New GABAergic Neurogenesis in the Hippocampal CA1 Region of a Gerbil Model of Long‐Term Survival after Transient Cerebral Ischemic Injury

Abstract

We investigated the probability of newly generated neurons that could survive and mature in the ischemic hippocampal CA1 region (CA1) of a gerbil model of transient cerebral ischemia. Neuronal death was shown in the stratum pyramidale (SP) from 4 days post‐ischemia, and a significant increase in NeuN‐positive (⁺) neurons was found in the SP at 180 days post‐ischemia. 5‐Bromo‐2‐deoxyuridine (BrdU)⁺ cells were co‐stained with NeuN and glutamic decarboxylase 67 (GAD67). Brain‐derived neurotrophic factor (BDNF) immunoreactivity and protein level was shown in nonpyramidal cells from 4 days post‐ischemia, and the immunoreactivity was strong at 30 days post‐ischemia and not significantly changed until 180 days post‐ischemia. Furthermore, TrkB immunoreactivity was co‐stained with GAD67 when we examined at 180 days post‐ischemia. Myelin basic protein (MBP)⁺ nerve fibers were reduced at 4 days post‐ischemia and maintained until 60 days post‐ischemia, and MBP immunoreactivity and levels were significantly increased at 180 days post‐ischemia. In the passive avoidance test, cognitive dysfunction was improved at 180 days post‐ischemia. These results suggest that the differentiation of neural progenitor cells into new GABAergic neurons may be promoted via BDNF in the ischemic CA1 and that the neurogenesis may partially mediate the recovery of cognitive impairments via increasing myelinated nerve fibers.

Introduction

Transient cerebral ischemia causes fatal delayed degeneration of specific vulnerable neurons in some regions of the brain including the hippocampal CA1 region 19. Neuronal death in the hippocampal CA1 region occurs some days after transient ischemic damage and the neuronal death is referred to as “delayed neuronal death” 19. The pronounced death of the CA1 pyramidal neurons is associated with severe impairments of hippocampal‐dependent brain functions, such as spatial learning and memory 13.

Neurogenesis is important in neurological repair and in the fundamental function of the central nervous system (CNS), which contains progenitor cells that are capable of generating new neurons, astrocytes and oligodendrocytes 9, 48. In adult mammals, persistent neurogenesis has been demonstrated in two specific brain areas; the subventricular zone (SVZ), which borders the lateral ventricles, and the subgranular zone (SGZ), which is in the hippocampal dentate gyrus (DG) 10, 30, 39. Previous studies have shown that cerebral ischemia stimulates neurogenesis and the neuronal regeneration takes place from endogenous neural progenitor cells in animal models of global ischemia 18, 29, 32, 52. Although the adult CA1 region is extremely vulnerable to ischemic insults, the production of new neurons appears to be very limited in the adult brain including the CA1 region after global cerebral ischemia 42. However, the origin and differentiation of neural progenitor cells, which give rise to new neurons in the adult CNS, has been a subject of debate.

The present study aims to address whether neurogenesis following cerebral ischemia would be able to appear in the adult gerbil CA1 region. We examined long‐term changes in neuronal distribution in the CA1 region of the gerbil induced by 5 minutes of transient global cerebral ischemia and determined whether newly born cells were differentiated into specific neuronal phenotypes. Moreover, we examined whether this neurogenesis in the CA1 region was related with the re‐myelination and the recovery of cognitive impairment.

Materials and Methods

Experimental animals

We used male Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Kangwon National University, Chunchon, South Korea. The gerbils were used at 24 weeks (body weighting 65–75 g) of age and maintained in pathogen‐free conditions under adequate temperature (23°C) and humidity (60%) control with a 12‐h light/12‐h dark cycle. The procedures for animal handling and care adhered to guidelines that are in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National Academies Press, 8th Ed., 2011) and they were approved by the Institutional Animal Care and Use Committee (IACUC) at Kangwon University.

Induction of transient cerebral ischemia

Transient cerebral ischemia was developed according to our previous method 25. In brief, the experimental animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. Ischemia was induced by occluding the arteries with nontraumatic aneurysm clips (Yasargil FE 723K, Aesculap, Tuttlingen, Germany). After 5 minutes of occlusion, the aneurysm clips were removed from the common carotid arteries. The body (rectal) temperature under free‐regulating or normothermic (37 ± 0.5°C) conditions was monitored with a rectal temperature probe (TR‐100; Fine Science Tools, Foster City, CA) and maintained using a thermometric blanket before, during and after the surgery until the animals completely were recovered from anesthesia. Thereafter, animals were kept on the thermal incubator (temperature, 23°C; humidity, 60%, Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature until the animals were sacrificed. Sham‐operated animals were exposed to similar surgery without carotid artery occlusion.

