Brain Research 922 (2001) 165–172www.elsevier.com/ locate /bres

Research report

Expression of glial cell line-derived neurotrophic factor induced bytransient forebrain ischemia in ratsa b a a ,*Hiroyuki Miyazaki , Kazuo Nagashima , Yasunobu Okuma , Yasuyuki Nomura

aDepartment of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, JapanbDepartment of Molecular and Cellular Pathology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan

Accepted 21 March 2001

Abstract

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNFproduction in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNFmRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNAincrease in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatumand brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after theischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidalneurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3–7 days after the ischemia. On the other hand, in otherregions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. Thesefindings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest thatischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important rolein ischemia-induced neuronal death in the brain. 2001 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Neurotrophic factors: expression and regulation

Keywords: Glial cell line-derived neurotrophic factor; Cerebral ischemia; Astrocyte; Hippocampus; Neuronal death

1. Introduction area, but is widely expressed in the brain [28,33]. How-ever, there have been no reports that GDNF is expressed in

Glial cell line-derived neurotrophic factor (GDNF) has glial cell astrocytes in vivo.been purified from glial cell line conditioned medium, It has been suggested that GDNF plays an importantbased on its ability to promote dopamine uptake in role, not only in the survival of dopaminergic neuronsembryonic midbrain cultures [18]. GDNF has been re- injured by neurotoxins, such as 6-OHDA [6,17,19,31],ported to promote the survival and mature phenotype of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [7,9,32], andcentral dopaminergic [4,11,18] and noradrenergic neurons N-methyl-4-phenylpyridinium [11,16], but also in prevent-[3], and motoneurons [26,35], as well as that of various ing pyramidal neuronal death in the hippocampus aftersubpopulations of peripheral sensory and sympathetic kainic acid administration [19]. We recently reported thatneurons [5]. GDNF is not restricted to the dopaminergic by modulating the expression of tyrosine hydroxylase,

GDNF protects against delayed neuronal death induced inthe hippocampal CA1 region by transient forebrain is-

Abbreviations: GDNF, glial cell line-derived neurotrophic factor; chemia [20,21]. Moreover, it has been reported that GDNFGFAP, glial fibrillary acidic protein; RT-PCR, reverse transcription and provides potent protection against the brain infarctionpolymerase chain reaction

[14,34] and edema [2,14] induced by middle cerebral*Corresponding author. Tel.: 181-11-706-3246; fax: 181-11-706-artery occlusion, and blocks the rise in nitric oxide levels4987.

E-mail address: nomura@pharm.hokudai.ac.jp (Y. Nomura). that accompanies middle cerebral artery occlusion and

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )03013-X

166 H. Miyazaki et al. / Brain Research 922 (2001) 165 –172

reperfusion [34]. These observations suggest that GDNF GTACATTGTCTCGGCCGC-39 (corresponding to nucleo-possesses potent neuroprotective effects against neuronal tides 60–77 and 500–517 of the cDNA sequence, respec-injury, including delayed neuronal apoptotic death, induced tively), and the expected size of the PCR product was 458by ischemia. base pairs.

This study examined the temporary GDNF mRNA RT-PCR of rat RNA with GDNF primer yielded a majorexpression and the cellular localization of GDNF pro- band of the predicted size (approximately 450 base pairs),duction induced in adult rats subjected to transient fore- after 40 cycles of PCR. To determine the identity of thisbrain ischemia. Although, as determined by in situ hybridi- product, the band was purified and cloned directly intozation [28], GDNF is expressed mainly in neurons in the pCR3.1 vector (Invitrogen, The Netherlands). The se-hippocampus and cerebral cortex, we demonstrated that quence and orientation of the inserts were confirmed bytransient forebrain ischemia temporarily increases GDNF automated sequencing, which identified the major band asmRNA in the hippocampus and cerebral cortex, and that identical to the predicted 458-base-pair product from PCRischemia-induced reactive astrocytes produced GDNF. of GDNF cDNA.

