be more

easily cxpanded to facilitate CSF c~emmce when ICPs were elevated- -Q one possibility, these

authors suggested that CSF may be shunted into the subarachnoid space surrounding the spinal

cord. In sheep, we identified several nodes in the abdominal cavity and thorax that were

positioned dong Iynphatic routes that drain spinal CSF, with the intercostai and lumbar nodes

having the most dominant role (Boulton et al., 1996). Following the injection of radioactive

protein tracers into lwnbar CSF, high concentMtions of the tracer were demonstrated in thoracic

duct lymph. Nonetheless, even though the bony cniriform plate may have limited capacity to

expand it is possible that the number of open channels through the cribriform plate may increase

as ICP is elevated. Not al1 perineural spaces associated with the olfactory nerves may be open at

Iower ICPs. The expansion of some of these conduits may require a threshold pressure which is

reached only at high ICPs.

Another possibility relates to the contractile properties of the lymphatic vessels.

Lymphatics can be modeled as a series of hearts with each pumping unit or Iymphangion

containing an inflow and outflow valve (Benoit et al., 1989; Li et al., 1998). Lymphangion

pressure-volume anaiysis yields contraction loops similar to those of the kart with distinct

systolic and diastolic phases. As greater volumes of CSF are delivered to the cervical ducts, the

baseline or diastolic lymphatic pressure increases (Figure 32). An increase in t r ; tnsmd

L

pressure would enhance contractile parameters such as stroke volume as has been demonstrated

with iri sirtc ldvmphatic preparations (Li et al.. 1998) and the increased contractile performance

may facilitate CSF transport. The increase in the pulse size noted in chapter 3.3 of this thesis

may be indicative o f such a response. However, the pulses that we obsexved were the resuIt of

the total output fkom the vesse1 upstream to the site of cannulation, not the output fiom one

single Iymphangion. As such, the change in contractile pattern couid be representative of a

change in the strength of the pumping of the lymphmgions making up the vessel, or altemativeiy

the change could represent a shiR kom a peristaitic contraction pattern to a non-peristaitic

pattern. Whether or not each lymphmgion increases its stroke volume or contraction fkequency

in response to elevations of ICP carmot be detennined fiom this investigation, thou& M e r

study is warranted-

Whichever mechanism is recruited to explain tbis phenornenon, it may also explain an

interesting observation relating to resistance to CSF outfiow. The ease with which CSF is

removed Erom the cranial cornpartment (CSF outflow resistance - can be caicdated using a

number of infusion methods (reviewed in Gjerris et al., 1989). In several species, including

hurnans, the reiationship between ICP and outnow resistance is non-linear with &,, increasing as

ICP is raised until a point is reached where &,, b e g h to fa11 (Mann et al., 1978). Elevations of

ICP couid decrease system resistance by expanding CSF pathways within the cranium leadmg to

enhanced CSF transport to absorption sites. In this regard, Butler (Butler, 1989) has suggested

that the dechne in &,, is due to the formation of ïncreasing numbers of open transendothelial

channels through the arachnoid viIIi. However, this increased CSF delivery would apply not

only to arachnoid viIli, but also to sites accessible to the cervical lymphatic vessels, It is of

interest to note that in the studies of Mann et al, (Mann et al., 1978), the &,, declined in man, l.

dog, cat. rabbit and rat as KPs were elevared beond 30 cm H1O. in Our study, the change in the

dope of the ICP vs. c e ~ c a i lymph flow reiationship appeared to occur somewhere between 30

and 50 cm HzO. At this

increase. Therefore, it is

the decline in Ku,.

point, the abiliry of cervical Iymphatics to

possible that enhanced lymphatic transport

transport CSF appeared to

of CSF coufd contribute t*

1.4 Elevatiow of R,, afer sealing the cribrifonn plme

Having demonstraîed that elevating ICP increases cervical lpphatic pressure a ~ d flow,

the nexr Iogical step was to determine whether blocking the lymphatics had an upstrevn effect

on CSF clearance. Our early attempts to find such an effect by ligating cervical lymphatic

vessels and rnon i to~g ICP led to mixed results, due no doubt to some of the rasons discussed

in the Introduction to this thesis. We soon realised that there were too many confounding

variables to use such a simple approach. Instead, we decided thar blocking access of CSF to

lpphatic drainage bby sealing the cribrifom plare exmcranialiy mi& yield more consistent

results as we would be eiiminating al1 do~vnstream cornpartment cornpliance factors.

