| Objective:Strong clinical and experimental evidences suggest that hyperhomocysteinemia(HHCY) is an independent risk factor for stroke, vascular dementia, Alzheimer’s disease, Parkinson’s and other neuron-degenerative diseases. Cell senescence play an critical role in the pathogenesis of these conditions.But it is still note clarified how HHCY do a detrimental and harmful effect on cerebrovascular and neuron. Based on these conflicts in the fields of HHCY inducing neuron degenerative disease. We established HHCY mice model, observed the distribution and ultrastructural changes in astrocytes,neurons and brain micro-vessels using various morphological methods. Then, we detected the expression changes of the relative detrimental molecules. By these experiments, the detrimental and harmful effect on cerebrovascular and neuron of HHCY were explored. It may provide a sight on the mechanisms of the HHCY related neuron-degenerative diseases. This study were consisted of two parts. Part one is the study of HHCY inducing cerebrovascular damage in the Apo E- /- mice brain. The other part is the study of HHCY inhibiting brain astrocytes GFAP expression and neuron impairement in Apo E-/- mice.Mehtods:12 male wild-type mice and 36 male apolipoprotein E-deficient(Apo E-/-) mice on a C57BL/6J genetic background were kept in our laboratory for 16 weeks. The animals were purchased from Jackson Laboratory(Bar Harbor, ME) and bred to 5 weeks of age in the Animal Center of Beijing University(Beijing, China). After 1 week of acclimatization, Apo E-/- mice were randomly divided into three groups(n= 12 each).Mice of the Apo E-/-control group were fed with regular mouse diet for 18 weeks. The high-methionine group(HM)received the same diet but with a 1.7% methionine supplement. The high-methionine plus folate and vitamin B12 group(HM+FA+VB) received regular mouse diet plus 1.7% methionine, 0.006% folate, and 0.0004% vitamin B12. Wild-type C57BL/6J mice(n=12) were fed with a regular mouse diet as blank control group.After 18-week experimental diets, all the animals were euthanized.Blood was immediately collected to measure serum HCY levels and The brain were carefully removed. Half of The brain were stored at-80℃ until analysis and the other half of brain was prefixed in 10% neutral buffered formalin.Section the paraffin-embedded tissue block were analyzed by HE stain and ICH for GFAP,CD31 and iba1 expression. Mealwhile, the number of cortexvessels werecounted by HE staining. we observe the ultra-structural changes of brain microvascular, astrocytes and cortical neurons by electron microscope.We explored the protein expression of GFAP in brain and SIRT1 in cerebrovascular using western blotting.Results:Our study showed the following:(1) A high methionine diet for 18 weeks is sufficient to induce hyperhomocystinemia.(2) There is no significant difference in heart rate, blood pressure(pre-experiment, mid-experiment and post-experiment) and the number of microvascular after traced by ICH among various group(p>0.05).(3). HHCY can do a detrimental and harmful effect on cerebrovascular ultrastructure significantly by endothelial, basement membrane imparement and the organelles of endothelial vacuolar degeneration. Also, folic acid and vitamin intervention play a preventive role during this pathology.(4)When compared with the blank control and Apo E-/-group, the expression of SIRT1 in cerebrovascular is depressed(p<0.05).After folic acid and Vitamin B intervention, this harmful trend is reversed(p<0.05).(5). Normal brain tissue, usual distribution of neurons and evenly glial cells were found in various group without detectable differences of neurons and astrocyte by light microscopy HE staining. Pyknotic neurons were occasionally detected in methionine-fed group. There is no significant morphology change in astrocytes.(6). By electron microscope, Nuclear membrane shrinkage, mild chromatin condensation and constitutive organelles were observed in the cortical neurons of Apo E-/-control group with a slightly lipofuscin deposits. As expected, methionine diet accelerated detrimental effects of Apo E gene knock out by causing additional vacuolar degeneration of mitochondria and increased lipofuscin deposits.(7)HHCY decrease the expression of GFAP in the cortex and hippocampus by western blot and ICH(p<0.05). Folic acid and Vitamin B intervention increases the expression(p<0.05).(8) HHCY increases iba1 expression when compared with the blank control and Apo E-/-group. folic acid and vitamin B intervention can reversed this deleterious trend(p<0.05).Conclusion:The downregulation SIRT1 in cerebrovascular is a important mechanism that HCY involved in the cerebrovascular impairment. Futhermore, HCY inhibits the expression of GFAP, accompanied with the neuron aging and the activation of microglia. Importanty, folic acid play an preventive role in the pathology of HCY related brain impairment. |
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