RT-PCR with b-actin (a housekeeping gene) primers(59-ATGCCATCCTGCGTCTGGACCTGGC-39 and 59-AGCATTTGCGGTGCACGATGGAGGG-39, corre-

2. Material and methods sponding to nucleotides 597–617 and 1983–1203, respec-tively, of the cDNA sequence) confirmed the RNA integri-

2.1. Surgical four-vessel occlusion ty of all samples: all rat hippocampus, cerebral cortex,striatum, and brainstem samples yielded a 607-base-pair

Male Sprague–Dawley rats (Japan Laboratory Animals product, from amplification of a cDNA template after 20Inc., Tokyo, Japan) weighing 280–350 g were used. cycles of PCR.Transient forebrain ischemia was induced by a modi-fication of a previously described method [29]. Briefly,under sodium pentobarbital (40 mg/kg, i.p.) anesthesia, the 2.3. Western blot analysiscarotid arteries were exposed through a ventral mid-cervi-cal incision and separated from the surrounding nerves and The hippocampus was sliced at about 500 mm with aveins. A silk thread was loosely placed around each artery, tissue chopper, and CA1 regions were obtained by slicingwithout interrupting the carotid blood flow. The vertebral with a razor under a stereoscopic microscope. The tissuesarteries were electrocauterized with a monopolar were disrupted in 1 ml of 20 mM Tris–HCl (pH 7.4)coagulator through the alar foramina. After 2 days, the containing 1 mM MgCl and 0.5 mM phenylmethylsul-2

carotid arteries were re-exposed and occluded for 15 min fonyl fluoride (serine protease inhibitor), using 10 strokeswith small aneurysmal clips, to produce a four-vessel of a glass–Teflon homogenizer. The homogenate wasocclusion. Body temperature was maintained at dissolved in Laemmli’s buffer (62.5 mM Tris, 2% sodium37.560.58C via a thermostatically controlled infrared dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol,lamp. In sham-operated rats, the carotid arteries were 0.01% bromophenol blue, pH 7.4). Protein samples (10–30dissected, but not occluded. mg protein / lane) were fractionated by sodium dodecyl

For reverse transcription and polymerase chain reaction sulfate–polyacrylamide gel electrophoresis, and transferred(RT-PCR) and Western blot analyses, rats were killed by from the gel to a polyvinylidene difluoride membrane bydecapitation at various times. Each brain region was then electroelution at a constant voltage of 100 V for 1 h at 48C.rapidly removed and stored at 2808C until use. After the polyvinylidene difluoride membrane was incu-

All animal experiments were carried out in accordance bated with phosphate buffer containing 5% dehydratedwith the NIH Guide for the Care and Use of Laboratory skim milk (Snow Brand Milk Products Co. Ltd., Japan) toAnimals and approved by the Animal Care and Use block nonspecific protein binding, the membrane wasCommittee at Hokkaido University. incubated with anti-GDNF polyclonal antibody (diluted to

1:500, Santa Cruz Biotechnology, Inc., CA, USA) at 48Covernight; the membrane was then incubated with horse-

2.2. RT-PCR radish peroxidase-linked anti-rabbit (diluted to 1:2000,Amersham, Germany) for 30 min. In a preliminary study,

The acid guanidium thiocyanate /phenol /chloroform ex- we confirmed that anti-GDNF antibody cross-reacted withtraction procedure was used to prepare total cellular RNA GDNF using a positive-control GDNF antigen (sc-from samples of each brain region. To evaluate GDNF 4169WB, Santa Cruz Biotechnology, Inc.). Subsequently,mRNA expression, we initially used a highly sensitive the bands were detected using the enhanced chemilumines-RT-PCR technique. The 18-mer primers for rat GDNF cence detection system (ECL Kit, Amersham). The proteinmRNA were 59-GGGATGTCGTGGCTGTCT-39 and 59- bands that cross-reacted with antibodies could be detected

H. Miyazaki et al. / Brain Research 922 (2001) 165 –172 167

in X-ray films (X-Omat JB-1, Kodak, Japan) 5–30 s after 3. Resultsexposure.

3.1. GDNF mRNA expression induced by 15-mintransient forebrain ischemia

2.4. Immunohistochemistry

In sham-operated rats, almost no GDNF mRNA wasAt intervals after occlusion and recirculation, animals

detected in the hippocampal sample 6 h after the shamwere anesthetized with an overdose of sodium pentobarbi-

operation (Fig. 1A,B, lane S); this was similar to thetal (60 mg/kg, i.p.), and the brain was perfused with 4%

results in normal animals (data not shown). Ischemiaparaformaldehyde in 0.1 M phosphate-buffered saline (pH

induced transient (from 3 to 24 h, with peak expression at7.4) via the ascending aorta, as previously described [15].

