| Back ground:As the improvement of living standards in China, accelerating the process of industrial automation, increasing traffic accidents and natural disasters, eye contusion become more frequently, cause visual function defect, loss of vision, and one of the indispensable reason affecting the quality of life. Contusion caused by the high-speed objects in car accidents and war injuries are extremely common, and often leads to severe visual dysfunction after injury, even if no merger penetrating eye injury. Therefore, it became the focus of attention of the majority of medical workers and experimental researchers. Contusion of eyeball is a blunt object, a direct blow to the eye damage caused by the high-pressure liquid and gas on the impact of the eye, but does not cause the wall of the eye break, also known as the eye of non-perforating injury is the most common eye injuries. This damage can directly damage the combating area, also injury the corresponding parts and the entire eye when the force conduct through the eyes of the contents. the common injury:immediate physiological disorder caused by tissue injury, organizational change caused by vascular reactivity; severe cases can lead to ocular tissues mechanical tear and eye rupture.Scholars study the mechanism of contusion injury through animal models. Domestic research often choose rabbits for experiment, meanwhile select the gravity of injury and impact injury for preparing experimental animal models; Foreign research more choose the experimental pigs, mice and other animals to build experimental model building, and observe pathological changes and biochemical factors before and after injury, to evaluate mechanism of eye trauma injury. Taking energy as for standards, injury is divided into light-level and weight level using energy as for standards. Researchers always analysis the injury through the following aspects:the analysis of tissue biopsy; observing based on the experimental study of the cellular and molecular level:changes of antioxidant enzymes in the retinal, the level of Immunoreactive-endothelin (ir-ET) in the retina and choroid of injury eye by radioimmunoassay, detecting the level of retinal nerve growth factor (NGF) and Nogo, expression NGF and TrkA in the retina nerve cells, also some research study the protection mechanism of retinal ganglion cells (RGCs) through taking a variety of interventions to inhibit neuronal apoptosis, that can improve the survival rate of damaged RGCs and expected to help the regeneration of RGCs in the experiment of acute optic nerve injury.Glu serves as a major excitatory neutrotransmitter for the central nervous system (CNS) of mammals. Pathologically and excessively high levels of Glu likely cause damages and even kill neurons. The approach is known as the oxidation of glutamate toxicity (Oxidativetoxicity), and it has confirmed that glutamic acid metabolism abnormalities causes many central nervous system diseases and damage the nerve cells. In order to maintain the sensitivity of synaptic transmission, glutamate must be cleared. Extracellular clear glutamate completed by uptaken by the carrier-mediated. In addition, as early as1981, Costman proposed that may be the existence of glutamate-glutamine cycle between glutamatergic neurons and glial cells in the central nervous system. Glutamate, stored in presynaptic vesicles, is released into the synaptic cleft through the way of calcium-dependent exocytosis, combine the Ionic or metabotropic glutamate receptors in postsynaptic membrane, lead to activation of its coupled to sodium, calcium ion channels or G protein, produce the excitatory mpulses. Glutamate that is not binding by the receptor is transported by the other two pathways:one side is re-uptake into the presynaptic capsule; the other side is converted to glutamine by glutamine synthetase enzyme after spread to the synaptic cleft, then glutamine convers for glutamic acid and transferred to the presynaptic vesicle. This is glutamate-glutamine cycle theory.in the central nervous system. So any one aspect of the obstacles in the glutamate-glutamine cycle, may lead to nerve eye related by glutamate excitotoxicity. Therefore, cell membrane damage and disorders of the various metabolic pathways can cause neurotoxic damage by glutamate accumulation from different sources after eye contusion.In order to close to the actual situation of the trauma in daily life and clinic this study injure rabbit eye using a bullet shot (negligible mass). Considering the high-speed as primary factor, we simulate the human eye injured caused by high-speed low-mass objects, focus on observation of changes in the anterior segment of rabbits’eyes, changes of the important nerves and excitatory amino acids (glutamic acid) in the retina and vitreous, and analysis the correlation between Glu and retinal damage and between glutamate and glutamine. We descript such injury model preliminary, provide a reference for clinical study of the early treatment of eye contusion caused by high-speed objects, achieve research results that will eventually be used to improve clinical diagnosis and treatment.Objective:To verify that the low-energy high-speed can cause serious eye injury, we observe the changes in the anterior segment of rabbits’eyes, changes of the important nerves and excitatory amino acids (glutamic acid) in the retina and vitreous, and analysis the correlation between Glu and retinal damage and between glutamate and glutamine. Therefore, we confirmed that the high-speed is the vital factor in ocular trauma, and provide new ideas for the future and timely clinical treatment and protection.Methods:Fifty-four healthy two-month-old New Zealand rabbits(weighing2kg, regardless of sex) were selected from the experimental animal center of our hospital. They were in clean conditions with free access to water and food. Eighteen rabbits were treated with left eye damages, eighteen rabbits were treated with both eyes damages, and the other eighteen animals were used as controls without any treatment. The experimental rabbits were subjected to intramuscular anesthesia using25mg/kg ketamine hydrochloride, and they were lying on their sides on an experimental table. The rabbits’ eyelids were kept open using eye speculum, and TB bullets (weighing0.20122g on average) were shot at the central cornea approximately1cm from the rabbits’ eyes at a speed of90m/s. All the experimental models were established under same