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Impact of hyperthermia before and during ischemia–reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia
Journal of the Neurological Sciences 348 (2015) 101–110
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Impact of hyperthermia before and during ischemia–reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia Min Joung Kim a,b,1, Jun Hwi Cho b,1, Jeong-Hwi Cho c, Joon Ha Park c, Ji Hyeon Ahn c, Hyun-Jin Tae d, Geum-Sil Cho e, Bing Chun Yan f, In Koo Hwang g, Choong Hyun Lee h, Eun Joo Bae i, Moo-Ho Won c,⁎, Jae-Chul Lee c,⁎⁎ a
Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea Department of Emergency Medicine, Yonsei University College of Medicine, Seoul 120-752, South Korea c Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea d Department of Biomedical Science and Research, Institute of Bioscience and Biotechnology, Hallym University, Chuncheon 200-702, South Korea e Department of Neuroscience, College of Medicine, Korea University, Seoul 136-705, South Korea f Institute of Integrative Traditional & Western Medicine, Medical College, Yangzhou University, Yangzhou 225001, China g Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea h Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan 330-714, South Korea i Department of Pediatrics, Chuncheon Sacred Heart Hospital, College of Medicine, Hallym University, Chunchen 200-702, South Korea b
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 22 October 2014 Accepted 11 November 2014 Available online 20 November 2014 Keywords: Hippocampal subregions Ischemia–reperfusion Hyperthermic pre-condition Pyramidal neurons Delayed neuronal death Glial cells
a b s t r a c t Hyperthermia can exacerbate the brain damage produced by ischemia. In the present study, we investigated the effects of hyperthermia before and during ischemia–reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining. The animals were randomly assigned to 4 groups: (1) sham-operated animals with normothermia (normothermia + sham group); (2) ischemia-operated animals with normothermia (normothermia + ischemia group); (3) sham-operated animals with hyperthermia (hyperthermia + sham group); and (4) ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia (39.5 ± 0.2 °C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30 min before and during ischemia–reperfusion. In the normothermia + ischemia groups, a significant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5 days after ischemia–reperfusion. In the hyperthermia + ischemia groups, neuronal death in the SP of the CA1 occurred at 1 day post-ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2 days postischemia. In addition, we examined activations of astrocytes and microglia using immunohistochemistry for antiglial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia + ischemia groups than those in the normothermia + ischemia groups. Based on our findings, we suggest that an experimentally hyperthermic pre-condition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The brain is very vulnerable to ischemia because of its high metabolic rate, low oxygen stores and an insufficient reserve of high-energy ⁎ Correspondence to: M.H. Won, Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea. Tel.: +82 33250-8891; Fax: +82 33 256 1614. ⁎⁎ Corresponding author. Tel: +82-33-250-8892; Fax: +82 33 256 1614. E-mail addresses: [email protected] (M.-H. Won), [email protected] (J.-C. Lee). 1 Min Joung Kim and Jun Hwi Cho have contributed equally to this article.
http://dx.doi.org/10.1016/j.jns.2014.11.015 0022-510X/© 2014 Elsevier B.V. All rights reserved.
carbohydrates compared with the other tissues [1]. During global forebrain ischemia, the reduction of blood supply to the brain triggers a number of neuro-pathophysiological processes that result in irreversible neuronal damage in sensitive regions, such as the hippocampus [2]. In the hippocampus, the vulnerability differs from each hippocampal subregion: the hippocampal CA1 region is the most vulnerable to transient forebrain ischemia, whereas the CA3 region is the most resistant to the ischemia [3]. This unique process in the CA1 region is termed “delayed neuronal death”, which occurs from 4 days after 5 min of transient forebrain ischemia [2,4]. The delayed neuronal death is due to diverse cellular changes, such as DNA damage and oxidative stress
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following ischemia–reperfusion [5,6]. However, the precise mechanisms of the delayed neuronal death remain unclear. The Mongolian gerbil has been used as a good animal model to investigate mechanisms of selective delayed neuronal death following transient global forebrain ischemia [7–9], because about 90% of the gerbils lack the communicating vessels between the carotid and vertebral circulations. Thus, the bilateral occlusion of the common carotid arteries essentially and completely eliminates blood flow to the forebrain while completely sparing the vegetative centers of the brain stem. Body temperature is a major factor in neuronal survival/death after cerebral ischemia; hypothermia is neuroprotective and hyperthermia is damaging [10–12]. Hyperthermia (N38 °C) has been included as an independent prognostic marker for the prediction of mortality and functional outcome after an acute ischemic stroke [13,14]. Preclinical studies have provided several evidences for the harmful effects of elevated body temperature after post-ischemia on different animal models of ischemia, such as global forebrain ischemia [15], focal permanent ischemia [16] and focal transient ischemia [17,18]. Clinical data have confirmed that mild hyperthermia in ischemic stroke patients can enlarge infarct size and worsen the outcome of ischemic stroke [19,20]. The brain temperature can reach above 40.5 °C after transient forebrain ischemia. At this temperature, the ability of the hypothalamus becomes compromised to coordinate thermoregulation and there can be a further increase in brain temperature, low blood pressure, an increase in intracerebral pressure, monoamine overload, and multi-organ dysfunction [21,22]. Although it is well known that a rise in body temperature after experimentally induced transient forebrain ischemia produces more extensive brain damage [23], studies regarding neuronal damage/death in the hippocampus according to hyperthermic condition before ischemic insults are not reported. Therefore, in the present study, we examined whether hyperthermic condition before an ischemic insult is associated with neuronal damages and glial changes in the hippocampus of the gerbil, which is a good animal model of ischemic stroke [24–26], following transient forebrain ischemia. 2. Materials and methods 2.1. Experimental animals Male Mongolian gerbils (Meriones unguiculatus) were obtained from the Experimental Animal Center, Kangwon National University, Chuncheon, South Korea. Gerbils were used at 6 months (B.W., 65– 75 g) of age. The animals were housed in a conventional state under adequate temperature (23 ± 0.2 °C) and humidity (60%) control with a 12-h light/12-h dark cycle, and were provided with free access to food and water. The procedures for animal handling and care adhered to guidelines that are in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National Academies Press, 8th Ed., 2011), and they were approved by the Institutional Animal Care and Use Committee (IACUC) at Kangwon University. 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 forebrain ischemia Experimental animals (n = 7 at each time point per group) were divided into four groups: (1) sham-operated animals with normothermia (normothermia + sham group); (2) ischemia-operated animals with normothermia (normothermia + ischemia group); (3) sham-operated animals with hyperthermia (hyperthermia + sham group); and (4) ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia was induced by exposing the gerbils to a heating pad connected to a rectal thermistor while the animals were under anesthesia until their rectal temperature was elevated to
39.5 ± 0.2 °C, and the animals were maintained at this temperature for 30 min before and during ischemia–reperfusion. The animals of all the groups were anesthetized with a mixture of 2.5% isoflurane (Baxter, Deerfield, IL) in 33% oxygen plus 67% nitrous oxide gas. Bilateral common carotid arteries were isolated and occluded using non-traumatic aneurysm clips (Yasargil FE 723K, Aesculap, Tuttlingen, Germany). Bilateral common carotid arteries were occluded using non-traumatic aneurysm clips for 5 min. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope (HEINE K180®, Heine Optotechnik, Herrsching, Germany). During the surgery, the animals of the normothermia and hyperthermia groups were kept on the heating pad at 37 ± 0.2 °C and 39.5 ± 0.2 °C, respectively. Thereafter, the animals were kept in the thermal incubator (temperature, 23 °C; humidity, 60%) (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature on the normothermic level until they were euthanized. Sham-operated animals were exposed to similar surgery without carotid artery occlusion. The animals in each group were given recovery times of 1 day, 2 days, and 5 days, because pyramidal neurons in the hippocampal CA1 region do not die until 3 days and begin to die from 4 days after ischemia–reperfusion. 2.3. Tissue processing for histology All of 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 (CM1900 UV, Leica, Wetzlar, Germany) into 30 μm coronal sections, and they were then collected into six-well plates containing PBS. 2.4. Cresyl violet (CV) staining To examine neuronal damage/death in the hippocampus at each time point after transient forebrain ischemia using CV staining, the sections were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma-Aldrich, St. Louis, 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 Chemical, Tokyo, Japan). 2.5. NeuN immunohistochemistry To investigate the neuronal damage/death in the hippocampus at each time point after transient forebrain ischemia using anti-neuronal nuclei (NeuN, a marker for neurons), 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 (diluted 1:1000, Chemicon International, Temecula, CA) overnight at 4 °C. Thereafter the tissues were exposed to biotinylated goat anti-mouse IgG (Vector, Burlingame, CA) and streptavidin peroxidase complex (diluted 1:200, Vector). They were then 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 Chemical). 2.6. Fluoro-Jade B (F-J B) histofluorescence staining To confirm the neuronal death in the hippocampus at each time point after transient forebrain ischemia using F-J B (a high affinity fluorescent marker for the localization of neuronal degeneration)
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histofluorescence (Candelario-Jalil et al., 2003), the sections were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol, and followed in 70% alcohol. They were then transferred to a solution of 0.06% potassium permanganate, and transferred to a 0.0004% Fluoro-Jade B (Histochem, Jefferson, AR) staining solution. After washing, the sections were placed on a slide warmer (approximately 50 °C), and then examined using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450–490 nm) excitation light and a barrier filter. With this method neurons that undergo degeneration brightly fluoresce in comparison to the background [27].