Our experimental study involving the sealhg of the cribriform plate was desieed to

determine wheîher or not the &Ur was in part determined by the olfactory nerve / cervical

lymphatic pathway. If the pathway did not contnbute to the &,, then sealing it should have no

effect. However, out results indicate that kU, is signincantly affected by blocking this pathway.

These data seem to conflict with the claims of Marmarou and of Mann that outflow resistance is

dependent only on arachnoid villi and intracranial venous pressure (Marin et ai., 1978; Marmarou

et al., 1973; Marmarou et ai., 1978).

4.5 ImpCicatioas

Since it has been assumed that the major resistor to CSF outflow exists at the level of the

arachnoid villi. investigation of perceived anomalies in CSF drainage has quite naniraily focused

on these structures. Our observation that the outflow resistance can be dramaticdly increased by

blocking a pathway independent of the arachnoid villi would suggest the need for further study

of alternative explanations for the pathogenesis of various disease states.

in the p a s , the factors which

equation as foIIows (Marmarou et al.,

ICP where If is the CSF formation rate,

determine the ICP have been expressed in the f o m of an

1978):

&,, the resistance to outflow of CSF (the reciprocal of

conductance), and P, the sagittal sinus pressure- As mentioned previously, arachnoid villus

function was considered to be the prùnary factor iduencing kur. In our study, sealing the

cnbrïfom plate caused a mean increase in &., of aimost 300%, demonstrating that &,, can be

substantially altered through rnechanisms independent of arachnoid villi. This result implies a

porentially major role for extracraniai lymphatics in determinhg ICP and in the pathogenesis of

various diseases associated with an imbalance in extracellular fluid d y n e c s in the CNS. It

should also be noted that these experiments were conducted in adult sheep, where the drainage of

CSF is carried out half by lymphatics and half by arachnoid villi (Boulton et al., 1997). If, as

discussed earlier, there are no arachnoid villi in the fetus, sealing the fetal cnbriform plate may

block essentiaily al1 CSF clearance and lead to hydrocephalus,

For a given physiological pathway to be considered of import in a disease state,

disrupting the pathway should cause a physiological perturbation which resembles the disease

state. Most CSF cases of hydr6cephalus are thought to be caused by impaired flow or drainage

of CSF rather than its increased formation (reviewed in Fishman, 1992). The exception to this

mie is the exceedingly rare choroid plexus papilIoma which is associated with overproduction of

CSF. In cases where there is a deficienc:~ in absorption of CSF, the arachnoid villi and the veinç

into which these drain were thouat to be the only nvo possible responsible factors. It seems

clear that the perineural sheath/extracranial lymphatic pathway rnay be a third alternative factor

which, when impaired. may connibute independently to deficient drainage of CSF.

An exampie of a disease thou@t to be associated with Unpaired arachnoid villi drainage

is postraumatic hydrocephaius fo1Iowing the retease ofwhole blood hto the subarachnoid space.

The blood is supposed to piug the arachnoid villi with a rneshwork of fibrin and red blood cells,

acutely increasing &,,, presumably by impeding flow throught the vilLi (Butler et al., 1980), and

increasing the ICP to a new equilibrïurn point. Subsequent fibrosis of the villi is proposed to

chronically maintain the unusually large &,, value and lead to a posttraumatic hydrocephalic

state. Similar blockqe and chronic fibrosis of per ined CSF drainage pathways leading to

extracranÏal Ipphatics is not measonable, given the reports of perineural drainage of red blood

cells W C ) in humans fkom the subarachnoid space to the nasal submucosa (Lowhagen et al.,

1994), the a p p e m c e of RBCs in cervical lymph nodes following intracranial bleeding (Csanda

et al., l983), as well as the known inhibitory effect of oxyhemoglobin (found in CSF foflowing

subarachnoid hemorrhage) on lymphatic pumping (Elias et ai., 1992; Wandolo et al., 1992; Elias

et al., 1990; Eisenhoffer et al., 1995). Indeed. in one experiment we conducted in a sheep,

indroducing whole blood into the cerebral ventricles reduced cervical Iymphatic pressure

(unpublished data). The radical departure fiom accepted CSF physiology the results of these

studies represent suggests the need for a comprehensive review of cument understandings of the

pathogenesis of CSF drainage disorders. L

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