Rehabilitative therapies differentially alter proliferation and survival of glial cell populations...

神经损伤与功能重建 ·2008年 3月 ·第 3卷 ·第 2期 1O9

· 《GLIA 》优秀论文推荐 ·

Rehabilitative Therapies Differentially Alter

Proliferation and Survival of Glial Cell Populations

in the Perilesional Zone of Cortical Infarcts

SILKE KEINER，FANNY W URM ，ALBRECHT KUNZE．

oTTo W ．W ITTE，AND CHRISToPH REDECKER

Department of Neurology，Friedrich—Schiller—University，Erlanger Allee 101，Jena，Germany

[Abstract] Rehabilitative therapies after stroke are designed to improve remodeling of neuronal circuits and to pmmote

functional recovery．Only very little is known about the underlying cellular mechanisms．In particular。the effects of rehabilita

tive training on glial cells，which play an important role in the pathophysiology of cerebral ischemia，are only poorly under—

stood．Here，we examined the effects of rehabilitative therapies on proliferation and survival of distinct glial populations in the

perilesional arP_~t of photochemieally induced focal ischemic infarcts in the forelimb sensorimotor cortex in rats． Immediatelv after

the infarct，one group of animals housed in standard cages received daily sessions of skilled reaching training of the impaired

forelimb；a second group was transferred tO an enriched environment，whereas a third control group rema ined in standard cages

without further treatment． Functional recovery was assessed in a sensorimotor walking task．To label proliferating cells，bro—

modeoxyuridine(BrdU)was administered from day 2 until day 6 postinfarct．Proliferation and survival of astrocytes。micro—

glia／ma erophages，and immature and ma ture oligodendrocytes in the perilesional zone were immunocytochemically quantified at

day 10 and 42．Using this approach，we demonstrate that enriched environment and reaching training both significantly improve

functional recovery of the impmred forelimb．Furthermore，these therapies strongly reduce the proliferation of mieroglia／maero—

phages in the perilesional zone，and daily training of the impMred forelimb significantly increased the survival of newly generated

astrocytes．Our data，therefore，demonstrate that reha bilitative therapies after cortical infarcts not only improve the functional

recovery but also significantly influence the glial response in the perilesional zone．．

[Key words] astrocytes；enriched environment；plasticity；microglia；sensorimotor cortex；stroke

[Classification numbers of Chinese library] R741；R681．55 [Document code] A [Article ID| 1001—117X

