- Email: [email protected]
Differences in neuronal damage and gliosis in the hippocampus between young and adult gerbils induced by long duration of transient cerebral ischemia
Journal of the Neurological Sciences 337 (2014) 129–136
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Differences in neuronal damage and gliosis in the hippocampus between young and adult gerbils induced by long duration of transient cerebral ischemia Bing Chun Yan a,1, Taek Geun Ohk b,1, Ji Hyeon Ahn c, Joon Ha Park c, Bai Hui Chen d, Jae-Chul Lee c, Choong Hyun Lee e, Myoung Cheol Shin f, In Koo Hwang g, Seung Myung Moon h, Jun Hwi Cho f,i,⁎, Moo-Ho Won c,i,⁎⁎ a
Department of Integrative Traditional & Western Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu 225001, China Department of Emergency Medicine, Kangnam Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 150-950, South Korea c Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea d Department of Physiology, College of Medicine, Institute of Neurodegeneration and Neuroregeneration, Hallym University, Chuncheon 200-702, South Korea e Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan 330-714, South Korea f Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea g Department of Anatomy and Cell Biology, College of Veterinary Medicine, Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, South Korea h Department of Neurosurgery, Dongtan Sacred Heart Hospital, College of Medicine, Hallym University, Hwaseong 445-170, South Korea i Institute of Medical Sciences, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea b
a r t i c l e
i n f o
Article history: Received 13 September 2013 Received in revised form 29 October 2013 Accepted 20 November 2013 Available online 1 December 2013 Keywords: Young gerbil Ischemia–reperfusion injury Pyramidal neurons Delayed neuronal death Astrocytes Microglia
a b s t r a c t Response to cerebral ischemia in young animals was very different from that in the adult. The aim of this study was to investigate differences in neuronal death and gliosis in the hippocampal CA1 region (CA1) between adult and young gerbils following 5 and 15 min of transient cerebral ischemia. Delayed neuronal death (DND) of pyramidal cells occurred in the CA1 was similar in all the adult gerbils after 5 and 15 min of ischemia: the DND occurred 4 days after ischemia. In the young groups, DND of pyramidal cells in the CA1 region occurred 7 and 3 days after 5 and 15 min of ischemia, respectively. On the other hand, the activation of GFAP-immunoreactive (+) astrocytes and Iba-1+ microglia was different in the young groups from the adult groups after ischemia. The change pattern of GFAP immunoreactivity in the adult groups was similar in both the adult groups after ischemia; in the young groups, the activation of GFAP+ astrocytes after 5 min of ischemia was much delayed than that after 15 min of ischemia. Activated Iba-1+ microglia were aggregated in the stratum pyramidale 4 days after ischemia in all the adult ischemia-operated groups; in the young groups, activated Iba-1+ microglia were aggregated in the stratum pyramidale 7 days after 5 min of ischemia and 3 days after 15 min of ischemia. These observations indicate that DND in young animals is very different from the adult according to different duration of transient cerebral ischemia and glial activation is very different in young animals after different duration of transient ischemia. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Stroke is the third leading cause of death and can be subdivided into two types, ischemic and hemorrhagic. Ischemic stroke, accounting for approximately 87% of all cases, is more common than hemorrhagic stroke [1,2]. Recently, it has been reported that stroke has been ⁎ Correspondence to: J.H. Cho, Department of Emergency Medicine, College of Medicine, Kangwon National University, Chuncheon 200-701, South Korea. Tel.: +82 33 258 2378; fax: +82 33 258 2451. ⁎⁎ Correspondence to: M.-H. Won, Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chuncheon 200-702, South Korea. Tel.: +82 33 248 2522; fax: +82 33 256 1614. E-mail addresses: [email protected] (J.H. Cho), [email protected] (M.-H. Won). 1 Bing Chun Yan and Taek Geun Ohk contributed equally to this article. 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.11.034
increased in young people. As a high risk of serious morbidity for survivors, childhood stroke is one of the top ten causes of death among childhood disease in the U.S.A. [3,4]. Stroke or ischemic damage shows various characteristics according to age [5,6]. In both humans and animals, transient cerebral ischemia, which is induced by the deprivation of blood flow via both carotid arteries occlusion to the brain, damages some specific vulnerable regions such as the hippocampus [7]. The CA1 region of the hippocampus is the most vulnerable to transient cerebral ischemia [8]. Especially, neuronal death in the CA1 region is called “delayed neuronal death” because the death occurs very slowly [9,10]. Gliosis of astrocytes and microglia is easily induced in the hippocampus after transient cerebral ischemia in the adult gerbil [4,11]. Both astrocytes and microglia participate in the repair and regeneration of damaged neurons and cause neuronal death or dysfunction [12].
130
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
The gerbil is commonly used for making a good animal model of transient cerebral ischemia [13–15]. Many studies have focused on neuronal death using adult gerbils [16,17], and some reports regarding ischemia in the aged gerbil have demonstrated that neuronal death induced by transient cerebral ischemia occurs much later in the aged than in the adult [16–18]. Some studies have shown that the young gerbil is resistant to ischemic damage [19–21]. Recently, we compared neuronal damage in the ischemic CA1 region between the young and adult gerbils after 5 min of transient cerebral ischemia and found that the neuronal death in the hippocampal CA1 region of the young was more delayed and less than that in the adult [22]. However, in the young, few studies on neuronal damage according to longer duration of ischemia in the brain are reported. Therefore, the present study was undertaken to identify comparably damaged degree and glial changes in the young and adult gerbil hippocampus following 5 and 15 min of transient cerebral ischemia, respectively.
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. 2.4. Staining for neuronal damage 2.4.1. CV staining To investigate the delayed neuronal death in the CA1 after ischemia– reperfusion, sham-operated and ischemia-operated young and adult gerbils were used at designated times for CV staining. In brief, the sections were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma, MO) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid was added to this solution. The sections were stained and dehydrated by immersing in serial ethanol baths, and they were then mounted with Canada balsam (Kanto, Tokyo, Japan).