6 h) expression of GDNF mRNA in the hippocampus; thisThe brain was removed and fixed with the same solution

expression was no longer evident after 7 days (Fig. 1A,B).for at least 24 h more. Coronal brain sections were made

Changes of GDNF mRNA expression in various brainafter embedding the brain in paraffin.

regions are shown in Fig. 2. GDNF mRNA expression inThe streptavidin–biotin–peroxidase complex method

the cerebral cortex was similar to that in the hippocampus,was used in immunohistochemical studies, as described

although slight GDNF mRNA expression was seen in the[23]. In brief, after deparaffinization, specimens were

sham-operated rats and at 7 days. On the other hand,treated with 0.3% H O –methanol to suppress endogenous2 2 GDNF mRNA expression in the striatum and brainstemperoxidase activity, incubated with 10% normal goat

were unchanged, even though it initially was expressed inserum, and allowed to react with anti-GDNF polyclonal

those brain regions of sham-operated rats.antibody (diluted to 1:100) diluted in 1% bovine serum

These results indicate that transient forebrain ischemiaalbumin at 48C overnight. Incubations with 1% BSA only

induces the expression of GDNF mRNA in the hippocam-were used as negative controls. The sections exposed to the

pus and, to a lesser extent, in the cerebral cortex.first antibody (anti-GDNF antibody) were reacted withbiotinylated goat anti-rabbit IgG. The streptavidin–biotin–

3.2. Change of GDNF production in the hippocampalperoxidase reaction products were visualized with 3,39-

CA1 region by Western blot analysisdiaminobenzidine tetrahydrochloride (Sigma, St Louis,MO, USA) and counter-stained with hematoxylin. We

When we first examined GDNF production in samplesconfirmed that anti-GDNF antibody recognized GDNF in a

from the whole hippocampus, no clear changes in GDNFpreliminary study, in which the primary antibody was

production were detected. Therefore, for the Western blotpre-incubated with GDNF.

analysis we chose the hippocampal CA1 region whereFor colocalization studies, the sections exposed to the

delayed neuronal death induced by ischemia was observed.first antibody (anti-GDNF antibody) were visualized with

Fig. 3 shows GDNF (|35 kDa) production at variousAlexa-594-conjugated goat anti-rabbit secondary antibody

times after the ischemic episode. In sham-operated rats,(Molecular Probes, Inc., Eugene, OR, USA) diluted 1:100

GDNF was originally observed (lane S) 6 h after thein phosphate buffer, and then the sections were incubated

operation. Between 3 and 24 h, GDNF production in thewith anti-glial fibrillary acidic protein (GFAP) polyclonal

hippocampal CA1 region was slightly induced by is-antibody (diluted to 1:100) containing 1% bovine serum

chemia; production declined to below the baseline levelsalbumin at 48C overnight. This was followed by visualiza-

thereafter (lane 7d). On the other hand, GDNF proteintion with Alexa-488-conjugated goat anti-rabbit secondary

levels in the cerebral cortex, striatum, and brainstem wereantibody (Molecular Probes, Inc.) diluted 1:100 in phos-

not changed markedly at any time (data not shown).phate buffer. The Alexa-594- and Alexa-488-conjugatedantibodies were visualized with Texas red and fluorescein,

3.3. Histopathological alteration of GDNF-producingrespectively. All images were photographed under a laser-

cellsscanning confocal microscope (LSM 510, Carl Zeiss,Germany).

In the hippocampus of sham-operated rats, almost allGDNF-positive cells were observed in the pyramidal cell

2.5. Statistical analysis layer (Fig. 4a). Although there was no marked alteration ofGDNF-positive cells in the CA1 region within 24 h of the

All data represent the mean6S.E.M. of at least three ischemia, the GDNF level was reduced and accompaniedindependent experiments. The results were analyzed using by neuronal death, beginning 2 or 3 days after ischemia,Student’s t-test or one-way ANOVA followed by Dunnett’s and GDNF-positive cells were observed diffusely aroundtest, with StatView (Abacus Concepts, Inc., Berkeley, CA, the hippocampal CA1 pyramidal cell layer (Fig. 4b). In theUSA) software for Windows. Differences were considered cerebral cortex, GDNF-positive cells were observed main-statistically significant at P,0.05 (*) and P,0.01 (**). ly in layers II to VI, most clearly in pyramidal cell layers

168 H. Miyazaki et al. / Brain Research 922 (2001) 165 –172

Fig. 1. Expression of GDNF mRNA in the hippocampus induced by transient forebrain ischemia as determined by RT-PCR methods. (A) cDNA samplesobtained from the hippocampus at various times (1, 3, 6, 12, 24 h and 3, 7 days) post-ischemia were amplified by 40 cycles of PCR for GDNF mRNA.Reaction products were electrophoresed in a 6% polyacrylamide gel and visualized by ethidium bromide fluorescence. Sample from a sham-operated rat(lane S) 6 h after the sham operation without occlusion. Data are from an experiment typical of four independent experiments. (B) Quantification of theRT-PCR data for GDNF was performed by densitometric analysis using image-analyzing software (NIH image, Ver. 1.61). Data are shown as themeans6S.E.M. of four independent experiments. Statistical analysis was performed using ANOVA followed by Dunnett’s test.

