动物造模,药效评价,干眼模型 z-bio 2024-07-02 570

　　1. Surgical removal of lacrimal gland, Harder's gland, and creation of a tear deficient rabbit dry eye model with the third eyelid. Specific operating methods for surgical removal of lacrimal gland, Harder's gland, and third eyelid: New Zealand white rabbits were given three consecutive days of antibiotic eye drops in both eyes before surgery, with 3% pentobarbital sodium 40mg/kg administered intraperitoneal anesthesia and fixed on an experimental table. The surgical eyes were routinely disinfected and covered with gauze; Push the eyeball forward, search for the lacrimal gland at the posterior wall of the eye socket, and remove it completely; The main duct of the Harder's gland can be seen on the inner side of the third eyelid, gradually and completely separating the Harder's gland along the duct. The gland is divided into two parts: the smaller part in front of the backrest side is white, and the larger part in front of the ventral side is light red, with a total length of about 2cm. The third eyelid and Harder's gland are completely removed; Apply antibiotics, steroid eye drops, and eye ointment after surgery.

　　The composition of rabbit tear film is similar to that of human tear film. The tear gland is an important source of tear film fluid layer, and the Hasselblad gland has a similar effect to the meibomian gland and sebaceous gland. The third eyelid has a protective effect on the ocular surface. Therefore, removing the tear gland and Hasselblad gland can cause a decrease in tear secretion (mainly the fluid layer) and eliminate the protective effect of the third eyelid on the ocular surface, resulting in tear deficient dry eye. Francois et al. confirmed the feasibility of this method. However, due to the development of goblet cells in rabbits and the fact that this method does not damage the accessory lacrimal gland, the formation of dry eyes often takes 2 months or even longer, and sometimes even cannot form obvious dry eyes. If the lacrimal gland is not completely removed, it can lead to the failure of making a dry eye model. In addition, due to the wide distribution of tear glands in rabbits and significant surgical damage, the quality of tear film in the early stages of this model is greatly affected by surgical interference. Gibard et al. made certain improvements to the above method. He did not remove the lacrimal gland, but instead used a slender probe to insert into the opening of the lacrimal duct and burn it, sealing it and preventing the discharge of tears. This method reduces the stimulation and interference of surgery on the ocular surface, and its effect is similar to that of tear gland removal. However, there are certain difficulties in finding the opening of the tear gland duct. Xie Hanping in China improved Francois et al.'s surgical method by removing the lacrimal gland, Hasselblad gland, and third eyelid. He used a fine cotton swab dipped in 50% trichloroacetic acid to apply to the conjunctiva 3mm away from the corneal edge. When the conjunctiva turned white, he slowly rinsed it with physiological saline. This method destroyed the accessory lacrimal gland and goblet cells, making dry eyes more likely to occur and more severe. However, this method has greater interference with the ocular surface in the early stages. Burning such a large area of conjunctiva with 50% trichloroacetic acid can easily cause necrosis and infection of the ocular surface epithelium in rabbits, ultimately leading to suppuration. Lang Lili and others improved Xie Hanping's method by dipping a fine cotton swab in 50% trichloroacetic acid and only applying it to the upper and lower conjunctiva, slightly reducing the burning area. The experimental results are still satisfactory. If we want to observe the therapeutic effect of drugs that promote the growth of goblet cells, using a concentration of trichloroacetic acid that is too high or burning for too long may cause complete damage to goblet cells and the therapeutic effect of such drugs cannot be observed. There have also been reports of simply removing the lacrimal gland without removing the Hasselblad gland and creating a tear deficient dry eye model with the third eyelid, but their success rate is lower.

　　2. Creating a tear deficient rabbit dry eye model by disrupting the tear secretion reflex arc. Gilbert used a radiofrequency probe to insert into the trigeminal ganglion of the rabbit and remove nerve innervation on the surface of the eye. It was found that there was a significant decrease in tear secretion, an increase in tear osmotic pressure, and a decrease in goblet cell density, exhibiting significant dry eye changes. The principle may be that the tear gland, ocular surface components, and their nerve innervation form a complete functional unit. If any link is damaged, the tear film used to maintain normal ocular surface structure will be affected. One drawback of this method is that it is difficult to accurately locate the trigeminal ganglia. In addition, if the temperature control is improper during operation, it may also damage the motor branches in the trigeminal nerve.

