Research On The Inhibitive Effects Of Right-Restoring Beverage For The Hippocampus Neurons Damaged By Dexamethasone In The Cerebral Ischemia

Objective:Stroke or cerebrovascular accident is ranked as in the third position to cause death and in the first place to cause disability, while in the case of China, it ranks the second to cause death. Over 80% of the stroke sufferers belong to ischemic stroke. There are a great number of inflammatory cells and mediators of inflammation. Although inflammation plays a great role in the tissue repair, researchers come to find out that inflammatory cells and mediators of inflammation can lead to secondary damages to the neurons. So it is a strategic way to cure ischemic stroke by eradicating or inhibiting inflammation. As a kind of strong and most effective anti-inflammatory drugs, glucocorticoids are thought to be controversial. On one hand, glucocorticoids can contract infarction volume, alleviate edema, improve blood supply and as a result protect the brain; On the other hand, endogenous or pharmacological glucocorticoids can lead to toxigenicity to the neurons, esp. apoptosis of postischemia hippocampus neurons. So most researchers would like to prove whether glucocorticoids can be used to protect the brain tissues on the prerequisite of not causing toxicity to the neurons.This experiment aims to probe whether glucocorticoids can be used to protect the brain tissues on the prerequisite of not causing toxicity to the neurons by the intervention of Right-Restoring Beverage or Right-Restoring Beverage which can warm and invigorate the kidney-Yang . This research will provide scientific proofs for the right clinical use of glucocorticoids accompanied by the use of Right-Restoring Beverage. Meanwhile, further concerns will be given to whether Right-Restoring Beverage has any effects on the ischemic focuses or spots while alleviating the toxicity to the neurons caused by glucocorticoids. This research is also expected to provide references for the use of warming and invigorating the Kidney-Yang and mutual supplementing of Yin-Yang in the treatment of postischemia hippocampus apoptosis, as will be valuable in both theoretical research and clinical use. Methods:This experiment contains 3 parts:1. MCAO or middle cerebral artery occlusion animal models were selected to conduct ethologic observations and score in sequence; Some brain tissues were taken out and dyed by the staining method of TTC, the effects of Dexamethasone on the infarction focuses as well as the intervention of Right-Restoring Beverage were investigated; Histological slices of hippocampus were dyed by HE before observation.2. Histological slices of hippocampus were marked by in situ TUNEL to observe apoptosis originated from hippocampus cells. We tested the expression of genes and proteins related to the apoptosis originated from hippocampus cells. This research was aimed to probe whether Right-Restoring Beverage has any effects on the ischemic focuses or spots while alleviating the toxicity to the neurons caused by glucocorticoids.3. Hippocampus neurons were cultivated in vitro. Morphological changes were observed with inverted microscope, purity coefficients of hippocampus neurons were tested by immnohistological methods, their survival rates by MTT; Built cerebral ischemia animal models under the condition of free sugar and oxygen and then supply of sugar and oxygen for 24 h each, tested the expression of genes and proteins related to the apoptosis originated from hippocampus cells by immnohistological methods; Observed how serum containing Right-Restoring Beverage acted on glucocorticoid as well as excitatory amino acids by Calcium ion imaging to probe the mechanism of Right-Restoring Beverage which might has any effects on the ischemic focuses or spots while alleviating the toxicity to the neurons caused by glucocorticoids.Results:1. Ethological score: There was no significant difference between treatment group 2 and treatment group3, but significant differences could be seen between treatment group 1 and group 2 or group 3. Meanwhile, ethological changes were alleviated with regard to group 2 and group 3.2. Measurement of infarction volume: There was significant difference between the model group and treatment group 1 .There was no significant difference between treatment group 2 and treatment group3, but significant differences could be seen between treatment group 1 and group 2 or group 3. Meanwhile, infarction volumes were declined with regard to group 2 and group 3.3. Histological changes of hippocampus and the density of hippocampus neuron: 24h after the damage duo to ischemia-reperfusion, pyramidal cells in CA1 area of hippocampus of the rats of the model group and group 1,2 and 3 shew swelling endochylema and karyopycnosis. Pyramidal cells in gyrus dentatus shew serious swelling, condensed nuclear chromatin, indistinct perinuclear membrane. Histological changes in group 2 and 3 were slighter than those in group 1. Survival rates of hippocampus neurons in group 2 and 3 