Effect Of NO/NOS System On The RGCs Apoptosis After Rat Optic Nerve Crushing Injury

[Objective] We want to establish a rat optic nerve crushing injury model in different time points; to study the expression of NO and the relationship with apoptosis of RGCs after optic nerve injury, to explore the function of three synthases to the content of NO after optic nerve injury, to provide a new idea and clinical treatment method for traumatic optic nerve injury.[Methods] 1) 170 adult SPF female SD rats was randomly divided into three groups:normal animal control group, sham-operated group and operated group. There are 2 rats in normal group,84 rats in sham-operated group,84 rats in operated group. Raised in the same conditions. The left eyes of rats in operated group were operated eyes and used for setting up optic nerve crushing injury model, we used a fixed 50g holding force forcep clamps 10s on optic nerve about 2mm set near the eye balls. The left eyes of rats in sham-operated group were only separated the optic nerve but not for clamping. The right eyes of the two growp rats and the normal group were not done any deal.2)4 left and right eyes of normal group were paraffin embedding. The operated group was divided to 7 subgroups by the time points after optic injury:6h group(12 rats,6 hours after injury); 12h group(12 rats,12 hours after injury); 24h group(12 rats,24 hours after injury); 3d group(12 rats,3 days after injury); 5d group(12 rats,5 days after injury); 7d group(12 rats,7 days after injury); 14d group(12 rats,14 days after injury); Injected excessive chloral hydrate anesthesia to rats, we use microscopic forceps and scissor separated the eye balls with about 5 mm optic nerve in these time points.3)4 rats’eyes of every time point in operated group and sham-operated group were paraffin embedding; 4 rats’eyes retina were used to detect the content of the retina NO and another 4 rats’eyes retina were taken homogenate to extract RNA. The normal, sham-operated group and operated group optic nerves and retinas were made into paraffin embedding slices and did HE staining, IHC staining, RGCs TUNEL apoptotic testing and counting and analyzing RGCs; 4 rats’eyes retina of every time point in operated group and sham-operated group were homogenated to detect the content of the NO with NO Kit; The RNA extracted from each group retina was reverse transcripted to cDNA, then detect the express of iNOS、eNOS and nNOS mRNA in different time points after optic nerve injury by Real-time PCR. All results were analysised by the SPSS 19.0 statistical software.[Results] 1) The rat optic nerve and retina HE staining shows:Sham-operated eyes were not different with the normal group; 12 h and 24 h after injury slices shows:retinal vasodilatation and swelling cells in RGC layer, RGCs begin to cell nucleus pycnosis, optic fibers sparse and glial cells disorder; 3 d and 5 d after injury slices shows:part of the RGCs vacuoles with nucleus chromatin gathered and sparse, some nucleus of RGC pycnosis and sparse with deep staining, optic fiber appear edema and degenerations appear in vacuoles, and glial cells arranged disorder; 7 d and 14d after injury slices shows:there was not obviously arranged very sparse、edema and vacuolar degeneration about RGCs.2) TUNEL apoptotic experiment shows:There was occasionally seen RGCs apoptosis between the normal group and sham-operated group; the operation group compared with sham-operated group, the RGCs apoptosis rate obviously increased at the time points of 3d(P < 0.05), peaked at 5 d (P< 0.05), steady decline at 7 d and 14 d.3) The content of NO detection results showed that retinal NO expression increased significantly after optic nerve clamping 6h and 12h, and the difference was statistically significant between operation group and control group (P< 0.05); dropped to a lower level at 24h (P<0.05); increased a lot and the difference was statistically significant compared with control group (P<0.05) after optic nerve clamping 3d later.4) The Real-time PCR detection results showed that retinal iNOS mRNA expression increased significantly after optic nerve clamping 24 h, and the difference was statistically significant between operation group and control group (P< 0.05), back to normal level at 3 d (P>0.05). The retinal nNOS mRNA expression increased significantly and the difference was statistically significant compared with control group (P<0.05) after optic nerve clamping 5 d. It was down a bit but the difference was statistical significance (P<0.05) after optic nerve clamping 7d, returned to normal levels (P>0.05) after optic nerve clamping 14d; eNOS mRNA expression was no significant change over time (P>0.05).5) The IHC staining results showed that the optic nerve and retina of sham group scattered in a small amount of iNOS and nNOS positive cells, but obviously expressed at 24h of iNOS positive cells and at 3d of nNOS positive cells in operation group, iNOS and nNOS positive staining cells in the retina are mainly concentrated in the GCL layer and RNFL layer.[Conclusion] 1) Optic nerve crushing injury can result in rat retina RGCs apoptosis, and can result in the increase of NO content of in ratina, to a certain extent, the increase of NO content maybe promote the apoptosis of RGCs.2) The NO changes in the retina after optic nerve injury may be caused by the result of iNOS and nNOS interwoven change, the activity of iNOS and nNOS change on TON damage has the key regulatory role.3) In the damage model, oxidative stress changes in local microenvironment promoted the apoptosis of RGCs, reducing the eye oxidative stress may be one of the ways to alleviate the RGCs apoptosis.