Journal of the Neurological Sciences 347 (2014) 179–187

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Effects of ischemic preconditioning on VEGF and pFlk-1 immunoreactivities in the gerbil ischemic hippocampus after transient cerebral ischemia Yoo Seok Park a,b,1, Jun Hwi Cho a,1, In Hye Kim c, Geum-Sil Cho d, Jeong-Hwi Cho c, Joon Ha Park c, Ji Hyeon Ahn c, Bai Hui Chen e, Bich-Na Shin e, Myoung Cheol Shin a, Hyun-Jin Tae f, Young Shin Cho a,g, Yun Lyul Lee e, Young-Myeong Kim h, 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 Neuroscience, College of Medicine, Korea University, Seoul 136-705, South Korea e Department of Physiology, College of Medicine and Institute of Neurodegeneration and Neuroregeneration, Hallym University, Chuncheon 200-702, South Korea f Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chuncheon 200-702, South Korea g Department of Emergency Medicine, Seoul Hospital, College of Medicine, Sooncheonhyang University, Seoul 140–743, South Korea h Department of Molecular and Cellular Biochemistry, 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 25 July 2014 Received in revised form 1 September 2014 Accepted 23 September 2014 Available online 2 October 2014 Keywords: Ischemia–reperfusion Ischemic preconditioning Delayed neuronal death Vascular endothelial growth factor Phospho-Flk-1 Pericytes

a b s t r a c t Ischemia preconditioning (IPC) displays an important adaptation of the CNS to sub-lethal ischemia. In the present study, we examined the effect of IPC on immunoreactivities of VEGF-, and phospho-Flk-1 (pFlk-1) following transient cerebral ischemia in gerbils. The animals were randomly assigned to four groups (sham-operated-group, ischemia-operated-group, IPC plus (+) sham-operated-group, and IPC + ischemia-operated-group). IPC was induced by subjecting gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operatedgroup, a significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) alone 5 days after ischemia–reperfusion, however, in all the IPC + ischemia-operated-groups, pyramidal neurons in the SP were well protected. In immunohistochemical study, VEGF immunoreactivity in the ischemiaoperated-group was increased in the SP at 1 day post-ischemia and decreased with time. Five days after ischemia– reperfusion, strong VEGF immunoreactivity was found in non-pyramidal cells, which were identified as pericytes, in the stratum oriens (SO) and radiatum (SR). In the IPC + sham-operated- and IPC + ischemia-operated-groups, VEGF immunoreactivity was significantly increased in the SP. pFlk-1 immunoreactivity in the sham-operated- and ischemia-operated-groups was hardly found in the SP, and, from 2 days post-ischemia, pFlk-1 immunoreactivity was strongly increased in non-pyramidal cells, which were identified as pericytes. In the IPC + sham-operatedgroup, pFlk-1 immunoreactivity was significantly increased in both pyramidal and non-pyramidal cells; in the IPC + ischemia-operated-groups, the similar pattern of VEGF immunoreactivity was found in the ischemic CA1, although the VEGF immunoreactivity was strong in non-pyramidal cells at 5 days post-ischemia. In brief, our findings show that IPC dramatically augmented the induction of VEGF and pFlk-1 immunoreactivity in the pyramidal cells of the CA1 after ischemia–reperfusion, and these findings suggest that the increases of VEGF and Flk-1 expressions may be necessary for neurons to survive from transient ischemic damage. © 2014 Elsevier B.V. All rights reserved.

1. Introduction When the blood supply to the brain is disrupted, the tissue deprivation of oxygen and glucose occurs and it may give irreversible brain

⁎ Corresponding author. Tel.: +82 33 250 8891; fax: +82 33 256 1614. ⁎⁎ Corresponding author. Fax: +82 33 256 1614. E-mail addresses: [email protected] (M.-H. Won), [email protected] (J.-C. Lee). 1 Yoo Seok Park and Jun Hwi Cho have contributed equally to this article.

http://dx.doi.org/10.1016/j.jns.2014.09.044 0022-510X/© 2014 Elsevier B.V. All rights reserved.

damage [1,2]. A brief period of global brain ischemia causes cell damage/death in vulnerable hippocampal CA1 region a few days after reperfusion, and is referred to as “delayed neuronal death” [3]. Many mechanisms related to ischemia-induced delayed neuronal death, including reactive oxygen species, oxidative stress and DNA damage, have been suggested [4–6]. However, the mechanisms underlying them have not been exactly elucidated yet. Ischemia preconditioning (IPC) represents an important adaptation of the central nervous system (CNS) to sub-lethal ischemia, which results in increased ischemia tolerance of the CNS to a subsequent longer or lethal period of

