research article statins promote long-term recovery after...

Research ArticleStatins Promote Long-Term Recovery after Ischemic Stroke byReconnecting Noradrenergic Neuronal Circuitry

Kyoung Joo Cho1 So Young Cheon2 and Gyung Whan Kim1

1Department of Neurology College of Medicine Yonsei University Seoul 120-752 Republic of Korea2Department of Anesthesiology and Pain College of Medicine Yonsei University Seoul 120-752 Republic of Korea

Correspondence should be addressed to Gyung Whan Kim gyungkimyuhsac

Received 13 February 2015 Revised 6 April 2015 Accepted 28 April 2015

Academic Editor Midori A Yenari

Copyright copy 2015 Kyoung Joo Cho et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Inhibitors of HMG-CoA reductase (statins) widely used to lower cholesterol in coronary heart and vascular disease are effectivedrugs in reducing the risk of stroke and improving its outcome in the long term After ischemic stroke cardiac autonomicdysfunction and psychological problems are common complications related to deficits in the noradrenergic (NA) system Thisstudy investigated the effects of statins on the recovery of NA neuron circuitry and its function after transient focal cerebralischemia (tFCI) Using the wheat germ agglutinin (WGA) transgene technique combined with the recombinant adenoviral vectorsystem NA-specific neuronal pathways were labeled and were identified in the locus coeruleus (LC) where NA neurons originateNA circuitry in the atorvastatin-treated group recovered faster than in the vehicle-treated group The damaged NA circuitry waspartly reorganized with the gradual recovery of autonomic dysfunction and neurobehavioral deficit Newly proliferated cells mightcontribute to reorganizing NA neurons and lead anatomic and functional recovery of NA neurons Statins may be implicated toplay facilitating roles in the recovery of the NA neuron and its function

1 Introduction

Therapeutic aims afterCNS injury are to prevent second com-plications in acute stage and to enhance functional recoveryof the damaged brain in chronic stage The neurological sec-ondary implications contain urinary disorders depressionor dementia [1] Cardiac complications including suddendeath are also frequent during recovery phase after stroke [2]Response to increased serum cardiac enzyme and high bloodpressure plasma noradrenaline (NA) levels are often elevated[3 4] Dysfunction of the noradrenergic system is a fre-quent feature in various neurodegenerative disorders such asAlzheimerrsquos disease andParkinsonrsquos disease aswell as in acuteischemic stroke At stroke onset ischemic sites and lesionsize may be related to different characteristics of autonomicdysfunction [5] Malfunction in the noradrenergic system isalso implicated in many psychiatric disorders manifestingabnormal social behavior attention deficit hyperactivityanxiety and depression Brain ischemia induces pathologicalsynaptic plasticity caused by delayed neuronal death andinduces physiological plasticity which leads to structural

reorganization resulting in functional recovery [6 7] NA hasbeen reported to regulate neural stem cell and also act asa positive modulator for hippocampal neurogenesis in vitroand in vivo [8] In adult mice alteration of noradrenergicsystem could affect neurogenesis and consequently improvethe performing ability to perceptual learning task [9]

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) reductase are widely used for secondary strokeprevention [10] Statin has been reported to have numerouseffects on neuron survival angiogenesis and neurogenesisas well as lipid-lowering activity [11 12] Along with thesetherapeutic usage it was been mainly investigated whetherstatin treatment HMG-CoA reductase inhibition couldpromote neurological recovery perilesional and contrale-sional neuronal plasticity in the post-acute stroke phasepromotes [13] Administration of rosuvastatin for 30 daysafter middle cerebral artery occlusion (MCAO) showed theeffect on reducing dementia [13] Similar results were alsoreported in treatment by simvastatin or atorvastatin [14]Additionally statin might be related to reducing risk ofdementia [15ndash17] and it received attention regarding playing

Hindawi Publishing CorporationNeural PlasticityVolume 2015 Article ID 585783 10 pageshttpdxdoiorg1011552015585783

2 Neural Plasticity

another role in neurodegenerative disease In Parkinsonrsquosdisease (PD) animal model simvastatin treatment showedvarious neuroprotective effects by interacting with NMDAreceptor [18] and also by regulatingmuscarinicM14 receptor[19] Additionally mevalonate pathway inhibited the neuritesoutgrowth by blocking the cell rounding rapidly induced [17]

However it is extremely poorly reported that statin affectsNA system which is deeply affected and altered by cerebralischemic stroke and closely related to chronic disabilities ofphysical and emotional typeThis current study presented thevisualized noradrenergic neuronal circuitry using a genetictracing method and investigated the effects of statins on thefunctional recovery after stroke by focusing on NA neuroncircuitry and its function We put the point on the behavioralimprovement in chronic stage by statin treatment

2 Materials and Methods

21 Animal Model and Drug Treatment To obtain focal cere-bral ischemia animal model adult male C57BL6 mice (23ndash26 g Orient Co Gyeonggi-do South Korea) were housedin a 12 h lightdark cycle and permitted food and waterThese mice were anesthetized by inhalation of isofluranein N2OO2( 70 30) and subjected to fFCI by MCA

occlusion with a surgical nylon suture (50 Silkam BBraumPA USA) for 1 hour A Laser Doppler flowmeter (TransonicSystem Inc New York USA) probe placed directly on theskull surface over the territory of the MCA (1mm posteriorand 5mm lateral to bregma) measured regional CBF beforeand after occlusion and immediately before sacrifice Bloodpressure was monitored by cannulating a femoral artery withPressure Transducer (Harvard Apparatus Inc HollistonMA USA) All procedures were approved by the animalcare committee at Yonsei University medical college Statin(atorvastatin) was dissolved to concentration of 1mgmL in5methanol (methanol 10 120583L in saline 20mL)The preparedstatin solution was treated with intraperitoneal injection with10mgkg from 1 day after MCAO

22 MRI Analysis and Infarct Size Measurement To obtainconsecutive brain damage images we performed MRI scansof mice For MRI analysis mice were put on a horizontalbore (400mm) after anesthetizing with isoflurane by 47Teslar MR scanner (Brucker Biospin Billerica MA USA)Images were obtained using a T2-weighted fast spin echosequence Diffusion images were also acquired Temperaturewas maintained and respiration was monitored throughoutthe entire scan Mice recovered quickly following the scanwith a 100 survival rate Among the obtained MR images4 cuts were used in infarct size measurement and werecombined Each damage region was measured by ImageJ andrepresented by relative value with area of contralateral sideversus damaged area of ipsilateral side

