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Intravenous Brain-Derived Neurotrophic Factor EnhancesPoststroke Sensorimotor Recovery and
Stimulates NeurogenesisWolf-Rudiger Schabitz, MD; Tobias Steigleder, MD; Christiana M. Cooper-Kuhn, PhD;
Stefan Schwab, MD; Clemens Sommer, MD; Armin Schneider, MD; H. Georg Kuhn, PhD
Background and Purpose—The discovery of spontaneous neuronal replacement in the adult brain has shifted experimentalstroke therapies toward a combined approach of preventing neuronal cell death and inducing neuronal plasticity.Brain-derived neurotrophic factor (BDNF) was shown to induce antiapoptotic mechanisms after stroke and to reduceinfarct size and secondary neuronal cell death. Moreover, in intact animals, BDNF is a potent stimulator of adultneurogenesis.
Methods—The current study analyzed the effects of BDNF on induction of neuronal progenitor cell migration andsensorimotor recovery after cortical photothrombotic stroke.
Results—Daily intravenous bolus applications of BDNF during the first 5 days after stroke resulted in significantlyimproved sensorimotor scores up to 6 weeks. At the structural level, BDNF significantly increased neurogenesis in thedentate gyrus and enhanced migration of subventricular zone progenitor cells to the nearby striatum of the ischemichemisphere. BDNF treatment could not, however, further stimulate progenitor cell recruitment to the cortex.
Conclusions—These findings consolidate the role of BDNF as a modulator of neurogenesis in the brain and as an enhancerof long-term functional neurological outcome after cerebral ischemia. (Stroke. 2007;38:2165-2172.)
Neurotrophic factors control intercellular and intracellularsignaling pathways that sculpt neuronal circuits during
brain development and fundamentally regulate plasticity aswell as cell survival in the adult brain.1 One of the prominentfactors and, within the central nervous system, one of themost widely distributed factors is brain-derived neurotrophicfactor (BDNF), which acts specifically via a high-affinity cellsurface receptor (TRKB). By activating intracellular proteinkinase B (ie, Akt), mitogen-activated protein kinases, and theextracellular signal–regulated kinases, BDNF effectively pre-vents neuronal cell death after various traumatic events suchas cerebral ischemia.2 BDNF also crucially promotes synapticand axonal plasticity associated with learning, memory, andsensorimotor recovery.3,4
In addition to qualitatively enhancing neural structuralplasticity, BDNF was shown to increase the number ofnewborn neurons in several brain areas. However, BDNF-induced neurogenesis in vivo was observed only in unle-sioned healthy animals.5–8 After global ischemia, blockade ofendogenously occurring BDNF as well as exogenous delivery
of BDNF counteracted neuronal differentiation in the affecteddentate gyrus.9,10 In the present study, we explored the effectof peripheral BDNF application on neurogenesis in a focalcortical ischemia model. We assessed functional sensorimo-tor outcome as well as the generation and migration ofneuronal progenitors in the cortex, striatum, andhippocampus.
Materials and MethodsPhotothrombotic Ischemia ModelAll animal experiments were performed in accordance with nationaland international regulations and were approved by governmentauthorities. Male Wistar rats weighing 280 to 320 g were anesthe-tized with ketamine hydrochloride (100 mg/kg body weight IM;Ketanest) and xylazine hydrochloride (8 mg/kg body weight IM;Rompun). A PE-50 tube was inserted into the right femoral artery formonitoring mean arterial blood pressure and blood gases. The rightfemoral vein was cannulated with a PE-50 tube for treatmentinfusion. During the experiment, rectal temperature was maintainedat 37°C by a thermostatically controlled heating pad (Fohr MedicalInstruments). Photothrombotic ischemia was induced in the rat
Received November 7, 2006; accepted January 19, 2007.From the Department of Neurology (W.-R.S.), University of Munster, Germany; the Department of Neurology (T.S., S.S.), University of Erlangen,
Germany; the Department of Neuropathology (C.S.), University of Mainz, Germany; Sygnis Pharma (A.S.), Heidelberg, Germany; and the Institute forNeuroscience and Physiology (C.M.C.-K., H.G.K.), Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden.
