| Corneal injury often occurs in canine clinical ophthalmology, which frequently induces blindness. Amniotic membrane has been confirmed to be an ideal biomaterial for transplantation due to its superior physical and chemical properties as well as its low immunogenicity. In order to explore the effective treatment of corneal injury, canine corneal alkali burn was induced in this study as a pathological model, and the amniotic membrane transplant (AMT) was used for the treatment. The clinical symptoms, histological changes and the relative expression of the TNF-α, VEGF and TGF-β1 genes in the corneas were observed.6 healthy adult Beagles were selected during the course of the experiment. The corneal injury model was made using circular filter paper of 6mm diameter with 1mol/L NaOH, which was placed onto the central cornea for 1 min, then rinsed with saline. The amniotic membrane was collected from fetal canine via caesarean section. The pieces of amniotic membrane were prepared and placed in DMEM glycerol solution, then stored at -80℃. The left eyes which received the alkali burn but no treatment were used as the control group, whereas the right eyes of the same canine were the experimental group, which received alkali burn and then treated using AMT. Corneal edema, neovascularization and eye secretions were observed daily post-surgery. The intraocular pressure (IOP) and tear volume were measured on days 3,7,14,21, 28 and 35 post-surgery and the corneal opacity, neovascularization and epithelial healing were scored on days 7,14,21,28 and 35. The corneal tissue was extracted on day 35 post-surgery and divided into 2 halves, one prepared for histopathology observation, including HE staining and Masson trichrome staining, and the other was used for qRT-PCR, to detect the relative expression levels of TNF-α, VEGF and TGF-β1 genes. The results are as follows:(1) Varying degrees of corneal edema and increased eye secretion were observed in the canine corneas of both groups. In the experimental group, the amniotic membrane was smooth and dissolved within one week. Neovascular buds appeared on day 5 post-surgery. Secretion volume returned to normal after 16 days. In the control group, brush-like gross neovascularization could be seen on day 5, one case of corneal perforation was detected on day 6, and excess secretions were observed in the eyes of two canines on day 28. The degree of corneal opacity and neovascularization was less in the experimental group compared to that of the control group on day 35 (P<0.05). Fluorescent staining on day 21 revealed a significant difference between the two groups (P<0.05), with the experimental group displaying a lesser degree of staining than that of the control group. On day 35, the experimental group showed no obvious staining unlike the control group, which still retained traces of dye. The IOP and tear volume increased in the experimental group on day 14, but remained within the normal range, whereas the control group had decreased IOP (P<0.01) and tear volume (P<0.05). By day 35, the indicators returned within normal parameters in both groups, however in the control group they remained in the lower limit.(2) On day 35 post-surgery, histopathological examination of the canine cornea of the control group presented a large number of inflammatory cells and new blood vessels containing mature red blood cells. Hypertrophic fibroblasts, swollen basal cells with increased intercellular gap and disorganized collagen fibers were also noted. The experimental group presented fewer inflammatory cells and new blood vessels as well as flat fibroblasts and relatively organized collagen fibers.(3) The relative expression level of TNF-a, VEGF and TGF-β1 genes was significantly lower in the corneas of the experimental group than those of the control group on day 35 (P< 0.01).The results suggested that amniotic membrane inhibited the inflammation and formation of new blood vessels and scar tissues, promoting the healing of the injured cornea. AMT is a clinically effective treatment for canine corneal alkali burn. |
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