2. Materials and methods 2.1. Experimental animals We used male Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Kangwon National University, Chuncheon, South Korea. Mongolian gerbils were used at 1 (B.W.: 25–30 g) and 6 months (B.W.: 65–75 g) of age for the young and 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 provided with free access to water and food. 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 the animal protocol used in the present study was reviewed and approved based on ethical procedures and scientific care by the Kangwon National University-Institutional Animal Care and Use Committee (KIACUC-12-0018). All of the experiments were conducted to minimize the number of animals used and the suffering caused by the procedures used in the present study. 2.2. 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 retina using an ophthalmoscope. After 5 and 15 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 on 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. 2.3. Tissue processing for histology For histology, sham- and ischemia-operated young and adult gerbils (n = 7 at each time point) at designated times (3, 4 and 7 days after reperfusion) were sacrificed. The animals were anesthetized with 40 mg/kg pentobarbital sodium and perfused transcardially with 0.1 M phosphatebuffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M
2.4.2. NeuN immunohistochemistry To investigate the neuronal death in the CA1 region after transient cerebral ischemia, the sham-operated and ischemia-operated young and adult gerbils were used for NeuN immunohistochemistry under the same conditions. 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). And they were visualized by staining with 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). 2.4.3. F-J B histofluorescence staining To investigate neuronal degeneration in the CA1 region after ischemia–reperfusion, F-J B (a high affinity fluorescent marker for the localization of neuronal degeneration) histofluorescence staining was performed with a previous method [16]. In brief, the sections were transferred to a solution of 0.06% potassium permanganate, and then transferred to a 0.0004% F-J B (Histochem, Jefferson, AR) staining solution. The sections were examined using an epifluorescent microscope (Carl Zeiss, Germany) with blue (450–490 nm) excitation light and a barrier filter. 2.5. Immunohistochemistry for astrocytes and microglia In order to examine the degree of reactive gliosis in the CA1 region in the young and adult gerbils after ischemia–reperfusion, we carried out immunohistochemical staining under the same conditions with mouse anti-GFAP (1:800, Chemicon, Temecular) for astrocytes and rabbit antiIba-1 (1:800, Wako) for microglia according to the above-mentioned method (see NeuN immunohistochemistry). The sections were exposed to biotinylated horse anti-mouse, goat anti-rabbit IgG (1:200, Vector) for GFAP and Iba-1, and streptavidin peroxidase complex (1:200, Vector). And they were visualized with 3,3′-diaminobenzidine tetrahydrochloride in 0.1 M Tris–HCl buffer and mounted on the gelatin-coated slides. After dehydration the sections were mounted in Canada balsam (Kanto Chemical, Japan). 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 3 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 mean intensity of immunoreactivity in each immunoreactive structure
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
131
Fig. 1. Low magnification of CV staining in the adult (left 2 columns) and young (right 2 columns) hippocampus of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. Low CV stainability is detected in the CA1 region (asterisks) of the adult 5 min and 15 min ischemia-operated groups 4 days after ischemia–reperfusion. In the young, CV stainability in the CA1 region (asterisks) is apparently reduced at 7 and 3 days after 5 and 15 min of ischemia, respectively. CA; cornus ammonis, DG; dentate gyrus. Scale bar = 400 μm.
was measured by a 0–255 gray scale system (white to dark signal corresponded from 255 to 0). Based on this approach, the level of immunoreactivity was scaled as −, ±, + or ++, representing no staining (gray scale value: ≥200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤99), respectively.
2.6. Statistical analysis Data are expressed as the mean ± SEM. The data were evaluated by a Tukey test for post-hoc multiple comparisons following one-way ANOVA. Statistical significance was considered at P b 0.05.
Fig. 2. High magnification of CV staining in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the adult, significant disappearance of CV+ cells (asterisks) is found in both the 5 and 15 min ischemia-operated groups 4 days after ischemia–reperfusion; however, in the young, CV+ cells (asterisks) are reduced at 7 and 3 days after 5 and 15 min of ischemia, respectively. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm.
132
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
Fig. 3. Immunohistochemistry for NeuN in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the adult, NeuN+ neurons are hardly detected in the adult 5 min and 15 min ischemia-operated groups (asterisks) 4 days after ischemia. In the young, NeuN+ neurons are apparently reduced 7 and 3 days after ischemia in the 5 and 15 min ischemia-operated group, respectively. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm. O: Relative analysis as percent in the number of NeuN+ neurons in the CA1 region (n = 7 per group; *P b 0.05, significantly different from the corresponding sham group, #P b 0.05, significantly different from the respective preceding group; †P b 0.05, significantly different from the same day of other groups). The bars indicate the means ± SEM.
3. Results
group were apparently decreased; however, their morphology was a little better than that in the other groups (Figs. 1M–P and 2M–P).
3.1. Cresyl violet (CV) positive (+) cells 3.2. Neuronal nuclei (NeuN)+ neurons Neuronal death in the ischemic CA1 region of the adult and young gerbils was examined by CV staining. In the sham-operated groups, CV + cells were easily observed in all the sub-regions of the hippocampus in the adult and young gerbils (Figs. 1A–D and 2A–D). Three days after ischemia–reperfusion, significant change in CV+ cells was found only in the young 15 min ischemia-operated group compared to those in the sham-operated groups. Most of CV+ cells in the stratum pyramidale of the CA1 region became much smaller than those of the other groups (Figs. 1E–H and 2E–H). Four days after ischemia–reperfusion, a few CV+ cells were detected in the stratum pyramidale of the CA1 region in the adult 5, 15 min and young 15 min ischemia-operated groups, not in the young 5 min ischemia-operated group (Figs. 1I, J, K, 2I, J and K). In the young 5 min ischemia-operated group, the morphology of CV+ cells in the stratum pyramidale of the CA1 region was similar to that in the shamoperated group (Figs. 1K and 2K). Seven days after ischemia–reperfusion, CV+ cells in the stratum pyramidale of the CA1 region in the young 5 min ischemia-operated
Neuronal death in the ischemic CA1 region was examined in the adult and young gerbils by NeuN immunohistochemistry. In all the sham-operated groups, NeuN+ neurons in the stratum pyramidale of the CA1 region were well observed (Fig. 3A-D). Three days after ischemia, NeuN+ neurons were not changed in the adult 5, 15 min and young 5 min ischemia-operated groups compared with those in the sham-operated groups (Fig. 3E–G); however, in the young 15 min ischemia-operated group, NeuN+ neurons in the stratum pyramidale of CA1 region were apparently decreased (Fig. 3H and Q). Four days after ischemia, NeuN+ neurons were hardly found in the stratum pyramidale of the CA1 region in the adult 5 and 15 min ischemia-operated groups (Fig. 3I and J). At this time point, NeuN+ neurons in the young 5 min ischemia-operated were similar to those in the sham-operated group (Fig. 3K and Q), and in the young 15 min ischemia-operated group, NeuN+ neurons were more decreased (Fig. 3L and Q). Seven days after ischemia, NeuN+ neurons in the stratum pyramidale of the CA1 region in the young 5 min ischemia-operated group were
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
133
Fig. 4. Histofluorescence staining for F-J B in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the adult, many F-J B+ cells are detected in the stratum pyramidale (asterisks) in the 5 min and 15 min ischemia-operated groups 4 days after ischemia. In the young, F-J B+ cells are found in the stratum pyramidale (asterisks) 7 and 3 days after ischemia in the 5 and 15 min ischemia-operated group, respectively. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm. O: Relative analysis as percent in the number of F-J B positive cells in the CA1 region (n = 7 per group; *P b 0.05, significantly different from the corresponding sham group, #P b 0.05, significantly different from the respective preceding group; †P b 0.05, significantly different from the same day of other groups). The bars indicate the means ± SEM.
significantly decreased compared with those in the sham-operated group; however, the mean number of NeuN+ neurons was much more than that in the other group (Fig. 3M-P and Q).
3.3. Fluoro-Jade B (F-J B)+ cells Neuronal degeneration in the ischemic CA1 region of the adult and young gerbils was examined by F-J B histofluorescence staining. F-J B+ Table 1 Semi-quantifications of GFAP immunoreactivity in the hippocampal CA1 region of the adult and young groups. Ischemic duration
Adult Young
5 min 15 min 5 min 15 min
Time after ischemia/reperfusion Sham
3d
4d
7d
+ + ± ±
++ ++ + ++
++ ++ ++ ++
++ ++ ++ ++
Immunoreactivity is scaled as −, ±, + or ++, representing no staining, weakly positive, moderate, or strong, respectively. SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum.
cells in the stratum pyramidale of the CA1 region were not observed in all the adult and young sham-operated groups (Fig. 4A–D). Three days after ischemia, many F-J B+ cells were easily detected only in the stratum pyramidale of the young 15 min ischemiaoperated group (Fig. 4E–H and Q). Four days after ischemia, F-J B+ cells were observed in the stratum pyramidale of all the adult ischemia-operated groups as well as the young 15 min ischemia–ischemia group; however, F-J B+ cells were not found in the stratum pyramidale of the young 5 min ischemiaoperated group (Fig. 4I-L and Q). Seven days after ischemia, F-J B+ cells in the stratum pyramidale were found in the young 5 min ischemia-operated group; the mean number of the F-J B+ cells was much smaller than that in the adult ischemia-operated groups (Fig. 4M–P and Q). 3.4. Astrocytes activation Changes of astrocytes activation in the ischemic CA1 region of the adult and young gerbils were examined by glial fibrillary acidic protein (GFAP) immunohistochemistry. In the sham-operated groups, GFAP+ astrocytes showed a rest form and distributed in all the layers of the CA1 region; especially, GFAP immunoreactivity in the young sham-
134
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
Fig. 5. Immunohistochemistry for GFAP in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the young sham-operated groups, GFAP immunoreactivity in astrocytes (arrows) is lower than that in the adult sham-operated groups. Astrocytes become larger and their GFAP immunoreactivity increase with time after ischemia in all the groups. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm.
