动物造模,认知功能障碍,血凝酶 z-bo 2020-09-10 348

　　Background: Cognitive dysfunction is the main cause of poor quality of life in stroke patients, and stroke is notorious for its high morbidity, disability, mortality and recurrence rate. However, due to its complicated pathological mechanism and narrow treatment time window, there is no effective treatment strategy in clinical practice. Therefore, it is necessary to further study its potential signal transduction mechanism and adopt new promising intervention measures to alleviate the cognitive dysfunction caused by stroke. New evidence suggests that ischemia-reperfusion (I/R) injury plays an important role in the development of stroke. When I/R damage occurs, the damage and dysfunction of endothelial cells will lead to the destruction of the blood-brain barrier, thereby exacerbating I/R damage. In addition, the free hem released by methemoglobin during brain I/R is a toxic component of endothelial cells. In established in vitro and in vivo models, free hem is toxic to cortical neurons and stellate cells. Hemagglutinin (HPX) is a 60 kDa serum glycoprotein that is locally synthesized by cells in the central nervous system. HPX has the highest binding affinity to the hem and can be used as an effective scavenger for excessive toxic hem. Our previous studies have shown that 24 hours after ischemia-reperfusion, the expression of penumbra neurons and stellate cells in the penumbra area increases. Within 7 days after MCAO, intracerebroventricular injection of HPX can reduce infarct volume and improve neurological function measurement. After ischemia and reperfusion, the neuroprotective effect of HPX lasted for 7 days. Oxygenase 1 (HO-1) is an enzyme that determines the rate at which free hem is broken down. New evidence shows that HO-1 plays an important role in protecting the blood-brain barrier from cerebral infarction. In addition, HO-1 can up-regulate the number of circulating endothelial precursor cells (EPC) and reduce the damage of ischemia-reperfusion injury to multiple organs. In this study, we investigated whether HPX can improve cognitive dysfunction associated with cerebral ischemia-reperfusion injury, and designed an experiment to determine whether it is related to HO-1.

　　Method: Animals: SD rats (7-8 weeks old, body weight 250-280g), all animals were randomly divided into groups (sham operation group, n=36; MCAOn=36; vehicle group, n=36). HPXn = 36; HPX + ZnPPIX, n = 30). Rats in the sham operation group underwent sham operation, the other group underwent MCAO, and they were euthanized by intraperitoneal injection of sodium pentobarbital (200mg/kg) after the experiment. Surgery: The middle cerebral artery occlusion (MCAO) method is used to induce a model of local cerebral ischemia reperfusion (I/R) injury. In short, rats must be fasted for 12 hours before surgery and anesthetized by intraperitoneal injection of chloral hydrate (10%, 3 ml/kg). Next, make an incision in the midline of the abdomen and determine the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA), and carefully dissect from the surrounding tissues. After ligating the proximal ends of the CCA and ECA, insert the surgical filament into the ICA until a slight resistance is felt. This indicates that the middle cerebral artery (MCA) is blocked, the blood flow to the MCA supply area temporarily stops, and then a cerebral infarction occurs. The skin was sutured with 4-0 suture and ischemia for 1 hour. Then the surgical wire is removed and the reperfusion process begins. During the whole process, the body temperature of the rat was kept at 37±0.5°C. Intraventricular injection: After reperfusion, the rat is anesthetized and placed in a stereotactic frame as before. The skull is fixed with four stainless steel screws. The scalp incision exposes the skull and front... Make a hole in the right skull (1.5 mm outside of Bregma and 0.8 mm behind). Slowly insert a 25μL microsyringe at 3.5 mm below the dural surface, and inject physiological saline (10μl, 0.9% sodium chloride for MCAO group rats), excipients (10μl), rat hemagglutinin reference serum (10μl)) 1.86 μg/L HPX dissolved in 0.16 sodium azide) or mixed solution (for rats in the HPX + ZnPPIX group, 10 μl, 1.86 g/L HPX + 20 μM ZnPPIX). Before stopping the drug, place the microsyringe for 5 minutes to observe the effect of the injection. After stopping the drug, the incision was sutured to allow the rat to recover from anesthesia. Shamrat underwent the same operation without any MCAO injury or intraventricular injection.

