7 nicotinic acetylcholine receptor agonism confers...

�7 Nicotinic Acetylcholine Receptor Agonism ConfersNeuroprotection Through GSK-3� Inhibition in a Mouse

Model of Intracerebral HemorrhagePaul R. Krafft, MD; Orhan Altay, MD; William B. Rolland, BS; Kamil Duris, MD;

Tim Lekic, MD, PhD; Jiping Tang, MD; John H. Zhang, MD, PhD

Background and Purpose—Perihematomal edema formation and consequent cell death contribute to the delayed braininjury evoked by intracerebral hemorrhage (ICH). We aimed to evaluate the effect of �7 nicotinic acetylcholine receptor(�7nAChR) stimulation on behavior, brain edema, and neuronal apoptosis. Furthermore, we aimed to determine the roleof the proapoptotic glycogen synthase kinase-3� (GSK-3�) after experimental ICH.

Methods—Male CD-1 mice (n�109) were subjected to intracerebral infusion of autologous blood (n�88) or sham surgery(n�21). ICH animals received vehicle administration, 4 or 12 mg/kg of �7nAChR agonist PHA-543613, 12 mg/kg of�7nAChR agonist PNU-282987, 6 mg/kg of �7nAChR antagonist methyllycaconitine (MLA), 15 �g/kg of phospha-tidylinositol 3-kinase (PI3K) inhibitor wortmannin, or PHA-543613 combined with MLA or wortmannin. Behavioraldeficits and brain water content were evaluated at 24 and 72 hours after surgery. Western blotting and immunofluo-rescence staining were used for the quantification and localization of activated Akt (p-Akt), GSK-3� (p-GSK-3�), andcleaved caspase-3 (CC3). Neuronal cell death was quantified through terminal deoxynucleotidyl transferase–mediateddUTP nick-end labeling (TUNEL).

Results—�7nAChR stimulation improved neurological outcome and reduced brain edema at 24 and 72 hours after surgery(P�0.05 compared with vehicle). Furthermore, PHA-543613 treatment increased p-Akt and decreased p-GSK-3� andCC3 expressions in the ipsilateral hemisphere (P�0.05, respectively), which was reversed by MLA and wortmannin.P-Akt, p-GSK-3�, and CC3 were generally localized in neurons. PHA-543613 reduced neuronal cell death in theperihematomal area (P�0.05).

Conclusions—�7nAChR stimulation improved functional and morphological outcomes after experimental ICH in mice.PHA-543613 reduced the expression of proapoptotic GSK-3� through the PI3K-Akt signaling pathway. (Stroke. 2012;43:844-850.)

Key Words: �7 nicotinic acetylcholine receptor � glycogen synthase kinase-3 � intracerebral hemorrhage � apoptosis� caspase-3 � PHA-543613 � PHA-282987

The prognosis of spontaneous intracerebral hemorrhage(ICH) is often less favorable than that of similar-sized

ischemic strokes.1 Approximately 15% of all cerebrovascularaccidents are hemorrhagic in nature, not including the con-siderable proportion of ischemic brain lesions (30%) that willeventually undergo hemorrhagic transformation.2,3 The ex-panding blood clot as well as the subsequent formation ofbrain edema induce cellular and molecular processes, whichprovoke neuronal apoptosis, thus aggravating the injury afterICH.3–5 Consequently, prospective treatments reducing brainedema or apoptotic cell death may provide a neuroprotectivestrategy for ICH patients.

�7 nicotinic acetylcholine receptors (�7nAChR) are ex-pressed in neurons, neuroglia, and endothelial cells of the

mammalian brain.6–8 In addition to their effects as ligand-gated ion channels, �7nAChR stimulation attenuates neuro-nal apoptosis in various disease models, particularly throughactivation of the phosphatidylinositol 3-kinase (PI3K)-Aktsignaling pathway.7–9