5‐bromo‐2′‐deoxyuridine (BrdU) administration and tissue processing

To examine the cumulative labeling of proliferative cells in the hippocampal CA1 region after transient cerebral ischemia, gerbils were received intraperitoneal BrdU injection (50 mg/kg in saline, Sigma‐Aldrich, St. Louis, MO) once a week for 2 months prior to 180 days after post‐ischemia. Sham‐ and ischemia‐operated animals (n = 7 at 4, 10, 30, 60 and 180 days after I‐R were anesthetized with pentobarbital sodium, and perfused transcardially with 0.1 M phosphate‐buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 phosphate‐buffer (PB, pH 7.4). The brains were removed and postfixed in the same fixative for 6 h. The brain tissues were embedded in tissue‐freezing medium and serially sectioned into 30 µm coronal sections on a cryostat (Leica, Wetzlar, Germany).

Cresyl violet staining

To investigate the morphological and neuronal changes in the hippocampus after transient cerebral ischemia, the hippocampal sections in each group were stained with cresyl violet (CV) as we descried previously 35. In brief, the sections were mounted on gelatin‐coated microscopy slides. CV acetate (Sigma, St. Louis, MO) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid (Sigma) was added to this solution. Before and after staining for 2 minutes at room temperature, the sections were washed twice in distilled water. The fixed brain tissues were dehydrated by immersing for 2 h in 50%, 70%, 80%, 90%, 95% and 100% ethanol baths in succession at room temperature. After dehydration, the sections were mounted with Canada Balsam (Kanto, Tokyo, Japan).

Immunohistochemistry and immunofluorescence for NeuN, GAD67 BDNF and MBP

To investigate the neuronal changes in the CA1 region after transient cerebral ischemia, according to previous study 25, the NeuN⁺, GAD67⁺, BDNF⁺ and MBP⁺ staining was performed in the present study. The sections were sequentially treated with 0.3% hydrogen peroxide (H₂O₂) in PBS for 30 minutes and 10% normal horse serum in 0.05 M PBS for 30 minutes. The sections were then incubated with diluted mouse anti‐NeuN (1:1000, Chemicon International, Temecula, CA), mouse anti‐GAD67 (1:50, Chemicon International) rabbit anti‐BDNF (1:1000; Abcam Incorporated) and rabbit anti‐MBP (1:1000; Abcam Incorporated) overnight at room temperature. Thereafter the tissues were exposed to biotinylated horse anti‐mouse (1:200, Vector, Burlingame, CA), anti‐goat (1:200, Vector) and anti‐rabbit IgG (1:200, Vector) and streptavidin peroxidase complex (1:200, Vector). Then, the sections were visualized with 3, 3′‐diaminobenzidine tetrachloride (DAB) in 0.1 M Tris‐HCl buffer and mounted on the gelatin‐coated slides. For myelin basic protein (MBP) detection, the sections were incubated in FITC‐conjugated goat anti‐rabbit IgG (1:600; Jackson ImmunoResearch, West Grove, PA). After dehydration the sections were mounted with Canada balsam (Kanto chemical, Tokyo, Japan).

Cell counts

As previously described 24, all the measurements were performed to ensure objectivity in blind conditions (three observers for each experiment) and control and experimental samples were assayed under the same conditions. According to anatomical landmarks corresponding to AP −1.4 to −2.2 mm of the gerbil brain atlas, the studied tissue sections were selected with 120‐μm interval, and cell counts were obtained by averaging the total cell numbers 15 sections taken from each animal per group. The number of NeuN‐positive (⁺) and GAD67⁺ neurons was counted in a 250 × 250 µm square, applied approximately at the center of the CA1 region in the stratum pyramidale using an image analyzing system (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ). Cell counts were obtained by averaging the total number of NeuN‐immunoreactive neurons from each animal per group.

To quantitatively analyze BDNF and MBP immunoreactivities, the corresponding areas of the CA1 region were measured from 15 sections per animal. Images of all BDNF‐immunoreactive structures were taken from three layers (strata oriens, pyramidale and radiatum in the hippocampus proper) through an AxioM1 light microscope (Carl Zeiss, Göttingen, Germany) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. Images were calibrated into an array of 512 × 512 pixels corresponding to a tissue area of 250 × 250 µm (40 × primary magnifications). The densities of all BDNF⁺ and MBP⁺ structures were evaluated by optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log (256/mean gray level). The background OD was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the OD of the image file was calibrated as % [relative optical density, (ROD)] using Adobe Photoshop version 8.0 and then analyzed using NIH Image 1.59 software.

