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. 2012 May 4;32(7):1127–1138. doi: 10.1007/s10571-012-9837-z

Comparison of Glial Activation in the Hippocampal CA1 Region Between The Young and Adult Gerbils After Transient Cerebral Ischemia

Bing Chun Yan 1, Joon Ha Park 1, Ji Hyeon Ahn 2, Jung Hoon Choi 3, Ki-Yeon Yoo 4, Choong Hyun Lee 5, Jun Hwi Cho 6, Sung Koo Kim 7, Yun Lyul Lee 8, Hyung-Cheul Shin 8,✉, Moo-Ho Won 1,✉
PMCID: PMC11498410  PMID: 22555669

Abstract

It has been reported that young animals are less vulnerable to brain ischemia. In the present study, we compared gliosis in the hippocampal CA1 region of the young gerbil with those in the adult gerbil induced by 5 min of transient cerebral ischemia by immunohistochemistry and western blot for glial cells. We used male gerbils of postnatal month 1 (PM 1) as the young and PM 6 as the adult. Neuronal death in CA1 pyramidal neurons in the adult gerbil occurred at 4 days posti-schemia; the neuronal death in the young gerbil occurred at 7 days post-ischemia. The findings of glial changes in the young gerbil after ischemic damage were distinctively different from those in the adult gerbil. Glial fibrillary acidic protein-immunoreactive astrocytes, ionized calcium-binding adapter molecule (Iba-1), and isolectin B4-immunoreactive microglia in the ischemic CA1 region were activated much later in the young gerbil than in the adult gerbil. In brief, very less gliosis occurred in the hippocampal CA1 region of the young gerbil than in the adult gerbil after transient cerebral ischemia.

Keywords: Ischemia/reperfusion, Pyramidal neurons, Delayed neuronal death, Astrocytes, Microglia

Introduction

Stroke, which is induced by a lacking of cerebral blood flow, is the third leading cause of death. Recently, stroke has been increased in young people. Childhood stroke is one of the top ten causes of death in the U.S., and it has a high risk of serious morbidity for survivors (Ganesan et al. 2000). In India, 10–15 % of strokes occur in young people (Tripathi and Vibha 2010). Stroke or ischemic damage varies according to age (Blanco et al. 2007; Montori et al. 2010a; Nagayama et al. 1999; Saucier et al. 2007; Schaller 2007; Won et al. 2006).

Transient cerebral ischemia induced by the deprivation of blood flow via both carotid arteries occlusion to the brain causes delayed neuronal death in some specific vulnerable regions such as hippocampus and neocortex (Buiatti de Araujo et al. 2008; Himeda et al. 2007; Hu et al. 2007; Lorrio et al. 2009; Satoh et al. 2011). It has been reported that young animals appear less vulnerable to ischemia (Chen et al. 2009; Oguro et al. 2004; Tortosa and Ferrer 1994). Many studies have focused on neuronal death using adult gerbils (Brahma et al. 2009; Shiraishi et al. 2011; Yamashita et al. 2010). In addition, in aging studies, neuronal death induced by ischemia occurs much later in the aged than in the adult (Crain et al. 1988; Lee et al. 2010; Tamagaki et al. 2000). The gerbil is one of useful animal models of transient cerebral ischemia for the assessment of potential therapeutic strategy for human stroke (Canistro et al. 2010; Crain et al. 1988; Lin et al. 1990; Szilagyi et al. 2009; Zhang et al. 2009). Some studies have shown that developing gerbil is resistant to ischemic damage (Kusumoto et al. 1995; Oguro et al. 2004; Tortosa and Ferrer 1994). Kusumoto et al. (1995) has reported that less neuronal death occurred in young gerbils after 5 min of ischemia/reperfusion (I/R). Recently, we compared neuronal damage in the ischemic CA1 region between the young and adult gerbils at various times after I/R (Yan et al. 2011).