Activated Akt inhibits the glycogen synthase kinase-3�(GSK-3�) in neurons and nonneuronal cells by serine-9phosphorylation.10 Several proapoptotic stimuli, includingoxygen-glucose deprivation and growth factor withdrawal,have been reported to increase the activity—as well as thetyrosine-216 phosphorylation—of GSK-3�.10–12 Phosphory-lated GSK-3� (p-GSK-3�, Tyr216) induces caspase-3-activation, an early step in the execution phase of cellularapoptosis.13 Furthermore, stimulated GSK-3� has been found

Received September 26, 2011; accepted November 8, 2011.From the Department of Physiology and Pharmacology (P.R.K., O.A., W.B.R., K.D., T.L., J.T., J.H.Z.) and the Department of Neurosurgery (J.H.Z.),

Loma Linda University School of Medicine, Loma Linda, CA.Correspondence to John H. Zhang, MD, PhD, Department of Neurosurgery, Loma Linda University, Loma Linda, CA 92354. E-mail

[email protected]© 2011 American Heart Association, Inc.

Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.111.639989

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to aggravate the brain injury after experimental ischemicstroke11 and subarachnoid hemorrhage14; however, the role ofGSK-3� in ICH remains unexplored.

In the present study, we aim to investigate 2 hypotheses:(A) Selective �7nAChR agonists, PHA-543613 and PNU-282987, ameliorate behavioral and morphological outcomes(brain edema) after experimental ICH in mice and (B)�7nAChR stimulation reduces activated GSK-3� through thePI3K-Akt signaling pathway, eventually decreasing neuronalapoptosis.

MethodsAnimals and Intracerebral Blood InfusionAll procedures were conducted following an institutionally approvedprotocol in accordance with the NIH Guide for the Care and Use ofLaboratory Animals. Male CD-1 mice (n�109, weighing 35–45 g;Charles River, Wilmington, MA) were housed in a light- andtemperature-controlled environment with unlimited access to foodand water.

Hemorrhagic stroke in mice was induced by using a modifieddouble infusion model of autologous whole blood (30 �L) aspreviously reported.15,16 Briefly, mice were anesthetized through theuse of intraperitoneal coinjection of ketamine (100 mg/kg) andxylazine (10 mg/kg) in a 2:1 vol/vol ratio. With the mouse positionedprone onto a stereotactic head frame (Kopf Instruments, Tujunga,CA), a craniotomy was performed and a 27-gauge needle wasinserted into the right hemisphere (stereotactic coordinates frombregma: 0.2 mm anterior, 2.0 mm lateral and 3.0 mm in depth).Autologous whole blood (5 �L), collected from the central tailartery, was infused at a rate of 2 �L/min. The needle was thenlowered to the target position of 3.7 mm in depth. After a waitingperiod of 5 minutes, 25 �L of autologous whole blood was infusedinto the right basal ganglia. The needle was left in place for anadditional 10 minutes after the completed infusion, before beingwithdrawn at a rate of 1 mm/min. The craniotomy was sealed withbone wax and the scalp was sutured. All mice were allowed to fullyrecover under observation. Sham-operated animals were subjected toneedle insertion only.

Experimental Groups andPharmacological InterventionsExperimental groups consisted of mice subjected to ICH (n�88) orsham surgery (n�21). After ICH-induction, animals were randomlyselected and treated with either 4 or 12 mg/kg of �7nAChR agonistPHA-543613 (PHA-4 mg, PHA-12 mg) or with 12 mg/kg of�7nAChR agonist PNU-282987 (PNU-12 mg). All treatments wereadministered as an intraperitoneal injection at 1 hour after surgery.Alternatively, ICH mice received 6 mg/kg of the �7nAChR antag-onist methyllycaconitine (MLA) intraperitoneally, at 15 minutes, orthe PI3K inhibitor wortmannin (15 �g/kg) intravenously, at 90minutes before PHA-543613 (12 mg/kg) injection (PHA�MLA,PHA�Wort). MLA and wortmannin were also administered withoutconsecutive �7nAChR agonist treatment (MLA-6 mg, Wort-15 �g).Sham and vehicle animals received an equivalent volume of normalsaline, as given to animals in the intervention groups (0.2 mL). Alldrugs were purchased from Sigma-Aldrich (St Louis, MO) andprocessed according to previously published protocols.8,17

