Effect And Mechanism Of Homer1a On Ischemia-Reperfusion Brain Injury In Mice

Ischemic brain injury is a common clinical disease, ranked the second cause of death in the world. According to the World Health Organization report, each year about 5.7 million people die or disable as a result of ischemic brain injury, placing a huge economic burden to society and family. The etiology of the disease is mainly induced by brain blood supply occlusion, and without reperfusion in a short time. In clinic, the early thrombolytic reperfusion is the basic treatment strategies. However, studies suggest that restoring blood supply over 4.5 h after brain ischemia, can stimulate cells to produce cascading effects leading to ischemia-reperfusion injury. It is regrettable that most clinical patients have missed the window of time. So ischemia-reperfusion injury is the most common ischemic brain injury. The pathogenesis of cerebral ischemia-reperfusion injury is mainly contributed to excitatory amino acid toxicity, free radical damage, calcium overload, inflammatory response, apoptosis and other pathological mechanism. Further study on the mechanism of ischemic brain injury become an urgent problem for treatment.In recent years, studies found that Homer protein is a scaffold protein, which belonged to postsynaptic density in central nervous system. Homer protein plays an important role in many diseases of central nervous system, involved in synaptic structure and mediated neural signal transduction. Homer 1 is a member of the Homer protein family which was widely studied. Both an immediate-early gene encoding a Homer 1 variant and a constitutively expressed Homer 1 variant, Homer 1 consists of Homer 1a and Homer 1b/c proteins. Homer 1a protein belongs to the immediate-early gene expression. There is almost no expression under physiological condition, but high expression after the neurons were activated. However, Homer 1b/c protein belongs to a constitutively expressed protein that has regular expressions in vivo. Two kinds of protein has negative regulatory role in the function each other. It was reported that Homer 1 proteins can regulate receptor clustering and trafficking, intracellular calcium homeostasis, and intracellular molecule complex formation. Preclinical laboratory studies have suggested that Homer 1 proteins are associated with many diseases in central nervous system. In previous experimental studies, it was found that Homer 1a protein is early expressed, and Homer 1b/c protein is constantly expressed in traumatic brain injury or neuronal oxygen-glucose deprivation model. And further study showed that the high expression of Homer1 a proteins can be through the negative regulation and dismantling Homer1b/c proteins involved in various protein complex formations. Homer 1a protein may play a neuro-protective role by alleviating toxicity of excitatory amino acids, intracellular calcium overloading damage and so on. Therefore, we presumed that Homer 1a may also exist the similar mechanisms in focal cerebral ischemia-reperfusion injury. Nevertheless, this hypothesis was not identified in animal vivo. Thus, we investigated the protective effects of Homer1 a in ischemia-reperfusion injury by applying Homer 1a gene knockout mice. Our aim was to determine the role of Homer 1 proteins in transient focal cerebral ischemia, as well as the mechanism of the Homer 1a effect. Partâ… : Homer 1a gene knockout mice breeding and gene identificationObjective: Homer 1a gene knockout mice breeding and gene identification. Methods: Homer 1a gene knockout mice were generous gifts from Professor Paul F. Worley in Johns Hopkins University. These mice were matched breeding, and extracted DNA from the mice tails. Agarose gel electrophoresis using PCR products from the extracted DNA. Results: 142 mice were bred, 42 of which were wild-type(WT) mice, 79 were heterozygous(HET) mice and 21 were Homer 1a gene knockout(KO) mice. Conclusion: The results showed that we successfully reproduced and identified different gene-type mice. It provided a reliable source of experimental animals for the following tests. Partâ…¡: Protective effects and activation of astrocytes of Homer 1a in focal cerebralischemia-reperfusion injury in vivo.Objective: To identify whether Homer 1a has a protective effect in ischemia-reperfusion brain injury,on the other side,to investigate the influence of astrocyte activation by comparing the Homer 1a gene knockout mice with the wild-type mice. Methods: Male Homer 1a knockout mice and wild-type mice were obtained from Partâ… . Male Homer 1a gene KO mice and wild-type mice were randomly divided into four groups: Gene knockout mice sham operated group(SKO); wild-type mice sham operated group(SWT); Gene knockout mice operated with cerebral ischemia-reperfusion injury group(MKO); wild-type mice with cerebral ischemia-reperfusion injury group(MWT). Mice were subjected to transient middle cerebral artery occlusion(MCAO) for 2 h, followed by 24 h reperfusion. Sham groups were subjected to the same surgical procedure without MCAO. We evaluated the mice neurological function by the modified neurological severity scores(m NSS); the percentage of mice cerebral infarct volume were detected with 2,3,5 triphenyltetrazolium chloride(TTC); we observed the brain cell morphology through HE staining; the neuronal cell apoptosis were observed by terminal deoxynucleotidyl transferase(Td T)-mediated deoxyuridine triphosphate(d UTP) nick end labeling(TUNEL); activation of astrocytes(Ast) was measured by immunofluorescence with the specific markers glial fibrillary acidic protein(GFAP). Results: Homer 1a gene knockout raised the m NSS score of mice with cerebral ischemia-reperfusion injury. The results indicated that Homer 1a gene knockout aggravated neurological defect of mice with cerebral ischemia-reperfusion injury. HE staining of brain tissue sections showed partial cell death, neuronal loss, cell shrinkage, nuclear condensation, and fragmentation. TTC staining showed that Homer 1a gene knockout increased the percentage of infarct volume. We could observe more cell apoptosis in KO mice group comparing to WT mice group. Moreover, the number of TUNEL-positive cells was more in MKO group than MWT group. Cerebral ischemia-reperfusion injury led to activation of astrocyte cells. GFAP fluorescence staining showed that the expression of GFAP was higher in MWT group than MKO group. But GFAP positive staining could hardly be observed in sham group. Conclusion:Homer 1a gene knockout exacerbated brain ischemia-reperfusion injury in vivo. It was implied that Homer1 a could play a protective role in cerebral ischemia-reperfusion injury. In the meanwhile, Homer 1a gene knockout reduced the activation of astrocytes. It suggested that astrocytes might be involved and play as a complex role.