Clioquinol Attenuates Zinc-Induced Neuronal Death After Global Ischemia In The Gerbil

PrefaceZinc(Zn²⁺) is the second most abundant transition metal in the human body.And it's required for normal growth,gene expression,protein metabolism,and immune function.Short time cerebral ischemia gives rise to delayed neuronal death of pyramidal neurons in the CA1 region of the hippocampus in rodents.Evidence for the early Zn²⁺ translocation from presynaptic terminals into postsynaptic neuronal cell bodies has also been demonstrated in rat cortex subjected to focal ischemia,as occurs in stroke.Excessive release of synaptic Zn²⁺ may contribute to the pathogenesis of delayed selective neuronal cell death after transient global ischemia.Many researches have shown that chelation of zinc in vivo puts a protective effect on neurological disorders in animal models.For instance,injection of the zinc chelator,Ca-EDTA, significantly reduced neuronal death resulting from experimentally-induced forebrain ischemia.It has been verified that late injection of Ca-EDTA could rescue CA1 neurons from damaging in global-ischemia Mongolian gerbils.In the recent study,it has been suggested that clioquinol(iodochlorhydroxy-quin,5-chloro-7-iodo-8-hydroxyquinoline, CQ) is a membrane-permeable and hydrophobic zinc chelator.So it can easily cross the blood-brain barrier.In the present study,we examine the protective effects of CQ on the ischemia gerbil brains,to address whether administration of CQ after ischemia can rescue the CA1 pyramidal cells.Materials and Methods1.Experimental animals and TreatmentsAdult Mongolian gerbils were randomly divided into three groups:(1) sham,(2) vehicle-treated(1d,3 d,7d) ischemia groups,(3) CQ-treated(1d,3d,7d) ischemia groups.DMSO was used as the vehicle for CQ. To verify the chelation of CQ,adult gerbils were randomly divided into normal and CQ 2h-normal groups.ZnSe AMG was used for checking the chelation of CQ.2.Induction of Global Cerebral IschemiaThe gerbils were anesthetized with pentobarbital(40mg/kg,i.p.).Ventral neck incision was made;the bilateral common carotid arteries were separated carefully from vagus nerve and were occluded bilaterally for 10 min with non-traumatic aneurysm clips.Ten minutes later,the aneurysm clips were removed and the complete reperfusion of the arteries was verified by direct visual observation.Then the neck incision area was sutured by 4-0 nylon and ethanol for disinfection was applied.The gerbils were kept under heating lamp for 2 h until recovery.The sham-operated non-ischemia gerbils underwent the same surgical procedures,except bilateral common carotid arteries were not occluded.CQ and the vehicle were administered immediately after ischemia and then were given everyday(i.p.,10mg/kg) till sacrificed(1d,3d,7d).3.Morphological and molecular biological methodsThe chelation of CQ was evaluated by the ZnSe autometallography(AMG).TSQ fluorescence was used to detect the chelation of CQ in CQ-treated animals.We used Nissl staining to detect the loss of pyramidal neurons in the CA1 region of the hippocampus between vehicle-treated groups and CQ-treated groups after the surgery. TUNEL staining was used to examine whether CQ administered after ischemia can rescue the CA1 neurons.We also used in situ hybridization to detect the expression of Caspase-3 and Caspase-9 mRNA between vehicle-treated 3d groups and CQ-treated 3d groups.Furthermore,Western blot was used to detect the expression of Caspase-3 and AIF between vehicle-treated groups and CQ-treated after the surgery(1d,3d,7d).Results1.ZnSe AMG resultUnder light microscope,compared with non-CQ group,the CQ group ZnSe AMG staining intensity was decreased seriously,and the stain was lower in the region of DG and mossy fiber in hippocampus.2.TSQ fluorescenceTSQ fluorescence results showed that the fluorescence in the CQ-treated group was lower than sham and vehicle-treated group after 3d of surgery.And vehicle-treated group was higher than the sham group.3.Nissl staining and hippocampal CA1 cell countUnder light microscope,there was no any cell damage in sham group after 1d,3d, 7d of surgery.Cell degeneration,cell death and cellular swelling were observed in CA1 pyramidal neurons in the vehicle-treated groups.Cell number of CA1 in the CQ-treated groups was raised than that of vehicle-treated groups,the arrangement was more regularity and the shape was more integrity.4.TUNEL assayUnder light microscope,there were few TUNEL positive cells in sham group after 3d of surgery.TUNEL positive cell count of CA1 pyramidal neurons in the CQ-treated groups was decreased than that of vehicle-treated groups;the TUNEL positive cells were brown.The vehicle-treated 3d group was statistically different from the CQ-treated 3d group:P<0.01.5.Caspase-3 and Caspase-9 in situ hybridization assayUnder light microscope,in situ hybridization results revealed that Caspase-3 and Caspase-9 mRNA positive cells were brown.There were few Caspase-3 and Caspase-9 mRNA positive cells in sham group after 3d of surgery.Caspase-3 and Caspase-9 mRNA positive cell number of CA1 pyramidal neurons in the CQ-treated a groups was decreased than that of vehicle-treated groups.The vehicle-treated 3d group was statistically different from the CQ-treated 3d group:P<0.01.6.Western blotThe expression levels of Caspase-3 and AIF protein were measured using Western blot analysis.The semiquantitative analysis of immuno-blots showed statistically significant elevations of Caspase-3 and AIF in the hippocampi of vehicle-treated groups(1d,3d,7d) compared with CQ-treated groups(1d,3d,7d).Caspase-3(17KD): the vehicle-treated groups were statistically different from the CQ-treated groups: P<0.01.Procaspase-3(32KD):the vehicle-treated groups were statistically different from the CQ-treated groups:P<0.05(1d,7d);P<0.01(3d).AIF(67KD):the vehicle-treated groups were statistically different from the CQ-treated groups:P<0.05 (3d,7d);P<0.01(1d).Conclusion1.AMG staining and TSQ fluorescence results showed that CQ had the effect of chelation of zinc.2.Nissl staining showed that the cell number of CA1 pyramidal neurons in the CQ-treated groups was raised than that of vehicle-treated groups.TUNEL staining showed that the TUNEL positive cell number of CA1 pyramidal neurons in the CQ-treated groups was decreased than that of vehicle-treated groups.3.The detection of apoptosis factors Caspase-3/Caspase-9 and AIF showed the CQ could protect the CA1 pyramidal neurons death after global ischemia in the gerbil.