| The purpose of this study is to determine the optimal preparation of canine amniotic supernatant collected fresh amnion preservation condition in dogs;canine deep corneal ulcer model,curative effect of conjunctival flap combined with canine amniotic supernatant instillation to treat deep corneal ulcers.Test method:(1)the fresh canine amniotic membrane was collected to prepare the supernatant of the dog.To determine the preservation conditions,PBS and DMEM groups were divided into 4,-20,-80℃,and 1,20,40 and 80 days to detect the content of b FGF in the amnion supernatant.(2)By using alkali burnand mechanical injury establishd the canine deep layer corneal ulcer model(n=15),randomly divided into 3 groups: deep layer corneal ulcer(C group),conjunctival covering group(T1 group),conjunctival covering combined amnion liquid supernatant eyedrop group(T2 group).A model of canine deep corneal ulcer was made by using alkali burn combined with mechanical injury.The contents of IL-1,IL-8,VEGF,MMP-1,MMP-2 and MMP-9 in canine aqueous humor were examined on days of-3,1,7,14,21 and 45;The total number of blood leukocytes was detected at-3,1,4,7,10,13,16,19,21 days;In-3—21 days,eye pressure and tear secretion were measured every day.At 21、45 days,one group was randomly selected to collect corneal tissue for histopathological observation.Test results :The content of b FGF in the supernatant of amniotic membrane in dogs of group DMEM in the twentieth day was significantly higher than that of PBS group(p<0.01);On the twentieth day,the-80℃was significantly higher than the 4℃(p<0.05)for40,80 days,and the-80℃was significantly higher than the-20℃ and the 4℃(p<0.01).At the same time,there was also a significant difference between the-20℃ and the 4℃(p<0.05)on the eightieth day.(2)Compared with the T1 group,the content of IL-1,IL-8,VEGF,MMP-1,MMP-2 and MMP-9 in the eye aqueous humor of group T2 was significantly reduced(p<0.05);The total number of white blood cells in the blood was significantly decreased in the medium term(6-12 days)(p<0.05);Tear secretion increased significantly in the mid-term(6 and 11-13 days),and intraocular pressure increased significantly at the later stage(12-21 days).Compared with the C group,the content of IL-1,IL-8,VEGF,MMP-1,MMP-2 and MMP-9 in the dog eye aqueous humor of group T2 was significantly decreased(p<0.01).(3)On the twenty-first day,compared with the T1 group,the T2 group had less subcutaneous vessels in the cornea,but the vessels were more abundant between the conjunctiva and the corneal epithelium.Compared with the C group,the cornea was more complete,and the lower cornea was less and thinner.On the forty-fifth day,compared with group T1,the inflammatory cells in group C had severe infiltration,more blood vessels and rupture of ciliary body.In the T2 group,the corneal thickness was more moderate,the lower cornea vessels were less,and the infiltration of inflammatory cells was lower.Test conclusion:(1)the content of b FGF in the preservative solution was greatly reduced with the prolongation of the preservation time.DMEM preservation solution and-80℃can effectively reduce the decrease of b FGF content,providing a guarantee for short-term storage.(2)Conjunctiva combined canine amniotic supernatant can effectively reduce the canine eye deep corneal ulcer inflammatory cell infiltration,reduce corneal injury,improve the corneal transparency,Accelerate the recovery of intraocular pressure,effectively maintain the secretion of tears to reduce the occurrence of dry eye. |
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