Spatiotemporal expression of RCAN1 and its isoform RCAN1-4 in the mouse hippocampus after pilocarpine-induced status epilepticus

Regulator of calcineurin 1 (RCAN1) can be induced by an intracellular calcium increase and oxidative stress, which are characteristic features of temporal lobe epilepsy. Thus, we investigated the spatiotemporal expression and cellular localization of RCAN1 protein and mRNA in the mouse hippocampus after pilocarpine-induced status epilepticus (SE). Male C57BL/6 mice were given pilocarpine hydrochloride (280 mg/kg, i.p.) and allowed to develop 2 h of SE. Then the animals were given diazepam (10 mg/kg, i.p.) to stop the seizures and sacrificed at 1, 3, 7, 14, or 28 day after SE. Cresyl violet staining showed that pilocarpine-induced SE resulted in cell death in the CA1 and CA3 subfields of the hippocampus from 3 day after SE. RCAN1 immunoreactivity showed that RCAN1 was mainly expressed in neurons in the shammanipulated hippocampi. At 1 day after SE, RCAN1 expression became detected in hippocampal neuropils. However, RCAN1 signals were markedly enhanced in cells with stellate morphology at 3 and 7 day after SE, which were confirmed to be reactive astrocytes, but not microglia by double immunofluorescence. In addition, real-time reverse transcriptase–polymerase chain reaction showed a significant upregulation of RCAN1 isoform 4 (RCAN1-4) mRNA in the SE-induced hippocampi. Finally, in situ hybridization with immunohistochemistry revealed astrocytic expression of RCAN1-4 after SE. These results demonstrate astrocytic upregulation of RCAN1 and RCAN1-4 in the mouse hippocampus in the acute and subacute phases of epileptogenesis, providing foundational information for the potential role of RCAN1 in reactive astrocytes during epileptogenesis.

Expression of RCAN1 and CnA in tissues of in-stent restenosis after intervention of lower extremity arteriosclerosis obliterans and its significance / 上海交通大学学报（医学版）

Objective · To investigate the expression of the regulator of calcineurin 1 (RCAN1) and calcineurin A (CnA) in tissues of in-stent restenosis after intervention of arteriosclerosis obliterans (ASO), and to explore the relationship between their expression levels and the occurance of in-stent restenosis. Methods · Superficial femoral arterial tissues were collected from 15 ASO patients undergoing lower extremity amputation for in-stent restenosis in Department of Vascular Surgery, The First Affiliated Hospital of Chongqing Medical University from September 2013 to June 2016. H-E staining and Masson staining were performed on the stenosis tissues, as well as on the proximal and distal tissues, and the morphological changes of these tissues were observed under optical microscope. Western blotting was used to detect the protein levels of RCAN1, CnA and proliferating cell nuclear antigen (PCNA). The distribution of RCAN1 and CnA proteins was observed by immunohistochemistry and immunofluorescence methods. In addition, co-immunoprecipitation was used to validate the protein-protein interaction between RCAN1 and CnA in vascular tissues. Results · The expression of RCAN1 in the distal tissues was significantly elevated compared with the proximal tissues and the stenosis tissues (P<0.05). The expression of RCAN1 in the proximal tissues was higher than that in the stenosis tissues (P <0.05). The expression of CnA and PCNA in the stenosis tissues was significantly elevated compared with the proximal tissues and the distal tissues (P<0.05). Immunohistochemistry and immunofluorescence analyses showed that RCAN1 and CN proteins were mainly expressed in the cytoplasm of vascular smooth muscle cells. Co-immunoprecipitation analysis showed there is protein-protein interaction between RCAN1 and CnA in arterial tissues. Conclusion · The low expression of RCAN1 and the high expression of CnA are probably related to the occurrence of in-stent restenosis.