Fig. 6. Immunohistochemistry for Iba-1 in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the young sham-operated groups, Iba-1 immunoreactivity in microglia (arrows) is similar to that in the adult sham-operated groups. Iba-1+ microglia become larger with time, and they are aggregated in the stratum pyramidale (SP, asterisks) 4 days after ischemia in both the adult groups, 7 days after ischemia in the young 5 min ischemia-operated group and 3 days after ischemia in the young 15 min ischemia-operated group. SO, stratum oriens; SR, stratum radiatum. Scale bar = 100 μm.
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136 Table 2 Semi-quantifications of Iba-1 immunoreactivity in the hippocampal CA1 region of the adult and young groups. Ischemic duration
Adult Young
5 min 15 min 5 min 15 min
Time after ischemia/reperfusion Sham
3d
4d
7d
+ + + +
++ ++ + +++
+++ +++ + +++
+++ +++ ++ ++
Immunoreactivity is scaled as −, ±, +, ++ or +++ representing no staining, weakly positive, moderate, strong or very strong, respectively. SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum.
operated groups was much lower than that in the adult sham-operated groups (Table 1, Fig. 5A–D). Three days after ischemia, GFAP+ astrocytes were larger than those in the sham-operated groups, and their GFAP immunoreactivity was increased; at this time, GFAP immunoreactivity in the young 5 min ischemia-operated group was a little lower than that in the other groups (Table 1, Fig. 5E–G). Four and 7 days after ischemia, the distribution pattern of GFAP+ astrocytes in the ischemic CA1 region were similar in all the groups, and they became a little larger in size (Table 1, Fig. 5E–G). 3.5. Microglia activation In the sham groups, ionized calcium-binding adapter molecule (Iba-1)+ microglia were easily detected in all the layers of the CA1 region. We did not find any difference in the distribution pattern of Iba-1 + microglia between the adult and young sham-operated group (Fig. 6A–D). Three days after ischemia, Iba-1+ microglia were larger in size, and their Iba-1immunoreactivity was a little increased in all the groups (Table 2, Fig. 6E–H). At this time point, many Iba-1+ microglia were aggregated in the stratum pyramidale only in the young 15 min ischemiaoperated group (Table 2, Fig. 6H). Four days after ischemia, the aggregation of strong Iba-1+ microglia in the stratum pyramidale was found in all the adult ischemia-operated groups and young 15 min ischemia-operated group, not in the young 5 min ischemia-operated group (Table 2, Fig. 6I, J and L). Seven days after ischemia, strong Iba-1+ microglia were a little aggregated in the stratum pyramidale like the other groups (Table 2, Fig. 6M-P). At this time, Iba-1+ microglia were larger in size than that in the other groups. 4. Discussion Kusumoto et al. 21 previously reported that developing gerbils showed a greater tolerance to various periods of transient cerebral ischemia using 2-, 3-, 4-, 5-, and 12-week-old gerbils. It is necessary to exactly investigate neuronal death in young gerbils after 5 and 15 min of cerebral ischemia. We recently reported that neuronal death in the gerbil hippocampus was much more delayed and less in the young hippocampal CA1 region following 5 min of ischemia compared to that in the adult [4,23,24]. Also, it was reported that the different degree of neuronal death in the hippocampal subregions induced by various durations (5, 10, 15 20 min) of cerebral ischemia in the adult gerbil [2]. In present study, we comparably studied neuronal death in the hippocampal CA1 region between the adult and young gerbil induced by 5 and 15 min of cerebral ischemia using CV staining, NeuN immunohistochemistry and F-J B staining. We observed that the time point of neuronal death in the adult ischemic CA1 region was similar after 5 and 15 min of ischemia: the neuronal death occurred 4 days after ischemia. However, in the young ischemic CA1 region, neuronal death occurred 7 and 3 days after 5 and 15 min of ischemia, respectively.
135
Reactive astrogliosis was also observed in the present study. GFAP+ astrocytes were activated more quickly in the young hippocampal CA1 region following 15 min of cerebral ischemia compared with that in the adult, although GFAP+ astrocytes were much slowly activated in the young 5 min ischemia-operated gerbils. Reactive astrogliosis is a key component of the cellular response to CNS injury [25]: the entire process of reactive astrogliosis, as a uniformly negative and maladaptive phenomenon, unavoidably causes neurotoxicity, inflammation or chronic pain. Furthermore, the activation of GFAP+ astrocytes in the ischemic hippocampal CA1 region is related with neuronal degeneration [17,26]. In addition, some studies demonstrated that total inhibition of reactive astrogliosis could be regarded as a therapeutic strategy [27,28]. Therefore, our present results indicate that the accelerated/severe GFAP immunoreactivity in the young 15 min ischemia-operated group may be associated with the possibility of a secretion of harmful substances with different degrees induced by 15 min of ischemia. We, in the present study, also observed that the morphological change and immunoreactivity of Iba-1+ microglia in the young hippocampal CA1 region following 5 and 15 min of cerebral ischemia. We found that Iba-1+ microglia in the young were much earlier aggregated in the stratum pyramidale after 15 min of ischemia compared with the young 5 min ischemia-operated group. It has been reported that changes in the morphology and function of microglia are involved in response to various neural environments [29,30]. Microglia are markedly increased in infarct areas of the brain following transient focal ischemia in rats and in the hippocampal CA1 region following transient cerebral ischemia in gerbils [11,31]. Especially, it was recently reported that extensively increased astrocytes and microglia activation occurred in the hippocampal subregions and striatum with severe neuronal damage induced by much prolonged ischemic duration [2,10]. Therefore, we suggest that the excess of microglia activation in the young 15 min ischemia-operated group may be related with the accelerated neuronal death in the hippocampal CA1 region. In the CNS, the gliosis, including the overexpression of astrocytes and microglia, as a ubiquitous hallmark of different neural pathological states, is important in forming an environment that contributes either to successful repair of damaged brain tissue or to severe injury of bystander cells [32]. It is well known that astrocytes communicate with microglia and this interaction is very important in pathological conditions [17,33,34]. In conclusion, accelerated neuronal death occurred in the young gerbil CA1 region following 15 min of cerebral ischemia, and glia activation in the ischemic CA1 region was much faster and severer than that in the adult gerbil. These results indicate that, in the young brain, neuronal death and gliosis are very different from those in the adult brain according to the duration of transient cerebral ischemia. Overall, the novelty in the present study is that young gerbils undergo neuronal damage/death earlier in the hippocampal CA1 region induced by longer period (15 min) of cerebral ischemia.
