| Objective: As a new non-invasive treatment technique in vitro,high intensity focused ultrasound(HIFU)has been widely used in gynecology,hepatobiliary surgery,urology and other medical fields,especially in the clinical treatment of solid tumors.Clinical trials in the 1980 s and 1990 s found that HIFU could be used to treat glaucoma in ophthalmology.However,there are few reports on its security when applied to treat related diseases in ophthalmology.Our early experiment has proved that HIFU can change the refractive state in living rabbit cornea,so this paper discusses the effects of HIFU on living rabbit cornea(retina and optic nerve)and provides theoretical support for safely applying HIFU in clinical treatment of living cornea.Methods: 1.The living animal models of rabbit cornea for HIFU irradiation were established,and the treatment parameters for CZF ultrasonic therapeutic apparatus were set as: power 1W;frequency 10.20MHz;pulse 1000Hz;focal length 4.5mm;irradiation time 6s(the time for uniformly rotating the device head in a circle driven by an electric motor at the speed of 10r/min);irradiation ring diameter about 9mm;and irradiation pattern was designed to locate and irradiate living rabbit corneal stroma in a circular ring to form the focal ring of cornea.7 healthy New Zealand White rabbits without eye diseases were selected.Right eyes of each rabbit were treated as the experimental group and left eyes were treated as the control group.Ring positioned HIFU irradiation of corneal stroma was applied in the experimental group and no intervention was done in the control group.The rabbits were examined with slit-lamp microscope and taken photos in 1,7,14,30,60,90 day(s),postoperatively;5 rabbits were randomly selected out of the 7 healthy New Zealand White rabbits,and their corneal curvatures were measured by automatic computer refractometer in 1,7,14,30,60,90 day(s)and before operation.2.Histomorphological and biological changes in retina of HIFU irradiated cornea in living rabbits: 36 rabbits(72 eyes)were randomly selected out of the 41 healthy New Zealand White rabbits,and among them,we randomly put 6 rabbits(12 eyes)to death,removed eyeballs and fixed them in 1,7,14,30,60,90 day(s)postoperatively to observe the retina cell layers,their arrangement and histomorphological changes of optic nerve under the light microscope;we observed the structure changes of optic nerve under the electron microscope;we detected Nogo-A expression in retina with immunohistochemical method;we investigated the apoptosis in each retina cell layer with Td T-mediated d UTP nick-end labeling(TUNEL);and the SOD activity and MDA content in retinal tissues were also measured.The remaining 5 rabbits consisted of the OCT group,which underwent OCT scanning for retinal morphology and thickness in 1,7,14,30,60,90 day(s)before and after operation.Results:1.The living animal models of rabbit cornea for HIFU irradiation were successfully established.2.Slit lamp microscope: part of the corneal epithelium defected in irradiated area after operation immediately,gray-white focal ring formed in stroma,clearly distinguished with the surrounding normal tissue,with sodium fluorescein staining dyeing in the corresponding parts.HIFU irradiation rings gradually became weak with the extension of observation time after operation until disappeared.Sodium fluorescein staining was not dyed and cornea was transparent again.3.The average curvature of the cornea was significantly increased at 1,7 and 14 days after irradiation with HIFU,and the increase of the 7th day was the most significant.The corneal curvature was decreased at 30,60 and 90 days after operation;the corneal curvature was slightly increased at 90 days after operation,but it was not greater than that of preoperative corneal curvature.4.The retinal HE staining under the light microscope: retinal layers arranged neatly and densely in the control group,with clear retinal ganglion cell nuclei and relatively clear boundaries between the layers.There was no significant change of retinal HE staining in each time point compared with that before irradiation.5.HE staining of optic nerve under light microscope: nerve fibers arranged in neat rows in cross-section of optic nerve,stroma slightly stained in red,no inflammatory cell infiltration,uniform interval and good vascular morphology.There was no significant change in HE staining of optic nerve in each time point compared with that before operation.6.Ultrastructure of optic nerve under electron microscope: myelin sheath was intact,dense and smooth and regularly arranged without demyelination in the control group.There was no siginificant change in the ultrastructure of the optic nerve in each time point compared with that before operation.7.Retinal immunohistochemistry: a little Nogo-A positive expression was visible in ganglion cell layer,the inner nuclear layer and the outer nuclear layer of normal retina.Nogo-A expression was visible in ganglion cell layer,the inner nuclear layer and the outer nuclear layer of the experimental group in each time point after the operation,and there was no significant enhancement compared with that of the control group.8.Retinal cell apoptosis detection: no positive apoptotic cells,nuclei collapse or nuclear fragmentation,chromatin condensation,etc.were visible in retinal ganglion cells(RGCs)of the control group and the experimental group.9.Superoxide dismutase activity and malondialdehyde content in retinal tissue: SOD activity of the experimental group 1 and 7 day(s)after operation was not significantly lower(P>0.05)than that of the control group.There were no significant differences between the experimental groups(P>0.05)in each time point.After statistical analysis,MDA content of the experimental group was higher than or close to that of the control group,but there were no significant differences at the same time point between the experiment and the control group(P>0.05).There were no significant differences in each time point between the experimental groups(P>0.05).10.OCT retina scan: retinal structure was normal with clear inner layer structure,well-defined and clear imaging in each time point of the experimental group and the control group.Preoperative retina was thicker than postoperative one,yet after statistical analysis,the differences between the control group and the experimental group at the same time or different time points were not statistically significant(P>0.05).Conclusions:1.Appropriate dose of HIFU can be accurately positioned in the corneal stromal layer,and it causes no obvious damage to normal corneal tissue outside of the irradiation area.HIFU also can make corneal collagen fibers shrink and increase corneal curvature by using its thermal mechanism.Thus,the application of HIFU in changing corneal refractive state is featured by safe and feasibility.2.Histomorphological and bioactive factor changes were not significant in retina and optic nerve of HIFU irradiated cornea in living rabbits,so HIFU is safe and reliable to retina and optic nerve in treating relevant diseases and changing the refractive state in ophthalmology.It suggests that HIFU has great prospects in the field of refractive surgery in ophthalmology. |
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