Establishment Of Mild, Medium And Severe Optic Nerve Crush Animal Model

ObjectiveTraumatic optic neuropathy(TON), as a severe complication of brain, orbit and prosopo injury, often results in serious vision deterioration even permanent visual loss. For a long time, there have been arguments about the treatments of TON. The difference of injury degrees were recognized as the key for the reaction of treatment. Establishing a reliable, convenient and reproducible animal model with optic nerve injury which can be graded will be beneficial for the research of effects of different treating methods.Previous animal models of optic nerve injury were qualitative or semi-quantitative, there was not a unified standard calibrated model. In this experiment, mild, medium and severe optic nerve crush injury models were made in rabbits by using 3 kinds of vascular clamp of permanent pressure, as the animal model for the evaluation of effects of different treating methods.Materials and Methods1 Animal and grouping48 healthy adult albino rabbits without eye diseases were divided into 5 groups randomly: normal control group, false injury group, injury group I , injury group II, and injury group III. Among of them, 24 eyes of 12 rabbits were randomly assigned as the normal control group and the false injury group, another 36 rabbits were divided into 3 injury group, 12 rabbits in each group. Optic nerve crush injury was only made in unilateral eye of each animal.2 Measurement about pressure and injury intensity (average impulse) of vascular clampThe pressure power of the vascular clamp was measured by using the hang heavy tensile force, and the injury intensity (average impulse) was calculated according to the area of vascular clamp and the pressure power. The pressure power of small, moderate and big vascular clamp were respectively 32g, 98g and 148g, and the average impulse were 397.52g.s/mm², 1209.88g.s/ mm² and 1549.74 g.s/mm², respectively.3 Establishment of animal modelThe crush injury of optic nerve was made by using different vascular clamp in every group for twenty seconds, 32g for injury group I, 98g for injury group II and 148g for injury group III. The rabbits of false injury group only received optic nerve dissociation and exposure without optic nerve crush.4 Observation items and timePathologic changes of the optic nerve crush injury were examined at 3d, 1w and 2w after injury. The changes of tissue morphology of optic nerve were observed by routine HE stain, the integral light density of optic nerve fiber and myelin were measured by using Glee-sliver stain and ponceau red G-light green stain.At 3d, 1w and 2w after injury, pathologic changes of retina and the apoptosis of RGC were examined by routine HE stain and terminal deoxynucleotidyl transferase mediated DIG-dUTP nick end labeling method (TUNEL). The number of RGC and the rate of apoptotic RGC were calculated.F-VEP was examined at 1h, 6h,1d, 3d and 1w after injury, the latency and amplitude of P₂ wave were analyzed.5 Statistical analysisAll the data were analyzed statistically by ANOVA and t test. Results1 Focal pathological changes of optic nerve crush injury1.1 HE stain and focal morphological observation of optic nerve crush injuryIn normal group and false injury group, the optic nerve fiber was intensive, regular and stained evenly.No obvious changes were found in injury group I at 3d, 1w and 2w after injury.Optic nerve edema and focal infarct were seen in injury group II at 3d after injury. These changes deteriorated gradually within 2w.Optic nerve edema and infarct in injury group III were more obvious than that in injury group III. At 2w after injury, the frame of optic nerve bundles disappeared. The pathological change of injury group III showed more serious than injury group II in any time.1.2 Glee-sliver stain and the examination of integral light density of optic nerve fiberNo significant difference of the integral light density of optic nerve fiber was found among the normal control group, the false injury group and injury group I (P >0.05).Optic nerve fiber of injury group II and III were sparse. The value of integral light density was lower at 3d after injury. Pathological change deteriorated gradually within 2w. At each point, the change in injury group III was more obvious than that in injury group II. At each point, the integral light density of optic nerve fiber in injury group II and IIIwere lower than normal group(P<0.05), and degraded gradually. At the same point, the difference of the integral light density of optic nerve fiber among these injury group was statistically significant(P<0.01).1.3 Ponceau red G-light green stain and the examination of integral light density of myelinThe morphology of optic nerve of false injury group and injury group I were nearly identical with the normal group, and the difference of the integral light density of myelin showed no statistical significance, when we compared respectively false group and injury group I with normal group (P > 0.05). At 3d after injury, demyelination was observed in injury group II and III. These changes deteriorated gradually with the lapse of time. At each point, the change in injury group III was more obvious than that in injury group II. At each point, the integral light density of myelin in injury group II and III were lower than normal group(P<0.05), and degraded gradually. At the same point, the difference of the integral light density of myelin among these injury group was statistically significant(P< 0.01). 2 pathological observation of retina2.1 HE stain and morphological observation of retinaIn normal group and false injury group, the layers of retina were clear. Distribution of RGC was regular intensively, nucleus was distinct. The changes of injury group I were slight. At 3d after injury, nucleus pyknosis of RGC was seen in injury group II and injury group III, then RGC loss were seen within 2w, and the thickness of retina become thin. These pathological changes were more obvious in injury group III. With the lapse of time, these changes deteriorated quickly. At each point, the changes of injury group III were more serious than the injury group II.2.2 Count of RGCAt each point, the number of RGC in false injury group and injury group I were approximate to the normal control group(P>0.05). The number of RGC both in injury group II and III were fewer than normal group(P<0.01), and count of RGC decreased continuously. At the same point, the difference of count of RGC among each injury group showed statistically significant(P<0.05).2.3 Examination of RGC apoptosis and rate of RGC apoptosisThere were few apoptotic RGC in normal control group and false injury group. In each injury group, apoptotic RGC distributed in ganglion cell layer.In injury group I , there was less apoptotic RGC at each point, but the difference of the rate of apoptotic RGC showed no statistically significant(P>0.05) at each time point.In injury group II, the rate of apoptotic RGC increased gradually within 3d～2w after injury, however, the rate of apoptotic RGC among different time points showed statistically significant(P<0.05).In injury group III, there were more apoptotic RGC at 3d after injury, the number of apoptotic RGC increased gradually within 2w, and among different time points, the rate of apoptotic RGC showed statistically significant(P<0.05).At the same point, the difference among different injury groups showed extremely statistical significance.3 The result of F-VEPIn normal group, the latency of P₂ wave was (71.72±3.66)ms, and the amplitude of P₂ wave was (20.53±4.15)μv. At each point, the difference of latency and amplitude between false injury group and normal group showed no statistical significance(P> 0.05).The delay of P₂ wave latency was found in all three injury groups at 1h after injury compared with normal group (P>0.05). and the amplitude was lower than normal group(P<0.05).At 1d after injury, latency and amplitude of injury group I had recovered to normal(P>0.05). But latency and amplitude of injury group II and III deteriorated gradually within 2w.Conclusions1 Stable and reproducible animal model of mild, medium and severe optic nerve crush was established by using the vascular clamp which pressure power was 32g, 98g and 148g, respectively.2 In mild injury group, changes of morphology were slight, and visual function was fine. In medium injury group, pathological changes of optic nerve were obvious, and the injury deteriorated gradually within 2w observing time, however, part visual function remained. In severe injury group, changes of morphology were irreversible sooner after injury, and the visual function lost completely3 The medium optic nerve crush model can be used to observe the therapy effect of different treatment methods of TON.