On the other hand, astrocytes play important roles during several processes, such as neurotransmission and neuronal functions, in the central nervous system (Horner and Palmer 2003). Microglia, as principle immune cells in the brain, defend the brain against pathological events in the central nervous system. They participate in the repair and regeneration of damaged neurons and cause neuronal death or dysfunction (Boje and Arora 1992; Nagayama et al. 1999). The gliosis of astrocytes and microglia are easily induced in the hippocampus after transient cerebral ischemia in the adult gerbil (Bonnekoh et al. 1990; Hwang et al. 2006). In the present study, we compared gliosis in the hippocampal CA 1 region at various time points after 5 min of ischemia/reperfusion between the young gerbil at postnatal month (PM) 1 and the adult gerbil at PM 6.

Materials and Methods

Experimental Animals

We used the progeny of male Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. Mongolian gerbils were used at 1 month (young, B.W. 25–30 g) of age for the young group and 6 months (adult, B.W. 65–75 g) of age for the adult group. The animals were housed in a conventional state under adequate temperature (23 °C) and humidity (60 %) control with a 12-h light/12-h dark cycle, and were provided with free access to water and food. Procedures involving animals and their care conformed to the guidelines, which are in compliance with current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996). All experiments were conducted to minimize the number of animals used and suffering caused.

Induction of Transient Cerebral Ischemia

The animals were anesthetized with a mixture of 2.5 % isoflurane in 33 % oxygen and 67 % nitrous oxide. A midline ventral incision was then made in the neck, and bilateral common carotid arteries were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. The restoration of blood flow (reperfusion) was observed directly using the ophthalmoscope. Body (rectal) temperature was maintained under free-regulating or normothermic (37 ± 0.5 °C) conditions with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA) and a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. Thereafter, animals were kept in the thermal incubator (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature until the animals were euthanized. Sham-operated animals were subjected to the same surgical procedures except that the common carotid arteries were not occluded.

Tissue Processing for Histology

For histology, sham- and ischemia-operated young and adult gerbils (n = 7 at each time point) at designated times (1, 4, 7, 10, and 15 days after reperfusion) were euthanized. The animals 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 M phosphate-buffer (PB, pH 7.4). The brains were removed and postfixed in the same fixative for 6 h. The brain tissues were cryoprotected by infiltration with 30 % sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Germany) into 30-μm coronal sections, and they were then collected into six-well plates containing PBS.

Hematoxylin-Eosin (H-E) Staining

To investigate the morphological changes and neuronal changes in the CA1 region after I/R, H-E staining was performed. In brief, the sections were mounted on gelatin-coated microscopy slides and stained with H-E according to the general method. After they were stained, they were dehydrated and mounted with Canada balsam (Kanto, Tokyo, Japan).

NeuN Immunohistochemistry

To investigate the neuronal changes in the CA1 region after I/R, NeuN was used in this study. The sections were sequentially treated with 0.3 % hydrogen peroxide (H2O2) in PBS for 30 min and 10 % normal goat serum in 0.05 M PBS for 30 min. The sections were next incubated with diluted mouse anti-NeuN (1:1000, Chemicon, Temecula, CA) overnight at 4 °C. Thereafter, the tissues were exposed to biotinylated goat anti-mouse IgG (Vector, Burlingame, CA) and streptavidin peroxidase complex (1:200, Vector); they were visualized by staining using 3,3′-diaminobenzidine tetrahydrochloride in 0.1 M Tris–HCl buffer (pH 7.2) and mounted on gelatin-coated slides. After dehydration, the sections were mounted with Canada balsam (Kanto, Tokyo, Japan).

Cell Counts

All measurements were performed to insure objectivity in blind conditions, by two observers for each experiment, carrying out the measures of control and experimental samples under the same conditions. The studied tissue sections were selected according to anatomical landmarks corresponding to AP from −1.4 to −1.8 mm of gerbil brain atlas (Loskota et al. 1974). The number of NeuN-immunoreactive neurons and H-E positive cells were counted in a 250 × 250-μm square, applied approximately at the center of the CA1 region in the stratum pyramidale. Cell counts were obtained by averaging the total cell numbers from each animal per group: A ratio of the count was calibrated as %.

