Influence Of Gap Junction Communication On Neuronal Cell Death In Perilesional Zones And Hippocampus After Focal Cerebral Ischmemia In Mice

Part One To make photothrombosis and middle carotid arteryocclusion model in heterozygous connexin 43 null miceObjective: Gap junctions assemble astrocytes into syncytia, allowing exchange ofmetabolites, catabolites, and second-messenger molecules. Connexin 43 is the predominantconnexin of astrocytic gap junctions. Astrocytic gap junctions remain open during ischemicconditions. Gap junctions therefore may link ischemic astrocytes in an evolving infarct withthe surroundings. In this part, we used the heterozygous connexin 43 null mice, whichexhibit reduced cx43 expression, to made photothrombosis and MCAO model.Materials and Methods: Heterozygous connexin 43 null mice mice were buyed fromThe Jackson Laboratory. The genotypes of the newborns were judged by protocols fromThe Jackson Laboratory. According to their genotype, mice were divided into 2 groups:cx43+/+ group and cx43+/- group. Middle carotid artery occlusion (MCAO) model andphotothrombosis model were made. Behavior test were carried out 24h after ischemia andthe infarct volume were calculated by TTC staining.Results: According to protocol from The Jackson Laboratory, the genotypes ofnewborn mice were judged successfully. Although cx43-/- die immediately after birth,heterozygous cx43 null mice cx43+/- are viable. Middle carotid artery occlusion (MCAO)model and photothrombosis model are successfully made in heterozygous cx43 null miceand the wildtyp ones. The infracted tissue are white with TTC staining.Conclusion: Middle carotid artery occlusion (MCAO) model and photothrombosismodel are successfully made in heterozygous cx43 null mice. Part Two Effect of gap junction communication on delayedneuronal cell death in mouse hippocampus after MCAOObjective: Delayed neuronal death (DND) was found in hippocampus, which is notsupplied by middle carotid artery, after middle carotid artery occlution (MCAO). In brain,astrocytes are coupled extensively by gap junctions, which are highly conductive channelsthat allow the direct transfer of intracellular messengers such as Ca²⁺ and inositoltriphosphate (IP3) between interconnected cells. In this part, we observed the effect ofblocking gap junction communication after MCAO on the delayed neurnal death inipsilateral hippocampus and explored the mechanism.Materials and Methods: Mice were divided into CX43+/+ sham group, CX43+/-sham group, CX43+/+ MCAO group and CX43+/- MCAO group, according to theirgenotypes, 1d after surgery, behavior test and TTC staining were carried out. Then micewere killed at 3d and 7d post ischemia respectively, and the samples were harvested forfurther use. Neuronal cell popotosis were measured by TUENL; the calcium in neurons ofhippocampus were measured by two-photon calcium imaging; the calpain activity weremeasured by Calpain Activity Assay Kit and water maze were carried out 30d afterischemia.Results: Our results showed that compared with CX43+/+ MCAO group, behaviortest scores and infarct volum were low in CX43+/- MCAO group. DND were detected infewer mice in CX43+/- MCAO group and the calcium concentration and calpain activitywere low in this group. 30 days after ishchmia, mice of CX43+/- MCAO groupperformaced better in water maze task than those of CX43+/+ MCAO group.Conclusion: Blocking gap junction communication may reduce the infart volume and incidence rate of delayed neuronal death in hippocampus after middle carotid arteryocclusion. The free exchange of intracellular calcium between dying cells and those inremote region might contribute to expansion of ischemic damage. Part Three Effect of gap junction communication on neuronalcell death in perilesional zone after focal cerebral ischemia in miceObjective: To explore the effect of gap junction communication on neuronal cell deathin perilesional zone after focal cerebral ischemia in mice and the mechanism.Methods: Mice were divided into CX43+/+ sham group, CX43+/- sham group,CX43+/+ ischemia group and CX43+/- ischemia group, according to their genotypes.Photothrombosis were made. Mice were killed at 2h, 6h, 12h, 1d, 3d and 7d post ischemiarespectively, and then the samples were harvested for further use. calpain,Bax,caspase 12,and actived-caspase3 were detected by immunofluorescence.Results: (1) Actived-caspase3 protein increased post ischemia and actived-caspase3positive cells were fewer in CX43+/- ischemia group. (2) Calpain protein increased postischemia and reach the summit at 1 day post ischemia. Calpain-positive cells were fewer inCX43+/- ischemia group. (3) Caspase 12 protein increased post ischemia and reached thesummit at 2h post ischemia in CX43+/+ ischemia group, but the time in CX43+/- ischemiagroup was 6h. Caspasel2-positive cells were fewer in CX43+/- ischemia group. (4) Baxprotein increased post ischemia and reached the summit at 1d post ischemia in CX43+/+ischemia group, but the time in CX43+/- ischemia group was 3d. Bax-positive cells werefewer in CX43+/- ischemia group.Conclusion: Apopotosis neurons increased in prelesional zone post focal ishchemia.Calpain, Bax and caspase12 take part in the neurnal apoptosis. The Bax- andcaspase12-positive cells were fewer in CX43+/- ischemia group.