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ischemia [7,8]. IPC induces the expression of a diverse family of genes involved in cytoprotection, which, in turn, encodes proteins that serve to enhance brain resistance to ischemia [9,10]. This phenomenon has been termed “ischemic tolerance”, and its basic mechanisms underlying ischemic tolerance are not fully understood yet [11]. Vascular endothelial growth factor (VEGF) is well known as an angiogenic and vascular permeability factor [12,13], and plays a regulatory role in the nervous system [14,15]. In the developing nervous system, VEGF acts as a neurotrophic factor, regulates axonal outgrowth and increases neuronal survival [16,17]. On the other hand, VEGF protects hippocampal neurons from glutamate toxicity in vitro [18]. Moreover, it has been well known that VEGF improves cognitive deficits and neuronal protection against ischemic injury, and it is suggested that VEGF may participate in the brain's endogenous response to ischemic injury [19–22]. In addition to VEGF itself, two main classes of receptors for the VEGF have been identified: the tyrosine kinase and nontyrosine kinase receptors. The former contains three structurally related receptors, VEGFR-A (Flt-1), VEGFR-B (Flk-1), and VEGFR-C. The nontyrosine receptors consist of neuropilin-1 (NP-1) and neuropilin-2 (NP-2). Flk1 is a major mediator of angiogenic and permeability-enhancing effects of VEGF [23,24], and its expression is up-regulated in neurons, glial cells, and endothelial cells by focal cerebral ischemia [25–27] and global cerebral ischemia [28]. Furthermore, we recently reported that VEGF, which was expressed in the gerbil hippocampus, might be related with neuronal loss after transient global cerebral ischemia, and its expression was different according to aging [29]. However, little is known regarding expression patterns of VEGF and Flk-1 and phenotypes of cells expressing VEGF and Flk-1 in the brain of IPC-mediated animals induced by transient cerebral ischemia. This study, therefore, was performed to investigate temporal changes and cellular localization of VEGF and phospho-Flk-1 (pFlk-1) in the gerbil hippocampus following transient global cerebral ischemia. We also examined changes in VEGF and pFlk-1 expressions in the brain of IPCmediated gerbils. 2. Materials and methods 2.1. Experimental animals We used the male Mongolian gerbils (Meriones unguiculatus) 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 °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 cerebral ischemia and animal groups Transient cerebral ischemia was developed following our previous method. [6]. The experimental animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. Under an operating microscope, ischemia was induced by occluding the common carotid arteries with non-traumatic aneurysm clips (Yasargil FE 723 K, Aesculap, Tuttlingen, Germany) for 2 min or 5 min. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope (HEINE K 180®, Heine Optotechnik, Herrsching, Germany). The body (rectal) temperature was monitored with a rectal temperature probe (TR-100; Fine Science Tools, Foster

City, CA) and maintained under free-regulating or the normothermic (37 ± 0.5 °C) conditions using a thermometric blanket for the surgery. Thereafter, animals were kept on the thermal incubator (temperature, 23 °C; humidity, 60%) (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature of animals until the animals were euthanized. The animals were divided into four groups: (1) shamoperated-group (n = 7 at each time point): the bilateral common carotid arteries were exposed, no ischemia was given (sham-operation) in the animals; (2) ischemia-operated-group (n = 7 at each time point): the animals were given a 5 min lethal ischemic insult 24 h after shamoperation (3) IPC plus (+) sham-operated-group (n = 7 at each time point): the animals were subjected to a 2 min sublethal ischemic insult; and (4) IPC + ischemia-operated-group (n = 7 at each time point): the animals were pretreated with a 2 min sublethal ischemia 1 day prior to a 5 min lethal ischemia. The animals in groups 2) and 4) 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 4 days after ischemia–reperfusion. This preconditioning paradigm has been proven to be very effective at protecting neurons against ischemia in this ischemic model [30]. 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 (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 the neuronal death in the hippocampal CA1 region in each group 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. Neuronal nuclei immunohistochemistry To examine the neuronal changes in the hippocampal CA1 region after transient cerebral 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 (a neuron-specific soluble nuclear antigen) (diluted 1:1000, Chemicon International, Temecula, CA) overnight at 4 °C. Thereafter the tissues were exposed to biotinylated horse antimouse IgG and streptavidin peroxidase complex (Vector, Burlingame, CA). And they were visualized with 3,3′-diaminobenzidine in 0.1 M Tris–HCl buffer and mounted on the gelatin-coated slides. After dehydration the sections were mounted with Canada balsam (Kanto chemical). 2.6. Fluoro-Jade B (F-J B) histofluorescence To examine neuronal death in the CA1 region at each time point after ischemia using F-J B (a high affinity fluorescent marker for the localization of neuronal degeneration) histofluorescence [31], the sections were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol, and followed in 70% alcohol. They were then

