Journal of Chemical Neuroanatomy 66–67 (2015) 40–51

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Differential expression of the calcium-sensing receptor in the ischemic and border zones after transient focal cerebral ischemia in rats Jeong Sook Noh a, Ha-Jin Pak a, Yoo-Jin Shin a, Tae-Ryong Riew a, Joo-Hee Park a, Young Wha Moon b, Mun-Yong Lee a,* a

Department of Anatomy, Catholic Neuroscience Institute, Cell Death Disease Research Center, College of Medicine, The Catholic University of Korea, 137-701 Seoul, South Korea Department of Natural Sciences, College of Medicine, The Catholic University of Korea, 137-701, Seoul, South Korea

b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 January 2015 Received in revised form 20 April 2015 Accepted 15 May 2015 Available online 23 May 2015

G-protein-coupled calcium-sensing receptor (CaSR) has been recently recognized as an important modulator of diverse cellular functions, beyond the regulation of systemic calcium homeostasis. To identify whether CaSR is involved in the pathophysiology of stroke, we studied the spatiotemporal regulation of CaSR protein expression in rats undergoing transient focal cerebral ischemia, which was induced by middle cerebral artery occlusion. We observed very weak or negligible immunoreactivity for CaSR in the striatum of sham-operated rats, as well as in the contralateral striatum of ischemic rats after reperfusion. However, CaSR expression was induced in the ischemic and border zones of the lesion in ischemic rats. Six hours post-reperfusion there was an upregulation of CaSR in the ischemic zone, which seemed to decrease after seven days. This upregulation preferentially affected some neurons and cells associated with blood vessels, particularly endothelial cells and pericytes. In contrast, CaSR expression in the peri-infarct region was prominent three days after reperfusion, and with the exception of some neurons, it was mostly located in reactive astrocytes, up to day 14 after ischemia. On the other hand, activated microglia/macrophages in both the ischemic and border zones were devoid of specific labeling for CaSR at any time point after reperfusion, despite their massive infiltration in both regions. Our results show heterogeneity in CaSR-positive cells within the ischemic and border zones, suggesting that CaSR expression is regulated in response to the altered extracellular ionic environment caused by ischemic injury. Thus, CaSR may have a multifunctional role in the pathophysiology of ischemic stroke, possibly in vascular remodeling and astrogliosis. ß 2015 Elsevier B.V. All rights reserved.

Keywords: Pericytes Receptors, calcium-sensing Reactive astrocytes Corpus striatum Stroke Endothelial cells Microglia Vascular remodeling

Introduction Calcium-sensing receptor (CaSR), which is coupled to Gprotein, was initially associated with the maintenance of calcium homeostasis by regulating the secretion of the parathyroid hormone (Brown et al., 1993; Brown and MacLeod, 2001). However, the identification of calcium as an external ligand has generated a special interest in the function of CaSR, unrelated to systemic calcium homeostasis (see as reviews Conigrave and Ward, 2013; Smajilovic and Tfelt-Hansen, 2007; Ward et al., 2012). Particularly, cumulative evidence has shown that CaSR is involved in modulating wide-ranging aspects of cellular function in the central nervous system, by sensing changes in the extracellular * Corresponding author. Tel.: +82 2 2258 7261; fax: +82 2 536 3110. E-mail address: [email protected] (M.-Y. Lee). http://dx.doi.org/10.1016/j.jchemneu.2015.05.001 0891-0618/ß 2015 Elsevier B.V. All rights reserved.

Ca2+ levels (Bandyopadhyay et al., 2010; Bouschet and Henley, 2005). CaSR is present in almost all brain areas, with high expression in the subfornical organ, olfactory bulb, and hypothalamus, suggesting a role for CaSR in region-specific neuronal functions (Bandyopadhyay et al., 2010; Bouschet and Henley, 2005; Chen et al., 2010; Mudo et al., 2009; Ruat and Traiffort, 2013; Yano et al., 2004). In addition, expression of CaSR in nerve terminals suggests its involvement in synaptic plasticity and neurotransmission, while its presence in glial cells (i.e., oligodendrocytes, astrocytes, and microglia), suggests a role for CaSR in local ionic homeostasis in the brain (Bandyopadhyay et al., 2010; Bouschet and Henley, 2005; Ruat and Traiffort, 2013). However, the role of CaSR in glia and neurons remains unclear. There have been several reports regarding the regulation of CaSR expression in a variety of pathological conditions, including epileptic seizures (Mudo et al., 2009), Alzheimer’s disease (Armato et al.,