were significantly higher than that of group 1.4. Apoptosis of hippocampus slices: few positive cell could be seen in both normal and sham operation group; Dispersal apoptosis cells could be seen in the model group and 3 treatment groups. The number of positive cells in model group and treatment group 1 were more than those in treatment group 2 and 3. there were statistical differences between them. The number of positive cells in treatment group 2 was less than those in treatment group 1. there was statistical difference between them.5. Immuno-histo-chemical tests:Test of Bcl-2: Bcl-2 positive cells in treatment group 1 were increased compared to those in normal group and sham operation group; Bcl-2 positive cells in treatment group 1 and 2 were significantly increased compared to those in model group and treatment group 1.Test of Bax: Bax positive cells in model group and sham operation group were increased; Bax positive cells in treatment group 2 and 3 were significantly decreased compared to those in model group and treatment group 1.Test of Caspase-3: Caspase-3 positive cells in treatment group 1 and model group were increased compared to those in normal group and sham operation group; Caspase-3 positive cells in treatment group 2 and 3 were significantly decreased compared to those in model group and treatment group 1.6. Cultivation and evaluation of in situ hippocampus neurons: 24-hour-old SD rats were chosen; 6 days after separation and cultivation of hippocampus neurons, hippocampus neurons accounted for 91% or more under the evaluation of NSE immunohistochemisty.7. Activity of hippocampus neurons by MTT: the survival rate of cells in the model group was significantly lower than that in the model group; There was no difference between treatment group 1 and 2. The survival rates of cells in group 1 and 2 were significantly lower than that in the model and normal groups; The survival rates of cells in group 3 were significantly higher than that in treatment group 2; The survival rates of cells in group 4 were significantly higher than that in model group and treatment group 2 and 3.8. Test of hippocampus neurons by immunohistochemisty:Test of Bcl-2: Bcl-2 positive cells in model group and treatment group 1 were increased compared to those in normal group; there was no significant difference between group 1 and 2; Bcl-2 positive cells in treatment group 3 and 4 were significantly increased compared to those in treatment group 2.Test of Bax: Bax positive cells in model group were significantly increased compared to those in normal group. There was no significant difference between group 1 and 2; Bax positive cells in treatment group 3 and 4 were significantly decreased compared to those in model group and treatment group 2.Test of Caspase-3: Caspase-3 positive cells in treatment group 1 and model group were increased compared to those in normal group; There was no significant difference between group 1 and 2;Caspase-3 positive cells in treatment group 2 and 3 were significantly decreased compared to those in model group and treatment group2.9. Test of free Ca²⁺ hippocampus neurons: the concentration of Ca²⁺ in model group was increased compared to that in normal group; There was no significant difference between group 1 and 2; the concentration of Ca²⁺ in treatment group 1 and 2 was increased compared to that in normal group and model group; the concentration of Ca²⁺ in group 3 was decreased compared to that in group 2; the concentrations of Ca²⁺ in group 4 were decreased compared to those in model group and group 2 and 3;Conclusion:1. Reperfusion after cerebral ischemia can lead to the increase of Bax and Caspase-3 in hippocampus neurons, change the permeability of mitochondrial membrane, induce cytochrome C to release and cause cell apoptosis.2. High level of glucocorticoid caused by stress accompanied by cerebral ischemia may play a great role in the damage of reperfusion after cerebral ischemia. Its mechanism might be that glucocorticoid can activate the calcium channels affiliated to the cell membrane, promote the inflow of calcium, cause calcium overload in hippocampus neurons; Meanwhile, it can also down-regulate the expression of Bcl-2, promote the expression of Bax, as a result, Bcl-2/Bax might go down, as inhibit Bcl-2 which can protect cells and accelerate apoptosis indirectly.3. At the acute stage of reperfusion after cerebral ischemia, glucocorticoid can hardly improve the symptoms related to cerebral ischemia, on the contrary, aggravate the damages to the neurons, so glucocorticoids should be cautiously used. At nearly the late stage of cerebral ischemia, small doses of glucocorticoids should be used to fight against inflammation and improve cerebral edema.4. Right-Restoring Beverage can sustain calcium homeostasis of hippocampus neurons, upregulate the expression of Bcl-2, downregulate the expression of Bax and Caspase-3, as a result, it can relieve the damages caused by the use of glucocorticoids or Dexamethasone in case of reperfusion after cerebral ischemia.