23 Recombinant Adenoviral Construction andViral InfectionThe procedure for generation of recombinant adenoviruseswas described and performed previously [20 21] The PRS-WGA adenovirus was friendly gifted from Dr Huh KyungHee University A gene cassette containing the WGA gene

downstream of the PRS2x8 promoter was inserted into thepAdTrack plasmid and finally into the pAdEasy1 Recombi-nant adenoviral DNA was cut with Pac1 and transfected intoHEK293 cells using Lipofectamine (Invitrogen CA USA)Viruses were harvested 5 days after transfection from thetransfected HEK293 cells and amplified on dishes The viralparticles were purified by cesium chloride density gradientultracentrifugation dialyzed and tittered

Animals were anesthetized and placed in a stereotaxicinstrument After incision of the skin a small burr hole wasmade directly at LC (54 mm posterior 10 mm lateral and38mm deep to bregma) and the viral vector was unilaterallyinjected into the LCof ischemic hemisphere using aHamiltonsyringe (Hamilton Co Nevada USA) A single injectionof 02sim03 120583L of the concentrated adenovirus suspension(about 2 times 1013 cfumL) was used in this study The scalp wasthen sutured and then the mouse was returned to standardhousing After 2 days of virus injection mice were perfusedwith 4 formaldehyde and the brains were removed forhistological analysis

24 Immunohistochemistry Fixed mouse brains were sec-tioned with a cryostat to obtain 40 120583m sections respectivelyThe sections were pretreated for 20min with 1H

2O2in PBS

containing 03 Triton X-100 for inactivation of endogenousperoxidase activity and permeabilization of cellsThe sectionswere then incubated for 30min with 5 normal rabbitserum in PBS to block nonspecific protein-binding sitesand incubated with anti-WGA polyclonal antibody (1 2000Vector Laboratory CA USA) and anti-BrdU monoclonalantibody (1 200 AbD Serotec UK) in PBS containing 03Triton X-100 with 2 normal rabbit serum overnight at 4∘Cand for 2 hours at room temperature After washing thesections were incubated with biotin-labeled anti-goat IgG(1 200 Vector Laboratory) followed by Vectastatin ABCelite kit (Vector Laboratory) TSA kit (Perkin Elmer BostonUSA) and Vectastatin ABC elite kit again Signals werevisualized with Ni2+-intensified diaminobenzidineperoxidereaction kit (Vector Laboratory) Specimens were observedwith a microscope and computerized digital camera system(Olympus Tokyo Japan Provis) and an image analysissystem and program (Adobe Photoshop San Jose CA)

25 BrdU Labeling and Tissue Processing Cell proliferationwas measured by the incorporation of the thymidine ana-logue 51015840-bromo-2-deoxyuridine (BrdU) that is incorporatedinto the DNA of dividing cells in immunohistochemicallydetectable quantities during the S phase of cell division AfterMCAO the animals were twice injected intraperitoneallywith BrdU (Roche) (50mgkg at a concentration of 10mgmLin 09 NaCl) at 24 hours and 1 hour before sacrificeFor immunohistochemical detection of incorporated BrdUdouble-stranded DNA was denatured to a single-strandedform suitable for immunohistochemical detection on sec-tions Sections were incubated in 50 formamide in standardsodium citrate at 65∘C for 2 hours and treated further with2M HCl at 35∘C for 30min After being rinsed for 10minat room temperature in 01M boric acid the sections werewashed with PBS and then incubated with 003 H

2O2in

Neural Plasticity 3

PRSx8p BGHpA CMVp SV40pAGFPWGA

(a) (b)

(c)

Figure 1 Expression of WGA and GFP in LC detecting PRS-WGA adenovirus (a) Schematic diagram representing the structure of thetransgene (b) WGA immunoreactivity in the noninjured mouse brain After 2 days of PRS-WGA adenovirus injection in the LC LC-originated WGA protein was detected in NA neurons and other areas Scale bars = 500120583m

methanol for 5min The sections were incubated with aprimary antibody against BrdU (1 1000 Roche Diagnostics)at room temperature for 30 minutes washed with PBS andreacted with a FITC-conjugated secondary antibody (1 200Jackson ImmunoResearch PA USA) for 30min at roomtemperature After washing stained tissue samples weremounted using Vectashield mounting medium (Vector LabBurlingame CA USA)

26 Behavioral Assessment-LightDark Transition Test andResident-Aggression-Intruder Test

261 LightDark Transition Test The test is based on theaversive nature of mice to light and on their spontaneousexploratory behavior in new environments [22] The appa-ratus for the test consisted of a dark chamber and a brightchamber There are gates to pass between two chambers andmice are allowed to move freely between the two chambersThe test is performed in the bright illuminated chamber andthe number of entries into the dark chamber evaluated for5min Besides the number of transitions latent time in darkchamber was evaluated

262 Resident-Aggression-Intruder Test Mice were habitu-ated in the same cage for at least over 1 week Aggressionlevel was assessed with three sessions at three-day intervalEvaluating itemswere duration of aggressive behavior and thenumber of aggression Duration of aggression indicates thesum of time that determine resident animal the duration toshow active move action toward intruder social explorationlateral threat or clinch attack Latency to attackwas expressedby measuring the time between the introduction of theintruder and the first clinch attack

27 Data Analysis The data were expressed as mean plusmn SDThe statistical comparisons were performed by unpaired 119905-test and one-wayANOVA (StatView SAS Institute Inc CaryNC USA)The significance between the groups was assignedat lowast119901 lt 005 and lowastlowast119901 lt 0001

3 Results31 Adenovirus-Mediated NA Neuron Expressing WGA inthe Locus Coeruleus (LC) Adenoviral construct used in thisstudy contains the wheat germ agglutinin (WGA) gene undercontrol of the eight copies of PRS promoter and the greenfluorescent protein (GFP) gene under the control of thecytomegalovirus (CMV) promoter which makes it possibleto detect viral delivery to the target sites (Figure 1(a)) Afterinjection of the PRS-WGA adenovirus to the LC localizationof GFP expression and WGA protein was examined onadjacent coronal sections Colocalization of GFP and WGAdemonstrates that WGA protein was synthesized in NA neu-rons of the LC in our previous report [21] In sagittal sectionsof noninjured brain intense WGA immunoreactivity wasdetected in the pontine reticular nucleus motor and sensorytrigeminal nucleus thalamic nucleus lateral hypothalamicarea deep mesencephalic nucleus superior colliculus cau-date putamen corpus callosum anterior commissure andolfactory bulb (Figure 1(b)) WGA immunoreactivity wasalso observed in the parabrachial nucleus A5 NA cells sub-stantia nigra lateral habenular nucleus hippocampus optictract and piriform cortex in coronal sections (Figure 1(b))In adenovirus infected mice there was no evidence of cellloss or tissue damage due to the viral infection in LC All ofthe experimental animals that recovered after the infectionremained healthy until being sacrificed without exhibitingany behavioral abnormalities

4 Neural Plasticity

Vehicle

Statin

3 days after tFCI

(a)

VehicleStatin VehicleStatin VehicleStatin

8 months after tFCI6 months after tFCI1 month after tFCI

aci

LHb

OpInWh

hf DG

(b)

