动物实验,新生血 z-bo 2020-09-10 538

　　Abstract: The purpose of this study is that neurokinin 1 receptor (NK-1R) antagonists induce the degradation of established corneal angiogenesis (CNV)? Calcium hydrogen superphosphate?

　　Methods: Alkaline burns were performed on 20 C57BL/6 mice. After 7 days, when corneal angiogenesis was formed, 10 mg/ml fosartanone was locally injected into the right eye 6 times a day for 10 days. At the same time, a control group was established and the eyes with corneal neovascularization were treated with saline. To evaluate the safety of fosaptan in 10 healthy mice, they received the same topical treatment for 10 days. The Vesselj plug-in is used to measure blood production and lymphatic production. Secondary endpoints such as leukocyte infiltration, corneal opacity and corneal fluorescein staining were also evaluated. Determine the composition of inflammatory cells by flow cytometry. Use unpaired t-test, Mann-Whitney U test or (in some cases) two-way analysis of variance to assess differences between groups. Results: Local injection of formracetam can significantly reduce (i) CD31 + blood corneal angiogenesis, (ii) lyve1 + lymphocorneal angiogenesis, and (iii) CD45 + leukocyte infiltration. In addition, posapretan-treated keratin showed reduced opacity, damage to corneal fluorescein staining, and reduced neutrophil (-72%) and macrophage infiltration. Topical application of fosaptan is non-toxic to the ocular surface: no signs of conjunctivitis, opacity, perforation or corneal fluorescein staining were found. Similarly, corneal TUJ1 + nerve density is not affected. Conclusion: Our data suggest that NK-1R antagonists (such as fosapretan) may be a new promising therapeutic tool to inhibit established CNV.

