Contribution of TRPC Ion Channels to Brain Injury in the Cerebellum

Abstract

Ischemic brain injury is a major cause of morbidity and mortality, with no neuroprotective treatment available. Cerebellar strokes are particularly devastating and show the highest mortality rate of any brain region, although their incidence is less common than for cerebral strokes. This study investigated the putative contribution of the canonical transient receptor potential (TRPC) ion channels to ischemic injury in the cerebellum. TRPC ion channels are calcium permeable, non-selective cation channels which are coupled to metabotropic glutamate receptors (mGluR) and are highly expressed in the cerebellum. The contribution of sustained activation of the mGluR1-TRPC3 pathway to Ca2+ influx was assessed by comparing Ca2+ entry in the Purkinje neurons from wildtype (WT) and TRPC3 knockout (KO) mice in acute brain slices. MGluR1-mediated Ca2+ responses were assayed in response to application of glutamate or the mGluR1 agonist dihydroxyphenylglycine (DHPG), using the genetically encoded Ca2+ indicator GCaMP5G. Fluorescent Ca2+ signals in the WT Purkinje neurons typically consisted of an initial peak followed by a sustained elevation. In Purkinje neurons from TRPC3KO mice the amplitude of the initial transient Ca2+ signal was significantly reduced and the sustained elevation completely missing, suggesting that mGluR1- mediated Ca2+ influx through TRPC3 ion channels may contribute to sustained neuronal Ca2+ loading. To assess how this sustained Ca2+ loading response may contribute to cerebellar ischemia-derived injury in vivo, the photothrombotic model of stroke was adapted to deliver focal ischemic lesions in the cerebellar cortices of adult WT, TRPC3KO and TRPC1/3/6/7 KO (TRPC QKO) mice. A longitudinal analysis of the photothrombotic injury development was initially performed in WT mice to characterise infarct volumes, neuronal loss and glial activation at 2 hours post injury, and 1, 4, 7, 14, and 30 days post injury (DPI). The penumbra expansion was greatest at 1 DPI and tissue remodelling was apparent at 7 DPI. The technique of darkfield imaging was developed to assess infarct volumes from unstained brain cryosections and was found to be more efficient compared to the conventional haematoxylin and eosin staining technique. In a blinded-study, the infarct volumes in the TRPC3KO and TRPC QKO were significantly smaller than the WT mice at 4 DPI, thus confirming the involvement of TRPC ion channels in ischemic injury in the cerebellum