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transferred to a solution of 0.06% potassium permanganate, and transferred to a 0.0004% F-J 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 [7]. 2.7. Cell counts 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. According to anatomical landmarks corresponding to AP − 1.4 to − 1.9 mm of the gerbil brain atlas, the studied tissue sections were selected with 300-μm interval, and cell counts were obtained by averaging the total cell numbers 15 sections taken from each animal per group. The number of NeuN- and F-J B-positive cells was counted in a 200 × 200 μm square, applied approximately at the center of the CA1 region using an image analyzing system (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ). Cell counts were obtained by averaging the total number from each animal per group.

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fibrillary acidic protein (GFAP) (diluted 1:200, Chemicon International) for astrocytes or goat anti-platelet-derived growth factor receptor-β (PDGFR-β) (diluted 1:50, Santa Cruz Biotech.) for pericytes. The sections were incubated in the mixture of antisera overnight at room temperature. After washing 3 times for 10 min with PBS, they were then incubated in a mixture of both FITC-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA), Cy3-conjugated donkey anti-mouse IgG (1:200; Jackson ImmunoResearch), and Cy3-conjugated donkey anti-goat IgG (diluted 1:200; Jackson ImmunoResearch) for 2 h at room temperature. The immunoreactions were observed under the confocal MS (LSM 510 META NLO, Carl Zeiss, Germany). Negative control test was also carried out using preimmune serum instead of primary antibody in order to establish the specificity of the immunostaining. The negative control resulted in the absence of immunoreactivity in all structures. 2.10. Statistical analysis All data are presented as mean ± S.E.M. A multiple-sample comparison was applied to test the differences between groups (ANOVA and the Tukey multiple range test as post hoc test using the criterion of the least significant differences). Statistical significance was considered at P b 0.05.

2.8. Immunohistochemistry for VEGF and pFlk-1 3. Results To obtain the accurate data for immunoreactivity, the sections from sham-operated, IPC-operated-sham, ischemia-operated and IPC + ischemia-operated-group (n = 7 at each time point) were used at designated times under the same conditions. The sections collected into six-well plates containing PBS after tissue processing 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. They were next incubated with diluted rabbit anti-VEGF (diluted 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-pFlk-1 (diluted 1:1000, Santa Cruz Biotechnology) overnight at 4 °C and subsequently exposed to biotinylated horse anti-rabbit IgG and streptavidin peroxidase complex (diluted 1:200, Vector, Burlingame, CA). They were then visualized by staining with 3,3′-diaminobenzidine (Sigma-Aldrich) 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). In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. The negative control resulted in the absence of immunoreactivity in any structures. Twenty sections per animal were selected to quantitatively analyze VEGF and pFlk-1 immunoreactivity. Cellular immunoreactivity of VEGF and pFlk-1 was graded in the hippocampal CA1 region. Digital images of the hippocampal CA1 region (strata oriens, pyramidale and radiatum in the hippocampus proper) were captured with an AxioM 1 light microscope (Carl Zeiss) equipped with a digital camera (AxioCam, Carl Zeiss) connected to a PC monitor. Semi-quantification of the intensity of NeuN- and F-J B-positive structures was evaluated with digital image analysis software (MetaMorph 4.01, Universal Imaging Corp.). 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 [32]. 2.9. Double immunofluorescence staining To confirm the cell type containing VEGF and pFlk-1 immunoreactivity, the sections at 5 days after the ischemic surgery were processed by double immunofluorescence staining. Double immunofluorescence staining was performed using rabbit anti-pFlk-1 (1:25, Santacruz Biotec.)/goat anti-ionized calcium-binding adapter molecule 1 (Iba-1) (diluted 1:100, Santacruz Biotec) for microglia or mouse anti-glial