Immunohistochemistry for GFAP, Iba-1, and Isolectin B4 (IB4)

In order to examine the change of astrocytes and microglia in the CA1 region after I/R, we carried out immunohistochemical staining with rabbit anti-glial fibrillary acidic protein (GFAP, 1:800, Chemicon, Temecular) for astrocyte, rabbit anti-ionized calcium-binding adapter molecule (Iba-1, 1:500, Wako, Japan) and anti-peroxidase-conjugated isolectin B4 (1:50, Sigma) for microglia, and biotinylated goat anti-rabbit IgG (Vector, Burlingame, CA) for secondary antibody according to the above mentioned-method (see the NeuN immunohistochemistry). In order to quantitatively analyze GFAP, Iba-1, and IB4 immunoreactivity, the corresponding areas of the hippocampal CA1 region were measured from 15 sections per animal. Images of all GFAP- and Iba-1-immunoreactive structures were taken from three layers (strata oriens, pyramidale, and radiatum in the hippocampus proper) through an AxioM1 light microscope (Carl Zeiss, 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 140 × 140 μm (40 × primary magnification). The densities of all GFAP- and Iba-1-immunoreactive structures were evaluated on the basis of optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log (256/mean gray level). The OD of background was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the optical density of image file was calibrated as % (relative optical density, ROD) using Adobe Photoshop version 8.0 and then analyzed using NIH Image 1.59 software.

Western Blot Analysis

In order to examine the protein levels of glia in the ischemic CA1 region, the animals (n = 7 in each group) were used for western blot analysis sham 4 and 7 days after the ischemic surgery in the young and adult group. After euthanizing them and removing the hippocampus, it was serially and transversely cut into a thickness of 400 μm on a vibratome (Leica, Germany), and the hippocampal CA1 region was then dissected using a surgical blade. The tissues were homogenized in 50 mM PBS (pH 7.4) containing EGTA (pH 8.0), 0.2 % NP-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM PMSF, and 1 mM DTT. After centrifugation, and the protein level in the supernatants was determined using a Micro BCA protein assay kit using bovine serum albumin as a standard (Pierce Chemical, USA). Aliquots containing 50 μg of total protein were boiled in loading buffer containing 250 mM Tris (pH 6.8), 10 mM DTT, 10 % SDS, 0.5 % bromophenol blue, and 50 % glycerol. The aliquots were then loaded onto a suitable polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Crop, East Hills, NY, USA). In order to incubate antibodies, the same nitrocellulose membrane strips were used. To reduce background staining, the membranes were incubated with 5 % non-fat dry milk in TBS containing 0.1 % Tween 20 for 45 min, followed by incubation with GFAP (diluted 1:400, Chemicon, Temecular) and Iba-1 (diluted 1:200, Wako) overnight at 4 °C and subsequently exposed to peroxidase-conjugated goat anti-mouse IgG (Santa Cruz, USA), goat anti-rabbit IgG (Santa Cruz, USA), and an ECL kit (Amersham, UK).

Statistical Analysis

Data are expressed as the mean ± SEM. The data were evaluated by an one-way ANOVA SPSS program, and the means were assessed by Duncan’s multiple-range test. Statistical significance was considered at P < 0.05.

Results

Neuronal Damage

H-E Staining

Morphological changes in the ischemic CA1 region of the adult and young and gerbils were examined by H-E staining. One day after I/R, cells in the CA1 region were similar to those in the adult-ischemia group (Fig. 1c, c′, m). However, 4 days after I/R, H-E positive cells were apparently damaged, and the mean percentage of H-E positive cells was significantly decreased compared to that of the adult-sham group (Fig. 1e, e′, m). Thereafter, the findings and the number of H-E positive cells were similar to those in the 4 days post-ischemia group until 15 days after I/R (Fig. 1g, g′, i, i′, k, k′, m).

Fig. 1.

