动物造模 z-bo 2020-09-08 437

　　Introduction: Clinicians will treat severe facial and eyelid burns, leading to severe eye injuries, loss of vision and/or blindness. Not only these injuries but also widespread alkaline or acidic substances, accounting for 11.5-22.1% of ocular trauma, chemical and thermal injuries are the most difficult to treat clinically. In the case of chemical burns, alkali burns are more severe and harsh because the alkali agent penetrates the eye tissues more quickly due to the lipophilic nature. This leads to tissue necrosis and ischemia. From a molecular point of view, exposure to alkaline compounds can cause the saponification of fatty acids in the cell membrane because they penetrate the corneal matrix and destroy proteoglycans and collagen. When the corneal alkali burn heals, the damaged tissue secretes proteolytic enzymes, causing additional and continuous tissue damage. For these reasons, the clinical rehabilitation of corneal alkali burn patients is often very challenging, due to the long-term complications of corneal epithelial erosion and chronic inflammation leading to vision loss. Patients with destructive facial burns often cause damage to the periorbital tissues (ie conjunctiva, periorbital fat, glands, and eyelids). Regardless of whether the patient has suffered chemical burns or thermal burns, when the ocular surface does not heal in time, the visual loss that may occur due to these injuries is the same. Therefore, clinicians need more advanced treatment methods to treat these patients. Although amniotic membrane, buccal skin transplantation, tarsal suture (partial suture of the eyelid), and bandage contact lenses can be used as treatments, these treatments fail for the most severe eye burn patients. In severe trauma, topical treatments such as ascorbic acid drops, citrate drops, corticosteroids or bandage contact lenses can be combined with amniotic membrane transplantation, limbal stem cell transplantation or corneal transplantation to resolve any corneal scars or opacity. When limbal stem cell transplantation cannot be used to repair a stable ocular surface, corneal transplantation may be required. However, despite interventions, undesirable results often occur. In addition, relying on topical eye drops management is not enough. The topical eye drops dissipate almost completely after instillation and must be used frequently. A new drug has been developed to eliminate the need for wound administration while promoting wound healing. The drug delivery system is a proprietary sulfhydryl cross-linked hyaluronic acid (CMHA) polymer for ocular use and has been shown to accelerate wound closure after photorefractive keratectomy. In addition, when used as a liquid gel, due to the unique engineering, this polymer stays on the ocular surface for more than 2 hours. However, this polymer can also be manufactured as a thin, flexible film that can be inserted into the lower fornix to provide sustained release of the therapeutic agent. Cross-linked CMHA-S is a multifunctional biocompatible polymer that combines the recorded wound healing properties of HA with drug delivery capabilities to provide an innovative ophthalmic treatment when quickly installed on the ocular surface. Due to the inherent healing properties of this polymer, we studied this new CMHA-S treatment to treat the most challenging ocular chemical burns and alkali chemical damage.

　　Method: Animals: male New Zealand rabbits (2.5-4 kg), randomly divided into groups (5 per group). The three treatment groups consisted of animals with uninjured eyes receiving CMHA-S membrane, animals with corneal alkali burns receiving CMHA-S membrane treatment, and animals with corneal alkali burns not receiving CMHA-S membrane treatment. All animal operations are performed on anesthetized animals.

　　Instant membrane removal: Remove the instant membrane three weeks before corneal alkali burn wound or membrane placement. Before the nictitating membrane was removed, ketamine hydrochloride (35～45 mg/kg), buprenorphine (0.5 mg/kg) and xylazine (5 mg/kg) were administered by intramuscular injection (IM). Fluoroether (1～3.5%). The left eye of the rabbit was anesthetized with 0.5% tetracaine hydrochloride. Disinfect the surgical site with a povidone-iodine solution (5% to 10%) diluted in PBS. Clamp the nictitating membrane, and inject 0.1-0.2 ml lidocaine hydrochloride and 1,100,000 USP of epinephrine into the posterior and anterior base of the nictitating membrane. Westcott scissors are used to remove the nictitating membrane. Use WEK-CEL sponge to remove blood and cauterize the surgical site with high temperature cautery. Apply bacitracin zinc and polymyxin B sulfate eye ointment to the surgical site. The animal is placed in an oxygen chamber (39-40% oxygen) to recover. The animals were examined twice a day, treated for three days, treated for pain, and healed in three weeks.

　　Corneal alkali burn: Soak 52, 175μm thick, 0.015% ash, round filter paper in 1N NaOH for 60 seconds, then place them on the central cornea of the anesthetized rabbit for 30 seconds. The corneal alkali burn is 5.5 mm in diameter. Measure the cornea with a caliper to ensure the central burn wound. The wound was immediately washed and debrided with 10 ml of balanced salt solution.

　　Placement of CMHA-S membrane: Combine the mercaptocarboxymethyl HA solution in PBS (final concentration of 16 mg/ml) and polydiacrylate solution (PBS) (final concentration of 15 mg/ml) to prepare CMHA －S film. The obtained solution was introduced into a silicone resin mold (3 mm wide × 9.9 mm × 1 mm thick), cross-linked for 2 hours, and dried at room temperature. . Upon rehydration, the CMHA-S film exhibited swelling, resulting in a rectangular, transparent and flexible film with a width of 4 mm and a length of 15 mm. The dried CMHA-S membrane was partially rehydrated in a sterile BSS for two minutes and then placed in the lower left fornix. Some membranes were exposed to 0.1% Rose Bengal dye before placement for visualization.

