Protective Effect Of Leukemia Inhibitory Factor On The Retinal Injury Induced By Acute Ocular Hypertension In Rat

Purpose:To investigate the effect of leukemia inhibitory factor(LIF)on the rat retinal injury induced by acute ocular hypertension.Methods:1.Sprague-Dawley(SD)rats were divided into 3 groups.Ocular hypertension was induced by the anterior chamber infusion with saline after 60 minutes.Intraocular pressure(IOP)was increased above 90mmHg.After that,injections were immediately conducted with LIF(5μL,1μg/μL)in one group and PBS in another one.The eyeballs were extracted from the executed rats 24 hours and 3 days after the ocular hypertension.2.After paraffin embedding,the embedded tissue was used for morphological analysis.(1)H&E staining was conducted in each group for 24 hours and 3 days after acute ocular hypertension to evaluate the morphological change in retinal cells.(2)TUNEL staining was performed in each group at 24 hours after the ocular hypertension was halted to assess the apoptosis of retinal cells.3.Seven days before establishing acute ocular hypertension model,normal SD rats were randomly selected for FG retrogradely labeling RGCs in the superior colliculus.After successfully maintaining ocular hypertension for 60 minutes,intravitreal injections were immediately carried out with LIF in one group and PBS in another one.The flat-mounted retinas were harvested by excavating the experimental eyes 3 days after acute ocular hypertension to evaluate the quantity of RGCs.Statistical analysis was conducted in each group.4.After acute ocular hypertension for 24 hours,Western blot was carried out to evaluate the relationship between the effect of LIF and apoptotic pathway by detecting the expression of cleaved-caspase 3 and PARP.5.Western blot was also carried out after acute ocular hypertension for 3 days to evaluate the role of LIF in the relationship between JAK/STAT3 signaling pathway and AKT/mTOR/p70S6k signaling pathway.STAT3,p-STAT3,AKT,p-AKT,mTOR,p-mTOR,p70S6k and p-p70S6k may act as crucial roles in the neurodegenerative diseases.Results:1.After ocular hypertension,a great number of RGCs were much more slightly staining in larger sizes and an increasing number of karyopyknosis from 24 hours to 3 days’ group in H&E staining compared to the normal group.Edema existed in the inner retina especially 24 hours after ocular hypertension with markedly increasing thickness in the untreated group(P<0.05).The thickness of the inner retina was significantly decreased(P<0.001)in the untreated group after finishing the ocular hypertension operation for 3 days to contrast with the LIF treatment group(P<0.001).2.The quantity of fluorogold-labeled RGCs in LIF treatment group(2131.2 ± 85.6/mm2)were significantly increased compared with untreated group(1572.6±21.3/mm2,P<0.001),and non-significantly decreased compared with normal control group(2390.4±68.8/mm2).3.The TUNEL-positive cells were obviously increased especially in the RGC layer and inner nuclear layer in the untreated retina(127.6±30.0/mm2)compared with the normal retina and the LIF treatment group(26.9± 13.3/mm2,P<0.001)at 24 hours after acute ocular hypertension was completed.4.After acute ocular hypertension was completed for 24 hours,the expressions of cleaved-caspase-3(P<0.01)and PARP(P<0.001)protein in LIF treatment group were significantly decreased compared with untreated retina,with no significantly difference from the normal retina.5.When AOH was finished for 3 days,the level of phosphorylation of mTOR and its downstream factor p70S6k in LIF treatment group was significantly increased compared with untreated retina(P<0.05),with no significantly difference from the normal retina.But the level of phosphorylation of AKT,the upstream factor of mTOR signaling pathway,was no significantly difference from the other 2 groups(P>0.05).The level of phosphorylation of STAT3 was significantly increased compared with untreated retina(P<0.001),with no significant difference from the normal retina.Conclusion:The changes in the morphology and signaling pathway suggested that LIF may possibly play an important role in the neuroprotection of impaired retina induced by the acute ocular hypertension model via its anti-apoptotic effect and mTOR/p70S6k signaling activation.