Fig. 1

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H-E staining in the CA1 region of the adult (left two columns) and young (right two columns) of the sham- (a–b′) and ischemia- (c-l′) groups. In the adult group, significantly decreased H-E positive cells are shown in the stratum pyramidale (SP) (asterisks) 4 day after I/R. In the young group, many H-E positive cells are lost in the SP (asterisks) 7 days after I/R. SO stratum oriens, SR stratum radiatum. Scale bar = 200 μm (low magnification photos, b–l), 50 μm (high magnification photos, a′–l′). M Relative analysis as percent in the number of H-E positive cells in the CA1 region (n = 7 per group; * P < 0.05, significantly different from the corresponding sham group, # P < 0.05, significantly different from the respective preceding group; + P < 0.05, significantly different from the corresponding adult-group). The bars indicate the means ± SEM

In the young-ischemia groups, the morphology and the number of H-E positive cells was not changed 1 to 4 days after I/R (Fig. 1d, d′, f, f′, m). However, 7 days after I/R, H-E positive cells in the ischemic CA1 region were damaged and the number of the cells was significantly decreased; the number was much more than that of the corresponding adult-ischemia group (Fig. 1h, h′, m). Ten and 15 days after I/R, the number of H-E positive cells was similar to that at 7 days post-ischemia (Fig. 1j, j′, l, l′, m).

NeuN-Immunoreactive Cells

Numerous NeuN-immunoreactive cells were well distributed in the CA1 region of the adult- and young-sham groups (Fig. 2a, a′, b, b′). One day after I/R, no change in NeuN-immunoreactive neuronal numbers was found in the CA1 region of the adult-ischemia group (Fig. 2c, c′, m). At 4 days after I/R, a significant loss of NeuN-immunoreactive neurons was observed in the CA1 region in the adult group: The mean percentage of NeuN-immunoreactive neurons 12 % of that in the adult-sham group (Fig. 2e, e′, m). Thereafter, the number of NeuN-immunoreactive neurons was not changed in the adult-ischemia group (Fig. 2h, h′, j, j′, l, l′, m).

Fig. 2.

Fig. 2

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Immunohistochemistry for NeuN in the CA1 region of the adult (left 2 columns) and young (right 2 columns) of the sham- (a–b′) and ischemia- (c–l′) groups. In the adult group, a few NeuN immunoreactive cells are shown in the stratum pyramidale (SP) 4 days after I/R (asterisk), However, in the young group, many NeuN immunoreactive cells are lost in the SP 7 days after I/R (asterisks). SO stratum oriens, SR stratum radiatum. Scale bar = 200 μm (low magnification photos, a–l), 50 μm (high magnification photos, a′–l′). M Relative analysis as percent in the number of NeuN positive cells in the CA1 region (n = 7 per group; * P < 0.05, significantly different from the corresponding sham group, # P < 0.05, significantly different from the respective preceding group; + P < 0.05, significantly different from the corresponding adult-group). The bars indicate the means ± SEM

In the young-ischemia group at 1 and 4 days post-ischemia, NeuN-immunoreactive neurons in the CA1 region was similar to those in the young-sham group (Fig. 1d, d′, f, f′, m). However, at 7 days after I/R, the number of NeuN-immunoreactive neurons in the CA1 region was significantly decreased (mean percentage, 41 % of the young-sham group) (Fig. 2h, h′ m). Thereafter, in the young-ischemia groups, the number of NeuN-immunoreactive neurons was similar to that in the 7 days post-ischemia group (Fig. 2j, j′, l, l′, m).

Glial Activation

GFAP-Immunoreactive Astrocytes

In the adult- and young-sham group, GFAP-immunoreactive astrocytes, which showed a resting form (a small body with thread like thin process), were distributed throughout the CA1 region (Fig. 3a–b′).

Fig. 3.

Fig. 3

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Immunohistochemistry for GFAP in the CA1 region of the adult (left 2 columns) and young (right 2 columns) of the sham- (a–b′) and ischemia- (c–l′) groups. Many reactive astrocytes (arrows) are detected from 4 and 7 days after I/R, respectively, in the adult- and young-group. SO stratum oriens, SP stratum pyramidale, SR stratum radiatum. Scale bar = 200 μm (low magnification photos, a–l), 50 μm (high magnification photos, a′–l′). M: Relative optical density as % of GFAP-immunoreactive structures in the adult- and young-group (n = 7 per group; * P < 0.05, significantly different from the young-sham group, # P < 0.05, significantly different from the respective preceding group; + P < 0.05, significantly different from the corresponding adult-group). The bars indicate the means ± SEM

