Preparation On The Rat Model Of Stroke. Xingnaojing Protective Effect And Mechanism Studies

Cerebral ischemic stroke is one of serious diseases that threaten human being, which with high incidence, especially in the senile. Due to its high morbidity and mortality, cerebral ischemic stroke imposes a heavy burden on individuals, families and society. In previous reports, the vast majority of patients with stroke suffer from unrecoverable neurological defects even when they are treated, such as limb dysfunction, mental retardation, memory loss, depression, and so on. These symptoms may have troubled the patients all the time. With no appropriate measures of treatment and intervention, they may continue and aggravate over time. Therefore, treatment in sub-acute phase is essential.Traditional Chinese medicines are effective for cerebral ischemia, which always act through various pathways. They are usually mild with lasting effect, with few side effects. They can treat both symptoms and causes of diseases, with regulation on generalized condition. In China, there are many famous classic formulas in ancient times, which can be prepared into Chinese patents by modern techniques. These patents are effective as ancient formulas in clinic, and can be applied for more symptoms because of their convenience for administration.Xingnaojing Injection is commonly used in clinic, which is composed of she xiang (Moschus), bing pian (Borneolum Synthcticum), yu jin (Radix Curcumae Wenyujin), and zhi zi(Fructus Gradeniae). It is commonly applied to nervous system diseases, such as carbon monoxide poisoning, coma hepaticum, and so on. It is effective significantly for acute cerebral ischemia. However, injection is usually applied for acute symptoms. Because of its dosage form, it can not be administered out of hospitals. Development of peroral preparation of Xingnaojing injection will ensure its wide application. Peroral preparation of Xingnaojing is made from extract of identical Chinese medicinals of Xingnaojing injection. Based on previous study, effect of peroral preparation of Xingnaojing has been proved.In this study, neuroprotective effect of Xingnaojing peroral preparation was evaluated in MCAT (Middle Cerebral Artery Thrombosis) model rats. We observed improvements in neuro-functions and the size of cerebral infarction to make sure both injection and peroral preparations have neuroprotective effect in acute phase of cerebral ischemia. In addition, we further explored action of peroral preparation in convalescence in pMCAO (permanent middle artery occlusion) model rats, and evaluated its effect as improvement on neurological defects and relief on oxidative stress. In addition, in this study, SHRSP was used to evaluate antioxidant action of Xingnaojing peroral preparation.Studies have shown that the protection of Xingnaojing injection for ischemic injury is related to its anti-inflammatory, sedative, anti-oxidatant and other effects. However, whether its neuroprotective action is related to synaptic function has not been explored yet. We evaluated protective effect of Xingnaojing injection on neurons and synapse in acute and sub-acute phases in rat model of pMCAO. Post-ischemia synaptic structure and function was observed, and action of Xingnaojing Injection on synaptic structure and function was evaluated.This study is composed of the two following parts:1 Effect of Xingnaojing peroral preparation on rat model of focal cerebral ischemia and stroke-prone spontaneously hypertension rat1.1 Neuroprotection of Xingnaojing peroral preparationin on rat model of focal cerebral ischemia1.1.1 Protection from two preparations of on Xingnaojing on rat model of FeCl3-induced focal cerebral ischemiaObjective:it is to evaluate effects of preparations of Xingnaojing in acute phase of cerebral ischemia, and further demonstrate neuroprotection of Xingnaojing peroral preparation.Methods:SD rats were randomly divided into model group, Xingnaojing injection 3 mL/kg group and 9 mL/kg group (equal to 9- and 27-time clinical dosage of adult),40 mg/kg group and 120 mg/kg group of peroral preparation, and the sham group. Rats were administered with peroral preparation two days earlier before the establishment of model, or intraperitoneally administered with Xingnaojing injection, once per day. One hour later administration on the third day, MCAT model in rats was established with FeCl3 Rats were administered with medicines 6 hours later after establishment