Figure 2 Immunoreactivity of WGA in the tFCI mouse brain WGA immunoreactivity at 30 180 and 240 days after tFCI in the vehicle-and atorvastatin-treated groups showed that NA circuitry in the atorvastatin-treated group recovered faster than in the vehicle-treated groupat 180 days aci anterior commissure intrabulbar part hf DG hippocampal fissure and dentate gyrus LHb lateral habenular nucleus Opoptic nerve layer of the superior colliculus InWh intermediate gray layer of the superior colliculus Scale bar = 500 120583m

32 Transsynaptic Transfer of WGA in Noradrenergic Neuronsof Vehicle-Treated and Statin-Treated Mice after MCA Occlu-sion After transient focal cerebral ischemia (tFCI) WGAimmunoreactivity showed the transsynaptic pattern of NAneurons according to day in each vehicle-treated or statin-treated mouse (Figure 2) NA circuits containing thalamicarea lateral hypothalamic area cortex and hippocampuswere destroyed with severe infarction (data not shown)Although a large infarction was formed in theMCA territorymice that survived slowly and gradually recovered the dam-aged area in the long-term stage (Figure 2(a)) When statinwas given for a long period the convalescence periods werefaster than the vehicle-treated mice (Figure 2(b)) WhereasWGA immunoreactivity was not detected in some regionsuch as superior colliculus from 1 month to 8 months

in vehicle-treated mice after tFCI a very strong WGAimmunoreactivity was observed in the statin-treated micein the intrabulbar part of the anterior commissure (aci) andlateral habenular nucleus (LHb)These features indicated thatdamagedNA circuits were partly reorganized over time in thearea of previous lesion

33 Ischemic Brain Damage and Convalescent Process afterMCAO in Vehicle- or Statin-Treated Mice Physiological dataand regional cerebral blood flow (rCBF) were measuredTherewere no statistically significant differences in rCBFdur-ing ischemic condition between the group before occlusionand the group after reperfusion (10min before occlusion100 plusmn 0 10min after occlusion 231 plusmn 53 10min afterreperfusion 962 plusmn 37 mean plusmn SD of each value 119899 = 9 each

Neural Plasticity 5Ve

hicle

-trea

ted

1 month

Over 6 months

1 month

Over 6 months

Stat

in-tr

eate

d

1 month Over 6 months

Lesio

n siz

e (au

)

0

20

40

60

80

100

VehicleStatin

lowast

lowast

Figure 3 Ischemic brain damage and convalescent processes shown by magnetic resonance imaging (MRI) after MCAO

group) Presented in the magnetic resonance images (MRI)the infarcted area after reperfusion from MCAO was grad-ually reduced and significantly statin-treated mice showedsmaller infarct area than vehicle-treated mice (Figure 3)After a longer period the damaged brain regionwas naturallyameliorated even in the vehicle-treatedmice but the recoveryshowed earlier in statin-treated mice than vehicle-treatedmice The differences in the recovery time determined thebehavioral improvement and related to NA neuron circuitryas shown in the result of the behavior test (Figure 5)

34 Neurogenesis after Ischemic Injury in Normal and Vehicle-or Atorvastatin-Treated Mice In postischemic brain injuryneuronal cells derived from neural precursor cells are newlyborn and the cells can be detected by bromodeoxyuridine(BrdU) The BrdU-immunopositive cells were observed inthe postischemic cortex striatum and subventricular zone(SVZ) on the ipsilateral side of the ischemic infarct at 1month and 6 months (Figures 4(a) and 4(b)) The BrdU-positive cells were more frequently observed in the ipsilateralside of the statin-treated mice than in the contralateral sideEspecially at 1 month after tFCI many BrdU- and NeuN-immunopositive cells were detected in the lesioned cortex

and striatum at the direct site to ischemic damage by MCAO(Figure 4(c)) It could be implicated that newborn cells havearisen from the SVZ and migrated into striatum and eveninto cortical area Moreover the proliferation of neural cellsis promoted by long-term statin treatment At 6months and 8months after fFCI the incidence of BrdU-positive cells in theipsilateral striatum and cortex was significantly diminishedcompared to that of 1 month after fFCI (data not shown)TheBrdU-positive cells were not detected in either striatum orcortex but a few BrdU-positive cells were detected in SVZand corpus callosum

35 Neurological Outcome and Physiological Parametersaccording to NA Circuitry The mean arterial blood pressure(MABP) slightly increased until 4 months after fFCI andthen gradually returned to the initial level at 8 months(Figure 5(a)) In contrast statin treatment for a long periodsustained the constant MABP level overall duration aftertFCI The body temperature and weight governed by NAsystem were also monitored according to month but theyjust slightly oscillatedwith no significant changes To evaluatethe NA system alteration after tFCI and effects of statin onthe NA system after tFCI mice performed the lightdark

6 Neural Plasticity

Stat

inVe

hicle

Nor

mal

(a)

StatinVehicle

BrdU

cells

num

ber i

n SV

Z

500

400

300

200

100

0

lowast

(b)

Stat

inVe

hicle

Nor

mal

Cortex Striatum

(c)

Figure 4Neurogenesis after ischemic injury in normal and vehicle- or atorvastatin-treated group (a)Cell proliferation in SVZof the ischemichemisphere is identified by BrdU (green) and NeuN (red) immunoreactivity Many BrdU-positive cells are shown in the SVZ of both groupswith increasedBrdU-positive cells in the striatumof the atorvastatin-treated group (b)Aquantitative graph representing the number of BrdU-positive cells which is increased in the atorvastatin-treated group compared with the vehicle-treated group in the SVZ (c) The migration ofthe proliferated cells is identified in the cortex and striatum each stained with BrdU (green) and NeuN (red) Scale bars = 20 120583m

transition test and aggression-intruder test In the lightdarktransition test the number entering into the bright chamberfrom the dark chamber was convalescent at 6months onward(Figure 5(b)) However the duration of time spent in thelight chamber for vehicle-treated mice was longer over twotimes the normal until 6 months and then it got back tonormal at 8 months Whereas the vehicle-treated mice hada fluctuation the statin-treated mice were stable from theinitial to final stage The duration of aggression swung inthe vehicle-treated mice however the duration in statin-treated mice did not vacillate (Figure 5(c)) The tendency toshow aggression to new intruder was slightly higher in statin-treated mice than vehicle-treated mice in later stages (ie at

8 months) In general statin-treated mice presented peacefulcondition without upset through the observed duration

4 Discussion

This study demonstrated that the NA neurons were transsy-naptically labeled by the WGA-expressing adenoviral vectorBy simply injecting the virus into the unilateral LC the NAneural pathways were clearly visualized with great accuracyand high reproducibility from the LC to its projectionareas of interest The current study shows the following (1)Transsynaptic tracing visualized NA circuitry during long-term recovery after ischemic damage and it is promoted