(ANOVA) with a post hoc Bonferroni's multiple comparison tests with SPSS program in order to elucidate ischemia-related differences among experimental groups. In order to compare two independent variables between normothermia and hyperthermia, and their interaction, two-way ANOVA was used with the Bonferroni post hoc. Statistical significance was considered at P b 0.05.
2.7. Immunohistochemistry for astrocytes and microglia
CV+ cells were well distributed in the hippocampus of the normothermia + sham group (Fig. 1a). In the normothermia + ischemia groups, the distribution pattern of CV+ cells was not changed until 2 days after ischemia–reperfusion (Fig. 1c and e). At 5 days after ischemia–reperfusion, CV+ cells were apparently damaged; CV stainability was significantly decreased in the stratum pyramidale of the CA1 region compared to that of the normothermia + sham group (Fig. 1g). In the hyperthermia + sham group, the distribution pattern of CV+ cells in the hippocampus was similar to that in the normothermia + sham group (Fig. 1b). In the hyperthermia + ischemia groups, the morphological damage of CV+ cells began to occur in the stratum pyramidale of the CA1 region 2 days after ischemia–reperfusion (Fig. 1d and f). At 5 days after ischemia–reperfusion, CV stainability was markedly reduced in the stratum pyramidale of the CA1 region; in addition, CV+ cells in the stratum pyramidale of the CA2/3 region showed weak CV stainability (Fig. 1h).
In order to examine the degree of reactive gliosis at each time point after transient forebrain ischemia, we carried out immunohistochemical staining under the same conditions with rabbit anti-GFAP (diluted 1:1000, Chemicon International) for astrocytes and rabbit anti-Iba-1 (diluted 1:500, Wako, Osaka, Japan) for microglia overnight at 4 °C and subsequently exposed to biotinylated goat anti-rabbit IgG (diluted 1:200, Vector) for a secondary antibody and streptavidin peroxidase complex (diluted 1:200, Vector). They were then visualized according to the above-mentioned method (see the NeuN immunohistochemistry). In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of the primary antibody. The negative control resulted in the absence of immunoreactivity in any of the structures.
3. Results 3.1. Cresyl violet-positive (CV+) cells
2.8. Cell counts 3.2. NeuN+ cells All measurements were performed to insure objectivity in blind conditions, by three observers for each experiment, carrying out the measures of experimental samples under the same conditions. The studied tissue sections were selected with a 120 μm interval according to anatomical landmarks corresponding to AP from AP − 1.4 to −1.8 mm of the gerbil brain atlas, and cell counts were obtained by averaging the counts from 20 sections taken from each animal. All NeuNand F-J B-positive structures were taken from 3 layers (strata oriens, pyramidale and radiatum) in the CA1 and CA2/3 through an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. The number of NeuN+ and F-J B+ cells was counted in a 200 × 200 μm square applied approximately at the center of the CA1 and CA2/3 regions. Cell counts were obtained by averaging the total cell numbers from each animal per group. The staining intensity of GFAP and Iba-1 immunoreactivity was evaluated from 20 sections taken from each animal. Their immunoreactivity was graded in the CA1 and CA2/3 regions. Digital images of the middle area of the hippocampal CA1 and CA2/3 were captured with an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Carl Zeiss) connected to a PC monitor. Video images were digitized into an array of 512 × 512 pixels corresponding to a tissue area of 140 × 140 μm (20× primary magnification). Each pixel resolution has 256 gray levels (white to dark signal corresponded from 255 to 0). With a previous method [15], the density of all immunoreactive structures was 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 the background was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the OD of an image file was calibrated in Adobe Photoshop 8.0 and then analyzed as a percent (relative optical density, ROD), with the normothermia + sham group designated as 100% in NIH Image 1.59. 2.9. Statistical analysis Data are expressed as the mean ± SEM. Differences of the means among the groups were statistically analyzed by analysis of variance
3.2.1. CA1 region Pyramidal neurons in the CA1 region were well immuno-stained with NeuN in the normothermia + sham group (Fig. 2a). In the normothermia + ischemia groups, no change in the distribution pattern of NeuN+ neurons in the stratum pyramidale of the CA1 region was found from 1 day to 2 days after ischemia–reperfusion (Table 1, Fig. 2e, i). At 5 days after ischemia–reperfusion, however, a significant loss of NeuN+ neurons was observed in the stratum pyramidale of the CA1 region (14 ± 5.4% of the normothermia + sham group, Table 1, Fig. 2m). In the hyperthermia + sham group, pyramidal neurons in the stratum pyramidale of the CA1 region were also well immuno-stained with NeuN (Fig. 2c). In the hyperthermia + ischemia groups, the distribution pattern of NeuN+ neurons in the stratum pyramidale was not significantly changed 1 day after ischemia–reperfusion (Table 1, Fig. 2g); however, 2 days after ischemia–reperfusion, a significant loss of NeuN+ neurons was observed in the stratum pyramidale of the CA1 compared with that in the normothermia + ischemia group at 2 days post-ischemia (17 ± 7.4% of the normothermia + sham group, Table 1, Fig. 2k). At 5 days after ischemia–reperfusion, many neurons in the stratum pyramidale showed NeuN immunoreactivity; however, the NeuN+ neurons were small and shrunken in shape compared with those in the hyperthermia + sham group (36 ± 9.8% of the normothermia + sham group, Table 1, Fig. 2o). 3.2.2. CA2/3 region Neurons in the stratum pyramidale of the CA2/3 region in the normothermia + sham group were well stained with NeuN (Table 1, Fig. 3a). In the normothermia + ischemia groups, we did not find a significant change in the distribution pattern of NeuN+ neurons in the stratum pyramidale of the CA2/3 region until 5 days post-ischemia (Table 1, Fig. 3e, i, m). In the hyperthermia + sham group, NeuN+ neurons in the stratum pyramidale of the CA2/3 region were also well stained with NeuN (Table 1, Fig. 3c). In the hyperthermia + ischemia groups, 1 day after ischemia–reperfusion, the distribution pattern of NeuN+ neurons in the stratum pyramidale was similar to that in the normothermia + sham
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Fig. 1. CV staining in the hippocampus of the normothermia + ischemia (left column) and hyperthermia + ischemia (right column) groups at sham (a, b), 1 (c, d), 2 (e, f) and 5 days (g, h) after ischemia–reperfusion. In the normothermia + ischemia groups, a significant loss of CV+ cells (arrows) is shown in the stratum pyramidale (SP) 5 days after ischemia– reperfusion. In the hyperthermia + ischemia groups, CV+ cells (arrow) are decreased in the SP of the CA1 region from 2 days post-ischemia. Especially, CV+ cells in the SP of the CA2/ 3 region are apparently decreased 5 days after ischemia–reperfusion. CA; cornu ammonis, DG; dentate gyrus, H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 800 μm.
group (Table 1, Fig. 3g). Two days after ischemia–reperfusion, the decrease of NeuN+ neurons was found in the stratum pyramidale compared with the hyperthermia + sham group (77 ± 3.1% of the normothermia + sham group, Table 1, Fig. 3k). At 5 days postischemia, NeuN+ neurons were more decreased in the stratum pyramidale compared with those at 2 days post-ischemia (59 ± 4.0% of the normothermia + sham group, Table 1, Fig. 3o). 3.3. F-J B+ cells 3.3.1. CA1 region No F-J B+ cells were observed in the CA1 region of the normothermia + sham group (Table 1, Fig. 2b). In the normothermia + ischemia
groups, we did not find any F-J B+ cells in the CA1 region from 1 day to 2 days after ischemia–reperfusion (Table 1, Fig. 2f, j). At 5 days postischemia, the significant increase of F-J B+ cells was observed in the stratum pyramidale of the CA1 region (67 ± 7.8% of NeuN+ neurons in the normothermia + sham group, Table 1, Fig. 2n). In the hyperthermia + sham group, F-J B+ cells were not observed in the CA1 region (Table 1, Fig. 2d). In the hyperthermia + ischemia groups, F-J B+ cells, which were weakly stained with F-J B, were observed in the stratum pyramidale 1 day after ischemia–reperfusion (36 ± 6.2% of the normothermia + ischemia group or 24 ± 4.1% of NeuN+ neurons in the normothermia + sham group, Table 1, Fig. 2h). Thereafter, many strong F-J B+ cells were found in the stratum pyramidale of the CA1 region 2 days (88 ± 8.4% of the normothermia +
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Fig. 2. Immunohistochemistry for NeuN (1st and 3rd columns) and F-J B (2nd and 4th columns) in the CA1 region of the normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia groups, only a few NeuN+ neurons (black arrows) and many F-J B+ cells (white asterisk) are detected in the stratum pyramidale (SP) 5 days after ischemia–reperfusion. In the hyperthermia + ischemia groups, NeuN+ neurons are decreased in the SP at 2 days post-ischemia; however, NeuN immunoreactivity is shown again in the SP at 5 days post-ischemia. Many F-J B+ cells are easily detected in the SP from 1 day after ischemia–reperfusion. H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.
ischemia group or 59 ± 5.6% of NeuN+ neurons in the normothermia + sham group) and 5 days (116 ± 9.5% of the normothermia + ischemia group or 77 ± 6.3% of NeuN in the normothermia + sham group) postischemia (Table 1, Fig. 2l, p).
neurons in the normothermia + sham group), and their number was significantly increased in the stratum pyramidale of the CA2/3 region at 5 days post-ischemia (37 ± 4.6% of NeuN+ neurons in the normothermia + sham group, Table 1, Fig. 3l, p).