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2012; Chiarini et al., 2009; Conley et al., 2009; Dal Pra et al., 2014), and traumatic brain injury (Kim et al., 2013). In addition, Kim et al. (2011, 2013) showed that after traumatic and ischemic brain injury, CaSR overexpression and concurrent down-regulation of metabotropic g-aminobutyric acid receptor (GABABR) occurred before apparent neurodegeneration, suggesting that alteration of CaSR expression contributes to brain injury. Considering that calcium overload due to an excitotoxic mechanism contributes to neuronal injury induced by cerebral ischemia (Candelario-Jalil, 2009; Mehta et al., 2013), the induction of CaSR in the ischemic brain is of great interest. However, the temporal regulation and identification of the precise cell phenotypes expressing CaSR in the ischemic brain remain to be established. In the present study, we determined the spatiotemporal expression pattern of CaSR in response to a disruption of ionic homeostasis caused by ischemic injury. For this purpose, we used a rat model of focal cerebral ischemia-reperfusion, induced by the occlusion of the middle cerebral artery (MCA). Our results clearly showed that CaSR expression in the ischemic zone (which is destined for tissue destruction) presented a different pattern than in the peri-infarct border zone (which has the potential for full recovery). Thus, we focused our attention on identifying the phenotypes of CaSR-positive cells in the ischemic and border zones using double-labeling techniques for various cell type-specific markers. Materials and methods Animal preparation All experimental procedures were conducted in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Survival Surgery, and were approved by the IACUC (Institutional Animal Care and Use Committee) in the College of Medicine, The Catholic University of Korea (Approval Number: CUMC-2014-0006-01). Adult male Sprague-Dawley rats (250–300 g) were used in this study. Transient focal ischemia was induced by the intraluminal thread method described previously (Longa et al., 1989). Briefly, the right external carotid artery was ligated with a 4-0 silk suture, and a 3-0 rounded tip nylon suture was introduced into the right common carotid artery. The suture was advanced through the internal carotid artery to occlude the MCA. Reperfusion was performed by withdrawing the surgical suture from the common carotid artery after 60-min ischemia. Interruption of blood flow distal to ligation and the restoration of blood flow in the right common carotid artery, external carotid artery, and internal carotid artery were confirmed under a dissecting microscope. Body temperatures (measured rectally) were maintained at 37.5  0.3 8C with a heating lamp during and after ischemia. Sham-operated rats underwent the same experimental procedure except that the MCA was not occluded. In this model, ischemia for 60 min consistently resulted in a large infarct confined to the territory of the right MCA, whereas no morphological ischemic injury was detected in the territory of the left MCA. Animals were allowed to live for 6 h, or 3, 7, or 14 days after reperfusion. At each of the four time points following reperfusion, animals (n = 5 per time point for ischemic group; n = 3 per time point for the sham-operated group) were deeply anesthetized with 16.9% urethane (10 mL/kg i.p.) and sacrificed by transcardial perfusion with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). In addition, at 3 (n = 7) and 7 (n = 7) days following MCA occlusion (MCAO), ischemic animals were killed for quantitative analysis. For western blot analysis, rats from 4 groups (sham controls, experimental rats 6 h, or 3, 7, or 14 days

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after reperfusion (n = 5 per group) were killed by decapitation under anesthesia (16.9% urethane; 10 mL/kg i.p.). The cortical and striatal tissues from the ipsilateral (ischemic) and contralateral hemispheres were carefully dissected under stereoscopic microscope and immediately frozen in liquid nitrogen. Brain samples were stored at 70 8C until further processing. To evaluate tissue injury in animals subjected to 1 h MCAO, rats (n = 3) were deeply anesthetized with 16.9% urethane (10 mL/kg i.p.) at 3 days after reperfusion. Following decapitation, brains were quickly removed and were sliced at 1-mm thickness. Brain slices were incubated at 37 8C for 30 min in a 2% solution of 2,3,5triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA). Histochemistry and immunohistochemistry To simultaneously detect apoptotic cells and the CaSR protein, we performed immunostaining for CaSR and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Free-floating sections (25-mm thick) were incubated at 37 8C for 60 min with biotinylated 20 -deoxyuridine-50 -triphosphate (dUTP), according to the manufacturer’s protocol (Roche Diagnostics Corporation, Indianapolis, IN, USA). The sections were then immunostained with monoclonal mouse anti-CaSR antibody (Sigma-Aldrich; 1:100), followed by 1-hr incubation with fluorescein isothiocyanate (FITC)conjugated donkey anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:50) for CaSR staining and Cy3-conjugated streptavidin (Jackson ImmunoResearch; 1:1500) for the TUNEL method. The specificity of immunoreactivity was confirmed by the absence of immunohistochemical reaction in sections from which primary or secondary antibodies were omitted. Counterstaining of cell nuclei was carried out with DAPI (40 ,6-diamidino-20 -phenyindole, Roche, Mannheim, Germany; 1:1000) for 10 min. Slides were viewed with a confocal microscope (LSM 700; Carl Zeiss Co. Ltd., Oberkochen, Germany) equipped with four lasers (Diode 405, Argon 488, HeNe 543, HeNe 633). Images were converted to the TIFF format, and contrast levels were adjusted using Adobe Photoshop v. 7.0 (Adobe System, San Jose, CA, USA). Immunohistochemistry and double labeling For double-immunofluorescence histochemistry, sections were incubated at 4 8C overnight with a mixture of monoclonal mouse anti-CaSR antibody (Sigma-Aldrich; 1:100) and one of following antibodies: polyclonal rabbit antibody to glial fibrillary acidic protein (GFAP; Millipore, Temecula, CA, USA; 1:1500), ionized calcium-binding adaptor molecule 1 (Iba1; Wako Pure Chemical Industries, Ltd., Osaka, Japan; 1:500), NG2 chondroitin sulfate proteoglycan (NG2; Millipore; 1:500), laminin (Sigma-Aldrich; 1:100), von Willebrand factor (Sigma-Aldrich; 1:200), Ki67 (Novocastra Laboratories Ltd., Newcastle Upon Tyne, UK; 1:1000), and biotin-conjugated mouse monoclonal anti-neuronal nuclei (NeuN; Millipore; 1:500). Antibody staining was visualized using the following secondary antibodies: Cy3-conjugated donkey antimouse (Jackson ImmunoResearch; 1:2000), Alexa Fluor 488 goat anti-mouse (Molecular Probes, Eugene, OR, USA; 1:300), Alexa Fluor 488 goat anti-rabbit (Molecular Probes; 1:300), and Cy3-conjugated streptavidin (Jackson ImmunoResearch; 1:1500). Control sections were prepared as described above. Counterstaining of cell nuclei was carried out by incubating the sections with DAPI for 10 min. Slides were viewed with a confocal microscope. Cell counting To count the number of CaSR-positive cells and CaSR/GFAP or CaSR/NeuN double-labeled cells in the border zone at days 3 and