Assessment of Behavioral OutcomeBehavioral outcomes were assessed in a blinded fashion at 24 and 72hours after surgery. The sensorimotor Garcia test18 has been modi-fied for use in mice after experimental hemorrhagic stroke. Thiscomposite assessment consists of 7 individual tests evaluatingspontaneous activity (I), axial sensation (II), vibrissae proprioception(III), symmetry of limb movement (IV), lateral turning (V), forelimboutstretching (VI), and climbing (VII). Each test received a scorebetween 0 (worst performance) and 3 (best performance), and a total

Garcia score was calculated as the sum of all subtests (maximum�21points). For the corner turn test,19 mice were allowed to advance intoa 30° angled corner. To exit the corner, mice could turn either to theright or to the left. The choice of turning side was recorded for eachout of 10 to 15 trials, and a score was calculated as number of leftturns/all turns �100 (%). Reflexive motor ability of the animal’scontralateral (left) forelimb, to respond to a vibrissae-elicited exci-tation, was assessed through the forelimb placing test.19 The scorewas expressed as number of successful paw placements out of 10consecutive vibrissae stimulations. Spatial exploratory and cognitiveperformances in mice were evaluated with the T-maze task aspreviously reported.20 This procedure relies on the rodent’s naturalbehavior to explore novelty and thus alternate between the right andthe left arm of a T-maze. Spontaneous alternations were recorded asa percentage of all trials. All behavioral scores were converted as apercentage of average sham performance (�100%).

Measurement of Brain Water ContentBrain water content was measured at 24 and 72 hours after surgeryas previously reported.16 Briefly, mice were decapitated under lethalisoflurane anesthesia, and brains were quickly removed. A coronalbrain section of 4-mm thickness was separated 2 mm anterior andposterior of the needle tract and then further divided into ipsilateraland contralateral cortex and basal ganglia. The cerebellum wasadditionally collected as an internal control. All brain specimenswere weighed using an analytic microbalance (APX-60, DenverInstrument, Bohemia, NY) to obtain the wet weight. Samples werethen dried at 100°C for 24 hours before determining the dry weight.The brain water content (%) was calculated as (wet weight�dryweight)/wet weight�100.

Western BlottingMice were euthanized at 24 hours after surgery, and ipsilateralhemispheres were isolated and processed as previously described.16

Equal amounts of protein (50 �g) were separated by SDA-PAGE,then transferred onto nitrocellulose membranes and incubated withthe respective primary and secondary antibodies (1:1000). Thefollowing primary antibodies were obtained from Cell SignalingTechnology: anti–p-Akt (Ser473), anti-Akt, anti–GSK-3�, andanti– cleaved caspase-3 (CC3). Anti–p-GSK-3� (Tyr216), anti–�-Actin, and all secondary antibodies were purchased from SantaCruz Biotechnology. Immunoblots were visualized with the ECLPlus chemiluminescence reagent kit (Amersham Bioscience, Ar-lington Heights, IL) and densitometrically quantified, using thesoftware ImageJ (National Institutes of Health). Results areexpressed as relative density ratio, normalized to the mean valueof the sham group.

Immunofluorescence StainingMice were euthanized at 24 hours after surgery, and brain specimenswere processed as previously described with minor modifications.21

Triple immunofluorescence was performed using the neuronalmarker anti-NeuN (1:100, Millipore, Temecula, CA) and anti–p-GSK-3� (Tyr216, 1:100) in combination with terminal deoxynu-cleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) (Insitu Cell Death Detection Kit, Fluorescein, Roche Inc, Mannheim,Germany). Double immunofluorescence staining was performedwith anti–NeuN and anti-CC-3 (1:1000). Microphotographs wereanalyzed with the use of a fluorescent microscope and Magna FireSP system (Olympus). TUNEL-positive neurons were counted at�400 magnification in 4 perihematomal areas (500�500-�m grids),and the data were expressed as cells/mm2.

