动物造模,大鼠腔隙性脑梗死模型 z-bio 2021-10-21 301

　　Introduction: Sudden brain injury leads to the release of damage-related molecular patterns (DAMPs) and subsequent activation of local immune responses. After an ischemic stroke, astrocytes are activated in a short time. They change shape, swell, increase calcium signal, and change their function. Microglia is another type of cell involved in the main response after ischemic stroke. Activated astrocytes and microglia produce pro-inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-α, chemokines and metalloproteinases, leading to destruction of the blood-brain barrier (BBBD) and inducing inflammatory changes in the brain endothelium , And further develop into nerve tissue damage in the brain. BBBD promotes the infiltration of white blood cells into nerve tissue from peripheral blood. After ischemic stroke, neutrophils, monocytes, dendritic cells, T and B lymphocytes and natural killer cells are mainly seen in the brain. Infiltrating cells produce pro-inflammatory cytokines, such as tumor necrosis factor-α, interferon-γ, chemokines and metalloproteinases, leading to the progression of nerve cell damage and nerve tissue destruction. However, neuroinflammation is not the same as an adverse result of the central nervous system. Previous studies have shown that new neurons are produced in damaged brain tissue. Ischemic stroke induces endogenous neurogenesis. Different studies have shown that the onset of ischemic stroke induces the proliferation of neural stem cells (NSC) in the subventricular zone (SVZ) and promotes the expansion of the NSC pool. In addition, it has been demonstrated that neuroblasts may migrate from SVZ to areas damaged by ischemic stroke, thereby enhancing the process of neurogenesis. The process of endogenous neurogenesis involves the maturation of neural stem cells, which leads to the formation of new neurons. In addition, enhanced synapse formation was observed after ischemic stroke. A lacunar cerebral infarction model was prepared by injecting Na/katpase pump inhibitor ouabain into the brain parenchyma, which provided damage to deep brain structures. The chemical block of the pump causes changes in the metabolism and structure of the brain to simulate ischemic injury. Observe the core of the lesion in the early stage of lacunar infarction, observe the local immune response and the components of neurogenesis after brain injury. "Establishment of a rat model of deep lacunar infarction: Adult male Wistar rats of approximately 7-8 weeks of age (250 g body weight) were used in all experiments. A total of 36 animals were used in the study. Animals eat and drink freely. Rats were intraperitoneally injected with ketamine (53.6 mg/kg) and medetomidine (0.4 mg/kg) and placed in a stereotactic device. The focal brain injury model is as described above. Make a hole in the skull, connect the needle with a 10μl syringe, and place it in the right striatum. Then, a micro infusion pump was used to inject 1 μl of 5 nmol ouabain into the brain at a rate of 1 μl/min. After the injection, the needle was removed and the skin was sutured with sutures. After surgery, all animals were treated with antibiotics (0.4 mg/ml) and analgesics (5 mg/ml). Rats that did not suffer from focal brain injury served as control animals.

　　Brain tissue collection and preparation: Three days, five days and nine days after focal brain injury, rats were deeply anesthetized by intraperitoneal injection of ketamine (53.6 mg/kg) and medetomidine (0.4 g/kg) and decapitated. Separate the brain, freeze on dry ice, and store at -70°C. For immunohistochemical analysis, the brain was cut into 25 μm thick coronal tissue sections on a cryostat, placed on a microscope slide, and frozen at -80°C. To detect cytokine expression, homogenize the brain in RIPA lysis buffer (20 mM Tris-HCl (pH=7.5), 150 mM NaCl, 1 mM PMSF, 0.05 Tween-20, and a mixture of protease and phosphatase inhibitors. In Centrifuge at 11500 rpm for 10 minutes at 4°C to clarify the lysate; collect the supernatant, resuspend in RIPA lysis buffer, and store at -80°C.

　　Evaluation of deep lacunar infarct damage in the brain of rats: After injection of ouabain, hematoxylin-eosin (HE) staining was performed to detect brain damage. On days 3, 5, and 9 after injury, rats were intraperitoneally injected with ketamine (53.6mg/kg) and medetomidine (0.4g/kg), perfused with phosphate buffered saline (PBS) through the heart, and then treated with 4% paraformaldehyde ( PFA) fixed. The brain was taken out, fixed with 4% PFA, dehydrated in a series of ethanol, embedded in paraffin, and cut into tissue sections with a thickness of 3 μm. Three animals were analyzed per group.

　　Immunohistochemistry: Perform immunohistochemical analysis on 25μm-thick coronary brain tissue sections. The tissue sections were rinsed in PBS, and then incubated in 10% goat serum, 0.25% Triton X-100 and 0.1% bovine serum albumin in PBS for 60 minutes. ; Then rinse the sections with PBS and incubate with primary antibody overnight at 4°C. Mouse anti-rat monoclonal CD4 (1:300), mouse anti-rat monoclonal CD45 (1:100), mouse anti-rat monoclonal CD8 (1:300), rabbit anti-rat polyclonal Doublecortin ( 1:800) Mouse anti-rat monoclonal ED1 (1:100) and mouse anti-rat monoclonal Nestin (1:200). On the second day, wash the tissue sections with PBS, and add secondary antibodies conjugated with Alexa Fluor 488 nm: goat anti-mouse IgG1 (1:500), goat anti-mouse IgG2a (1:500) or goat anti-rabbit IgG (H +L) (1:500) Incubate for 60 min under dark conditions at room temperature. The nuclei were then labeled with 5 μM Hoechst 33258 fluorescent dye.

