Neurochem Res (2013) 38:1045–1054 DOI 10.1007/s11064-013-1015-2
ORIGINAL PAPER
Reduced Beta-Catenin Expression in the Hippocampal CA1 Region Following Transient Cerebral Ischemia in the Gerbil Jeong-Hwi Cho • Bing Chun Yan • Young Joo Lee • Joon Ha Park • Ji Hyeon Ahn • In Hye Kim • Jae-Chul Lee • Young-Myeong Kim • Bonghee Lee • Jun Hwi Cho • Moo-Ho Won
Received: 3 December 2012 / Revised: 8 February 2013 / Accepted: 5 March 2013 / Published online: 16 March 2013 Ó Springer Science+Business Media New York 2013
Abstract Beta-catenin, a transcription factor, plays a critical role in cell survival and degradation after stroke. In this study, we examined changes of expression in betacatenin in the hippocampal CA1 region of the gerbil following 5 min of transient cerebral ischemia. We observed neuronal damage using cresyl violet staining, neuronal nuclei immunohistochemistry and Fluro-Jade B immunofluorescence. Four days after ischemia-reperfusion (I-R), most of pyramidal cells in the CA1 region were damaged. In addition, early damage in dendrites was detected 1 day after I-R by immunohistochemical staining for microtubuleassociated protein 2 (MAP-2), and MAP-2 immunoreactivity was hardly detected in the CA1 region 4 days after I-R. We found that beta-catenin (a synapse-enriched cell Jeong-Hwi Cho and Bing Chun Yan have contributed equally to this article. J.-H. Cho B. C. Yan J. H. Park I. H. Kim J.-C. Lee M.-H. Won (&) Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea e-mail:
[email protected]
adhesion molecule) was well expressed in dendrites before I-R. Its immunoreactivity was well colocalized with MAP-2. Chronological change of beta-catenin immunoreactivity was novelty in the present study. Twelve hours after I-R, its immunoreactivity was decreased in the stratum radiatum of the CA1 region, however, its immunoreactivity was increased 1 and 2 days after I-R, and decreased sharply 4 days after I-R. However, we did not find any change in beta-catenin immunoreactivity in the CA2 and CA3 region. In brief, we suggest that early change of beta-catenin expression in the stratum pyramidale of ischemic hippocampal CA1 region is associated with early dendrite damage following transient cerebral ischemia. Keywords Ischemia-reperfusion injury Dendrite damage Microtubule-associated protein 2 Synaptic plasticity Beta-catenin B. Lee Center for Genomics and Proteomics, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon 406-840, South Korea
Y. J. Lee Department of Emergency Medicine, Seoul Hospital, College of Medicine, Sooncheonhyang University, Seoul 140-743, South Korea
J. H. Cho (&) Department of Emergency Medicine, College of Medicine, Kangwon National University, Chuncheon 200-701, South Korea e-mail:
[email protected]
J. H. Ahn Laboratory of Neuroscience, Department of Physical Therapy, College of Rehabilitation Science, Daegu University, Gyeongsan 712-714, South Korea
J. H. Cho M.-H. Won Institute of Medical Sciences, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
Y.-M. Kim Vascular System Research Center and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
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Introduction Delayed neuronal death (DND) in the hippocampal CA1 region is caused by transient cerebral ischemia induced by the deprivation of blood flow to the brain. Especially, the CA1 region, not CA2 and CA3 region, in the hippocampus proper is a most vulnerable region and DND occurs at several days after ischemia-reperfusion [1]. In addition, it has been well known that selective dendrite damage occurs in the stratum pyramidale of the CA1 region after transient cerebral ischemia in the rat [2]. Microtubule associated protein-2 (MAP-2), a high molecular weight protein, is mainly present in mature neurons, and it is selectively concentrated in the dendrites. Therefore, MAP-2 has been considered as a specific neural dendritic phosphoprotein [3, 4]. In addition, MAP-2 is one of the most vulnerable cytoskeletal proteins in the situation of neuronal injury [5]. Furthermore, MAP-2 plays an important role in priming microtubule assembly and maintaining microtubule stability [6], maintains the structural integrity of the cytoskeleton and acts in synapse formation and dendrite plasticity [7]. Synaptic plasticity is highly important for functional recovery after brain injury and is partially regulated by a group of synapse-enriched cell adhesion molecules such as catenin [8]. Catenins, a family of proteins found in complexes with cadherin cell adhesion molecules in cells, are identified as several kinds including alpha-, beta, delta and gamma-catenin [9, 10]. Among them, beta-catenin, as a transcription factor, plays a critical role in cell survival and keeps other functions, such as forming synapses, in neurons [11]. It has been reported that beta-catenin is degraded in the brain after focal ischemic damage in the rat, however, few studies have focused on the changes of beta-catenin in the dendrites of the brain after transient cerebral ischemia [11, 12]. Therefore, in the present study, we investigated changes in beta-catenin distribution in the dendrites in the hippocampal CA1 region following 5 min of transient cerebral ischemia in the gerbil, which is a good animal model of transient cerebral ischemia [13–15].