3.1. Cresyl violet-positive (CV+) cells We examined whether IPC was associated with an increase in neuronal cell survival in the hippocampal CA1 region after ischemia. In the present study, neurons were identified morphologically by their larger and pale nuclei surrounded by darkly stained cytoplasm containing Nissl bodies (diameter N 10 μm). In the sham-operated-group, CV+ cells were easily observed in all the subregions of the hippocampus (Fig. 1A, B). Neurons in the stratum pyramidale were relatively large, pyramid-like or round in shape. In the ischemia-operated-groups, the morphology and the number of CV+ cells were not changed until 2 days after ischemia–reperfusion (Fig. 1E, F). However, in the ischemiaoperated-group at 5 days after ischemia–reperfusion, the number of CV+ cells was significantly decreased in the stratum pyramidale of the CA1 region compared with those of the sham-operated-group (Fig. 1I, J); in high magnification, the damaged cells were shrunken and contained dark and polygonal nuclei (Fig. 1J). In the IPC + sham-operated-group, CA1 pyramidal cells were well stained with CV (Fig. 1C, D). In the IPC + ischemia-operated-groups, the distribution pattern of CV+ cells in the stratum pyramidale was similar to that in IPC + sham-operated-group at all times after ischemia– reperfusion (Fig. 1G, H, K, L). 3.2. NeuN+ and F-J B+ neurons The protection afforded by IPC against delayed neuronal death in the CA1 region of the hippocampus was assessed with NeuN immunohistochemistry and F-J B histofluorescence staining (Fig. 2). In the shamoperated-group, pyramidal neurons in the CA1 region were well stained with NeuN (Table 1, Fig. 2A); however, there were no F-J B+ neurons (Table 1, Fig. 2B). In the ischemia-operated-group at 2 days postischemia, the distribution pattern of NeuN+ and F-J B+ neurons in the CA1 was similar to that in the sham-operated- group (Table 1, Fig. 2E, F). At 5 days post-ischemia, NeuN+ neurons were markedly decreased in the stratum pyramidale of the CA1 region (Table 1, Fig. 2I), and many F-J B+ cells were detected in the CA1 region (Table 1, Fig. 2J). In the IPC + sham-operated-group, pyramidal neurons in the CA1 region were also well stained with NeuN (Table 1, Fig. 2C), and F-J B+ cells were not observed (Table 1, Fig. 2D). In the IPC + ischemiaoperated-groups from 2 days to 5 days post-ischemia, the distribution

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Fig. 1. Histochemical staining for cresyl violet in the CA1 of the ischemia-operated- (left two columns) and IPC + ischemia-operated- (right two columns) groups at sham (A–D), 2 days (E–H) and 5 days (I–L) post-ischemia. In the ischemia-operated groups, a few CV+ neurons (black arrows) are shown in the stratum pyramidale (SP) at 5 days post-ischemia; however, abundant CV+ neurons (white asterisk) are observed in the IPC + ischemia-operated-group. SO; stratum oriens, SP; stratum pyramidale, SR; stratum radiatum. Scale bar = 800 (A, C, E, G, I, K) and 50 (B, D, F, H, J, L) μm.

Fig. 2. NeuN immunohistochemistry (the first and third longitudinal columns) and F-J B histofluorescence staining (the second and fourth longitudinal columns) in the CA1 of the ischemiaoperated- (left two columns) and IPC + ischemia-operated- (right two columns) groups at sham (A–D), 2 days (E–H) and 5 days (I–L) after ischemia–reperfusion. In the sham-operatedgroups, many NeuN+ neurons and no F-J B+ cells are detected in the stratum pyramidale (SP). In the ischemia-groups, only a few NeuN+ neurons (black arrows) and many F-J B+ cells (white asterisk) are detected in the SP at 5 days post-ischemia. However, in the IPC + ischemia-operated-group, abundant NeuN+ neurons and few F-J B+ cells are detected in the SP at 5 days post-ischemia. SO; stratum oriens, SR; stratum radiatum. Scale bar = 50 μm.