Conflict of interest The authors have no financial conflict of interest.
Acknowledgments The authors would like to thank Mr. Seung Uk Lee for his technical help in this study. This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0010580), and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A 2001404).
136
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
References [1] Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, et al. Heart disease and stroke statistics—2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2007;115:e69-171. [2] Yu DK, Yoo KY, Shin BN, Kim IH, Park JH, Lee CH, et al. Neuronal damage in hippocampal subregions induced by various durations of transient cerebral ischemia in gerbils using Fluoro-Jade B histofluorescence. Brain Res 2012;1437:50–7. [3] Ganesan V, Hogan A, Shack N, Gordon A, Isaacs E, Kirkham FJ. Outcome after ischaemic stroke in childhood. Dev Med Child Neurol 2000;42:455–61. [4] Yan BC, Park JH, Ahn JH, Choi JH, Yoo KY, Lee CH, et al. Comparison of glial activation in the hippocampal CA1 region between the young and adult gerbils after transient cerebral ischemia. Cell Mol Neurobiol 2012;32:1127–38. [5] Blanco S, Castro L, Hernandez R, Del Moral ML, Pedrosa JA, Martinez-Lara E, et al. Age modulates the nitric oxide system response in the ischemic cerebellum. Brain Res 2007;1157:66–73. [6] Montori S, Martinez-Villayandre B, Dos-Anjos S, Llorente IL, Burgin TC, FernandezLopez A. Age-dependent modifications in the mRNA levels of the rat excitatory amino acid transporters (EAATs) at 48 hour reperfusion following global ischemia. Brain Res 2010;1358:11–9. [7] Lorrio S, Negredo P, Roda JM, Garcia AG, Lopez MG. Effects of memantine and galantamine given separately or in association, on memory and hippocampal neuronal loss after transient global cerebral ischemia in gerbils. Brain Res 2009;1254:128–37. [8] Schmidt-Kastner R, Freund TF. Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 1991;40:599–636. [9] Kirino T, Sano K. Selective vulnerability in the gerbil hippocampus following transient ischemia. Acta Neuropathol 1984;62:201–8. [10] Ohk TG, Yoo KY, Park SM, Shin BN, Kim IH, Park JH, et al. Neuronal damage using fluoro-jade B histofluorescence and gliosis in the striatum after various durations of transient cerebral ischemia in gerbils. Neurochem Res 2012;37:826–34. [11] Hwang IK, Yoo KY, Kim DW, Choi SY, Kang TC, Kim YS, et al. Ionized calcium-binding adapter molecule 1 immunoreactive cells change in the gerbil hippocampal CA1 region after ischemia/reperfusion. Neurochem Res 2006;31:957–65. [12] Amor S, Puentes F, Baker D, van der Valk P. Inflammation in neurodegenerative diseases. Immunology 2010;129:154–69. [13] Canistro D, Affatato AA, Soleti A, Mollace V, Muscoli C, Sculco F, et al. The novel radical scavenger IAC is effective in preventing and protecting against post-ischemic brain damage in Mongolian gerbils. J Neurol Sci 2010;290:90–5. [14] Szilagyi G, Simon L, Wappler E, Magyar K, Nagy Z. (−)Deprenyl-N-oxide, a (−)deprenyl metabolite, is cytoprotective after hypoxic injury in PC12 cells, or after transient brain ischemia in gerbils. J Neurol Sci 2009;283:182–6. [15] Zhang YB, Kan MY, Yang ZH, Ding WL, Yi J, Chen HZ, et al. Neuroprotective effects of N-stearoyltyrosine on transient global cerebral ischemia in gerbils. Brain Res 2009;1287:146–56. [16] Tamagaki C, Murata A, Asai S, Takase K, Gonno K, Sakata T, et al. Age-related changes of cornu ammonis 1 pyramidal neurons in gerbil transient ischemia. Neuropathology 2000;20:221–7.
[17] Lee CH, Yoo KY, Choi JH, Park OK, Hwang IK, Kim SK, et al. Neuronal damage is much delayed and microgliosis is more severe in the aged hippocampus induced by transient cerebral ischemia compared to the adult hippocampus. J Neurol Sci 2010;294:1–6. [18] Crain BJ, Westerkam WD, Harrison AH, Nadler JV. Selective neuronal death after transient forebrain ischemia in the Mongolian gerbil: a silver impregnation study. Neuroscience 1988;27:387–402. [19] Oguro K, Miyawaki T, Yokota H, Kato K, Kamiya T, Katayama Y, et al. Upregulation of GluR2 decreases intracellular Ca2+ following ischemia in developing gerbils. Neurosci Lett 2004;364:101–5. [20] Tortosa A, Ferrer I. Poor correlation between delayed neuronal death induced by transient forebrain ischemia, and immunoreactivity for parvalbumin and calbindin D-28k in developing gerbil hippocampus. Acta Neuropathol 1994;88:67–74. [21] Kusumoto M, Arai H, Mori K, Sato K. Resistance to cerebral ischemia in developing gerbils. J Cereb Blood Flow Metab 1995;15:886–91. [22] Yan BC, Park JH, Lee CH, Yoo KY, Choi JH, Lee YJ, et al. Increases of antioxidants are related to more delayed neuronal death in the hippocampal CA1 region of the young gerbil induced by transient cerebral ischemia. Brain Res 2011;1425:142–54. [23] Yan BC, Kim SK, Park JH, Ahn JH, Lee CH, Yoo KY, et al. Comparison of inflammatory cytokines changes in the hippocampal CA1 region between the young and adult gerbil after transient cerebral ischemia. Brain Res 2012;1461:64–75. [24] Yan BC, Park JH, Ahn JH, Lee YJ, Lee TH, Lee CH, et al. Comparison of the immunoreactivity of Trx2/Prx3 redox system in the hippocampal CA1 region between the young and adult gerbil induced by transient cerebral ischemia. Neurochem Res 2012;37:1019–30. [25] Zhu Z, Zhang Q, Yu Z, Zhang L, Tian D, Zhu S, et al. Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo. Glia 2007;55:546–58. [26] Ordy JM, Wengenack TM, Bialobok P, Coleman PD, Rodier P, Baggs RB, et al. Selective vulnerability and early progression of hippocampal CA1 pyramidal cell degeneration and GFAP-positive astrocyte reactivity in the rat four-vessel occlusion model of transient global ischemia. Exp Neurol 1993;119:128–39. [27] Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci 2009;32:638–47. [28] Hamby ME, Sofroniew MV. Reactive astrocytes as therapeutic targets for CNS disorders. Neurotherapeutics 2010;7:494–506. [29] Hailer NP, Jarhult JD, Nitsch R. Resting microglial cells in vitro: analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures. Glia 1996;18:319–31. [30] Schwartz M, Butovsky O, Bruck W, Hanisch UK. Microglial phenotype: is the commitment reversible? Trends Neurosci 2006;29:68–74. [31] Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol 2010;87:779–89. [32] Chen CJ, Ou YC, Lin SY, Raung SL, Liao SL, Lai CY, et al. Glial activation involvement in neuronal death by Japanese encephalitis virus infection. J Gen Virol 2010;91:1028–37. [33] Verderio C, Matteoli M. ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma. J Immunol 2001;166:6383–91. [34] von Bernhardi R, Ramirez G. Microglia-astrocyte interaction in Alzheimer's disease: friends or foes for the nervous system? Biol Res 2001;34:123–8.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Differences in neuronal damage and gliosis in the hippocampus between young and adult gerbils induced by long duration of transient cerebral ischemia Bing Chun Yan a,1, Taek Geun Ohk b,1, Ji Hyeon Ahn c, Joon Ha Park c, Bai Hui Chen d, Jae-Chul Lee c, Choong Hyun Lee e, Myoung Cheol Shin f, In Koo Hwang g, Seung Myung Moon h, Jun Hwi Cho f,i,⁎, Moo-Ho Won c,i,⁎⁎ a
Department of Integrative Traditional & Western Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu 225001, China Department of Emergency Medicine, Kangnam Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 150-950, South Korea c Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea d Department of Physiology, College of Medicine, Institute of Neurodegeneration and Neuroregeneration, Hallym University, Chuncheon 200-702, South Korea e Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan 330-714, South Korea f Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea g Department of Anatomy and Cell Biology, College of Veterinary Medicine, Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, South Korea h Department of Neurosurgery, Dongtan Sacred Heart Hospital, College of Medicine, Hallym University, Hwaseong 445-170, South Korea i Institute of Medical Sciences, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea b
a r t i c l e
i n f o
Article history: Received 13 September 2013 Received in revised form 29 October 2013 Accepted 20 November 2013 Available online 1 December 2013 Keywords: Young gerbil Ischemia–reperfusion injury Pyramidal neurons Delayed neuronal death Astrocytes Microglia
