Investigation Of Functions Of Na～+-Ca～(2+) Exchanger (NCX) In Cerebral Ischemia And Relevant Pharmacological Studies

The incidence of stroke in China is similar to that in the western country. Strokeis the secondary and third most common cause of death in China and in occident,respectively, and it is also the principal cause of postinjury disability. The percentageof ischemic stroke is about 85% in stroke. Ischemia-induced neuronal damageproduces infarct that is severely compromised and may not be repaired. Researchershave been contributing to the pathophysiological mechanisms of cerebral ischemiaand finding clinical effective drugs.Among the theoretical hypotheses of cerebral ischemia, Ca²⁺ overload is one ofthe most important mechanisms. Na⁺-Ca²⁺ exchanger (NCX) is a widely distributedmembrane protein, which can exchange three Na⁺ ions for one Ca²⁺ ion across the cellmembrane. Thus NCX played an important role in maintaining the intracellular Ca²⁺homeostasis in many cells, including neurons and myocytes. Many researches haveshown that NCX was closely related to cerebral and myocardial ischemia. There arethree NCX isoforms, including NCX1, NCX2, and NCX3, in mammals. NCXisoforms are distributed tissue-specifically. NCX1 is the only isoform in heart. NCX1,NCX2, and NCX3 exit selectively in central nervous system, which suggests NCXisoforms may have different functions in cerebral ischemia.The aim of our work is to further the research of NCX as a drug target and findnew clinical effective drugs. There are three parts in our research: 1) we observed thealteration of mRNA and protein expressions of NCX isoforms in transient andpermanent focal cerebral; 2) we built a cellular model specifically expressed NCX1.1isform and observed the effects of temperature and KB-R7943 on NCX1.1 currentwith whole-cell patch clamp; 3) we built a simple, stable, and repeatable animalmodel to mimic aneurysmal SAH and observed the effect of nimodopine on theneurological deficit in SAH rats. The results as follows: Partâ… Alteration of expressions of NCX isoforms in acute focal cerebralischemia in ratsMiddle cerebral artery occlusion (MCAO) is the most widely used animal modelof acute focal cerebral ischemia. MCAO was used to mimic the transient focalcerebral ischemia and permanent focal cerebral ischemia. We observed the mRNAand protein expressions of three NCX isoforms in brain cortex at different time pointsin these two pathophysiological conditions.1 Alteration of mRNA and protein expressions of NCX isoforms in transientmiddle cerebral artery occlusion (tMCAO) in ratsRT-PCR was used to investigate the mRNA levels of NCX1, NCX2, and NCX3at 2 h, 6 h, 12 h, and 24 h of reperfusion after 2 h of MCAO. Western blot wasemployed to measure the protein levels of NCX1, NCX2, and NCX3 at 2 h, 12 h, and24 h of reperfusion after 2 h of MCAO. Our results showed a transient and significantdecrease of NCX1 mRNA in ipsilateral cortex. NCX1 mRNA was decreased by42.1% and 27.8%, respectively, at 2 and 6 h of reperfusion. The mRNA levels ofNCX2 and NCX3 did not change significantly over time. NCX1 protein level inipsilateral cortex was decreased by 36.6% at 2 h of reperfusion, tMCAO did notinfluence the protein expressions of NCX2 and NCX3 at various time points.2 Alteration of mRNA and protein expressions of NCX isoforms in permanentmiddle cerebral artery occlusion (pMCAO) in ratsRT-PCR was used to investigate the mRNA levels of NCX1, NCX2, and NCX3after 2 h, 6 h, 12 h, and 24 h of MCAO. Western blot was employed to measure theprotein levels of NCX1, NCX2, and NCX3 at 2 h, 12 h, and 24 h of MCAO. Ourresults showed the mRNA levels of three NCX isoforms did not change at differenttime points. However western blot analysis showed NCX1 protein in the ipsilateralcortex was decrease by 58.6% at 2 h of MCAO and increased by 76% and 1.36 foldsat 12 h and 24 h of MCAO, respectively, pMCAO did not influence the protein expressions of NCX2 at various time points. NCX3 protein was decrease by 50% and59.6% at 12 h and 24 h of MCAO, respectively.Partâ…¡Specific-expression of NCX1.1 isoform in HEK293 cells and relatedpharmacological studiesIn this experiment we transfected rat NCX1.1 plasmid and green fluorescentprotein (GFP) plasmid into HEK293 cells by calcium phosphate precipitation method.After transfection for 48 h, we recorded NCX1.1 current (I_NCX1.1 in HEK293 cellswith green fluorescence with whole-cell patch clamp.Our results showed that most HEK293 cells had green fluorescence aftertransfection for 48 h. The current in blank HEK293 cells is very small. The current intransiently transfected HKE293 cells was much larger and was inhibited by 5 mMNiCl₂, which suggested the recorded current was I_NCX1.1. I_NCX1.1 was sensitive totemperature. I_NCX1.1 at 35℃was much larger than that at room temperature. 10μMKB-R7943 could inhibit outward and inward I_NCX1.1 by 68% and 62%, respectively.Partâ…¢Establishment and evaluation of animal model of subaraehnoidhemorrhage (SAH) in ratsThe aim of our experiment is to establish a simple and repeatable animal modelof aneurysmal SAH. A sharpening nylon thread was passed through the right internalcarotid artery and pierced a hole in the right anterior cerebral artery to produce SAH.Rats were randomly divided into three groups, including vehicle group treated withvehicle after SAH, nimodopine treated group (i.p 0.25mg·kg^-1 5 min, 6 h, 12 h afterSAH) and sham group. At 12 h and 24 h of SAH, the rats were evaluated with rotarodtest and the behavior scale (5-point scale).At the point of the perforation there was usually a capping clot. There was bloodin the basal cisterns with some spread over the hemisphere. After 12 h and 24 h ofSAH, the time of rotarod test of SAH rats decreased significantly and the rats hadserious neurological deficit. Nimodipine could alleviate the neurological deficit after24 h of SAH. Conclusion: Our results suggested that NCX1 was closely related to tMCAOand pMCAO and may play important but different roles in these twopathophysiological conditions, while NCX3 may have an important role in pMCAO.NCX1.1 isoforms was expressed specifically in the transiently transfected HEK293cells. I_NCX1.1 was sensitive to the temperature. The inhibitory effect of 10μM KB-R7943 on outward I_NCX1.1 paralleled to its effect on inward I_NCX1.1 which suggestedthe inhibitory effect of KB-R7943 on operation mode of NCX was lack of selectivity.We present a simple and reliable animal model of SAH in rats, which allowsevaluating novel compounds and new drugs for treatment of SAH.