3.3.2. CA2/3 region No F-J B+ cells were not found in the CA2/3 region in the normothermia + sham and ischemia groups at any time after ischemia–reperfusion (Table 1, Figs. 3b, 2f, 3j, 3n). In addition, F-J B+ cells were not found in the CA2/3 region in the hyperthermia + sham group and hyperthermia + ischemia groups 1 day after ischemia–reperfusion (Table 1, Fig. 3d, h). However, F-J B+ cells were easily detected in the stratum pyramidale at 2 days post-ischemia (16 ± 2.3% of NeuN+
3.4. Activation of glial cells 3.4.1. Astrocyte activation In the normothermia + sham group, GFAP+ astrocytes, which showed a resting form (a small body with thread like thin processes), were well distributed in all layers of the CA1 and CA2/3 regions (Tables 2, Fig. 4a, b). In the normothermia + ischemia groups, the morphology of GFAP+ astrocytes in the CA1 and CA2/3 regions at 1 day
Table 1 Change in the mean number of pyramidal neurons of the hippocampal CA1 and CA2/3 regions in the normothermia + ischemia and hyperthermia + ischemia groups. Time after I–R
CA1
CA2/3
NeuN+
Sham 1 day 2 days 5 days
F-J B+
NeuN+
F-J B+
N + Ischemia
H + Ischemia
N + Ischemia
H + Ischemia
N + Ischemia
H + Ischemia
N + Ischemia
H + Ischemia
87 86 82 12
91 84 15 31
0 0 1 ± 0.6 58 ± 6.8⁎
0 21 ± 3.6⁎,# 51 ± 4.9⁎,# 67 ± 5.5#
204 205 207 198
199 211 157 121
0 0 0 1 ± 0.4
0 0 33 ± 4.6⁎,# 76 ± 9.4⁎,#
± ± ± ±
7.3 6.1 6.9 4.7⁎
± ± ± ±
5.7 7.3 6.4⁎,# 8.5⁎,#
± ± ± ±
7.2 7.6 8.7 8.4
± ± ± ±
8.6 9.8 6.3⁎,# 8.1⁎,#
The mean numbers of NeuN+ and F-J B+ cells are counted in a 250 × 250 μm square of the stratum pyramidale of the CA1 and CA2/3 regions after ischemia–reperfusion. n = 7 at each time point per group. N + Ischemia, normothermia + ischemia group; H + Ischemia, hyperthermia + ischemia group. ⁎ P b 0.05, significantly different from the corresponding normothermia + sham group. # P b 0.05, significantly different from the corresponding normothermia + ischemia group at each time point.
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Fig. 3. Immunohistochemistry for NeuN (1st and 3rd columns) and F-J B (2nd and 4th columns) in the CA2/3 region of the normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia groups, the distribution pattern of NeuN+ and F-J B+ cells in the stratum pyramidale (SP) is similar to that in the normothermia + sham group. In the hyperthermia + ischemia groups, NeuN+ neurons (black arrows) are decreased and F-J B+ cells (white asterisk) are increased in the SP from 2 days after ischemia–reperfusion. H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.
post-ischemia was slightly changed: the processes of GFAP+ astrocytes were a little enlarged compared with those in the normothermia + sham group (Tables 2, Fig. 4e, f). At 2 days after ischemia–reperfusion, GFAP+ astrocytes in the CA1 and CA2/3 regions were similar to those at 1 day post-ischemia (Tables 2, Fig. 4i, j). At 5 days postischemia, GFAP+ astrocytes were highly activated in all layers of the CA1 and CA2/3 regions (Tables 2, Fig. 4m, n). In the hyperthermia + sham group, GFAP+ astrocytes in the CA1 and CA2/3 regions were similar to those in the normothermia + sham group and showed a resting form (Tables 2, Fig. 4c, d). In the hyperthermia + ischemia groups, GFAP+ astrocytes were much more changed in the CA1 region than in the CA2/3 region. In the CA1 region, GFAP+ astrocytes were apparently activated in all the layers from 1 day after ischemia–reperfusion and hardly found in the stratum pyramidale 5 days after ischemia–reperfusion (Tables 2, Fig. 4g, k, o).
However, in the CA2/3 region, GFAP+ astrocytes at 1 day postischemia were similar to those in the hyperthermia + sham group (Tables 2, Fig. 4h), and they were apparently activated in all the layers from 2 days after ischemia–reperfusion (Tables 2, Fig. 4i, p). 3.4.2. Microglia activation In the normothermia + sham group, Iba-1+ microglia were ubiquitously distributed in all layers of the CA1 and CA2/3 regions, and they had fine processes with web-like network characteristics of ramified microglia (Tables 3, Fig. 5a, b). In the normothermia + ischemia groups, Iba-1+ microglia were activated in the CA1 and CA2/3 regions from 1 day after ischemia– reperfusion (Tables 3, Fig. 5e, f). Iba-1+ microglia were more activated at 2 days after ischemia–reperfusion; they had an enlarged body with short and thickened processes, which are an activated form of microglia
Table 2 Change of the mean values of GFAP immunoreactivity in the hippocampal CA1 and CA2/3 regions in the normothermia + ischemia and hyperthermia + ischemia groups. Region
Groups
Time after ischemia–reperfusion Sham
CA1 CA2/3
N H N H
+ + + +
Ischemia Ischemia Ischemia Ischemia
100 100 100 100
± ± ± ±
1 day 3.0 1.1 5.0 3.2
107.5 115.1 103.2 106.3
2 days ± ± ± ±
3.0 4.5⁎ 2.2 3.7
114.2 127.7 111.4 110.9
± ± ± ±
5 days 5.0⁎ 4.8⁎,# 3.9⁎ 4.5⁎
130.1 136.4 119.1 128.1
± ± ± ±
7.4⁎ 7.8⁎ 2.4⁎ 3.2⁎,#
The relative optical density (ROD) values of GFAP immunoreactivity are calibrated as the mean percentage of GFAP immunoreactive structures with the normothermia + sham group designated as 100%. n = 7 at each time point. N + Ischemia, normothermia + ischemia group; H + Ischemia, hyperthermia + ischemia group. ⁎ P b 0.05, significantly different from the corresponding normothermia + sham group. # P b 0.05, significantly different from the corresponding normothermia + ischemia group.
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(Fig. 5i, j). At 5 days post-ischemia, especially, Iba-1+ microglia were aggregated near the stratum pyramidale of the CA1 region; however, they were not aggregated near the stratum pyramidale of the CA2/3 region (Tables 3, Fig. 5m, n). In the hyperthermia + sham group, Iba-1+ microglia in the CA1 and CA2/3 regions were similar to those in the normothermia + sham group (Tables 3, Fig. 5c, d). In the hyperthermia + ischemia groups, Iba-1+ microglia were also activated in the CA1 region than in the CA2/3 region from 1 day post-ischemia (Tables 3, Fig. 5g, h). In the CA1 region, Iba1+ microglia were apparently aggregated near the stratum pyramidale 2 days after ischemia–reperfusion and scattered in all layers 5 days after ischemia–reperfusion (Tables 3, Fig. 5o). In the CA2/3 region, at 5 days post-ischemia, Iba-1+ microglia were apparently activated in all the layers of the CA2/3 region; they did not show any aggregation near the stratum pyramidale (Tables 3, Fig. 5p). 4. Discussion We demonstrate that the extent of ischemia-induced pyramidal cell damage in gerbils is significantly affected by hyperthermia condition. Raising the body temperature for 30 min before and during transient global cerebral ischemia markedly augmented ischemic brain damage in the hippocampal CA1 and CA2/3 regions compared with that in normothermia condition, and we found that the morphological activation of astrocytes and microglia was found from 1 day post-ischemia. In healthy humans, the normal core body temperature is 37 °C and tightly controlled by the thermoregulatory center in the hypothalamus. Sometimes, however, the thermoregulatory control is impaired by serious diseases. Cases with abnormally high temperatures are grossly divided into two classes. The first class referred to as ‘fever’ denotes to
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temperature due to a change in the hypothalamic set point. Fever depends on the presence of pyrogens and immune system intervention as well as the coordination of autonomic, endocrinal and behavioral responses [28]. Fever is clinically characterized by vasoconstriction and shivering for heat production or vasodilatation and sweating for heat dissipation. Such fevers usually respond well to antipyretics, and the pathophysiology is well known. The other class is hyperthermia that is characterized by an unchanged setting of the thermoregulatory center. Uncontrolled heat production (e.g., during exercise), poor heat dissipation (e.g., wearing protective clothing in a hot and humid environment), or an external heat load (e.g., sunbathing or sauna) are the underlying mechanisms [29]. Conditions in this group include heat stroke, malignant hyperthermia, neuroleptic malignant syndrome, serotonin syndrome, cerebral hemorrhage, and drug-induced hyperthermia. In a gerbil model of transient cerebral ischemia, sensitive regions in the brain include the cerebral cortex, striatum and CA1 region of the hippocampus [30–34]. Hippocampal pyramidal neurons, particularly those in the CA1 region of the dorsal hippocampus, have long been known to be very sensitive to cerebral ischemic damage [2–4]. 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 with that in the adult [35–37]. Also, it was reported that the different degrees of neuronal death in the hippocampal subregions was apparent when transient cerebral ischemia was induced by various durations (5, 10, 15 and 20 min) in the adult gerbil [38]. In the present study, to elucidate neuronal death in the gerbil hippocampal sub-regions induced by 5 min of cerebral ischemia under hyperthermic condition, CV staining, NeuN immunohistochemistry and F-J B histofluorescence staining were carried out. Especially, F-J B has a good affinity for entirely degenerating neurons, and it
Fig. 4. Immunohistochemistry for GFAP in the CA1 (1st and 3rd columns) and CA2/3 (2nd and 4th columns) regions of normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia group, GFAP+ astrocytes (arrows) are activated in the CA1 region from 2 days after ischemia–reperfusion and in the CA 2/3 region at 5 days post-ischemia. In the hyperthermia + ischemia groups, GFAP+ cells (Arrows) are activated in the CA1 from 1 day after ischemia–reperfusion and in the CA2/3 region from 2 days post-ischemia. At 5 days post-ischemia, GFAP+ astrocytes are hardly found in the stratum pyramidale (SP, asterisk). H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.
