动物造模-,药效评价,遗传性白内障 z-bio 2024-08-07 375

　　In clinical practice, partial or complete opacity of the crystalline lens that exists or gradually forms after birth is called congenital cataract. It is the main cause of blindness and amblyopia in children, and congenital cataract is a disease caused by abnormal metabolism of the crystalline lens during the embryonic stage, leading to a decrease in its own transparency. Many factors can lead to the occurrence of congenital cataracts in fetuses, and the genetic loci and etiological mechanisms of such cataracts are diverse.

　　1. Nakano mice were the first animal model used for hereditary cataracts and have been widely used in biochemical research. It was first reported by Nakano et al. in 1961, and Iwata and Kinoshita began studying it in 1971. Afterwards, Kinoshita's laboratory conducted nearly 10 years of research on the Nakano mouse model, and they were the first scholars to systematically attempt to determine the pathogenesis of hereditary cataracts. Since then, NakanO mice have been used as animal models for hereditary cataracts. This type of mouse was introduced by Dr. Iwata to the Kinoshita Laboratory at Harvard University and crossbred with normal Charkes River mice, with an autosomal recessive inheritance pattern. Cataract first appears in the fourth week after birth, initially presenting as a needle like opacity, and eventually the entire lens becomes opaque. Turbidity is accompanied by significant morphological changes and inhibition of protein synthesis, impaired enucleation on postnatal day 6, accompanied by swelling of the fiber endings in the perinuclear zone of the crystalline lens. The cytoplasm of the affected crystalline fibers contains granular material and many microcellular organelles. In vitro studies have shown that at 15 days, cells elongate, aggregate, and transparent bodies form. Electron microscopy shows that these transparent bodies are composed of immature lens cells, with many large vacuoles appearing in the cytoplasm. Immunofluorescence studies have shown that these transparent bodies are positive for alpha crystallin. Biochemical studies have shown that there is a permeable cataract, and after 2 weeks, the growth of the lens slows down, accompanied by calcium pump defects and swelling of the fiber tips. Around 20 days after birth, rapid increase in sodium concentration can lead to cataracts, which are caused by a deficiency in Na+- K-ATPase activity and are believed to be related to the onset of cataracts. Philly mice are derived from the Swiss Webster mouse line, first introduced by Kador. In 1980, it was reported that at 15 days after birth, a slit lamp examination revealed slight opacity around the lens sutures, which gradually worsened thereafter; At 30 days, obvious turbidity appeared around the nucleus; At 35 days, it significantly worsened; At 45 days, dense opacities and subcapsular opacities appeared in the nucleus. This type of cataract is a penetrative cataract characterized by enhanced hydration, increased sodium and calcium concentrations, and unaffected transport systems before turbidity appears. However, at 15-20 days, due to increased membrane permeability, sodium accumulation decreases. The enhancement of membrane permeability is a fatal defect that leads to permeable cataracts. The protein metabolism in the crystalline lens undergoes significant changes in weeks 3 and 8, accompanied by a significant decrease in beta and gamma crystalline proteins, due to increased protein hydrolysis and decreased protein synthesis related to alpha crystalline protein synthesis. This synthesis reduction is related to changes in ion concentration within the crystalline body, and it has been found that an antigen component of β - crystallin is missing. Fraser mouse cataract is a dominant cataract that occurs before birth,In 1962, Fraser and Schantach first reported that it was caused by a mutated gene called shrivelled, which is now known as Fraser mice or CATFR mice. Pathological changes can be detected within 10 days of pregnancy, with abnormal mitosis causing significant degeneration of multiple layers of lens nucleus epithelium and fiber cells. It has been reported that this type of cataract lens lacks β H-crystallin condensation, reduces the concentration of γ - crystallin peptides, and increases protein hydrolysis. Muggleton Harris et al. reported in 1987 that the gene for Fraser cataract is located on chromosome 10, and this gene is closely linked to the (LOP) lens opacity gene reported by Lyon et al. in 1981. Due to the similarity in phenotype between this type of cataract and Fraser opacity, the Fraser gene and LOP gene are considered to be likely alleles. The SAM mouse was first reported by Takeda et al. in 1981. Hosokawa research found that the incidence rate of SAM-P3 strain was the highest, with 27.5% having cataracts at the age of one year and 70.6% having cataracts at the age of 16 months. Cataracts are usually single eyes. In 1988, Hosokawa bred the SAM-R/3 strain. Cataract of this kind of mice can occur in about 10 weeks, and the incidence rate is 50% at 20 weeks. At the age of one year, the incidence rate of cataract of female mice is as high as 90%, and the incidence rate of male mice is kept at 50%~60%. Most animals finally form binocular cataract. The cloudiness begins at the posterior pole, and the posterior surface and cortical area of the posterior nucleus gradually increase, and the posterior pole protrusion and nuclear dislocation appear at maturity. Compared with normal cataracts of the same age, mature cataracts have a 10% to 20% increase in water content and a 50% decrease in protein content.