Neural Plasticity 7

Days after tFCI0 50 100 150 200 250

Bloo

d pr

essu

re (m

mH

g)

70

75

80

85

90

95

100

lowast

(a)

Days0 50 100 150 200 250

Num

ber o

f tra

nsiti

ons

0

5

10

15

20

25

Days0 50 100 150 200 250

Late

ncy

in th

e lig

ht (s

)

0

50

100

150

200

250

300lowast

lowast

(b)

Dur

atio

n of

aggr

essio

n (s

)

0 50 100 150 200 2500

10

20

30

40

50

Days0 50 100 150 200 250

Late

ncy

to at

tack

(s)

0

2

4

6

8

10

12

14

VehicleStatin

VehicleStatin

Days

lowast

lowast

lowast

lowast

lowast

(c)

Figure 5 Physiological recovery and behavioral tests relating to the NA circuitry (a) Profiles of blood pressure in vehicle-treated oratorvastatin-treated mice after tFCI (b) Effects of atorvastatin on behavioral parameters in the lightdark test The lightdark test wasperformed at 30 180 and 240 days after tFCI in mice The effect on mouse behavior in the lightdark test was determined by p-test Datapresented are the mean value plusmn SEM lowast119901 lt 005 relative to the vehicle group (c) Effects of atorvastatin on behavioral parameters in theaggression-intruder test in mice Aggression-intruder test was performed at 30 180 and 240 days after tFCI in mice The effect on mousebehavior in the aggression-intruder test was determined by p-test Data presented are mean value plusmn SEM lowast119901 lt 005 relative to the vehiclegroup

8 Neural Plasticity

by statin (2) Eventually statin ameliorated the damagedbehavioral function of NAergic neuron during long-termrecovery period after stroke injury

The WGA protein injection method had been used tovisualize optic pathways in monkeys [23] olfactory systemsin rodents [24] common afferent projections to the LCin rat [25] and connections of the A5 noradrenergic cellgroup in rat [26] A genetic strategy employing cDNA forWGA as a transgene under the control of specific promoterelements was introduced in neural tracing study [27] Thepresent method successfully and reliably detected strongtranssynaptically transferred WGA protein which might berelated to efficient infection of the adenovirus to the NAneurons as well as the promoter elements (PRS promoter)being used for robust expression ofWGA PRS has previouslybeen shown to be an NA-specific cis element binding topaired-like homeodomain factor Phox2aPhox2b [28] Ithas been confirmed that increasing copies of PRS causesynergistic activation of reporter gene expression reachingmaximal efficacy at eight copies [20] which is in agreementwith successful visualization of the NA system in a mousebrain with a recombinant adenoviral vector expressingWGAunder the control of PRS promoter elements in this study(Figure 1) NA is the neurotransmitter being implicated inmany of these disorders and has been found to affect socialbehavior in both humans and animals [29 30] Thereforeinvestigating alterations of the NA systems in stroke mayprovide clues to the understanding of its symptomologyclinical courses and adequate management NA alterationmay help tomodulate the pathologically alteredmotor systemin stroke patients which resulted in upregulated couplingof damaged motor areas and consequently improved motorfunction [15] Actually d-Amphetamine has a role in multiplebrain transmitter systems improved functional recovery afterstroke and its effect has been attributed to its noradrenergicsystems [31] Intense WGA immunoreactivity was initiallydetected in the most contralateral side and the ipsilateralnonlesioned areas such as the thalamic nucleus lateralhabenular nucleus and amygdalaThese findings suggest thatcompensatory reinforcement of undamaged contralateral NAcircuits occurred shortly after the onset of tFCI and thenevolved into subsequent reorganization of NA circuitriesin severely damaged area Moreover this process can bepromoted by statin treatment When NA synaptic activityincreased pharmacologically symptoms derived by corticaldamage were recovered [32] Synaptic activity also representsthe functional recovery after a stroke in human Afterfocal cortical damage circuitry reorganization is related tofunctional recovery which takes place at the primary corticallevel [33] Therefore investigating alterations of NA systemsin stroke may provide clues to understand its symptomologyclinical courses and adequate management It has beenknown that an acute stroke induced by MCAO inducedcellular proliferation or neurogenesis in the ipsilateral SVZand that a large number of immature neurons migrate fromthe SVZ to ipsilateral infarcted areas at 2 weeks followinginsults [34 35] Statins are well-known drug to reducecholesterol level There are some reports that chronic statintreatment promotes endogenous neuronal cell proliferation

neurogenesis and new synapse formation as well as pro-tecting the neuron from cell death in acute stage [12 14]In a long-term stage we assume that statins are involved infacilitating long-term recovery after stroke by reconnectingthe NA circuit using the newly proliferated cells in SVZor subgranular zone (SGZ) BrdU immunohistochemistry athymidine analogue was used at 1 month and 6 months aftertFCI (Figure 4) It was also known that brain insults such ascerebral ischemia causing neuronal death are accompaniedby increased neurogenesis in the SGZ and SVZ [36 37]BrdU-positive cells were observed in cortical and striatallesions and increased numbers of BrdU-positive cells wereobserved in SVZ and corpus callosum which is consistentwith the process of initial neuronal proliferation in SVZ andthe subsequentmigration into the ischemic lesions as a repairmechanism

To assess the relationship of the remodeling process withfunctional recovery in the NA system the alteration of bloodpressure wasmeasured starting 1 month after tFCI (Figure 5)Blood pressure and behavior change following acute ischemicstroke were related with the anatomical reorganization from6-months after tFCI Besides blood pressure [38 39] dys-function of the NA system has been widely known to beassociated with body temperature [40 41] body weight [42]and anxiety [29 43] Previous experiments indicate thatblood pressure is significantly higher in mice having insularinfarction [38 39] The insular cortex is directly connectedwith central nucleus of the amygdala and the posterior lateralhypothalamus [44 45] belonging to NA system Our resultsindicated that altered physiological dysfunction by acuteischemic stroke injury gradually recovered with the reorga-nization of damaged processes in the mature NA systemsin those structures The behavioral test using the lightdarktest and the aggression-intruder test showed a significantdifference between vehicle-treated mice and statin-treatedmice (Figure 5) For longer period of up to 8 months afterstroke attack statin improved themalfunctioning anxiety andaggression from an earlier period Vehicle-treated mice hadaltered anxiety-like behavior in a time-dependent mannerfollowing tFCI as displayed by a significant increase in thetime spent in the light chamber In contrast to the fluctuatingpattern related to the NA system in vehicle-treated micestatin-treated mice were stable Increase in NA signaling isassociated with higher anxiety and decreased NA signalingwith lower anxiety [46 47] It has been reported that oxidativestress in the CNS is related to anxiety [48 49] and aggressivebehavior is linked to oxidative stress which is one of themain pathologies of ischemic stroke [50] Our results alsopresented that after initial dysfunction of NA activitiesgradual recovery followed along with reorganization of NAcircuits in the infarcted structures

