动物造模,大脑中动脉栓塞模型急性脑梗死 z-bio 2022-01-10 582

　　Introduction: Stroke is one of the most common diseases affecting human health. It is the second leading cause of death and the first leading cause of disability in China. In 1995, it was first reported that recombinant tissue plasminogen activator (rt-PA) was effective in patients with acute cerebral infarction (ACI). A recent study showed that rt-PA is still the first-line drug for revascularization in the treatment of ACI. Due to the limitation of the 4.5-hour thrombolytic time window, most patients do not have the opportunity to receive this treatment. Therefore, effective measures need to be taken in the treatment of acute ischemic stroke. In recent years, it has been reported that thrombolytic therapy for ACI patients can be performed 6 hours after ACI. Recent studies have shown that thrombolytic therapy 3-9 hours after ACI can significantly improve the results of magnetic resonance imaging or computed tomography and clinical results. However, the mechanism of thrombolytic therapy in the broadened treatment window is not obvious. Neovascularization is a key factor for the survival of ischemic nerve after ACI, and it is very important for the recovery of learning and memory and the improvement of prognosis. Ischemia and reperfusion can lead to a significant increase in free radicals and lipid peroxidation, thereby aggravating brain damage. Nitric oxide (NO) is a reactive gaseous free radical. Excessive release can cause neuronal damage. Malondialdehyde (MDA), a degradation product of lipid peroxidation, can also cause direct damage to the brain. Superoxide dismutase (SOD) is an important antioxidant enzyme in brain tissue, which plays an important role in brain protection by removing excessive free radicals in cells. Therefore, these molecules may be the key regulators of the influence of rt-PA on ACI. In this study, we used an improved rat middle cerebral artery embolism (MCAO) model to study the effects of rt-pa thrombolytic therapy on the infarct volume, microvessel density (MVD), and caspase- 3. The effects of nitric oxide, nitric oxide synthase (NOS), malondialdehyde (MDA) and superoxide dismutase (SOD), aiming to reveal middle cerebral artery embolism (MCAO) in rats 6 hours after myocardial infarction Mechanisms. Research on the mechanism of thrombolytic therapy within a widened treatment window provides a reliable theoretical basis for the clinical treatment of ACI.

　　Materials and methods: Animals: Adult male rats (320-350g), 162, 2 months old. All rats were kept in a room with a light cycle of 8:16, temperature (22±2°C) and humidity (58%-68%). They were randomly divided into three groups (54 rats in each group): sham operation group, conventional group (ie, conventional treatment infarction group), and thrombolysis group (ie, RT-PA treatment of infarction group 6 hours after ACI). According to the number of days after the model was established (the first day, the third day, or the seventh day), each group was further randomly divided into three groups (18 rats in each group).

　　Middle cerebral artery occlusion model: A rat MCAO model embolized with autologous blood clot was established: (a) Preparation of thrombus: 0.6 ml of venous blood extracted from the tail vein and 0.15 ml of thrombin (200 U/ml) were mixed, Quickly put it into the PE50 tube and let it stand for 4 hours. The thrombus was cut into small emboli (1 mm) and placed in a PE50 tube for use. (B) Establishment of embolization MCAO model: rats were anesthetized by intraperitoneal injection of 6% chloral hydrate (35 mg/100 g). Make a 2 cm long midline incision in the neck, and then dissect the right common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA), occipital artery (OA), superior thyroid artery (STA) and wing The palatine artery (PPA). Ligate the distal ends of OA, STA, PPA, and ECA, and temporarily cut off CCA and ICA. Insert the PE-50 tube containing the blood clot into the proximal end of the ECA, and then open the capillary clip for clot infusion (10-12 embolisms). Remove the CCA clip and suture the incision. (C) Intervention: The routine group was intraperitoneally injected with cytidine (500 mg/kg, once a day) 24 hours after MCAO. On the basis of the above-mentioned drugs, the thrombolytic group received intravenous rt-PA (10 mg/kg) 6 hours after MCAO; the sham operation group also underwent the same procedure, except that no clot was infused and intravenous thrombolytic agent was injected. As mentioned above, after the model is established, a 0-4 scoring scale is performed to obtain a neurological deficit scale. 0- No obvious neurological deficit; 1- Mild neurological dysfunction (that is, the contralateral forelimb cannot be fully extended when the rat is lifted by the tail); 2- Moderate focal neurological dysfunction (that is, to the right when walking Rotation); 3-severe focal neurological dysfunction (tilting to the right when walking). 4- Inability to walk or coma. Rats with a score of 1-3 are considered infarcts.