Double immunofluorescence for BrdU/NeuN or GAD67, BDNF/GFAP or Iba‐1 and TrkB/GAD67

To confirm the differentiation from newly generated cells to mature GABAergic neurons and the cell type containing TrkB immunoreactivity, the sections at 180 days after ischemia were used for double immunofluorescence staining with BrdU/NeuN, BrdU/GAD67 and TrkB/GAD67. In addition, we performed double immunofluorescence staining for BDNF/GFAP and BDNF/Iba‐1 to identify the cell type of nonpyramidal cells expressing BDNF at 30 days after ischemia. In brief, DNA denaturation was conducted, and then sections were incubated in 2N HCl and in boric acid (only for BrdU staining). The sections were incubated with the mixture of diluted mouse anti‐NeuN (1:400, Chemicon International), rat anti‐BrdU (1:250, Serotec, Bicester, UK), rabbit anti‐TrkB (1:50, Abcam Incorporated), mouse anti‐GAD67 (1:50, Chemicon International), rabbit anti‐BDNF (1:200, Santa Cruz Biotechnology), mouse anti‐glial fibrillary acidic protein (GFAP) (1:400, Chemicon International) and goat anti‐ionized calcium‐binding adapter molecule 1 (Iba‐1) (1:500, Santa Cruz Biotechnology) overnight at 4°C. After washing three times for 10 minutes with PBS, the sections were incubated in a mixture of both FITC‐conjugated goat anti‐rabbit IgG (1:600; Jackson ImmunoResearch), FITC‐conjugated donkey anti‐rat IgG (1:600; Jackson ImmunoResearch), Cy3‐conjugated donkey anti‐mouse IgG (1:200; Jackson ImmunoResearch) and Cy3‐ conjugated donkey anti‐goat IgG (1:200; Jackson ImmunoResearch) for 2 h at room temperature. The immunoreactions were observed under the confocal microscope (LSM510 META NLO, Carl Zeiss). Negative control test was also performed using preimmune serum instead of primary antibody to establish the specificity of the immunostaining. The negative control resulted in the absence of immunoreactivity in all structures. For calculating the number of BrdU⁺/NeuN⁺ cells and BrdU⁺/GAD67⁺ cells in the CA1 region, z stack images were taken from the tissue area of 200 × 200 µm with 30 µm thick 10 sections per animal. Cell counts were performed following the above‐mentioned method.

Western blot analysis

To confirm changes in level of BDNF and MBP proteins in the hippocampus after transient cerebral ischemia, at designated times (4, 30 and 180 days after the surgery), the animals (n = 7 at each point in time) were sacrificed and used for the western blot analysis. After sacrificing them and removing the hippocampus, the tissues were homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM ethylene glycol bis (2‐aminoethyl ether)‐N,N,N′,N′ tetraacetic acid (EGTA) (pH 8.0), 0.2% Nonidet P‐40, 10 mM ethylendiamine tetraacetic acid (EDTA) (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β‐glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT). After centrifugation, the protein level was determined in the supernatants using a Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, Rockford, IL). Aliquots containing 20 µg of total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6% SDS, 0.3% bromophenol blue and 30% glycerol. The aliquots were then loaded onto a 10% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Crop, East Hills, NY). To reduce background staining, the membranes were incubated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 45 minutes, followed by incubation with rabbit anti‐BDNF antibody (1:2000; Abcam Incorporated) or rabbit anti‐MBP (1:2000; Abcam Incorporated), peroxidase‐conjugated goat anti‐rabbit IgG (Vector, Cat# PI‐1000) and ECL kit (Pierce Chemical). Loading controls were performed using antibodies against beta actin (Abcam Incorporated). The results of the western blot analysis were scanned, and densitometric analysis for the quantification of the bands was done using Scion Image software (Scion Corp., Frederick, MD), which was used to count ROD: A ratio of the ROD was calibrated as %, with sham‐group designated as 100%.