(2008)02—0109—10

【摘要】卒中后的康复治疗能改善神经环路的重塑，促进功能恢复。但人们对其潜在的细胞分子机制却知之

甚少。特别是康复训练对在大脑局部缺血的病理生理过程中扮演着重要角色的胶质细胞的影响，一直不甚明了。

现在，作者设计一项实验来检测康复训练对光化学诱导的局灶性脑缺血损伤灶周围(梗死灶位于大鼠前肢感觉运

动皮质功能区)的不同胶质细胞种群增殖和存活的影响。造模成功后，标准组小鼠即开始在标准笼中接受每日定

期的针对损伤前肢的功能训练，强化组大鼠转移到强化环境中饲养，抓取组大鼠仍然放在标准笼中未给予进一步

的治疗。作者通过感觉运动行走测试来评价功能恢复情况，并于梗死后 2—6 d给予 BrdU 以标记检测增殖的细

胞。分贝在梗死后的第 10天和第 42天，用免疫细胞化学方法对病灶周围增殖和存活的星形胶质细胞、小胶质细

胞／巨噬细胞和成熟或未成熟的少突胶质细胞进行定量检测。结果作者发现，强化环境和抓取训练都能显著提高

损伤前肢的功能恢复程度。此外，这些治疗能显著减少梗死灶周围小胶质细胞／巨噬细胞的增殖，其中对损伤前肢

的每日训练还能明显提高新生星形胶质细胞的存活率。因此，作者的数据证明，皮质梗死后的康复治疗不仅能促

进功能恢复，而且显著影响着病灶周围区胶质细胞的反应

【关键词】星形胶质细胞；强化环境；可塑性；小胶质细胞；感觉运动皮质卒中

l Introduction

Environmental stimuli and physical activity

摘自《GLIA~2008，56：516—527．

are capable to shape the functional and structural

circuits in the adult brain(Diamond，200 l；Johan—

sson。2004)．This form of environmentally induced

plasticity also Occurs after pathophysiological in—

suIts such as stroke (Biernaskie and Corhett，

维普资讯 http://www.cqvip.com

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110

2001；Keyvani and Schallert，2002)．For example，

exposure to an enriched environment after focal is—

chemic infarcts resulted in a better functional per—

formance in a battery of sensorimotor tasks(Bier—

naskie and Corbett，2001；Gonzalez et a1．，2004；

Johansson，1996)．Furthermore，specific rehabili—

tative training of the affected forelimb in animals

(Biernaskie et a1．，2005；Nudo et a1．，1996；Nu—

do，2007)or constraintinduced therapy in stroke

patients(Liepert et a1．，2005；Taub et a1．，1993；

W olf et a1．，2006)substantially improve the func—

tional outcome．Rehabilitative therapies，therefore，

favor the reorganization in the surrounding intact

tissue and contribute to the compensation and／or

recovery of the impaired function．Although sever—

al studies have begun to analyze the effects of reha—

bilitative training on functional sensorimotor recov—

cry (Biernaskie et a1．， 2005； Gharbawie et a1．，

2005； Kozlowski et a1．， 1996； Shanina et a1．，

2006)，only little is known about the underlying

cellular mechanisms and particularly the role of gli—

al cells during these processes．

Increasing evidence indicates that glial cells

crucially contribute to the degenerative and regen—

erative processes in the surrounding of ischemic

brain lesions (Nedergaard and Dirnagl， 2005；

Nilsson and Pekny，2007)．Focalischemia activates

astrocytes which undergo a characteristic hypertro—

phy of their cellular processes and thereby contrib—

ute to the formation of theglial scar．Astrocytes al-

so protect neurons by providing trophic support

(Anderson et a1．，2003；Trendelenburg and Dirna—

gl，2005)，for example，they release erythropoie—

tin as an inhibitor of neuronal apoptosis(Ruscher

et a1．，2002)or scavange free radicals(Trendelen—

burg et a1．，2002)．Furthermore，microglial cells，

the macrophage equivalent of the CNS．are activa—

ted by the ischemic infarct．A number of beneficial

functions of microglial cells have been identified．

They resolve the lesion by phagocytosis，produce

trophic factors but also release proinflammatory

cytokines，among others (Elkabes et a1．，1996；

yon Zahn et a1．，1997)． In addition，ischemic in-

farcts further increase the number of NG2一positive

glia(Komitova et a1．，2006)，which possess some

characteristics of muhipotent progenitor cells as

well as neuron—supporting functions(Aguirre and

Gallo，2004：Belachew et a1．，2003)．

To shed more light on this complex post—is—

chemic glial response，we quantified the prolifera—

tion and survival of distinct glial populations in the

perilesional zone of small cortical infarcts and ana—

lyzed the question whether rehabilitative training

Neural Iniury and Functional Rec0nstructi0n，March 2008，Vo1．3，No．2

influenced these processes．To this purpose，focal

ischemic infarcts were photochemically induced in

the forelimb sensorimotor cortex of adult rats．Im—

mediately after the infarcts，one group of animals

was transferred to an enriched environment；a sec—

ond group received daily sessions of skilled sensori—

motor training of the impaired forelimb，while a

third group remained in the standard cage during

the whole experiment． Using this approach， we

recently demonstrated that these rehabilitative

therapies increase postischemic dentate neurogene—

sis and improve functional recovery in the Morris

water maze(W urm et a1．，2007)．Here，we dem—

onstrate in the same animals that environmental

enrichment and daily training of the impaired fore—

limb also strongly influence the proliferation and

survival of distinct glial subpopulations in the per—

i1esiona1 zone．

2 MATERlALS AND METHoDs

2．1 Infarct Inductlon The experiments were

carried out on a total of 47 male W istar rats (3一

month-old，250—300 g)．At the time of the ische—

mic insult，all animals were preoperatively housed

in standard cages (4 rats per cage) under 12 h

light／1 2 h dark conditions with free access to food

and water． All animals received photothrombotic

cortical infarcts in the forelimb sensorimotor cortex

(FL—SMC)．These infarcts were induced using the

method initially described by W atson et a1．

(1 9 85)． Briefly，animals were anesthetized with

2．0 一3．5 enflurane in a mixture of oxygen／ni—

trous oxide(30 9／6／70 9／6)．Body temperature was

kept constant throughout the surgery at 36．5℃ ．A

fiberoptic bundle(3．0 mm diameter)connected to

the cold light source (KL 1500，Schott，Jena，

Germany)was positioned on the skull 0．5 mm an—

terior to bregma and 3．7 mm lateral to the mid—

line， aligned with stereotaxic coordinates corre—

sponding to the region of the forelimb sensorimotor

cortex(Paxinos，1995)．The beam had a light in—

tensity of 15 W／cm0 and the color temperature was

3200 K． No filter was used for the illumination

process．The light was turned on for 20 min．Dur—

ing the first minute，Rose Bengal(1．3 mg／0．1 kg

body weight at a concentration of 10 mg／ml in

0．9 NaCl；Sigma—Aldrich，Taufkirchen，Germa-

ny)was injected through a femoral vein catheter．

2．2 ExperlmentaI Deslgn The rats were ran—

domly assigned to one of the three experimental

conditions (see Fig． 1)． The first group was

housed under standard conditions(standard， 13)