　　Morris Water Maze (MWM) analysis: MWM analysis consists of two parts: space detector testing and hidden platform testing. In short, the test system consists of a four-quadrant circular water tank (210 cm in diameter and 50 cm in height) filled with water (the temperature is maintained at 19-22 degrees and the depth is 30 cm). .. The colored cards on the wall were used throughout the experiment. Continuously. In the space test, a black target platform was fixed 1 cm below the water surface in the center of the southwest grid, so the platform was invisible to rats. As shown below, each rat was tested four times a day for five consecutive days with an interval of 5 minutes between the two tests, as shown below, and was randomly released in the quadrant position. The maximum time for each test is 120 seconds, allowing the rat to swim freely in the water tank to find a hidden platform. After discovering the hidden platform, the mouse rested on the platform for 30 minutes and recorded the time spent on the search platform as an escape dive. If the mouse did not find the platform within 120 seconds, the researchers placed it on the platform for 30 seconds and recorded the escape delay as 120 seconds. After 5 days of testing, the hidden platform test was completed. In this test, we removed the platform hidden above to observe and record the time (percentage of time) spent in the target quad SW and the number of platform intersections. The computer video tracking system uses MWM to record the escape latency, time percentage and cross-platform number. Western blot analysis: 7 days after ischemia-reperfusion, brain tissue in the area surrounding the ischemia, which was quickly frozen with liquid nitrogen, was quickly harvested at the expense of rats. Then the ischemic penumbra brain tissue was ground with a radioimmunoprecipitation (ripa) analysis buffer (Sigma Aldrich) containing protease inhibitors to obtain protein extracts. Load an equal volume (40μg) of protein samples from each group of samples into each channel of a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel, separate by electrophoresis, and then electrotransfer to a PVDF membrane . After blocking with 5% skimmed milk for 2 hours at room temperature, the membrane was incubated with the following main antibodies: anti-HO-1 (rabbit polyclonal, 1:1000 dilution), anti-VE casein (rabbit polyclonal, 1:3000 Dilution) and anti-rabbit β-actin (rabbit polyclonal antibody, diluted 1:1000), shake gently overnight at 4°C. After washing the membrane 3 times with TBST (5 minutes each time), it was incubated with the corresponding goat anti-rabbit antibody (1:11000), and the bound antibody was observed at room temperature for 2 hours. Immerse the western blot in an enhanced chemiluminescence (ECL) reagent, observe the bands of a specific protein, and then expose it to ECL. Immunofluorescence staining: 7 days after reperfusion, rats were sacrificed after abdominal anesthesia. The rats were intraperitoneally anesthetized as described above, and extracardiac perfused with normal saline supplemented with heparin, and then extracardial perfused with 4% paraformaldehyde. After perfusion, the rats were sacrificed, the brain tissue was removed from the skull, implanted in OCT medium and stored at 80°C. Coronal sections (8 μm) of frozen brain tissue were stained with immunohistochemistry. CD31 and vWF are two endothelial cell markers. After dewaxing and dehydration, it was treated with 3% hydrogen peroxide in methanol for 30 minutes to inhibit the activity of non-specific endogenous peroxidase. The antigen was recovered by boiling the slices in 10 mM citrate buffer (pH 6.0) for 10 minutes. Next, CD31 (1:100) and vWF (1:100) were incubated with goat monoclonal antibody (mAb) overnight at 4°C. Then the sections were washed with PBS and incubated with biotinylated anti-goat IgG (1:100) at 37°C for 2 hours. The sections were washed and then incubated with avidin peroxidase binding solution (1:100) 1 hour. I will. Finally, it was rinsed with diaminobenzidine for 5 minutes, and the negative control group was treated similarly, but without using the primary antibody. CD31 and vWF measurement: Use vWFf and CD31 immunofluorescence staining to determine the blood vessel density and the number of CD31-positive cells after brain injury. Here, five 50 mm brain tissue sections were analyzed with an optical microscope (200x). Calculate the vWF-positive blood vessels in the boundary between the lesion and the damaged hippocampus. Divide the immunoreactive blood vessel by the corresponding area to determine the blood vessel density and the number of CD31-positive cells in these two areas. Brain blood barrier permeability: BBB permeability was evaluated using Evans Blue (EB) pigment spillover method. Two days after MCAO and one hour before euthanasia, EB (2.5%, 2 ml/kg) was injected via femoral vein. After perfusion with 4% paraformaldehyde, the brain was removed and incubated in dimethylformamide at 60°C for 24 hours. Then the brain tissue was homogenized and centrifuged at 1000pm for 5 minutes. Measure the absorbance of gradient dose Evans blue and draw a standard curve. The absorbance of the supernatant was detected at 625 nm, and the relative content of EB was calculated. As mentioned earlier, brain water content is measured by the wet-dry method and expressed as a percentage of wet weight. Fluorescence quantitative PCR: Trizol was used to extract total RNA from the above-mentioned frozen brain tissue. Smartspec? The total RNA concentration was measured with a Plus spectrophotometer at 260nm OD. Reverse transcription of RNA into cDNA using cDNA synthesis kit. Use ready-made qRT-PCR mixture to perform RT-PCR amplification in a volume of 50 μL. All operations are performed in three parallel tests.