Passive avoidance test

Short‐term memory ability was evaluated by assessing the latency of the passive avoidance test. This test was performed using passive avoidance apparatus (GEM 392, San Diego instruments) according to the method of Sharma et al 44 with slight modification. The apparatus is divided into two sections. One compartment is large, white and illuminated, the other is small, dark and without illumination. The two compartments communicate through a sliding door system and the floor is a steel grid in both compartments. The bars of the dark compartment floor are wired to a constant current scrambler circuit. The test was divided into a training and test session. The experimental session was divided into three phases: habituation trial, acquisition trial and retention trial. During the habituation trial, which was executed on the third day after ischemic insults, the gerbil was placed in the white and illuminated compartment. In this phase, the sliding door was initially closed and it opened after 5 s. The gerbil can then explore both compartments for 60 s and after this period it was taken off the apparatus. After 10 minutes, it was placed again in the white compartment. The sliding door opened after 5 s and successively closed when the gerbil crossed the cage, entering the dark room, where it remained for 10 s, then was removed from the cage. The acquisition trial was performed 60 minutes after the habituation trial. In this phase, the gerbil was replaced in the white room and when it crossed the sliding door entering the dark room, it received an electric shock (2 mA for 5 s), released from the grid. Twenty‐four hours later, the retention trial was performed. The gerbils were replaced in the white room and the sliding doors opened as the acquisition trial. The latency times for entering the dark compartment and the number of error times were recorded. When an animal did not enter the dark room within 180 s, the latency was recorded as 180 s. Response latency was measured as an index of learning ability. Passive avoidance test were performed at 1 day before ischemia‐reperfusion, 4, 10, 30, 60 and 180 days after I‐R. On the 3rd, 9th, 29th, 59th and 179th days, the retention trial was performed again.

Statistical analysis

Data are expressed as the mean ± standard error of the mean. The data of cell counts were evaluated by a one‐way ANOVA SPSS program and comparisons among groups were made using parametric two‐way ANOVA. Further comparisons were assessed using Duncan's multiple‐range test. The latency of entrance to the dark compartment of the passive avoidance test was analyzed using a Kruskal–Wallis nonparametric one‐way analysis of variance, followed by a two‐tailed Mann–Whitney U test. Statistical significance was set at P < 0.05.

Results

Changes of cresyl violet‐positive cells

Neuronal death in the hippocampus after transient cerebral ischemia was examined using CV staining. In the sham‐operated group, neurons stained with CV were easily observed in all of the subregions of the hippocampus. In the ischemia‐operated groups, a loss of cresyl violet‐positive (CV⁺) pyramidal neurons was observed in the stratum pyramidale of the entire CA1 region; however, neurons in the other subregions were well stained with CV (Figure 1A).

Figure 1.

Changes of CV‐positive (CV⁺) cells and NeuN‐immunoreactive (NeuN⁺) neurons in the hippocampal CA1 region after I‐R. A. CV staning in the hippocampus of the sham‐ and ischemia‐operated groups. CV⁺ cells are dramatically decreased only in the CA1 region after ischemia. CA, cornus ammonis; DG, dentate gyrus. Scale bar = 400 µm. B. Immunohistochemistry for NeuN in the CA1 region of the sham‐ and ischemia‐operated groups. SP, stratum pyramidale; SO, stratum oriens and SR, stratum radiatum. Scale bar = 50 µm. C. The mean number of NeuN⁺ neurons/250 × 250 µm of the CA1 region at sham, 4, 10, 30, 60 and 180 days after I‐R (n = 7 per group; *P < 0.05, significantly different from the sham‐group; ^#P < 0.05, significantly different from the former group). The bars indicate the means ± SEM. D. Double immunofluorescence staining for BrdU (green), NeuN (red) and merged images (yellow) in the CA1 region 180 days after I‐R. Most of the BrdU⁺ cells are colocalized with NeuN⁺ neurons (arrows). Scale bar = 50 μm. E. The mean number of NeuN⁺, BrdU⁺ and BrdU⁺/NeuN⁺ cells/200 × 200 µm of the CA1 region 180 days after I‐R. (n = 7 per group). The bars indicate the means ± SEM.

Changes of NeuN‐immunoreactive neurons

Delayed neuronal death in the CA1 region following ischemia‐reperfusion (I‐R) was examined using NeuN immunohistochemistry. In the sham‐operated group, neurons in the CA1 region were well stained showing the presence of NeuN (92 ± 4.52 cells/250 × 250 µm, Figure 1B,C). Four days after I‐R, NeuN‐immunoreactive (NeuN⁺) neurons were hardly detected in the CA1 region due to the delayed cell death of pyramidal neurons in the stratum pyramidale (7 ± 1.05 cells/250 × 250 µm, Figure 1B,C). Thereafter, there were no significant changes in NeuN immunoreactivity and in the number of NeuN⁺ neurons in the CA1 region for 60 days after I‐R (Figure 1B,C). However, there was a significant increase in the number of NeuN⁺ neurons in the CA1 region 180 days after I‐R (24 ± 3.32 cells/250 × 250 µm, Figure 1B,C); the NeuN⁺ neurons were generally distributed near or within the stratum pyramidale. To elucidate that BrdU⁺ cells were newly generated and transformed into mature neurons, double immunofluorescence staining was performed in the CA1 region 180 days after I‐R. All of the BrdU⁺ cells revealed co‐expression with NeuN⁺ cells in the CA1 region 180 days after I‐R (Figure 1D,E).