Statistical AnalysisData were expressed as mean�SEM and statistically analyzed with1-way ANOVA followed by Tukey post hoc test. All behavior datawere expressed as mean�SEM of sham performance and analyzedwith Kruskal-Wallis 1-way ANOVA on ranks, followed by theStudent-Newman-Keuls method. A probability value of �0.05 was

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considered statistically significant. All statistical analyses wereperformed using SigmaPlot version 10.0 for Windows.

ResultsPHA-543613–Mediated Attenuation of BehavioralDeficits and Brain Edema at 24 Hours After ICHIs Dependent on the PI3K-Akt Signaling PathwayBehavioral deficits were evaluated at 24 hours after surgery(n�6 per group). Mice subjected to ICH presented a signif-icantly worse Garcia test performance than sham-operatedanimals (P�0.05, Figure 1A); however, treatment with�7nAChR agonists PHA-543613 (PHA-4 mg, PHA-12 mg)or PNU-282987 (PNU-12 mg) improved the outcome signif-icantly (P�0.05, compared with vehicle). Mice within thepharmacological intervention groups additionally received aninjection of the �7nAChR antagonist MLA (PHA�MLA) orthe PI3K inhibitor wortmannin (PHA�Wort) before PHA-543613 (12 mg/kg) administration to assess whether the

observed behavioral improvements are dependent on�7nAChR-induced activation of the PI3K-Akt signalingpathway. Wortmannin, as anticipated, reversed the initialattenuation of behavioral deficits observed with PHA-543613. The 2 applied inhibitors, MLA (6 mg/kg) andwortmannin (15 �g/kg), did not worsen the behavioraloutcome of the Garcia test when administered alone (P�0.05,compared with vehicle). After experimental right-sided ICH,mice turned less frequently to the impaired (left) side whileperforming the corner turn test (P�0.05 compared withsham); however, no differences were found between treatedand untreated ICH animals (P�0.05). Furthermore, vehicleanimals demonstrated significantly impaired contralateral(left) forelimb function, evaluated through the forelimb plac-ing test, as well as a reduced number of spontaneousalterations during the T-maze assessment (P�0.05 comparedwith sham). Treatments of PHA-543613 (PHA-12 mg) orPNU-282987 (PNU-12 mg) improved the forelimb placing

Figure 1. Statistical analysis of behav-ioral outcome (A) and brain edema (B) at24 hours after intracerebral hemorrhageinduction or sham surgery. Data areexpressed as mean�SEM. *P�0.05compared with sham, †P�0.05 com-pared with vehicle, ‡P�0.05 comparedwith PHA, 12 mg, §P�0.05 comparedwith PNU, 12 mg; n�6 in each group.Garcia indicates Garcia test; CTT, cornerturn test; FLP, forelimb placing test; con-tra, contralateral; ipsi, ipsilateral; and NS,not significant.

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ability and increased the number of spontaneous alterations inICH animals (P�0.05 compared with vehicle). In contrast,mice receiving the �7nAChR agonist PHA-543613 (12 mg/kg) combined with MLA (PHA�MLA) or wortmannin(PHA�Wort) showed no significant differences comparedwith the respective vehicle group (P�0.05).

Brain edema was evaluated at 24 hours after surgery (n�6per group). Treated mice (PHA-12 mg) showed a signifi-cantly reduced brain water content in the ipsilateral basalganglia (P�0.05, compared with vehicle, Figure 1B); how-ever, coadministration of MLA (PHA�MLA) or wortmannin(PHA�Wort) reversed this effect entirely (P�0.05 comparedwith PHA-12 mg). The latter compounds did not increasebrain edema compared with the vehicle (P�0.05). No signif-icant differences were evident between all groups, in con-tralateral and ipsilateral cortex, contralateral basal ganglia, orin the cerebellum (P�0.05).

PHA-543613 Attenuates Behavioral Deficits andBrain Edema at 72 Hours After ICHSham-operated and vehicle and treated (PHA-12 mg) animals(n�6 per group) were used to evaluate behavioral outcomesand brain edema at 72 hours after surgery (Figure 2).PHA-543613 treatment significantly improved performancesof Garcia, corner turn, forelimb placing, and T-maze tests ascompared with the vehicle group (P�0.05).