a b s t r a c t Response to cerebral ischemia in young animals was very different from that in the adult. The aim of this study was to investigate differences in neuronal death and gliosis in the hippocampal CA1 region (CA1) between adult and young gerbils following 5 and 15 min of transient cerebral ischemia. Delayed neuronal death (DND) of pyramidal cells occurred in the CA1 was similar in all the adult gerbils after 5 and 15 min of ischemia: the DND occurred 4 days after ischemia. In the young groups, DND of pyramidal cells in the CA1 region occurred 7 and 3 days after 5 and 15 min of ischemia, respectively. On the other hand, the activation of GFAP-immunoreactive (+) astrocytes and Iba-1+ microglia was different in the young groups from the adult groups after ischemia. The change pattern of GFAP immunoreactivity in the adult groups was similar in both the adult groups after ischemia; in the young groups, the activation of GFAP+ astrocytes after 5 min of ischemia was much delayed than that after 15 min of ischemia. Activated Iba-1+ microglia were aggregated in the stratum pyramidale 4 days after ischemia in all the adult ischemia-operated groups; in the young groups, activated Iba-1+ microglia were aggregated in the stratum pyramidale 7 days after 5 min of ischemia and 3 days after 15 min of ischemia. These observations indicate that DND in young animals is very different from the adult according to different duration of transient cerebral ischemia and glial activation is very different in young animals after different duration of transient ischemia. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Stroke is the third leading cause of death and can be subdivided into two types, ischemic and hemorrhagic. Ischemic stroke, accounting for approximately 87% of all cases, is more common than hemorrhagic stroke [1,2]. Recently, it has been reported that stroke has been ⁎ Correspondence to: J.H. Cho, Department of Emergency Medicine, College of Medicine, Kangwon National University, Chuncheon 200-701, South Korea. Tel.: +82 33 258 2378; fax: +82 33 258 2451. ⁎⁎ Correspondence to: M.-H. Won, Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chuncheon 200-702, South Korea. Tel.: +82 33 248 2522; fax: +82 33 256 1614. E-mail addresses: [email protected] (J.H. Cho), [email protected] (M.-H. Won). 1 Bing Chun Yan and Taek Geun Ohk contributed equally to this article. 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.11.034
increased in young people. As a high risk of serious morbidity for survivors, childhood stroke is one of the top ten causes of death among childhood disease in the U.S.A. [3,4]. Stroke or ischemic damage shows various characteristics according to age [5,6]. In both humans and animals, transient cerebral ischemia, which is induced by the deprivation of blood flow via both carotid arteries occlusion to the brain, damages some specific vulnerable regions such as the hippocampus [7]. The CA1 region of the hippocampus is the most vulnerable to transient cerebral ischemia [8]. Especially, neuronal death in the CA1 region is called “delayed neuronal death” because the death occurs very slowly [9,10]. Gliosis of astrocytes and microglia is easily induced in the hippocampus after transient cerebral ischemia in the adult gerbil [4,11]. Both astrocytes and microglia participate in the repair and regeneration of damaged neurons and cause neuronal death or dysfunction [12].
130
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
The gerbil is commonly used for making a good animal model of transient cerebral ischemia [13–15]. Many studies have focused on neuronal death using adult gerbils [16,17], and some reports regarding ischemia in the aged gerbil have demonstrated that neuronal death induced by transient cerebral ischemia occurs much later in the aged than in the adult [16–18]. Some studies have shown that the young gerbil is resistant to ischemic damage [19–21]. Recently, we compared neuronal damage in the ischemic CA1 region between the young and adult gerbils after 5 min of transient cerebral ischemia and found that the neuronal death in the hippocampal CA1 region of the young was more delayed and less than that in the adult [22]. However, in the young, few studies on neuronal damage according to longer duration of ischemia in the brain are reported. Therefore, the present study was undertaken to identify comparably damaged degree and glial changes in the young and adult gerbil hippocampus following 5 and 15 min of transient cerebral ischemia, respectively.
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. 2.4. Staining for neuronal damage 2.4.1. CV staining To investigate the delayed neuronal death in the CA1 after ischemia– reperfusion, sham-operated and ischemia-operated young and adult gerbils were used at designated times for CV staining. In brief, the sections were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma, MO) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid was added to this solution. The sections were stained and dehydrated by immersing in serial ethanol baths, and they were then mounted with Canada balsam (Kanto, Tokyo, Japan).
2. Materials and methods 2.1. Experimental animals We used male Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Kangwon National University, Chuncheon, South Korea. Mongolian gerbils were used at 1 (B.W.: 25–30 g) and 6 months (B.W.: 65–75 g) of age for the young and 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 provided with free access to water and food. 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 the animal protocol used in the present study was reviewed and approved based on ethical procedures and scientific care by the Kangwon National University-Institutional Animal Care and Use Committee (KIACUC-12-0018). All of the experiments were conducted to minimize the number of animals used and the suffering caused by the procedures used in the present study. 2.2. 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 retina using an ophthalmoscope. After 5 and 15 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 on 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. 2.3. Tissue processing for histology For histology, sham- and ischemia-operated young and adult gerbils (n = 7 at each time point) at designated times (3, 4 and 7 days after reperfusion) were sacrificed. The animals were anesthetized with 40 mg/kg pentobarbital sodium and perfused transcardially with 0.1 M phosphatebuffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M
2.4.2. NeuN immunohistochemistry To investigate the neuronal death in the CA1 region after transient cerebral ischemia, the sham-operated and ischemia-operated young and adult gerbils were used for NeuN immunohistochemistry under the same conditions. 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). And they were visualized by staining with 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). 2.4.3. F-J B histofluorescence staining To investigate neuronal degeneration in the CA1 region after ischemia–reperfusion, F-J B (a high affinity fluorescent marker for the localization of neuronal degeneration) histofluorescence staining was performed with a previous method [16]. In brief, the sections were transferred to a solution of 0.06% potassium permanganate, and then transferred to a 0.0004% F-J B (Histochem, Jefferson, AR) staining solution. The sections were examined using an epifluorescent microscope (Carl Zeiss, Germany) with blue (450–490 nm) excitation light and a barrier filter. 2.5. Immunohistochemistry for astrocytes and microglia In order to examine the degree of reactive gliosis in the CA1 region in the young and adult gerbils after ischemia–reperfusion, we carried out immunohistochemical staining under the same conditions with mouse anti-GFAP (1:800, Chemicon, Temecular) for astrocytes and rabbit antiIba-1 (1:800, Wako) for microglia according to the above-mentioned method (see NeuN immunohistochemistry). The sections were exposed to biotinylated horse anti-mouse, goat anti-rabbit IgG (1:200, Vector) for GFAP and Iba-1, and streptavidin peroxidase complex (1:200, Vector). And they were visualized with 3,3′-diaminobenzidine tetrahydrochloride in 0.1 M Tris–HCl buffer and mounted on the gelatin-coated slides. After dehydration the sections were mounted in Canada balsam (Kanto Chemical, Japan). 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 3 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 mean intensity of immunoreactivity in each immunoreactive structure
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
131
Fig. 1. Low magnification of CV staining in the adult (left 2 columns) and young (right 2 columns) hippocampus of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. Low CV stainability is detected in the CA1 region (asterisks) of the adult 5 min and 15 min ischemia-operated groups 4 days after ischemia–reperfusion. In the young, CV stainability in the CA1 region (asterisks) is apparently reduced at 7 and 3 days after 5 and 15 min of ischemia, respectively. CA; cornus ammonis, DG; dentate gyrus. Scale bar = 400 μm.