Materials and Methods Experimental Animals We used the progeny of male Mongolian gerbils (body weight 65–75 g, 6 months) obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were housed in a conventional state under adequate temperature (23 °C) and humidity (60 %)
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control with a 12-h light/12-h dark cycle, and provided with free access to water and food. Procedures involving animals and their care conformed with the guidelines, which are in compliance with current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996). The animal protocol used in the present study was reviewed and approved based on ethical procedures and scientific care by the Kangwon National University-Institutional Animal Care and Use Committee (KIACUC12-0018). All experiments were conducted to minimize the number of animals used and suffering caused. Induction of Transient Cerebral Ischemia The animals were anesthetized with a mixture of 2.5 % isoflurane in 33 % oxygen and 67 % nitrous oxide. A midline ventral incision was then made in the neck, and bilateral common carotid arteries were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. The restoration of blood flow (reperfusion) was observed directly using the ophthalmoscope. Body (rectal) temperature was maintained under free-regulating or normothermic (37 ± 0.5 °C) conditions with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA, USA) and a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. Thereafter, animals were kept in a thermal incubator (Mirae Medical Industry, Seoul, South Korea) until the animals were euthanized. Sham-operated animals were subjected to the same surgical procedures except that the common carotid arteries were not occluded. Tissue Processing for Histology For histology, sham- and ischemia-operated gerbils (n = 7 at each time point) at designated times (12 h, 1, 2, and 4 days after reperfusion) were sacrificed. The animals were anesthetized with pentobarbital sodium and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4 % paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The brains were removed and postfixed in the same fixative for 6 h. The brain tissues were cryoprotected by infiltration with 30 % sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Germany) into 30-lm coronal sections, and they were then collected into six-well plates containing PBS.
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Staining for Neuronal Damage
F-J B Histofluorescence
CV Staining
To investigate the neuronal death in the hippocampus CA1 region after transient cerebral ischemia, Fluro-Jade B (F-J B) histofluorescence staining, a high affinity fluorescent marker for the localization of neuronal degeneration, was conducted according to the method by previous study [16]. In brief, 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 % F-J B (Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were placed on a slide warmer (approximately 50 °C), and then examined using an epifluorescent microscope (Carl Zeiss, Germany) with blue (450–490 nm) excitation light and a barrier filter [17].
To investigate the morphological changes and neuronal changes after ischemia treatment, CV staining was performed. In brief, the sections were mounted on gelatincoated microscopy slides. Cresyl violet acetate (Sigma, MO) was dissolved at 1.0 % (w/v) in distilled water, and glacial acetic acid was added to this solution. The sections were stained and dehydrated by immersing in serial ethanol baths, and they were then mounted with Canada balsam (Kanto, Tokyo, Japan). Immunohistochemistry for NeuN, MAP-2 and BetaCatenin 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. Then they were next incubated with diluted mouse anti-NeuN (1:800, Chemicon), anti-MAP-2 (1:500, Chemicon) and rabbit anti-betacatenin (1:1,000, abcam) overnight at 4 °C. Thereafter the tissues were exposed to biotinylated horse anti- mouse and goat anti-rabbit IgG (Vector, Burlingame, CA, USA) and streptavidin peroxidase complex (1:200, Vector). And they were visualized by staining with 3,30 -diaminobenzidine tetrahydrochloride in 0.1 M Tris-HCl buffer (pH 7.2) and mounted on gelatin-coated slides. After dehydration, the sections were mounted with Canada balsam (Kanto, Tokyo, Japan). In order to establish the specificity of the immunostaining, a negative control test was carried out with preimmune serum instead of primary antibody. The negative control resulted in the absence of immunoreactivity in all structures. In order to quantitatively analyze beta-catenin immunoreactivity, the corresponding areas of the hippocampal CA1 region were measured from 15 sections per animal. Images of all beta-catenin-immunoreactive structures were taken from the stratum radiatum of the hippocampus proper through an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. Images were calibrated into an array of 512 9 512 pixels corresponding to a tissue area of 250 9 250 lm. The densities of beta-catenin-immunoreactive structures were evaluated on the basis of a optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log (256/mean gray level). The OD of background was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the optical density of image file was calibrated as % (relative optical density, ROD) using Adobe Photoshop version 8.0 and then analyzed using NIH Image 1.59 software.
