Establishing a Standard Animal Model of Traumatic Optic【推薦論文】 .doc

精品論文establishing a standard animal model of traumatic opticneuropathy with fpiyu jinguo1, yu rongguo2, shen zhansheng1, wang xing1, zhang wei2, lu yingjuan3,5yan hua1(1. department of ophthalmology, tianjin medical university general hospital , tianjin 300052;2. department of ophthalmology, tianjin huanhu hospital, tianjin 300060;3. tianjin medical university eye hospital, tianjin 300384)abstract: objective: to establish an animal model of traumatic optic neuropathy (ton) using fluid10percussion injury (fpi) that resembles the clinical state with or without lens injury, and observe the different progress of repair using flash visual evoked potential (f-vep). methods: chinese white rabbits were used as the animal research subjects. sixty-four healthy chinese white rabbits (128 eyes) were divided into two groups according to right and left eyes. the right eyes were selected as theexperimental group (optic nerve injury and lens injury) and the left eyes were used as the control group15(optic nerve injury). the traumatic optic neuropathy was made in both eyes using fpi, meanwhile penetrating lens injury was performed by acupuncture needle. according to different observing times, every group was divided into 8 sub-groups. the function of optic nerve was evaluated using f-vep, and the results were compared between groups and observing times. results: at one day post injury, the latencies of p100 in both groups were longer, and the amplitudes of p100 in both group were lower20than before injury (p0.05). the duration of longer latency in experimental group was shorter than in the control group (p0.05). the latency in experimental group restored more quickly than the controlgroup. conclusions：this animal model is made with high success rate. macrophage accumulates inretina and optic tissue because of lens injury. there is a good correlation between f-vep and pathophysiology.25keywords: traumatic optic neuropathy; lens injury; animal model; flash visual evoked potential0introductiontraumatic optic neuropathy (ton) is an important cause of severe visual loss following blunt or penetrating head trauma 12, which can occur in 5% of cases after head injury 3. lee v 430estimated the minimum incidence was 1.005 per million, the leading causes included falls (25.6%), road traffic accidents (rtas) (21.5%), and assaults (20.7%). for the past decade, corticosteroids and/or optic canal decompression surgery have been widely embraced as therapeutic paradigms for the treatment of ton. however, there is little clinical evidence to support the effectiveness of these therapeutic approaches, and spontaneous improvement is definitely possible, raising35questions about the efficiency of current therapy for improving visual outcomes 5627. atpresent, treatment of ton remains controversial 89, and most of the published data are either retrospective or presented in case reports 10. on the other hand, the knowledge concerning the pathophysiologic mechanisms of traumatic optic neuropathy is limited 1112. it is therefore necessary to build a ton animal model, which would be suitable to study the therapy and injury40repair mechanisms of ton, and for the development of novel and effective clinical treatments.in the past, it was thought that optic nerve can not be regenerated post injury. however, dezawa 13 found that after transectional optic nerve injury, autologous sciatic nerve transplantation can promote rgc axon regeneration. when the lens was injured, a large amount of macrophages were detected in retina, leon 14 believed that this infiltration of monocytes and45macrophages could promote rgc survival and axon regeneration. visual evoked potentials (vep)foundations: research fund for the doctoral program of higher education of china (no.20091202110008) brief author introduction:yu jinguo, (1975-), male, attending doctor.main research: the basic and clinical research of ocular trauma and optic nerve injury.correspondance author: yan hua, (1965-), male, professor.main research: the basic and clinical research of ocular trauma and optic nerve injury. e-mail: - 11 -have been used for many years in the objective evaluation of disorders of visual pathways and in the electrophysiological research of visual procession 15. it is an objective and susceptive physiological index in traumatic optic nerve injury 16. the wave shape of rabbits flash visual evoke potentials (f-vep) is stable and reproducible, and the amplitude and latency of f-vep have50no significant statistical difference in left and right eyes of female and male rabbits 1718. thechange of amplitude and latency of f-vep are related to the severity of ton, and it is useful in diagnosis.for these reasons, we undertook an experimental study to establish a stable rabbits tonusing fluid percussion brain injury device (fpi) combined with or without lens injury, which could55reveal regular change of f-vep in two groups and try to establish the fundamentals for further study of ton repair mechanisms. such a model could be promising for the development of novel and effective treatments for optic nerve injury.1materials and methods1.1 preparation of animals60thirty-two male and thirty-two female adult rabbits (spf grade) with a body weight between2-2.5kg were used, which were provided by tianjin medical university experimental animal center. all the right eyes were