Brain water content of the ipsilateral basal ganglia wassignificantly higher in the vehicle group than in sham animals(P�0.05, Figure 2B), and PHA-543613 treatment (PHA-12 mg) reduced the amount of brain edema significantly(P�0.05, compared with vehicle). No significant differenceswere evident between all groups, in contralateral and ipsilat-eral cortex, contralateral basal ganglia, or in the cerebellum(P�0.05).

PHA-543613 Activates the PI3K-Akt SignalingPathway and Decreases Protein Expressions ofp-GSK-3� and CC-3 at 24 Hours After ICHTo further confirm that PHA-543613–mediated neuroprotec-tion is dependent on the antiapoptotic PI3K-Akt signalingpathway, Western blot analysis was conducted and thechanges in protein levels of phosphorylated Akt (p-Akt,Ser473), phosphorylated GSK-3� (p-GSK-3�, Tyr216), andCC3 were quantified. The treatment (PHA-12 mg) increasedphosphorylation of Akt significantly, compared with both thesham and the vehicle group (P�0.05, n�5 per group, Figure3A). MLA and wortmannin (PHA�MLA, PHA�Wort) sig-nificantly decreased the level of p-Akt at 24 hours after ICH(P�0.05, compared with PHA-12 mg). GSK-3� phosphory-lation was significantly reduced by PHA-543613 (P�0.05,compared with vehicle, Figure 3B); however, MLA andwortmannin reversed this effect entirely. PHA-12 mg, brainspecimens contained significantly decreased quantities ofCC3 (P�0.05, compared with vehicle, Figure 1C), andwortmannin (PHA�Wort) abolished the antiapoptotic effectof the treatment (P�0.05, compared with PHA-12 mg).

PHA-543613 Reduces Neuronal Apoptosis at 24Hours After ICHImmunofluorescence staining and neuronal cell death quan-tification was performed in sham, vehicle, and PHA-12 mg,

animals (n�4 per group). All mice were euthanized at 24hours after ICH or sham surgery, and immunoreactivities ofNeuN, p-GSK-3�, TUNEL, and CC3 were evaluated in theperihematomal area or near the needle tract in sham animals.Consistent with the Western blot results, the immunofluores-cence analysis revealed decreased expressions of p-GSK-3�(Figure 4A) and CC3 (Figure 4B) in the treatment group(PHA-12 mg). TUNEL-positive cells (Figure 4A) were lessapparent after PHA-543613 treatment. Immunoreactivities ofp-GSK-3�, TUNEL, and CC3 were generally colocalizedwithin NeuN-positive cells (neurons).

The total number of TUNEL and NeuN double-stainedcells (TUNEL�neurons) in the perihematomal area wassignificantly decreased after PHA-543613 treatment com-pared with the vehicle group at 24 hours after ICH (P�0.05,Figure 4C).

DiscussionThe first aim of the present study was to investigate whetherthe �7nAChR agonists PHA-543613 and PNU-282987 ame-liorate behavioral deficits and brain edema after experimentalICH in mice. Hemispheric hemorrhage, affecting the basal

Figure 2. Statistical analysis of behavioral outcome (A) andbrain edema (B) at 72 hours after intracerebral hemorrhageinduction or sham surgery. Data are expressed as mean�SEM.*P�0.05 compared with sham, †P�0.05 compared with vehicle;n�6 in each group. Garcia indicates Garcia test; CTT, cornerturn test; FLP, forelimb placing test; contra, contralateral; andipsi, ipsilateral.




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ganglia, has been reported to cause debilitating sensorimotoras well as cognitive deficits in humans.22,23 Intending toachieve a translational perspective, we included sensorimotor(Garcia, corner turn, and forelimb placing test) and cognitive(T-maze) assessments in our experiment. The compositeGarcia test sensitively detects sensorimotor consequences ofunilateral hemorrhagic or ischemic brain injury.8,18 Both thecorner turn and the forelimb placing test have been widelyused to evaluate lateralizing behaviors as well as sensorimo-tor impairments in preclinical ICH studies.19 During theT-maze assessment, rodents normally select alternating armson consecutive trials (spontaneous alterations).20 A decreasednumber of those spontaneous alterations correlates well withthe severity of the cognitive impairment in injured animals.24