was measured by a 0–255 gray scale system (white to dark signal corresponded from 255 to 0). Based on this approach, the level of immunoreactivity was scaled as −, ±, + or ++, representing no staining (gray scale value: ≥200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤99), respectively.
2.6. Statistical analysis Data are expressed as the mean ± SEM. The data were evaluated by a Tukey test for post-hoc multiple comparisons following one-way ANOVA. Statistical significance was considered at P b 0.05.
Fig. 2. High magnification of CV staining in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the adult, significant disappearance of CV+ cells (asterisks) is found in both the 5 and 15 min ischemia-operated groups 4 days after ischemia–reperfusion; however, in the young, CV+ cells (asterisks) are reduced at 7 and 3 days after 5 and 15 min of ischemia, respectively. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm.
132
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
Fig. 3. Immunohistochemistry for NeuN in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the adult, NeuN+ neurons are hardly detected in the adult 5 min and 15 min ischemia-operated groups (asterisks) 4 days after ischemia. In the young, NeuN+ neurons are apparently reduced 7 and 3 days after ischemia in the 5 and 15 min ischemia-operated group, respectively. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm. O: Relative analysis as percent in the number of NeuN+ neurons in the CA1 region (n = 7 per group; *P b 0.05, significantly different from the corresponding sham group, #P b 0.05, significantly different from the respective preceding group; †P b 0.05, significantly different from the same day of other groups). The bars indicate the means ± SEM.
3. Results
group were apparently decreased; however, their morphology was a little better than that in the other groups (Figs. 1M–P and 2M–P).
3.1. Cresyl violet (CV) positive (+) cells 3.2. Neuronal nuclei (NeuN)+ neurons Neuronal death in the ischemic CA1 region of the adult and young gerbils was examined by CV staining. In the sham-operated groups, CV + cells were easily observed in all the sub-regions of the hippocampus in the adult and young gerbils (Figs. 1A–D and 2A–D). Three days after ischemia–reperfusion, significant change in CV+ cells was found only in the young 15 min ischemia-operated group compared to those in the sham-operated groups. Most of CV+ cells in the stratum pyramidale of the CA1 region became much smaller than those of the other groups (Figs. 1E–H and 2E–H). Four days after ischemia–reperfusion, a few CV+ cells were detected in the stratum pyramidale of the CA1 region in the adult 5, 15 min and young 15 min ischemia-operated groups, not in the young 5 min ischemia-operated group (Figs. 1I, J, K, 2I, J and K). In the young 5 min ischemia-operated group, the morphology of CV+ cells in the stratum pyramidale of the CA1 region was similar to that in the shamoperated group (Figs. 1K and 2K). Seven days after ischemia–reperfusion, CV+ cells in the stratum pyramidale of the CA1 region in the young 5 min ischemia-operated
Neuronal death in the ischemic CA1 region was examined in the adult and young gerbils by NeuN immunohistochemistry. In all the sham-operated groups, NeuN+ neurons in the stratum pyramidale of the CA1 region were well observed (Fig. 3A-D). Three days after ischemia, NeuN+ neurons were not changed in the adult 5, 15 min and young 5 min ischemia-operated groups compared with those in the sham-operated groups (Fig. 3E–G); however, in the young 15 min ischemia-operated group, NeuN+ neurons in the stratum pyramidale of CA1 region were apparently decreased (Fig. 3H and Q). Four days after ischemia, NeuN+ neurons were hardly found in the stratum pyramidale of the CA1 region in the adult 5 and 15 min ischemia-operated groups (Fig. 3I and J). At this time point, NeuN+ neurons in the young 5 min ischemia-operated were similar to those in the sham-operated group (Fig. 3K and Q), and in the young 15 min ischemia-operated group, NeuN+ neurons were more decreased (Fig. 3L and Q). Seven days after ischemia, NeuN+ neurons in the stratum pyramidale of the CA1 region in the young 5 min ischemia-operated group were
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
133
Fig. 4. Histofluorescence staining for F-J B in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the adult, many F-J B+ cells are detected in the stratum pyramidale (asterisks) in the 5 min and 15 min ischemia-operated groups 4 days after ischemia. In the young, F-J B+ cells are found in the stratum pyramidale (asterisks) 7 and 3 days after ischemia in the 5 and 15 min ischemia-operated group, respectively. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm. O: Relative analysis as percent in the number of F-J B positive cells in the CA1 region (n = 7 per group; *P b 0.05, significantly different from the corresponding sham group, #P b 0.05, significantly different from the respective preceding group; †P b 0.05, significantly different from the same day of other groups). The bars indicate the means ± SEM.
significantly decreased compared with those in the sham-operated group; however, the mean number of NeuN+ neurons was much more than that in the other group (Fig. 3M-P and Q).
3.3. Fluoro-Jade B (F-J B)+ cells Neuronal degeneration in the ischemic CA1 region of the adult and young gerbils was examined by F-J B histofluorescence staining. F-J B+ Table 1 Semi-quantifications of GFAP immunoreactivity in the hippocampal CA1 region of the adult and young groups. Ischemic duration
Adult Young
5 min 15 min 5 min 15 min
Time after ischemia/reperfusion Sham
3d
4d
7d
+ + ± ±
++ ++ + ++
++ ++ ++ ++
++ ++ ++ ++
Immunoreactivity is scaled as −, ±, + or ++, representing no staining, weakly positive, moderate, or strong, respectively. SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum.
cells in the stratum pyramidale of the CA1 region were not observed in all the adult and young sham-operated groups (Fig. 4A–D). Three days after ischemia, many F-J B+ cells were easily detected only in the stratum pyramidale of the young 15 min ischemiaoperated group (Fig. 4E–H and Q). Four days after ischemia, F-J B+ cells were observed in the stratum pyramidale of all the adult ischemia-operated groups as well as the young 15 min ischemia–ischemia group; however, F-J B+ cells were not found in the stratum pyramidale of the young 5 min ischemiaoperated group (Fig. 4I-L and Q). Seven days after ischemia, F-J B+ cells in the stratum pyramidale were found in the young 5 min ischemia-operated group; the mean number of the F-J B+ cells was much smaller than that in the adult ischemia-operated groups (Fig. 4M–P and Q). 3.4. Astrocytes activation Changes of astrocytes activation in the ischemic CA1 region of the adult and young gerbils were examined by glial fibrillary acidic protein (GFAP) immunohistochemistry. In the sham-operated groups, GFAP+ astrocytes showed a rest form and distributed in all the layers of the CA1 region; especially, GFAP immunoreactivity in the young sham-
134
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
Fig. 5. Immunohistochemistry for GFAP in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the young sham-operated groups, GFAP immunoreactivity in astrocytes (arrows) is lower than that in the adult sham-operated groups. Astrocytes become larger and their GFAP immunoreactivity increase with time after ischemia in all the groups. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 100 μm.