神经损伤与功能重建 ·2008年 3月 ·第 3卷 ·第 2期

in ordinary cages(3— 4 animals／cage。54 cm X

38 cm×19 cm，)．The second group of animals was

transferred into the enriched environment (en—

riched：nl 6)at day 1 after the infarct and housed

in a large cage(8 animals／cage，85 cm×75 cm ×

40 cm)containing various objects such as horizon—

tal and vertical boards，climbing platforms，plastic

tubes and tunnels，chains，and small houses(Fig．

1B)．The configuration of the objects was changed

every day．A third group of animals was kept in a

standard cage and received one daily session of

skilled reaching training of the impaired forelimb in

a Plexiglas reaching box (reaching；n18)starting

at day 1 after the surgery(Fig．1C)．The rats had

to grasp 50 pellets (Dustless Precision Pellets

45 mg。BioServ。USA)．After 1 week，the mum—

ber of pellets was increased to 1 00 per day．During

the daily training session．the number of successful

reachings was counted which were defined as effec—

tive courses of movements including the grasping

and consuming of the food pellets． A drop of the

food pellet was assessed as an unsuccessful reac—

hing．The animals of all experimental groups were

food deprived to 95 of their initial body weight．

To maintain body weight，additional food was giv—

en in their home cages after skilled reaching train—

ing every day．All animals received daily single i．

P．injections(BrdU，50 mg／kg，Sigma—Aldrich

Taufkirchen，Germany) starting 2 days after sur—

gery and followed for 5 days． The animals were

sacrified 1 0 or 42 days after the photothrombotic

lesion (Fig．1A)．

A

KEfNl Ef L̂

re酏hing

p目．inlI

l

0 10 20 3o 柏 (d)