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is a very useful marker for study on neuronal degeneration after ischemic injury [27]. We, in the present study, increased the body temperature to 39.5 ± 0.2 °C (hyperthermia) for 30 min before and during the induction of transient global cerebral ischemia and examined much more exacerbated cell death in the CA1 region after ischemia– reperfusion. This finding is consistent with the previous studies that showed that an increase in brain temperature, during and after the ischemic insult, exacerbated ischemia-induced neuronal death in the hippocampal CA1 neurons [15,39]. Hara et al. (2000) also evaluated the effects of brain temperature (37–39 °C) on specific DNA fragmentation in gerbil CA1 pyramidal neurons following transient cerebral ischemia, and they indicated that hyperthermia accelerated the DNA fragmentation in the gerbil CA1 pyramidal neurons. We, in this study, found that neuronal death in the hyperthermia-mediated ischemic CA1 region occurred rapidly (1 day after 5 min of ischemia) compared with that in the normothermia-mediated ischemic CA1 region. In addition, we found that this hyperthermic condition exacerbated neuronal death in the CA2/3 region from 2 days after ischemia–reperfusion; this neuronal death never occurs in the normothermia-mediated ischemic CA2/3 region. Elevation of body temperature leads to physiological and structural changes, including the alteration of enzyme activity and damage of cytoskeletal proteins [40,41]. In addition, the release of neurotoxic excitatory neurotransmitters and reactive oxygen species [42], calcium influx into neurons [43] and vascular permeability [44] have been proposed as mechanisms through which hyperthermia leads to tissue injury. In the hippocampus, the vulnerability differs from each hippocampal subregion. The CA1 region is the most vulnerable to ischemia, whereas the CA2/3 regions are resistant to ischemic insults [3,38]. Thus,
hyperthermic condition before and during transient cerebral ischemia must increase the extent of pyramidal cell death not only in the CA1 region, which is the most vulnerable to ischemia, but also in the CA2/3 region, which is much more resistant to ischemic insults. On the other hand, in the central nervous system, gliosis including activations of astrocytes and microglia, as a ubiquitous hallmark of different neural pathological states, is very important in forming an environment that contributes either to successful repair and regeneration of damaged neurons or to severe injury of bystander cells [45]. Gliosis of astrocytes and microglia is easily induced in the hippocampus after transient cerebral ischemia in the adult gerbil [31,35]. Glial cells are far less susceptible to ischemic injury than neurons. For example, astrocytes are able to maintain ATP levels longer than neurons; they provide metabolic and trophic support to neurons and modulate synaptic activity [46]. That is why their impaired functions can critically influence neuronal survival. It is well known that astrocytes respond to ischemia by an increase in their number and size together with elongation of cytoplasmic processes [47], an increase in the expression of GFAP of the intermediate filaments [48], reorganization of their gap junctions supporting a functional syncytium [49] and transient accumulation of glycogen [47]. It was reported that a change in GFAP immunoreactivity in the CA1 region was related with neuronal degeneration and that astrocyte activation was associated with local tissue damage and neuronal loss [50,51]. Reactive astrogliosis was also observed in our present study. GFAP+ astrocytes were more quickly activated in the hyperthermia + ischemia groups compared with that in the normothermia + ischemia groups. Therefore, our present results indicate that accelerated/severe GFAP immunoreactivity in the hyperthermia + ischemia groups may be associated with a possibility of the
Fig. 5. Immunohistochemistry for Iba-1 in the CA1 (1st and 3rd columns) and CA2/3 (2nd and 4th columns) regions of the normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia groups, Iba-1+ cells are activated in all layers of the CA1 and CA 2/3 regions from 1 day after ischemia–reperfusion (black arrows) and aggregated in the stratum pyramidale (SP) of the CA1 region at 5 days post-ischemia (black asterisk). In the hyperthermia + ischemia groups, Iba-1+ cells also are activated in all layers of the CA1 and CA2/3 regions from 1 day after ischemia– reperfusion; however, they are aggregated in the SP of the CA1 region at 2 days post-ischemia (black asterisk) and markedly increased in the SP of the CA2/3 region at 5 days postischemia. H, hyperthermia; N, normothermia; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 50 μm.
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Table 3 Change of the mean values of Iba-1 immunoreactivity in the hippocampal CA1 and CA2/3 regions in the normothermia + ischemia and hyperthermia + ischemia groups. Region
Groups
Time after ischemia–reperfusion Sham
CA1 CA2/3
N H N H
+ + + +
Ischemia Ischemia Ischemia Ischemia
100 100 100 100
± ± ± ±
1 day 4.5 4.0 3.0 3.1
101.8 109.3 103.9 103.4
2 days ± ± ± ±
2.1 3.8 2.9 5.4
103.0 115.5 111.7 109.5
± ± ± ±
5 days 2.4 2.60⁎ 2.6⁎ 5.4⁎
113.2 124.8 120.4 124.4
± ± ± ±
3.0⁎ 3.3⁎,# 2.5⁎ 4.0⁎
The relative optical density (ROD) values of Iba immunoreactivity are calibrated as the mean percentage of Iba immunoreactive structures with the normothermia + sham group designated as 100%. n = 7 at each time point. N + Ischemia, normothermia +ischemia group; H + Ischemia, hyperthermia + ischemia group. ⁎ P b 0.05, significantly different from the corresponding normothermia + sham group. # P b 0.05, significantly different from the corresponding normothermia + ischemia group.
secretion of harmful substances with different degrees induced by hyperthermia. It is well known that changes in the morphology and function of microglia are involved in response to various neural environments [52,53]. We, in the present study, compared the morphological change and immunoreactivity of Iba-1+ microglia in the hippocampal sub-regions following 5 min of cerebral ischemia between the normothermia + ischemia and hyperthermia + ischemia groups. Iba-1+ microglia in the hyperthermia + ischemia-groups were much earlier aggregated in the stratum pyramidale after 5 min of ischemia compared with that in the normothermia + ischemia groups. This finding coincides with a previous study that showed that a stronger and more intense glial reaction was related with post-ischemic tissue damage in aged gerbils [54]. Especially, it was recently reported that extensively increased microglia activation occurred in the hippocampal sub-regions and striatum with severe neuronal damage induced by much prolonged ischemic duration [33,38]. Therefore, we suggest that the excess of microglia activation in the hyperthermia + ischemia groups may be related with the accelerated neuronal death in the hippocampus. In conclusion, hyperthermia (39.5 ± 0.2 °C) for 30 min before and during ischemic insult markedly augmented ischemic neuronal damage in the hippocampal sub-regions compared with normothermia (37 ± 0.2 °C) and more accelerated activations of astrocytes and microglia under hyperthermia. These results indicate that neuronal death and gliosis are very different in hyperthermia-mediated brains from those in normothermia-conditioned brains following transient cerebral ischemia. These suggest a wide window of opportunity that is available for the prevention and management of ischemic damage under hyperthermia. Acknowledgements The authors would like to thank Mr. Seung Uk Lee for his technical help in this study. This study was supported by a 2014 Research Grant from Kangwon National University (No. 120140271), and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2001404). Conflict of interest The authors have declared that there is no conflict of interest. References [1] Sims NR, Zaidan E. Biochemical changes associated with selective neuronal death following short-term cerebral ischaemia. Int J Biochem Cell Biol 1995;27:531–50. [2] Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 1982;239:57–69. [3] Schmidt-Kastner R, Freund TF. Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 1991;40:599–636. [4] 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.