procedure.Three experimental rabbits were sacrificed three hours, six hours, one day, three days, seven days, and14days after model establishment, respectively.A sclera puncture incision was made3mm from the corneascleral limbus using a puncture needle. A portion of0.5ml of vitreous was extracted from the incision using a No.18sterile syringe, and the vitreous was immediately stored at-80degrees. The eyeballs were fixed in4%formaldehyde.The vitreous samples were mixed with acetonitrile, centrifuged, and then1u1of supernate was prepared for the detection of Glu and glutamine levels, using HPLC. Conventional HE staining was performed following the formaldehyde fixation.SPSS13.0statistical analysis system were adopted. Quantitative data were descripted by the number of cases, mean, standard deviation. More independent groups were compared using single factor analysis of variance (One-Way ANOVA), If the variance is homogeneity, LSD test were adopted in group mean differences between any two, or Dunnett’s T3test were adopted. P≤0.05was considered statistically significant.Result:No ocular rupture was observed after bullet-shot treatment. Conjunctival edema and hyperemia became more apparent one day after the treatment. Three days following treatment, bulbar conjunctival edema, hyperemia, and eyelid swelling were relatively ameliorated. Fourteen days after the contusions, The corneal haze was slightly improved. Neovascularization was observed at the peripheral cornea. A blood clot was found in the anterior chamber, was not absorbed until the end of experiment. The pupil was traumatic dilated.10rabbits eyes appear irreversible deformation of the pupil, and crystal turbidity.Ocular changes under light microscope:In the control group, the eyes presented complete and distinct layer structures; the inner and outer segments of photoreceptor cells were distributed in order. Consecutive and regular pigments in the epithelial layer were observed under light microscopy, Three hours after the injuries, the retinal tissue edema and intercellular edema, among the inner nuclear layer cells, were observed. The number of cells distributed in the ganglion cell layer became scarce. The internal limiting membrane was damaged. The inner segment of the photoreceptor layer showed a loose and disordered structure. Pigment epithelial layer edema was noted. An exudative retinal detachment could be seen. Pigment cell degeneration was found in the choroid membrane. Six hours after the injuries, the retinal tissue edema was aggravated; the continuity of the internal limiting membrane was disrupted. A relatively large amount of vacuolar degeneration cells were irregularly distributed in the inner nuclear layer. The outer nuclear layer cells were arranged in disorder. The outer segment of the photoreceptor layer presented abnormal structure, and the membrane disk fragments were distributed between the inner segment and the pigment epithelial cells. The continuity of the pigment epithelial layer was destroyed, and the partial cells were disrupted. One day after the injuries, the amount of cells located in the ganglion cell layer and the inner and outer nuclear layers were significantly decreased. The inner segment of the photoreceptor layer cells was intact, whereas the outer segment was disrupted, shortened, and arranged irregularly. Limited cytolysis was found in the pigment epithelial layer, which was partially destroyed. Three days after the injuries, the number of inner and outer nuclear cells was reduced. Normal plexiform structures disappeared. Both inner and outer segments of the photoreceptor layer cells were disrupted and disintegrated. The structure of the pigment epithelial layer was basically disrupted. Seven days after the injuries, retinal edema was further exacerbated. Both the inner and outer nuclear layers presented irregular structures. Small blood vessels in the choroid membrane were basically unobservable. A blood clot and a large amount of red blood cells had accumulated in vessels. Fourteen days after the injuries, normal retinal tissues were destroyed. Choroidal membrane detachment occurred. The elasticity of the vascular wall of the choroidal membrane vessels had decreased.Glu concentration in vitreous body:The level of Glu in the experimental rabbits was elevated at6h,3d,7d, and14d after the injury, compared with that in normal controls, which presented extremely slight variations in terms of Glu content (P=0.039、P=0.024、P=0.038、P=0.041). The level of Glu in unilateral injury group and in binoculus injury group are both higher than in control group at6h,3d,7d, and14d, but no significant difference between unilateral injury group and binoculus injury group. The Glu levels in the each time point was statistically significant (P=0.046, P=0.026) in the unilateral injury group and the binoclius injury group; the difference of glutamic acid content at all time points was not statistically significant in the control group (P=0.217).Gln concentration in vitreous body:The level of glutamine in the experimental rabbits was elevated at6h, Id,3d,7d, and14d after the injury, compared with that in normal controls, which presented slight variations in terms of glutamine content (P=0.044\P=0.039、P<0.001、P=0.03、P=0.049). The level of glutamine in unilateral injury group and in binoculus injury group are both higher than in control group, but no significant difference between unilateral injury group and binoculus injury group. the Gln levels in the each time point was statistically significant (P=0.043, P=0.001) in the unilateral injury group and the binoclius injury group; the difference of glutamic acid content at all time points was not statistically significant in the control group (P=0.160).Conclusion:This study confirmed the high-speed bullet can cause serious injury of the eye structure, refractive media, and more severe retinal damage. Contusion could increase glutamate concentration in vitreous. This study reflected that the level of Glu increased in the retinal synaptic cleft with time, we speculated that the nerve excitatory amino acids was a major factor to increase the retinal structural damage, and then retinal ultrastructure damage increased the levels of Glu and glutamine. This vicious circle caused serious visual impairment and vision irreversible damage after eye trauma. Therefore, the impact of high-speed is essential in eye trauma. |
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