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7 after reperfusion, three coronal sections per animal (n = 7 per time point), cut at 100-mm intervals, were obtained from the invariable region between bregma levels 0.7 mm and 1.20 mm dorsal (Paxinos and Watson, 2006). Three areas (160  160 mm per field) along the outside borders adjacent to the ischemic zone of each section were chosen and captured at 400 magnification using a confocal laser microscope. CaSR-positive cells were scored when their nuclei could be clearly seen, and among them, cells clearly showing NeuN-positive nuclei or GFAP-positive processes were counted, separately. Statistical significance was assessed by unpaired t test, in which P < 0.05 was accepted as significant. All values are given as means and SEM. Western blot analysis For the immunoblot analysis, protein was isolated from the ipsilateral or contralateral hemispheres using boiling lysis buffer (1% sodium dodecyl sulfate (SDS), 1.0 mM sodium orthovanadate, 10 mM Tris, pH 7.4). Equal amounts (20 mg) of total protein were separated by SDS polyacrylamide gel electrophoresis (10%) and transferred to PVDF membrane. Immunostaining of the blots was performed using monoclonal mouse anti-CaSR antibody (SigmaAldrich; 3 mg/mL) and anti-b-actin (Sigma-Aldrich; 1:10000). Membranes were then incubated with peroxidase-coupled secondary antibodies (Millipore; 1:1000) for 1 h at room temperature. Blots were developed using the Amersham ECL prime western blotting detection reagent (GE healthcare. Little Chalfont, UK). Densitometric analysis was performed using the Eagle Eye TMII Still Video System (Stratagene, La Jolla, CA, USA). Statistical significance was determined by analysis of variance (ANOVA) followed by Dunnett’s t test; P < 0.05 was regarded as significant. Results CaSR expression is induced in the ischemic hemisphere after MCAO The mortality rate of MCAO rats was 25.4%, and all shamoperated rats survived after the operation. TTC staining in rats killed on day 3 after 1-h MCAO revealed a prominent and comparable ischemic zone confined to the ipsilateral MCA territory including the majority of the striatum and cerebral cortex (Fig. 1A). To examine the induction of CaSR protein expression after transient MCAO, we performed western blot analysis with protein extracted from the ischemic or non-ischemic hemisphere (Fig. 1B and C). Immunoblotting revealed that a band of about 130 kDa corresponding to the CaSR protein was clearly induced in the ipsilateral hemisphere. The intensity of CaSR protein expression significantly increased at 6 h and peaked at 3 days in the ischemic hemisphere compared with the contralateral control hemisphere. Thereafter, the expression level declined, although the enhanced expression was maintained until at least day 14, which was the latest time point examined. CaSR expression is induced in the ischemic zone 6 h after reperfusion Immunofluorescence labeling revealed a substantial increase in CaSR labeling in the ischemic brain, including the parietaltemporal cortex and striatum, as early as 6 h after reperfusion (Fig. 2A). However, no prominent positive profiles were identified in the corresponding contralateral (i.e., non-ischemic) areas (Fig. 2A), or in the striatum of sham-operated rats (data not shown). Double labeling for CaSR and TUNEL revealed an overlapping regional distribution confined to the ischemic zone, resulting in a clear demarcation of the ischemic and peri-infarct border zones at this time point (Fig. 2B). As shown at higher magnification (Fig. 2C,D), CaSR expression was induced in some

