| Background and objectiveChronic cerebral ischemia is a common chronic pathophysiological process, which is caused by a variety of reasons resulting in cerebral artery stenosis or low perfusion, leading to insufficient cerebral blood flow that cannot satisfy the physiological needs. It can cause the diffuse or localized dysfunction of the brain, such as pathologic partial or complete loss of the ability to recall past experiences or to form memories, intellectual deterioration, dizziness, headache and insomnia, and so on. What's more, it is associated with vascular dementia(VD) and Alzheimer's disease(AD), et al. Up to now, most countries around the world moved in the ageing society. With the coming acceleration of global population ageing, chronic cerebral ischemia-related diseases increase, therefore, it is more important to reveal the general and specific mechanisms of chronic cerebral ischemic injury.Recent studies have found that sodium-calcium exchanger 3(NCX3) play an important role in nervous system diseases. Apoptosis-inducing factor(AIF) is a phylogenetically old and caspase-independent effector of cell death. Dl-butylphthalide(NBP) have a better therapeutic effect on ischemic cerebrovascular disease.At present, it was not reported that whether NBP could exert neuroprotective effects by affecting NCX3 and AIF after chronic cerebral ischemia in rats. The purpose of this study is to explore the general and specific mechanisms of NBP exerting neuroprotective effects on chronic cerebral tissues in aged rats by observing the effects of NBP on the expressions of NCX3 and AIF in the cortex and hippocampus after chronic cerebral ischemia in rats.Materials and methods80 male Wistar rats weighing 350-450g were used to carry out the experiments. The rats were adapted to housing conditions for 7 days before experiments, then they underwent the Morris water maze test to evaluate the ability of learning and memory. All of the rats were randomly divided into 4 groups, they were group A, B, C and D, that were, control group(sham-operated+solvent), model group(surgery+solvent) and low-dose NBP-treated group(surgery+low-dose NBP+solvent) as well as high-dose NBP-treated group(surgery+high-dose NBP+solvent). Chronic cerebral ischemia was induced by permanent bilateral common carotid artery ligation(BCAL) for 2 months. Rats in control group were subjected to the same surgical procedure, except that the bilateral common carotid arteries were only isolated and were not ligated. During the surgery, their body temperature was maintained at (37.5±0.5)℃until the rats recovered to thermal homeostasis.2 months later, rats in group A and B were both given solvent by gastric irrigation, and rats in group C and D were given NBP at the dose of 60mg/kg/d and 120mg/kg/d by gastric irrigation respectively for 1 month.1 moth later, all rats alive underwent the Morris water maze test, then they were anesthetized, abdominal aorta was clipped, sterile saline and paraformaldehyde were perfused through heart respectively. The brain was carefully removed, fixed, dehydrated and then embedded in paraffin blocks. Brains were sectioned at 5μm coronally for HE and immunohistochemistry staining. Slices were observed under microscopes, pictures were taken by imaging analysis equipment and detected by imaging analysis system which recorded morphological changes and the mean value of IOD of each slice with immunohistochemistry technique. Data were presented as mean±SEM, and analyzed by SPSS 13.0 for windows software. A one-way analysis of variance(ANOVA) was performed for differences among the 4 groups, LSD-t test was conducted between every two groups. Values of P<0.05 were considered to be significant.Results1 General behavior of rats:Rats were in poor spirit, unresponsive and anorexic after the surgeries. Followed by group A, D, C and B, rats gradually recovered, eating back to normal.2 The results of learning-memory ability:Before BCAL the differences between group A and group B were not significant (p>0.05). After BCAL two months the differences were significant (p<0.05). The learning-memory ability of group C and D were improved comparing with group B, moreover, group D was more obvious than group C. All the difference were significant (p<0.05). 3 HE staining:There was not obvious ischemic infarction in each slice. The morphology of the hippocampus and temporal cortex of most slices from group A was normal, except few degenerative and dead neurons. Neurons from group B showed ischemic changes that most neurons disappeared, nuclei became irregular pyknotic, fragmental or karyolitic, bubble appeared in cytoplasm and the number of glial cells increased. Both the number of neurons and glial cells in the hippocampus and temporal cortex from group C decreased, although some of them was degenerated or dead. Compared with group B, the morphology of group D significantly improved, compared to group C, the morphology of group D improved partly.4 Effect of NBP on the expression of NCX3 after BCAL:The immunoreactive product for NCX3 was brown or brown-yellow, which mainly located on the membrance and in the cytoplasm. The expression of NCX3 in the hippocampus and cortex was significantly decreased in group B, compared with that in group A, but increased in group C and D, compared with that in group B. The expression level of NCX3 in group D was higher than that in group C.The mean value of IOD of NCX3 in group A, B, C and D were as follows:hippocampus:132.84±3.04,118.64±5.90, 123.48±3.64,127.06±3.96; cortex:148.37±4.84,114.44±8.14,127.51±11.51, 147.61±5.14. In the hippocampus group, the value of F was 28.77, P<0.01. In the cortex group, the value of F was 61.20, P<0.015 Effect of NBP on the expression of AIF after BCAL:The immunoreactive product for AIF was brown or brown-yellow, which mainly located in cytoplasm and nuclei of neurons. The expression of AIF in the hippocampus and temporal cortex was significantly increased in group B, compared with that in group A, but decreased in group C and D compared with that in group B. The expression level of AIF in group D was lower than that in group C. The mean value of IOD of AIF in group A, B, C and D were as follows:hippocampus:108.91±8.91,154.91±8.49,131.59±5.09, 115.23±6.19; cortex:93.76±6.56,145.71±4.52,131.46±8.77,123.71±11.07. In the hippocampus group, the value of F was 101.85, P<0.01. In the cortex group, the value of F was 103.93, P<0.01.Conclusion NBP can reduce loss of nervous tissue and promoted functional recovery from chronic cerebral ischemic injury in rats. NBP may exert neuroprotective effects by increasing the expression of NCX3 and decreasing the expression of AIF in the hippocampus and temporal cortex after chronic cerebral ischemia in rats, which inhibits cell injury and death.
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