Changes of glutamic acid decarboxylase 67 cells

In the sham‐operated group, glutamic acid decarboxylase 67 (GAD67⁺) neurons were detected near the stratum pyramidale of the CA1 region (4.3 ± 0.51 cells/250 × 250 µm, Figure 2A,B). In the ischemia‐operated groups, four days after I‐R, GAD67 immunoreactivity and GAD67⁺ cells were similar to that the sham‐operated group (3.9 ± 0.42 cells/250 × 250 µm, Figure 2A,B). Until 60 days after I‐R, GAD67 immunoreactivity and its pattern were not changed to those 4 days after I‐R (Figure 2A,B). At 180 days after I‐R, the GAD67 immunoreactivity and GAD67⁺ cells were significantly increased near the stratum pyramidale compare with those 4 days after I‐R (11.3 ± 0.53 cells/250 × 250 µm, Figure 2A,B). In addition, when the double labeling of BrdU and GAD67 at180 days after I‐R was performed, all of the BrdU⁺ cells in the ischemic CA1 region were co‐labeled with GAD67 (Figure 2C,D).

Figure 2.

Changes of GAD67‐immunoreactive (GAD67⁺) neurons in the hippocampal CA1 region after I‐R. A. Immunohistochemistry for GAD67 in the CA1 region of the sham‐ and ischemia‐operated groups. SP, stratum pyramidale; SO, stratum oriens and SR, stratum radiatum. Scale bar = 50 µm. B. The mean number of GAD67⁺ neurons/250 × 250 µm of the CA1 region at sham, 4, 10, 30, 60 and 180 days after I‐R (n = 7 per group; ^*P < 0.05, significantly different from the sham‐group; ^#P < 0.05, significantly different from the former group). The bars indicate the means ± SEM. C. Double immunofluorescence staining for BrdU (green), GAD67 (red) and merged images (yellow) in the CA1 region 180 days after I‐R. All of the BrdU+ cells are co‐labeled with GAD67⁺ neurons (arrows). Scale bar = 50 μm. D. The mean number of BrdU⁺, GAD67⁺ and BrdU⁺/GAD67⁺ cells/200 × 200 µm of the CA1 region 180 days after I‐R. (n = 7 per group). The bars indicate the means ± SEM.

Changes of brain‐derived neurotrophic factor immunoreactivity and protein level

In the sham‐operated group, BDNF immunoreaction was presented in the neurons of stratum pyramidale in the CA1 region (Figure 3A,B). Four days after I‐R, a conspicuous reduction of BDNF immunoreactivity was noted in the stratum pyramidale, and strong BDNF immunoreaction was expressed in nonpyramidal cells in the strata oriens and radiatum 30 days after I‐R (Figure 3A,C). To identify the cell type of nonpyramidal cells expressing BDNF 30 days after I‐R, double immunofluorescence staining for BDNF/GFAP or BDNF/Iba‐1 was performed and we found that BDNF immunoreactive cells were colocalized with GFAP⁺ astrocytes (Figure 3B); however, BDNF immunoreactivity was not found in the Iba‐1⁺ microglia (data not shown). Thereafter, the distribution pattern of the distribution of BDNF⁺ cells until 180 days after I‐R was similar those 4 days after I‐R, although BDNF immunoreactivity was a little changed with time (Figure 3A,C).

Figure 3.

Changes of BDNF‐immunoreactive (BDNF⁺) neurons in the hippocampal CA1 region after I‐R. A. Immunohistochemistry for BDNF in the CA1 region of the sham‐ and ischemia‐operated groups. SP, stratum pyramidale; SO, stratum oriens and SR, stratum radiatum. Scale bar = 50 µm. B. Double immunofluorescence staining for BDNF (green), GFAP (red) and merged images (yellow) in the CA1 region 30 days after I‐R. BDNF immunoreactivity is shown in GFAP⁺ positive astrocytes (arrows). Scale bar = 50 μm. The bars indicate the means ± SEM. C. Relative optical density (ROD) of BDNF immunoreactivity of the CA1 region at sham, 4, 10, 30, 60 and 180 days after I‐R. The ROD as % values of sham is represented (n = 7 per group; ^*P < 0.05, significantly different from the sham‐group). The bars indicate the means ± SEM. D. Western blot analysis of BDNF in the CA1 region of the sham‐ and ischemia‐operated groups. ROD as % values of immunoblot band is also represented (n = 7 per group; ^*P < 0.05, significantly different from the sham‐group; ^#P < 0.05, significantly different from the former group). E. Double immunofluorescence staining for TrkB (green), GAD67 (red) and merged images (yellow) in the CA1 region 180 days after I‐R. Most of the TrkB⁺ cells are colocalized with GAD67⁺ cells (arrows). Scale bar = 50 μm. The bars indicate the means ± SEM.