Fig. 1. Temporal regulation of CaSR protein expression after ischemic injury. (A) Representative brain slice stained by 2,3,5-triphenyltetrazolium chloride (TTC) 3 days after MCAO. TTC staining showed a sharp border between undamaged (red colored) and infarcted tissue (pale). Asterisks indicate the ischemic zone, and its border is demarcated by a broken line. (B) Representative results of western blot analysis for CaSR protein expression in the ipsilateral (i) and contralateral (c) hemisphere extracts from sham controls and rats killed at 6 h, 3, 7, and 14 days after reperfusion. Note that a band of about 130 kDa corresponding to CaSR protein was clearly induced in the ipsilateral ischemic hemisphere. (C) Quantification of CaSR protein expression. Data were obtained by densitometry and were normalized using b-actin as the loading control. Results are expressed in relative optical density and represent means  SEM. The intensity of CaSR protein expression significantly increased at 6 h, peaked at 3 days, and declined thereafter, but the enhanced expression was maintained until at least day 14. # P < 0.05 compared to the contralateral hemispheres at the same time point; * P < 0.05 compared to the ipsilateral hemispheres of the sham controls.

TUNEL-positive cells, but most CaSR-positive cells were devoid of TUNEL staining, and vice versa. In the ischemic zone, some CaSRpositive cells presented fusiform or spindle-shaped cell bodies with processes; these were NeuN-positive neurons; however, most neurons did not express CaSR (Fig. 2E and F). In addition, nearly all neurons in the contralateral non-ischemic striatum were devoid of CaSR labeling (Fig. 2G). CaSR labeling was located in the cytoplasm but was not observed in the nucleus (Fig. 2C–F). CaSR-positive cells in the ischemic zone were located in close vicinity to blood vessels, including capillaries and vessels of larger diameter labeled with laminin, which is localized in the basal lamina of blood vessels (Eriksdotter-Nilsson et al., 1986) (Fig. 3A– C). Double labeling with CaSR and the endothelial cell marker von Willebrand factor revealed an overlapping expression within vascular structures (Fig. 3D–F). Observation of the double-stained sections at higher magnification revealed that CaSR expression was induced in most endothelial cells (Fig. 3G–I). However, double labeling for CaSR and GFAP or Iba1 showed no significant immunoreactivity for CaSR in astrocytes or microglia in the ischemic zone, despite the evident glial responses at this time point (Fig. 3J,K,M and N). In contrast, in the corresponding contralateral (i.e., non-ischemic) areas of the brain, where GFAP

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Fig. 2. Localization of CaSR expression and its relation with the cell death marker TUNEL in the ischemic zone of the striatum 6 h after ischemic injury. (A) There was a remarkable increase in CaSR immunoreactivity in the ischemic region, but neither in the peri-infarct nor in the contralateral (i.e., non-ischemic) region. The ischemic region is indicated by asterisks, and its border is demarcated by a broken line. (B) Double-labeling for CaSR and TUNEL showing that OPN and TUNEL staining was restricted to the infarcted tissue (left side of the broken line). (C, D) Higher magnification views of the ischemic region (boxed areas in B). Note that some CaSR-positive cells were dying cells (arrowheads in C), but most TUNEL-positive cells were not positive for CaSR, or vice versa. (E–G) Double-labeling for CaSR and NeuN in the ischemic region (E, F) and in the contralateral (non-ischemic) striatum (G). A subset of neurons in the ischemic region (arrowheads in E and F) showed CaSR expression. Note that no significant staining for CaSR was detected in neurons in the contralateral striatum. Cell nuclei are stained with DAPI. Abbreviations: cc, corpus callosum; LV, lateral ventricle. Scale bars = 500 mm in A; 200 mm in B; 50 mm in C–G.

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Fig. 3. Characterization of CaSR-positive cells in the ischemic zone six hr after reperfusion. (A–C) Double-labeling for CaSR and laminin showing that CaSR expression was localized to, or in close vicinity to, laminin-positive blood vessels. (B, C) Higher-magnification views of the boxed area in A. (D–I) Double-labeling for CaSR and von Willebrand factor (vWF) endothelial cell marker showing that CaSR-positive cells frequently coexpressed vWF. (G–I) Higher-magnification views of the boxed area in D–F, respectively. (J–O) Double-labeling for CaSR and GFAP (K, L), or Iba1 (N, O) in the ischemic region (J, K, M, N) and in the contralateral striatum (L, O). Note that CaSR-positive cells in the ischemic region were negative for astroglial and microglial markers. Note also that no specific staining for CaSR was detected in the contralateral striatum, where GFAP and IBa1 staining were very weak or negligible. Cell nuclei are stained with DAPI. Scale bars = 100 mm in A, D–F, J–L; 50 mm in B, C, G–I, M–O.

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and Iba1 staining were very weak or negligible, no specific staining for CaSR was detected (Fig. 3L and O).

only a few neurons presented CaSR immunoreactivity at this time point (data not shown).

