单眼局限性感光细胞变性模型 z-bo 2020-12-02 588

　　Background: Retinitis pigmentosa (RP) is a degenerative disease of the retina that causes night blindness and eventual loss of peripheral and central visual fields. Some 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 cause hallucinations by stimulating the remaining retinal neurons and have been studied as a potential tool to restore vision in these patients. Various animal models of rodent photosensitivity have been developed to study visual function. RCS rats carry the spontaneous mutation of Mertk gene and cause photoreceptor degeneration. Continuous light irradiation is another technique used to investigate the mechanism of photodenaturation. The rabbit model of photosensitive degeneration will undertake complex behavioral tasks and more closely simulate human visual functions. We report 2 methods to generate a rabbit model of monocular photoreceptive degeneration. Verteporfin is a photosensitive dye that is used clinically without any side effects. In preclinical studies, verteporfin and/or extended exposure induced damage to photoreceptors and retinal pigment epithelial cells. Verteporfin is used to induce degeneration of local retinal photoreceptors. Sodium Nitroprusside (SNP) is used to treat high blood pressure. SNP breaks down and releases nitric oxide (NO) in the blood, acting as a vasodilator. In the retina, excessive NO induces photoreceptor degeneration. Intravitreal injection of sodium nitroprusside induces extensive photodegeneration. Two-dimensional Teporfin and SNP-induced photoreceptor degeneration had no obvious effect on the contralateral eye. The benefits of the photodegeneration monocular model include the use of the same animal eye as a control, which reduces the number of experimental animals used.

　　Method: Use verteporfin according to the instructions. In our research, we used a halogen reflector lamp as the light source. 2 mg of verteporfin was dissolved in 7 ml of sterile distilled water, and the concentration was adjusted to 0.1 mg/ml with 5% glucose solution. The solution was injected intravenously at 0.5 mg/kg at a speed of 1 ml/min. SNP was dissolved in physiological saline, and SNP of different concentrations was injected into the vitreous cavity of rabbit eyes. Eighteen male Dutch rabbits were used. The rabbits were anesthetized by intramuscular injection of ketamine (66 mg/ml) and xylazine (33 mg/kg). Verteporfin was used to induce retinal degeneration. After pupil dilation with 1% atropine and 2.5% phenylephrine hydrochloride, the retina was exposed to a halogen reflector lamp 10 mm from the cornea. In the second model, obecaine hydrochloride was applied locally to the eye, using an operating microscope, a small incision was made to expose the sclera, conjunctiva and a 30-gauge needle to inject 100 μL of SNP solution into the vitreous. Two weeks later, fundus photography was taken using a handheld fundus camera. Obtain ERG records 1 month after treatment. The white rabbits were dark adapted overnight, and were dilated with 1% atropine, 2.5% phenylephrine hydrochloride, and 0.5% proparacaine hydrochloride corneal anesthesia. The gold wire ring small contact lens is placed on the cornea, and the silver wire reference electrode is placed between the eyes under the skin. A white LED pulse is activated to produce a flash, and the light stimulation lasts for 10 milliseconds. Record the full-field dark vision ERG, band-pass filter from 0.3 to 500 Hz, and take the average of 5 responses for each light intensity. The ground electrode is clamped on the tail. White light LED (7500Kelvin) is used for white stimulation. The analysis of verteporfin and SNP-treated eye retinal morphology was performed as previously described by Tomita et al. The animals were sacrificed by intravenous injection of sodium pentobarbital. The eyeballs were removed and fixed, embedded in paraffin, and a three-micron thick retina was cut and stained with hematoxylin-eosin. Statistical analysis was performed using GraphPad Prism software, and the standard of statistical significance was P<0.05.

　　Result: There was no significant change in the untreated group and the verteporfin group without light exposure. After exposure, the verteporfin treatment group or intravitreal injection of sodium nitroprusside induced retinal atrophy. The degree of atrophy depends on the exposure duration or SNP concentration. When the verteporfin treatment retina is exposed to light, even for only 10 minutes, the pigmentation of retinal pigment epithelial cells (RPE) can be clearly observed. With the prolongation of the light time, the pigmentation is obviously marked as RPE atrophy. Verteporfin plus light exposure clearly limits the area exposed to light. However, SNP can cause peripheral retinal degeneration. Fluorescein angiography showed bleeding around the optic nerve looking at 1 millimolar SNP. The amplitude of ERGs (waves a and b) was slightly lower in verteporfin-treated eyes than in untreated eyes. The b-wave amplitude decreases as the exposure time continues. SNP treatment eyes, the amplitude decreases with the increase of SNP concentration. Even if only 0.1 mM SNP was injected, the b wave amplitude of ERG was significantly reduced. Verteporfin treatment without light irradiation does not induce photoreceptor degeneration. However, neurodegeneration of the retina (mainly photoreceptor cells) takes at least 10 minutes of exposure. In the SNP-induced degeneration model, the lesions include the peripheral inner retina and the photosensitive layer.

　　 Discussion: The photosensitizing dye verteporfin is widely used as a part of photodynamic therapy (PDT) for the treatment of CNV. However, PDT is not a pure choice of choroid and may cause damage to the retina. PDT induces dose-dependent damage to photoreceptor cells and retinal pigment epithelial cells. With the absorption of specific wavelengths, oxygen free radicals are generated, which are toxic to photoreceptor cells and retinal pigment epithelial cells. Fundus photography showed that after intravenous injection of verteporfin, the photoreceptor damage caused by exposure to light was confined to the illuminated area. Through a photochemical reaction, SNP releases nitric oxide, which reduces metabolites including thiols and microsomal biological organelles in various ways. NO participates in a variety of retinal functions. Endogenous NO enhances the pyramidal response during light adaptation, sometimes causing retinal toxicity and contributing to damage caused by ischemia. Under normal circumstances, in ischemia or continuous intense light exposure, the severity of retinal degeneration depends on the local NO level.

　　 Conclusion: Our results emphasized two methods of inducing rabbit retinitis pigmentosa: through light and SNP. In verteporfin-exposure mode, photoreceptor degeneration can limit the area exposed to light. This model is useful for inducing local photosensitive lesions, such as age-related macular degeneration. In contrast, SNP induces degeneration of photoreceptors throughout the retina.