　　2,3,5-triphenyltetrazolium chloride staining: 6 rats were randomly selected from the above three groups on the first, third and seventh days after the model was established. The rat was deeply anesthetized to death. The brain was quickly excised, the coronal section was 2 mm, and stained with 2% 2,3,5-triphenyltetrazolium chloride dissolved in 0.9% saline at 37°C in the dark for 30 minutes. The sections were fixed in 10% neutral formalin overnight, and then photographs were taken with a digital camera. The infarct size was standardized as edema. The infarct volume of each brain is calculated as I%=(ipsilateral contralateral-normal volume)/contralateral brain volume.

　　Immunohistochemistry: 6 rats were randomly selected from the above three groups on the first, third and seventh days after the model was established. The animals were sacrificed and perfused with 0.9% saline and then with 4% paraformaldehyde. The brain was quickly removed, fixed with 4% paraformaldehyde for 24 hours, and embedded in paraffin wax. Perform serial frozen section (3μm) and check with immunohistochemistry. The main antibodies used are rabbit anti-mouse CD34 antibody (1:100), anti-nNOS antibody (1:100) and anti-caspase-3 antibody (1:50). Phosphate buffered saline was used as a negative control. Observe the reaction with diaminobenzidine. MVD, nNOS positive cells and caspase-3 positive cells in the ischemic area of the infarct group were observed under a microscope (400 x). Five areas were randomly selected for each slice to count the number of microvessels or positive cells. The average value is used as the expression of mvd or nnos or caspase-3 of the slice. The average value of the counts of microvessels or positive cells in all sections of each group was used as the MVD, or the expression of nNOS or caspase-3 in this group.

　　The content of nitric oxide, malondialdehyde and the activity of inosine and superoxide dismutase: 6 rats were randomly selected from the above three groups on the first, third and seventh days after the model was established. The animal was put to death and the brain was quickly removed. The infarcted cortex was quickly frozen in liquid nitrogen and then stored at ~80°C. The skin tissue samples were homogenized in cold saline to obtain a 10% homogenate. It was then centrifuged at 1248 g, and the supernatant was collected and stored at -80°C. According to the manufacturer’s instructions, use nitric oxide analysis kit, malondialdehyde analysis kit (TBA method), NOS type analysis kit (color measurement method), and SOD analysis kit (WST-1 method) to measure monoxide. Nitrogen and malondialdehyde content and iNOS and SOD activity.

　　Result: Thrombolysis reduces the infarct volume after MCAO: there is no obvious infarct area in the sham operation group, and the obvious infarct area (white) in the infarct group. At each time point, the infarct volume of the conventional group was significantly higher than that of the sham operation group. Compared with the conventional group, thrombolysis can effectively reduce the infarct volume at each time point.

　　Thrombolysis increased the MVD after MCAO: The MVD of the sham operation group did not change at any point in time. The expression of MVD in the infarction group, especially the thrombolysis group, was significantly increased at each time point. In addition, the expression of MVD in the thrombolysis group was significantly higher than that in the routine group at all time points (P<0.05), especially on the 7th day.

　　Thrombolysis reduces the expression of nNOS and caspase-3 after MCAO: nNOS positive cells and caspase-3 positive cells are stained with brown or tan. There were fewer positive cells in the sham operation group. One day after MACO, the expression of nNOS and caspase-3 in the ischemic area of the routine group was higher than that of the sham operation group. In addition, the expression of the thrombolytic group was lower than that of the conventional group.

　　Thrombolysis reduced the free radical activity and SOD activity after MCAO: Compared with the sham operation group, the cortex nitric oxide, malondialdehyde content and iNOS activity of the infarct side were significantly increased at all time points (P<0.05), but dissolved The suppository treatment group was significantly lower than the conventional treatment group. On the contrary, compared with the sham operation group, the SOD activity of the infarct side cortex decreased at all time points (p<0.05), and the SOD activity of the thrombolytic group was significantly higher than that of the conventional group.

　　Conclusion: Thrombolytic therapy with rt-PA within a widened treatment window (6 hours) can significantly reduce the infarct volume after ACI, which may reduce the oxidative stress by increasing MVD, reducing the expression of apoptotic molecules, reducing the activity of free radicals and enhancing the activity of SOD. Irritating.