Double Immunofluorescence Staining The sections were processed by double immunofluorescence staining. Double immunofluorescence staining was performed using diluted rabbit anti-beta-catenin (1:1,000, abcam)/mouse anti-MAP-2 (1:500, chemicon). 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 Cy3conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch) and FITC-conjugated rabbit anti-goat IgG (1:200; Jackson ImmunoResearch) for 2 h at room temperature. Lastly, they were incubated in a solution of DraQ5 (1:1,000, Biostatus Limited) for 30 min. DraQ5 is a highly cell permeable DNA-interactive agent, with fluorescence signature extending into the infra-red region of the spectrum. It is a good choice for nuclear staining of cells. The immunoreactions were observed under the confocal MS (LSM510 META NLO, Carl Zeiss, Germany). Western Blot Analysis In order to examine the protein levels of beta-catenin in the hippocampus, the animals (n = 7 in each group) were used for western blot analysis at sham, 12 h, 1, 2 and 4 days after the ischemic surgery group. After sacrificing them and removing the hippocampus, it was serially and transversely cut into a thickness of 400 lm on a vibratome (Leica, Germany), and the hippocampal CA1 region was then dissected with a surgical blade. The tissues were homogenized in 50 mM PBS (pH 7.4) containing EGTA (pH 8.0), 0.2 % NP-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM b-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthvanadate, 1 mM PMSF and 1 mM DTT. After centrifugation, and the protein level
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Fig. 1 CV (a–f), NeuN (g–l) staining and F-J B histofluorescence (m–r) in the CA1, CA2 and CA3 region of the sham- and ischemiagroups 4 days after I-R. In the ischemia-group, a few CV? and
NeuN? cells, and many F-J B? cells are shown in the stratum pyramidale (SP, asterisk) of the CA1 region of the ischemia-group. SO, stratum oriens; SR, stratum radiatum. Scale bar 50 and 100 lm
in the supernatants was determined using a Micro BCA protein assay kit with bovine serum albumin as a standard (Pierce Chemical, USA). Aliquots containing 50 lg of total protein were boiled in loading buffer containing 250 mM Tris (pH 6.8), 10 mM DTT, 10 % SDS, 0.5 % bromophenol blue and 50 % glycerol. The aliquots were then
loaded onto a suitable polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Crop, East Hills, NY, USA). In order to incubate antibodies, the same nitrocellulose membranes striped were used. To reduce background staining, the membranes were incubated with 5 % non-fat dry milk in
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Fig. 2 Immunohistochemistry for MAP-2 in the stratum radiatum of the CA 1 region of the sham—(a) and ischemia— (b–e) groups. MAP-2 immunoreaction (arrows) is hardly found 4 days after I-R. Scale bar 20 lm. f Relative optical density as % of MAP-2? structures in the sham- and ischemia-groups (n = 7 per group; *P \ 0.05, significantly different from the sham-group, # P \ 0.05, significantly different from the respective preceding group). The bars indicate the mean ± SEM
TBS containing 0.1 % Tween 20 for 45 min, followed by incubation with rabbit anti-beta-canetin (1:500, abcam) overnight at 4 °C and subsequently exposed to peroxidaseconjugated goat anti-rabbit IgG (Santa Cruz, USA), and an ECL kit (Amersham, UK). The result of western blot analysis was scanned, and densitometric analysis for the quantification of the bands was done using Scion Image software (Scion Corp., Frederick, MD), which was used to count relative optical density (ROD): A ratio of the ROD was calibrated as %, with the sham-group designated as 100 %. Statistical Analysis Data are expressed as the mean ± SEM. Differences of the means among the groups were statistically analyzed by analysis of variance (ANOVA) with a post hoc Bonferroni’s multiple comparison test in order to elucidate ischemia-related differences among experimental groups. Statistical significance was considered at P \ 0.05.
