动物造模,兔单眼局限性感光细胞变性模型 z-bo 2021-01-08 365

　　Background: Retinitis Pigmentosa is a degenerative disease of the retina that can cause night blindness and eventually loss of peripheral and central vision. Several genes responsible for RP have been identified, most of which are related to the light transmission pathway. These findings have not yet led to the discovery of effective treatment or prevention strategies. Retinal prostheses have been studied as a potential tool to stimulate the remaining retinal neurons in these patients to cause hallucinations and restore vision. Various animal models of rodent photosensitivity have been developed to study visual function. CS rats have spontaneous mutations in the Mertk gene, resulting in degeneration of photoreceptor cells. Continuous light irradiation is another technique used to study the mechanism of light denaturation. The light-sensitive rabbit model undertakes complex behavioral tasks and more closely simulates human visual functions. We report two methods to generate a rabbit model of monocular photoreceptivity degeneration. Berteporfin is a photosensitive dye with no clinical side effects. In preclinical studies, verteporfin and/or prolonged exposure caused damage to photoreceptors and retinal pigment epithelial cells. Bethpofin is used to induce local degeneration of retinal photoreceptor cells. Sodium nitroprusside (SNP) is used to treat high blood pressure. SNP decomposes, releases nitric oxide (NO) in the blood, and acts as a vasodilator. In the retina, excessive NO can induce degeneration of photoreceptor cells. The intravitreal injection of sodium nitroprusside causes extensive photodenaturation. The degeneration of photoreceptor cells induced by two-dimensional teporphin and SNP has no obvious effect on the contralateral eye. The benefits of the photodenaturation monocular model include using the same animal eye as a control. This reduces the number of experimental animals used.

　　Method: Use Berteporfin according to the instructions. In our research, we use halogen reflector lamps as the light source. Dissolve 2 mg of Verteporfin in 7 ml of sterile distilled water and adjust the concentration to 0.1 mg/ml with 5% glucose solution. The solution was injected intravenously with 0.5 mg/kg at a rate of 1 ml/min. SNP was dissolved in physiological saline, and different concentrations of SNP were injected into the vitreous cavity of rabbit eyes. 18 Dutch rabbits were used. The rabbits were anesthetized by intramuscular injection of ketamine (66 mg/ml) and xylazine (33 mg/kg). After inducing retinal degeneration with Verteporfin and dilating pupils with 1% Atropin and 2.5% phenylefrin hydrochloride, the retina was exposed to a halogen reflector lamp 10 mm from the cornea. In the second model, obexetine hydrochloride was locally applied to the eye using a surgical microscope, a small incision was made to expose the tube, conjunctiva, and 30-gauge needle, and 100 μL LSNP solution was applied to the vitreous. injection. Two weeks later, a fundus photograph was taken with a portable fundus camera. ERG records were obtained one month after treatment. The rabbits were acclimatized overnight in the dark, and were anesthetized with 1% atropine, 2.5% phenyleffrin hydrochloride and 0.5% propakin hydrochloride. A small contact lens with a gold wire loop is placed on the stratum corneum, and a silver wire reference electrode is placed between the eyes under the skin. The white LED pulse is activated to produce a flash, and the light stimulation lasts for 10 milliseconds. Record full-field scotopic ERG, 0.3-500 Hz band-pass filter, and average 5 responses for each light intensity. The ground electrode is fixed at the tail. White light LED (7500 Kelvin) is used for white light stimulation. As described by Tomita et al., the retinal morphology of the eyes treated with Verteporfin and SNP was analyzed. The animals were sacrificed by intravenous injection of sodium pentobarbital. The eyeball was removed, fixed, embedded in paraffin, and a 3 micron thick retina was cut out and stained with hematoxylin-eosin. Use GraphPad Prism software for statistical analysis, the statistical significance standard is P\u003c0.05,

　　Result: There was no significant change in the untreated and unexposed Berteporfin groups. After exposure, the Verteporfin treatment group or intravitreal injection of sodium nitroprusside can cause retinal atrophy. The degree of atrophy depends on the exposure time or SNP concentration. With Berteporfin treatment, when the retina is only exposed for 10 minutes, the pigmentation of retinal pigment epithelial cells (RPE) can be clearly observed. Under a longer period of light, the pigmentation is obviously marked as RPE atrophy. Berteporfin and exposure clearly limit the exposed area. However, SNP can cause peripheral retinal degeneration. Fluorescein angiography showed bleeding around the optic nerve, and 1 mmol SNP was observed. The ERG amplitudes (waves a and b) in the eyes treated with Verteporfin were slightly lower than those in the untreated eyes. As the exposure time continues, the amplitude of the b wave decreases. In SNP-treated eyes, the amplitude decreases with increasing SNP concentration. Even if only 0.1 mM SNP is injected, the b-wave amplitude of ERG will be significantly reduced. Verteporfin treatment without light does not cause photoreceptor cell degeneration. However, neurodegeneration of the retina (mainly photoreceptor cells) requires an exposure time of at least 10 minutes. In the SNP-induced degeneration model, the lesions include the surrounding internal retina and photosensitive layer.

　　 Discussion: The photosensitizer verteporfin is widely used as part of photodynamic therapy (PDT) for the treatment of CNV. However, PDT is not a pure choice for choroid, it can damage the retina. PDT induces dose-dependent damage to photoreceptor cells and retinal pigment epithelial cells. The absorption of certain wavelengths produces oxygen radicals. Toxic to photoreceptor cells and retinal pigment epithelial cells. The funding photo shows that after intravenous administration of weltiporfin, the photoreceptor damage caused by exposure to light is limited to the irradiated area. SNP releases nitric oxide through a photochemical reaction and reduces metabolites such as mercaptans and microsomal biological cells in a variety of ways. O participates in various retinal functions. Intrinsic NO can enhance the pyramidal response during light adaptation, causing retinal toxicity and ischemic damage. Under normal circumstances, ischemia or continuous exposure to strong light, the severity of retinal degeneration depends on the local NO level.

　　 Conclusion: Our results highlight two methods of inducing rabbit retinitis pigmentosa: through light and SNP. In the Berteporfin exposure mode, the photoreceptor degeneration will limit the exposure area. This model helps induce local photosensitive lesions, such as age-related luteal degeneration. In contrast, SNP induces degeneration of photoreceptor cells throughout the retina. These two models may be useful for regeneration research such as iPS transplant development and gene therapy of retinal prostheses.