The role of astrocytic calcium signaling in brain damage after photothrombosis

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Ischemic stroke is the third leading cause of death in industrialized countries. Our previous study found elevated [Ca[superscript 2+]]i signals in astrocytes after photothrombosis-induced ischemia. However, the role of astrocytic Ca[superscript 2+] signaling in ischemia is still poorly understood. In this study, we generated a stable and repeatable photothrombosis-induced ischemia model so that its ischemic infarction could be controlled by regulating the intensity of the output light and the size of the irradiated area in the cortex, which could be used to study the mechanisms of tissue damage and neuronal protection. Secondly, we investigated the effect of the Pleckstrin Homology (PH) domain of Phospholipase C (PLC)-like protein p130 (p130PH) on Ca[superscript 2+] signaling in astrocytes in vivo. We used the serotype 2/5 recombinant adeno-associated virus (rAV2/5) vectors to introduce p130PH fused with a tagged protein monomer red fluorescent protein at the N-terminal (i.e., transgene mRFP-p130PH). In order to selectively disrupt the Ca[superscript 2+] signaling pathway in astrocytes, the transgene was driven by a novel astrocyte-specific promoter gfaABC1D. Our results show that mRFP-p130PH is exclusively expressed in astrocytes with a high efficiency and a stable expression level. In vivo imaging using two-photon microscopy demonstrated reduced Ca[superscript 2+] signal in transduced astrocytes in response to ATP simulation. As Ca[superscript 2+] signaling is a characteristic form of cellular excitability in astrocytes that can mediate chemical transmitter release and contribute to neuronal excitotoxicity, the current study provides an in vivo approach to better understand Ca[superscript 2+]-dependent gliotransmission and its involvement in glia related diseases (i.e., ischemia). In order to study the role of astrocytic Ca[superscript 2+] signaling in ischemia, we tested the photothrombosis ischemic model on IP[subscript 3]R2 knockout mice. First, we demonstrated that IP[subscript 3]R2 knockout mice depleted astrocytic IP[subscript 3]R in vivo, but also abolished IP[subscript 3] mediated astrocytic [Ca[superscript 2+]]i signaling in response to ATP using in vivo imaging. Between WT and IP[subscript 3]R2 knockout mice, there is no difference in the number of astrocytes number and expression of specific astrocytic proteins which have been demonstrated to be crucial in ischemia damage. At 24 hours after photothrombosis-induced ischemia, we found a similar size of infarct volume and neuronal response between WT and IP[subscript 3]R2 knockout mice. The same phenomena were observed at 2 days and 7 days after ischemia. Until day 14 after ischemia, we discovered smaller infarct volume in IP[subscript 3]R2 knockout mice than WT mice, with more shrinking tissue around the ischemic core in the IP[subscript 3]R2 knockout mice. Further investigation demonstrated that the IP[subscript 3]R2 knockout mice have more severe glia scar formation and microglia activation during the time period between day 7 and 14. The neurogenesis study suggests that more migration of proliferating cells in IP[subscript 3]R2 knockout mice might result in dense and thick glia scar after 14 days ischemia. However, investigation should be done to test the process of migration. These observations suggest that IP3 mediated astrocytic Ca[superscript 2+] has an active role in ischemia recovery.