Fig．1 Infarct model and experimental design．(A)Lo—

cation of the photothrombotic infarct(dotted line) in the

forelimb sensorimotor cortex and Nissl—stained section at day

111

t 0 post surgery．Prior to the surgery all animals received a

handling．The schematic illustration demonstrates three dif—

ferent groups of lesioned animals comprising each two groups

with a t0 day or 42 day surviva1：standard housing (stand—

ard)．enriched environment(enriched)，and skilled reaching

training (reaching)． Daily intraperitoneal BrdU—injections

were applied at day 2— 6 post infarct．(B)Configuration of

the environmenta1 enrichment．Obj ects were changed every

day to promote exploratory behavior．(C)Skilled daily reac—

hing training in the Plexiglas chamber． The animals had to

grasp small food pellets with their impaired forelimb．

A

B

· 口 d

enrkl’ed

hing

Fig．2 Sensorimotor forelimb function in the ladder

rung walking task．(A)Photograph illustrating a rat walk—

ing on the ladder with irregularly placed rungs(minimum of

0．5 cm between the rungs)．(B)Percentage of correct place—

ments of the contralateral(impaired) and ipsilateral fore-

limb．Skilled reaching training as well as enriched environ—

ment improves the sensorimotor performance of the impaired

contralateral forelimb when compared with the standard

housing conditions．Reaching training of the impaired fore—

limb further reduced the correct placements of the ipsilateral

forelimb．Bars represent mean 6 SEM． Asterisks indicate

significant differences(P<0．05)．

2．3 BehavioPaI Test1ng The ladder rung

walking task was used to assess sensorimotor func—

tion．Prior to training and testing，all animals were

handled daily． The horizontal rung walking appa—

ratus had side walls of clear Plexiglas and metal

rungs(diameter：0．3 cm)，which were arranged in

an irregular pattern with a distance varying from

0．5 to 5 cm． The animals had to cross the ladder

rung 5 times per testing session (Fig．2A)．A dig—

ital camera was placed a lateral position at a

slight ventral angle to record both sides of the body

and paw positions．The video recordings were ana—

lvzed frame—by-frame in order to examine limb

placements．The foot placement on the rung was

∞ 帅 ∞ 蚰 ∞ 0

母̂一一 { _ l



112

measured according to the position that occurred in

placement accuracy． Only correct placements of

the forelimbs with its mid position on the rung and

digits of the forepaw flexed around the rung were

measured．The number of correct placements was

counted for each forelimb separately but also the

total number of all steps in five crossing trials．

From these data，the percentage of correct place—

ments was calculated for each forelimb． The rats

were tested prior and 2 9 days after surgery to de—

tect the chronic phase of deficits．The examination

was done blinded to the experimental group．

2．4 Infarct Volumetry Using a charge—COU—

pied device(CCD)camera and National Institute of

Health(NIH )，USA image software。the area of

the cortical infarct as well as that of both hemi—

spheres(mm )was measured by tracing these re—

gions on the computer screen．The infarct area was

quantified on every eighth section (40 ttm)．Vol—

umes(mm。)were determined by the integration of

the appropriate region with the section interval

thickness(320 um)．

2．5 TIssue PreparatIon and ImmUn0hIsto—

chemIstry The animals were deeply anesthetized

with diethylether and perfused through the ascend—

ing aorta with 4 paraformaldehyde in phosphate

buffer(0．15 mol／L，PH 7．4)．The brains were

removed immediately after the perfusion and post—

fixed in 4 paraformaldehyde in phosphate buffer

overnight．All samples were then cryoprotected in

phosphate-buffered saline containing 30 sucrose

for 24 h and stored at一 75℃ for further process—

ing．Sequential sections were cut using a(reezing

microtome(40 um)and stored at一 20℃ until fur—

ther processing．

Free—floating 40 gtm sections were treated with

0．6 H2 O2 in Tris-buffered saline(0．15 M NaCl，

0．1 M Tris HCl，PH 7．5)for 30 min rio block en—

dogenous peroxidase．After washing， the sections

were incubated in 2 N HCl at 37℃ for 30 min．

Sections were rinsed in boric acid(pH 8．5)and af—

ter several rinses in Tris—buffered saline containing

0．25 Triton X一100 (Tris—Triton)，、sections were

incubated overnight at 4X2 in primary rat mono—

clonal anti—BrdU (1：500)in TBS—Triton containing

3 normal donkey serum．The next day，sections

were incubated in biotinylated donkey anti—rat anti—

sera (1：500， Jackson Immunoresearch， West

Grove，PA)for 2 h，rinsed and incubated in avidin—

biotin—peroxidase complex (Vector Laboratories，

Burlingame，CA) for 60 min．Finally，they were

reacted for peroxidase detection in a solution of 3．

3-diaminobenzidine(0．25 mg／ml；Sigma—Aldrich，

Neural Inj ury and Functional Reconstruction，March 2008，Vo1．3，No．2

Munich，Germany)containing 0．0 1 H2 O2． Sec—

tionswere thoroughly washed，mounted，and COV—

erslipped．

For triple immunofluorescence staining， the

brains were treated in the same way as described

for the immunoperoxidase staining． Sections were

then incubated for 24 h with the primary antibodies

rat anti—BrdU (1：500；Immunologicals Direct—Ox—

ford Biotechnology，Oxfordshire，UK)；guinea pig

anti—glial fibrillary acidic protein (1：1，000；Ad—

vanced Immunochemistry， Los Angeles， CA ．

USA)，rabbit anti—S100b (1：2500；Swant，Bell—

inzona，Switzerland)，and goat anti—vimentin for

astrocytes(1：500；Santa Cruz，CA)，rabbit anti—

NG2 for oligodendrocytes precursor (1： 500；

Chemicon，CA)；rabbit anti—M B．P for mature oli—

godendrocytes(1：500；Biomeda，CA)；mouse anti—

CD68 clone ED1 for macrophages and microglia

(1：500；Serotec，Oxford，UK)． Immunofluores—

cent triple labeling was performed using the fol—

lowing secondary antibodies：Rhodamine anti—rat

(1：250；Dianova，Hamburg，Germany)，CY5 anti—

mouse (1：250；Dianova， Hamburg，Germany)，

CY5 anti-rabbit(1：250；Dianova，Hamburg，Ger—

many)，Alexa Fluor 488 antirabbit(1：250；M olec—

ular Probes，Leiden， Netherlands)， Alexa Fluor

488 anti—guinea pig (1：250； Molecular Probes，

Leiden，Netherlands)，and Alexa Fluor 488 anti—

goat(1：250；Molecular Probes，Leiden，Nether—

lands)．

To assess the apoptotic cell death．the termi—

nal deoxynucleotidvl transferase—mediated dUTP in

situ nick end labeling (TUNEL)method was used

according to the protocol supplied by the manufac—

turer(Apoptag Peroxidase In situ apoptosis detec—

tion kit， Sterologicals Corp．， A H Diagnostics，

Schweden)．

2．6 StereoIogy and Quantification To de—

termin~the total numbers of BrdU—labeled cells in

the perilesional area，the optical—fractionator meth—

od was used as implemented in the semiautomatic

stereology system (StereoInvestigator， Micro—

BrightField， Colchester，USA )． Phenotypes of

lhese quantified cells were analyzed with the confo—

cal laser scanning microscope．Every 1 2th sections

from the appearance to the disappearance of the in—

farct area were analyzed，resulting in approximate—

ly 8— 10 coronal sections per animals(Fig．3A)．

These anterior and posterior landmarks correspond ’