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Table 1 Changes in the mean number of pyramidal neurons of the hippocampal CA1 region in the ischemia-operated- and IPC + ischemia-operated-groups. Time after I–R

Group Ischemia NeuN

Sham 1d 2d 5d

356 361 354 41

+

± ± ± ±

IPC + ischemia F-J B

14.88 12.27 10.25 7.28⁎

+

0 0 5 ± 3.17⁎ 122 ± 7.33⁎

NeuN+ 367 354 361 315

± ± ± ±

F-J B+ 10.11 12.69 13.11 11.98⁎#

0 0 0 14 ± 5.36⁎#

The mean number of NeuN+ and F-J B+ cells is counted in a 200 × 200 μm square of the stratum pyramidale of the CA1 region after ischemia–reperfusion (I–R)(n = 7 per group. ⁎ P b 0.05, significantly different from the corresponding sham-group. # P b 0.05, significantly different from the respectively preceding group). IPC, ischemic preconditioning.

patterns of NeuN+ and F-J B+ neurons in the stratum pyramidale were not significantly changed compared with those in the IPC + shamoperated-group (Table 1, Fig. 2G, H, K, L). 3.3. VEGF immunoreactivity in the CA1 region In the sham-operated-group, weak VEGF immunoreactivity was detected in somata of pyramidal cells in the stratum pyramidale of the CA1 region (Table 2, Fig. 3A). Moderate VEGF immunoreactivity was also found in the dendrites of pyramidal cells in the stratum radiatum (Fig. 3A). In the ischemia-operated-groups, VEGF immunoreactivity was apparently increased in the stratum pyramidale 1 day after ischemia–reperfusion (Table 2, Fig. 3C), and, at 2 days post-ischemia, VEGF immunoreactivity was decreased in the stratum pyramidale (Table 2, Fig. 3E). At 5 days post-ischemia, strong VEGF immunoreactivity was observed in non-pyramidal cells in the strata oriens and radiatum (Table 2, Fig. 3G). In the IPC + sham-operated-group, VEGF immunoreactivity was markedly increased in the pyramidal cells of the CA1 region; in this group, moderate VEGF immunoreactivity was found in the neuropil of the stratum oriens and radiatum (Table 2, Fig. 3B). Thereafter, VEGF immunoreactivity in the pyramidal cells of the CA1 region was maintained until 5 days after ischemia–reperfusion (Table 2, Fig. 3D, F, H), and, in the stratum oriens and radiatum, VEGF immunoreactivity was increased in non-pyramidal cells from 1 day after ischemia–reperfusion (Table 2, Fig. 3F, H). 3.4. pFlk-1 immunoreactivity in the CA1 region In the sham-operated-group, pFlk-1 immunoreactivity was hardly detected in the pyramidal cells of the stratum pyramidale in the CA1 region, and moderate pFlk-1 immunoreactivity was shown in the neuropil of the strata oriens and radiatum (Table 2, Fig. 4A). This pattern was

Table 2 Semi-quantifications of VEGF and pFlk-1 immunoreactivities in the hippocampal CA1 region in the ischemia-operated- and IPC + ischemia-operated-groups. Antibody

Groups

Category

VEGF

Ischemia

CSP CSOR CSP CSOR CSP CSOR CSP CSOR

IPC + ischemia pFlk-1

Ischemia IPC + ischemia

Time after ischemia/reperfusion Sham

1d

2d

5d

+ ± ++ ± − + ++ +

++ ± ++ + − + ++ +

+ ± ++ + − ++ ++ +

± ++ ++ + − ++ ++ ++

Immunoreactivity is scaled as −, ±, + or ++, representing no staining, weakly positive, moderate or strong, respectively. CSP, cells in stratum pyramidale; CSOR, cells in stratum oriens and radiatum. IPC, ischemic preconditioning.

Fig. 3. Immunohistochemistry for VEGF in the CA1 region of the ischemia-operated- (left columns) and IPC + ischemia-operated- (right columns) groups at sham (A, B), 1 day (C, D), 2 days (E, F) and 5 days (G, H) after ischemia–reperfusion. VEGF immunoreactivity is increased in the stratum pyramidale (SP) 1 day after ischemia–reperfusion, and decreased at 2 days post-ischemia. At 5 days post-ischemia, strong VEGF immunoreactivity is found (black arrows) in the stratum oriens (SO) and radiatum (SR). In the IPC + shamoperated-group, VEGF immunoreactivity is significantly increased in the SP (black asterisk), and maintained until 5 days post-ischemia. At 5 days post-ischemia, VEGF immunoreactivity is increased in the SO and SR. Scale bar = 50 μm.