III and V. There was no difference between sham-operated induced by transient forebrain ischemia, immunohistoch-and ischemic rats (Fig. 4c,d). In layer I, which is mainly emical double staining was performed using anti-GDNFcomposed of astrocytes, GDNF-positive cells were not and -GFAP antibody. At 6–12 h post-ischemia (corre-observed at any time. sponding to the period of maximum response of GDNF

On the other hand, although there were only a few in the production), GDNF-positive cells differed from GFAP-striatum, strongly GDNF-positive sells were observed in positive cells (Fig. 5a–c). Interestingly, 3 days after theneurons of the caudate putamen, ventrolateral and reticular ischemic episode, GDNF-positive except for pyramidalthalamic nucleus, and the paraventricular hypothalamic neurons and GFAP-positive were the same cells (Fig.nucleus. There were no changes in GDNF-positive cells in 5d–f). These findings were clearly evident in the hip-these areas in either the treated or sham-operated rats at pocampal CA1 region from approximately 3 days post-any time (data not shown). ischemia.

To confirm the cell localization of GDNF production These findings indicate that although GDNF was gener-

H. Miyazaki et al. / Brain Research 922 (2001) 165 –172 169

ipsilateral cerebral cortex induced by ischemia followingmiddle cerebral artery occlusion, although the peak wasearlier (3 h) and the effect was almost dissipated by 24 h[1]. Moreover, kainate-induced transient upregulation ofGDNF mRNA in the hippocampus has been reported[12,33]. These findings suggest that GDNF plays animportant role in brain injury.

It has also been reported that GDNF protects against thebrain infarction and edema induced by middle cerebralartery occlusion [2,14,34]. Additionally, we previouslyreported that intrahippocampal microinjection of GDNF(1.0 mg/2 ml) significantly protects against delayed neuro-nal death induced by transient forebrain ischemia [20].Therefore, expression of GDNF might be related toischemic brain injury, especially to delayed neuronal deathin the hippocampal CA1 region, or to the recovery ofneuronal functions, such as neuronal activity and thesurvival of neuronal networks still containing energymetabolites.

In the Western blot analysis, ischemia induced anincrease in GDNF (accompanying increased GDNFmRNA) in the hippocampal CA1 region from 3 to 24 hpost-ischemia; GDNF subsequently declined to belowbaseline levels. These results suggest that increased GDNFproduction was transient and served to rescue neuronsfrom neuronal death; then production subsequently de-clined and was accompanied by decreased energy metabo-lites in neurons and neuronal death in the hippocampusduring post-ischemia days 3–7. On the other hand, in ourpreliminary experiments, GDNF did not change signifi-cantly in other regions, such as the striatum and brainstem.

Thus, these findings suggest that GDNF plays animportant role in inhibition of neuronal apoptotic death invivo, as it has been previously reported that GDNF inhibitsapoptosis in human embryonic dopaminergic neurons [8]

Fig. 2. GDNF mRNA expression in the cerebral cortex, striatum and and cerebral cortical neurons [25] in vitro.brainstem induced by transient forebrain ischemia. cDNA samplesobtained from each brain region at various times (1, 3, 6, 12, 24 h and 3, 4.2. Immunohistochemical study in GDNF-producing7 days) post-ischemia were amplified by 40 cycles of PCR for GDNF

cellsmRNA. Sample of a sham-operated rat (lane S) 6 h after the shamoperation without occlusion. Data are from an experiment typical of fourindependent experiments. Using in situ hybridization, Nosrat et al. [24] identified

GDNF mRNA expression in the hippocampus in pyramidalally produced in neurons, it was also produced in the cell layers in embryonic rats at days 15–21 and atreactive astrocytes and accompanied by the progress of postnatal day 0, but not at postnatal day 1 to week 4,neuronal death induced by transient forebrain ischemia. suggesting that it plays important developmental roles in

neurons. Additionally, Pochon et al. [28] reported thatGDNF mRNA is expressed mainly in neurons and that its

4. Discussion synthesis is not restricted to dopaminergic neurons. In thepresent study, although GDNF mRNA expression was not

4.1. Expression of GDNF induced by transient forebrain detected in the hippocampus of sham-operated rats, asischemia determined by RT-PCR method (Fig. 1), in agreement with

previously described findings [12], an immunohistochemi-In this study, we found that transient forebrain ischemia cal technique confirmed that GDNF protein is produced in

transiently induced GDNF mRNA in the hippocampus and the hippocampus, mainly in pyramidal neurons.cerebral cortex, with a peak at 6 h. These observations No alteration in the location of GDNF-producing cells inwere similar to the expression of GDNF mRNA in the various regions was induced within 24 h of ischemia. In