Western blot analysis showed that the pattern of change in BDNF protein level in the CA1 region after I‐R was similar to that observed in the immunohistochemical data. In the ischemia‐operated groups, BDNF protein level was markedly increased 30 days after I‐R compared with that in the sham‐operated group. Thereafter, BDNF protein level was not significantly changed until 180 days post‐ischemia (Figure 3D). In addition, to elucidate that newly generated cells express BDNF receptor, TrkB, double immunofluorescence staining was performed in the CA1 region 180 days after I‐R. Double labeled TrkB⁺/GAD67⁺ cells were examined near the stratum pyramidale of the CA1 region 180 days after I‐R (Figure 3E).

Changes of myelin basic protein immunoreactivity and protein level

The assessment of myelin was evaluated by immunohistochemistry and western blot for MBP in the CA1 region after I‐R. In the sham‐operated group, MBP⁺ myelinated nerve fibers were easily observed in all of the layers of the CA1 region, especially, in the strata oriens and radiatum (Figure 4A). In the ischemia‐operated groups, MBP⁺ myelinated nerve fibers were decreased in the CA1 region from 4 days after I‐R (Figure 4A,B). At 10 days post‐ischemia, MBP⁺ myelinated nerve fibers were significantly lower (28% vs. sham) than those 4 days after I‐R, and MBP⁺ myelinated nerve fibers were not significantly changed until 60 days after I‐R (Figure 4A,B). However, 180 days after I‐R, MBP⁺ myelinated nerve fibers were significantly increased (44% vs. sham) in the CA1 region compared with those at 60 days post‐ischemia (Figure 4A,B).

Figure 4.

Changes of MBP‐immunoreactive (MBP⁺) neurons in the hippocampal CA1 region after I‐R. A. Immunohistochemistry for MBP in the CA1 region of the sham‐ and ischemia‐operated groups. SP, stratum pyramidale; SO, stratum oriens and SR, stratum radiatum. Scale bar = 50 µm. B. Relative optical density (ROD) of MBP⁺ myelinated fibers of the CA1 region at sham, 4, 10, 30, 60 and 180 days after I‐R. ROD as % values of Sham is represented (n = 7 per group; ^*P < 0.05, significantly different from the sham‐group; ^#P < 0.05, significantly different from the former group). The bars indicate the means ± SEM. C. Western blot analysis of MBP in the CA1 region of the sham‐ and ischemia‐operated groups. The ROD as % values of immunoblot band is also represented (n = 7 per group; ^*P < 0.05, significantly different from the sham‐group; ^#P < 0.05, significantly different from the former group). The bars indicate the means ± SEM.

Conversely, western blot analysis showed that the pattern of change in MBP protein level in the CA1 region after I‐R was similar to that observed in the immunohistochemical data (Figures 4C).

Passive avoidance response after global ischemia

To examine whether the formation of new CA1 neurons was related with any functional recovery, we observed hippocampal‐dependent learning and memory performance 1 day before I‐R, and 4, 10, 30, 60 and 180 days after I‐R. The mean latency time of the passive avoidance response in the retention trial was 52.2 ± 12.4 s 1 day before I‐R (Figure 5). Transient global cerebral ischemia significantly induces shorter escape latencies. At 4, 10, 30 and 60 days after I‐R, the latency time is decreased by 14.1 ± 9.5 s, 12.8 ± 6.2 s, 11.5 ± 5.4 s, 11.7 ± 4.1 s (P < 0.05 vs. 1 day before I‐R); however, 180 days after I‐R, the latency time was significantly increased by 24.5 ± 9.2 s (Figure 5).

Figure 5.

Escape latency of entrance to the dark compartment of passive avoidance apparatus during memory retention test 1 day before I‐R, 4, 10, 30, 60 and 180 days after I‐R. Although a significant reduction in response latency after I‐R is observed, there is a significant improvement in response latency 180 days after I‐R. (n = 7 per group; ^*P < 0.05, significantly different from the sham‐group; ^#P < 0.05, significantly different from the former group). The bars indicate the means ± SEM.