maintained at 1 day post-ischemia (Table 2, Fig. 4C). Two days after ischemia–reperfusion, strong pFlk-1 immunoreactivity was observed in non-pyramidal cells, which were located in the strata oriens and radiatum (Table 2, Fig. 4E), and, this pattern in the ischemia CA1 region was maintained until 5 days after ischemia–reperfusion (Table 2, Fig. 4G). In the IPC + sham-operated-group, strong pFlt-1 immunoreactivity was detected in the pyramidal neurons of the CA1 region, and, moderate pFlk-1 immunoreactivity was found in non-pyramidal cells in the stratum oriens (Table 2, Fig. 4B). In the IPC + ischemia-operated-groups, the pattern of pFlk-1 immunoreactivity in the CA1 region was similar to that in the IPC + sham-operated-group until 2 days post-ischemia (Table 2, Fig. 4D, F). However, 5 days after ischemia–reperfusion, pFlk1 immunoreactivity in the stratum oriens and radiatum was increased, although the pFlk-1 immunoreactivity in the stratum pyramidale was similar to that in the IPC + sham-operated-group (Table 2. Fig. 4H) 3.5. Double immunofluorescence staining for VEGF and pFlk-1 in the CA1 region To elucidate the type of VEGF+ and pFlk-1+ cells that were found in the stratum radiatum, we conducted double immunofluorescence

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staining in the CA1 region using VEGF or pFlk-1/Iba-1, VEGF or pFlk-1/ GFAP, and VEGF or pFlk-1/PDGFR-β 5 days after ischemia–reperfusion. We found that VEGF+ and pFlk-1+ structures were colocalized with PDGFR-β+ pericytes (Fig. 5), not Iba-1+ microglia and GFAP+ astrocytes (data not shown), in the ischemic CA1 region of the IPC + ischemiaoperated-group (Fig. 5). 3.6. VEGF and pFlk-1 immunoreactivities in the CA3 region Moderate VEGF and pFlk-1 immunoreactivities were detected in the stratum pyramidale of the CA3 region (Fig. 6A and E). VEGF and pFlk-1 immunoreactivities in the CA3 region were barely changed in the ischemia-operated-group (Fig. 6C and G). In the IPC + sham-operated-group, the pattern of VEGF and pFlk-1 immunoreactivities was similar to that in the sham-operated-group (Fig. 6B and F). In addition, the pattern of the immunoreactivities in the IPC + ischemia-operated-groups was not changed after ischemia– reperfusion (Fig. 6D and H). 4. Discussion

Fig. 4. Immunohistochemistry for pFlk-1 in the CA1 of the ischemia-operated (left columns) and IPC + ischemia-operated- (right columns) groups at sham (A, B), 1 day (C, D), 2 days (E, F) and 5 days (G, H) after ischemia–reperfusion. pFlk-1 immunoreactivity is barely detected in the stratum pyramidale (SP) of the sham-operated-group, and, moderate pFlk-1 immunoreactivity is shown in the stratum oriens (SO) and stratum radiatum (SR). pFlk-1 immunoreactivity is strongly increased (black arrows) in the SO and SR from 2 days after ischemia–reperfusion. In the IPC + sham-operated-group, pFlk-1 immunoreactivity is markedly increased in the SP (black asterisk), and maintained until 5 days after ischemia–reperfusion; at 5 days post-ischemia, pFlk-1 immunoreactivity in the SO and SR is increased. Scale bar = 50 μm.

In our present study, we found that and CA1 pyramidal neurons did not die in the IPC-induced gerbil hippocampus 5 days after ischemia– reperfusion and that IPC increased VEGF and pFlk-1 immunoreactivities in the CA1 pyramidal neurons after ischemia–reperfusion injury. Thus, the increases of VEGF and pFlk-1 immunoreactivities by IPC may be a key factor in protecting neurons from ischemia–reperfusion damage. Transient cerebral ischemia following the deprivation of blood flow of the forebrain develops delayed neuronal death in specific vulnerable regions of the brain, such as the hippocampus and neocortex [3]. The Mongolian gerbil has been used as a good animal model to investigate mechanisms of the delayed neuronal death following transient cerebral ischemia, because about 90% of gerbils lack the communicating vessels between the carotid and vertebral circulations [33]. An important feature of global brain ischemia is the vulnerability of specific neuronal populations. Especially, pyramidal neurons in the hippocampal CA1 region do not die immediately but rather survive over several days [34–36]. However, the exact mechanisms underlying neuronal damage and delayed neuronal death in ischemic conditions have not been elucidated yet. In the present study, we found a significant loss of CV positive cells and NeuN-immunoreactive neurons in the pyramidal layer of the ischemic CA1 region 5 days after ischemia–reperfusion. CV has been widely used for a histological method to identify cell damage in the nervous system, because it binds to acidic components in the cytoplasm

Fig. 5. Double immunofluorescence staining for VEGF (green, A), pFlk-1 (green, D), PDGFR-β (red, B and E) and merged images (C and F) in the ischemic CA1 region at 5 days post-ischemia. VEGF+ and pFlk-1+ cells are colocalized with PDGFR-β+ pericytes. Scale bar = 50 μm.