to 2．0 mm anterior to bregma and 21 mm posterior

to bregma of the stereotaxic atlas by Pax．

inos

(1 9 95)．The perilesional zone was defined a n ap—

proximately 500 Ilm wide cortical zone directly r一



神经损伤与功能重建 ·2008年 3月 ·第 3卷 ·第 2期

rounding the infarct(Fig． 3B)． The size of the

counting frame was 60／,m × 60 ／,m ．the thickness

of the tissue averaged was 40／,m ，with a height of

the dissector cube of 15／,m and a grid of 95／,m ×

145 um． Section thicknesses were measured，and

appropriate guard zones at the top and the bottom

of the section were defined to avoid over sampling．

To trace the perilesional area we used a 5×objec-

tive．The numbers of the BrdU positive cells(per—

oxidase method)were counted through a 63×ob—

jective．Positive cells，which intersected the upper—

most foca1 plane(exclusion plane)and the 1atera1

exclusion boundaries of the counting frame，were

not counted． The perilesional area reference vol—

ume was determined by adding the traced perile—

siona1 area for each section multiplied by the dis—

tance between sections sampled． The number of

BrdUlabeled cells was then related to 1／ram。sec—

tiona1 volume to estimate the tota1 number of Br—

dU—positive cells．

The phenotypes of BrdU—positive cells for ev—

ery 1 2th section were analyzed using confocal laser

scanning microscopy (I SM M eta 5 1 0，Zeiss，Ger—

many)． For each series， 90— 1 00 BrdU—labeled

cells were phenotyped using a z-scan through the

cel1 soma by allowing the definite assessment of o—

verlap between the cell specific antigens．The abso—

lute number of a certain phenotype was calculated

per animal by multiplying the absolute number of

BrdU—positive cells with the percentage of the phe—

notype of interest assessed by the co—localization

studies．

2．7 StatisticaI AnaIysIS All cel1 cohnts are

expressed as mean 6 S．D．，the behaviora1 data are

given as mean 6 S．E．M 。Statistical analysis of cell

counts was performed using the one way—analysis

of variance (ANoVA ) followed by an unpaired

two tailed t-test． Differences in the sensorimotor

test between the groups were assessed with the

M ann—W hitney U—test． A11 numerica1 analyses

were performed using SPSS for W indows 1 1．5

(Standard Version)．A 1eve1 of P< 0．05 was con-

sidered statistically significant．

3 RESULTS

3．、1 MOrpho1ogy of the Photothrombot1o In—

fa rC tS Histologica1 processing revealed well—de—

fined cortica1 infarcts in the forelimb sensorimotor

cortex(FL—SMC)with a slight involvement of the

primary motor and somatosensory cortex which is

already described in detai1 in the study by W urm et

a1．(2007)(Figs．1 and 3)．Briefly，the 1esion af一

113

fected al1 cortica1 1ayers 1eaving the subcortica1

white matter intact． Ten days after the photo—

thrombotic 1esion，the infarct volume showed no

statistically significant differences between the ex—

perimental groups(standard，15．28± 2．41 mm。；

enriched，18．32± 3．13 mm。；reaching， 18．44±

2．72 mm。)．At day 42，the 1esion size was consid—

erably reduced in all the experimental groups：

一 68．8 in the standard group，一 70．7 in the

enriched group， and 一 63．3 in the reaching

group． No significant differences in the absolute

volume of the 1esion were detected (standard，

4．77± 1．16 mm。； enriched，5．37 4-2．55 mm。，

reaching，6．76 4-1．58 mm。)．

A

Fig 3 Quantification of the number of BrdU-po sitive cells

in the perilesional zone at day 10 and 42 post surgery．(A)Defini—

tion of the perilesiona1 area on the brain surface and a sequence of

corona1 sections．The perilesiona1 zone is marked with a dotted 1ine

and the area of the infaret is hatched． (B)Corona1 sections

through he perilesiona1 area(dotted 1ine)immunohistochemically

stained th antibodies against the proliferation ma rker BrdU，The

number of BrdU-po sitive cells was quantified using a semiautomat—

ic stereology system and the optical-fractionator method。(C)To—

ta1 numbers of BrdU-positive cells in the perilesional area at day 10

and 42 po stinfarct。Enriched environment(enriched)as wel1 as

daily reaching training(reaching)significantly decreased the peril—

esiona1 proliferation at day 10，whereas training of the impaired

forelimb(reaching)significantly increased the survival of the Br—

dU-po sitive cells at day 42。Bars represent mean 6 SD．Asterisks

indicate significant differences(P<0，05)。

3．2 EnrIohed Env1 ronment and Reach1ng

T ra I n 1 ng All animals，which were transferred to

the enriched environment，actively used the stimu—

lation obj ects．The rats climbed the ladders and

boards，run through the tunnels，played with the



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s u r v i v a l t o 3 5 ％ a n d r e a c h i n g t r a i n i n g u p t o 6 0 ％．

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f e c t e d b y r e a c h i n g t r a i n i n g o r e n v i r o n n l e n t a l e n—

r i c h m e n t ， t h e s e d i f f e r e n c e s i n n u m b e r o f B r d U

p o s i t i v e c e l l s p e r v o h l m e w e r e n o t c o n f o u n d e d b y

a l I e r a t i o n s i n t h e r e f e r e n c e v o I u m e ． T U N E I ． s t a i —

n i n g r e v e a l e d n o d i f f e r e n c e s i n n u m b e r s o f a p o p t o t

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a f t e r t h e i n f a r c t ( s t a n d a r d ， 1 ％ ± 1 ％； r e a c h i n g ，

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s u p p r e s s i o n o f B r d U p o s i t i v e c e l l s i n t h e r e a c h i n g

a n d e n r i c h e d g r o u p w a s n o t c a u s e d b y a n i n c r e a s e

i n a p o p t o s i s ．

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d i f f e r e n t e x p e r i m e n t a l g r o u p s ． ( A ) C o n f o c a l i m a g e s o f d o u b —

l e l a b e l e d s e c t i o n s s t a i n e d w i t h a n t i b o d i e s a g a i n s t B r d U

( r e d ) a n d C D 6 8 ( g r e e n ) ． S c a l e b a r s r e p r e s e n t 1 0 u m ． T h e

r i g h t l o w e r i m a g e d i s p l a y s a l o w e r m a g n i f i c a t i o n ． ( 13 ) Q u a n

t i f i c a t i o n o f t h e B r d U — — C D 6 8 — —

p o s i t i v e c e i l s i n t h e p e r i l e s i o n a l

z o n e a t d a y 1 0 a n d 4 2 a f t e r t h e i n f a r c t ． E n v i r o n m e n t a l e n—

r i c h m e n t a n d d a i l y r e a c h i n g t r a i n i n g b o t h ， s i g n i f i c a n t l y s u p

p r e s s e d t h e p r o l i f e r a t i o n o f m i c r o g l i a ／m a c r o p h a g e s i n t h e

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m e n t a l g r o u p s ． ('

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s t a i n e d w i t h a n t i b o d i e s a g a i n s t B r d U ( r e d ) a n d N G 2 ( g r e e n )

( A ) a s w e l l a s B r d U ( r e d ) a n d M B P ( g r e e n ) ( C ) ． ( }{ )