In the adult-ischemia group, 1 day after I/R, the morphology of GFAP-immunoreactive astrocytes and their GFAP immunoreactivity were similar to those in the adult-sham group (Fig. 3c, c′, m). However, at 4 days post-ischemia, GFAP-immunoreactive astrocytes with reactive form were markedly increased, and their GFAP immunoreactivity was higher than that in the adult-sham group (Fig. 3e, e′, m). Thereafter, GFAP immunoreactivity in the CA1 region was still increased until 10 days after I/R (Fig. 3g, g′, i, i′, m). GFAP immunoreactivity, then, was slightly decreased 15 days after I/R (Fig. 3k, k′, m).

GFAP-immunoreactive astrocytes in the young-ischemia group were not markedly changed until 4 days after I/R (Fig. 3d, d′, f, f′). Seven days after I/R, many of the GFAP-immunoreactive astrocytes showed reactive form: A punctuated cytosol with thick processes was observed in the CA1 region, and their GFAP immunoreactivity was increased at this time point (Fig. 3h, h′, m). Thereafter, reactive GFAP-immunoreactive astrocytes were increased until 10 days after I/R, and GFAP immunoreactivity and reactive processes in astrocytes were decreased 15 days after I/R in the young-ischemia group (Fig. 3j, j′, l, l′, m).

Iba-1-Immunoreactive Microglia

In the adult- and young-sham groups, Iba-1-immunoreactive microglia cells were ubiquitously distributed in all the layers of the CA1 region, and they had fine processes with web-like network characteristics of ramified or resting microglia (Fig. 4a–b′).

Fig. 4.

Fig. 4

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Immunohistochemistry for Iba-1 in the CA1 region of the adult (left 2 columns) and young (right 2 columns) of the sham- (a–b′) and ischemia- (c–l′) groups. Iba-1-immunoreactive microglia activation (arrows) is observed from 1 day after I/R in both the groups. The aggregation (asterisks) of Iba-1-immunoreactive microglia in the stratum pyramidale (SP) is observed 4 and 7 days after I/R, respectively, in the adult and young group. SO stratum oriens, SR stratum radiatum. Scale bar = 200 μm (low magnification photos, a–l), 50 μm (high magnification photos, a′–l′). M Relative optical density as % of Iba-1-immunoreactive structures in the adult- and young-groups (n = 7 per group; * P < 0.05, significantly different from the corresponding sham group, # P < 0.05, significantly different from the respective preceding group; + P < 0.05, significantly different from the corresponding adult-group). The bars indicate the means ± SEM

In the adult-ischemia group, morphological change in microglia was detected 1 day after I/R: Iba-1-immunoreactive microglia had an enlarged body with short and thicken processes, which was an activated form of microglia (Fig. 4c, c′); however, the immunoreactivity of Iba-1-immunoreactive structures was not markedly changed in this group (Fig. 4m). At 4 days post-ischemia, Iba-1-immunoreactive microglia were aggregated near the stratum pyramidale, and their Iba-1 immunoreactivity was significantly increased: This pattern continued until 10 days after I/R (Fig. 4e, e′, g, g′, i, i′, m). At 15 days post-ischemia, activated Iba-1 immunoreactive microglia in the stratum pyramidale were decreased and their Iba-1 immunoreactivity was slightly decreased (Fig. 4k, k′, m).

In the young-ischemia group, the morphological changes of Iba-1-immunoreactive microglia were also observed in the CA1 region from 1 day to 4 days after I/R; however, the immunoreactivity of Iba-1-immunoreactive structures was not markedly changed until 4 days post-ischemia compared to that in the young-sham group (Fig. 4f, f′, m). At 7 days post-ischemia, activated Iba-1-immunoreactive microglia were markedly aggregated near the stratum pyramidale, where neuronal degeneration occurred in the young-ischemia group, and their Iba-1 immunoreactivity was significantly increased (Fig. 4h, h′, m). Ten days after I/R, the immunoreactivity of Iba-1-immunoreactive microglia was more increased (Fig. 4j, j′, m). At 15 days post-ischemia, the number of activated Iba-1-immunoreactive microglia was reduced in the stratum pyramidale, and its immunoreactivity was slightly decreased compared to that in the 10 days young-ischemia group (Fig. 4l, l′, m).

