Neuroprotective Effect Of The PPAR-γ Ligand Pioglitazone In The Rat Ischemia/reperfusion Model

An Rat Model of Retinal Ischemia/Reperfusion InjuryObjective:Establish a rat model of retinal ischemia/reperfusion Injury and investigate the acute elevation of IOP and ischemia insult on retinal morphology, function and retinal neuron apoptosis, further evaluate the I/R model for neuroprotection research.Methods:Retinal ischemia was induced in30rats by increasing the intraocular pressure to110mmHg for60minutes,21for both eyes and12for the right eye,9normal rats served as control. Retinal damage was quantified by measuring the thickness of the retina, the functional changes of visual evoked potential (VEP) and electroretinography (ERG), and the retinal ganglion cells (RGCs) number was determined both by Nissl staining and retrograde labeling,1,3,7days after double-eye injury. The VEP, ERG, RGC number and the retina thickness was detected in the sigle-eye injury group7days after I/R and compared with those received I/R in both eyes.Results:The retina thickness of Normal group was180.8±3.33um and the IPL was48.71±1.05um. One day after I/R the retina thickness was172.9±4.71um and IPL decreased slightly (43.58±1.88um, p<0.05vs. Control). After3days the whole retina and the IPL thickness was142.3±3.55um and28.23±0.64um respectively (both p<0.01vs. Control). Seven days later the retina thickness reduced to124.8±2.13um and IPL was13.56±0.52um (both p<0.01vs. Control). Three days after I/R RGCs decreased to about88%(p<0.05vs. Control) and7days later drop to57.18%(p<0.01vs. Control) by Nissl staining. The retrograde labeled RGC was about46%of control 7days after I/R. ERG amplitudes were significantly decreased1day after injury (a, b p<0.01vs. Control). Seven days later the a-wave amplitude reduced by53%and b-wave amplitude decreased up to71%(p<0.01vs.Control). VEP also showed significant decrease in N1P1、P1N2、N2P2amplitudes1d after I/R (p<0.01vs.Control), and worsened with time. The number of TUNEL labeled cells in retina including RGC, INL, and ONL was significantly increased24h after I/R. A few TUNEL labeled cells scattered in retina3day after I/R and barely any TUNEL positive cells were observed7days after the insult. No significant differences were observed between the rats that received I/R operation in both eyes or in single eye.Conclusions:I/R caused thinning of the retina, destruction of visual function and great lose of vital RGC. Retrograde labeling might be better reflecting the RGC survival. The I/R model could cause apoptosis in retinal neuron and might be a useful animal model for retinal neuron injury. Since one eye injured and the contralateral eye was not affected the unharmed eye could serve as control.Chapter TwoNeuroprotective Effect of the PPAR-y Ligand Pioglitazone in the rat ischemia/reperfusion modelObjective:To investigate the protective effect of the PPAR-y Ligand pioglitazone in the rat retina after ischemia/reperfusion (I/R) injury.Methods:Eighty rats were divided randomly into four groups:I/R group, I/R with pioglitazone1.0mg/kg intraperitoneal injection, I/R with pioglitazone0.125mg periocular injection, and Control. Only the right eye of each animal was included in the experiment. Pioglitazone was delivered by periocular injection and intraperitoneal injection3hours before I/R. Cell apoptosis in the retina was detected by TUNEL assay24hours after reperfusion. Retinal damage was quantified by measuring the thickness of the retina, the functional changes of visual evoked potential (VEP) and electroretinography (ERG), and the retinal ganglion cells (RGCs) number7days after I/R injury. Results:I/R caused severe destruction of the retina (retina thickness:119.3±3.593um; IPL:13.45±0.476um; both p<0.001vs. Control). Pioglitazone either delivered by periocular injection (retina thickness:158.2±5.848um, p<0.01vs. I/R; IPL:34.84±1.429um, p<0.01vs. I/R;) or intraperitoneal injection (retina thickness:143.7±7.967um,p<0.05vs. I/R; IPL:25.54±2.018um p<0.01vs. I/R) maintained the thickness of the whole retina and IPL after I/R. Seven days after I/R, the number of RGC was1141.14±82.89/mm2, a decrease of approximately53%compared with the control group (p<0.01). While under treatment of pioglitazone, the surviving RGC number was1954.43±38.16/mm2(p<0.01vs. I/R) in