5 Conclusion

Taken together the present study visualized the NA circuitryofmice by tracingwith the PRS-WGAadenoviral system aftertFCI for a long duration Our result showed the reorganiza-tion of damaged NA circuit after tFCI In a chronic stage afterstroke statins were involved in long-term recovery of the NA

Neural Plasticity 9

circuitry and promoted the neurons to be proliferated andmigrate to the infarcted area Statins could ameliorate theNA-related malfunction after stroke in a chronic period

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Kyoung Joo Cho and So Young Cheon equally contributed tothis work

References

[1] A Członkowska and M Lesniak ldquoPharmacotherapy in strokerehabilitationrdquo Expert Opinion on Pharmacotherapy vol 10 no8 pp 1249ndash1259 2009

[2] F L Silver J W Norris A J Lewis and V C Hachinski ldquoEarlymortality following stroke a prospective reviewrdquo Stroke vol 15no 3 pp 492ndash496 1984

[3] M G Myers J W Norris V C Hachinski and M J SoleldquoPlasma norepinephrine in strokerdquo Stroke vol 12 no 2 pp200ndash204 1981

[4] S L Tokgozoglu M K Batur M A Topcuoglu O SaribasS Kes and A Oto ldquoEffects of stroke localization on cardiacautonomic balance and sudden deathrdquo Stroke vol 30 no 7 pp1307ndash1311 1999

[5] D Sander KWinbeck J Klingelhofer T Etgen and B ConradldquoPrognostic relevance of pathological sympathetic activationafter acute thromboembolic strokerdquoNeurology vol 57 no 5 pp833ndash838 2001

[6] M Hallett ldquoPlasticity of the human motor cortex and recoveryfrom strokerdquo Brain Research Reviews vol 36 no 2-3 pp 169ndash174 2001

[7] M Rijntjes and C Weiller ldquoRecovery of motor and languageabilities after stroke the contribution of functional imagingrdquoProgress in Neurobiology vol 66 no 2 pp 109ndash122 2002

[8] V Meneghini B Cuccurazzu V Bortolotto et al ldquoThe nora-drenergic component in tapentadol action counteracts 120583-opioid receptor-mediated adverse effects on adult neurogene-sisrdquoMolecular Pharmacology vol 85 no 5 pp 658ndash670 2014

[9] MMMoreno K Bath N Kuczewski J Sacquet A Didier andN Mandairon ldquoAction of the noradrenergic system on adult-born cells is required for olfactory learning inmicerdquoThe Journalof Neuroscience vol 32 no 11 pp 3748ndash3758 2012

[10] J Chen Z G Zhang Y Li et al ldquoStatins induce angiogenesisneurogenesis and synaptogenesis after strokerdquo Annals of Neu-rology vol 53 no 6 pp 743ndash751 2003

[11] Z Zheng and B Chen ldquoEffects of Pravastatin on neuro-protection and neurogenesis after cerebral ischemia in ratsrdquoNeuroscience Bulletin vol 23 no 4 pp 189ndash197 2007

[12] M Rodriguez-Yanez J Agulla R Rodriguez-Gonzalez T So-brino and J Castillo ldquoStatins and strokerdquoTherapeutic Advancesin Cardiovascular Disease vol 2 no 3 pp 157ndash166 2008

[13] E Kilic R Reitmeir A Kilic et al ldquoHMG-CoA reductaseinhibition promotes neurological recovery peri-lesional tissueremodeling and contralesional pyramidal tract plasticity afterfocal cerebral ischemiardquo Frontiers in Cellular Neuroscience vol8 article 422 2014

[14] K Karki R A Knight Y Han et al ldquoSimvastatin and atorvas-tatin improve neurological outcome after experimental intrac-erebral hemorrhagerdquo Stroke vol 40 no 10 pp 3384ndash3389 2009

[15] Q Wang J Yan X Chen et al ldquoStatins multiple neuroprotec-tive mechanisms in neurodegenerative diseasesrdquo ExperimentalNeurology vol 230 no 1 pp 27ndash34 2011

[16] Y-Q Xu L Long J-Q Yan et al ldquoSimvastatin induces neu-roprotection in 6-OHDA-Lesioned PC12 via the PI3KAKTCaspase 3 pathway and anti-inflammatory responsesrdquo CNSNeuroscience andTherapeutics vol 19 no 3 pp 170ndash177 2013

[17] M Hughes V Snetkov R-S Rose S Trousil J E Mermoudand C Dingwall ldquoNeurite-like structures induced by meval-onate pathway blockade are due to the stability of cell adhesionfoci and are enhanced by the presence of APPrdquo Journal ofNeurochemistry vol 114 no 3 pp 832ndash842 2010

[18] J Yan Y Xu C Zhu et al ldquoSimvastatin prevents dopaminergicneurodegeneration in experimental parkinsonian models theassociation with anti-inflammatory responsesrdquo PLoS ONE vol6 no 6 Article ID e20945 2011

[19] QWang XWei H Gao et al ldquoSimvastatin reverses the down-regulation of M14 receptor binding in 6-hydroxydopamine-induced parkinsonian rats the association with improvementsin long-term memoryrdquo Neuroscience vol 267 pp 57ndash66 2014

[20] D-Y Hwang W A Carlezon Jr O Isacson and K-S KimldquoA high-efficiency synthetic promoter that drives transgeneexpression selectively in noradrenergic neuronsrdquo Human GeneTherapy vol 12 no 14 pp 1731ndash1740 2001

[21] H J Kim H W Kim B I Lee et al ldquoTracing of noradrenergicneuronal circuitry and functional recovery after permanentfocal cerebral ischemia in micerdquoNeurocritical Care Journal vol1 no 1 pp 31ndash42 2008

[22] M Bourin and M Hascoet ldquoThe mouse lightdark box testrdquoEuropean Journal of Pharmacology vol 463 no 1ndash3 pp 55ndash652003

[23] S K Itaya and G W van Hoesen ldquoWGA-HRP as a transneu-ronal marker in the visual pathways of monkey and ratrdquo BrainResearch vol 236 no 1 pp 199ndash204 1982

[24] S K Itaya ldquoAnterograde transsynaptic transport of WGA-HRPin rat olfactory pathwaysrdquo Brain Research vol 409 no 2 pp205ndash214 1987

[25] H S Lee M-A Kim and B D Waterhouse ldquoRetrogradedouble-labeling study of common afferent projections to thedorsal raphe and the nuclear core of the locus coeruleus in theratrdquo Journal of Comparative Neurology vol 481 no 2 pp 179ndash193 2005

[26] C E Byrum and P G Guyenet ldquoAfferent and efferent connec-tions of the A5 noradrenergic cell group in the ratrdquo Journal ofComparative Neurology vol 261 no 4 pp 529ndash542 1987