170 H. Miyazaki et al. / Brain Research 922 (2001) 165 –172

Fig. 3. Transient forebrain ischemia-induced GDNF production in the hippocampal CA1 region. Total homogenates of the hippocampal CA1 regions atvarious times (1, 3, 6, 12, 24 h and 3, 7 days) post-ischemia were separated by SDS–polyacrylamide (12%) gel electrophoresis followed by immunoblotanalysis using anti-GDNF or anti-a-tubulin antibody, as described in the Material and methods section. The sample from a sham-operated rat (lane S) wasobtained within 12 h after the operation without occlusion. Data are from an experiment typical of two independent experiments in duplicate.

the hippocampus, however, GDNF-positive cells appeared they are larger and more apparent in the CA1 region thandiffusely, and seemed to involve cells besides the neurons in other regions of the hippocampus at day 2 or 3around the pyramidal cell layer (Fig. 4b) at 3 days (when [10,27,30]. Moreover, it has been shown that the microgli-delayed neuronal death begins). Ischemia-induced reactive al reaction induced by ischemia is observed in the entireastrocytes are present from about 24 h after ischemia and hippocampus as early as 20 min after reperfusion, with a

Fig. 4. Immunohistochemistry for GDNF in the hippocampal CA1 region (a and b) and cerebral cortex (c and d) 3 days after the ischemia or shamoperation. GDNF-positive cells were stained brown with 3,39-diaminobenzidine tetrahydrochloride, and nuclei were counter-stained purple withhematoxylin. Microphotographs are from a typical experiment and are representative of three independent experiments. Scale bar, 20 mm.

H. Miyazaki et al. / Brain Research 922 (2001) 165 –172 171

Fig. 5. Colocalization of GDNF and ischemia-induced reactive astrocytes in the hippocampal CA1 region. Microphotographs of hippocampal CA1 regionat 12 h (a–c) and 3 days (d–f) post-ischemia are shown. Although GDNF (red) and GFAP (green) were not co-expressed 12 h after the ischemia (c), GDNFwas colocalized at reactive astrocytes 3 days after that (yellow, f). Microphotographs are from a typical experiment and are representative of threeindependent experiments. Scale bar, 20 mm.

peak at 4–6 days in the CA1 and dentate gyrus [22]. On duced by astroglial cells [25]. Thus, our present findingsthe other hand, neuronal death in the cerebral cortex, suggest that GDNF plays a protective role in brain injury,striatum, and brainstem is not conspicuous, and reactive including neuronal apoptotic death.astrocytes are not readily observed, in contrast to thesituation in the hippocampus. Therefore, these findings

4.3. Conclusionsuggest that diffusely located GDNF observed 3 days afterpost-ischemia involves GDNF production in reactive glial

GDNF mRNA is regulated in part via ischemia-inducedcells around the CA1 layer.

neuronal degeneration, and ischemia-induced reactive as-Next, double immunohistochemical staining was used to

trocytes may produce GDNF as protection against neuronalidentify the cells responsible for post-ischemic GDNF

death. GDNF may play an important role in ischemia-production. Interestingly, we found that GDNF was pro-

induced neuronal death in the brain. This suggests thatduced in the ischemia-induced reactive astrocytes, which

GDNF could be a useful novel therapy for neurodegenera-were more clearly observed approximately 3 days after the

tive disorders, such as subarachnoid hemorrhage or occlu-ischemia. GDNF-positive cells around the CA1 pyramidal

sive cerebrovascular diseases.layer were colocalized with GFAP-positive astrocytes, andthese reactions were strong in the CA1 region.

Therefore, production of GDNF in neurons in the earlypost-ischemic phase may protect against brain injury, Acknowledgementsincluding neuronal death, and neuronal death might beprotected by GDNF produced in ischemia-induced reactive We thank Drs Fujii and Shimizu (Department of Molec-astrocytes in the late post-ischemic phase, which is ob- ular and Cellular Pathology, Graduate School of Medicine,served as delayed neuronal death in the hippocampus. In Hokkaido University, Sapporo, Japan) for their advice.surviving neurons, GDNF seems to protect against neuro- This work was supported by the Joint Research Project fornal death and strong staining was observed 7 days after the Regional Intensive from the Japan Science and Technologyischemia. It has been suggested that separation from glial Corporation, and by a Grant-in-Aid for Scientific Researchcells causes rat cortical neurons to die through apoptosis from the Japanese Ministry of Education, Science, Sportsresulting from deprivation of neurotrophic factors pro- and Culture.

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