Discussion

Over the past several years, stroke‐stimulated endogenous neurogenesis has been applied as one of effective strategies for the treatment of stroke associated with aging 2, 36. Cerebral ischemia is known to stimulate the proliferation and differentiation of neural precursor cells, which have a potential to enable regeneration and restoration; however, the functional relation of this phenomenon is not clear because of the poor survival and low neuronal differentiation rate of newly born cells in the brain, especially, in the aged brain 8, 15. Furthermore, it has been reported that hypoxia could induce neurogenesis in the hippocampus of the rodent 5, 43. Conversely, it has been demonstrated that transplanted neural precursor cells could increase endogenous neurogenesis after cerebral ischemia in the aged rat and suggested that cell based therapy for stroke might be able to increase endogenous adaptive processes including neurogenesis 16. On the basis of the above reports, in this study, we examined neurogenesis in the gerbil hippocampal CA1 region for a long time after transient cerebral ischemia.

In the present study, a massive regeneration of GAD⁺ neurons was found in the CA1 region very long time later following transient cerebral ischemic insult, which might be due to the lack of the process of spontaneously endogenous neurogenesis. Interestingly, we found that newly born NeuN⁺, BrdU⁺ and GAD67⁺ cells, which were co‐localized with TrkB, were distributed near the stratum pyramidale 180 days after I‐R and that the neurogenesis might be stimulated by increasing the production of endogenous BDNF. Moreover, this neurogenesis might mediate new myelination and the recovery of cognitive impairments.

Several studies have documented that experimental ischemia in the adult brain can trigger neurogenesis as a compensatory mechanism for neural cell death 27. Liu et al 29 reported an increase in cell proliferation in the hippocampal DG after transient global ischemia in the adult gerbil. A 12‐fold increase in cell birth in the DG was observed 1–2 weeks after 10 minutes of global ischemia; in this case, the neurogenesis represented an amplification of normal neurogenesis rather than the induction of neurogenesis in a normally non‐neurogenic area. However, such neurogenesis does not serve to replace the loss of the CA1 pyramidal neurons induced by transient ischemia. Nevertheless, it is of interest to note that the spontaneous regeneration of CA1 pyramidal neurons occurs after ischemic injury. Nakatomi et al 32 demonstrated the spontaneous regeneration of CA1 neurons after global cerebral ischemia using both BrdU administration and retroviral injection into the periventricular region. In addition, many studies have shown that an increased proliferation of neural progenitor cells starts at 3–4 days post‐ischemia, peaks at 7–10 days post‐ischemia and returns to the control level by 3–5 weeks after global cerebral ischemia 29, 47, 50, 52. However, Bendel and coworkers 37 reported that about 40% of CA1 neurons had reappeared 90 days after transient ischemic insult, whereas some researchers reported that the tertiary cell death had appeared between 90 and 125 days after ischemic damage 3, 51. Furthermore, Bueters et al 4 reporetd that newly formed CA1 cells had partly disappeared 250 days after ischemia. Considering their researches, we could not tell how new neurogenesis occurs in ischemic region with time after I‐R. In this study, we observed that many NeuN⁺ neurons were found near the stratum pyramidale of the CA1 region 180 days post‐ischemia and the NeuN⁺ neurons were colocalized with BrdU⁺ cells. Although BrdU immunocytochemistry is a powerful method in studies regarding adult neurogenesis, it has some shortcomings that can complicate studies on adult neurogenesis following brain injuries 33, 38. It is of more concern that damaged neurons in ischemic regions may re‐enter the cell cycle in a process of abortive DNA synthesis prior to dying via apoptosis 21. Thus, BrdU⁺ neurons in ischemic region cannot be assumed to be newly born. However, it has been thought that such damaged cells always die within 28 days 21, so analyses at longer points in time should eliminate such concerns. In addition, recent data suggest that some postmitotic neurons may survive long after taking up BrdU by DNA synthesis 6. We, in this study, found the significant increase of BrdU⁺/NeuN⁺ neurons near the stratum pyramidale of the CA1 region 180 days after I‐R. In addition, we recently reported that F‐J B⁺ cells in the stratum pyramidale were hardly observed from 60 days after I‐R 24. These findings are in agreement with the report by Teramoto et al 49, which showed a gradual increase of BrdU/NeuN double‐positive neurons up to 13 weeks after focal ischemia. Taken together, these findings suggest that NeuN⁺ cells are produced during the division of neural progenitor cells rather than through the repair process of preexisting neurons. Therefore, our present study indicates that neurogenesis in the ischemic CA1 region occurs very late after transient cerebral ischemia.