W.R.S., T.S., and S.S. were formerly affiliated with the Department of Neurology, University of Heidelberg, Germany.Correspondence to W.-R. Schabitz, Neurologische Klinik, Universitatsklinikum Munster, Albert-Schweitzer-Strasse 33, 48149 Munster, Germany.
parietal cortex (right side) according to the method of Watson et al.11
In brief, after injection of Bengal rose (0.133 mL/kg body weightIV), the skull was illuminated with a cold, white light beam (150 W)for 20 minutes at 4 mm posterior to and lateral from the bregma.Sham-operated animals received vehicle infusion and were notilluminated.
Before surgery, animals were assigned in a fully randomized andblinded fashion to the following groups: ischemia�vehicle (n�10),sham�vehicle (n�7), ischemia�BDNF (n�10), or sham�BDNF(n�7). The randomization code was broken after all data wereacquired, including those from morphological analysis. Animalswere treated with 20 �g BDNF IV or vehicle 1 hour after inductionof ischemia. Treatment was repeated on days 2 to 5. Four hours aftertreatment, dividing cells were labeled with bromodeoxyuridine(BrdU; 50 mg � kg�1 � d�1 IP). Animals were perfused 37 days afterthe last injection, and 6 animals per group were randomly chosen forimmunohistochemistry.
Sensorimotor MeasurementsIn all animals, sensorimotor tests were performed during the lightcycle 1 to 3 days before ischemia after a training period of 3 days andat 2, 3, 4, 5, and 6 weeks after ischemia by an investigator blindedto the experimental groups.3 Tests such as the Rotorod, adhesive taperemoval, and beam balance were done as described in detail.3 Byusing the previously described Neurological Severity Score (NSS),3
neurological function was graded on a scale of 0 to 16 (normalscore�0, maximal deficit score�16). The NSS is a composite ofmotor, sensory, and reflex tests, including beam balance.
Neurogenesis Detection In VivoNeuronal progenitor cells and new neurons were visualized byimmunofluorescence against double cortin and by double labeling ofBrdU with the mature neuronal marker NeuN. Antibodies used wereas follows: rat anti-BrdU (1:500, Accurate); mouse anti-NeuN(1:500, Chemicon); goat anti–double cortin (DCX) C-18 (1:500,Santa Cruz); and anti-rat fluorescein isothiocyanate, anti-goat rho-damine X, and anti-mouse CY5 (Jackson ImmunoResearch). Free-floating sections were treated with 0.6% H2O2 in Tris-buffered saline(TBS�0.15 mol/L NaCl, 0.1 mol/L Tris-HCl; pH 7.5) for 30minutes. BrdU-labeled nuclei were detected by DNA denaturationsteps after a 2-hour incubation in 50% formamide/2� standard salinecitrate (2� standard saline citrate is 0.3 mol/L NaCl and 0.03 mol/Lsodium citrate) at 65°C, a 5-minute rinse in 2� standard salinecitrate, a 30-minute incubation in 2 mol/L HCl at 37°C, and a10-minute rinse in 0.1 mol/L boric acid, pH 8.5. Incubation inTBS/0.1% Triton X-100/3% normal donkey serum for 30 minuteswas followed by incubation with primary antibodies (48 hours, 4°C).After being washed in TBS/0.1% Triton X-100/3% normal donkeyserum, sections were incubated with secondary antibodies for 2hours, washed in TBS, and mounted on glass slides. Fluorescencewas detected by confocal scanning laser microscopy (Leica).
Histological QuantificationA systematic random-counting procedure was used as previouslydescribed.12 The dentate gyrus was analyzed on a series of every 10thsection (400-�m interval), which yielded �6 or 7 sections peranimal spanning the hippocampus �2.4 to �6.48 mm from thebregma.13 All BrdU-positive cells in the granule cell layer werecounted. For colabeling with NeuN, 100 randomly selected BrdU-positive cells per animal were analyzed. Multiplying the totalnumber of BrdU-positive cells by the percentage of NeuN/BrdUdouble-positive cells yielded the number of new neurons in thedentate gyrus. Because the migratory path of progenitor cells fromthe lateral ventricle wall to the lesion site was limited to a fewsections, we selected 2 sections per animal (400 �m apart) forsystematic analysis of the cortical area between the subventricularzone (SVZ) and the lesion site, the corpus callosum, and theunderlying striatum. The tissue was systematically analyzed bycounting the cells in adjacent fields of view (25 fields total, 5�5,
250�250 �m) starting adjacent to the lesion for the cortex andadjacent to the ventricle wall for the striatum.
