The Effect Of High Intensity Focused Ultrasound On Rabbit Anterior Segment

Purpose: To evaluate the safety of high-intensity focused ultrasound(HIFU)keratoplasty as a treatment for presbyopia by examining its effect on the rabbit anterior segment.Methods: In the first part,the right corneas of 18 New Zealand rabbits were treated with HIFU keratoplasty and the left eyes were used as the control group.1,7,15,30,60 and 90 days postoperatively,the corneas were observed with slit lamp and stained with sodium fluorescein.The animals were sacrificed at each time point.The sections of corneas were stained with hematoxylin–eosin(HE).Collagen type I,Matrix Metalloproteinase-2(MMP-2)and Matrix Metalloproteinase-9(MMP-9)were evaluated using immunohistochemistry.For the detection of apoptosis,the TUNEL method was applied.Collagen fibers and cell structure were observed by transmission electron microscope(TEM).In the second part,the right corneas of 18 New Zealand rabbits were treated with HIFU keratoplasty and the left eyes were used as the control group.1,7,15,30,60 and 90 days postoperatively,the lenses were observed with slit lamp.The animals were sacrificed at each time point.The sections of lenses were stained with HE.The SOD and MDA levels were analyzed with assay kits.The apoptosis of epithelial cells of the lenses was tested by TUNEL.Results: In the first part,1.During the study period,all corneas were stable without infection and perforation.A white irradiated ring was observed in the peripheral cornea 1 day postoperation.The rings faded from 7 to 90 days gradually.2.Corneal sodium fluorescein staining 1 day after the operation,part of the corneal epithelium was defective in the irradiated area.7 days postoperation,a small amount of the corneal epithelium was defective in the irradiated area.15 days after the operation,no sodium fluorescein staining was observed.3.1 day after the operation,the focal zone is deeply stained.From 7 days to 60 days,the focal zone disappeared gradually,and the epithelium was hyperplastic.90 days postoperation,epithelial hyperplasia was significantly reduced,and the matrix fibers were arranged in order.4.1 day and 7 days after operation,there was no expression of collagen type I in the focal zone.15 days after the operation,the expression of collagen type I increased.30 days postoperation,the expression of collagen type I returned to normal state.On day 1,MMP-2 staining was found in the corneal epithelium and increased on day 7.On day 15 and 30,the expression of MMP-2 reached a maximum value.On day 60,the expression of MMP-2 was reduced.On day 90,there was no expression of MMP-2 in the epithelium or stoma.The expression of MMP-9 was observed on day 7 and peaked on day 15.From 30 days to 60 days,the expression of MMP-9 decreased gradually.On day 90,there was no expression of MMP-9 in the epithelium or stoma.5.On day 1,there was a large number of corneal apoptotic cells in the focal zone.On day 7,15 and 30,the number of apoptotic cells decreased.On day 60 and 90,there was no apoptosis in the cornea.6.Under the TEM,on day 1,corneal stromal cells were swollen and ruptured,matrix fibers were fractured.From 7 days to 60 days,the mitochondria dissolved,and the number of rough endoplasmic reticulum was increased.The matrix fibers were distorted.On day 90,corneal stromal cells returned to normal and matrix fibers were arranged neatly.In the second part,1.During the study period,all lenses remained transparent.2.At each time point,the crystalline capsule was complete,and the epithelial cells of lens were arranged neatly.The fibers of the lens were arranged closely and orderly.3.There were no significant differences in the SOD or MDA levels in the lenses between the treated group and the control group at any time point.4.No apoptosis was observed in the epithelial cells of the lenses.Conclusion: 1.After HIFU keratoplasty,the histomorphology of the cornea changed form 1 day to 60 days,and returned to normal on day 90.All corneas were stable without infection and perforation.It suggested that HIFU keratoplasty was safe and feasible.2.The corneal collagen type I levels decreased,the corneal MMP-2 and MMP-9 levels increased,the corneal cells underwent apoptosis and the electron microscopic structure of corneas changed for a period of time.The thermal effect of HIFU induced fibroblasts resynthesized new collagen fibers and apoptosis of corneal cells.All the changes returned to normal on day 90.HIFU is safe applied in rabbits corneas.3.After operation,all lenses remained transparent.The histomorphology and the SOD or MDA levels of lenses were normal.No apoptosis was observed in the epithelial cells of the lenses.Thus,lenses were not affected by HIFU keratoplasty.HIFU presents a good biological safety for eyes.