动物造模,视网膜缺血大鼠模型 z-bio 2021-12-29 718

　　Background: Ischemia is considered to be the main pathogenic disease of various eye diseases, such as diabetic retinopathy, retinal artery/vein occlusion and glaucoma. Ischemia leads to cell apoptosis and increases the production of certain neurotoxic mediators by activating glial cells. The inflammatory cytokine tumor necrosis factor-α (TNFα) increased after ischemic injury. After inflammation, infection and traumatic reactions, its secretion increases. TNF-α binds to receptors and induces neuronal apoptosis through various intracellular signaling events. Previous studies have shown that the production of TNF-α is increased in in vitro and in vivo ischemia models. After intravitreal injection of TNF-α antibody, tumor necrosis factor α can significantly retain retinal function through neutralization. Etanercept (Enbrel) is a TNF-α inhibitor that can be used as a decoy receptor to bind to tumor necrosis factor-α. It is the first fusion monoclonal antibody against tumor necrosis factor alpha currently on the market for clinical use. Etanercept reduces the inflammatory effect of TNF-α and is used in the clinical treatment of various autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis. In addition, recent studies have shown that systemic injection of etanercept can effectively prevent the loss of retinal ganglion cells in glaucoma rats. High levels of TNF-α and its compounds can be detected in glaucoma and uveal rat models. Receptor. In this study, the widely used TNF-α inhibitor etanercept was injected subcutaneously after acute ischemic injury to determine its effectiveness in preventing axonal ischemic injury. The purpose of this study was to determine whether etanercept can reduce optic nerve degeneration and microglia activation in a rat model of acute hypertensive retinal ischemia. Animal model of retinal ischemia: 36 male SD rats weighing 250-300 grams were used in the experiment. All animals were placed in a light-dark cycle at 21°C for 12 hours. Thermotherapy facilities and animal testing facilities where you can get food and water for free. Before inducing ischemia, 30 mg/kg thiacycline (Salta; Vic, Fort Worth, Texas) and 10 mg/kg xylazine (Lombon 2%; Bayer, Peoria) were injected intraperitoneally. , IL) Rats are anesthetized. It induces ischemia by increasing intraocular pressure (IOP), thereby blocking the blood supply from the retinal artery to the retina. Connect the anterior chamber cannula of the right eye to a silicone rubber tube with a 30-gauge needle and a pressure gauge for 0.9% sterile saline infusion. When the saline container rises above the arterial pressure of the whole body, the intraocular pressure rises. The intraocular pressure was 130mmHg for 60 minutes. The disappearance of iris albinism and retinal red reflex confirmed retinal ischemia. For retinal perfusion, intraocular pressure is monitored every 5 minutes. The injection was then stopped to allow reperfusion of the retinal blood vessels, as evidenced by the replicated red reflex. As a non-ischemic control, the left eye was inserted into the anterior chamber with 30 needles through the contralateral cornea. The animals were sacrificed at different times and their eyes were removed for morphological and immunohistochemical studies. Etanercept treatment: Mix Etanercept with sterile water (0.3 or 1 mg/kg), and divide the rats into 3 groups. One day after the induction of acute ischemia or sham injection, until the day, groups 1 and 2 were injected with 0.3 mg/kg (N=6) and 1 mg/kg (N=15) etanercept subcutaneously every week. The second group was treated with 1 mg/kg etanercept, 3 animals were sacrificed after 3 days of treatment, 6 animals were sacrificed after 2 weeks of treatment, and 6 animals were sacrificed after 4 weeks of treatment. In the same way, the third group (n = 15) was injected with the same amount of 1 mg/kg phosphate buffered saline (PBS). Three animals were sacrificed after 3 days of treatment, and 6 animals were sacrificed after 2 weeks of treatment. Six animals were sacrificed after 4 weeks of treatment. The dose selection is based on previous research, which shows the efficacy of the drug in other disease models.

　　Histological evaluation and immunohistochemistry: Remove the eyeballs under anesthesia. Minimize the stretching damage during the extraction process. The optic nerve orbit was dissected through a lateral incision of the conjunctiva and a lateral musculotomy. When the perineum is sufficiently visualized to obtain sufficient nerve length, the optic nerve is incised approximately 3 mm from the root and then removed from the eyeball. The resulting axons were fixed with Karnovsky's fixative, stained with 1% osmotic acid, and then embedded in conventional paraffin. 10μm continuous slices. Fix the short part of the myopic nerve in 2.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M phosphate buffer (pH 7.4) for 24 hours at 4°C. Then add 1% overnight acid overnight and wash with buffer at room temperature. The tissue is then dehydrated and embedded in epoxy resin through a gradual series of alcohol. The cross-section (1 μm) of the optic nerve was stained with 1% toluidine blue and 1% sodium borate, and the cross-sectional area was measured. An ultra-thin (60m) cross-section was created and observed with a transmission electron microscope (TEM). The optic nerve obtained 3 days after the induction of ischemic injury was used for the immunohistochemical evaluation of microglia activity. The connected optic nerve retinal section (10 mm) was pre-incubated with PBS containing 10% goat serum, 0.5% gelatin, 3% bovine serum albumin and 0.2% Tween 20, and then incubated with anti-iba1 rabbit antibody (1:500). Done. ) As a marker for microglia.