　　2. White Jie et al. selected 24 adult female Wistar rats in estrus with a body weight of 200-220g; 16 male Wistar rats weighing 260-280g. Randomly select 4 male rats and 6 female rats, with a male to female ratio of 3:2, and keep them in the same cage in a regular breeding room. On the second day at 8 o'clock, observe the female mice and mark those with vaginal plugs or sperm found under the microscope as day 0 of pregnancy. Afterwards, postpone and record the date of conception. After conception, female mice were randomly divided into a control group (Group A) and three experimental groups, namely, blocking uterine blood flow for 5 minutes (Group B), 10 minutes (Group C), and 20 minutes (Group D). Six experimental group rats were randomly selected each time. Experimental groups A, B, and C were clamped with hemostatic forceps on both uterine arteries for 5, 10, and 20 minutes, respectively, and blood flow was restored. The surviving mice in each group were euthanized 25 days after birth, and the lenses were removed and immediately fixed in glutaraldehyde at room temperature. Fresh fixative was replaced daily. After 3 days, the specimen was taken out and washed with phosphate buffer solution for 3 times, each time for 10 minutes. A cylindrical lens structure with a cross-sectional area of 1mm2 was cut along the equator under a microscope as a transmission electron microscope specimen. Due to the low risk of miscarriage in rats during the middle and late stages of pregnancy, surgery was performed on the 19th day of pregnancy as the optimal time to study the interruption of uterine blood supply. By comparing the intraoperative conditions, stillbirth rate, and lens transmission electron microscopy observation, the results showed that blocking the uterine artery for 10 minutes can successfully establish a congenital cataract model in fetal mice. Compared with blocking blood flow for 20 minutes, the surgical time is shorter, the required amount of anesthesia is relatively less, the postoperative recovery time is shorter, and the impact of anesthesia on pregnant mice and their fetuses is relatively reduced. In addition, this experimental method is simple to operate, cost-effective, and highly feasible. The duration of intrauterine hypoxia in fetal mice is easy to grasp and has good reproducibility. It provides a new experimental method for preparing animal models of congenital cataracts and exploring the occurrence and development of human congenital cataracts. ICR rat cataracts were first reported by Ihara in 1983, and at approximately 2 months, mild diffuse opacities appeared under the anterior capsule of the lens due to swelling of the lens fibers. After 3 months, dense opacities appeared in the posterior pole, quickly affecting the entire surface of the lens cortex and nucleus. By 4 months, opacities also appeared in the central area of the nucleus, with only the anterior surface and equatorial cortex transparent.After 3 months, there was significant swelling and degeneration of the fibrous cells in the posterior cortex and nucleus, showing autosomal recessive inheritance, and an increase in serum lipid peroxides, reaching its peak at 13 months. Electron microscopy showed the appearance of vacuoles in the posterior subcapsular lens, and abnormal oxidative stress occurred before the lens became cloudy. Ihara used slit lamp microscopy and histological methods to study ICR rats, and slit lamp examination showed that the lenses of these rats were completely transparent at one month; At 2 months, there was a slight diffuse opacity under the posterior capsule, especially around the suture line, while the rest remained normally transparent; After 3 months, dense opacities appeared in the posterior pole, which quickly extended to the entire cortex and the subnuclear area in the posterior region, then to the perinuclear area in the anterior region, and finally to the entire nucleus. At about 4 months, except for the anterior inferior and equatorial cortex, the rest of the lens appears completely cloudy. Upon histological examination, at one month, the lens fibers in the cortex (excluding the equatorial region) showed spindle shaped or spherical swelling, and no proliferation of lens epithelial cells or localized fiber necrosis was observed. At 2 months, a large number of swollen fibers appeared in the nucleus, with obvious degeneration. The nuclear fibers dissociated, atrophied, fused, disintegrated, and had diffuse swelling. Finally, many structures of different sizes appeared, indicating liquefaction. Calcium deposition occurs in the anterior part of the nucleus after complete maturation. Emory rats were first reported by kuck in 1982. They were bred from the Carworth Form Webster breed. The typical changes of lens occurred in 5 to 6 months. Cataracts were autosomal dominant inheritance. The age of onset, incidence rate and morphological changes of cataracts were very different. In the early stage of cataract formation, there is a decrease in GSH and SH protein, and in the late stage, there is an increase in lipid peroxidation, indicating the occurrence of oxidative stress response.

　　3.Compared to rodents, dogs have the closest lens to eye ratio to humans, which may be the most important reason for using dogs as an animal model for cataracts. Hereditary cataracts are either congenital or postnatal, and the lens can be partially affected or completely opaque, but the final outcome of cataracts is similar. Cataract in small bearded dogs appears 2 weeks after opening the eyes, with turbidity mainly appearing in the lens nucleus and posterior cortex, accompanied by microphthalmia. The cortical vacuoles and water gaps in the anterior and equatorial regions mainly appear from April to June. Cataracts mature between the ages of 1 and 3. The lens of this cataract reduces free GSH levels and increases protein type GSH levels.

　　4. Guinea pig (Dutch pig) 13/N was first reported by Stone and Amsbaugh in 1984, and nuclear cataract appeared at birth. This cataract is transmitted by a single autosomal dominant gene, accompanied by a loss or downregulation of zeta crystallin concentration. This protein is a class specific crystallin protein that appears in guinea pig lenses. A new protein closely related to zeta crystallin has been synthesized in cataract animals, which appears to be a product of gene variation in zeta crystallin.