Another fundamental feature is that adult neural progenitor cells can regenerate into specific neuronal subtypes, which are appropriate for damaged sites. In the hippocampal DG, most of newly formed dentate granule cells differentiate into NeuN⁺ neurons by 3–4 weeks after ischemia 20. In the striatum, partial progenitor cells migrate into the striatum and develop into GAD67⁺ GABAergic and acetyltransferase⁺ cholinergic neurons 26. Furthermore, in the hippocampus proper, neurogenesis in adults may include some hippocampal cell types, which contain GABAergic neurons 41. On the basis of these findings, in this study, we found observed a significant increase of GABAergic neurons in the CA1 region 180 days after I‐R using GAD67 that is well known as one of excellent immunohistochemical markers for identifying GABAergic neurons 45, 46. In addition, we found that all of the BrdU⁺ cells were colocalized with GAD67 in the ischemic CA1 region 180 days after I‐R via double immunofluorescence staining for BrdU/GAD67. This finding indicates that a renewal of GABAergic neurons would contribute to plasticity and help to maintain the integrity of neural networks in the adult brain 11, because it was reported that newly generated neurons could develop into mature neurons displaying functional neurotransmission after cerebral ischemia in the adult brain 14.

Interestingly, BDNF acts as a powerful trophic agent for GABAergic population. Actually, BDNF increases the differentiation of hippocampal and neocortical inhibitory interneurons, promotes a GABAergic phenotype in hippocampal granule cells 12, 28, and enhances the expression of GAD67 in primary hippocampal cultures obtained from adult mice 1. In addition, BDNF stimulates neurogenesis in the SGZ or SVZ regions following cerebral ischemia 7, 40. In the present study, we investigated the change of BDNF immunoreactivity and its level in the ischemic CA1 region to explain its relationship with the increase of neurogenesis after I‐R. We found a significant increase of BDNF expression in the nonpyramidal cells, which were colocalized with GFAP⁺ astrocytes, at 30 days post‐ischemia, and the increased BDNF expression was maintained until 180 days post‐ischemia. It was reported that BDNF was expressed in astrocytes in the mouse hippocampus following transient cerebral ischemia and suggested that the increased BDNF production in astrocytes might be related with the enhancement of hippocampal neurogenesis after brain ischemia 34. Therefore, we state that BDNF mediates, at least in part, the enhancement of hippocampal neurogenesis induced by an ischemic insult, although neural progenitor cells may not produce BDNF; newly generated cells express BDNF receptor (TrkB) and can respond to BDNF 22. Conversely, to better understand the mechanism controlling cell differentiation in the ischemic CA1 region, we found that GAD67⁺ cells were co‐localized with TrkB when we examined 180 days post‐ischemia. Therefore, our observation suggests that BDNF may act on neural progenitor cells to promote their differentiation into GABAergic neurons via TrkB.

The pronounced deletion of CA1 pyramidal neurons is associated with severe impairments of hippocampal‐dependent brain functions 23. Cognitive function in ischemic animals is impaired at 14 days post‐ischemia, and the cognitive dysfunction subsides at 90 or 125 days post‐ischemia 3, 51. In particular, ischemic hippocampal damage leads to cognitive dysfunction as an evident from reduction in response latency in passive avoidance test 17. As expected, we found that cognitive dysfunction, as evaluated by the passive avoidance test, appeared from 4 days to 60 days after ischemia and improved significantly at 180 days post‐ischemia. This possibility is supported by a significant increase in NeuN⁺ neurons that were found at 180 days after post‐ischemia as compared with those at 60 days post‐ischemia. In addition, recent studies have demonstrated that enhanced learning and memory functions as well as behavioral effects are associated with neurogenesis in the hippocampus following cerebral ischemia 3.

Conversely, myelin is responsible not only for the protection and insulation of axons but also for the production of supportive neurotropic factors. Thus, a loss of myelin displays an adverse effect on axonal function and neuronal survival, and myelination may play an important role in cognitive function 31. In the present study, MBP staining showed that MBP immunoreactivity in the ischemic CA1 region was decreased from 4 days after I‐R, persisted until at least 60 days post‐ischemia and the immunoreactivity was restored to about 40% of the sham‐group 180 days after I‐R. Therefore, some renewal of myelinated nerve fibers in the ischemic CA1 region could contribute to the recovery of cognitive impairments, which suggests that the new CA1 neurons can restore disrupted neuronal circuits following cerebral ischemia.

In conclusion, our study provides the first evidence in the development of newly‐generated GABAergic neurons in the ischemic CA1 region and may enhance our understanding of neurogenesis in ischemic adult brain. Taken together, our findings suggest that increasing BDNF can enhance the induction of GABAergic neurons in the ischemic CA1 region very long time later after I‐R and that the renewal of GABAergic neurons in the ischemic CA1 region can partially restore disrupted neuronal circuit following cerebral ischemia.