Data and Statistical AnalysisValues are presented as mean�SEM. Thionin/basic fuchsin-stainedslices (400-�m interval) were obtained from the infarcted region.The slice with the largest length, diameter, and height was used forinfarct volume calculation (see supplemental Figure I, availableonline at http://stroke.ahajournals.org). Infarct volumes were calcu-lated from the equation for ellipsoids: 4/3�r1r2r3, where r1 is 1/2 thelength, r2 is 1/2 the diameter, and r3 is 1/2 the height.3 A 2-tailed t testwas used to determine significant differences in infarct volumes.Sensorimotor measurements were analyzed with a 1-way ANOVA orKruskal-Wallis test for each time point. The area under the curve(AUC) was analyzed by 1-way ANOVA and a post hoc Duncan’stest. One-way ANOVA and post hoc Tukey’s test were used todetermine the statistical significance of differences in physiologicalparameters and neurogenesis. Statistical calculations and analyseswere done with NCSS statistical software and Prism 4 (GraphpadInc). A probability value �0.05 was considered significant.
Results
Peripheral Application of BDNF ImprovesFunctional Outcome After Cerebral IschemiaSystemic BDNF treatment (20 �g IV for 5 postischemicdays) in the present study was tailored to enhance poststrokerecovery without affecting infarct size,2 and indeed, infarctvolumes between BDNF-treated animals and controls werenot statistically different (27.7�9.5 versus 37.8�9.4 mm3;see supplemental Figure I). Physiological parameters (rectaltemperature, pH, PCO2, PO2, and mean arterial pressure duringsurgery; see supplemental Table I, available online at http://stroke.ahajournals.org) as well as body weight and mortality(no animals died during the experiment) were not differentbetween the groups.
Sensorimotor deficits were statistically significant invehicle-treated ischemic animals (lesion�vehicle) comparedwith sham-operated rats (sham�vehicle) in the Rotorod test,adhesive tape removal test, and NSS for up to 6 weeks afterthe insult (Figure 1, a–d; compare yellow versus blue).BDNF-treated ischemic rats (lesion�BDNF) performed sig-nificantly better than did vehicle-treated animals in theoverall NSS as well as in the adhesive tape removal test bycomparing group means per time point (Figure 1, b–d, greenversus yellow curves; P�0.05 by 1-way ANOVA, Kruskal-Wallis test). By analyzing the even more appropriate timeseries measurements in subjects (comparison of individualAUCs over time; see Figure 1, a–d, bar graphs), the datademonstrated a robust improvement with BDNF treatmentversus vehicle in all test paradigms, including the Rotorodtest (Figure 1, a–d, green versus yellow bars; P�0.01, AUCand Duncan’s test). Interestingly, the treatment effect wasmost pronounced in the adhesive tape removal paradigm onthe affected, ie, paretic, left forepaw, consisting of a substan-tial sensory component in contrast to the motor function–focused NSS (compare Figures 1c versus 1b; P�0.0001,AUC and Duncan’s test). No difference was noted betweensham�vehicle and sham�BDNF treatment (data not shown).
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Cortical Ischemia Stimulates Recruitment ofNeuronal Progenitor Cells From the SVZ Towardthe Lesion Site, but BDNF Fails to Increase SVZProgenitor Cell Recruitment to the CorticalLesion SiteIn intact animals, migration of neuronal progenitor cellsoriginating in the SVZ was restricted to chain migrationtoward the olfactory bulb (Figure 2a), with few cells oriented
toward the cortex. In the corpus callosum of sham�vehicleanimals, we very rarely detected DCX-positive cells (Figure2b) and no BrdU/NeuN double-positive cells. We observed atrend toward more DCX-positive cells passing through thecorpus callosum in lesion�vehicle and lesion�BDNF ani-mals, not significant due to the high intragroup variability(Figure 2b; ANOVA F(3,20)�0.87, P�0.47). In ischemicanimals, the cortical area between the lesion site and the
Figure 1. BDNF treatment improves long-term functional outcome after cortical ischemia. a, BDNF-treated ischemic animals had betterrunning function compared with controls (green bar vs yellow bar). b, BDNF-treated ischemic animals (green bar and line) displayedbetter motor performance (NSS including beam balance) compared with controls (yellow bar and line). c and d, BDNF-treated ischemicanimals (green bar and line) displayed better sensorimotor function (adhesive tape removal test) compared with controls (yellow bar andline). Line graphs are group comparisons over time (1-way ANOVA, post hoc Kruskal-Wallis test, *P�0.05). Bar graphs are comparisonfor each rat over time per group (AUC, post hoc Duncan’s test, *P�0.01). Yellow indicates ischemia�vehicle; green, ischemia�BDNF;and blue, sham-lesion�vehicle.