　　Introduction: Corneal angiogenesis (CNV) is an important medical field. The second reason for global blindness is the limited current treatment options. Many compounds, including anti-vascular endothelial growth factor, have been shown to be effective in patients with recent CNV attacks, and their role in inducing vascular degeneration is limited to reducing the caliber of blood vessels. From a clinical perspective, it is very beneficial to treat blood vessels that invade the cortex instead of preventing them from growing in the cornea. The purpose of this study is to test the degenerative effect of NK-1R antagonist phosapipitant in inducing corneal angiogenesis and lymphangiogenesis. A wide range of corneal diseases, including trauma, infection and loss of limbal epithelial stem cells, are usually associated with CNV, which is directly related to: (i) loss of vision and (ii) loss of corneal immune privilege. In addition, the degree of existing CNV is directly related to the risk of corneal transplant rejection. Treatment for CNV is limited. Topical steroids are widely used because they inhibit the migration of inflammatory cells and the secretion of inflammatory cytokines. Long-term use of steroids is the most effective method and can cause side effects of visual disturbances, including cataracts, glaucoma and wound healing disorders. Surgical operations or surgical treatments such as photodynamic therapy, fine needle diathermy, laser-induced photocoagulation, conjunctival, limbal or amniotic membrane transplantation have been used to treat mature CNVs with convertible capabilities. Anti-vascular endothelial growth factor drugs are used in clinical trials to treat existing CNV. Local administration effectively reduces the diameter of blood vessels, but does not result in a significant reduction in the corneal area (ie, the invasive area) where angiogenesis invades. Finally, recent CNV treatments may cause side effects, such as epithelial defects, corneal parenchymal thinning, and neurotoxicity. Anti-vascular endothelial growth factor is very effective on new blood vessels before they cover subcutaneous cells, but stable CNV is not. Corneal angiogenesis (CNV) is usually associated with inflammation. Neurogenic peptides play an important role in corneal inflammation and CNV. This is because the keratin receives the densest sensory nerves in the entire body. Specifically, substance P (SP) is a neuropeptide that contains 11 amino acids of the takinin family and is secreted by various immune cells in sensory nerve endings (including corneal endings) and in the process of inflammation. Substance P acts by binding to the high-affinity neurokinin 1 receptor (NK-1R). The receptor is expressed in nerves, immune cells and epithelial cells. The activation of K-1R promotes lymphocyte proliferation and induces the release of inflammatory mediators, such as interleukins and growth factors required to initiate and maintain CNV. It has previously been proposed that NK-1R antagonists may have therapeutic applications in angiogenesis-related eye diseases. Local administration of highly compatible and competitive NK-1R antagonist (Lampitant) can effectively prevent the onset of CNV. After blood vessels invaded at least 50% of the corneal surface, we tested whether selective NK-1R antagonists block the existing CNV by blocking SP. To this end, we used the NK-1R antagonist fosapretan, which is a clinically approved water-soluble drug that prevents nausea and vomiting. Fosaprepitant is a prodrug that can be easily converted into active aprepitant in the body by the ubiquitous phosphatase expressed in epithelial cells, endothelial cells and corneal cells in the cortex. Materials and methods: Corneal alkali burn model: All experiments (80 mice in total) used female C57BL/6 mice aged 6-8 weeks. CNV was induced in the cornea of the right eye of 60 mice. In short, after general anesthesia, 2μl of 0.15 mMaOH in the center of the stratum corneum will cause alkaline burns on the stratum corneum, which can be rinsed thoroughly with 20 ml of normal saline. Next, the corneal epithelium is scraped off with the corneal parenchyma in a rotational motion parallel to the valve annulus. To prevent infection, treat the eyes with antibiotic ointment once a day for the first three days. Seven days later, the cornea was examined and photographed with a slit lamp microscope SL990. Clinical measurement of corneal angiogenesis. The keratin is divided into 4 quadrants, and the score ranges from 0 points (no blood vessels) to 4 points (4 quadrant blood vessels). Of the 60 eyes, 20 were excluded from the study. Five eyes have holes, two eyes have cataracts, and thirteen eyes have angiogenesis in one quadrant. According to CNV's clinical scoring system, the remaining eyes were equally divided into two groups (20 cases in each group). In the second independent experiment, 30 mice with 10 eyes were used in each group. Haloperidone treatment: The two selected groups received a total volume of 10 μl phosphate buffered saline (PBS) or 10 mg/ml fosapidan dissolved in 10 μl vehicle control, and the right eye was locally 6 times a day for 10 days. injection. Treatment begins 7 days after corneal injury. Once CNV is established, at least 50% of the corneal surface should be covered. We decided to give 10 times the intravenous concentration (1 mg/ml) used to treat nausea and vomiting. In a preliminary study, 1 mg/ml fosaptan could not reduce CNV and inflammation. Ten healthy animals (20 mice in total) in the other two groups received topical Fosapretan or 10 μl PBS at 10 mg/ml 6 times a day for the right eye for 10 consecutive days to assess the toxicity of Fosapretan. After each treatment, clinical examination was performed under a narrow gap microscope to blindly check the overall pathological changes and drug toxicity. As mentioned earlier, the scoring system (0-4, 0=fully transparent, 4=fully opaque) is used to evaluate the opacity of the cornea. In vivo corneal fluorescein staining is used to evaluate corneal epithelial defects in healthy eyes and eyes with alkaline burns after treatment with fosapretan. The eyes were photographed under the blue light of the SL990 slit lamp microscope. Use IMAGEJ1.44P software to analyze the image to evaluate the ratio of the green fluorescent area to the total corneal area. On day 7 and day 17 (before and after treatment, respectively), the slit lamp image was analyzed in image J to determine the CNV entry area in the body. Analysis of corneal angiogenesis and leukocyte infiltration: At the end of the treatment, the cornea was excised, fixed and immunostained as described above, and labeled as: rat anti-CD31, goat anti-Lyve1 and rabbit anti-. CD45, contrast the cornea with DAPI, lay it flat and photograph it with a fluorescence microscope. In order to obtain a two-dimensional reconstruction of the entire stratum corneum, a set of six adjacent overlapping images will be collected and remapped to the editing configuration. These digital images were analyzed using the recently developed ImageJ plug-in called Vesselj. It can semi-automatically quantify corneal blood and lymphangiogenesis. A confocal microscope was used to count the double positive (DAPI + CD45 +) cells in each field of view to quantify the infiltration of CD45 + cells; the healthy keratin was processed with local Fostar dam and nerves as described above. The treatment marker TUJ1 (rabbit anti-β3 tube protein polyclonal antibody) was immunostained to evaluate the potential neurotoxicity of the drug. The imaging method was used to quantify the peripheral basal plexus in the fosapitan and vehicle treatment groups. Flow cytometry analysis: remove twelve keratins/group and combine on day 7 (before treatment) and day 17 (after treatment with Fosapidem or excipient). After removing the corneal epithelium with 20 mM EDTA, the single cells were treated with collagenase IV (2 mg/ml) and DNase I (0.5 mg/ml) in a dissolving HBSS solution containing Ca2 +/Mg2 + medium. form. suspension. After shaking at 37°C for 60 minutes, the cell suspension was filtered at 70 μm and washed with cold PBS containing 0.5% BSA/2 mM EDTA. Incubate all cell suspensions with rat anti-FcγIII/II receptor blocking antibody (1:50) on ice for 20 minutes. Then, stain with the following primary antibodies for 30 minutes at 4°C. The main antibodies previously titrated are rat anti-CD45-PB (1:200), rat anti-CD11B-APC-eFluor780 (1:800), rat anti-Ly6G/Ly6C (GR1)-PE/Cy7 (1:200) . ) And rat anti-F4/80-FITC (1:100). The cells were washed and resuspended in buffer containing 7-aminoactinomycin D (1:100).