Differential expression of CaSR in the ischemic and border zones at 3 and 7 days after reperfusion

Discussion

On day 3 after the induction of stroke, CaSR immunoreactivity increased preferentially in the peri-infarct border zone, which was clearly demarcated from the ischemic zone by the absence of TUNEL-positive cells (Fig. 4A). In the ischemic zone, however, CaSR-positive cells seemed to be less prominent compared to those at 6 h, and dying or dead cells were devoid of immunolabeling for CaSR (Fig. 4A,B). Double labeling with CaSR and laminin revealed that the CaSR-positive cells in the ischemic zone were still associated with vasculature (Fig. 4C and D), although the microvessels had become fragmented and were disintegrated compared with those at 6 h, as has been observed previously (Burggraf et al., 2008; Li et al., 2012). CaSR expression was colocalized in part with von Willebrand factor-positive endothelial cells (Fig. 4E–G). In addition, some CaSR-positive cells were positive for NG2, a marker for microvascular pericytes (Dore-Duffy, 2003) (Fig. 4H–J), or for Ki67, which labels cells at all phases of the cell cycle (Gerdes et al., 1983) (Fig. 4K–M). We further characterized CaSR-positive cells in the border zone by colabeling with several markers. Double labeling for CaSR and NeuN revealed that only some neurons were positive for CaSR (Fig. 5A and B). In addition, double labeling for CaSR and GFAP indicated that the majority of CaSR-positive cells in the border zone were reactive astrocytes, while the ischemic zone was virtually devoid of GFAP immunoreactivity (Fig. 5C–F). We also performed double immunofluorescence labeling for CaSR and Iba1. Almost no colocalization for CaSR and Iba1 was detected in both the ischemic and border zones, where large, round, amoeboid-like brain macrophages, and activated stellate microglial cells with thick and short processes were observed, respectively (Fig. 5G–J). This pattern of labeling was maintained 7 days after reperfusion, but there was a decrease in the intensity of CaSR immunoreactivity in the ischemic zone (Fig. 6A). In the periinfarct zone, a subset of neurons also showed CaSR immunoreactivity (Fig. 6A–D), but most CaSR-positive cells were reactive astrocytes (Fig. 6E–G). On the contrary, Iba1-positive microglia/ macrophages were devoid of specific labeling for CaSR (Fig. 6H–J). To determine the relative proportion of CaSR-positive astrocytes or neurons in the border zone, phenotype-specific markerpositive cells expressing CaSR were counted at 3 and 7 days after reperfusion. The increase in the number of CaSR-positive cells in the border zone was observed between 3 and 7 days after reperfusion, but the proportion of phenotype-specific markerpositive cells was similar in both groups (Fig. 6K and L). CaSRpositive cells that co-expressed GFAP were abundant in the border zone; at 3 and 7 days, 62.7% and 61.4% of all CaSR-positive cells were astrocytes, respectively (Fig. 6K). By contrast, neurons comprised 23.6% and 21.1% of all CaSR-positive cells at 3 and 7 days, respectively (Fig. 6L). These results indicated that the majority of CaSR-positive cells in the border zone were reactive astrocytes, although a subset of neurons were labeled. CaSR expression and astrogliosis in the peri-infarct border zone 14 days after reperfusion On day 14, CaSR immunoreactivity was still detected in the border zone, particularly in areas immediately adjacent to the ischemic zone. Virtually all CaSR labeling was located in GFAPpositive astrocytes (Fig. 7A–E). In contrast, Iba1-positive microglia/ macrophages within the ischemic and border zones were still devoid of specific CaSR immunoreactivity (Fig. 7F–J). In addition,