was found between the sham- and ischemia-groups 4 days after I-R (Fig. 1f, l). No Fluoro-Jade B (F-J B)? cells were found in all the hippocampal subregions of the sham-group (Fig. 1m–o); however, many F-J B? cells were detected in the CA1 region, not in the CA2 and CA3 region, 4 days after I-R (Fig. 1p–r). MAP-2 Immunoreactivity In the sham-group, MAP-2? structures were easily observed in dendrites of pyramidal cells in the hippocampus, and they were beaded in morphology (Fig. 2a). MAP2 immunoreactivity was changed in the CA1, not CA2 and CA3, region. Twelve hours after I-R, MAP-2 immunoreactivity was decreased (Fig. 2b), and a significant decrease in MAP-2 immunoreactivity was found 1 day after I-R (Fig. 2c, f). However, MAP-2 immunoreactivity was increased 2 days after I-R (Fig. 2d, f), and MAP-2 immunoreactivity was dramatically decreased in the stratum radiatum of the CA1 region 4 days after I-R (Fig. 2e, f). Beta-Catenin Immunoreactivity
Results Neuronal Damage Cresyl violet (CV)-positive (?) and neuronal nuclei (NeuN)-immunoreactive (?) cells were easily observed in the hippocampal subregions, such as the CA1, CA2 and CA3 region, of the sham-group (Fig. 1a–c, g–i). Four days after ischemia-reperfusion (I-R), a significant loss of CV? and NeuN? cells was observed in the CA1 region (Fig. 1d, e, j, k); however, in the CA2 and CA3 region, no difference
In the sham-group, beta-catenin? structures were observed in the stratum radiatum of the hippocampus. Dot-like betacatenin? structures were scattered throughout the stratum radiatum (Fig. 3A, a). Beta-catenin immunoreactivity was changed only in the CA1 region. Beta-catenin immunoreactivity was decreased 12 h after I-R (Fig. 3B, b, G); however, its immunoreactivity was increased 1 and 2 days after I-R (Fig. 3C, c, D, d, G). Four days after I-R, betacatenin immunoreactivity was dramatically decreased in the stratum radiatum of the CA1 region (Fig. 3E, e, G).
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Fig. 3 Immunohistochemistry (A–E) and immunofluroescence (a–e) for beta-catenin in the stratum radiatum (SR) of the CA 1 region of the sham-and ischemia-groups. Beta-catenin immunoreactivity is apparently changed with time after I-R. Its immunoreactivity increases 1 and 2 days after I-R and decreases dramatically 4 days after I-R. Scale bar 20 lm. F Negative control test shows the absence of beta-catenin immunoreactivity in the hippocampus. G Relative optical density as % of betacatenin? structures in the shamand ischemia-groups (n = 7 per group; *P \ 0.05, significantly different from the sham-group, # P \ 0.05, significantly different from the respective preceding group). The bars indicate the mean ± SEM
Changes in MAP-2/Beta-Catenin Immunoreactivity In this study, we could find that beta-catenin was colocalized with MAP-2 and that they were changed with time after I-R. In the sham-group 4 days after I-R, MAP-2 and beta-catenin were well detected in the CA1, CA2 and CA3 region of the hippocampus (Figs. 4a, b, 5a, b); however, in the ischemia-group, a significantly decreased beta-catenin immunoreactivity was found in the MAP-2? cells in the ischemic CA1 region (Fig. 4e, f). On the other hand, we found that beta-catenin immunoreactivity in the MAP-2? cells was not obviously decreased in the CA2 and CA3 region of the ischemia-group (Fig. 5e, f).
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We also found that beta-catenin immunoreactivity was localized in DraQ? cells in the CA1 CA2 and CA3 regions and that the change of beta-catenin immunoreactivity in DraQ? cells after I-R was very similar to those in the MAP-2? cells in the ischemia-group (Figs. 4d, h, 5d, h). Change in the Level of Beta-Catenin In this study, we examined the level of beta-catenin protein in the hippocampus after I-R (Fig. 6). Twelve hours after I-R, the level of beta-catenin was much lower than that in the sham-group; however, its level was much increased 1 and 2 days after I-R, and significantly decreased 4 days after I-R.