High-dose treatment (12 mg/kg) of PHA-543613 or PNU-282987 significantly improved the outcome of all conductedbehavioral tests, with the exception of the corner turn testperformance at 24 hours after surgery (compared with vehi-cle). Even mild unilateral brain injuries may cause a lateral-izing behavior in rodents, and the outcome of the corner turntest after experimental ICH was previously reported toimprove to a lesser extent than the outcome of the forelimbplacing test.19 Brain edema, defined as an increase in thewater content of brain tissue, is observed in acute and delayedstages after ICH.25 Several studies suggest a close associationbetween the degree of perihematomal brain edema and pooroutcome in patients.25,26 Our results showed significantlyreduced brain water content of the ipsilateral basal ganglia inthe PHA-12 mg, group, at 24 and 72 hours after ICHinduction (compared with vehicle). The aforementioned find-ings support our first hypothesis that the �7nAChR agonistsPHA-543613 and PNU-282987 ameliorate behavioral andmorphological outcomes (brain edema) after ICH in mice.PHA-543613, in a concentration of 12 mg/kg, ameliorated thebrain injury to a greater extent than did 4 mg/kg of PHA-543613 and 12 mg/kg of PNU-282987 (Figure 1) and wastherefore exclusively applied in the following experiments(Figures 2, 3, and 4). We previously reported that short-termactivation of the �7nAchR through PNU-282987 did not alterphysiological parameters (blood pressure, heart rate) orplasma concentrations of Na�, K�, Cl�, and glucose inrodents.8 The observed amelioration of the brain injury in thetreatment groups (PHA-4 mg, PHA-12 mg, and PNU-12 mg)were unlikely to be caused by changes of physiologicalparameters.

Aiming to further examine the dependence between�7nAChR-induced neuroprotection and the PI3K-Akt signal-ing pathway, we administered either MLA or wortmanninbefore PHA-543613 (12 mg/kg).

MLA is a potent competitor, interfering with the[125I] �-bungarotoxin binding site of the �7nAChR(Ki�1.4 nm).27,28 MLA is quickly redistributed from thevasculature to the brain, where it extensively inhibits thereceptor activation by PHA-543613 (Ki�8.8 nm).17,27,28

Wortmannin inhibits PI3K irreversibly and is commonly usedto prevent downstream phosphorylation of Akt.8 Animalsreceiving these interventions in addition to the treatment didnot show functional or morphological improvements at 24hours after surgery. Furthermore, MLA or wortmannin ad-

Figure 3. Representative Western blots and densitometricquantification of p-Akt/Akt (A), p-GSK-3�/GSK-3� (B), andCC3/�-Actin (C) at 24 hours after intracerebral hemorrhageinduction or sham surgery. Data in bar graphs are expressedas mean�SEM. *P�0.05 compared with sham, †P�0.05compared with vehicle, ‡P�0.05 compared with PHA, 12 mg;n�5 in each group.




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ministered alone did not worsen the injury compared with thevehicle. These results indicate that �7nAChR agonists exerttheir neuroprotective effects through the PI3K-Akt signalingpathway. Consistent with these findings, earlier studies dem-onstrated the ability of �7nAChR agonists to trigger PI3Kactivation, possibly through stimulation of the receptor’scatalytic intracellular domain; however, the exact mechanismof this process remains yet to be discovered.7,29 Akt, aserine/threonine kinase, which is directly activated by PI3K-mediated phosphorylation, stimulates several antiapoptoticmechanisms, among them the inhibition of GSK-3�.10 Be-longing also to the serine/threonine kinase family, GSK-3�has been described to activate proapoptotic caspase-3,13 thusaggravating neuronal injury after experimental ischemicstroke11 and subarachnoid hemorrhage.14

In the present study, we additionally intended to show theprotective effect of GSK-3� inhibition. PHA-543613 treat-ment significantly increased the protein expression of acti-vated Akt (p-Akt, Ser473) in the ipsilateral hemisphere at 24hours after surgery, which successively reduced the expres-sion of activated GSK-3� (p-GSK-3�, Tyr216) and CC3.MLA or wortmannin, in combination with PHA-543613,showed similar levels of the respective proteins as the vehiclegroup. These results support the proposed antiapoptotic effectof �7nAChR agonism through PI3K-Akt activation andresulting GSK-3�-inhibition.