of model. On 6 h and 24 h after establishment of model, scores on neurological defects were measured. And 24 hours after establishment of model, coronal sections of brain tissue was taken and stained with TTC. Weight of white infarction and whole brain tissues was measured, taking percentage weight of infarction to the total weight of brain as degree of cerebral infarct.Results:after MCAT, rats got some neurological defects, such as curled body, poor balance, rotation and falling to the left side, inability to walk, and so on. Compared with model group, 9 mL/kg group of Xingnaojing Injection and 120 mg/kg group of Xingnaojing peroral preparation got lower score on neurological defects at 6 h post-MCAT (P<0.05); 24 h post-MCAT, Xingnaojing Injection 3 mL/kg and 9 mL/kg groups got significantly improved on neurological defects (P<0.05,P<0.01),120 mg/kg group of peroral preparation of Xingnaojing also got milder defects (P<0.01) than control group. In the sham group, there was no infarction on brain tissue, while white infracted area in the model group. In the medicated group, infarction size was more reduced than the model group. In the 3 mL/kg and 9 mL/kg groups of Xingnaojing Injection,120 mg/kg group of peroral preparation of Xingnaojing, infarction size was significantly smaller (P<0.05,P<0.01).Conclusion:both Xingnaojing Injection and peroral preparation of Xingnaojing had significant neuroprotective effects within 24 hour after cerebral ischemia.1.1.2 Protective effects of peroral preparation of Xingnaojing on neurological defects and oxidative damage in pMCAO model rats in early convalescenceObjective:it is to evaluate peroral preparation of Xingnaojing in pMCAO model rats in early convalescenceMethod:pMCAO model was established with suture. Rats were randomly divided into model group, sham group, peroral preparation of Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group, taking Butylphthalide 70 mg/kg group as positive control group. All animals were administered medicines 3-14 d post-ischemia, rats in the sham group were administered with same volume of oleum olivae. Neurological deficit scores, tactile stimulation experiments, beam walking test, strength test were operated to observe protective effects of peroral preparation of Xingnaojing on neural function in pMCAO model rats on day 3,6,9, 12, and 14; and measure SOD activity and MDA content in ischemic brain tissue.Results:compared with the sham group, rats in the model group got obvious symptoms of neurological defects. After treatment, Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group got significantly improvement on their neurological functions, manifesting improvement on scores of neurological defect (P<0.01), beam walking ability (P<0.05, P<0.01), and recovery of forelimbs strength (P<0.05,P<0.01), and enhancement of tactile sensitivity (P<0.01). Peroral preparation of Xingnaojing was able to significantly reduce the MDA content of ischemic brain tissue.Conclusion:peroral preparation of Xingnaojing had protective action on pMCAO rat, by reducing oxidative damage.1.2 Study on protection of Xingnaojing peroral preparation on SHRSP and related mechanism1.2.1 Action of of Xingnaojing peroral preparation on general status in SHRSPObjective:it is to explore impacts of peroral preparation of Xingnaojing on body weight, blood pressure, heart rate, and general condition in SHRSP.Method:all SHRSP were given salt-loaded diet, and were randomly divided into SHRSP group, the AA group (vitamin C,1000 mg/d), Xingnaojing 25 mg/kg group and 50 mg/kg group. All rats were intragastrically administered with medicines for 6 weeks. Food intake, water intake, urine output, and defecation output within 24-hour were measured on the 2nd,4th and 6th weeks respectively.Results:there were no significant differences on body weight and general condition between groups pre-medication (0 week). Since second week, given the salt-loaded food, water intake, urine output, the amount of defecation of SHRSP increased significantly. Compared with the SHRSP group, food intake, water intake, and urine output in AA group was significantly reduced (P<0.01), with great body weight loss (P<0.01). In the 6th week of medication, there were no significant differences on food intake between groups, but the body weight in the AA group was still significantly less than others'(P<0.01). There was no significant difference on body weight, food intake Xingnaojing-treated groups (P>0.05). Given