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Fig. 4 Double immunofluorescence staining for MAP-2 (green), betacatenin (red), DraQ (blue) and merged images in the stratum radiatum of the CA1 region of the sham—(a–d) and ischemia—(e–h) groups. Decreased MAP-2, beta-catenin and DraQ immunoreactivity (arrows)
are found 4 days after I-R compared to those in the sham-group. Betacatenin immunoreaction is easily detected in MAP-2? and DraQ? cells. Scale bars 10 lm (Color figure online)
Discussion
leading to cell death using some animal models of transient brain ischemia [2, 20]. It is well known that dendrites are major sites of synaptic input from partner neurons, and their development is linked to the formation of a functional network [21]. Synapses are gained and lost in continuing basis in adult brains. Generally, the formation of new synapses is induced by increased neuronal activity, and neuronal inactivity results in the loss of synaptic connections [22]. Synaptic pathology is observed during cerebral injured events that develops changes in dendrite structures and conductance [23, 24]. Beta-catenin as a transcription factor and adhesion molecule plays an important role in cell survival and keeps
In the present study, we firstly observed ‘delayed neuronal damage/death’ that occurred in the hippocampal CA1 pyramidal neurons 4 days after I-R. The result is consistent with previous studies [18, 19]. In addition, the early dendrite damage in the stratum radiatum of the ischemic CA1 region was observed by MAP-2 immunohistochemistry. The result showed that MAP-2? dendrites were significantly decreased from 1 day after I-R. Some previous studies have shown that ischemia-induced loss of MAP-2 in the hippocampus is one of the earliest pathogenic events that dendritic breakdown may be one of initial steps
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Fig. 5 Double immunofluorescence staining for MAP-2 (green), beta-catenin (red), DraQ (blue) and merged images in the stratum radiatum of the CA2 and CA3 region of the sham—(a–d) and
ischemia—(e–h) groups. MAP-2, beta-catenin and DraQ immunoreactivity (arrows) 4 days after I-R are similar to those in the shamgroup. Scale bars 10 lm (Color figure online)
the functions of synapses formation in neurons, and it is an important factor in synaptic plasticity or remodeling [25]. In the present study, the chronological change of betacatenin immunoreactivity was novelty. Its immunoreactivity in the stratum radiatum of the CA1 region were increased at 1 and 2 days after I-R, and decreased sharply at 4 days after I-R. Similarly, some researchers reported that some protein were increased at early time and degraded with neuronal death at once, such as SODs and BDNF, and suggested that it might play a critical role in cell survival [26, 27]. These may be associated with the compensatory mechanisms against ischemic damage. In addition, it was reported that beta-catenin was degraded in the brain after focal ischemia by western blot analysis [12].
On the other hand, Yu and Malenka reported that betacatenin stabilization was shown to influence dendritic branching and to keep a crucial role in morphogenesis [28]. We, in the present study, found the significant decrease of beta-catenin? structures in the stratum radiatum of the hippocampal CA1, not CA2 and CA3 region after 4 days after transient cerebral ischemia in the gerbil. This finding is associated with the CA1 region, which is the most vulnerable region to transient ischemic damage in the hippocampus, not the CA2 and CA3 region, which are resistant to transient ischemic damage, after 5 min of transient cerebral ischemia. In the present study, we found that numerous beta-catenin? structures were shown in and surrounding MAP-2?
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References
Fig. 6 Western blot analysis of beta-catenin in the hippocampal CA1 region of the sham- and ischemia-groups (n = 7 per group). Its protein level is significantly decreased 4 days after I-R (*P \ 0.05, significantly different from the sham-group, #P \ 0.05, significantly different from the respective preceding group). The bars indicate the mean ± SEM
dendrites in the stratum radiatum of all the CA regions before transient cerebral ischemia, however, at early time after I-R, with breakdown of MAP-2? dendrites, the significant decrease of beta-catenin? structures was examined in the dendrites. Furthermore, few dendrites with very weak beta-catenin expression were detected in the stratum oriens of the CA1 region, not CA2/3, 4 days after I-R. It was reported that the loss of beta-catenin in postsynapses blocked presynaptic homeostatic adaptation [29]. In addition, some reports showed that beta-catenin was a central component of Wnt/beta-catenin signaling pathway, which is known as an important mediator of dendritic development [30, 31]. Therefore, these findings indicate that betacatenin stabilization or loss in dendrites may contribute to selective dendrite survival or damage in the hippocampal subregions following transient cerebral ischemia. In brief, our findings showed that the significant decrease of beta-catenin immunoreactivity began at early time after I-R in the hippocampal CA1 region, not in the CA2 and CA3 region after transient cerebral ischemia. Acknowledgments The authors would like to thank Mr. Seung Uk Lee for his technical help in this study. This work 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 the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0010580).
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