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Fig. 6. Immunohistochemistry for VEGF and pFlk-1 in the CA3 region of the ischemia-operated (left columns) and IPC + ischemia-operated- (right columns) groups at sham (A, B, E, F) 5 days (C, D, G, H) after ischemia–reperfusion. Moderate VEGF and pFlk-1 immunoreactivities are shown in the stratum pyramidale (SP) of the sham-operated-group and IPC + sham-operatedgroup. The pattern of these immunoreactivities was not changed after ischemia–reperfusion. SO; stratum oriens, SR; stratum radiatum. Scale bar = 50 μm.

[37]. Damaged cells show various features including a shrunken cell body with pyknosis and chromatolysis. However, this method is insufficient to discriminate neuronal degeneration, because the presence of argyrophilic dark neurons simply reflects exposure to an insult that would ultimately result in the neurons either dying or recovering [38]. On the other hand, it is important to count neuronal, not glial, loss in a damaged brain, because NeuN immunohistochemistry shows an apparent neuronal loss. In addition, we used F-J B histofluorescence to elucidate the degree of neuronal damage in the hippocampus of the gerbil brain after ischemia. F-J B has a good affinity for entirely degenerating neurons (cell bodies, dendrites, axons and axon terminals), and it is a very useful marker for study on neuronal degeneration after ischemic injury [7]. IPC, which does not lead to neuronal death, is able to induce neuronal tolerance to a subsequent longer or lethal period of ischemia. The first description of IPC in the brain was demonstrated by Kitagawa et al. [39] in a gerbil model of global ischemia. As further studies have been widely described in other animal models, including global and focal cerebral ischemia [9,40–44]. The preconditioning time period was determined by a previous paper [30] that indicates that at least 1 day interval between sublethal 2 min ischemia and lethal 5 min ischemia was necessary for the induction of the neuroprotection of the CA1 pyramidal cells. In this study, we examined the protective effect of IPC against delayed neuronal death in the hippocampal CA1 region after 5 min of transient cerebral ischemia in gerbils. Five days after ischemic insult, the CA pyramidal neurons showed a feature of neuronal death; we found a significant reduction of CV+ and NeuN+ neurons and appearance of F-J B+ neurons, however, the numbers of viable neurons in the CA1 region were significantly increased by IPC. Because IPC often provides profound protection against ischemic brain injury, understanding its mechanisms may have important significance in the development of therapeutic strategies for cerebral ischemia. VEGF is well known as an angiogenic and permeability regulator [12,13,45], and possesses neurotrophic and neuroprotective activities [46–49]. However, previous studies regarding the effects of exogenous VEGF on experimental brain injury models have produced conflicting results. It is documented that VEGF can increase cerebral vascular permeability, induce brain edema [50–52] and pro-inflammatory cell recruitment [53], which may be deleterious. In fact, the systemic delivery of VEGF increases blood–brain barrier (BBB) leakage and tissue

damage after cerebral ischemia [54,55], whereas the inhibition of VEGF or its receptor improves neurological outcomes as well as reduces BBB permeability after cerebral ischemia [56–60], especially during acute injury phase. On the other hand, it has been demonstrated that the induction of endogenous VEGF directly plays neuroprotective role against cerebral ischemia [61–63], and its neuroprotective mechanism may be related with angiogenesis [64] or vessel protection [63]. In addition, VEGF may play a direct cytoprotective role through the activation of VEGF receptor [61,65]. The activation of VEGF receptor-mediated phosphatidylinositol 3′-kinase (PI3-K)/Akt pathway can up-regulate anti-apoptotic proteins by global cerebral ischemia and down-regulate activated caspase-3, and, therefore, participate in endogenous neuroprotective responses to cerebral ischemia [66]. Furthermore, a recent investigation showed that VEGF was mainly expressed in neurons, endothelial cells and astroglia after hypoxia–ischemia in the neonatal rat and piglet brains [67,68]. In addition, VEGF/Flk-1 pathway has been shown to be involved in the protective mechanism of neonatal murine models of hypoxic preconditioning and ligation-preconditioning [68,69]. A low oxygen environment triggers VEGF synthesis by the pathway leading to transcription factor HIF-1, and induces the upregulation of Flk-1 [70]. Studies in vitro showed that Flk-1 was crucial for the survival of neuronal cells and endothelial cells [71,72]. Therefore, increased expressions of VEGF and its receptor following IPC could reflect survival mechanisms of neurons to prevent themselves from death. In the present study, we compared changes of the endogenous expressions of both VEGF and pFlk-1 in the CA1 region between the ischemia-operated-group and IPC + ischemia-operated-group. VEGF immunoreactivity in the ischemia-operated-group was increased in the SP at 2 days post-ischemia, and decreased at 5 days post-ischemia; meanwhile, any CA1 pyramidal neurons did not show pFlk-1 immunoreactivity at any time after ischemia–reperfusion. However, both VEGF and pFlk-1 immunoreactivities were significantly increased in nonpyramidal cells, which were identified as pericytes after ischemic insult. On the other hand, the immunoreactivities of both VEGF and pFlk-1 in the IPC + sham-operated-group were significantly higher than those in the sham-operated-group, and, in the IPC + ischemia-operatedgroups, the immunoreactivities of VEGF and pFlk-1 were maintained in the CA1 pyramidal neurons after ischemic insult. Therefore, our in vivo findings are compatible with the reports that showed that VEGF mediated the protective effect of hypoxic preconditioning in

