Contributions Of T-Type Ca²⁺ Channels Cav3.3 In The Experimental Subarachnoid Hemorrhage Induced Brain Injury

Background: The research of the mechanism of subarachnoid hemorrhage(SAH) induced brain injury has been lasted for several decades but it remaines unclear till now. Previous studies focused on the cerebral vasospasm and little attention have been paid to the changes of function brain region. It is indicated in experimental and clinical studies that nimodipine ,the only effective drugs which is evidence based , could do effect on protection of neurons and meliorate SAH induced demage in some extent or in the early phase.Although the effect and mechanism of nimodipine is still in debate, it means the intervention of calcium sighal would improve the outcome of SAH. The ability of T-type channels to open at similar potentials at which they inactivate suggests that they might generate current under steady-state conditions.This is commonly referred to as a window current,which likely involved in sustained burst firing, is operationally defined as the overlap region between the activation and steady-state inactivation curves,that they play an important role in signal amplification and induce depolarization and AP (action potential) .It is still short of the research in the mechanism of T-type channels in subarachnoid hemorrhage.But previous studies have showed there is inflammation , free radical activation , hypoxia in SAH,which would induce expression alteration and function swtch of T-type channels. Cortical spreading depression-like depolarizations was found in patients or animals post SAH. And Hippocampus and hypothalamus is ischemic sensitive .So, the alteration of T-type channels in these brain regions may make sense after SAH.Object : Reasonable animal SAH model were founded to observe the expression alteration and function swtch of T-type channels Cav3.3 after SAH. And to investigate the contributions of T-Type Channels Cav3.3 in the experimental subarachnoid hemorrhage by means of interven the expression and function of Cav3.3 after SAH .Thus ,clarify the possible mechanism of subarachnoid hemorrhage induced brain injury.Methods:1. SAH was induced in rats via blood injection into the cisterna magna (cisterna magna injection SAH model), or blood injection into the prechiasmatic cistern (prechiasmatic cistern injection SAH model). The mortality rate and physiological parameters was measured. Furthermore, by the means of neuroethology evaluation(Garcia score,Beam Walking Test,Morris water Maze Test)and Nissl's staining ,the degree of brain after subarachnoid hemorrhage was evaluated .2. Immunohistochemistry and RT-PCR were used to evaluate the expression of T-type channels Cav3.3 in adult rats brain( Cortical,Hippocampus and hypothalamus). By means of immunofluorescence and confocal microscopy to show the expression of Cav3.3 in neurons .3. SAH was induced in rats via blood injection into the cisterna magna into the prechiasmatic cistern. The expression of T-type channels Cav3.3 in SAH rats brain was evaluated by immunofluorescence . Furthermore, Mibefradil, Verapamil and Cav3.3 AS ODN were used to treat SAH. Mortality rate,physiological parameters and neuroethology evaluation were used to evaluate the effect.Results:1. The mortality rate of prechiasmatic cistern injection SAH model was 20%. Garcia score shows that the prechiasmatic cistern injection SAH model was lower than control group and sham group(P<0.01). Beam Walking Test shows that all the SAH groups have poor Skilled Movement score(P<0.01). Morris water Maze Test shows that all the SAH groups have poor space leanning performance., The prechiasmatic cistern injection SAH model is worse in all neuroethology evaluation.;2. Cav3.3 is almost exclusively expressed in adult rats brain and is high in olfactory bulb, striatum, cerebralcerebralcortex, hippocampus, reticular nucleus, lateral habenula,and cerebellum. Few Cav3.3 is found in hypothalamus neurons, But it is abundant in the nerve plexus .And the RT-PCR showed the similar results.3. Beam Walking Test shows that all the SAH groups have poor Skilled Movement score(P<0.01). Mibefradil, Verapamil and Cav3.3 AS ODN would improve the performance in Beam Walking Test . Morris water Maze Test shows the the similar results. Mibefradil and Cav3.3 AS ODN is some better than Verapamil .And Cav3.3 AS ODN may take effect from day3.Conclusion:1. Two SAH model are also reasonable models to mimic the clinical situation .But the prechiasmatic cistern injection SAH model was better .It is more close to the Willis circulation and more poor performance in neuroethology evaluation.It is seemed more close to reality.2. Cav3.3 is expressed in adult rats brain and is high in cerebralcortex, hippocampus. Few Cav3.3 is found in hypothalamus neurons, But it is abundant in the nerve plexus .3. There is overexpression of T-type channels Cav3.3 in SAH rats brain .And by means of interven the expression and function of Cav3.3 would reduce the brain injury after SAH...