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Impact of hyperthermia before and during ischemia–reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia Min Joung Kim a,b,1, Jun Hwi Cho b,1, Jeong-Hwi Cho c, Joon Ha Park c, Ji Hyeon Ahn c, Hyun-Jin Tae d, Geum-Sil Cho e, Bing Chun Yan f, In Koo Hwang g, Choong Hyun Lee h, Eun Joo Bae i, Moo-Ho Won c,⁎, Jae-Chul Lee c,⁎⁎ a
Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea Department of Emergency Medicine, Yonsei University College of Medicine, Seoul 120-752, South Korea c Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea d Department of Biomedical Science and Research, Institute of Bioscience and Biotechnology, Hallym University, Chuncheon 200-702, South Korea e Department of Neuroscience, College of Medicine, Korea University, Seoul 136-705, South Korea f Institute of Integrative Traditional & Western Medicine, Medical College, Yangzhou University, Yangzhou 225001, China g Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea h Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan 330-714, South Korea i Department of Pediatrics, Chuncheon Sacred Heart Hospital, College of Medicine, Hallym University, Chunchen 200-702, South Korea b
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 22 October 2014 Accepted 11 November 2014 Available online 20 November 2014 Keywords: Hippocampal subregions Ischemia–reperfusion Hyperthermic pre-condition Pyramidal neurons Delayed neuronal death Glial cells
a b s t r a c t Hyperthermia can exacerbate the brain damage produced by ischemia. In the present study, we investigated the effects of hyperthermia before and during ischemia–reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining. The animals were randomly assigned to 4 groups: (1) sham-operated animals with normothermia (normothermia + sham group); (2) ischemia-operated animals with normothermia (normothermia + ischemia group); (3) sham-operated animals with hyperthermia (hyperthermia + sham group); and (4) ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia (39.5 ± 0.2 °C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30 min before and during ischemia–reperfusion. In the normothermia + ischemia groups, a significant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5 days after ischemia–reperfusion. In the hyperthermia + ischemia groups, neuronal death in the SP of the CA1 occurred at 1 day post-ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2 days postischemia. In addition, we examined activations of astrocytes and microglia using immunohistochemistry for antiglial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia + ischemia groups than those in the normothermia + ischemia groups. Based on our findings, we suggest that an experimentally hyperthermic pre-condition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The brain is very vulnerable to ischemia because of its high metabolic rate, low oxygen stores and an insufficient reserve of high-energy ⁎ Correspondence to: M.H. Won, Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea. Tel.: +82 33250-8891; Fax: +82 33 256 1614. ⁎⁎ Corresponding author. Tel: +82-33-250-8892; Fax: +82 33 256 1614. E-mail addresses: [email protected] (M.-H. Won), [email protected] (J.-C. Lee). 1 Min Joung Kim and Jun Hwi Cho have contributed equally to this article.
http://dx.doi.org/10.1016/j.jns.2014.11.015 0022-510X/© 2014 Elsevier B.V. All rights reserved.
carbohydrates compared with the other tissues [1]. During global forebrain ischemia, the reduction of blood supply to the brain triggers a number of neuro-pathophysiological processes that result in irreversible neuronal damage in sensitive regions, such as the hippocampus [2]. In the hippocampus, the vulnerability differs from each hippocampal subregion: the hippocampal CA1 region is the most vulnerable to transient forebrain ischemia, whereas the CA3 region is the most resistant to the ischemia [3]. This unique process in the CA1 region is termed “delayed neuronal death”, which occurs from 4 days after 5 min of transient forebrain ischemia [2,4]. The delayed neuronal death is due to diverse cellular changes, such as DNA damage and oxidative stress
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following ischemia–reperfusion [5,6]. However, the precise mechanisms of the delayed neuronal death remain unclear. The Mongolian gerbil has been used as a good animal model to investigate mechanisms of selective delayed neuronal death following transient global forebrain ischemia [7–9], because about 90% of the gerbils lack the communicating vessels between the carotid and vertebral circulations. Thus, the bilateral occlusion of the common carotid arteries essentially and completely eliminates blood flow to the forebrain while completely sparing the vegetative centers of the brain stem. Body temperature is a major factor in neuronal survival/death after cerebral ischemia; hypothermia is neuroprotective and hyperthermia is damaging [10–12]. Hyperthermia (N38 °C) has been included as an independent prognostic marker for the prediction of mortality and functional outcome after an acute ischemic stroke [13,14]. Preclinical studies have provided several evidences for the harmful effects of elevated body temperature after post-ischemia on different animal models of ischemia, such as global forebrain ischemia [15], focal permanent ischemia [16] and focal transient ischemia [17,18]. Clinical data have confirmed that mild hyperthermia in ischemic stroke patients can enlarge infarct size and worsen the outcome of ischemic stroke [19,20]. The brain temperature can reach above 40.5 °C after transient forebrain ischemia. At this temperature, the ability of the hypothalamus becomes compromised to coordinate thermoregulation and there can be a further increase in brain temperature, low blood pressure, an increase in intracerebral pressure, monoamine overload, and multi-organ dysfunction [21,22]. Although it is well known that a rise in body temperature after experimentally induced transient forebrain ischemia produces more extensive brain damage [23], studies regarding neuronal damage/death in the hippocampus according to hyperthermic condition before ischemic insults are not reported. Therefore, in the present study, we examined whether hyperthermic condition before an ischemic insult is associated with neuronal damages and glial changes in the hippocampus of the gerbil, which is a good animal model of ischemic stroke [24–26], following transient forebrain ischemia. 2. Materials and methods 2.1. Experimental animals Male Mongolian gerbils (Meriones unguiculatus) were obtained from the Experimental Animal Center, Kangwon National University, Chuncheon, South Korea. Gerbils were used at 6 months (B.W., 65– 75 g) of age. The animals were housed in a conventional state under adequate temperature (23 ± 0.2 °C) and humidity (60%) control with a 12-h light/12-h dark cycle, and were provided with free access to food and water. The procedures for animal handling and care adhered to guidelines that are in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National Academies Press, 8th Ed., 2011), and they were approved by the Institutional Animal Care and Use Committee (IACUC) at Kangwon University. 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 forebrain ischemia Experimental animals (n = 7 at each time point per group) were divided into four groups: (1) sham-operated animals with normothermia (normothermia + sham group); (2) ischemia-operated animals with normothermia (normothermia + ischemia group); (3) sham-operated animals with hyperthermia (hyperthermia + sham group); and (4) ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia was induced by exposing the gerbils to a heating pad connected to a rectal thermistor while the animals were under anesthesia until their rectal temperature was elevated to
39.5 ± 0.2 °C, and the animals were maintained at this temperature for 30 min before and during ischemia–reperfusion. The animals of all the groups were anesthetized with a mixture of 2.5% isoflurane (Baxter, Deerfield, IL) in 33% oxygen plus 67% nitrous oxide gas. Bilateral common carotid arteries were isolated and occluded using non-traumatic aneurysm clips (Yasargil FE 723K, Aesculap, Tuttlingen, Germany). Bilateral common carotid arteries were occluded using non-traumatic aneurysm clips for 5 min. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope (HEINE K180®, Heine Optotechnik, Herrsching, Germany). During the surgery, the animals of the normothermia and hyperthermia groups were kept on the heating pad at 37 ± 0.2 °C and 39.5 ± 0.2 °C, respectively. Thereafter, the animals were kept in the thermal incubator (temperature, 23 °C; humidity, 60%) (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature on the normothermic level until they were euthanized. Sham-operated animals were exposed to similar surgery without carotid artery occlusion. The animals in each group were given recovery times of 1 day, 2 days, and 5 days, because pyramidal neurons in the hippocampal CA1 region do not die until 3 days and begin to die from 4 days after ischemia–reperfusion. 2.3. Tissue processing for histology All of 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 (CM1900 UV, Leica, Wetzlar, Germany) into 30 μm coronal sections, and they were then collected into six-well plates containing PBS. 2.4. Cresyl violet (CV) staining To examine neuronal damage/death in the hippocampus at each time point after transient forebrain ischemia using CV staining, the sections were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma-Aldrich, St. Louis, 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 Chemical, Tokyo, Japan). 2.5. NeuN immunohistochemistry To investigate the neuronal damage/death in the hippocampus at each time point after transient forebrain ischemia using anti-neuronal nuclei (NeuN, a marker for neurons), 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 (diluted 1:1000, Chemicon International, Temecula, CA) overnight at 4 °C. Thereafter the tissues were exposed to biotinylated goat anti-mouse IgG (Vector, Burlingame, CA) and streptavidin peroxidase complex (diluted 1:200, Vector). They were then 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 Chemical). 2.6. Fluoro-Jade B (F-J B) histofluorescence staining To confirm the neuronal death in the hippocampus at each time point after transient forebrain ischemia using F-J B (a high affinity fluorescent marker for the localization of neuronal degeneration)
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histofluorescence (Candelario-Jalil et al., 2003), the sections were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol, and followed in 70% alcohol. They were then transferred to a solution of 0.06% potassium permanganate, and transferred to a 0.0004% Fluoro-Jade B (Histochem, Jefferson, AR) staining solution. After washing, the sections were placed on a slide warmer (approximately 50 °C), and then examined using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450–490 nm) excitation light and a barrier filter. With this method neurons that undergo degeneration brightly fluoresce in comparison to the background [27].