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neurons [69,73]. In our present study, VEGF and pFlk-1 immunoreactivities were not changed in the CA3 region after ischemia–reperfusion. The CA3 region is known as the most tolerant region to ischemia in the hippocampus [1,2]. This finding indicates that the expression of VEGF and pFlk-1 in the CA3 region may be necessary for neurons to survive after ischemic damage, although little is known about the role of VEGF and pFlk-1 in the ischemic CA3 region. In addition, our present finding is supported by a previous study that showed that hypoxic preconditioning resulted in the increased expressions of VEGF and its receptor after hypoxia–ischemia in the neonatal piglet brain [67]. Taken together, these results suggest that up-regulations of VEGF and pFlk-1 in the CA1 pyramidal neurons display important roles in IPCinduced neuroprotection after ischemia–reperfusion. It was reported that, in a rat model of transient global ischemic injury, Flk-1 receptor was up-regulated in activated glial cells and vascular endothelial cells, but not in any neurons in ischemic brain and that activated glial cells expressing Flk-1 were mostly reactive astrocytes, plus some microglial cells [28]. In addition, some researchers showed that VEGF and Flk-1 receptors were expressed in glial and vascular endothelial cells after focal cerebral ischemia [25,62]. Furthermore, after traumatic insults, Flk-1 receptor is up-regulated in neurons [68,71,74]. In the present study, both VEGF and pFlk-1 immunoreactivities were observed in non-pyramidal cells, which were located in the strata oriens and radiatum, after ischemia–reperfusion. We also found that both VEGF and pFlk-1 immunoreactivities were colocalized with PDGFR-β, not in Iba-1 and GFAP, in the strata oriens and radiatum of the CA1 region 5 days after ischemic insult; however, in the IPC + ischemiaoperated-group, pFlk-1 immunoreactivity in the pericytes was weaker than that in the ischemia-operated-group. Pericytes in the brain are known to express high levels of VEGF and their corresponding receptor [75,76]. These pericytes are located at the periphery of microvessel wall and wrap around endothelial cells with their processes, and they provide structural support, stability, and integrity to the vessel wall [77,78]. In addition, pericytes can help to repair injured brain after ischemic insults, because the pericytes have multiple functions in the pathogenesis of ischemic injury [79–81]. In conclusion, main findings of this study are that VEGF and pFlk-1 immunoreactivities are significantly increased in pericytes in the CA1 region induced by transient ischemia and that IPC protects CA1 pyramidal neurons form ischemic damage with increases of VEGF and pFlk-1 immunoreactivities in the CA1 pyramidal neurons. These suggest that VEGF and Flk-1 may be necessary for neurons to survive from ischemic damage. Conflict of interest The authors have no financial conflict of interest. Acknowledgments The authors would like to thank Mr. Seung Uk Lee for their technical help in this study. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2001404), and by 2013 research grant from Kangwon National University (no. 120131326). References [1] Schmidt-Kastner R, Freund TF. Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 1991;40:599–636. [2] Munekata K, Hossmann KA. Effect of 5-minute ischemia on regional pH and energy state of the gerbil brain: relation to selective vulnerability of the hippocampus. Stroke 1987;18:412–7. [3] Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 1982;239:57–69. [4] Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab 2001;21:2–14.

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