Figure 2. Cortical ischemia stimulatesrecruitment of neuronal progenitor cellsfrom the SVZ toward the lesion. a, DCX-positive cells leave the rostral migratorystream (rms) to penetrate the corpus cal-losum (cc) toward the overlying neocor-tex. Green indicates DCX; red , NeuN.Scale bar in a�25 �m. b, Quantificationof DCX-positive cells. Cell counts repre-sent 1000 cells/�m2, mean�SEM.
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ventricle wall displayed a substantial amount of DCX-positive progenitors (Figure 3a), whereas no DCX-positivecells were present in the corresponding cortical region ofsham�vehicle or sham�BDNF animals (data not shown).The area and intensity of DCX immunoreactivity inlesion�BDNF animals were comparable to those inlesion�vehicle animals (Figures 3c and 3d; t test t�0.4135,df�10, P�0.68 for area and t�0.3566, df�10, P�0.73 forintensity). Using confocal microscopy and 3-dimensionalanalysis, we found no incorporation of BrdU into NeuN-expressing cells. Instead, BrdU-positive nuclei were fre-quently detected in very close proximity but were distinctlydifferent from neuronal cell bodies, indicative of newlyformed satellite cells (Figure 4d). As an indicator ofbeginning neuronal differentiation of DCX-positive cells,we frequently detected the coexpression of DCX and NeuNin cells surrounding the lesion site and in the striatum(Figure 4, a– e).
BDNF Increases SVZ Progenitor Cell MigrationInto the Ipsilateral Striatum AfterCortical StrokeA significant increase in the density of BrdU-positive cells inthe striatum of lesion�vehicle and lesion�BDNF animalscompared with sham-lesioned animals (Figure 5c; ANOVAP�0.001, F(3,20)�8.5, Tukey’s post hoc test: sham�vehicleversus lesion�vehicle q�5.8, P�0.01; sham�vehicle versus
lesion�BDNF q�4.1, P�0.05; sham�BDNF versus lesion�vehicle q�5.7, P�0.01; sham�BDNF versuslesion�BDNF q�4.0, P�0.05) was observed, but nodifference between lesion�vehicle and lesion�BDNF wasfound. More important, however, the number of DCX-positive neuronal progenitor cells in the striatum wasincreased only in lesion�BDNF animals (Figure 5d,ANOVA P�0.001, F(3,20)�8.5, Tukey’s post hoc test:sham�vehicle versus lesion�BDNF q�6.6, P�0.001;sham�BDNF versus lesion�BDNF q�5.7, P�0.01;lesion�vehicle versus lesion�BDNF q�7.2, P�0.001; allother comparisons P�NS). Similar to the situation in thecortex, we observed colabeling of DCX with NeuN in thestriatum, indicating that neuronal progenitor cells began toexpress mature neuronal markers at 6 weeks after lesioninduction (Figure 4e). Although present, BrdU/NeuNdouble-positive cells were so infrequently detectedthat quantification of this observation was not possiblebecause of low counts (total of 8 BrdU/NeuN cells in allanimals).
Hippocampal Neurogenesis Is Increased inBDNF-Treated Animals After Cortical StrokeFirst we analyzed the total number of BrdU-positive cells inthe hippocampus, which represents all new cells includingneurons, astrocytes, oligodendrocytes, microglia, and macro-phages. Animals subjected to cortical photothrombosis had an
Figure 3. Progenitor cell recruitmenttoward the ischemic lesion. a and b, Thearea between the lateral ventricle andlesion displays a substantial number ofDCX-positive neuronal progenitors(green). Green indicates DCX; red, NeuN;LV, lateral ventricle; and Str, striatum.Scale bar in b�100 �m. c and d, Quanti-fication of DCX immunoreactivity: tracingthe area covered by DCX immunoreactiv-ity in 1000 �m2, mean�SEM (c) and im-munofluorescence intensities as meangray values �SEM (d).
