动物实验 z-bo 2020-09-05 425

　　Background: Corneal diseases and injuries can damage the cornea and eventually lead to blindness. The main treatment for corneal injury is corneal transplantation. However, two main problems restrict the clinical application of transplantation. These complications include severe postoperative complications, leading to graft rejection and insufficient supply of donor cornea. In the 1980s, penetrating keratoplasty (PKP) was the main transplant operation. Postoperative immune rejection is a serious complication. Following PKP, it can lead to graft endothelial dysfunction and reduced long-term survival. The survival rates at 1, 3, 5 and 10 years after transplantation were 84%, 69.3, 60.7%, and 59%, respectively. The most common cause of transplant failure is immune rejection. Followed by delayed endothelial failure and ocular surface complications, accounting for more than 74% of all failed cases. In recent years, under the condition that endothelial cells are not involved in corneal diseases, the thin-film technology that produces good clinical effects has rapidly replaced penetrating grafts. The purpose of this technology is to selectively replace diseased corneal stromal tissues to minimize unnecessary replacement of healthy endothelial layers. Lamellar keratoplasty (LK) is difficult and time-consuming, requiring conversion to PKP in 2.3-3.5% of cases. However, similar to visual penetrating keratoplasty, other limiting factors include corneal surface deformation, which is caused by differences in suture tension and graft host interface. After receiving femtosecond laser-assisted mosaic autologous or allogeneic transplantation, the rhesus monkeys did not suffer corneal rejection. In addition, the corneal lamellar mosaic model avoids irritating the surface of the eyeball and endothelial cells, and extends the survival rate of the graft to a certain extent. In addition, femtosecond laser cutting is a high-precision, safe and predictable technology, and it is a new choice for corneal refractive surgery and corneal transplantation. Femtosecond laser cutting can always achieve consistent depth and thickness in each part of the corneal stroma, and precisely match the donor and recipient tissues. Compared with ordinary laser cutting, femtosecond laser cutting offers the greatest advantage because it allows a high level of control and greatly reduces the invasiveness of surgery. Another clinical difficulty of corneal transplantation is the shortage of donors. The application of heterogeneous donors may help alleviate the insufficiency of the donor cornea because of its unique characteristics compared to other tissues and organs. . Recent studies have shown that low antigenic grafts can be used as support materials for corneal tissue regeneration. Glycerin dehydration has shown that the antigenicity of cells and donor corneas is greatly reduced, especially in the stroma. Therefore, glycerol dehydrated corneal sheet may provide a possible alternative source for inlay xenografts. This is currently an emerging research field. We have developed a new small incision femtosecond laser-assisted surgery to reduce the invasiveness of surgery and improve the matching ability of transplantation and donor tissue. We use femtosecond laser-assisted surgery to form inlay grafts, and dehydrate xenografts in glycerol to reduce their antigenicity. This study aimed to evaluate the biocompatibility of a small incision femtosecond laser-assisted surgery after implanting a heterogeneous corneal sheet and a fresh foreign body sheet into the cornea of rhesus monkeys. We also evaluated the biocompatibility of the glycerol dehydrated corneal lamellar as a corneal inlay.

　　Method: Research design: Femtosecond laser assisted surgery was used to obtain corneal lamina from three animals (human, rabbit, and monkey). Two types of xenografts (human and rabbit) were dehydrated with glycerol for 1 week. All transplantation and implantation of rhesus monkey corneal small incision stromal lens removal operation uses auxiliary surgery. Four monkeys received fresh allograft as a control group. The other 4 monkeys received the preserved human glycerol grafts, called the "human monkey group (H-M group). The other 4 monkeys received the rabbit glycerol preserved transplants, called the rabbit monkey group (RM group). After the operation, we were called every day The eyes of the monkeys were observed, and 12 monkeys at 1 week, 1, 3 months, and 6 months after transplantation were examined.

　　Animals: 5 adult New Zealand white rabbits and 16 5-year-old rhesus monkeys were used in this study. These animals were housed individually and received free food and water throughout the study.

　　Rhesus monkey allotransplantation: In the experiment, 4 rhesus monkeys chose to use Shutai 50 intramuscular injection (5 mg/kg) for anesthesia, and use femtosecond laser small incision matrix lens extraction (SMILE) to randomly select one eye for diopter-4 myopia treatment Correct. The femtosecond laser parameters are set as follows: 135nJ, 120μM cap thickness and 7.5mm diameter cap. A small incision (3 mm) with a side incision angle of 90° at the 140° position. Separate the corneal lamella (81-μm) as a fresh allograft.

