Elsevier

Journal of Chemical Neuroanatomy

Volumes 66–67, July–September 2015, Pages 40-51
Journal of Chemical Neuroanatomy

Differential expression of the calcium-sensing receptor in the ischemic and border zones after transient focal cerebral ischemia in rats

https://doi.org/10.1016/j.jchemneu.2015.05.001Get rights and content

Highlights

  • We show the spatiotemporal regulation of CaSR expression in ischemic rats.

  • CaSR was induced mostly in cells associated with vasculature in the ischemic region.

  • The peri-infarct region presented long-lasting CaSR expression in astrocytes.

  • CaSR appeared only on a subset of neurons in the ischemic and border regions.

  • CaSR induction was glia-type specific and was absent from microglia/macrophages.

Abstract

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.

Introduction

Calcium-sensing receptor (CaSR), which is coupled to G-protein, 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 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., 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, Kim et al., 2013 showed that after traumatic and ischemic brain injury, CaSR overexpression and concurrent down-regulation of metabotropic γ-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.

Section snippets

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

CaSR expression is induced in the ischemic hemisphere after MCAO

The mortality rate of MCAO rats was 25.4%, and all sham-operated 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

Discussion

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

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.Mun-Yong 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 (NRF-2014R1A2A1A11050246).

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