the group of pioglitazone periocular injection, and1890.53±68.96/mm2(p<0.01vs. I/R) in the group of pioglitazone intraperitoneal injection respectively. There was no statistical difference in RGC number between the two pioglitazone-pretreated groups. Pioglitazone pretreatement significantly attenuate the destruction of VEP and ERG caused by I/R (p<0.01vs. I/R) and the amplitude of N1-P1and P2-N2components in pioglitazone periocular injected group was higher than the intraperitoneal injected group (p<0.01in N1-P1and p<0.05in N2P2).24hours after the I/R the TUNEL labeled cells were increase in the retina (13.85±1.67cells/field vs. Control0.33±0.21cells/field,p<0.01,). The TUNEL-positive cells were6.2±0.51cell/field in the group of pioglitazone intraperitoneal injection (p<0.01vs. I/R) and8±0.58cell/field in the group of pioglitazone periocular injection (p<0.01vs. I/R) respectively. No significant difference was observed in the number of TUNEL-labeled cells between the two routes of pioglitazone administration.Conclusions:Pioglitazone maintained the retina thickness, promoted the survival of retinal ganglion cells, and attenuated the destruction of ERG and VEP caused by the I/R and inhibited the apoptosis in the retina neuron after I/R. The drug delivered by periocular injection acquired better effect than systemic delivery.Chapter ThreeThe Mechanisms of Pioglitazone in Neuroprotection during the Retinal Ischemia/Reperfusion InjuryObjective:To further reveal the mechanisms of pioglitazone in neuroprotection during the retinal ischemia/reperfusion (I/R) injury. Methods:Ninety-six rats were divided in to four groups randomly:I/R group, I/R with pioglitazone1.0mg/kg intraperitoneal injection, I/R with pioglitazone0.125mg periocular injection, and Control. Only the right eye of each animal was included in the experiment. Pioglitazone was delivered by periocular injection and intraperitoneal injection3hours before I/R. Western Blot was used to detect the expression of Bax, Bcl-2, Phospho-ERK (1/2), ERK (1/2), TNF-a and PPAR-y in the retinal tissue. Immunochemistry and Western blot analysis were performed to measure glial fibrillary acidic protein (GFAP) expression and activation of Nuclear factor-kappaB (NF-κB) in the retina. Real-time PCR were performed to measure the expression of GFAP mRNA after I/R.Results:I/R cause significant increase of Bax:Bcl-2ratio, upregulation of ERK phosphorylation level and decrease of PPAR-y expression compared to control group (all p<0.01)24h after the insult. Pioglitazone pretreatment up regulated the PPAR-y expression and suppressed the upregulation of p-ERK. The Bax:Bcl-2ratio was much lower in the retinas in groups pretreated with pioglitazone than that in the I/R group(p<0.05vs. I/R). I/R caused activation of glia and GFAP expression was observed in the end feet of Miiller cell24after I/R and through out the retina7days later. Western blot analysis also showed increased GFAP expression after I/R (p<0.01vs. control). Pioglitazone significant inhibited the upregulation of GFAP expression caused by I/R thus inhibit glia activation. Real-time PCR analysis presented similar trend in the GFAP mRNA level as that with the GFAP protein expression. NF-κB p65-positive cells were observed24h after I/R and there was no significant difference between all the operated groups. Seven days after I/R the expression of NF-κB p65was significantly upregulated with high phosphorylation level of p65(p<0.01vs. control), and TNF-a expression was also upregulated. Pioglitazone pretreatment by both the periocular (p<0.05vs. I/R) and intraperitoneal injections (p<0.05vs. I/R) significantly attenuated the p65subunit and TNF-a expression, and the phosphorylation level of NF-κB p65was reduced (both p<0.01vs. I/R). No significant difference in the Phospho-NF-KB p65subunit and NF-κB p65subunit expression was observed between the two different routes of pioglitazone administration.Conclusions:Pioglitazone could protect the retina from subsequent cellular damage caused by retinal I/R and upregulated the Bcl-2expression thus inhibit the apoptosis. It could inhibit glia activation and the interaction with NF-κB pathway, which contributes to the attenuation of retinal inflammation after ischemia. MAPK signaling might also be involved.