salt-loaded food, SBP of rats significantly increased since 2nd week of treatment; in the 4th week, SBP in the AA group, and Xingnaojing 50 mg/kg group slightly reduced, but with no significant differences compared with SHRSP group. This tendency did not vanish unitl the 6th week, in which, SBP in AA group was lower than in the SHRSP group, while was slightly higher in the Xingnaoj ing-treated groups. However, there was no significant difference between groups. Since 2nd week of medication, HR decreased in every group slightly, while increased until 6th week, with no significant difference between groups.Conclusion:peroral preparation of Xingnaojing had no significant impact on body weight, general condition, blood pressure, and heart rate.2.2 Antioxidant action of Xingnaojing peroral preparation of in SHRSPObjective:to investigate antioxidant action and related mechanism of Xingnaojing peroral preparation in SHRSP.Method:all SHRSP given the high salt load diet were randomly divided into SHRSP group, the AA group (1000 mg/d), Xingnaojing 25 mg/kg, and 50 mg/kg group. Rats were administered with medicine for 6 weeks. Plasma TAS was measured respectively in 2nd,4th and 6th week, and MDA content in the brain tissue and plasma was measured on 6th week, and expression of SOD and TNF-αmRNA was measured by RT-PCR.Results:in 2nd week, Xingnaojing peroral preparation was able to increase level of plasmaTAS. It had no significant action in 4th week. However, it was able to increase level of plasma TAS. It was able to reduce content of MDA in the plasma. There was only tendency of increased level of SOD and decreased TNF-αmRNA expression in medicated group, but no significant difference.Conclusion:Xingnaojing peroral preparation of had protective action on SHRSP, which might relate to reduction on oxidative damage.2 Study on protection of Xingnaojing injection on synapse and mechanism2.1 Protection of Xingnaojing injection on neurons in pMCAO model rat Objective:to investigate neuronal protection of XNJ injection in pMCAO model rats in acute and sub-acute phase.Methods:pMCAO model was established with suture. Rats were randomly divided into sham group, model group, XNJ Injection 1mL/kg,2.5 mL/kg, the 5 mL/kg groups, and taking Ginkgo injection 2.5 mL/kg group as control. Postoperative 6 h-7 d rats were administered respectively, once per day. H&E staining and Nissl staining was applied to observe morphology changes on neurons on 2 d and 7 d after ischemia. Microstructure of neurons was observed by transmission electron microscope.Results:on 2nd d post-ischemia, in visual fields of optical microscope, cortical neurons in the sham group were round with complete form. It was rich in cytoplasm in neurons with plenty of Nissl bodies evenly distributed. In the model group, there were a great number of degenerated and necrotic neurons in the cortex, manifesting as shrunken and darkly stained nucleus, and great loss of Nissl bodies. In the treatment groups, there were more complete neurons in the cortex, with clearly arranged neurons in the hippocampus areas. There were less dead neurons, less damage to neurons, and less loss of Nissl bodies. In the field of transmission electron microscope, nuclear morphology in the sham group was clear with complete nuclear membrane, unabroken karyosome, and evenly distributed chromatin. There were plenty of organelles in the cytoplasm, of which itochondria was oval or round. However, in the model group, there were pycnotic neurons, around which, there were vacuolars. There was less organelle in the cytoplasm. In the treatment groups, morphology of neurons was normal with clear nuclear membrane and plenty of organelles.Conclusion:in acute phase of cerebral ischemia, Xingnaojing Injection has protective effect on neurons by preventing neuronal death, loss of Nissl bodies, and ultrastructure damage of neurons induced by ischemia.2.2 Effect of Xingnaojing injection on synaptic structure in pMCAO model ratsObjective:to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 1.2 mL/kg group,2.5 mL/kg group, and 5mL/kg group, taking Gingko injection 2.5mL/kg group as the control group, and the sham group. Rats were administered with medicines 6 h-7 d post-operation. On 2 d and 7 d after ischemia, synapse-related proteins, synapsin-Ⅰ, PSD-95, andα-synuclein were detected with immunohistochemical staining.Results:compared with the sham group, on 2nd day post-ischemia, in the model group, expression of