IB4-Immunoreactive Microglia

In the adult- and young-sham groups, IB4-immunoreactive microglia was not found in any layers of the CA1 region (Fig. 5a–b′).

Fig. 5.

Fig. 5

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Immunohistochemistry for IB4 in the CA1 region of the adult (left 2 columns) and young (right 2 columns) of the sham- (a–b′) and ischemia- (c–l′) groups. IB4-immunoreactive microglia activation and aggregation (asterisks) in the stratum pyramidale (SP) are observed 4 and 7 days after I/R, respectively, in the adult and young group. SO stratum oriens, SR stratum radiatum. Scale bar = 200 μm (low magnification photos, a–l), 50 μm (high magnification photos, a′–l′). M Relative optical density as % of IB4-immunoreactive structures in the adult- and young-groups (n = 7 per group; * P < 0.05, significantly different from the corresponding sham group, # P < 0.05, significantly different from the respective preceding group; + P < 0.05, significantly different from the corresponding adult-group). The bars indicate the means ± SEM

In the adult-ischemia group, morphological change in microglia was detected 4 day after I/R: IB4-immunoreactive microglia were aggregated near the stratum pyramidale and their immunoreactivity was significantly increased (Fig. 5e, e′, m). However, IB4-immunoreactive microglia in the stratum pyramidale were significantly decreased from 7 days after I/R, and their immunoreactivity was dramatically decreased (Fig. 5g, g′, i, i′, k, k′, m).

In the young-ischemia group, IB4-immunoreactive microglia were hardly observed in the CA1 region 4 days after I/R (Fig. 5f, f′, m). Seven days after I/R, IB4-immunoreactive microglia were markedly increased and they were aggregated in the stratum pyramidale where neuronal degeneration occurred (Fig. 5h, h′, m). Thereafter, the immunoreactivity of IB4-immunoreactive microglia was dramatically decreased (Fig. 5j, j′, l, l′, m).

Levels of GFAP and Iba-1

In this study, we examined the levels of GFAP and Iba-1 proteins in the CA1 region after I/R (Fig. 6). The levels of GFAP and Iba-1 in the young-sham group were similar to those in the adult-sham group.

Fig. 6.

Fig. 6

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Western blot analysis of GFAP and Iba-1 in the CA1 of the young and adult groups after I/R. At 4 and 7 days post-ischemia, the levels of GFAP and Iba-1 are much lower in the young-ischemia group than those in the adult-ischemia group (n = 7 per group; * P < 0.05, significantly different from the corresponding sham-group, # P < 0.05, significantly different from the respective preceding group; + P < 0.05, significantly different from the corresponding adult-group). The bars indicate the means ± SEM

In the ischemia groups, the levels of all the proteins were changed with time. At 4 days post-ischemia, all the levels of the proteins in the adult-ischemia group were apparently increased in the ischemic CA1 region; however, in the young-ischemia group, their levels were not distinctively changed. Seven days after I/R, GFAP levels in both the groups were increased: The level in the adult-ischemia group was much higher than that in the young-ischemia group. At this time after I/R, Iba-1 levels in both the groups were slightly increased compared to those in the 4 days post-ischemia groups.

Discussion

In the present study, we compared neuronal damage in the CA1 region of the young gerbil with that in the adult gerbil after 5 min of I/R by H-E staining and NeuN immunohistochemistry. The delayed neuronal death of the CA1 pyramidal neurons was found in the adult gerbil 4 days after I/R: This finding is coincident with previous studies (Kirino 1982; Lee et al., 2011). Recently, we reported that the pattern of neuronal damage in the ischemic CA1 region of the young gerbil was very different from that in the adult gerbil at various times after I/R (Yan et al. 2011). In the present study, we also observed that the delayed neuronal death in the CA1 region of the young gerbil after I/R occurred much later than in the adult gerbil: Neuronal death was not observed in the CA1 region of the young group until 4 days after I/R; however, distinctive neuronal death was observed in many CA1 pyramidal neurons at 7 days post-ischemia. In addition, the number of the survived CA1 neurons in the young-ischemia group was much higher than that in the adult-ischemia group: About 35 % of CA1 pyramidal neurons remained in the CA 1 region until 15 days after I/R. This result indicates that young gerbils may be more resistant to ischemic insult compared with adult gerbils.

