| Objectivesl.To determine the best parameters for producting severe corneal thermal burn (SCTB) in rabbit model in homeothermic condition.2. To discuss relationship between the concentration of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) in the supernatant of rabbit corneal tissue and aqueous humor with the degree of thermal corneal damage at different times after SCTB.3. To determine the optimal timing of penetrating keratoplasty (PKP) after SCTB in rabbit in the early stage and to investigate the relationship between the concentration of MMP-2and MMP-9in supernatant of corneal tissue with the result of PKP.4. To investagate the safety and practicality of simulate corneal long distance transportation (LDT) for PKP in SCTB in rabbit.Methods1.Thirty-six healthy adult New Zealand white rabbits, male or female, weighing2.0-2.5kg were randomly divided into four groups with9rabbits (9eyes) in each group with different burning temperatures of100℃,200℃,300℃, and400℃in group A,B, C, and D, respectively. The burning duration of4s,7s,10s were set for every3rabbits (6eyes) in each group. The burning degree, morphology of burned area, number of eyes experiencing corneal dissolve or penetration were observed and quantified based on the degree of thermal burn in corneas. The relationship of burning temperature and burning duration with burning degree was statistically analyzed, determining the optimized burning temperature and duration for studying SCTB (the thermal burn degree reached grade three or grade four) in a rabbit model. Animals were killed3days after the injury. The corneal tissues were immunohistochernically stained (HE staining), and their morphology was observed at different degrees, identifying the reliability for preparing rabbit SCTB model under our experimental conditions. 2. Twenty-one healthy adult New Zealand white rabbits, male or female, weighing2.0-2.5kg were randomly divided into7groups (A-G) with3rabbits in each group. Given Group A functioning as a control, SCTB was prepared using the rabbits in the rest of the6groups. Animals from which SCTB were prepared were killed after the injury, with different time-points thereafter in different groups, as follows:Group B,2h; Group C,6h; Group D,12h; Group E,24h; Group F,48h and Group G,72h. Corneas and aqueous humor was collected, and rabbit corneal tissue was equally cut along the vertical middle axis into halves. Using each half cornea the concentration and expression of MMP-2and MMP-9were determined in the supernatant of corneal tissue and aqueous humor by enzyme-linked immunosorbent assay (ELISA) and injured corneal tissues was characterized by immuneohistochemistry (IHC), respectively.3. Forty-eight healthy adult New Zealand white rabbits were randomly divided into4groups with12rabbits in each group. SCTB was prepared only using the left eye of the animals and PKP was received at different time points after injury in different groups. Group A:12h after injury; Group B:24h after injury; Group C:48h after injury; Group D:72h after injury. Transplanted corneal surviving, transparency, corneal neovascularization (CNV) after surgery were observed.6rabbits were randomly selected and killed in each group on the20th day and40th day after the PKP treatment. Corneas and aqueous humor was collected, and rabbit corneal tissue was equally cut along the vertical middle axis into halves. Concentration of MMP-2and MMP-9in supernatant of rabbit corneal tissue and aqueous humor was determined by ELISA and the expression of MMP-2and MMP-9in rabbit corneal tissue was characterized by IHC, respectively.4. Thirty donor corneas from healthy adult New Zealand white rabbits were randomly divided into5groups with6corneas in each group. Group A:control; Group B, C:donor corneas were kept for24h using moist-chamber storage; Group D, E:donor corneas were kept for24h using moist-chamber storage as the same time by LDT simulation for24h. Central area of the corneal tissue was isolated with trephine from Group A, B and D, and cut into halves. Concentration of MMP-2and MMP-9in supernatant of rabbit corneal tissue was determined by ELISA and the expression of MMP-2and MMP-9in rabbit corneal tissue was characterized by IHC. The remaining corneal endothelial tissue in cornea after trephining was stained with trypan blue and alizarin red, followed by the calculation of density of CEC. In addition, the density of corneal endothelia, concentration of MMP-2and MMP-9in supernatant of rabbit corneal tissue and expression of MMP-2 and MMP-9in corneal tissues were observed.12healthy adult New Zealand white rabbits were randomly divided into Group F and Group G, with6rabbits in each group. Left eyes of rabbits were used for SCTB and the PKP was performed24hours after injury. Group F received donor corneas from Group C, and Group G received donor corneas from Group E. After40days, the transparency of corneal graft in the animals was observed and the area of CNV in corneal graft was measured. After that, animals were sacrificed and corneal grafts were isolated