　　3. Using atropine to inhibit tear secretion to create a tear deficient rabbit dry eye model. The lacrimal gland is innervated by the parasympathetic nerve, and excitation of the parasympathetic nerve can cause an increase in tear secretion, while parasympathetic receptor blockers can lead to a significant decrease in tear secretion. Atropine is a common parasympathetic receptor blocker. Burgalassi et al. applied 1% atropine eye drops locally and quickly produced dry eye signs such as fluorescein staining, tiger red staining, and Schirmer's test decline, but the authors only observed them for 6 days. Xiao Qiguo et al. used the same method to observe the dry eye model for 35 days and found that the dry eye signs were most obvious about one week after medication. With the extension of time, the performance of the dry eye gradually decreased, indicating that this dry eye model cannot be maintained in an ideal state for a long time. Moreover, throughout the entire experimental process, there was no significant decrease in goblet cell density observed in the Western blot cytology examination, and no abnormalities were observed in the pathological sections of the cornea, conjunctiva, and eyelids. This suggests that local administration of atropine eye drops may not cause significant damage to the surface epithelial cells of the rabbit eye in the short term. Therefore, this dry eye model is not suitable for long-term experimental observation research. In the evaluation of the efficacy of dry eye drugs, this model should be carefully selected based on the duration of medication to avoid the occurrence of false positives.

　　4. Zhu et al. removed one eye of a rabbit's lacrimal gland and cultured autologous peripheral blood lymphocytes in the excised lacrimal gland epithelial cells. After 5 days, the mixed cell fluid of the cultured lacrimal gland lymphocytes was injected into the contralateral lacrimal gland to induce the production of autoimmune response in the contralateral lacrimal gland tissue. Two weeks later, a dry eye model similar to Sj ö gren syndrome was induced. This method is more complex to operate, and both lacrimal glands are damaged at the same time, which is not conducive to self control. Due to the limited availability of monoclonal antibodies for anti rabbit use in the current market, further examination of relevant inflammatory factors or cytokines is limited after dry eye formation.

　　5. Specific method for making a castrated male rabbit dry eye model: The experimental rabbit was anesthetized with 0.05g/ml ketamine 50mg/kg intramuscular injection, and the scrotum was subcutaneously anesthetized with 2% lidocaine. One side of the testicle was squeezed into the scrotum through the abdominal cavity and pinched tightly, without sliding it. A disinfection blade was used to make a small incision in the scrotum, and the testicles were forcefully squeezed out. The spermatic vein and vas deferens were ligated, and the testicles and epididymis were removed. After continuous suturing of the scrotal skin, iodine tincture was applied locally to prevent infection. The other testicle and epididymis were removed using the same method.

　　Numerous studies have shown that androgen levels play a crucial role in the secretion function of the tear gland and meibomian gland, as well as in the inflammation and cell apoptosis processes during dry eye disease. Research by Luo Fengnian and others has shown that castration in male rabbits can lead to a decrease in testosterone levels, atrophy of lacrimal gland epithelial cells, disappearance of glandular vesicular mucus, and reduction of conjunctival goblet cells, resulting in changes in the quality and quantity of tear secretion, dryness and inactivation of ocular surface epithelium, and decreased tear film stability. Ma Yiqun et al. found that after 3 months of castration, the tear secretion of male rabbits significantly decreased, and the cornea became dry and thin, with punctate ulcers. There were scattered apoptotic cells in the corneal epithelial cells, and the number was significantly higher than that of the normal control group. Therefore, it is believed that cell apoptosis may be one of the important reasons for corneal thinning, cell shrinkage, and death in patients with dry eye disease, and a decrease in androgen levels may induce apoptosis. The advantage of this model is that it does not damage normal eye tissue, the technique of testicular resection surgery is easy to master, the dry eye symptoms of the model are obvious, the model is relatively stable and not easy to repeat.