synapsin-Ⅰ, PSD-95 in hippocampal in CA1 and CA3 area, and cerebral cortex were significantly decreased (P<0.01);α-synuclein in hippocampal CA1 and CA3 area, and cortical expression were significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area in Xingnaojing 1.2 mL/kg group, in CA3 and cortex of 2.5 mL/kg group, in CA1 area, CA3 area, and the cortex of 5 mL/kg group were significantly increased (P<0.05,P<0.01); PSD-95 expression was significantly increased in cortex and CA1 area in Xingnaojing 1.2 mL/kg group, CA1 area in 2.5 mL/kg group, and the cortex in 5 mL/kg group (P<0.05,P<0.01); expression of a-synuclein was decreased in the CA3 area in 1.2 mL/kg group, cortex in 2.5 mL/kg group, and CA1 and CA3 area in 5 mL/kg group (P<0.05.P<0.01).Compared with the sham group, on 7th d post-ischemia, expression of synapsin-Ⅰin the model group in the CA1 area and cerebral cortex was still significantly lower (P<0.05, P<0.01), expression PSD-95 in CA1, CA3 areas and cortex remained significantly lower (P<0.01) than the sham group; expression ofα-synuclein in CA1 area, CA3 area, and cerebral cortex was still significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were significantly increased (P<0.05,P<0.01); expression of PSD-95 on CA3 and cerebral cortex in Xingnaojing 1.2 mL/kg group was increased (P<0.05, P<0.01), in CA1 area in 2.5 mL/kg group was increased (P<0.05), in CA1, CA3 area, and cerebral cortex in 5 mL/kg group was increased significantly (P<0.05,P<0.01); expression ofα-synuclein in CA1, CA3, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were decreased (P<0.05,P<0.01).Conclusion:after pMCAO, expression of synaptic function related proteins, synapsin-Ⅰ, PSD-95,α-synuclein were significantly changed, indicating damage of synaptic function. On 2nd d after ischemia, Xingnaojing Injection was able to increase expression PSD-95 and synapsin-Ⅰ, and to reduce the accumulation ofα-synuclein. Part of this action lasted until 7th d post ischemia, indicating that Xingnaojing injection had a certain effect on neuronal synaptic function.2.3 Effect of Xingnaojing injection on synaptic function in pMCAO model ratsObjective:it is to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 2.5 mL/kg group and 5mL/kg group, taking Gingko injection 2.5 mL/kg group as the control group, taking normal SD rats as the sham group. Rats were administered with medicines 6 h-7 d post-operation. At 2 d and 7 d after ischemia, expression PSD-95, p-CaMKⅡ, NR2B proteins were detected with western blotting.Results:on 2nd d post-ischemia, in model group, PSD-95 protein expression in the cortex and hippocampus was significantly lower (P<0.01); compared with model group, in treatment group, protein expression of PSD-95 was significantly increased (P<0.05,P<0.01). On 7th day post-ischemia, compared with the sham group, protein expression of PSD-95 was still significantly lower (P<0.05,P<0.01); PSD-95 protein expression in cortex in treatment groups increased (P<0.05,P<0.01), PSD-95 protein expression also increased in Gingko group, Xingnaojing Injection 5 mL/kg group in hippocampal area, but there was no significant difference.On 2nd d post ischemia, compared with the sham group, in model group, protein expression of p-CaMKII in the cortex and hippocampus areas significantly decreased (P<0.05); compared with model group, protein expression of p-CaMKⅡwas significantly increased in Gingko group (P<0.05). On 7th day after ischemia, in model group, there was no significant difference on protein expression p-CaMKII compared with the sham group. There was no significant difference on protein expression of p-CaMKII between the mdoel group and treatment groups.At 2nd d post ischemia, in model group, protein expression of NR2B was significantly lower (P<0.01) than in the control group; compared with model group, NR2B protein expression in Xingnaojing treatment groups significantly increased (P<0.05,P<0.01). On 7th d post ischemia, there was no significant difference on NR2B protein expression between the sham group and model group. Compared with model group, the expression level of Xingnaojing treatment group has increased (P<0.05,P<0.01).Conculsion:Xingnaojing injection was able to affect protein expression in both cortex and hippocampus areas in rat model of pMCAO in the acute and sub-acute phase. It was able to prevent protein loss related to ischemia-induced synaptic dysfunction; this might be one of the mechanisms for its neuroprotective effect.