The Role And Mechanisms Of Calcium-sensing Receptor In Cerebral Ischemic Stroke In Mice

Part I Establishment and evaluation on the Model of Focal CerebralIschemia/Reperfusion induced by monofilament inKunming Mice with different body weightObjective:Prevention and treatment of cerebrovascular disease has become an importantsubject in the world. Stroke is a widespread disease affecting human health, especiallyIschemic Cerebrovascular Disease, ICVD, accounted for a high proportion. Therefore,an ideal model of middle cerebral artery occlusion in annimal is an important method tostudy such diseases. To establish the mouse focal cerebral ischemia/reperfusion modelwith suture method,study the stability of the model of middle cerebral artery（MCAO）and provide a basis for further experiments.Methods：One hundred KM mice, weighing20-39g, half male and half female, were dividedinto10groups according to the body weight: Sham group (♂), Sham group (♀), groupA(♂,20-24g), group B (♂,25-29g), group C (♂,30-34g),group D (♂,35-39g),group E(♀,20-24g), group F (♀,25-29g), group G (♀,30-34g),group H(♀,35-39g)（.n=10）Nylonthread was introduced into the internal carotid artery to establish the Model of FocalCerebral Ischemia/Reperfusion in mice. The reliability of the MCAO model wasestimated by the degree of neurological deficit score and area of cerebral infarction.Results： 1. Success rates and mortality rate：Compared with female mice，the successrates of MCAO model were increased considerably in male mice while the mortalityrates were decreased gradually. Group A and Group B had a higher success rate(70%and80%respectively).Besides, the mortality rate in Group B was lowest.2. Neurological function in mice after ischemia/reperfusion：Other groupsexcept sham groups all emerged the change in nervous behavioral function aftercerebral ischemic reperfusion. Neurological score was slightly higher in male KM micethan that in female KM mice. Compared with group C and group D, there were dramaticrise of behavior scores in group A and group B.And also the neural deficit scores ofgroup E and group F were much higher than that in group G and group H (P<0.01). Theneurological scores in group E were observably lower than that in group A (P<0.01).Meanwhile, there were dramatic drop of behavior scores in group F (P<0.01),compared with group B.3. Infarct area of cerebral focal ischemia/reperfusion models in mice：Theexperimental groups came out clear infarction area after cerebral ischemic reperfusion.There were considerable rise of infarct area in group A and group C (P <0.01), incomparison with group C and group D. In addition, the area of cerebral infarctionenhanced sharply (P<0.05) in group E and group F compared with group G and groupH.Besides, there were significant falls of infarct area in group B (P <0.01), compared togroup F.Conclusion：The mouse focal cerebral ischemic/reperfusion model with suture method is stableand simple. There are many crucial factors to guarantee the success of mouse focalcerebral ischemic/reperfusion model induced by nylon monofilament, such as the miceweight matching the diameter of monofilament, the depth of line, sex selection,operation time, and suitable temperature(25-28℃) during experiment. Experimental investigation shows that weighing about25g KM male mice are fit for the studies offocal cerebral ischemic/reperfusion. Part II the role and mechanisms of Calcium-sensing receptor incerebral ischemic stroke in miceObjective:At present, the disease of cerebral vascular which can upset millions of familieshas a high death and teratogenic rate with scant therapies. This study was conducted toinvestigate the effect of CaSR activation on cerebral ischemic stroke.Methods:Focal cerebral ischemia/reperfusion model in mice were established with suturemethod. We examined the role of the CaSR activator gadolinium chloride (GdCl3) andthe inhibitor of CaSR (NPS2390) in mice model of focal cerebral ischemia-reperfusion.Healthy male adult Kun-ming mice weighing23g to25g were randomly assigned to4groups (n=16for each group):Sham group,I/R group,I/R+Gdcl3group,I/R+NPS2390group.Expression of the CaSR protein was observed by Western blot. Neurologicaldeficit scores were evaluated and Infarct area was determined by TTC(triphenyltetrazolium chloride) staining. The morphology of neurons in the brainsections was examined by HE staining, JNK/p-JNK, P38/p-P38, Bax and Bcl-2expression were examined by using Western Blotting.Results 1. Scale of neural function and infarct area: The model group emerged thechange in nervous behavioral function and clear infarction area in TTC after cerebralischemic reperfusion in comparison with sham group. Compared with I/R group, Gdcl3could significantly worsen the grade of nerve function, increased the cerebral infarctarea. And this effect of GdCl3was inhibited by NPS2390.2. Expression of CaSR in mice after I/R: There were significant increases inexpression of CaSR in I/R group in the cortex and hippocampus compared to shamgroup. The results showed that GdCl3increased the expression of CaSR, and this effectof GdCl3was inhibited by NPS2390.3. HE staining: Sham group had no evident pathological change. Thehistopathological analysis showed that focal cerebral ischemia-reperfusion caused themorphological abnormalities, including degeneration and necrosis of neurons, focalcerebral edema, and infiltration of inflammatory cells in comparison with the shamgroup. And in I/R+Gdcl3group, the number of neuron which appeared degeneration andnecrosis increased in comparation to model group. And this increase was inhibited byNPS2390.4. Expression of Bax and Bcl-2in mice after I/R: There were significantincreases in expression of Bax in I/R group in the cortex and hippocampus compared tosham group. Treatment with Gdcl3increased Bax expression and decreased Bcl-2expression compared with I/R group in the cortex and hippocampus. While theexpression of Bax was significantly decreased in NPS2390group, and administration ofNPS2390inhibited the decrease of Bcl-2.5. Expression of p-JNK/JNK in mice after I/R: A very small amount of thephosphorylation of c-JunNH2-terminal protein kinases (JNK), and P38were detected inbrain tissue of the sham group, which significantly increased in I/R group. Comparedwith I/R group, GdCl3could further increase the expression of p-JNK and p-P38. Andthis increase was inhibited by NPS2390. Conclusion:Activation of CaSR could contribute to ischemic stroke and promote apoptosis inmice model of focal cerebral ischemia-reperfusion through activation of the JNK/P38MAPK pathway, as well as up-regulation of Bax and down-regulaiton of Bcl-2expression.