Our study shows the expression profile and cellular distribution of CaSR in the rat brain after transient focal cerebral ischemia. Very weak or negligible immunoreactivity for CaSR was observed in the corpus striatum of sham-operated rats, as well as in the contralateral non-ischemic striatum after reperfusion. CaSR expression was induced in both the ischemic and border zones but showed different patterns in these two regions. CaSR expression in the ischemic zone was induced preferentially in vessel-associated cells and in some neurons six hr after reperfusion. This was followed by a gradual and sustained induction of CaSR in reactive astrocytes located in the border zone of the postischemic brain (3–14 days after reperfusion). CaSR labeling was observed only in the cytoplasm. Cytoplasmic expression of CaSR has been previously shown in several types of cells with enhanced CaSR expression (Xi et al., 2010; Xing et al., 2010), and intracellular trafficking and localization of CaSR has been reported as essential regulators of CaSR functions (Bouschet and Henley, 2005; Bouschet et al., 2008). Thus, it seems possible that the cytoplasmic localization of CaSR in the present study reflects a functional status of CaSR activation. In addition, further studies are needed to obtain detailed and precise information on the subcellular localization of the CaSR protein in the ischemic brain. Our study revealed an overlapping regional distribution of CaSR labeling and TUNEL staining in the ischemic zone, particularly in the early ischemic period. This finding is interesting in view of previous reports showing that ischemia/reperfusion markedly increased the expression of CaSR, and that an increase in intracellular calcium mediated by CaSR played an important role in apoptosis (Xing et al., 2011; Zheng et al., 2011). In addition, Kim et al. (2014) recently demonstrated that ischemic injury leads to CaSR overexpression and GABABR1 downregulation in injured neurons, suggesting a novel ischemia-induced injury cascade involving CaSR. However, our study showed that CaSR was induced only in a small fraction of lethally damaged or dead cells (and not in astrocytes and most other neurons), both of which were destined for cell death (Chen and Swanson, 2003). In addition, in our study three days after reperfusion, CaSR immunoreactivity was preferentially observed in healthy astroglial cells and in some neurons in the border zone, where no significant TUNEL staining was observed. Interestingly, Mudo et al. (2009) have suggested a potential role for CaSR in repairing processes after brain injury. Thus, it is not likely that induction of CaSR expression is accompanied by cell death, and our data suggest a phenotypic and functional heterogeneity of CaSR-positive cells in the ischemic and border zones. Induction of CaSR within vessel-like structures in the ischemic zone was evident within 6 h after reperfusion and was still prominent at 3 days but appeared to decrease over time. Most vessel-associated CaSR-positive cells showed co-expression with von Willebrand factor, indicating that, at least a proportion of, these cells were endothelial cells. This idea is supported by the fact that CaSR is functionally expressed on cells of the vascular wall (including endothelial cells), smooth muscle cells, and adventitial cells (Smajilovic et al., 2006; Weston et al., 2011; Ziegelstein et al., 2006). CaSR expression in both large and small blood vessels highlights its importance in the regulation of vascular tone and peripheral resistance, since an increase of CaSR expression contributes to vascular remodeling and hypertension induced by hypoxia (Li et al., 2011; Molostvov et al., 2009; Peng et al., 2014). In addition, the rapid induction of CaSR in blood vessels of the ischemic zone reported here coincides with the period of active

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Fig. 4. Characterization of CaSR-positive cells in the ischemic zone 3 days after reperfusion. (A) Double-labeling for CaSR and TUNEL showing that a number of CaSR-positive cells appeared in the border zone adjacent to the ischemic region. Ischemic region is indicated by asterisks, and its border are demarcated by a broken line. (B) Higher magnification view of the ischemic region (boxed areas in (A)). Note that CaSR-positive cells, which were closely associated with blood vessel, were negative for TUNEL. Note also that dying (or dead) neurons did not show CaSR immunoreactivity. (C, D) Double-labeling for CaSR and laminin showing that CaSR expression was associated with blood vessel profiles. (E–G) Double-labeling for CaSR and von Willebrand factor (vWF) in the ischemic region. Note that CaSR-positive cells frequently exhibited vWF immunoreactivity (arrowheads in E–G). (H–J) Double-labeling for CaSR and NG2 in the ischemic region. Note that CaSR staining in the ischemic region co-localized in part with NG2-positive pericytes (arrowheads in H–J). (K–M) Double-labeling for CaSR and Ki67 showing that most CaSR-positive cells in the ischemic region were positive for Ki67. (L, M) Higher magnification views of the boxed area in K. Cell nuclei are stained with DAPI. Scale bars = 100 mm in A, C, D, K; 50 mm in B, E–J, L, M.

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Fig. 5. Characterization of CaSR-positive cells in the ischemic and border zones 3 days after reperfusion. (A) Double-labeling for CaSR and NeuN showing the absence of neurons in the ischemic region (asterisks). The broken line indicates the border between the ischemic and border zones. (B) Higher magnification view of the border zone (boxed area in A) showing that CaSR immunoreactivity was present only in some neurons (arrowheads). (C) Double-labeling for CaSR and GFAP showing that GFAP immunoreactivity is absent from the ischemic region. (D–F) Higher magnification views of the border zone (boxed area in C) showing that most CaSR-positive cells were GFAP-positive astrocytes. (G) Double-labeling for CaSR and Iba1. (H–J) Higher magnification views of the boxed areas in G. Note that Iba1-positive microglia/macrophages in both the ischemic region (H), and peri-infarct region (I, J) were devoid of CaSR expression. Cell nuclei are stained with DAPI. Scale bars = 100 mm in A, C, G; 50 mm in B, D–F, H– J.

vascular remodeling after focal cerebral ischemia (Abumiya et al., 1999; del Zoppo and Mabuchi, 2003; del Zoppo et al., 2011). Thus, CaSR induction within the perivascular compartment of the ischemic zone may contribute to vascular remodeling after stroke.