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p o s i t i v e c e l l s i n t b e p e r i l e— -

s i o n a l z o n e a t d a y 1 0 a n d 4 2 a f t e r t h e i n f a r c t ． E n r i c h e d e n v i

r o n m e n t s ig n i f i c a n t l y i n c r e a s e d t h e p r o l i f e r a t i o n o f N G 2 — —

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t h e B r d U — - M B P — -

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t h e e x p e r i m e n t a l g r o u p s h a v e b e e n o b s e r v e d ． B a r s r e p r e s e n t

m e a n 6 S D ． A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s ( P <

() ． 0 5 ) ．

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c a l c h a r a c t e r i z a t i o n o f B r d U p o s i t i v e c e l l s r e v e a l e d

t h a t r e a c h i n g t r a i n i n g a s w e l l a S e n r i c h e d e n v i r o n

m e n t d i f f e r e n t i a l l y i n f l u e n c e d t 11 e P r e l i f e r a t i o n a n d

s u r v i v a l o f a c t i v a t e d a s t r o c y t e s ， m a c r o p h a g e s ／m i —

c r o g l i a ， a n d o h g o d e n d r o c y t c s i n t h e p e r i l e s i o n a l

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116

nocytochemical analysis of the GFAP—BrdU—posi—

tive cells demonstrated a differential expression of

the markers S100b and vimentin at both time

points(see Fig．4)． 10 days after the infarct，the

ratio of GFAP—BrdU—positive cells co—expressing

S100b or vimentin was significantly increased in

the reaching group compared with standard con—

trois and enriched animals．In the reaching group，

88 ± 1 8 of GFAP—BrdU—positive cells showed a

co—localization with S100b and 73 -+-19 with vi—

mentin． This ratio was significantly lower in the

standard controls(S100b：48 -+-17 ，P=0．025；

vimentin：30 ± 7 ，P < 0．024) and enriched

group (S100b：54 -+-8 ，P 一 0．034；vimentin：

15 ± 12 ，P 一 0．034)which showed no differ—

ences among each other． At day 42，the ratio of

GFAPBrdU——positive cells co——expressing SlOOb only

significantly increased in the standard group (day

10：48 ± 17 ；day 42：85 ± 3 ，P一 0．034)