　　Results: Evaluation of deep lacunar cerebral infarction in rats: HE staining was performed 3, 5, and 9 days after cerebral infarction caused by ouabain, and the area and location of the lesion were observed. OUA injection caused damage to the cell structure of striatum and cortex in all animals. Progressive loss of neurons and tissue damage were observed. In control animals, typical cerebral cortex and striatal tissue structures were observed. Three days after lacunar infarction, the number of nuclei increased. The histological changes are characterized by atrophy of the nucleus of neurons and eosinophilic cytoplasm. The HE stained sections 5 days after brain injury showed necrosis and cavitation, and the entire cortex and dorsolateral striatum were edema. The damaged area of lacunar cerebral infarction is vacuolated and spongy with sparse nerve fibers. In addition, there are more cells in the damaged part of the brain tissue and infiltration of white blood cells. In the damaged striatum, there are also infiltrating cells in the white matter tracts. Nine days after ischemic tissue injury, all animals showed a growing cavity area in the diffuse matrix of delicate connective tissue. The boundary between normal tissue and abnormal tissue is clear, and there is no tissue necrosis in the early stage. The average infarct area was 4.34 mm2, accounting for 8.28% of the brain area analyzed. In summary, within the first 9 days after the injection of ouabain, progressive neuron loss and tissue damage were observed, resulting in cerebral lacunar infarction damage in rats.

　　Analysis of immune cells in the brain of rats with lacunar infarction: On the 3rd, 5th, and 9th days after ouabain-induced focal brain injury in rats, immunohistochemical staining was used to detect the number of immune cells in the injured brain hemisphere. Studies have shown that on the 9th day after ouabain-induced injury, the number of positive microglia activated by ED1+ increased significantly. The analysis showed that at any time point after focal brain injury, the total number of white blood cell CD45+ did not change significantly. However, the number of CD4+ T lymphocytes increased significantly on day 5 after ouabain induction, and further increased on day 9 after brain injury. In addition, the number of CD8+ T lymphocytes increased significantly on the 9th day after focal brain injury. The results showed that on the 9th day after lacunar infarction, the proliferation of ED1 + microglia increased, and CD4 + T lymphocytes and CD8 + T lymphocytes were infiltrated.

　　Detection of brain cytokines and chemokines in rats with lacunar cerebral infarction: The concentration of various pro-inflammatory cytokines increased after deep lacunar infarction. Studies have shown that IL-1α levels increase 3 and 9 days after brain injury. The secretion of IL-1β and IL-18 increased after lacunar infarction in the deep brain, but it was statistically significant only 3 days after focal brain injury. Three and nine days after ouabain-induced brain injury, the level of TNF-α increased significantly. In addition, on day 9 after focal brain injury, the expression of IFN-γ increased significantly. Similarly, 9 days after brain injury, increased expression of TGF-β1 was observed. Therefore, lacunar cerebral infarction damage leads to increased release of pro-inflammatory cytokines in the rat brain. Studies have shown that levels of anti-inflammatory cytokines decrease after deep brain injury. The expression of IL-10 decreased at 3, 5, and 9 days after brain injury. Similarly, TGF-β2 levels decreased at every time point after ouabain injection. Therefore, lacunar cerebral infarction damage leads to a decrease in the expression of anti-inflammatory cytokines in the rat brain. According to our research, many chemokines/growth factors increase after cerebral infarction. Experiments show that CCL-5 levels increase 3 days, 5 days and 9 days after brain injury. GM-CSF levels increased significantly at each time point after focal brain injury. In addition, the expression of CXCL-1 and MCP-1 (CCL-2) also increased, but only 9 days after the injection of ouabain was statistically significant. Therefore, lacunar cerebral infarction damage activates the release of certain chemokines/growth factors in the rat brain. In summary, intraperitoneal injection of ouabain can enhance the pro-inflammatory immune response in the rat brain.

　　Analysis of neurogenesis after lacunar infarction in rats: Studies have shown that the expression of neural stem cell marker Nestin increases after deep lacunar infarction, but it is not statistically significant. Compared with the control group, the morphology of Nestin-positive cells changed after ouabain induction, from branching on day 3 after injury to spindle shape on day 9 after injury. Experiments show that the cells expressing DCX, a neuron migration protein marker, increase significantly at 3, 5, and 9 days after focal brain injury. The findings indicate that during the 9 days of observation, lacunar infarction damage stimulates neurogenesis in the rat brain.

　　Conclusion: Stereotactic injection of ouabain (Na/K-ATPase pump inhibitor) induced structural damage to the brain tissue of rats. During the following 9 days of observation, the core of the lesion increased. At the same time, it was accompanied by microglial activation and immune cell infiltration, and the CD4+ and CD8+ T lymphocytes of the ipsilateral cerebral hemisphere were significantly increased. Compared with normal rats, the pro-inflammatory cytokines IL-1α, IL-1β, TNF-α and IFN-γ increased, while the anti-inflammatory cytokines IL-10 and TGF-β2 decreased. Compared with control rats, the accompanying focal brain injury showed a significant increase in the levels of chemokines, namely CCL-2, CCL-5 and CXCL-1. Immunohistochemical analysis showed that neural progenitor cells and migrating neuroblasts can be seen in the ipsilateral striatum and cortex. The discovery provides a new understanding of the early inflammatory response after lacunar infarction in the deep brain. The results of local inflammation around the infarct may highlight a new treatment method that uses immune cell subpopulations for neurorestorative intervention.