　　Quantification of optic nerve axon damage: The premise of this study is that the cross section of optic nerve axon is round or oval. The degenerated axon loses roundness and must deviate from its normal size range. Axonal degeneration is characterized by swelling of the axons and dividing into layers of myelin (dense axons), destruction of myelin and extensive fibrosis in severely injured cases. Other methods reported in the literature are used to quantify optic nerve axon damage. Western blot analysis: 3 days after induction of acute ischemic injury, Western blot analysis was performed with microglia markers Iba1 and CD68. The same amount of protein was electrophoresed on a 10% SDS-polyacrylamide gel. The separated protein is electrotransferred to the PVDF membrane. After blocking with 5% skimmed milk powder, the membrane was incubated with anti-Iba1 and CD68 antibodies overnight. The membrane was incubated for 1 hour, and incubated with HRP-labeled secondary antibody against rabbit IgG (1:2000) at room temperature. The immune response area was observed by adding a chemiluminescent agent and exposing it to X-ray film.

　　Result: Etanercept reduced the pathological changes of axons: By comparing the axon morphology and optic nerve density under a transmission electron microscope. Four weeks after the induction of acute ischemia, the optic nerve axon damage in the experimental group was greater than that in the control group. The disease is characterized by swelling of the axons and separation of the myelin sheath, increased changes in axon size and shape, and decreased axon density. However, similar to the control group, axons treated with 1 mg/kg etanercept maintained their normal composition and density for 4 weeks. The proportions of conserved axons in the eyes of animals treated with 1 mg/kg etanercept and PBS control group for 2 weeks were 0.88±0.16 and 0.68±0.17, respectively. The proportions of conserved axons in the eyes of animals treated with 1 mg/kg etanercept and PBS control group for 4 weeks were 0.98±0.04 and 0.65±0.11, respectively. It also showed that the continuous treatment with 0.3 mg/kg etanercept 4 weeks. The axon protection was better than that of the PBS group. Etanercept reduces the activation of microglia: 3 days after acute ischemia induction, the rabbit anti-Iba1 antibody immunohistochemical staining of the 1 mg/kg etanercept treatment group resulted in significantly lower optic disc microglia cells than the control group. In the control group without ischemic damage to the eyes, a small number of Iba1 and CD68 positive cells appeared in the optic nerve. In the ocular ischemia group, the average molecular weights of the corresponding Iba1 and CD68 protein bands were 17 kDa and 75-110 kDa. However, the expression levels of Iba1 and CD68 markers in the eyes of the etanercept 1 mg/kg treatment group were significantly lower than those of the control group. Therefore, it seems that etanercept treatment of ischemic injury can significantly prevent the increase in the expression of Ibal and CD68. Discussion: Etanercept is a commercially available TNF-α inhibitor. This study confirmed its effectiveness in maintaining axon structure and reducing microglia activation in a rat model of retinal ischemia. Previous studies have shown that etanercept can reduce intraocular inflammation in rat uveitis models and reduce the level of TNF-α, thereby preventing the loss of retinal ganglion cells in glaucoma rats. In this study, 3 days after induction of acute ischemia, the development of microglia activation was confirmed using microglia markers. oh etc. It is reported that the increase of tumor necrosis factor-α level within 3 days after the increase of intraocular pressure in glaucoma model rats is related to the activation of related microglia within 7 days. Therefore, we evaluated microglia activation 3 days after ischemic injury and clarified the inflammatory response caused by microglia. Two microglia markers, Iba1 and CD68, were used in this study. Iba1 provides an indicator of static and activated microglia and microglia density, while the lysosomal antigen CD68 can measure the phagocytic activity of microglia. This study has some limitations. First of all, we did not directly evaluate the loss of retinal ganglion cells or the level of TNF-α. Conclusion: The over-the-counter TNF-α inhibitor etanercept is of great significance to axon damage after acute myocardial ischemia in rats and has protection effect. Other research on the neuroprotective effects of etanercept will help determine whether the drug is suitable for new treatments for neurological diseases.