　　Corneal imaging: at 0 hours, 48 hours, 96 hours, and on the 14th day after injury, images were obtained with a camera (7D) with a 100 mm macro lens. The corneal wound was stained with 100 μL sterile BSS sterilized sodium fluorescein eye drops USP. The fluorescein solution was allowed to remain on the eye for 10 seconds, and then rinsed with BSS. The image was taken after cobalt blue staining. Quantify the area of wound closure and the area of corneal opacity by using Image J software. Compare each time point with 0 hours after injury. After fluorescein imaging, an optical coherence tomography (OCT) image is obtained. Use Image J software to measure the OCT image of the widest part of the central cornea. In vivo confocal microscopy images were obtained on uninjured healthy control animals, but could not be obtained on the injured cornea due to corneal opacity.

　　Histology: The histology on the 14th day after the eyeballs of the euthanized rabbits were removed, fixed in a modified Davidson solution for 24 hours, and then transferred to 10% neutral buffered formalin. The tissues were embedded in paraffin, sectioned (3～4μm), stained with hematoxylin and eosin (H&E) or Masson trichrome.

　　Results: Lower fornix retains CMHA-S: Although previous studies have demonstrated the ability of cross-linked CMHA-S to promote corneal repair in drop formulations, whether CMHA-S in the hydrogel film is still retained in the lower fornix The effective treatment is unclear. Therefore, retention membrane is an important part of our research. After 96 hours of placement in the inferior fornix, the membrane is safely retained and has biocompatibility and structural integrity. Daily observations of the films indicate that although the films are not completely degraded or dissolved, some films show slight abrasion and tearing, which may be due to the mechanical force of eye movements. No animal observed any irritation, redness, or swelling of the eyes or ocular structures due to the placement of the membrane.

　　CMHA-S membrane is biocompatible: the CMHA-S membrane is placed in the inferior fornix of an uninjured healthy eye animal and compared with a healthy untreated control group (without CMHA-S membrane). 14 days after the placement of the CMHA-S membrane, white light imaging, fluorescein staining, in vivo confocal microscopy, and corneal surface histological examination were performed. White light imaging showed no signs of corneal opacity. Lack of fluorescein uptake indicates no corneal epithelial defects in any group. HE and Masson trichrome staining also did not reveal any pathological changes.

　　CMHA-S film treatment leads to a reduction in the area of corneal opacity: After placing CMHA-S on the burn wound, white light images are taken at 0 hours, 48 hours, 96 hours and 14 days to observe the corneal opacity. At 48 hours, our results showed that in the eyes treated with CMHA-S, the area of corneal opacity decreased by 17%, while untreated alkali burns decreased by 0%. At 96 hours, the area of corneal opacity in the CMHA-S treatment group decreased by 30%, and the corneal refractive power of the non-treatment group decreased by 4%. This difference continues to increase over time. On the 14th day after the injury, a 50% significant decrease in opacity was observed in the CMHA-S-treated eyes, while a 16% overall decrease was observed in the untreated injury. The results of the study showed that compared with untreated injuries, the area of corneal opacity in CMHA-S treated trauma was significantly reduced, and the difference between the two groups increased over time.

　　CMHA-S membrane promotes corneal epithelialization: Fluorescein images are taken at 0 hours, 48 hours, 96 hours, and 14 days after corneal alkali burn, and CMHA-S membrane is placed, and corneal epithelial defects are detected by fluorescein uptake. The uptake of fluorescein treated with CMHA-S membrane at 48 and 96 h and 14 days after injury was less. The uptake of fluorescein at 48 hours showed that CMHA-S treated wounds exhibited 99% RE epithelialization at 48 hours, compared with about 86% re-epithelialization of untreated control wounds. The upper epithelial re-epithelialization rate increased by 13%.

　　CMHA-S treated alkali burns showed reduced corneal swelling.

　　CMHA-S treatment of alkali burns showed reduced corneal swelling: In order to determine the effect of CMHA-S film treatment on the total corneal thickness, OCT images were taken at 0 hours and 14 days after injury. OCT images showed no difference in corneal thickness at 0 hours, and the average corneal thickness in the two groups was 400μm. On the 14th day, the corneal thickness of the CMHA-S treatment group in the burn group (779μm±70) and that of the untreated burn group were about (975μm±67), the difference was statistically significant.

　　The histology of the corneal surface after treatment in the two groups was normal: H&E staining after central burn showed that there was no significant difference in the pathology of the cornea between CMHA-S treatment and untreated burns at 14 days after the injury. No new blood vessels were observed, and inflammation was minimal. The H&E section showed that a thin layer of epithelium appeared when it was removed on the 14th day, but the two groups did not return to full thickness.

　　Conclusion: The hydrogel form of CMHA-S has been shown to be beneficial to the re-epithelialization of the cornea after alkali burn. CMHA-S membrane may be a feasible treatment to promote re-epithelialization while reducing corneal opacity and swelling.