whereas it remained stable in the enriched and

reaching group．At this late time point no differ—

ences of GFAP—S100b—BrdU—positive cells were ob—

served between the enriched group and reaching

group(enriched：44 -+-17％；reaching：65 ±

6 ；P一 0．065)． The ratio of GFAP—BrdU—posi—

tive cells which showed a co—localization with vim—

entin was significantly reduced between day 10 and

42 in the reaching group (day 10：73 ± 19 ；day

42：46 ± 6 ，P一 0．02)，whereas the standard

and enriched animals showed no alterations．At day

42 the ratio of vimentin——expressing GFAPBrdU——

positive cells varied between 26 -+-8 in the an—

riched，39 -+-13 in the standard，and 46 ±

6 in the reaching group，with a significant differ—

ence between the enriched and reaching groups

(P一 0．024)．

Enriched environment and daily reaching

training also influenced the number of proliferating

CD68_。positive macrophages／microglia in the perile——

sional zone at day 10 after the infarct(Figs． 5A，

B)． Compared with standard controls (38306±

10506 mm。)，the total number of CD68一BrdU—pos—

itive microglia was significantly reduced in the

reaching (16112-+-8583 mm。；P一0．042)as well as

in the enriched groups(20838± 6364 mm。；P一

0．024)． This activity—dependent differences were

absent at day 42 post infarct。and the total number

of microglia was strongly reduced in all groups

(standard， 467 ± 212 mm。； enriched， 383±

160 mm。：reaching，807± 631 mm。)．

Analysis of BrdU—positive immature oligoden—

drocytes expressing the marker NG2 and mature

oIigodendrocytes expressing M BP revealed a signif—

Neural Injury and Functional Rec0nstructi0n，March 2008，Vo1．3，No．2

icant increase in number of NG2一BrdU—positive

cells in the enriched group (3928± 364 mm。)corn—

pared with standard controls (1786± 1278 mm0：

P一 0．018)and reaching animals(906± 390 mm。；

P< 0．001) at day 10 after the infarct，while the

number of MBP—BrdU—positive cells was not influ—

enced by the housing and training conditions

(standard，1 1097-+-4648 mm。；enriched，8000 4-

3800 mm。；reaching，12176--+-_ 9327 mm。) (Figs．

6A ，B)．At day 42 post surgery，the numbers of

NG2一BrdUpositive cells remained stable in all ex—

perimental groups (standard，4269--+-_ 2051 mm。：

enriched，3507± 1935 mm。； reaching， 4104±

3470 mm。)． Only 12 一 32 of the MBP-BrdU_

positive mature oligodendrocytes survived at this late

time point with no significant differences between the

groups(standard，2627± 1190 mm。；enriched，2569±

984 mm3；reaching，1443--+-_ 6602 mm3)．

Taking these findings together， we provide

evidence that enriched environment as well as daily

reaching training strongly decrease proliferation of

microglia and macrophages in the perilesional zone．

Furthermore， specific training of the impaired

forelimb promoted the survival of astrocytes in the

direct infarct surround．

4 DISCUSSlON

Our study demonstrates that rehabilitative

training following small cortical infarcts in the

forelimb representation cortex not only improve

functional recovery but also significantly alters the

proliferation and survival of distinct glial subpopu—

lations in the perilesional area．Environmental an—

richment as well as daily training of the impaired

forelimb，both suppresses cellular proliferation in

the perilesional zone at day 10 after the infarct．

This decrease in perilesional proliferation was pri—

marily caused by a significant reduction of prolifer—

ating microglia and macrophages，while the hum—

ber of GFAP--positive astrocytes was not signifi——

cantly changed at this early time point．However，

at 6 weeks after the infarct，the survival of the new

born cells was strongly increased in animals which

received daily reaching training and most of these

surviving cells were GFAP—positive astrocytes．De—

tailed analysis of the expression of vimentin and

S100b in GFAP——positive astrocytes further demon——

strated that these markers were also differentially

influenced by environmental enrichment and reac—

hing training．NG2一positive glia was only transient—

ly increased in the enriched group at day 10 but no

differences in the number of mature oligodendro—



神经损伤与功能重建 ·2008年 3月 ·第3卷 ·第2期

cytes were found at day 42．

Enriched environment and skilled reaching

training influence the proliferative response and

survival of BrdU—positive cells in the perilesional

area after photothrombotic stroke． The finding

that environmental enrichment increased the sur—

vival of BrdU—positive cells was also described by

Komitova et a1． (2006)．They analyzed the effects

of enriched environment 5 weeks after focal ische—

mic infarcts(M CAO)and described an increase of

BrdU—positive cell survival when the animals lived

in an enriched environment starting 24 h or 7 days

after the infarct． Under physiological conditions，

Ehninger and Kempermann (2003) reported that

wheel running caused a better survival of BrdU—

positive cells in the motor cortex 4 weeks after the

last BrdU iniection，while this effect was absent in

mice housed in an enriched environment． In ac—

cordance with these previous reports，we here de—

scribe that the survival of newborn cells was also

increased by specific training of the impaired fore—

limb following cortical infarcts．Animals which re—

ceived a nonspecific stimulation in an enriched envi—

ronment showed the same tendency but these alter—

ations did not reach significance in our experi—

ments．In opposite to these changes in the chronic

phase，daily skilled reaching training and nonspe—

cific environmentalstimulation both strongly de—

crease cellular proliferation during the subacute

phase as described at day 1 0 in the perilesional

zone．Phenotype analysis of the BrdU—positive cells

provides some evidence that this lOSS of prolif—erat—

ing cells is at least in part caused by a significant

suppression of microglial proliferation．

To our knowledge，this is the first study dem—

onstrating that enriched environment and skilled

reaching training of the impaired forelimb signifi—

cantly reduce the number of newly generated mi—

croglia in the perilesional zone．After focal cerebral

ischemia。 CD68一positive microglia and macropha—

ges were found not only in the direct vicinity of the

lesion but also in remote brain regions (Block et

a1．，2005；Jander et a1．，1998)．Immediately after

the ischemic insult，resting microglia change their

morphology from a ramified to an activated hyper—

ramified phenotype and express the CD68 antigen

(M orioka et a1．， 1993)． The activated microglia

migrate towards the lesion， remove the necrotic

tissue by phagocytosis，and thereby become macro—

phages(Streit et a1．，1999；Streit，2000)．Some

macrophages derive from monocytes which cross

the blood—brain barrier after the ischemic lesion

(Bechmann et a1．，2005；Jander et a1．，2007)．Be一

117

sides the degradation of the necrotic cells，activa—

ted microglia and macrophages release several

growth factors and scavenge free radicals(Elkabes

et a1．，1996；Imai et a1．，2006)．But activated mi—

croglia could also harm the ini ured brain(Dheen et

a1．，2007：von Zahn et a1．，1997)by the synthesis

of deleterious molecules like nitric oxide，reactive

oxygen radicals，and the release of glutamate(As—

chner et a1．，1999；Barger et a1．，2007；Boie and