Fig. 6. Immunohistochemistry for Iba-1 in the adult (left 2 columns) and young (right 2 columns) CA1 region of the sham- (A–D), 5 min and 15 min ischemia-operated (E–P) groups. In the young sham-operated groups, Iba-1 immunoreactivity in microglia (arrows) is similar to that in the adult sham-operated groups. Iba-1+ microglia become larger with time, and they are aggregated in the stratum pyramidale (SP, asterisks) 4 days after ischemia in both the adult groups, 7 days after ischemia in the young 5 min ischemia-operated group and 3 days after ischemia in the young 15 min ischemia-operated group. SO, stratum oriens; SR, stratum radiatum. Scale bar = 100 μm.
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136 Table 2 Semi-quantifications of Iba-1 immunoreactivity in the hippocampal CA1 region of the adult and young groups. Ischemic duration
Adult Young
5 min 15 min 5 min 15 min
Time after ischemia/reperfusion Sham
3d
4d
7d
+ + + +
++ ++ + +++
+++ +++ + +++
+++ +++ ++ ++
Immunoreactivity is scaled as −, ±, +, ++ or +++ representing no staining, weakly positive, moderate, strong or very strong, respectively. SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum.
operated groups was much lower than that in the adult sham-operated groups (Table 1, Fig. 5A–D). Three days after ischemia, GFAP+ astrocytes were larger than those in the sham-operated groups, and their GFAP immunoreactivity was increased; at this time, GFAP immunoreactivity in the young 5 min ischemia-operated group was a little lower than that in the other groups (Table 1, Fig. 5E–G). Four and 7 days after ischemia, the distribution pattern of GFAP+ astrocytes in the ischemic CA1 region were similar in all the groups, and they became a little larger in size (Table 1, Fig. 5E–G). 3.5. Microglia activation In the sham groups, ionized calcium-binding adapter molecule (Iba-1)+ microglia were easily detected in all the layers of the CA1 region. We did not find any difference in the distribution pattern of Iba-1 + microglia between the adult and young sham-operated group (Fig. 6A–D). Three days after ischemia, Iba-1+ microglia were larger in size, and their Iba-1immunoreactivity was a little increased in all the groups (Table 2, Fig. 6E–H). At this time point, many Iba-1+ microglia were aggregated in the stratum pyramidale only in the young 15 min ischemiaoperated group (Table 2, Fig. 6H). Four days after ischemia, the aggregation of strong Iba-1+ microglia in the stratum pyramidale was found in all the adult ischemia-operated groups and young 15 min ischemia-operated group, not in the young 5 min ischemia-operated group (Table 2, Fig. 6I, J and L). Seven days after ischemia, strong Iba-1+ microglia were a little aggregated in the stratum pyramidale like the other groups (Table 2, Fig. 6M-P). At this time, Iba-1+ microglia were larger in size than that in the other groups. 4. Discussion Kusumoto et al. 21 previously reported that developing gerbils showed a greater tolerance to various periods of transient cerebral ischemia using 2-, 3-, 4-, 5-, and 12-week-old gerbils. It is necessary to exactly investigate neuronal death in young gerbils after 5 and 15 min of cerebral ischemia. We recently reported that neuronal death in the gerbil hippocampus was much more delayed and less in the young hippocampal CA1 region following 5 min of ischemia compared to that in the adult [4,23,24]. Also, it was reported that the different degree of neuronal death in the hippocampal subregions induced by various durations (5, 10, 15 20 min) of cerebral ischemia in the adult gerbil [2]. In present study, we comparably studied neuronal death in the hippocampal CA1 region between the adult and young gerbil induced by 5 and 15 min of cerebral ischemia using CV staining, NeuN immunohistochemistry and F-J B staining. We observed that the time point of neuronal death in the adult ischemic CA1 region was similar after 5 and 15 min of ischemia: the neuronal death occurred 4 days after ischemia. However, in the young ischemic CA1 region, neuronal death occurred 7 and 3 days after 5 and 15 min of ischemia, respectively.
135
Reactive astrogliosis was also observed in the present study. GFAP+ astrocytes were activated more quickly in the young hippocampal CA1 region following 15 min of cerebral ischemia compared with that in the adult, although GFAP+ astrocytes were much slowly activated in the young 5 min ischemia-operated gerbils. Reactive astrogliosis is a key component of the cellular response to CNS injury [25]: the entire process of reactive astrogliosis, as a uniformly negative and maladaptive phenomenon, unavoidably causes neurotoxicity, inflammation or chronic pain. Furthermore, the activation of GFAP+ astrocytes in the ischemic hippocampal CA1 region is related with neuronal degeneration [17,26]. In addition, some studies demonstrated that total inhibition of reactive astrogliosis could be regarded as a therapeutic strategy [27,28]. Therefore, our present results indicate that the accelerated/severe GFAP immunoreactivity in the young 15 min ischemia-operated group may be associated with the possibility of a secretion of harmful substances with different degrees induced by 15 min of ischemia. We, in the present study, also observed that the morphological change and immunoreactivity of Iba-1+ microglia in the young hippocampal CA1 region following 5 and 15 min of cerebral ischemia. We found that Iba-1+ microglia in the young were much earlier aggregated in the stratum pyramidale after 15 min of ischemia compared with the young 5 min ischemia-operated group. It has been reported that changes in the morphology and function of microglia are involved in response to various neural environments [29,30]. Microglia are markedly increased in infarct areas of the brain following transient focal ischemia in rats and in the hippocampal CA1 region following transient cerebral ischemia in gerbils [11,31]. Especially, it was recently reported that extensively increased astrocytes and microglia activation occurred in the hippocampal subregions and striatum with severe neuronal damage induced by much prolonged ischemic duration [2,10]. Therefore, we suggest that the excess of microglia activation in the young 15 min ischemia-operated group may be related with the accelerated neuronal death in the hippocampal CA1 region. In the CNS, the gliosis, including the overexpression of astrocytes and microglia, as a ubiquitous hallmark of different neural pathological states, is important in forming an environment that contributes either to successful repair of damaged brain tissue or to severe injury of bystander cells [32]. It is well known that astrocytes communicate with microglia and this interaction is very important in pathological conditions [17,33,34]. In conclusion, accelerated neuronal death occurred in the young gerbil CA1 region following 15 min of cerebral ischemia, and glia activation in the ischemic CA1 region was much faster and severer than that in the adult gerbil. These results indicate that, in the young brain, neuronal death and gliosis are very different from those in the adult brain according to the duration of transient cerebral ischemia. Overall, the novelty in the present study is that young gerbils undergo neuronal damage/death earlier in the hippocampal CA1 region induced by longer period (15 min) of cerebral ischemia.