selected as experimental group (optic nerve injury and lens injury), and all the left eyes were used as control group (only optic nerve injury). according to different observing time points post injury, all the rabbits were randomly divided into 1, 2, 4, 7, 10, 14, 21,6528 day subgroups, eight eyes in every sub-group. all rabbits were individually housed on a12/12-hr light/dark schedule and were allowed free access to food and water. all experimental procedures employing animals adhered to the association for research in vision and ophthalmology resolution on the use of animals in research, and were approved by the animal care committee in tianjin medical university general hospital. the ethics council of tianjin70medical university general hospital authorized the study, which adhered to the tenets of thehelsinki declaration.1.2 methods1.2.1establishment of animal modela local anesthetic (10% chloral hydrate) was injected at a concentration of 4ml/kg into75abdomen, and a surface anesthesia was applied with 4% oxybuprocaine hydrochloride eye drops.both eyes fornix conjunctiva was cut at a curve track from 2 oclock to 10 oclock. the home-made hitting pipage was inserted into orbit 1.5cm through conjunctival incision along the outside wall of sclera. at the same time, the head of rabbits were properly fixed on the headflame of fpi (fig.1a, 1b). the strength of hitting power was set at 6.90.7atm. in the experimental80group, dragging superior rectus muscle to reveal the posterior part of eyeball with the strabismus hook, pricking vertically into the eyeball 5-6mm at the equator with the acupuncture needle to damage the lens (fig.1c). in the control group, only the optic nerve was injured. all eyes were given tenebrimycin eye-drop to prevent infection three times per day.85fig.1 establishment of animal model of traumatic optic neuropathy with fpi and lens injury. a) in experimental and control groups, the head of rabbits were properly fixed on the headflame of fpi and the strength of hitting power was set at fixed height to insure the uniformity of the traumatic optic neuropathy animal model. b) the home-made hitting pipage was inserted into orbit 1.5cm through conjunctival incision along the outside wall of sclera following full anesthesia. c) in the experimental group, the lens were injured by pricking vertically into90them with acupuncture needle, this caused the lens to become opaque.951001051101.2.2recording method and stimulating parameter of f-vepaccording to the standard of international clinical visual electrophysiology, american nicolet multifunctional electrophysiological diagnosis detection was used. a local anesthetic (10% chloral hydrate) was injected at a concentration of 4ml/kg into abdomen. all the needle electrodes were silver needle electrodes, which impedance were less than 15 k. the recording electrode was put 3mm from the internal occipital protuberance, reference electrode was put on the midposition of two eyes, ground electrode was connected with the same side of ala auris 18.led eyeshade flaring provocation was used, with 1.9hz frequency of stimulation, 5100 hz width of transmission, 0.2 ms width of wave, 200ms analyzed time, build up 70 times, amplify 20 times. every eye was measured continuously three times with 10 min intervals. while one eye was detected, the fellow eye was completely covered with light-proof black cloth. each eye was taken3 times stable waveshape. first, f-vep before injury of all the eight rabbits in the first model group were recorded, the range of the normal latencies and amplitudes of p100 were set. then, thelatencies and the amplitudes of p100 1、2、4、7、10、14、21、28 days post injury were recordedin the rabbits two eyes.1.2.3histopathologythe retina and optic nerve tissue section 1、2、4、7、14、21、28 days post injury were stained by immunohistochemistry. macrophages were labeled with antibody ed-1, and the number of macrophages was counted. the same histological section was stained by cresyl violet dyeing, and the survival retinal ganglion cells (rgcs) was labeled and counted. one slice was selected from115120125130135140145150every twenty-four slices, and four slices total were selected which took optic disc cross-section as central axis. the number of macrophages and survival rgcs were counted from ten random visual fields on high power lens per slice and then added. the average number of macrophages and survival rgcs of each sample were recorded and analyzed between the different subgroups of experimental and control groups.1.2.4analysis of statisticsspss 13.0 statistical software was used to do statistical analysis. the latencies and the amplitudes of p100 of different time points before and post injury were analyzed between the experimental and control groups with two factors analysis of variance. the latencies and the amplitudes of p100 among the different subgroups were also analyzed in a similar manner. snk-q test was used to analyze multiple comparisons among the different time points and subgroups. a pvalue of 0.05 was defined as being statistically significant.2results2.1 result of f-vep78.6% rabbits before injury could produce the typical waveform of npn, in which latencies and amplitudes of p100 were (42.595.14) ms and (8.723.32) v respectively (fig.2a).in experimental group, one day post injury, the latency of p100 was (103.5911.36) ms, andthe amplitudes were (6.583.39) v. compared with those