Arora，1992；Gibson et a1．，2005)．Several recent

studies showed that the suppression of activated

microglia and macrophages by application of the

antiinflammatory drugs indomethacin or minocyclin

significantly improved the functional recovery after

focal ischemic infarcts (Hewlett and Corbett。

2006；Hoehn et a1．，2005；Liu et a1．，2007)．It is，

therefore，conceivable that the reduction of prolif—

erating microglia and macrophages which was

found in the reaching and enriched group in our

study favors the better functional outcome in these

animals．However，there is up to now no proof of

causality and reactive alterations of additional glial

subpopulations as well as changes in the synaptic

reorganization might also contribute to the better

functional performance following environmental

enrichment and daily reaching training．

Specific rehabihtative training further in—

creased the survival of newly generated reactive as—

trocytes in the perilesional area．Astrocytes are es—

sential for neuronal function and synaptic homeo—

stasis and take an active part in synaptic sprouting

as well(Pekny et a1．，2007：Ridet et a1．，1997)．

After brain insults like stroke． astrocytes play a

multifaceted role(Nedergaard and Dirnagl，2005、．

They immediately proliferate in response to the le—

sion，increase their expression of GFAP，and con—

tribute to the formation of the glial scar (Kimel—

berg，2005)． Reactive astroglia might provide a

protective environment for neurons in the perile—

sional zone by production of antiapoptotic and

trophic factors and thereby promote neuronal sur—

vival，synaptic remodelling，and neurite outgrowth

(Nedergaard and Dirnagl， 2005； Trendelenburg

and Dirnagl，2005；W ilson，1997)．In our study，

we observed a significant increase in survival of

newly generated GFAP—positive astrocytes only in

the reaching group although animals housed in the

enriched environment showed a similar increase，

reaching no significance in our study． However，

reaching training also differentially modifies the co—

expression of the intermediate filament vimentin as

well as the Ca21一binding protein S100b in GFAP—

positive astrocytes． Increasing evidence indicated



118

that vimentin and S100b mark distinct subpopula—

tions of GFAP—positive astrocytes with heterogene—

OUS morphological and functional properties(Liu et

a1．，2006； Seifert et a1．， 2006)． Only little is

known about the role of these subtypes after ische—

mia． Knockout mice of GFAP and vimentin

showed a deterioration of wound healing。 an in—

crease of apoptotic neurons and oligodendrocytes

and reduction of functional recovery in a model of

spinal cord ini ury(Faulkner et a1．，2004；Menet et

a1．，2000)． Similar processes might also occur in

the perilesional zone． Additional evidence that re—

habilitative training influences S100b—and vimen—

tin—positive astrocytes after focal ischemia is pro—

vided by Nygren et a1．(2006)，who describe a re—

duction of both cell types in the striatum in animals

housed in an enriched environment after transient

middle cerebral artery occlusion． W e here demon—

strate that daily reaching training significantly in—

creased the survival of newly generated GFAP—pos—

itive astrocytes in the perilesional zone．

The proliferation and survival of immature and

mature oligodendrocytes is only slightly influenced

by the rehabilitative training．Solely the number of

proliferating NG2一positive cells was significantly

increased at day 1 0 after the infarct in the enriched

environment group．An activity—induced increase in

number of NG2一positive cells was also observed af—

ter middle cerebral artery occlusion in rats trans—

ferred into an enriched environment．but this in—

crease was located in the ipsi—and contralateral in—

tact cortex(Komitova et a1．，2006)．NG2一positive

cells are constitutively proliferating in the adult

brain and even after the photothrombotic infarct；

the number of BrdU—positive NG2一positive cells is

increasing during the first 6 weeks．W hereas NG2一

expressing glia give rise to myelin。·forming oligo——

dendrocytes under physiological conditions(Nish—

iyama et a1．，2002)only little is known about their

role in the lesioned brain． Some evidence from in

vitro and in vivo studies indicates that these prolif—

crating NG2一positive cells constitute a population

of multipotent progenitor cells possessing the ca—

pacity to build neuroblasts in the adult cortex

(Tamura et a1．，2007)．

In accordance with previous studies，we pro—

vide further evidence that rehabihtative training

promotes functional recovery after focal ischemic

infarcts(Biernaskie and Corhett，200 1；Biernaskie

et a1．，2004；Johansson and Ohlsson，1996；Ohls—

Neural Injury and Functional Reconstruction，March 2008．Vo1．3，No．2

son and Johansson， 1995； Will et a1．， 2004)．

Nonspecific activation of the animals by housing in

an enriched environment induced a very similar im—

provement of functional performance of the con—

tralesional forelimb in the skilled ladder rung walk—

ing test compared with a daily session of specific

reaching training of the impaired forelimb． Inter—

estingly，daily reaching training of the contrale—

sional forelimb deteriorated the performance of the

ipsilesional forelimb in the ladder rung walking

test．This finding provides further evidence for the

so—called learned nonuse of the ispilesional forelimb

following skilled reaching training of the contrale—

sional paw after cortical lesions which was previ—

ously described by Allred et a1．(2005)and Gonza—

lez et a1．(2004)．Johansson and Ohlsson(1996)

demonstrated that voluntary exercise in a running

wheel and housing in an enriched environment both

improved the functional outcome after middle cere—

bral artery occlusion．W hen enriched environment

and skilled reaching training was combined after

MCA0 the functional recovery could be further im—

proved (Biernaskie and Corhett，200 1)． The cellu—

lar consequences of such a combination of rehabili—

tative therapies were not the focus of the present

investigations but worthy to clarify in subsequent

studies．It is further important to note that com—

paratively young animals were used and age might

even influence the described cellular and functional

alterations．Although specific reaching training as

well as nonspecific stimulation of the animals in the

enriched enrichment both improve the functional

performance of the contralesional forelimb： it is

currently unfeasible to refer these functional alter—

ations to specific cellular subpopulations of the

complex glial response in the perilesional zone．But

it is crucial to emphasize that our findings provide

detailed stereological data that environmental stim—

uli and rehabilitative training significantly modify

the proliferation and survival of distinct glial sub—

populations in the direct vicinity of cortical in—

farcts．Further studies are required to elucidate the

functional role of these complex cellular alterations

in the perilesional zone．

4 ACKNOWLEDGMENTS

The authors thank J ulia Oberland for great

technical assistance．



Rehabilitative therapies differentially alter proliferation and survival of glial cell populations...

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