　　Statistical analysis: We compared the blood vessel density of MNSS, MWM and brain tissue in the three patient groups using unified analysis of diffusion (ANOVA) and Tukey's post-test. A P value of 0.05 is considered statistically significant.

　　Results: HPX improves the long-term spatial learning and memory ability of rats after local brain I/R injury: The baseline escape delay (? 24 hours) of the first five groups of spatial probe tests for sham surgery and local brain I/R injury is not too great difference. Compared with the sham operation group, the MCAO group has a higher latency in the space probe test 2-7 days after local probe I/R injury. The time spent by the MCAO group in the target quadrant and the number of cross-platforms in the hidden platform test were significantly lower than the fake operation group. Compared with the vehicle group, the HPX group significantly reduced the escape latency of the space probe test from the second day to the seventh day after the local probe I/R injury. The time spent in the target quadrant and the number of cross-platforms in hidden platform testing has increased significantly. 2-7 days after the local brain I/R injury, the escape time of the space probe test of the HPX + ZnPPIX group was significantly longer than that of the HPX group. The HPX + ZnPPIX group significantly reduced the time spent in the target quadrant and the number of cross-platforms in hidden platform testing. However, there were no significant differences between MCAO and excipient groups in terms of escape latency, time spent in the target quadrant, and the number of cross-platforms in hidden platform testing. The expression of HO-1 in the ischemic penumbra of the HPX treatment group was up-regulated: Compared with the sham operation group, the expression of HO-1 was up-regulated in the ischemic penumbra of the MCAO group for half a day after ischemia. The expression of HO-1 protein increased. In the HPX group, the expression of HO1 protein was significantly higher than that in the MCAO and carrier groups. However, there was no significant difference in the expression level of HO 1 protein between MCAO and vehicle groups.

　　HPX improves the formation of hippocampal angiogenesis in the ischemic penumbra of rats with regional brain I/R injury: Compared with the sham operation group, the ischemic penumbra becomes thicker in the MCAO group 7 days after the regional brain I/R injury. There are new blood vessels in the dark hippocampus. Seven days after the local brain I/R injury, patients receiving HPX treatment had a significantly increased density of hippocampal neovascularization around ischemia compared with the vehicle group. After administration of HO-1, ZnPPIX and HPX inhibitors to rats with local brain I/R injury, 7 days later in the local brain I/R and HPX groups, the angiogenesis density of the ischemic surrounding hippocampus increased. In contrast, the density was significant. To reduce. However, after 7 days in the MCAO group, in the vehicle group, there was no significant difference in local brain I/R injury and hippocampal neovascular density around ischemia.

　　HPX can prevent local I/R damage due to impaired blood-brain barrier (BBB) function: evaluate the permeability of the BBB, brain integrity and new blood vessels for stability 7 days after local I/R damage, we evaluate The effect of intracerebroventricular injection of HPX on reducing the structural and functional damage of BBB. The results showed that compared with the sham operation group, the exudation of Evans blue staining solution was significantly increased, and the blood-brain barrier permeability of the MCAO group was increased. In addition, in rats with local brain I/R injury, the degree of brain edema measured by water content (indicating the integrity of the BBB) was heavier than that in the sham operation group, and the ratio related to vascular stability Ang1/Ang2 has been greatly reduced. VE-cadherin plays an important role in endothelial cell migration and survival, angiogenesis, and maintenance of blood vessel integrity. The expression of VE-cadherin after MCAO was lower than that of the sham operation group. Compared with rats in the vehicle group, the leaching, water content and VE casein level of Evans blue dye in the HPX group were significantly higher than those in the vehicle group. Likewise, when comparing the MCAO and HPX groups, these changes are consistent. In rats treated with HPX and ZnPPIX (HO-1 antagonist), the positive effect of HPX on BBB function was blocked. Conclusion: HPX can reduce the cognitive dysfunction after focal cerebral ischemia-reperfusion injury through HO-1 pathway and prevent damage to the blood-brain barrier of rats.