[27] Y HannoM Nakahira K-I Jishage T Noda and Y YoshiharaldquoTracking mouse visual pathways with WGA transgenerdquo Euro-pean Journal of Neuroscience vol 18 no 10 pp 2910ndash2914 2003

[28] C Yang H-S Kim H Seo C-H Kim J-F Brunet and K-SKim ldquoPaired-like homeodomain proteins Phox2a and Phox2bare responsible for noradrenergic cell-specific transcription ofthe dopamine 120573-hydroxylase generdquo Journal of Neurochemistryvol 71 no 5 pp 1813ndash1826 1998

[29] M D Marino B N Bourdelat-Parks L Cameron Liles andD Weinshenker ldquoGenetic reduction of noradrenergic func-tion alters social memory and reduces aggression in micerdquoBehavioural Brain Research vol 161 no 2 pp 197ndash203 2005

10 Neural Plasticity

[30] M S Marcin and C B Nemeroff ldquoThe neurobiology ofsocial anxiety disorder the relevance of fear and anxietyrdquo ActaPsychiatrica Scandinavica Supplement vol 108 no 417 pp 51ndash64 2003

[31] C Breitenstein A Floel C Korsukewitz S Wailke S Bushu-ven and S Knecht ldquoA shift of paradigm from noradrenergicto dopaminergic modulation of learningrdquo Journal of the Neu-rological Sciences vol 248 no 1-2 pp 42ndash47 2006

[32] D M Feeney M P Weisend and A E Kline ldquoNoradrenergicpharmacotherapy intracerebral infusion and adrenal trans-plantation promote functional recovery after cortical damagerdquoJournal of Neural Transplantation and Plasticity vol 4 no 3 pp199ndash213 1993

[33] N S Ward and L G Cohen ldquoMechanisms underlying recoveryof motor function after strokerdquo Archives of Neurology vol 61no 12 pp 1844ndash1848 2004

[34] A Arvidsson T Collin D Kirik Z Kokaia and O LindvallldquoNeuronal replacement from endogenous precursors in theadult brain after strokerdquo Nature Medicine vol 8 no 9 pp 963ndash970 2002

[35] Z Kokaia and O Lindvall ldquoNeurogenesis after ischaemic braininsultsrdquo Current Opinion in Neurobiology vol 13 no 1 pp 127ndash132 2003

[36] W Jiang W Gu T Brannstrom R Rosqvist and P WesterldquoCortical neurogenesis in adult rats after transient middlecerebral artery occlusionrdquo Stroke vol 32 no 5 pp 1201ndash12072001

[37] K Jin M Minami J Q Lan et al ldquoNeurogenesis in dentatesubgranular zone and rostral subventricular zone after focalcerebral ischemia in the ratrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no8 pp 4710ndash4715 2001

[38] S Meyer M Strittmatter C Fischer T Georg and B SchmitzldquoLateralization in autononic dysfunction in ischemic strokeinvolving the insular cortexrdquoNeuroReport vol 15 no 2 pp 357ndash361 2004

[39] K E Smith V C Hachinski C J Gibson and J CirielloldquoChanges in plasma catecholamine levels after insula damage inexperimental strokerdquo Brain Research vol 375 no 1 pp 182ndash1851986

[40] D Corbett and J Thornhill ldquoTemperature modulation(hypothermic and hyperthermic conditions) and its influenceon histological and behavioral outcomes following cerebralischemiardquo Brain Pathology vol 10 no 1 pp 145ndash152 2000

[41] L Y Moy D S Albers and P K Sonsalla ldquoLowering ambientor core body temperature elevates striatal MPP+ levels andenhances toxicity to dopamine neurons inMPTP-treatedmicerdquoBrain Research vol 790 no 1-2 pp 264ndash269 1998

[42] B M King ldquoThe rise fall and resurrection of the ventromedialhypothalamus in the regulation of feeding behavior and bodyweightrdquo Physiology and Behavior vol 87 no 2 pp 221ndash2442006

[43] J Pandaranandaka S Poonyachoti and S Kalandakanond-Thongsong ldquoAnxiolytic property of estrogen related to thechanges of the monoamine levels in various brain regions ofovariectomized ratsrdquo Physiology and Behavior vol 87 no 4 pp828ndash835 2006

[44] E J Mufson M-M Mesulam and D N Pandya ldquoInsularinterconnections with the amygdala in the rhesus monkeyrdquoNeuroscience vol 6 no 7 pp 1231ndash1248 1981

[45] C B Saper ldquoConvergence of autonomic and limbic connectionsin the insular cortex of the ratrdquo Journal of Comparative Neurol-ogy vol 210 no 2 pp 163ndash173 1982

[46] M Cecchi H Khoshbouei and D A Morilak ldquoModulatoryeffects of norepinephrine acting on alpha1 receptors in the cen-tral nucleus of the amygdala on behavioral and neuroendocrineresponses to acute immobilization stressrdquo Neuropharmacologyvol 43 no 7 pp 1139ndash1147 2002

[47] M D S Lapiz Y Mateo S Durkin T Parker and C AMarsden ldquoEffects of central noradrenaline depletion by theselective neurotoxin DSP-4 on the behaviour of the isolated ratin the elevated plusmaze andwatermazerdquoPsychopharmacologyvol 155 no 3 pp 251ndash259 2001

[48] H Rammal J Bouayed C Younos andR Soulimani ldquoEvidencethat oxidative stress is linked to anxiety-related behaviour inmicerdquo Brain Behavior and Immunity vol 22 no 8 pp 1156ndash1159 2008

[49] H Rammal J Bouayed C Younos and R Soulimani ldquoTheimpact of high anxiety level on the oxidative status of mouseperipheral blood lymphocytes granulocytes and monocytesrdquoEuropean Journal of Pharmacology vol 589 no 1-3 pp 173ndash1752008

[50] H Rammal J Bouayed andR Soulimani ldquoA direct relationshipbetween aggressive behavior in the residentintruder test andcell oxidative status in adult male micerdquo European Journal ofPharmacology vol 627 no 1ndash3 pp 173ndash176 2010

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


BioMed Research International


Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


Neural Plasticity


Parkinsonrsquos Disease


Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Psychiatry Journal


Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases


Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

2 Neural Plasticity

another role in neurodegenerative disease In Parkinsonrsquosdisease (PD) animal model simvastatin treatment showedvarious neuroprotective effects by interacting with NMDAreceptor [18] and also by regulatingmuscarinicM14 receptor[19] Additionally mevalonate pathway inhibited the neuritesoutgrowth by blocking the cell rounding rapidly induced [17]

