动物造模,单眼局限性感光细胞变性模型 z-bio 2021-05-25 326

　　Background: Retinitis Pigmentosa (RP) is a degenerative disease of the retina that can cause night blindness and ultimately lead to loss of peripheral and central vision. Several genes responsible for RP have been identified, most of which are related to the light transmission pathway. The results of these studies have not yet led to the discovery of effective treatment or prevention strategies. The artificial retina has been studied as a potential tool to restore the vision of these patients by stimulating the remaining retinal neurons to cause hallucinations. Various animal models of rodent photosensitivity have been developed to study visual function. CS rats spontaneously mutated the Mertk gene, resulting in degeneration of photoreceptor cells. Continuous light irradiation is another technique used to study the mechanism of light denaturation. The rabbit photosensitivity degradation model undertakes complex behavioral tasks and more closely simulates the human visual function. 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 long-term 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 breaks down and releases nitric oxide (NO) in the blood, acting as a vasodilator. In the retina, excessive NO induces degeneration of photoreceptor cells. Intravitreal injection of sodium nitroprusside can cause extensive photodenaturation. Two-dimensional teporphin and SNP induced photoreceptor degeneration had no obvious effect on the contralateral eye. The benefits of the photodenaturation monocular model include using the eyes of the same animal as a control. This reduces the number of experimental animals used.

　　Method: Follow the instructions to use Berteporfin. 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 a 5% glucose solution. The solution was injected intravenously with 0.5 mg/kg at a rate of 1 ml/min. The SNP was dissolved in 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 retinal degeneration was induced with verteporfin and pupil dilation with 1% atropine and 2.5% phenylephrine hydrochloride, the retina was exposed to a halogen lamp 10 mm from the cornea. In the second model, obexetine hydrochloride was locally applied to the eye using an operating microscope, a small incision was made to expose the sclera, conjunctiva, and a 30-gauge needle, and then 100μLSNP solution was applied to the vitreous. . Two weeks later, a hand-held fundus camera was used for fundus photography. ERG records were obtained one month after treatment. The white rabbits adapt to the darkness overnight, and dilate their pupils with 1% atropine, 2.5% phenylephrine hydrochloride and 0.5% propakin hydrochloride for corneal anesthesia. Place a small contact lens with a gold wire loop on the cornea, and place the silver wire reference electrode between the eyes under the skin. The white LED pulse is activated, produces 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 previously described by Tomita et al., the retinal morphology of eyes treated with Verteporphin and SNPs was analyzed. The animals were sacrificed by intravenous injection of sodium pentobarbital. The eyeball was removed, fixed, embedded in paraffin, a 3 micron thick retina was cut, and stained with hematoxylin-eosin. Use GraphPad Prism software for statistical analysis, the statistical significance standard is P\u003c0.05,

　　Results: There was no significant change in the untreated group and the unexposed Berteporfin group. After exposure, intravitreal injection of verteporfin treatment group or sodium nitroprusside caused retinal atrophy. The degree of atrophy depends on the exposure time or SNP concentration. When the retina is exposed to light for 10 minutes, the pigmentation of retinal pigment epithelial cells (RPE) can be clearly observed. As the time of light becomes longer, the pigment epithelium is obviously marked as RPE atrophy. Verteporfin and the lesions caused by exposure clearly limit the exposed area. However, SNP may cause peripheral retinal degeneration. Fluorescein angiography showed bleeding around the optic nerve, and 1 mmol SNP was observed. Compared with untreated eyes, the ERG amplitude (waves a and b) of the eyes treated with verteporfin was slightly reduced. As the exposure time continues, the amplitude of the b-wave decreases. After treatment with SNP of the eye, the amplitude will decrease as the concentration of SNP increases. Even if only 0.1 mmol 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 at least 10 minutes of exposure time to develop. In the SNP-induced degradation model, the lesions include the lining and photosensitive layer of the surrounding retina. Discussion: The photosensitizer verteporfin is widely used as part of photodynamic therapy (PDT) to treat 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. It is toxic to photoreceptor cells and retinal pigment epithelial cells. Fundus photographs show that after intravenous administration of weltiporfin, the photoreceptor damage caused by light exposure is limited to the irradiated area. SNP releases nitric oxide through a photochemical reaction and reduces metabolites in a variety of ways, such as mercaptans and microsomal organelles. 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 macular degeneration. In contrast, SNPs induce 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 artificial retina).