Conflict of interest The authors have no financial conflict of interest.
Acknowledgments The authors would like to thank Mr. Seung Uk Lee for his technical help in this study. This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0010580), and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A 2001404).
136
B.C. Yan et al. / Journal of the Neurological Sciences 337 (2014) 129–136
References [1] Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, et al. Heart disease and stroke statistics—2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2007;115:e69-171. [2] Yu DK, Yoo KY, Shin BN, Kim IH, Park JH, Lee CH, et al. Neuronal damage in hippocampal subregions induced by various durations of transient cerebral ischemia in gerbils using Fluoro-Jade B histofluorescence. Brain Res 2012;1437:50–7. [3] Ganesan V, Hogan A, Shack N, Gordon A, Isaacs E, Kirkham FJ. Outcome after ischaemic stroke in childhood. Dev Med Child Neurol 2000;42:455–61. [4] Yan BC, Park JH, Ahn JH, Choi JH, Yoo KY, Lee CH, et al. Comparison of glial activation in the hippocampal CA1 region between the young and adult gerbils after transient cerebral ischemia. Cell Mol Neurobiol 2012;32:1127–38. [5] Blanco S, Castro L, Hernandez R, Del Moral ML, Pedrosa JA, Martinez-Lara E, et al. Age modulates the nitric oxide system response in the ischemic cerebellum. Brain Res 2007;1157:66–73. [6] Montori S, Martinez-Villayandre B, Dos-Anjos S, Llorente IL, Burgin TC, FernandezLopez A. Age-dependent modifications in the mRNA levels of the rat excitatory amino acid transporters (EAATs) at 48 hour reperfusion following global ischemia. Brain Res 2010;1358:11–9. [7] Lorrio S, Negredo P, Roda JM, Garcia AG, Lopez MG. Effects of memantine and galantamine given separately or in association, on memory and hippocampal neuronal loss after transient global cerebral ischemia in gerbils. Brain Res 2009;1254:128–37. [8] Schmidt-Kastner R, Freund TF. Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 1991;40:599–636. [9] Kirino T, Sano K. Selective vulnerability in the gerbil hippocampus following transient ischemia. Acta Neuropathol 1984;62:201–8. [10] Ohk TG, Yoo KY, Park SM, Shin BN, Kim IH, Park JH, et al. Neuronal damage using fluoro-jade B histofluorescence and gliosis in the striatum after various durations of transient cerebral ischemia in gerbils. Neurochem Res 2012;37:826–34. [11] Hwang IK, Yoo KY, Kim DW, Choi SY, Kang TC, Kim YS, et al. Ionized calcium-binding adapter molecule 1 immunoreactive cells change in the gerbil hippocampal CA1 region after ischemia/reperfusion. Neurochem Res 2006;31:957–65. [12] Amor S, Puentes F, Baker D, van der Valk P. Inflammation in neurodegenerative diseases. Immunology 2010;129:154–69. [13] Canistro D, Affatato AA, Soleti A, Mollace V, Muscoli C, Sculco F, et al. The novel radical scavenger IAC is effective in preventing and protecting against post-ischemic brain damage in Mongolian gerbils. J Neurol Sci 2010;290:90–5. [14] Szilagyi G, Simon L, Wappler E, Magyar K, Nagy Z. (−)Deprenyl-N-oxide, a (−)deprenyl metabolite, is cytoprotective after hypoxic injury in PC12 cells, or after transient brain ischemia in gerbils. J Neurol Sci 2009;283:182–6. [15] Zhang YB, Kan MY, Yang ZH, Ding WL, Yi J, Chen HZ, et al. Neuroprotective effects of N-stearoyltyrosine on transient global cerebral ischemia in gerbils. Brain Res 2009;1287:146–56. [16] Tamagaki C, Murata A, Asai S, Takase K, Gonno K, Sakata T, et al. Age-related changes of cornu ammonis 1 pyramidal neurons in gerbil transient ischemia. Neuropathology 2000;20:221–7.
[17] Lee CH, Yoo KY, Choi JH, Park OK, Hwang IK, Kim SK, et al. Neuronal damage is much delayed and microgliosis is more severe in the aged hippocampus induced by transient cerebral ischemia compared to the adult hippocampus. J Neurol Sci 2010;294:1–6. [18] Crain BJ, Westerkam WD, Harrison AH, Nadler JV. Selective neuronal death after transient forebrain ischemia in the Mongolian gerbil: a silver impregnation study. Neuroscience 1988;27:387–402. [19] Oguro K, Miyawaki T, Yokota H, Kato K, Kamiya T, Katayama Y, et al. Upregulation of GluR2 decreases intracellular Ca2+ following ischemia in developing gerbils. Neurosci Lett 2004;364:101–5. [20] Tortosa A, Ferrer I. Poor correlation between delayed neuronal death induced by transient forebrain ischemia, and immunoreactivity for parvalbumin and calbindin D-28k in developing gerbil hippocampus. Acta Neuropathol 1994;88:67–74. [21] Kusumoto M, Arai H, Mori K, Sato K. Resistance to cerebral ischemia in developing gerbils. J Cereb Blood Flow Metab 1995;15:886–91. [22] Yan BC, Park JH, Lee CH, Yoo KY, Choi JH, Lee YJ, et al. Increases of antioxidants are related to more delayed neuronal death in the hippocampal CA1 region of the young gerbil induced by transient cerebral ischemia. Brain Res 2011;1425:142–54. [23] Yan BC, Kim SK, Park JH, Ahn JH, Lee CH, Yoo KY, et al. Comparison of inflammatory cytokines changes in the hippocampal CA1 region between the young and adult gerbil after transient cerebral ischemia. Brain Res 2012;1461:64–75. [24] Yan BC, Park JH, Ahn JH, Lee YJ, Lee TH, Lee CH, et al. Comparison of the immunoreactivity of Trx2/Prx3 redox system in the hippocampal CA1 region between the young and adult gerbil induced by transient cerebral ischemia. Neurochem Res 2012;37:1019–30. [25] Zhu Z, Zhang Q, Yu Z, Zhang L, Tian D, Zhu S, et al. Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo. Glia 2007;55:546–58. [26] Ordy JM, Wengenack TM, Bialobok P, Coleman PD, Rodier P, Baggs RB, et al. Selective vulnerability and early progression of hippocampal CA1 pyramidal cell degeneration and GFAP-positive astrocyte reactivity in the rat four-vessel occlusion model of transient global ischemia. Exp Neurol 1993;119:128–39. [27] Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci 2009;32:638–47. [28] Hamby ME, Sofroniew MV. Reactive astrocytes as therapeutic targets for CNS disorders. Neurotherapeutics 2010;7:494–506. [29] Hailer NP, Jarhult JD, Nitsch R. Resting microglial cells in vitro: analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures. Glia 1996;18:319–31. [30] Schwartz M, Butovsky O, Bruck W, Hanisch UK. Microglial phenotype: is the commitment reversible? Trends Neurosci 2006;29:68–74. [31] Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol 2010;87:779–89. [32] Chen CJ, Ou YC, Lin SY, Raung SL, Liao SL, Lai CY, et al. Glial activation involvement in neuronal death by Japanese encephalitis virus infection. J Gen Virol 2010;91:1028–37. [33] Verderio C, Matteoli M. ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma. J Immunol 2001;166:6383–91. [34] von Bernhardi R, Ramirez G. Microglia-astrocyte interaction in Alzheimer's disease: friends or foes for the nervous system? Biol Res 2001;34:123–8.