(ANOVA) with a post hoc Bonferroni's multiple comparison tests with SPSS program in order to elucidate ischemia-related differences among experimental groups. In order to compare two independent variables between normothermia and hyperthermia, and their interaction, two-way ANOVA was used with the Bonferroni post hoc. Statistical significance was considered at P b 0.05.
2.7. Immunohistochemistry for astrocytes and microglia
CV+ cells were well distributed in the hippocampus of the normothermia + sham group (Fig. 1a). In the normothermia + ischemia groups, the distribution pattern of CV+ cells was not changed until 2 days after ischemia–reperfusion (Fig. 1c and e). At 5 days after ischemia–reperfusion, CV+ cells were apparently damaged; CV stainability was significantly decreased in the stratum pyramidale of the CA1 region compared to that of the normothermia + sham group (Fig. 1g). In the hyperthermia + sham group, the distribution pattern of CV+ cells in the hippocampus was similar to that in the normothermia + sham group (Fig. 1b). In the hyperthermia + ischemia groups, the morphological damage of CV+ cells began to occur in the stratum pyramidale of the CA1 region 2 days after ischemia–reperfusion (Fig. 1d and f). At 5 days after ischemia–reperfusion, CV stainability was markedly reduced in the stratum pyramidale of the CA1 region; in addition, CV+ cells in the stratum pyramidale of the CA2/3 region showed weak CV stainability (Fig. 1h).
In order to examine the degree of reactive gliosis at each time point after transient forebrain ischemia, we carried out immunohistochemical staining under the same conditions with rabbit anti-GFAP (diluted 1:1000, Chemicon International) for astrocytes and rabbit anti-Iba-1 (diluted 1:500, Wako, Osaka, Japan) for microglia overnight at 4 °C and subsequently exposed to biotinylated goat anti-rabbit IgG (diluted 1:200, Vector) for a secondary antibody and streptavidin peroxidase complex (diluted 1:200, Vector). They were then visualized according to the above-mentioned method (see the NeuN immunohistochemistry). In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of the primary antibody. The negative control resulted in the absence of immunoreactivity in any of the structures.
3. Results 3.1. Cresyl violet-positive (CV+) cells
2.8. Cell counts 3.2. NeuN+ cells All measurements were performed to insure objectivity in blind conditions, by three observers for each experiment, carrying out the measures of experimental samples under the same conditions. The studied tissue sections were selected with a 120 μm interval according to anatomical landmarks corresponding to AP from AP − 1.4 to −1.8 mm of the gerbil brain atlas, and cell counts were obtained by averaging the counts from 20 sections taken from each animal. All NeuNand F-J B-positive structures were taken from 3 layers (strata oriens, pyramidale and radiatum) in the CA1 and CA2/3 through an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. The number of NeuN+ and F-J B+ cells was counted in a 200 × 200 μm square applied approximately at the center of the CA1 and CA2/3 regions. Cell counts were obtained by averaging the total cell numbers from each animal per group. The staining intensity of GFAP and Iba-1 immunoreactivity was evaluated from 20 sections taken from each animal. Their immunoreactivity was graded in the CA1 and CA2/3 regions. Digital images of the middle area of the hippocampal CA1 and CA2/3 were captured with an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Carl Zeiss) connected to a PC monitor. Video images were digitized into an array of 512 × 512 pixels corresponding to a tissue area of 140 × 140 μm (20× primary magnification). Each pixel resolution has 256 gray levels (white to dark signal corresponded from 255 to 0). With a previous method [15], the density of all immunoreactive structures was 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 the background was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the OD of an image file was calibrated in Adobe Photoshop 8.0 and then analyzed as a percent (relative optical density, ROD), with the normothermia + sham group designated as 100% in NIH Image 1.59. 2.9. Statistical analysis Data are expressed as the mean ± SEM. Differences of the means among the groups were statistically analyzed by analysis of variance
3.2.1. CA1 region Pyramidal neurons in the CA1 region were well immuno-stained with NeuN in the normothermia + sham group (Fig. 2a). In the normothermia + ischemia groups, no change in the distribution pattern of NeuN+ neurons in the stratum pyramidale of the CA1 region was found from 1 day to 2 days after ischemia–reperfusion (Table 1, Fig. 2e, i). At 5 days after ischemia–reperfusion, however, a significant loss of NeuN+ neurons was observed in the stratum pyramidale of the CA1 region (14 ± 5.4% of the normothermia + sham group, Table 1, Fig. 2m). In the hyperthermia + sham group, pyramidal neurons in the stratum pyramidale of the CA1 region were also well immuno-stained with NeuN (Fig. 2c). In the hyperthermia + ischemia groups, the distribution pattern of NeuN+ neurons in the stratum pyramidale was not significantly changed 1 day after ischemia–reperfusion (Table 1, Fig. 2g); however, 2 days after ischemia–reperfusion, a significant loss of NeuN+ neurons was observed in the stratum pyramidale of the CA1 compared with that in the normothermia + ischemia group at 2 days post-ischemia (17 ± 7.4% of the normothermia + sham group, Table 1, Fig. 2k). At 5 days after ischemia–reperfusion, many neurons in the stratum pyramidale showed NeuN immunoreactivity; however, the NeuN+ neurons were small and shrunken in shape compared with those in the hyperthermia + sham group (36 ± 9.8% of the normothermia + sham group, Table 1, Fig. 2o). 3.2.2. CA2/3 region Neurons in the stratum pyramidale of the CA2/3 region in the normothermia + sham group were well stained with NeuN (Table 1, Fig. 3a). In the normothermia + ischemia groups, we did not find a significant change in the distribution pattern of NeuN+ neurons in the stratum pyramidale of the CA2/3 region until 5 days post-ischemia (Table 1, Fig. 3e, i, m). In the hyperthermia + sham group, NeuN+ neurons in the stratum pyramidale of the CA2/3 region were also well stained with NeuN (Table 1, Fig. 3c). In the hyperthermia + ischemia groups, 1 day after ischemia–reperfusion, the distribution pattern of NeuN+ neurons in the stratum pyramidale was similar to that in the normothermia + sham
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Fig. 1. CV staining in the hippocampus of the normothermia + ischemia (left column) and hyperthermia + ischemia (right column) groups at sham (a, b), 1 (c, d), 2 (e, f) and 5 days (g, h) after ischemia–reperfusion. In the normothermia + ischemia groups, a significant loss of CV+ cells (arrows) is shown in the stratum pyramidale (SP) 5 days after ischemia– reperfusion. In the hyperthermia + ischemia groups, CV+ cells (arrow) are decreased in the SP of the CA1 region from 2 days post-ischemia. Especially, CV+ cells in the SP of the CA2/ 3 region are apparently decreased 5 days after ischemia–reperfusion. CA; cornu ammonis, DG; dentate gyrus, H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 800 μm.
group (Table 1, Fig. 3g). Two days after ischemia–reperfusion, the decrease of NeuN+ neurons was found in the stratum pyramidale compared with the hyperthermia + sham group (77 ± 3.1% of the normothermia + sham group, Table 1, Fig. 3k). At 5 days postischemia, NeuN+ neurons were more decreased in the stratum pyramidale compared with those at 2 days post-ischemia (59 ± 4.0% of the normothermia + sham group, Table 1, Fig. 3o). 3.3. F-J B+ cells 3.3.1. CA1 region No F-J B+ cells were observed in the CA1 region of the normothermia + sham group (Table 1, Fig. 2b). In the normothermia + ischemia
groups, we did not find any F-J B+ cells in the CA1 region from 1 day to 2 days after ischemia–reperfusion (Table 1, Fig. 2f, j). At 5 days postischemia, the significant increase of F-J B+ cells was observed in the stratum pyramidale of the CA1 region (67 ± 7.8% of NeuN+ neurons in the normothermia + sham group, Table 1, Fig. 2n). In the hyperthermia + sham group, F-J B+ cells were not observed in the CA1 region (Table 1, Fig. 2d). In the hyperthermia + ischemia groups, F-J B+ cells, which were weakly stained with F-J B, were observed in the stratum pyramidale 1 day after ischemia–reperfusion (36 ± 6.2% of the normothermia + ischemia group or 24 ± 4.1% of NeuN+ neurons in the normothermia + sham group, Table 1, Fig. 2h). Thereafter, many strong F-J B+ cells were found in the stratum pyramidale of the CA1 region 2 days (88 ± 8.4% of the normothermia +
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Fig. 2. Immunohistochemistry for NeuN (1st and 3rd columns) and F-J B (2nd and 4th columns) in the CA1 region of the normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia groups, only a few NeuN+ neurons (black arrows) and many F-J B+ cells (white asterisk) are detected in the stratum pyramidale (SP) 5 days after ischemia–reperfusion. In the hyperthermia + ischemia groups, NeuN+ neurons are decreased in the SP at 2 days post-ischemia; however, NeuN immunoreactivity is shown again in the SP at 5 days post-ischemia. Many F-J B+ cells are easily detected in the SP from 1 day after ischemia–reperfusion. H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.
ischemia group or 59 ± 5.6% of NeuN+ neurons in the normothermia + sham group) and 5 days (116 ± 9.5% of the normothermia + ischemia group or 77 ± 6.3% of NeuN in the normothermia + sham group) postischemia (Table 1, Fig. 2l, p).
neurons in the normothermia + sham group), and their number was significantly increased in the stratum pyramidale of the CA2/3 region at 5 days post-ischemia (37 ± 4.6% of NeuN+ neurons in the normothermia + sham group, Table 1, Fig. 3l, p).