　　Result: Topical application of fosfan may reduce stable corneal neovascularization: 7 days after the establishment of CNV, the animals were divided into two groups (20 in each group) and the CNV and opacity were uniformly randomized according to the quadrant measurement. During the treatment period, perforation occurred in one eye of each group (Fosapidem on day 11 and vehicle on day 9) and was excluded from the analysis. Local injection of fosapantant for 10 days can significantly reduce (i) corneal angiogenesis and (ii) corneal lymphatic angiogenesis. In addition, the second experiment confirmed the data, showing that the angiogenesis of the eye treated with fosaptan was reduced by 33%, and the lymphangiogenesis was reduced by 51%. In addition, in vivo and in vitro, it was found that keratin treated with fosaptan has a reduced blood vessel diameter. The topical use of fosaptan can improve the transparency of the cornea and does not affect the healing of epithelial wounds. Fosaptan treatment can effectively reduce corneal opacity, but there is no significant difference compared with the second independent experiment. In addition, 47% of the eyes in the group treated with fosapyrdan (9 out of 19 corneas in total), compared with 21% of the eyes in the vehicle group (4 out of 21 corneas) There are blood vessels in the central area of the cornea. Not in these two experiments, topical application of fosaptan did not reduce the rate of epithelial wound closure. In only one experiment, fluorescein staining decreased slightly after 10 days of treatment. Fosapitant reduction of topical application

　　Corneal inflammatory cell infiltration: CD45 staining analysis showed that compared with the vehicle group, the leukocytes in the corneal stroma were significantly reduced after fosapitant treatment. This reduction was confirmed in the second experiment. Flow cytometry analysis showed that after drug treatment, the reduction rate of stratum corneum bone marrow infiltrating cells (CD45 + 7AAD-CD11B +) was high. In particular, phosphatidylinositol induced (i) macrophages, a significant reduction of GR1dimF4/80+ cells in the bone marrow cavity and (ii) GR1highF4/80-neutrophils. Sunburn is non-toxic to the surface of the eye: After puncture inspection and evaluation, topical foxatin (10 mg/ml, 6 times/day, continuous use for 10 days) is non-toxic to the eye surface of healthy eyes: congestion/conjunctivitis or perforation sign. In addition, the cornea remains transparent after treatment. In addition, fosapretan is non-toxic to corneal epithelium and is negative for fluorescein staining. K-1R antagonists are non-toxic to corneal nerves. K-1R antagonists are not toxic to corneal nerves because of the typical histology of the peripheral inferior basal nerve plexus immunostained with anti-TUJ1 antibody and quantification of TUJ1 + nerves. Discussion: Substance P promotes CNV by binding to the receptor NK-1R. Recently, we have shown that local injection of NK-1R antagonist brightener can prevent corneal hematoma and lymphangiogenesis. In these two independent experiments, it was found that the high-affinity NK-1R antagonist fosapidem 10 mg/ml can significantly reduce the established CNV. In addition to inhibiting CNV, the treatment of foxatin also reduced corneal inflammation. Immunostaining and flow cytometry data confirmed this. It is well known that white blood cells, especially macrophages, express NK1 receptor, and its activation leads to increased macrophage activity and chemotaxis, and release of angiogenic factors such as SP and VEGF. It has been shown that white blood cells contribute to corneal hematopoiesis and lymphangiogenesis, so reducing white blood cells after treatment may reduce the effect of CNV. Therefore, it can be inferred that fosapamide regulates the recruitment and activation of inflammatory cells, which ultimately leads to a decrease in blood vessel density. In addition, Fosapreitant also improved other clinical outcomes related to inflammation, such as corneal transparency and epithelial defect size. This is consistent with previous studies on various NK-1R antagonists. Finally, the topical use of fosapident is non-toxic to the ocular surface. When examined with healthy eyes, there were no signs of conjunctivitis, perforation, corneal opacity or epithelial defects. The treatment does not affect the lower cranial nerve plexus. This is consistent with previous observations about other NK-1R antagonists.

　　Conclusion: Fosapidan: (i) is currently the only water-soluble NK-1R antagonist approved for clinical use, (ii) there is no obvious ocular surface toxicity, (iii) the use of animal models can effectively reduce the growth of existing CNV. Therefore, Fosa Putan is considered an ideal candidate for clinical trials of human CNV.