动物造模 z-bio 2022-07-12 574

　　INTRODUCTION: Age-related macular degeneration, Stargardt disease, and retinitis pigmentosa are common RD disorders that often lead to irreversible blindness. Many of the treatments currently in use and in development, such as gene therapy and trophic factors, work only in the early stages of disease when host photoreceptors are damaged but still alive and can be rescued. Rescue treatment is unsuccessful when the photoreceptors degrade irreversibly. Because retinal ganglion cells survive severe photoreceptor cell loss, the most effective current treatment may be to replace photoreceptor cells and other retinal cells. Numerous studies have shown that, so far, fetal donor tissue has become a very effective option for CNS transplantation. Further research showed that this also applies to retinal transplants to replace cells from the degenerated retina. Later studies of transplanted photoreceptor precursor cells have not reported any satisfactory results in vision improvement. It is suspected that this may be due to the low number of photoreceptor precursors integrated into the retina. Isolated cell transplantation has advantages because the procedure is simpler than transplantation of aggregates and sheets. However, isolated cells cannot form lamellar tissue and have limited long-term survival. Transplantation of cell aggregates can form rosettes rather than parallel stacks. Previous work has shown that a degenerated retinal region is replaced by a fetal plate with or without RPE, forming lamellar grafts in rodents. In a rat model of RD, rat fetal retinal sheet transplantation restored visual responses in the superior colliculus region corresponding to the location of the retinal graft. Clinically, it is important to know whether to transplant human fetal retinal sheets together with their retinal pigment epithelial cells into the subretinal space. Studies using naked immunodeficient rats with normal vision showed that retinal pigment epithelium transplantation with RPE can form lamellar structures after prolonged survival, leading to the first clinical trial of combined transplantation. In a phase II clinical trial, 7 out of 10 patients had improved visual acuity one year after retinal pigment epithelium transplantation. However, improved visual function after retinal transplantation has not been tested in models of retinal degeneration. Since the ultimate goal is transplantation in human retinal degeneration patients, the ability of human retinal sheet transplantation to integrate into the remaining host architecture and the extent to which visual function is restored needs to be tested in animal models. Compared to rat tissue, human tissue develops and matures at a slower rate, necessitating an extended period of time required for experimental analysis. The current study aims to address these issues. Immunosuppression is commonly used when transplanting tissues such as rats. However, immunosuppression results are unreliable and can cause pain and damage to animals. For example, the immunosuppressant cyclosporine A may have nephrotoxic effects and reduce visual responses in RCS rats. Therefore, we developed a double mutant rat with FXN1-/- and Rho S334 Tor-/-Line-3 rhodopsin mutations that is both immunodeficient and retinal degeneration. This rat model eliminates the need for immunosuppression when transplanting xenografts and also eliminates chronic immune rejection of allografts. This study assessed visual function in a nude mouse model of RD following human retinal transplantation by extracellular recordings from the superior colliculus (SC). OCT and immunohistochemistry were used to study graft development and interactions between donor and host. The data suggest that human fetal retinal plate transplantation can improve visual function in a rat model of advanced retinal degeneration.

　　Animals: Animals were housed in IVC cages at a temperature of 21.5±0.8°C and a relative humidity of 50%. SD-Foxn1 Tg(S334ter)3Lav (RD nude mice) was obtained by crossing SD TG(S334TER)3LAV rats with NTac:NIH-Whn rats.

　　Donor tissue isolation and transplantation procedure: Human embryonic eyes (11 to 15.7 weeks after conception) were obtained from HSCRO-approved sources and shipped overnight in Hibernate E medium with B27 supplement. Donor human retinas were isolated in Hibernate E medium and processed as previously described. At 37°C, the donor eye was placed under the action of 37°C and 10% neutral protease for 30-40min, so that the retinal pigment epithelium and retina were detached from surrounding tissues. Donor tissue was taken from a different area of the fetal retina other than the macula, as the RPE is easily dissociated in this area. . Before transplantation, insert a small piece of donor tissue (average 0.7 mm × 1.2 μmm) into a custom-made graft instrument. Retinal sheet transplantation was performed as described above. Recipient rats were anesthetized with ketamine (40-55 mg/kg)/xylazine (6-7.5 mg/kg). A 1mm incision was made behind the flat part, followed by a localized mechanical retinal detachment. To prevent eyelid swelling, rats were injected subcutaneously with ketoprofen (4 mg/kg) before anesthesia. Dexamethasone eye drops + tetracaine and atropine in the treatment of common eye diseases. Donor retinal graft tissue was delivered into the subretinal space of the left eye using a custom implanter. The incision is closed with 10-0 sutures. The sham was injected with media only. For recovery, rats were injected subcutaneously with Ringer's saline solution and then placed in a heat-care incubator for recovery. The surgical group was treated with betatin and triple antibiotic ointment (neomycin/bacitracin/polymyxin). For pain management, animals also received butyl propyl ester (0.03 mg/kg) for analgesia.