3.3.2. CA2/3 region No F-J B+ cells were not found in the CA2/3 region in the normothermia + sham and ischemia groups at any time after ischemia–reperfusion (Table 1, Figs. 3b, 2f, 3j, 3n). In addition, F-J B+ cells were not found in the CA2/3 region in the hyperthermia + sham group and hyperthermia + ischemia groups 1 day after ischemia–reperfusion (Table 1, Fig. 3d, h). However, F-J B+ cells were easily detected in the stratum pyramidale at 2 days post-ischemia (16 ± 2.3% of NeuN+
3.4. Activation of glial cells 3.4.1. Astrocyte activation In the normothermia + sham group, GFAP+ astrocytes, which showed a resting form (a small body with thread like thin processes), were well distributed in all layers of the CA1 and CA2/3 regions (Tables 2, Fig. 4a, b). In the normothermia + ischemia groups, the morphology of GFAP+ astrocytes in the CA1 and CA2/3 regions at 1 day
Table 1 Change in the mean number of pyramidal neurons of the hippocampal CA1 and CA2/3 regions in the normothermia + ischemia and hyperthermia + ischemia groups. Time after I–R
CA1
CA2/3
NeuN+
Sham 1 day 2 days 5 days
F-J B+
NeuN+
F-J B+
N + Ischemia
H + Ischemia
N + Ischemia
H + Ischemia
N + Ischemia
H + Ischemia
N + Ischemia
H + Ischemia
87 86 82 12
91 84 15 31
0 0 1 ± 0.6 58 ± 6.8⁎
0 21 ± 3.6⁎,# 51 ± 4.9⁎,# 67 ± 5.5#
204 205 207 198
199 211 157 121
0 0 0 1 ± 0.4
0 0 33 ± 4.6⁎,# 76 ± 9.4⁎,#
± ± ± ±
7.3 6.1 6.9 4.7⁎
± ± ± ±
5.7 7.3 6.4⁎,# 8.5⁎,#
± ± ± ±
7.2 7.6 8.7 8.4
± ± ± ±
8.6 9.8 6.3⁎,# 8.1⁎,#
The mean numbers of NeuN+ and F-J B+ cells are counted in a 250 × 250 μm square of the stratum pyramidale of the CA1 and CA2/3 regions after ischemia–reperfusion. n = 7 at each time point per group. N + Ischemia, normothermia + ischemia group; H + Ischemia, hyperthermia + ischemia group. ⁎ P b 0.05, significantly different from the corresponding normothermia + sham group. # P b 0.05, significantly different from the corresponding normothermia + ischemia group at each time point.
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Fig. 3. Immunohistochemistry for NeuN (1st and 3rd columns) and F-J B (2nd and 4th columns) in the CA2/3 region of the normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia groups, the distribution pattern of NeuN+ and F-J B+ cells in the stratum pyramidale (SP) is similar to that in the normothermia + sham group. In the hyperthermia + ischemia groups, NeuN+ neurons (black arrows) are decreased and F-J B+ cells (white asterisk) are increased in the SP from 2 days after ischemia–reperfusion. H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.
post-ischemia was slightly changed: the processes of GFAP+ astrocytes were a little enlarged compared with those in the normothermia + sham group (Tables 2, Fig. 4e, f). At 2 days after ischemia–reperfusion, GFAP+ astrocytes in the CA1 and CA2/3 regions were similar to those at 1 day post-ischemia (Tables 2, Fig. 4i, j). At 5 days postischemia, GFAP+ astrocytes were highly activated in all layers of the CA1 and CA2/3 regions (Tables 2, Fig. 4m, n). In the hyperthermia + sham group, GFAP+ astrocytes in the CA1 and CA2/3 regions were similar to those in the normothermia + sham group and showed a resting form (Tables 2, Fig. 4c, d). In the hyperthermia + ischemia groups, GFAP+ astrocytes were much more changed in the CA1 region than in the CA2/3 region. In the CA1 region, GFAP+ astrocytes were apparently activated in all the layers from 1 day after ischemia–reperfusion and hardly found in the stratum pyramidale 5 days after ischemia–reperfusion (Tables 2, Fig. 4g, k, o).
However, in the CA2/3 region, GFAP+ astrocytes at 1 day postischemia were similar to those in the hyperthermia + sham group (Tables 2, Fig. 4h), and they were apparently activated in all the layers from 2 days after ischemia–reperfusion (Tables 2, Fig. 4i, p). 3.4.2. Microglia activation In the normothermia + sham group, Iba-1+ microglia were ubiquitously distributed in all layers of the CA1 and CA2/3 regions, and they had fine processes with web-like network characteristics of ramified microglia (Tables 3, Fig. 5a, b). In the normothermia + ischemia groups, Iba-1+ microglia were activated in the CA1 and CA2/3 regions from 1 day after ischemia– reperfusion (Tables 3, Fig. 5e, f). Iba-1+ microglia were more activated at 2 days after ischemia–reperfusion; they had an enlarged body with short and thickened processes, which are an activated form of microglia
Table 2 Change of the mean values of GFAP immunoreactivity in the hippocampal CA1 and CA2/3 regions in the normothermia + ischemia and hyperthermia + ischemia groups. Region
Groups
Time after ischemia–reperfusion Sham
CA1 CA2/3
N H N H
+ + + +
Ischemia Ischemia Ischemia Ischemia
100 100 100 100
± ± ± ±
1 day 3.0 1.1 5.0 3.2
107.5 115.1 103.2 106.3
2 days ± ± ± ±
3.0 4.5⁎ 2.2 3.7
114.2 127.7 111.4 110.9
± ± ± ±
5 days 5.0⁎ 4.8⁎,# 3.9⁎ 4.5⁎
130.1 136.4 119.1 128.1
± ± ± ±
7.4⁎ 7.8⁎ 2.4⁎ 3.2⁎,#
The relative optical density (ROD) values of GFAP immunoreactivity are calibrated as the mean percentage of GFAP immunoreactive structures with the normothermia + sham group designated as 100%. n = 7 at each time point. N + Ischemia, normothermia + ischemia group; H + Ischemia, hyperthermia + ischemia group. ⁎ P b 0.05, significantly different from the corresponding normothermia + sham group. # P b 0.05, significantly different from the corresponding normothermia + ischemia group.
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(Fig. 5i, j). At 5 days post-ischemia, especially, Iba-1+ microglia were aggregated near the stratum pyramidale of the CA1 region; however, they were not aggregated near the stratum pyramidale of the CA2/3 region (Tables 3, Fig. 5m, n). In the hyperthermia + sham group, Iba-1+ microglia in the CA1 and CA2/3 regions were similar to those in the normothermia + sham group (Tables 3, Fig. 5c, d). In the hyperthermia + ischemia groups, Iba-1+ microglia were also activated in the CA1 region than in the CA2/3 region from 1 day post-ischemia (Tables 3, Fig. 5g, h). In the CA1 region, Iba1+ microglia were apparently aggregated near the stratum pyramidale 2 days after ischemia–reperfusion and scattered in all layers 5 days after ischemia–reperfusion (Tables 3, Fig. 5o). In the CA2/3 region, at 5 days post-ischemia, Iba-1+ microglia were apparently activated in all the layers of the CA2/3 region; they did not show any aggregation near the stratum pyramidale (Tables 3, Fig. 5p). 4. Discussion We demonstrate that the extent of ischemia-induced pyramidal cell damage in gerbils is significantly affected by hyperthermia condition. Raising the body temperature for 30 min before and during transient global cerebral ischemia markedly augmented ischemic brain damage in the hippocampal CA1 and CA2/3 regions compared with that in normothermia condition, and we found that the morphological activation of astrocytes and microglia was found from 1 day post-ischemia. In healthy humans, the normal core body temperature is 37 °C and tightly controlled by the thermoregulatory center in the hypothalamus. Sometimes, however, the thermoregulatory control is impaired by serious diseases. Cases with abnormally high temperatures are grossly divided into two classes. The first class referred to as ‘fever’ denotes to
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temperature due to a change in the hypothalamic set point. Fever depends on the presence of pyrogens and immune system intervention as well as the coordination of autonomic, endocrinal and behavioral responses [28]. Fever is clinically characterized by vasoconstriction and shivering for heat production or vasodilatation and sweating for heat dissipation. Such fevers usually respond well to antipyretics, and the pathophysiology is well known. The other class is hyperthermia that is characterized by an unchanged setting of the thermoregulatory center. Uncontrolled heat production (e.g., during exercise), poor heat dissipation (e.g., wearing protective clothing in a hot and humid environment), or an external heat load (e.g., sunbathing or sauna) are the underlying mechanisms [29]. Conditions in this group include heat stroke, malignant hyperthermia, neuroleptic malignant syndrome, serotonin syndrome, cerebral hemorrhage, and drug-induced hyperthermia. In a gerbil model of transient cerebral ischemia, sensitive regions in the brain include the cerebral cortex, striatum and CA1 region of the hippocampus [30–34]. Hippocampal pyramidal neurons, particularly those in the CA1 region of the dorsal hippocampus, have long been known to be very sensitive to cerebral ischemic damage [2–4]. 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 with that in the adult [35–37]. Also, it was reported that the different degrees of neuronal death in the hippocampal subregions was apparent when transient cerebral ischemia was induced by various durations (5, 10, 15 and 20 min) in the adult gerbil [38]. In the present study, to elucidate neuronal death in the gerbil hippocampal sub-regions induced by 5 min of cerebral ischemia under hyperthermic condition, CV staining, NeuN immunohistochemistry and F-J B histofluorescence staining were carried out. Especially, F-J B has a good affinity for entirely degenerating neurons, and it
Fig. 4. Immunohistochemistry for GFAP in the CA1 (1st and 3rd columns) and CA2/3 (2nd and 4th columns) regions of normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia group, GFAP+ astrocytes (arrows) are activated in the CA1 region from 2 days after ischemia–reperfusion and in the CA 2/3 region at 5 days post-ischemia. In the hyperthermia + ischemia groups, GFAP+ cells (Arrows) are activated in the CA1 from 1 day after ischemia–reperfusion and in the CA2/3 region from 2 days post-ischemia. At 5 days post-ischemia, GFAP+ astrocytes are hardly found in the stratum pyramidale (SP, asterisk). H, hyperthermia; N, normothermia; SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.