However it is extremely poorly reported that statin affectsNA system which is deeply affected and altered by cerebralischemic stroke and closely related to chronic disabilities ofphysical and emotional typeThis current study presented thevisualized noradrenergic neuronal circuitry using a genetictracing method and investigated the effects of statins on thefunctional recovery after stroke by focusing on NA neuroncircuitry and its function We put the point on the behavioralimprovement in chronic stage by statin treatment

2 Materials and Methods

21 Animal Model and Drug Treatment To obtain focal cere-bral ischemia animal model adult male C57BL6 mice (23ndash26 g Orient Co Gyeonggi-do South Korea) were housedin a 12 h lightdark cycle and permitted food and waterThese mice were anesthetized by inhalation of isofluranein N2OO2( 70 30) and subjected to fFCI by MCA

occlusion with a surgical nylon suture (50 Silkam BBraumPA USA) for 1 hour A Laser Doppler flowmeter (TransonicSystem Inc New York USA) probe placed directly on theskull surface over the territory of the MCA (1mm posteriorand 5mm lateral to bregma) measured regional CBF beforeand after occlusion and immediately before sacrifice Bloodpressure was monitored by cannulating a femoral artery withPressure Transducer (Harvard Apparatus Inc HollistonMA USA) All procedures were approved by the animalcare committee at Yonsei University medical college Statin(atorvastatin) was dissolved to concentration of 1mgmL in5methanol (methanol 10 120583L in saline 20mL)The preparedstatin solution was treated with intraperitoneal injection with10mgkg from 1 day after MCAO

22 MRI Analysis and Infarct Size Measurement To obtainconsecutive brain damage images we performed MRI scansof mice For MRI analysis mice were put on a horizontalbore (400mm) after anesthetizing with isoflurane by 47Teslar MR scanner (Brucker Biospin Billerica MA USA)Images were obtained using a T2-weighted fast spin echosequence Diffusion images were also acquired Temperaturewas maintained and respiration was monitored throughoutthe entire scan Mice recovered quickly following the scanwith a 100 survival rate Among the obtained MR images4 cuts were used in infarct size measurement and werecombined Each damage region was measured by ImageJ andrepresented by relative value with area of contralateral sideversus damaged area of ipsilateral side

23 Recombinant Adenoviral Construction andViral InfectionThe procedure for generation of recombinant adenoviruseswas described and performed previously [20 21] The PRS-WGA adenovirus was friendly gifted from Dr Huh KyungHee University A gene cassette containing the WGA gene

downstream of the PRS2x8 promoter was inserted into thepAdTrack plasmid and finally into the pAdEasy1 Recombi-nant adenoviral DNA was cut with Pac1 and transfected intoHEK293 cells using Lipofectamine (Invitrogen CA USA)Viruses were harvested 5 days after transfection from thetransfected HEK293 cells and amplified on dishes The viralparticles were purified by cesium chloride density gradientultracentrifugation dialyzed and tittered

Animals were anesthetized and placed in a stereotaxicinstrument After incision of the skin a small burr hole wasmade directly at LC (54 mm posterior 10 mm lateral and38mm deep to bregma) and the viral vector was unilaterallyinjected into the LCof ischemic hemisphere using aHamiltonsyringe (Hamilton Co Nevada USA) A single injectionof 02sim03 120583L of the concentrated adenovirus suspension(about 2 times 1013 cfumL) was used in this study The scalp wasthen sutured and then the mouse was returned to standardhousing After 2 days of virus injection mice were perfusedwith 4 formaldehyde and the brains were removed forhistological analysis

24 Immunohistochemistry Fixed mouse brains were sec-tioned with a cryostat to obtain 40 120583m sections respectivelyThe sections were pretreated for 20min with 1H

2O2in PBS

containing 03 Triton X-100 for inactivation of endogenousperoxidase activity and permeabilization of cellsThe sectionswere then incubated for 30min with 5 normal rabbitserum in PBS to block nonspecific protein-binding sitesand incubated with anti-WGA polyclonal antibody (1 2000Vector Laboratory CA USA) and anti-BrdU monoclonalantibody (1 200 AbD Serotec UK) in PBS containing 03Triton X-100 with 2 normal rabbit serum overnight at 4∘Cand for 2 hours at room temperature After washing thesections were incubated with biotin-labeled anti-goat IgG(1 200 Vector Laboratory) followed by Vectastatin ABCelite kit (Vector Laboratory) TSA kit (Perkin Elmer BostonUSA) and Vectastatin ABC elite kit again Signals werevisualized with Ni2+-intensified diaminobenzidineperoxidereaction kit (Vector Laboratory) Specimens were observedwith a microscope and computerized digital camera system(Olympus Tokyo Japan Provis) and an image analysissystem and program (Adobe Photoshop San Jose CA)

25 BrdU Labeling and Tissue Processing Cell proliferationwas measured by the incorporation of the thymidine ana-logue 51015840-bromo-2-deoxyuridine (BrdU) that is incorporatedinto the DNA of dividing cells in immunohistochemicallydetectable quantities during the S phase of cell division AfterMCAO the animals were twice injected intraperitoneallywith BrdU (Roche) (50mgkg at a concentration of 10mgmLin 09 NaCl) at 24 hours and 1 hour before sacrificeFor immunohistochemical detection of incorporated BrdUdouble-stranded DNA was denatured to a single-strandedform suitable for immunohistochemical detection on sec-tions Sections were incubated in 50 formamide in standardsodium citrate at 65∘C for 2 hours and treated further with2M HCl at 35∘C for 30min After being rinsed for 10minat room temperature in 01M boric acid the sections werewashed with PBS and then incubated with 003 H

2O2in

Neural Plasticity 3


(a) (b)

(c)








4 Neural Plasticity

Vehicle

Statin

3 days after tFCI

(a)



aci

LHb

OpInWh

hf DG

(b)






hicle

-trea

ted

1 month

Over 6 months

1 month

Over 6 months

Stat

in-tr

eate

d


Lesio

n siz

e (au

)

0

20

40

60

80

100

VehicleStatin

lowast

lowast






6 Neural Plasticity

Stat

inVe

hicle

Nor

mal

(a)

StatinVehicle

BrdU

cells

num

ber i

n SV

Z

500

400

300

200

100

0

lowast

(b)

Stat

inVe

hicle

Nor

mal

Cortex Striatum

(c)




4 Discussion


Neural Plasticity 7

Days after tFCI0 50 100 150 200 250

Bloo

d pr

essu

re (m

mH

g)

70

75

80

85

90

95

100

lowast

(a)

Days0 50 100 150 200 250

Num

ber o

f tra

nsiti

ons

0

5

10

15

20

25

Days0 50 100 150 200 250

Late

ncy

in th

e lig

ht (s

)

0

50

100

150

200

250

300lowast

lowast

(b)

Dur

atio

n of

aggr

essio

n (s

)

0 50 100 150 200 2500

10

20

30

40

50

Days0 50 100 150 200 250

Late

ncy

to at

tack

(s)

