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Clioquinol Attenuates Zinc-Induced Neuronal Death after Global Ischemia in the Gerbil

Author: ZhouJiaMin
Tutor: WangZhanYou
School: China Medical University
Course: Human Anatomy and Embryology
Keywords: Zinc Clioquinol (CQ) Cerebral ischemia Neuroprotective Metal autometallography TSQ fluorescence Nissl staining TUNEL staining In situ hybridization Western blot
CLC: R96
Type: Master's thesis
Year: 2009
Downloads: 71
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Abstract

Preface zinc metal is a transition metal element of the second bit of the content of the human body, also normal growth element necessary in the process of development, gene expression, protein metabolism, and immune function. Rodents, transient global cerebral ischemia can hippocampal CA1 pyramidal cells appear delayed neuronal death. There is evidence that zinc ions from presynaptic neurons \Neuron axon terminals release of excess zinc ions may be one of the important reasons for brain selective delayed neuronal death after transient global cerebral ischemia. Many studies have shown a protective effect on ischemia-induced neurological dysfunction in ischemic animal models in vivo injection of zinc ion chelator. For example, in the model of global cerebral ischemia given zinc chelator Ca-EDTA can significantly reduce neuronal death given Ca-EDTA can mitigate the damage of the CA1 neurons in the Mongolian gerbil cerebral ischemia model. The clioquinol (Clioquinol, CQ, 5 - chloro-7 - iodo-8 - hydroxyquinoline, MW: 305.5) is a membrane permeability and hydrophobic zinc ion chelating agent, and can pass through the blood-brain barrier. This study produced global cerebral ischemia model and to give CQ, explore the protective effect and mechanism of CQ hippocampal CA1 pyramidal cells. Materials and methods Experimental animals and groups healthy adult (2) cerebral ischemia solvent control group (1d, 3d, 7d); (2-month-old Mongolian gerbils were randomly divided into three groups: (1) the sham control group; ) cerebral ischemia CQ treatment group (1d, 3d, 7d). Dimethylsulfoxide (DMSO) as a solvent of the CQ. Chelated zinc ions in order to verify the CQ normal 2-month-old gerbils were randomly divided into two groups: (1) normal group; (2) injection the CQ2 hour group. ZnSe metal autoradiography (autometallography, of AMG) assay CQ zinc ion chelation. Second, the establishment of global cerebral ischemia model in gerbils with 4% sodium pentobarbital anesthesia (40mg/kg, ip), neck midline incision free vagus nerve fibers exposed to bilateral common carotid artery, noninvasive arterial folder blocking bilateral common carotid artery (sham-operated group clipping), resulting in global cerebral ischemia, release clamping 10min artery clip, see bilateral carotid artery blood filling, bilateral carotid artery resume blood supply. Neck incision stitched with nylon lines, alcohol disinfection. Postoperative gerbils heat lamp irradiation insulation 2h until recovery. Cerebral ischemia solvent control group in the immediate postoperative intraperitoneal injection of the solvent DMSO (10mg/kg/day), cerebral ischemia CQ treatment group in the immediate postoperative intraperitoneal injection of the CQ solution (10mg/kg/day), in accordance with 1d, 3d, 7d point in time is based. Third, the detection indicators application of zinc selenide metal from developing AMG and TSQ zinc ion staining detection CQ chelated zinc ion. Apply Nissl staining after cerebral ischemia, solvent control group and CO treatment group between the loss of hippocampal CA1 pyramidal cells. The TUNEL staining brain ischemia 3d CQ protective effect on CA1 pyramidal cells after cerebral ischemia. Using in situ hybridization to detect cerebral ischemia 3d groups Caspase-3 and Caspase-9 mRNA gene expression. Western Blot detection of cerebral ischemia 1d, 3d and 7d groups Caspase-3 and apoptosis-inducing factor (apoptosis inducing factor, AIF) protein content changes. Experimental results, ZnSe AMG staining results of in normal sand injection of zinc ion chelator CQ mouse in this slice throughout the hippocampal region see the the sand mouse in this slice of the of the AMG positive reaction with uninjected zinc chelator staining decreased compared weakened in the dentate gyrus and mossy fiber at the positive reaction with the uninjected zinc chelator CQ the gerbils slices phase. TSQ zinc ion staining results fluorescent under the microscope of gerbil hippocampal mossy fiber and dentate gyrus site to enhance cerebral ischemia 3d after TSQ staining compared with the sham control group, and the injection of the zinc chelator CQ gerbils TSQ staining compared to the control group compared with sham group and solvent weakest. Nissl staining results and CA1 region of hippocampal CA1 region neurons count hippocampal CA1 zone, the sham control group no significant neuronal damage change. The cerebral ischemia 1d, 3d, 7d solvent control group of gerbil hippocampal CA1 region visible significant pyramidal cell degeneration and necrosis. Compared with the solvent control group, cerebral ischemia 1d, 3d, 7d CQ treatment group hippocampal CA1 pyramidal cell death, reduction in the number of pyramidal cells than arranged, complete morphology. , TUNEL staining results of the sham-operated group gerbil hippocampal CA1 region only see a small amount of TUNEL positive cells; cerebral ischemia 3d CQ treatment group of gerbil hippocampal CA1 pyramidal cells dark brown number of TUNEL-positive cells was significantly higher than the solvent control group decreased. Significant difference (P lt; 0.01) between cerebral ischemia 3d solvent control group hippocampal CA1 region of TUNEL-positive cell counts and CO treatment group. Caspase-3 and Caspase-9 in situ hybridization staining results Caspase-3 and Caspase-9 in situ hybridization showed a positive reaction product of dark brown yellow light microscope. The sham control group of gerbil hippocampal CA1 region only see a small amount of Caspase-3 and Caspase-9 in situ hybridization positive cells. Cerebral ischemia 3d CQ treatment group was dark brown and yellow Caspase-3 and Caspase-9 in situ hybridization-positive cells significantly reduced the number of gerbil hippocampal CA1 pyramidal cells. Cerebral ischemia 3d solvent control group hippocampal CA1 region of Caspase-3 and Caspase-9 in situ hybridization-positive cell counts with CQ treatment group comparison between a significant difference (P lt; 0.01). Caspase-3 and AIF Western blot result of cerebral ischemia and postoperative 1d, 3d, 7d solvent control group Caspase-3 and AIF expression in the hippocampus than CQ treatment group animals. Caspase-3 (17KD) solvent control group and the CQ treatment group, 1d, 3d, 7d time point in each group (P lt; 0.01); solvent control group Procaspase-3 (32 kD) and compared to the CQ treatment group, 1d, 7d time point (P lt; 0.05), in the the the 3d time point (P lt; 0.01); AIF (67KD) solvent control group and the CQ treatment group compared 1d time point (P lt; 0.01), 3d, 7d time point (P lt; 0.05). Conclusion 1 application of AMG staining and TSQ fluorescence techniques confirmed CQ zinc ion chelation. 2, 1d, 3d and 7d CQ treatment group CA1 pyramidal cells after cerebral ischemia number increased compared with the solvent control group. TUNEL staining confirmed 3d CQ treatment group CA1 pyramidal cells after cerebral ischemia TUNEL-positive cells reduced the number compared with the solvent control group. 3, zinc chelators CQ has a protective effect by affecting apoptotic factors caspase-3 expression and activity of Caspase-9 and AIF, global cerebral ischemic nerve cell death

Clioquinol Attenuates Zinc-Induced Neuronal Death after Global Ischemia in the Gerbil

Abstract

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