| Objective:We systematic analyzed the dynamical changes of all the mitochondrial complexes’activities and levels of oxidative stress of the retina and astrocyte under chronic high pressure. Try to reveal the mechanism of oxidative stress injury in retina of high intraocular pressure.Methods:In vivo, we use two kinds of animal model, one is Brown Norway rats whose chronic ocular hypertension were induced by injecting magnetic beads in the anterior chamber of, the other is pigment disseminated glaucoma model of DBA/2J mice. Get retinas at different time of high intraocular pressure and prepare tissue homogenate.In vitro, optic nerve head astrocytes were incubated under elevated hydrostatic pressure (30mmhg) for different time to simulate chronic high intraocular pressure. Collect the cells at different time points and prepare cell homogenates.Use the biochemical way to test the activity of all mitochondrial complexes, T-SOD, Cu,Zn-SOD, Mn-SOD and the content of MDA,ROS. Use SPSS16.0statistical software analysis data。Results:1. We achieved a sustained IOP increase in90%of Brown Norway rats.2. Elevated hydrostatic pressure induced changes of the mitochondrial complexes activities. ∥Brown Norway ratsi. Mitochondrial complexes Ⅰ activities in9-week-old Brown Norway mainly presented a downward trend during the first4weeks after high intraocular pressure (p<0.05), they reached the minimum at the4th week(0.0333±0.0056μmol·min·mgpro-1,p<0.001) and then gradually recovered to almost the normal level at the6th week (0.1071±0.0056μmol·min·mpro-1); However, mitochondrial complexes Ⅰ activities in22-week-old Brown Norway mainly presented a upward trend and reached the maximum at the4th week (0.2485±0.0029μmol·min·mgpro-1,p<0.05) before they dropped sharply, and almost reached the normal level at the6th week (0.0800±0.0018μmol·min·mgpro-1, p<0.001).ii. Mitochondrial complexes Ⅱ activities in two age groups decreased after high intraocular pressure. Activities of9-week-old age group reached the minimum at the2th week (0.0143±0.0003μmol·min·mgpro-1,p<0.001);while activities of9-week-old age group reached the minimum at the first week (0.0092±0.0023μmol·min·mgpro-1, p<0.05), although they had a small recovery latterly, but still significantly lower than normal (p<0.05)iii. Mitochondrial complexes Ⅲactivities in two age groups decreased immediately after high intraocular pressure, they respectively reduced by63.06%,80.04%(p<0.001) at the first week. Then they gradually recovered with the prolonging of pressurization time and were significantly higher than normal at the6th week(0.0694±0.0000μmol·min·mgpro-1,p<0.05;0.0599±0.0022μmol·min·mpro-1,p<0.001)iv. Mitochondrial complexesIVactivities in two age groups increased gradually in the first3weeks and reached the maximum at the3th week (0.1294±0.0076vs0.1249±0.0126μmol·min·mgpro-1, p<0.001),Then they began dropped sharply, and almost reached the normal level at the6th week (0.0800±0.0018μmol·min·mgpro-1, p<0.001). Though fell afterward, they were still higher than normal at the6th week(0.0330±0.0033VS0.0263±0.0031μmol·min·mgpro-1, p<0.05)v. Mitochondrial complexes V activities in9-week-old Brown Norway continued to slow down after high intraocular pressure and reduced by65.63%(p<0.001) at the6th week.But the activities in22-week-old groups continued rising slowly at the first4weeks and had risen by1.3times (p<0.05) in the4th week, then they fell rapidly and reduced by68.80%(p<0.05)until the6th week which were significantly lower than the normal group.②DBA/2J miceMitochondrial complexes Ⅰ activities were significantly elevated (p<0.001) while the other four mitochondrial complexes activities all reduced (p<0.01)③Astrocytes incubated under elevated hydrostatic pressureMitochondrial complexes Ⅴ activities actually increased overtime after high intraocular pressure and had risen by7.69times in the120th hour (p<0.001) Meantime the other four mitochondrial complexes activities continued to decline, complexes Ⅱ and IVactivities were on a gentle, but steady, downward slide while I and Ⅲ declined rapidly and sharply, they respectively reduced by88.66%(p<0.001) and68.39%(p<0.01) in the24th hours. Although complexes Ⅰ activities picked up slowly afterward (at the120th hour they reached to0.4236±0.0018μmol·min·mgpro-1),still well below the normal (p<0.001); complexes Ⅲactivities continued to slow down and reached the minimum at96h(reduced by82.11%, p<0.01)3. Mn-SOD activities showed a sustained downward trend under elevated hydrostatic pressure.①Brown Norway rats Activities of T-SOD, Cu,Zn-SOD in22-week-old Brown Norway respectively increased to71.9914±0.0025(p<0.001)and65.1074±3.9882(p<0.01)at the2th week after elevated hydrostatic pressure and then reduced rapidly during the3th and4th week, but they went up again in the6th week, respectively increased to72.1602±0.0006(p<0.001) and68.5408±0.2388(p<0.01).However, activities of Mn-SOD kept a sustained downward trend, they reduced by75.36%(p<0.05) until the6th week. Content of MDA changed correspondingly with the changes of T-SOD activities, at the2th,4th,6thweek after chronic ocular hypertension, they respectively increased by2.03times (p<0.001).0.93times (p<0.001)、1.48times (p<0.001) Activities of T-SOD in9-week-old Brown Norway respectively reduced by24.65%(p<0.001) and69.29%in the first2weeks after chronic ocular hypertension. Their activities began to rise again, but not untill the6th week (76.5028±1.2215U/mgprot, p<0.05, significantly higher than the normal level) they still well below the normal level and they reduced by38.35%(p<0.001) and28.78%(p<0.001) respectively. Content of MDA changed correspondingly with the changes of T-SOD activities, at the4th and6thweek after chronic ocular hypertension, they respectively increased by57.58%(p<0.01) and92.16%(p<0.05)。②DBA/2J miceActivities of T-SOD, Cu,Zn-SOD and Mn-SOD was respectively10.77%,10.49%,43.29%(p<0.05) and content of MDA declined by19.44%(p<0.05)③Astrocytes incubated under elevated hydrostatic pressure④Activities of T-SOD, Cu,Zn-SOD and Mn-SOD and content of MDA all declined steadily overtime.4. Content of ROS was significantly increased under elevated hydrostatic pressure.Conclusions:1. High intraocular pressure leads to changes of electron transport chain (ETC) enzymes activities and oxidative stress in the retina.2. Reactivity, compensatory ability and features in change of mitochondrial complexes activities in rats of different ages to stress injury were differently under elevated intraocular pressure.3. Mn-SOD activities showed a sustained downward trend under elevated hydrostatic pressure, lead oxidative imbalance in mitochondrial in early stage.4. MRCC Ⅲ is the main site of retina for ROS generation under high intraocular pressure. |
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