0

2

4

6

8

10

12

14

VehicleStatin

VehicleStatin

Days

lowast

lowast

lowast

lowast

lowast

(c)


8 Neural Plasticity





5 Conclusion


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of


Neural Plasticity 3


(a) (b)

(c)








4 Neural Plasticity

Vehicle

Statin

3 days after tFCI

(a)



aci

LHb

OpInWh

hf DG

(b)






hicle

-trea

ted

1 month

Over 6 months

1 month

Over 6 months

Stat

in-tr

eate

d


Lesio

n siz

e (au

)

0

20

40

60

80

100

VehicleStatin

lowast

lowast






6 Neural Plasticity

Stat

inVe

hicle

Nor

mal

(a)

StatinVehicle

BrdU

cells

num

ber i

n SV

Z

500

400

300

200

100

0

lowast

(b)

Stat

inVe

hicle

Nor

mal

Cortex Striatum

(c)




4 Discussion


Neural Plasticity 7

Days after tFCI0 50 100 150 200 250

Bloo

d pr

essu

re (m

mH

g)

70

75

80

85

90

95

100

lowast

(a)

Days0 50 100 150 200 250

Num

ber o

f tra

nsiti

ons

0

5

10

15

20

25

Days0 50 100 150 200 250

Late

ncy

in th

e lig

ht (s

)

0

50

100

150

200

250

300lowast

lowast

(b)

Dur

atio

n of

aggr

essio

n (s

)

0 50 100 150 200 2500

10

20

30

40

50

Days0 50 100 150 200 250

Late

ncy

to at

tack

(s)

0

2

4

6

8

10

12

14

VehicleStatin

VehicleStatin

Days

lowast

lowast

lowast

lowast

lowast

(c)


8 Neural Plasticity





5 Conclusion


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of


4 Neural Plasticity

Vehicle

Statin

3 days after tFCI

(a)



aci

LHb

OpInWh

hf DG

(b)






hicle

-trea

ted

1 month

Over 6 months

1 month

Over 6 months

Stat

in-tr

eate

d


Lesio

n siz

e (au

)

0

20

40

60

80

100

VehicleStatin

lowast

lowast






6 Neural Plasticity

Stat

inVe

hicle

Nor

mal

(a)

StatinVehicle

BrdU

cells

num

ber i

n SV

Z

500

400

300

200

100

0

lowast

(b)

Stat

inVe

hicle

Nor

mal

Cortex Striatum

(c)




4 Discussion


Neural Plasticity 7

Days after tFCI0 50 100 150 200 250

Bloo

d pr

essu

re (m

mH

g)

70

75

80

85

90

95

100

lowast

(a)

Days0 50 100 150 200 250

Num

ber o

f tra

nsiti

ons

0

5

10

15

20

25

Days0 50 100 150 200 250

Late

ncy

in th

e lig

ht (s

)

0

50

100

150

200

250

300lowast

lowast

(b)

Dur

atio

n of

aggr

essio

n (s

)

0 50 100 150 200 2500

10

20

30

40

50

Days0 50 100 150 200 250

Late

ncy

to at

tack

(s)

0

2

4

6

8

10

12

14

VehicleStatin

VehicleStatin

Days

lowast

lowast

lowast

lowast

lowast

(c)


8 Neural Plasticity





5 Conclusion


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of



hicle

-trea

ted

1 month

Over 6 months

1 month

Over 6 months

Stat

in-tr

eate

d


Lesio

n siz

e (au

)

0

20

40

60

80

100

VehicleStatin

lowast

lowast






6 Neural Plasticity

Stat

inVe

hicle

Nor

mal

(a)

StatinVehicle

BrdU

cells

num

ber i

n SV

Z

500

400

300

200

100

0

lowast

(b)

Stat

inVe

hicle

Nor

mal

Cortex Striatum

(c)




4 Discussion


Neural Plasticity 7

Days after tFCI0 50 100 150 200 250

Bloo

d pr

essu

re (m

mH

g)

70

75

80

85

90

95

100

lowast

(a)

Days0 50 100 150 200 250

Num

ber o

f tra

nsiti

ons

0

5

10

15

20

25

Days0 50 100 150 200 250

Late

ncy

in th

e lig

ht (s

)

0

50

100

150

200

250

300lowast

lowast

(b)

Dur

atio

n of

aggr

essio

n (s

)

0 50 100 150 200 2500

10

20

30

40

50

Days0 50 100 150 200 250

Late

ncy

to at

tack

(s)

0

2

4

6

8

10

12

14

VehicleStatin

VehicleStatin

Days

lowast

lowast

lowast

lowast

lowast

(c)


8 Neural Plasticity





5 Conclusion


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of


6 Neural Plasticity

Stat

inVe

hicle

Nor

mal

(a)

StatinVehicle

BrdU

cells

num

ber i

n SV

Z

500

400

300

200

100

0

lowast

(b)

Stat

inVe

hicle

Nor

mal

Cortex Striatum

(c)




4 Discussion


Neural Plasticity 7

Days after tFCI0 50 100 150 200 250

Bloo

d pr

essu

re (m

mH

g)

70

75

80

85

90

95

100

lowast

(a)

Days0 50 100 150 200 250

Num

ber o

f tra

nsiti

ons

0

5

10

15

20

25

Days0 50 100 150 200 250

Late

ncy

in th

e lig

ht (s

)

0

50

100

150

200

250

300lowast

lowast

(b)

Dur

atio

n of

aggr

essio

n (s

)

0 50 100 150 200 2500

10

20

30

40

50

Days0 50 100 150 200 250

Late

ncy

to at

tack

(s)

0

2

4

6

8

10

12

14

VehicleStatin

VehicleStatin

Days

lowast

lowast

lowast

lowast

lowast

(c)


8 Neural Plasticity





5 Conclusion


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of


Neural Plasticity 7

Days after tFCI0 50 100 150 200 250

Bloo

d pr

essu

re (m

mH

g)

70

75

80

85

90

95

100

lowast

(a)

Days0 50 100 150 200 250

Num

ber o

f tra

nsiti

ons

0

5

10

15

20

25

Days0 50 100 150 200 250

Late

ncy

in th

e lig

ht (s

)

0

50

100

150

200

250

300lowast

lowast

(b)

Dur

atio

n of

aggr

essio

n (s

)

0 50 100 150 200 2500

10

20

30

40

50

Days0 50 100 150 200 250

Late

ncy

to at

tack

(s)

0

2

4

6

8

10

12

14

VehicleStatin

VehicleStatin

Days

lowast

lowast

lowast

lowast

lowast

(c)


8 Neural Plasticity





5 Conclusion


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of


8 Neural Plasticity





5 Conclusion


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of


Neural Plasticity 9






References
































































Neural Plasticity










Psychiatry Journal









Journal of




































Neural Plasticity










Psychiatry Journal









Journal of


research article statins promote long-term recovery after...

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