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increased number of BrdU-positive cells in the hippocampusof the infarcted side (Figure 6d; ANOVA P�0.001,F(3,20)�9.7, Tukey’s post hoc test: sham�vehicle versuslesion�vehicle q�5.8, P�0.01). However, there was nostatistically significant increase in the number of new cellsdue to BDNF treatment under control or lesion conditions
(Figure 6d; ANOVA P�0.001, F(3,20)�9.7, Tukey’s posthoc test: sham�vehicle versus sham�BDNF q�2.38,P�NS and lesion�vehicle versus lesion�BDNF q�0.83,P�NS).
We determined the neuronal fraction among the newcells (BrdU/NeuN double-positive) by confocal micros-
Figure 4. a–c, Coexpression of DCX(green) and NeuN (red) in cells surround-ing the lesion. b and c, Color separationof a; arrows indicate double-positivecells. d, Maximum projection of a confo-cal image stack depicting DCX/NeuNdouble-positive cells (white arrow) andBrdU-positive satellite cells (blackarrows) in the striatum. e, xyz reconstruc-tion and color separation of the confocalimage stack in d. Image stack represents12.5-�m depth and consists of 25images with 0.5-�m-thick optical slicesand a pinhole size of airy 1.0 for a 63�oil objective. The hairline crosses in eshow the reconstruction position of x-y,y-z, and x-z. Scale bar in a�25 �m; in dand e�20 �m.
Figure 5. BDNF increases neuronal progenitor cells in the striatum. a and b, Confocal images of BrdU, DCX immunolabeling in the stri-atum of ischemia�vehicle and ischemia�BDNF animals. BrdU in red, DCX in green, white arrows. Scale bar in a�50 �m. c, Quantifica-tion of BrdU-positive cells. d, Quantification of DCX-positive cells. Data are presented as mean cells/mm3 striatal tissue �SEM.
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copy. Compared with all other groups, only thelesion�vehicle animals had a significantly smaller fractionof neurons (38% for lesion�vehicle versus 78% forsham�vehicle, 67% for sham�BDNF, and 64% forlesion�BDNF; ANOVA F(3,20)�11.9, Tukey’s post hoc test:sham�vehicle versus lesion�vehicle q�7.5, P�0.001;sham�BDNF versus lesion�vehicle q�7.0, P�0.01;lesion�vehicle versus lesion�BDNF q�5.2, P�0.01; all othercomparisons P�NS).
To compare whether the total amount of hippocampalneurogenesis was altered, the number of BrdU-positive cellswas multiplied by the percentage of NeuN/BrdU double-positive cells and expressed as the total number of newneurons per dentate gyrus (Figure 6e). BDNF treatment afterlesion induction significantly increased hippocampal neuro-genesis compared with all other groups (Figure 6e; ANOVAF(3,20)�6.98, P�0.002; Tukey’s post hoc sham�vehicle ver-sus lesion�BDNF q�6.3, P�0.01; sham�BDNF versuslesion�BDNF q�4.3, P�0.05; lesion�vehicle versuslesion�BDNF q�4.1, P�0.05; all other comparisonsP�NS). However, ischemia alone or BDNF alone wasinsufficient to increase the level of neurogenesis abovecontrol levels (Figure 6e).
DiscussionThe treatment paradigm and dose of BDNF in the presentstudy were chosen on the basis of previous evidence, with theidea of allowing brain penetration by the growth factor aftersystemic application, leading to enhanced poststroke recoverywithout changing stroke size. Therefore, BDNF was admin-istered as a low-dose intravenous infusion (20-�g repetitivebolus on 5 consecutive days) that should exert no protective
effects.2,3 This procedure indeed left infarct volumes ofBDNF-treated animals and controls unchanged, whereaspoststroke sensorimotor function was enhanced. Sensorimo-tor function gradually improved from weeks 3 to 6 in theRotorod test (running function), the NSS (walking andbalancing function, reflexes) as well as in the adhesive taperemoval test (sensory awareness of the hand, grip function).These results clearly suggest a true recovery-enhancing effectfor BDNF.