before injury, there were statistically significant differences between groups (p0.05). there was also a statistically significantdifference between the latencies of 1 day and that of 10 days post injury（p0.05）, as well asbetween the latency of 10 days and that of 28 days post injury（p0.05）. the latency of p100 in 10 days post injury was gradually extended（p0.05）and then gradually shortened (p0.05). therewere statistically significant differences between the amplitudes of 1 day and that of 7 days post injury（p0.05）, and between the amplitudes of 7 days and that of 28 days post injury（p0.05）. the amplitude of p100 in 7 days post injury was gradually decreased（p0.05） then gradually increased（p0.05）(fig.2b,2c,2d).in the control group, one day post injury, the latency of p100 was 105.5316.59ms and the amplitude of it was 6.322.12 v. compared with those before injury, there were statistically significant differences（p0.05）. there were statistically significant differences between the latencies of p100 1 days and that of 14 days post injury （p0.05）, and between 14 days and 28days post injury （p0.05）. there was statistically significant differences between amplitudes of1 day and that of 10 days post injury（p0.05）.there was no statistically significant differences among the amplitudes of the consecutive time points（p0.05）. the latency of p100 in 14 days post injury was gradually extended （p0.05）, and then gradually shortened（p0.05）. the amplitude of p100 was gradually decreased in ten days post injury（p0.05 ）(fig.2e,2f,2g).155160165fig.2 result of f-vep. f-vep was recorded before injury and used as the baseline range for normal latencies and amplitudes of p100. f-vep were subsequently then recorded in the rabbits two eyes at 1、2、4、7、10、14、21、28 days post injury in experimental and control groups. a) typical waveform of npn before injury, the latencies and amplitudes of p100 were (42.595.14)ms and (8.723.32)v respectively. b) latencies and amplitudes of p1001 day post injury in experimental groups were (103.59+/-11.36)ms and (6.58+/-3.39)v respectively. c) latencies and amplitudes of p100 7 days post injury in experimental groups were (122.7516.32)ms and (4.201.57)v respectively. d) latencies and amplitudes of p100 10 days post injury in experimental groups were (135.8019.69)ms and (5.221.51)v respectively. e) latencies and amplitudes of p100 1 day post injury in control groups were (135.8019.69)ms and (6.322.12)v respectively. f) latencies and amplitudes of p100 10 days post injury in control groups were (138.7621.11)ms and (4.081.62)v respectively. g) latencies and amplitudes of p10014 days post injury in control groups were (146.5822.90)ms and (4.141.26)v respectively.there were statistically significant differences between the latency of p100 21 days post injury in experimental group and that in control group（p0.05）, as well as at the 28 days post-injurytimepoin（tp0.05）. there was statistically significant difference between the amplitude of p100 28days post injury in experimental group and that in control grou（pp0.05）. however, the latency in170experimental group recovered faster than that in control group（p0.05）and the tendency became more obvious 14 days post injury in experimental group, which was similar to the level before injury. there was no statistically significant difference between the amplitude of 28 days postinjury in experimental group and that in control group（p0.05）（fig.3a,3b）.ab175180185190fig3. comparison of latency and amplitude of p100 between experimental and control groups. a) in the experimental group, the latency of p100 10 days post injury was gradually extended（p0.05）and then graduallyshortened (p0.05). in the control group, the latency of p100 in 14 days post injury was gradually extended（p0.05），and then gradually shortened（p0.05). b) in the experimental group, the amplitude of p100 in 7 dayspost injury was gradually decreased（p0.05）and then gradually increased（p0.05). in the control group, theamplitude of p100 gradually decreased 10 days post injury（p0.05）.2.2 histopathological results2.2.1count of macrophage labeled with ed-1 antibodyin the experimental group, there was statistically significant difference between the number of macrophages 4 days and 7 days post injury（p0.05）, and between 4 days and 10 days, 4 daysand 21 days, 7 days 10 days, 7 days and 21 days, 10 days and 14 days, 10 days and 21 days, 14 days and 21 days post injury（p0.05）. in control group, there were statistically significant differences between the number of macrophages at 4 days and 7 days post injury（p0.05）, as wellas between 4 days and 10 days, 4 days and 14 days, 7 days and 10 days, 7 days and 14 days, 7 days and 21 days, 10 days and 14days, 10 days and 21 days, 14 days and 21 days post injury195200205210215220225（p0.05）.the macrophages appeared in the retina 4 days post injury in both experimental and control groups and then gradually increased with a peak number at 10 days post injury. after that, they decreased gradually and disappeared 28 days post injury. the number of macrophage in experimental group was substantially more than that in control group at all observing time points（p0.05）(fig.4)（fig.5a）.fig4. comparison of macrophages and survival rgcs between experimental and control groups. the macrophages appeared in the retina 4 days post injury in both experimental and control groups and then gradually increased, and reached a peak nu