However, it should be noted that CaSR staining on the vascular structures appeared to decrease over the time of reperfusion, with concomitant loss of von Willebrand factor staining. Considering that blood vessels degenerate and endothelial cells in the ischemic

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Fig. 6. Characterization of CaSR-positive cells in the border zone 7 days after ischemic injury. (A) Low-magnification view of double-labeling for CaSR and NeuN showing the absence of neurons in the ischemic zone (asterisks). The broken line indicates the border between the ischemic and the border zone. (B–D) Higher magnification views of the border zone (boxed area in (A)) showing that only a subset of neurons (arrows) was positive for CaSR. (D) Orthogonal view of confocal z-stack. (E–G) Double-labeling for CaSR and GFAP. Note that almost all CaSR-positive cells in the border zone were reactive astrocytes. Arrows in E–G indicate a CaSR-positive astrocyte shown by orthogonal view of confocal z-stack (G). (H–J) Double-labeling for CaSR and Iba1 showing that microglia/macrophages were devoid of specific CaSR staining. Cell nuclei are stained with DAPI. (K, L) Absolute numbers of CaSR single-labeled cells and CaSR/GFAP (K) or CaSR/NeuN (L) double-labeled cells in the border zone at days 3 and 7 after reperfusion. The data are expressed as the mean  SEM. * P < 0.05; ** p < 0.01 versus 3 days. Scale bars = 100 mm for A; 50 mm for B–J.

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Fig. 7. Characterization of CaSR-positive cells in the peri-infarct border zone 14 days after ischemic injury. (A–E) Double-labeling for CaSR and GFAP. Note that CaSR immunoreactivity was observed in almost all reactive astrocytes in the border zone adjacent to the ischemic zone (asterisks). (C–E) Higher magnification views of the boxed areas in A and B. (F–J) Double-labeling for CaSR and Iba1 showing that neither amoeboid-like brain macrophages in the ischemic zone (asterisks in F and G), nor activated stellate microglial cells in the border zone presented CaSR immunolabeling. (H–J) Higher magnification views of the boxed areas in F and G. Cell nuclei are stained with DAPI. Scale bars = 100 mm in A, B, F, G; 50 mm in C–E, H–J.

core undergo cell death after focal cerebral ischemia (Burggraf et al., 2008; Li et al., 2012), it is likely that the change of CaSR expression simply reflects the alteration of vascular integrity during ischemia. In addition, some CaSR-positive cells in the ischemic zone coexpressed NG2, a marker of microvascular pericytes (DoreDuffy, 2003), and exhibited proliferative activity, suggesting that a subset of CaSR-positive cells in the ischemic zone showed a pericyte-like phenotype. However, specific immunochemical markers for pericytes are lacking (Fisher, 2009), and NG2 chondroitin sulfate proteoglycan has been reported to be expressed in diverse cells including oligodendrocyte progenitor cells, multipotent stem cells, and macrophage/monocyte lineage (Hill and Nishiyama, 2014; Matsumoto et al., 2008). In addition, the presence of CaSR-positive cells in perivascular regions appeared to correlate temporally and spatially with the presence of neural

stem/progenitor cells potentially originating from perivascular cells/microvascular pericytes (Nakagomi et al., 2011; Nakagomi et al., 2009; Nakano-Doi et al., 2010). Thus, we cannot exclude the possibility that the CaSR/NG2 double-labeled cells within the perivascular compartment may represent a pool of perivascular stem/progenitor cells. Considering the previous findings and our observations, we speculate that CaSR induction in the ischemic zone might be associated with specific, yet unknown, functions associated with vasculature in the ischemic brain. In the peri-infarct zone, reactive astrocytes were the predominant population of CaSR-positive cells. We did not observe expression of CaSR in astroglia either in the sham-operated striatum, or in the contralateral striatum of the ischemic rat. CaSR expression in astrocytes located in the peri-infarct region was evident three days after reperfusion, and this expression was maintained at least up to day 14, when CaSR labeling was