Blood-brain barrier (BBB) penetration by BDNF wasbeyond the scope of the present study, because passage ofBDNF through the intact BBB is a well-characterized phe-nomenon.14,15 A much smaller dose of intravenously injected125I-BDNF (2 ng compared with 20 �g as single bolus in ourstudy) had an early and rapid influx into the brain. Most ofthis BDNF was later (10 minutes) associated with the brainparenchyma of the cortex, demonstrating complete passageacross the BBB.14 BDNF influx into the brain is likely evenfurther augmented by the photothrombotic stroke model,which leads to rapid disruption of the BBB that persists fordays.16
When analyzing the distribution of DCX-expressing pro-genitor cells in the ventricle wall and the overlying corpuscallosum and cortex, we found robust recruitment of progen-itor cells into the peri-ischemic area of the neocortex. Thisobservation is in accordance with other studies describing themigration of neuronal progenitor cells from the lateral ven-tricle wall in response to a cortical lesion.17,18 BDNF treat-ment, however, failed to further increase the recruitment ofDCX-positive cells into the ischemic cortex. Although a largenumber of DCX-positive cells were detected in the immediateperiphery of the ischemic lesion, the colabeling of BrdU with
Figure 6. BDNF treatment increasesneurogenesis in the dentate gyrus of thehippocampus. a–c, Confocal image ofBrdU/NeuN double labeling in the den-tate gyrus. Identification of newborn neu-rons (white arrows) by color separation(b and c) to confirm the overlap of theBrdU-positive signal (green) and theNeuN signal (red). d, Quantification ofBrdU-positive cells and NeuN/BrdUdouble-labeled cells (e). Data are pre-sented as mean cells per dentate gyrus�SEM. Scale bar in a�50 �m.
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a marker for mature neurons (NeuN) analyzed by confocalmicroscopy did not indicate differentiation of progenitor cellsinto cortical neurons. Induction of neurogenesis in the le-sioned cortex was reported only by Magavi and colleagues,19
who used a very selective apoptotic elimination model ofindividual cortical neurons. Among the possible reasons forour inability to detect cortical neurogenesis are (1) the lesionmodel, because Arvidsson and colleagues,20 using the middlecerebral artery occlusion stroke model, were also unable todetect cortical neurogenesis, and (2) the possibility that theventricle wall produces new neurons for the cortex not duringthe first 5 days after lesion but rather at a later time. However,we frequently observed that DCX-positive cells were cola-beled with NeuN (Figure 4), a characteristic that indicatesthat these cells progressed from a migrating to a differenti-ating progenitor cell stage. Whether these cells fully differ-entiate into neurons requires additional experiments with adelayed BrdU labeling paradigm.
In contrast to the neocortex, neurogenesis in the striatumis a well-described though relatively rare phenomenon.20
As shown here, peripheral delivery of BDNF increasedSVZ progenitor cell migration to the ipsilateral striatumafter cortical stroke, which corresponds to previous find-ings that intrathecal infusion or viral vector–mediateddelivery of BDNF increased the number of striatal progen-itor cells.5,7,21 Our lesion paradigm produced no BrdU/NeuN-positive cells in the striatum, although we observedDCX/NeuN double labeling.
A prominent effect of BDNF on neurogenesis was seen inthe dentate gyrus. Though distant from the site of stroke, thisregion typically responds to ischemia with induction ofneurogenesis through glutamatergic stimulation. Thisexcitation-neurogenesis coupling hypothesis suggests thatadult hippocampal neural stem cells/progenitor cells possesstheir activity-sensing capability by acting in part viaN-methyl-D-aspartate receptors to adapt the adult neuralnetwork both to physiological demands and to pathologicalinsults.22,23 In our study, BDNF, which is endogenouslyexpressed by granule cells of the hippocampus,24 significantlyincreased the number of newly generated granule cells inischemic brains. Both ischemia alone and combined ische-mia/BDNF treatment increased the number of newly gener-ated cells (Figure 6d); however, under ischemic conditions, asignificant reduction in the neuronal portion of the newlygenerated cells was observed, leading to only slightly ele-vated hippocampal neurogenesis levels after cortical stroke(Figure 6e). BDNF, on the other hand, reversed this lesioneffect on neurogenesis by increasing the neuronal differenti-ation of new cells (Figure 6f). Direct intrahippocampaldelivery of high doses of BDNF (12 or 36 �g/d), however,has been reported to stimulate the formation of newlygenerated cells in the unlesioned dentate gyrus.8 Thisfinding is supported by similar results in BDNF-deficientmice.25 In the present study, BDNF failed to increaseneurogenesis in the intact dentate gyrus of sham-lesionedand nonischemic animals, possibly due to the relativelylow (20 �g/d) and systemically (intravenous) administereddose of BDNF.