　　Human corneal lamellar transplantation: 10 grafts were collected from 10 people who underwent SMILE surgery. The patient's equivalent spherical lens was 6-9 D. All patients agreed to participate in this study in writing. All surgical operations are performed by a surgeon. Femtosecond laser parameters are as follows: 135 nJ, 120 μM cap thickness and 7.5 mm diameter cap. The human corneal lamina (2.5mm) is extracted through the incision. The corneal sheet was soaked in sterile glycerin and dehydrated at 4°C for 1 week. In addition, they can be used as human corneal lamellar grafts.

　　Rabbit corneal lamellar transplantation: The animals were sacrificed with an overdose of anesthetics (Shutai 50, Virbac, 50 mg/kg, intramuscular injection). Using femtosecond laser assisted treatment to make rabbit corneal lamellar incision. A small curved interface cone is located in the center of the cornea. The surgical parameters are as follows: the incision diameter is 7.5 mm, the incision is located at a depth of 320 μm, and the side cut angle is 90°. Next, an excimer laser refractive keratectomy was performed to achieve −4 D myopia. Then we separate the corneal sheet from the stromal bed. The corneal lamella is soaked in sterile glycerin and dehydrated at 4°C for 1 week as a rabbit corneal lamella graft.

　　Femtosecond laser-assisted corneal transplantation: 12 macaques were anesthetized with Shutai 50 intramuscular injection (5mg/kg). Then, they received SMILE surgery to achieve a −0.75D correction for myopia in a randomly selected eye. Femtosecond laser parameters were used. : 135 nJ, 160μM cap thickness, cap diameter 7.8-mm, and lamellar cornea diameter 7.8-mm. A small incision was made at 140°, and the incision angle was 90°. The microlenses (28μm thick) are separated, creating a space similar to a "matrix pocket". The graft is implanted in the matrix pocket. During the implantation process, we avoided the turnover of the graft surface. After the operation, all monkeys received tobramycin and dexamethasone eye drops 4 times a day for 1 week. The eyes of the monkeys were observed every day, and the eyes of 12 monkeys at 1 week after transplantation and 1, 3 weeks and 6 months after transplantation were checked.

　　Clinical examination: Slit lamp examination is performed after surgery to evaluate changes in implant color, peripheral corneal epithelial transparency, neovascularization, corneal edema, rejection and implant degradation. The characteristics of the anterior segment of the eye, including corneal thickness, were evaluated using an anterior segment optical coherence tomography (OCT). The central thickness of the rhesus monkey donor cornea is about 450μm, and the graft thickness of 80μm is used to achieve a residual stromal thickness (RST) close to 250μm to stabilize the intraocular pressure of the monkey. In the allogeneic transplantation group, the predicted change was 81–28 = 53 μM.

　　Tear fluid collection and inflammatory mediator determination: Non-irritating tear fluid samples are collected using a disposable 10-l straw. Tear collection is performed in the lower border area, conjunctiva or eyelid margin. The kit was used to measure IL-1β, IL-6, IL-17, TGF-β1, TNF-α and IFN-γ in tears.

　　Result: Physical properties of the corneal lamella: A general observation showed that all corneal lamella remained transparent after glycerin dehydration.

　　Clinical observation: During the 6-month observation period, all macaques survived in both the allotransplantation group and the transplantation group without infection or bleeding complications. Within 6 months after the operation, there was no obvious blepharospasm, increased conjunctival redness, corneal neovascularization, signs of graft degeneration, and no noticeable reaction in the anterior chamber. The host corneal tissue remains transparent.

　　OCT observation: The corneal stroma was clearly visible after corneal transplantation in each group. During the 6-month observation period, the graft remained stable in the "matrix pocket". The corneal thickness increased 1 week after the operation, and there was no significant change in the corneal thickness 1 month after the operation.

　　Inflammatory mediators of tears: Measure the value of inflammatory mediators after operation and compare with those before operation. All tear inflammatory mediators changed significantly in the "rabbit-monkey" group. The change trend of tear media in the "human-monkey" group was similar to that of the "rabbit-monkey" group. The level of inflammatory mediators was low 1 month after operation, except for IL-1β, TGF-β1 and IFN-γ.

　　Conclusion: Studies have shown that glycerin dehydrated corneal transplantation remains transparent. After the operation, all monkeys survived without implant rejection or any adverse complications. Allografts and xenografts remain transparent and cause minimal inflammation. In addition, the inlay sheet matches the host tissue and is stable. Glycerin dehydrated corneal sheet has potential application prospects as a substitute for inlay heterogeneous materials. In addition, femtosecond laser assisted corneal transplantation has minimal invasion and can improve surgical efficiency. The host cornea also maintains a high degree of biocompatibility. In this study, we provide a new method for the treatment of high hyperopia, presbyopia, keratoconus, corneal bulging, and other diseases. We will also further study the potential xenogeneic immune mechanism and changes in corneal refractive status after implantation, in order to lay the foundation for future clinical applications.