In the present study, we compared the changes of astrocytes and microglia in the ischemic CA1 region between the young and adult gerbils after I/R. The hallmark of astrocytes’ response to a brain injury is an increase in the level of expression of GFAP (Kato et al. 1994; Petito and Halaby 1993). It was reported that changes in GFAP immunoreactivity in the ischemic CA1 region was associated with neuronal death (Ordy et al. 1993; Petito and Halaby 1993; Steward et al. 1992; Stoll et al. 1998). In the present study, marked increases of GFAP immunoreactivity and reactive GFAP-immunoreactive astrocytes after I/R were observed in the young and adult groups. However, time points of the increases were different (7 and 4 days after I/R in the young- and adult-ischemia group, respectively). Some researchers reported that the down-regulation of astrocyte level, as a marker decreasing the cerebral ischemic inflammation, offered a neuroprotective mechanism (Montori et al. 2010b). In addition, our previous studies showed that decreased astrocyte activation, which was produced by some extracts from plants, could protect against neuronal damage induced by cerebral ischemia (Park et al. 2011; Yoo et al. 2011). Therefore, we can postulate that more delayed astrocyte activation in the young-ischemia group must be related to the time of neuronal death that occurred in the ischemic CA1 region of the young gerbil.

Morphological and functional changes in microglia are involved in the response to various neural environments (Hailer et al. 1996; Schwartz et al. 2006). It was reported that microglia activation was significantly increased in the infarct region following focal ischemia and in the CA1 region following global ischemia (Mabuchi et al. 2000; Stoll et al. 1998; Sugawara et al. 2002). Iba-1 and IB4 have been widely used as reliable markers for microglia (Gehrmann et al. 1992; Lee et al. 2010; Yu et al. 2010; Zemtsova et al. 2011). We previously reported that Iba-1 and IB4 microglia were apparently aggregated in the stratum pyramidale of the ischemic CA1 region and their immunoreactivity was significantly increased from 4 days post-ischemia (Lee et al. 2010; Yu et al. 2010). In the present study, many activated Iba-1 and IB4-immunoreactive microglia were aggregated in the stratum pyramidale of the CA1 region in both the young and adult groups. However, the peak time of microglia activation and their aggregation in the CA1 region was 7 days post-ischemia and 4 days post-ischemia, respectively, in the young- and adult-ischemia group. In addition, the number of activated microglia and their immunoreactivity in the young-ischemia group were much lower than those in the adult-ischemia group. It was reported that the inhibition of microglia activation was beneficial for the treatment of brain disorder such as Parkinson’s disease and cerebral ischemia (Chung et al. 2011; Lee et al. 2003). In addition, Yu et al. (2010) showed that decreased microglia activation contributed to neuronal survival. Therefore, our present results, including more delayed, lower microglia activation and aggregation, must be associated with more delayed and lower neuronal death in the young gerbil.

In conclusion, we found that the activation of astrocytes and microglia in the young gerbil was much lower and occurred much later than those in the adult gerbil. These results indicate that the young gerbil must be much more resistant to an ischemic insult than the adult gerbil.

Acknowledgments

The authors would like to thank Mr. Seung Uk Lee and Ms. Hyun Sook Kim for their technical help in this study. This work was supported by a Grant (2010K000823) from the Brain Research Center of the twentyfirst Century Frontier Research Program funded by the Ministry of Education, Science and Technology, the Republic of Korea, and by the Technology Innovation Program funded by the Ministry of Knowledge Economy (MKE, Korea).

Footnotes

Bing Chun Yan and Joon Ha Park contributed equally to this article.

Contributor Information

Hyung-Cheul Shin, Phone: +82-33-248-2580, Email: hcshin@hallym.ac.kr.

Moo-Ho Won, Phone: +82-33-250-8891, FAX: +82-33-256-1614, Email: mhwon@kangwon.ac.kr.

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