The question remains how BDNF-induced neurogenesis inthe dentate gyrus corresponds to improved sensorimotorbehavior after cortical ischemia. It is well known that BDNFinteracts significantly with acute stroke pathophysiology andhas particularly potent antiapoptotic functions. For example,a reduction in BDNF’s endogenous biological activity byreplacement of the coding sequence in a single allele of theBDNF gene resulted in markedly enlarged infarctions.26 Afterhypoxia and/or ischemia, BDNF was shown to induce theextracellular signal–regulated kinase signaling pathway, tocounteract proapoptotic caspase 3 and bax, and to upregulatethe antiapoptotic bcl-2.3,27 BDNF may, however, not unani-mously exert neuroprotective or antiapoptotic effects in thehypoxic and/or ischemic brain as observed in a cardiac arreststudy in rats.28 Although it cannot be excluded that BDNF-induced prevention of apoptosis may have improved post-stroke recovery, similar infarct sizes in BDNF-treated ani-mals and controls makes this primary mechanism quiteunlikely. More important in this context are BDNF’s well-known stimulatory effects on neuroplasticity at several levels,including axonal growth, dendritic branching, and synaptictransmission.3,29,30 BDNF treatment after stroke, for instance,enhances MAP1B expression as a marker of axonal growth inthe ischemic border zone and synaptophysin expression as amarker of synaptogenesis in the contralateral cortex.3 Thus,BDNF’s strong trophic activity after stroke likely contributedto enhancement of sensorimotor recovery, independent of thepossible involvement of hippocampal neurogenesis. Never-theless, there are also data supporting a possible role forhippocampal structures in functional recovery from motordeficits. The hippocampus produces a rhythmic slow-waveactivity, which is involved in sensorimotor integration andmovement initiation31,32 that can be disrupted by corticallesions.32 Preservation of sensorimotor integration after cor-tical stroke as measured here by the adhesive tape removaltest could therefore be related to hippocampal function.Indeed, BDNF’s treatment effects were particularly pro-nounced in this test paradigm (see Figures 1c and 1d). Apossible connection between the motor system and hip-pocampal neurogenesis is further supported by the findingthat voluntary exercise is a strong activator of neurogen-esis and hippocampal upregulation of BDNF in mice.33
However, the causal relation would be reversed: ie, im-proved motor function leads to more neurogenesis and notvice versa.
Previous reports as well as our current study are limited bythe fact that enhancement of poststroke neurogenesis is so farjust correlative to an improvement in sensorimotor andcognitive recovery. Currently unavailable experiments areneeded to specifically impair hippocampal neurogenesis todissociate neurogenesis effects from other structural plasticityeffects during poststroke functional recovery.
SummaryWe have demonstrated that systemic application of BDNFduring the first 5 days after cortical stroke induces hippocam-pal neurogenesis and improves functional recovery during a6-week period in several sensorimotor tasks. These findingsconsolidate the role of BDNF as a modulator of neurogenesis
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in the brain and as an enhancer of long-term functionalneurological outcome after cerebral ischemia. Our studycould influence poststroke recovery trials and calls for furtherstudies that specifically target trophic factor–enhanced post-stroke physical therapy.
AcknowledgmentsThe authors thank Prof Dr W. Pan, Pennington Biomedical ResearchCenter, Baton Rouge, La, for advice regarding BBB penetration ofBDNF and Stephan Hennes, Frank Malischewsky, and Margit Wolffor excellent technical assistance.
Sources of FundingH.G.K. was supported by the Volkswagen Stiftung, Vetenskapsrådet,LUA/ALF Goteborg, and Stroke-Riksforbundet. W.R.S. was sup-ported by BMBF “tissue engineering” 0312133.
DisclosuresNone.
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