　　OCT: Rats were anesthetized with ketamine/xylazine, pupils were dilated with atropine, and SD-OCT images of the retina were acquired with a PixiGEN EnVISU-R2200 spectral domain ophthalmic imaging system as previously described. Transplanted rats were imaged every 1–2 months starting 2 weeks after surgery, with the last scan as close to the end of the experiment (SC recording) as possible.

　　Histology and immunofluorescence: Rats were perfused with cold 4% paraformaldehyde in 0.1 μm sodium phosphate buffer. Eyeballs were removed and embedded. Cryosections were prepared and HE stained.

　　Superior Colliculus (SC) Electrophysiology: Visual responses from the SC were recorded as previously described. Briefly, the responses of transplanted RD nude mice were recorded from 6.6 months to 9.5 months of age (5.8 months to 8.6 months post-surgery) and compared with those of age-matched non-transplanted nude mice and sham-operated rats. Reactions are compared. Multi-unit electrophysiological recordings were performed from 50-60 locations on the SC surface, approximately 200-400 μm. For each location, light stimuli (20 ms duration) were delivered approximately 10 times at 10 s intervals at intensities ranging from 0.58 to -6.13 log cd/m2. To detect response thresholds, when a response was found, the intensity of the light stimulus was reduced until there was no response.

　　RESULTS: Development and identification of human fetal retina: Donor tissues were isolated from five fetuses at gestational age 11-15.7 weeks (WKS). The cells in the tissue are retinal progenitors, which are still developing. In tissue from gestational week 12, strong CHX10 staining revealed retinal progenitors throughout the neuroblast layer. The ganglion cell layer visualizes ganglion cells by strong MAP2 (microtubule-associated protein 2) staining and NeuN (neuronal nucleus staining). A layer of differentiated cone photoreceptors, strongly stained by retinal RAX and OTX2 antibodies in the outer neuroblastoid layer (NBL). There was no rhodopsin staining at this stage (12 weeks). RPE is also immunoreactive to OTX2. At 11 weeks, only 1 layer of developing photoreceptors were stained in the outer NBL, but were recovered at 15.7 weeks. OTX2 and CHX10 were co-expressed in retinal NBL at 15.7 weeks.

　　OCT: Thirteen of 22 rats were successfully transplanted. Transplantation can be detected by OCT, as shown by OCT in Figure 2, human fetal retinal slices 1 month after transplantation are immature and only the IPL and outer neuroblastoid layer can be identified. About 3 months after surgery, the graft developed into the visible retinal layer. The grafts maintained the same structure until the end of the experiment, and in some cases external segments could be detected. A mixture of stacks and garlands is sometimes seen.

　　Photoreceptors: After transplantation into the subretinal space, fetal retinal sheets develop into mature, lamellar structures that can be identified as IPL, INL, OPL, and ONL layers. Mature photoreceptor cells were visible in the transplants 6 months after surgery. Revivin staining showed that part of the transplanted layer had obvious photosensitive layer. Although most transplants formed rosette structures (spheroidally arranged retinal layers centered on photoreceptors, n = 7/13), lamination within rosettes was still evident. There was no rhodopsin or cone transductin staining in the host, indicating complete loss of photoreceptors in the host at the time tested (5.8-8.6 months).

　　Bipolar cells: To study rod and pyramidal bipolar cells, PKCα and secretin antibodies were used, respectively. As shown in Figure 5, strongly PKCa-positive bipolar cells were found in the host retina. Co-localization of PKCα and SC121, a human cytoplasmic marker, after transplantation indicated that donor progenitor cells developed into mature rod-shaped bipolar cells after transplantation. Even in some grafts that only rosettes, the rosettes were in close proximity to the host rod bipolar cells and bound to the host. Also, after transplantation, some of the cells in the transplant developed into secretagogue-secreting pyramidal bipolar cells. Compared with rod bipolar cells, transplanted pyramidal bipolar cells were much less abundant than the host.

　　Superior colliculus (SC) electrophysiological recording to evaluate the improvement of visual function: 5.8-8.6 months after transplantation, the visual electrophysiological analysis of the transplanted rats was performed by electrophysiological recording method. Of the seven transplanted rats transplanted for SC electrophysiological recordings, four showed a response to a flash (0.58 log CD/m2) within a limited area of the SC after transplantation. In contrast, age-matched sham-operated rats or age-matched control non-operated rats showed no flash response.

　　CONCLUSIONS: This study demonstrates that transplanted human fetal retinal sheets continue to develop and differentiate into mature retinal cells in a severely degenerated environment and improve vision through direct connection to host retinal circuits. These findings provide important implications for the use of fetal retinal slice transplantation as a therapeutic strategy to restore vision. Ethically obtained fetal retinas can be used to improve vision in RD patients, while other approaches such as stem cell transplantation are still in development.