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prominent in hypertrophic reactive astrocytes confined to regions adjacent to the ischemic zone. This temporal pattern of CaSR expression overlaps with the initiation of astroglial activation, which precedes astroglial hypertrophy in the peri-infarct region. Interestingly, astrocytes in the ischemic zone did not show specific labeling for CaSR six hr after ischemia, despite the evident increase in GFAP immunoreactivity (see Fig. 2H). Considering that in the ischemic zone, astrocytes are irreversibly damaged (Chen and Swanson, 2003) and rapid aquaporin 4 loss occurs in perivascular astrocytic end-feet (Friedman et al., 2009), the increased GFAP immunoreactivity may not necessarily be followed by an increased expression of in CaSR, but, rather, CaSR expression might be upregulated during reactive astrocytosis in the less-damaged peri-infarct region. In addition to astrocytes, some, but not most, neurons were CaSR-positive cells. It is unclear why CaSR expression is induced in a subset of neurons in the border zone, and thus, characterization of these neurons using neurotransmitters or calcium-binding proteins is needed to further elucidate the functional significance of neuronal CaSR expression. However, in contrast to our findings, Mudo et al. (2009) reported the expression of CaSR mRNA in neurons and oligodendrocytes (but not in astrocytes and microglia), in normal animals and in those with kainate-induced seizures. In addition, Kim et al. (2011) demonstrated the induction of CaSR in hippocampal neurons, but not in astrocytes and microglia/macrophages, after global forebrain ischemia. These findings are in accord with our results showing that activated microglia/macrophages were devoid of any specific labeling for CaSR in both the ischemic and border zones. However, their results differ from ours in that we found CaSR immunolabeling in reactive astrocytes. Although the reasons for this discrepancy are unclear, this could be due to the use of different experimental models, the specific brain area studied, or the assessment of mRNA versus protein expression. In addition, in our study, we did not specifically assess for colocalization of CaSR and oligodendroglial markers. However, except for some neurons, the majority of CaSR-positive cells in the border zone were astrocytes with a reactive phenotype, indicating that the induced expression of CaSR was glia-subtype specific (i.e., astroglial). In addition, our findings are in accordance with previous studies demonstrating that CaSR is functionally expressed in human astrocytes and astrocytoma cell lines (Bouschet and Henley, 2005; Chattopadhyay et al., 2000; Yano et al., 2004). Furthermore, several studies have suggested that Ca2+ signaling stimulated by amyloid-beta peptide through CaSR in astrocytes plays a role in the progression of Alzheimer’s disease (Armato et al., 2012; Chiarini et al., 2009; Dal Pra et al., 2014; Ward et al., 2012). It is therefore likely that the induction of CaSR expression is related to the astroglial reaction in the ischemic stroke. Although our data provides evidence that CaSR may be involved in the pathogenesis of ischemic stroke, the present study has limitations since it used transient models restricted to relatively short time windows, in order to avoid risks of fatal edema and hemorrhage (see as reviews Bahjat et al., 2013; Fisher et al., 2009; Macrae, 2011). Recent studies suggest that CaSR cannot only be activated by extracellular calcium but also by various stimuli, such as polyvalent cations, pH, and ionic strength (Riccardi et al., 2009; Sun and Murphy, 2010; Tyler Miller, 2013). Astrocytes contribute to cell survival in the ischemic penumbra during stroke, where astrocytes are involved in a number of activities that profoundly influence tissue viability during ischemia, including glutamate homeostasis, ion homeostasis (i.e., buffering of extracellular potassium), water balance, and cerebral blood flow regulation (Chen and Swanson, 2003; Kimelberg and Nedergaard, 2010; Zhao and Rempe, 2010). Thus, the enhanced expression of CaSR in

reactive astrocytes in the border zone suggests that CaSR is upregulated in response to an altered extracellular ionic environment caused by ischemic injury, in an attempt to support and maintain neuronal viability. In support of this, it has been shown that CaSR in human astrocytes and astrocytoma cell lines could participate in local ionic homeostasis within the brain (Armato et al., 2012; Chattopadhyay et al., 2000; Yano et al., 2004). On the other hand, a subsequent CaSR activation in reactive astrocytes could signal astrocytes to form the glial scar, as suggested by Bouschet and Henley (2005). However, the precise role of astroglial CaSR induction in response to ischemic insults remains to be determined. Conclusions Our results indicate a phenotypic and functional heterogeneity of CaSR-positive cells, suggesting a multifunctional role for CaSR in the pathogenesis of ischemic stroke, possibly in the vascular remodeling and astrogliosis as response to ischemic injury. Author’s contributions All authors have contributed significantly to the research and the article preparation: Jeong Sook Noh and Ha-Jin Pak contributed to the treatment of the experimental animals, immunohistochemistry, histochemistry, and cell counting. Yoo-Jin Shin, Tae-Ryong Riew, and Joo-Hee Park worked on the immunohistochemistry, histochemistry, and photography. Young Wha Moon worked on the immunoblot assays.MunYong Lee worked on the design of the study, data analysis, and final manuscript preparation. Ethical statement All experimental procedures were conducted under and approved by the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Survival Surgery provided by the IACUC (Institutional Animal Care and Use Committee) in the College of Medicine, The Catholic University of Korea. Conflict of interest statement The authors have no conflict of interest including any financial, personal, or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence this work. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF2014R1A2A1A11050246). References Abumiya, T., Lucero, J., Heo, J.H., Tagaya, M., Koziol, J.A., Copeland, B.R., del Zoppo, G.J., 1999. Activated microvessels express vascular endothelial growth factor and integrin alpha(v)beta3 during focal cerebral ischemia. J. Cereb. Blood Flow Metab. 19, 1038–1050. Armato, U., Bonafini, C., Chakravarthy, B., Pacchiana, R., Chiarini, A., Whitfield, J.F., Dal Pra, I., 2012. The calcium-sensing receptor: a novel Alzheimer’s disease crucial target? J. Neurol. Sci. 322, 137–140. Bahjat, F.R., Gesuete, R., Stenzel-Poore, M.P., 2013. Steps to translate preconditioning from basic research to the clinic. Transl. Stroke Res. 4, 89–103.

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