and cut, where the expression and concentration of MMP-2and MMP-9was characterized as previously described.Result1.Different burning temperature and duration led to different degree of thermal burn in corneas. SCTB could be reached when burning temperature is above300℃and burning duration is longer than7s. At400℃,the corneal tissue was easily charred, while the boundaries of the corneal burn area were unclear if burned for more than7s. The optimized combination for SCTB is300℃and7s. The results of IHC showed that mild corneal thermal burns only damaged the corneal epithelium, moderate thermal burns damaged superficial corneal stroma cortex, severe thermal burns damaged full-thickness corneal tissue, which was consistent with the standard classification of corneal thermal burn.2.Compared with the control, the concentration of MMP-2and MMP-9in the supernatant of corneal tissues was significantly increased12hours after the SCTB and peaked24hours after the burn. They were maintained at a high level until72hours after injury (P<0.01). Compared with control, there is not significant difference2hours and6hours after injury (P>0.05) but it is significantly different between12hours and24hours after injury. However, the comparison between24h,48h and72h indicated no significant difference (P>0.05). Regarding the concentration of MMP-2and MMP-9in aqueous humor, it is significantly increased24hours after corneal thermal burn, and persisted at a high level until72hours after burn (P<0.01). There is not significant difference between2h,6h and12h after injury (P>0.05) but it is significantly different between12hours and24hours after injury. However, the comparison between24h,48h and72h indicated no significant difference (P>0.05). IHC indicated that MMP-2was detected at full-thickness of corneal stroma2hours after SCTB, enriched as time proceeding after the burn injury and highly expressed24hours thereafter. MMP-9started to be expressed in the basal cell layer of the cornea2hours after SCTB and extended into the superficial stroma later6hours after injury, expression appeared in full-thickness stroma and showed a high level24 hours after the burn injury.3.All the corneal grafts in rabbits of SCTB survived after PKP treatment. Due to the different timing receiving PKP treatment after SCTB, the transparency of corneal graft was significantly different between20days and40days after PKP treatment (P<0.01). The area of CNV in corneal graft in Group A and B are significantly smaller than that of Group C and D (P<0.01). On the20th day and40th day after surgery, the concentration of MMP-2and MMP-9in the supernatant of corneal tissue in Group A and B is significantly smaller than that of Group C and D (P<0.01) but there is no significant difference in aqueous humor among these groups (P<0.05). IHC indicated that corneal stromal fibers were organized in order with high density in Group A and B, but not in Group C and D. MMP-2and MMP-9were expressed in all the layers of corneal tissues, with a higher level in Group C and D.4.Twenty four hours after moist-chamber storaged and LDT simulation, there is no significant difference in term of CEC density (P>0.05) and concentration of MMP-2and MMP-9in corneal tissue supernatant (P>0.05) in Group B and D, compared with control. IHC indicated MMP-2and MMP-9were not largely expressed. All the corneal grafts survived in Group F and G. After40days following surgery, there was no significant difference between Group F and G regarding to the transparency and the area of CNV (P>0.05), and concentration of MMP-2and MMP-9in corneal tissue supernatant was not signifycantly changed between Group F and G (P>0.05). IHC indicated MMP-2and MMP-9were expressed in all the layers of corneal grafts.Conclusion1.When preparing the rabbit SCTB in homeothermic condition, the higher the burning temperature is, or the longer burning duration is, the severer corneas injury would be. The optimized parameters for preparing rabbit SCTB is:300℃burning temperature and7s burning duration. In our experiment the rabbit SCTB model shows a good effect when SCTB was completed, all layers of cornea were destroyed.2.The expression of MMP-2and MMP-9peaked24hours after SCTB, when corneal tissue was heavily destroyed. MMP-2and MMP-9were involved into the process of rabbit SCTB, and highly related with corneal tissue dissolving after the burn injury.3.PKP treatment within24hours after rabbit SCTB brought the best therapeutic result. MMP-2and MMP-9may participate in the postoperative repair process of corneal graft, its high expression may aggravate the postoperative damage. 4.The quality of donor corneas provided by moist-chamber storage and LDT is reliable and doesn’t affect the result of PKP treatment for rabbits of SCTB. The application of LDT is safe and reliable for rabbit of SCTB in early corneal transplant surgery. Therefore, LDT technology is able to provide donor corneas for rabbit of SCTB in PKP surgery, and is very valuable in clinic therapeutics. |
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