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is a very useful marker for study on neuronal degeneration after ischemic injury [27]. We, in the present study, increased the body temperature to 39.5 ± 0.2 °C (hyperthermia) for 30 min before and during the induction of transient global cerebral ischemia and examined much more exacerbated cell death in the CA1 region after ischemia– reperfusion. This finding is consistent with the previous studies that showed that an increase in brain temperature, during and after the ischemic insult, exacerbated ischemia-induced neuronal death in the hippocampal CA1 neurons [15,39]. Hara et al. (2000) also evaluated the effects of brain temperature (37–39 °C) on specific DNA fragmentation in gerbil CA1 pyramidal neurons following transient cerebral ischemia, and they indicated that hyperthermia accelerated the DNA fragmentation in the gerbil CA1 pyramidal neurons. We, in this study, found that neuronal death in the hyperthermia-mediated ischemic CA1 region occurred rapidly (1 day after 5 min of ischemia) compared with that in the normothermia-mediated ischemic CA1 region. In addition, we found that this hyperthermic condition exacerbated neuronal death in the CA2/3 region from 2 days after ischemia–reperfusion; this neuronal death never occurs in the normothermia-mediated ischemic CA2/3 region. Elevation of body temperature leads to physiological and structural changes, including the alteration of enzyme activity and damage of cytoskeletal proteins [40,41]. In addition, the release of neurotoxic excitatory neurotransmitters and reactive oxygen species [42], calcium influx into neurons [43] and vascular permeability [44] have been proposed as mechanisms through which hyperthermia leads to tissue injury. In the hippocampus, the vulnerability differs from each hippocampal subregion. The CA1 region is the most vulnerable to ischemia, whereas the CA2/3 regions are resistant to ischemic insults [3,38]. Thus,
hyperthermic condition before and during transient cerebral ischemia must increase the extent of pyramidal cell death not only in the CA1 region, which is the most vulnerable to ischemia, but also in the CA2/3 region, which is much more resistant to ischemic insults. On the other hand, in the central nervous system, gliosis including activations of astrocytes and microglia, as a ubiquitous hallmark of different neural pathological states, is very important in forming an environment that contributes either to successful repair and regeneration of damaged neurons or to severe injury of bystander cells [45]. Gliosis of astrocytes and microglia is easily induced in the hippocampus after transient cerebral ischemia in the adult gerbil [31,35]. Glial cells are far less susceptible to ischemic injury than neurons. For example, astrocytes are able to maintain ATP levels longer than neurons; they provide metabolic and trophic support to neurons and modulate synaptic activity [46]. That is why their impaired functions can critically influence neuronal survival. It is well known that astrocytes respond to ischemia by an increase in their number and size together with elongation of cytoplasmic processes [47], an increase in the expression of GFAP of the intermediate filaments [48], reorganization of their gap junctions supporting a functional syncytium [49] and transient accumulation of glycogen [47]. It was reported that a change in GFAP immunoreactivity in the CA1 region was related with neuronal degeneration and that astrocyte activation was associated with local tissue damage and neuronal loss [50,51]. Reactive astrogliosis was also observed in our present study. GFAP+ astrocytes were more quickly activated in the hyperthermia + ischemia groups compared with that in the normothermia + ischemia groups. Therefore, our present results indicate that accelerated/severe GFAP immunoreactivity in the hyperthermia + ischemia groups may be associated with a possibility of the
Fig. 5. Immunohistochemistry for Iba-1 in the CA1 (1st and 3rd columns) and CA2/3 (2nd and 4th columns) regions of the normothermia + ischemia (left two columns) and hyperthermia + ischemia (right two columns) groups at sham (a–d), 1 (e–h), 2 (i–l) and 5 days (m–p) after ischemia–reperfusion. In the normothermia + ischemia groups, Iba-1+ cells are activated in all layers of the CA1 and CA 2/3 regions from 1 day after ischemia–reperfusion (black arrows) and aggregated in the stratum pyramidale (SP) of the CA1 region at 5 days post-ischemia (black asterisk). In the hyperthermia + ischemia groups, Iba-1+ cells also are activated in all layers of the CA1 and CA2/3 regions from 1 day after ischemia– reperfusion; however, they are aggregated in the SP of the CA1 region at 2 days post-ischemia (black asterisk) and markedly increased in the SP of the CA2/3 region at 5 days postischemia. H, hyperthermia; N, normothermia; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 50 μm.
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Table 3 Change of the mean values of Iba-1 immunoreactivity in the hippocampal CA1 and CA2/3 regions in the normothermia + ischemia and hyperthermia + ischemia groups. Region
Groups
Time after ischemia–reperfusion Sham
CA1 CA2/3
N H N H
+ + + +
Ischemia Ischemia Ischemia Ischemia
100 100 100 100
± ± ± ±
1 day 4.5 4.0 3.0 3.1
101.8 109.3 103.9 103.4
2 days ± ± ± ±
2.1 3.8 2.9 5.4
103.0 115.5 111.7 109.5
± ± ± ±
5 days 2.4 2.60⁎ 2.6⁎ 5.4⁎
113.2 124.8 120.4 124.4
± ± ± ±
3.0⁎ 3.3⁎,# 2.5⁎ 4.0⁎
The relative optical density (ROD) values of Iba immunoreactivity are calibrated as the mean percentage of Iba immunoreactive structures with the normothermia + sham group designated as 100%. n = 7 at each time point. N + Ischemia, normothermia +ischemia group; H + Ischemia, hyperthermia + ischemia group. ⁎ P b 0.05, significantly different from the corresponding normothermia + sham group. # P b 0.05, significantly different from the corresponding normothermia + ischemia group.
secretion of harmful substances with different degrees induced by hyperthermia. It is well known that changes in the morphology and function of microglia are involved in response to various neural environments [52,53]. We, in the present study, compared the morphological change and immunoreactivity of Iba-1+ microglia in the hippocampal sub-regions following 5 min of cerebral ischemia between the normothermia + ischemia and hyperthermia + ischemia groups. Iba-1+ microglia in the hyperthermia + ischemia-groups were much earlier aggregated in the stratum pyramidale after 5 min of ischemia compared with that in the normothermia + ischemia groups. This finding coincides with a previous study that showed that a stronger and more intense glial reaction was related with post-ischemic tissue damage in aged gerbils [54]. Especially, it was recently reported that extensively increased microglia activation occurred in the hippocampal sub-regions and striatum with severe neuronal damage induced by much prolonged ischemic duration [33,38]. Therefore, we suggest that the excess of microglia activation in the hyperthermia + ischemia groups may be related with the accelerated neuronal death in the hippocampus. In conclusion, hyperthermia (39.5 ± 0.2 °C) for 30 min before and during ischemic insult markedly augmented ischemic neuronal damage in the hippocampal sub-regions compared with normothermia (37 ± 0.2 °C) and more accelerated activations of astrocytes and microglia under hyperthermia. These results indicate that neuronal death and gliosis are very different in hyperthermia-mediated brains from those in normothermia-conditioned brains following transient cerebral ischemia. These suggest a wide window of opportunity that is available for the prevention and management of ischemic damage under hyperthermia. Acknowledgements The authors would like to thank Mr. Seung Uk Lee for his technical help in this study. This study was supported by a 2014 Research Grant from Kangwon National University (No. 120140271), and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2001404). Conflict of interest The authors have declared that there is no conflict of interest. References [1] Sims NR, Zaidan E. Biochemical changes associated with selective neuronal death following short-term cerebral ischaemia. Int J Biochem Cell Biol 1995;27:531–50. [2] Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 1982;239:57–69. [3] Schmidt-Kastner R, Freund TF. Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 1991;40:599–636. [4] 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.
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