Given the fact that �7nAChR are not only expressed inneurons but also in neuroglia and endothelial cells of themammalian brain,6 we used immunofluorescence staining toshow colocalizations of p-GSK-3� and CC3 with NeuN(neuronal marker). Both target proteins were generally local-ized in neurons, and PHA-543613 treatment reduced theintensity of p-GSK-3� and CC3 immunofluorescence in theperihematomal area. We furthermore used TUNEL to detectapoptotic cell death and observed a significantly reduced

density of apoptotic neurons in the treatment group (com-pared with vehicle). Added together, these findings supportour second hypothesis, suggesting that �7nAChR-stimmulation reduces activated GSK-3� through the PI3K-Akt signaling pathway, thus decreasing the incidence ofneuronal apoptosis after ICH in mice.

In the present study, we investigated the antiapoptoticeffects of �7nAChR; however, previously reported anti-inflammatory properties of this receptor subtype may alsoparticipate in neuroprotection after hemorrhagic stroke.30

Inflammatory mechanisms, mediated by cellular componentssuch as resident immune cells of the brain (microglia), areinvolved in the progression of the ICH-induced brain injury.31

Activation of �7nAChR in microglial cells inhibits theproduction of proinflammatory cytokines but not the produc-tion of anti-inflammatory cytokines, thereby reducing thelocal inflammatory response.30 These anti-inflammatory ef-fects of �7nAChR may additionally protect the blood-brainbarrier integrity after experimental ICH. Inhibition ofGSK-3� will also increase the expression of its downstreamprotein �-catenin, an important component of the blood-brainbarrier.32,33 Furthermore, �7nAChR stimulation resulted inenhanced cognition and attention in animals as well ashumans, and �7nAChR agonists have been represented aspromising treatments for schizophrenia and Alzheimerdisease.6,9,17,27

In conclusion, the present study found that �7nAChRstimulation improved functional and morphological outcomesafter experimental ICH in mice. We demonstrated thatPHA-543613 treatment reduced the expression of proapoptot-ic GSK-3� through the PI3K-Akt signaling pathway, and thepharmacological reversal thereof obliterated this effect. Ad-ditional preclinical studies are needed to investigate potentialanti-inflammatory effects of �7nAChR agonists, and it is alsoessential to elucidate further mechanisms on how �7nAChR

Figure 4. A, Representative microphoto-graphs of immunofluorescence stainingshowing colocalization of NeuN (DAPI/blue), p-GSK-3� (Texas red/red), andTUNEL (FITC/green) at 24 hours aftersurgery. PHA-543613 treatmentdecreased perihematomal immunoreac-tivities of p-GSK-3� and TUNEL. B, Rep-resentative microphotographs showingcolocalization of NeuN (DAPI/blue) andCC3 (Texas Red/red) at 24 hours aftersurgery. PHA-543613 treatmentdecreased perihematomal immunoreac-tivity of CC3. C, Neuronal cell deathquantification. PHA-543613 treatmentdecreased the number of TUNEL-positive neurons in the perihematomalarea at 24 hours after surgery. Data inbar graphs are expressed asmean�SEM. *P�0.05 compared withsham, †P�0.05 compared with vehicle,‡P�0.05 compared with PHA, 12 mg;n�4 in each group.




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stimulation reduces brain edema and neuronal apoptosis afterexperimental ICH, as shown in the present study.

Sources of FundingThis study was supported by National Institutes of Health grantRO1NS053407 (J.H.Z.).

DisclosuresNone.

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H. ZhangPaul R. Krafft, Orhan Altay, William B. Rolland, Kamil Duris, Tim Lekic, Jiping Tang and John

Inhibition in a Mouse Model of Intracerebral Hemorrhageβ7 Nicotinic Acetylcholine Receptor Agonism Confers Neuroprotection Through GSK-3α

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