The Role Of Different Oxygen Inhalation Modes In Pathogenesis Of Retinopathy In Neonatal Mice

PART I The sensitivity of oxygen therapy to retina blood vesselin different postnatal day neonatal miceObjective To observe the sensitivity of oxygen therapy to postnatal day by different age mice inhaling oxygen , further study relationship between the developmental degree of the retina blood vessel and neovascularization .Methods A total of 48 postnatal day (P)4 ,7,9 ,11-day-old C57BL/6J mice were respectively divided into Hyperoxia group and Control group . The Hyperoxia group mice were exposed to 75% oxygen for 5 days and then to room air for another 5 days . The Control group mice were raised in room air for 10 days . The proliferative neovascular response was appraised by observing the vascular pattern in adenosine diphosphate-ase(ADPase) stained retina flat-mounts and quantitated by counting the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections .Results In the Control group the retina vessel graw to a majority of retine at P9 , be covered with entire retine at P11 , the peripheral vascular pattern was clear , and a few avascular area were seen at P12 . At P14 the avascular area disappeared . From P16 to P21 , the entire vascular pattern became completely normal , deep and shallow two layers vascular net were seen . In the Hyperoxia groups in 4, 7, 9-day-old mice , the central vessels become tortuous and constricted and central avascular area increased after hyperoxia for 5 days.After return to room air for 2 days, neovascularization was seen , after 5 days neovascularization peaked . In 11 -day -old Hyperoxia Group , the central avascular area increased and neovascularization was seen after hyperoxia for 5 days . But the central avascular area was reduced and abnormal neovascularization disappeared , with slight constriction of deep vessels after return to room air for 2 days . Five days later the vascular pattern become almost normal . The average number of neovascular nuclei extending into the vitreous per cross-section in 4, 7, 9, 11-day-old Hyperoxia group was 25.0±3.7, 47.7±5.0, 18.7±2.0, 2.6±1.4 respectively , the Control group was 0.7±1.1, 1.2±1.2, 0.8±1.0, 1.6±1.0 respectively. There were significant differences between 4, 7, 9-day old Hyperoxia group and the Control group 1, 2, 3 (P< 0.001), no differences between 11-day-old Hyperoxia group and the Control group (P=0.07).Conclusions Neonatal blood vessel in 4, 7, 9-day-old C57BL/6J mice is sensitive to oxygen therapy, neovascularization was formed by inhaling 75% oxygen for 5 days, 11-day-old mice no neovascularization. It was relative between the developmental extent of the retina vessel and neovascularization, the more immature retinal vessel was , the more easier neovascularization is, mature retinal vessel can not form pathogenesis of retinopathy. Therefore, for low brith weigh and small gastation age infants, we strictly check the retinopathy of prematurity, in order to discover and treat earlierly. PART II The effects of different oxygen inhalation modes on retinal vesselsdevelopment and the role in pathogenesis of retinopathy in neonatal mice Objective To study the effects of different oxygen inhalation modes on retinal vessels development and the role of different oxygen inhalation modes in pathogenesis of retinopathy in neonatal mice, in order to provide experimental data for proper oxygen therapy for premature infants.Methods A total of 720 postnatal day (P)7 C57BL/6J mice were randomly assigned into 6 groups according to different oxygen inhalation modes. Experimental group 1 was exposed to 30%, 40%, 50%, 60% and 75% oxygen in turn for one day respectively, followed by room air exposure for 5 days. Experimental group 2 was exposed to 75%, 60%, 50%, 40% and 30% oxygen in turn for one day respectively , followed by room air exposure for 5 days . Experimental group 3 was exposed to 75 % oxygen for 5 days , followed by room air exposure for 5 days . Experimental group 4 was exposed to 75% oxygen for 5 days , 50% for 2 days and 30% for 2 days, then room air exposure for 6 days . The supplemental 75% oxygen and room air recovering was performed alternately for the mice in the Experimental group 5 for 3 times and then room air exposure for 5 days . The control group was exposed to room air for consecutive 10 days . The retinal vascular development and proliferation were appraised by the retinal flat-mounts (ADPase stained retina) and cross-section . VEGF PEDF and IGF-1 mRNA expression was determined by real-time polymerase chain reaction , VEGF and PEDF proteins were determined by immunohistochemistry. Results The peripheral vascular pattern was clear , and a few avascular area were seen in the Control group at P12 . At P14 the avascular area disappeared . At P17 , the entire vascular pattern becomne completely normal . In the Experimental groups 1,3 and 5 , the central vessels become tortuous and constricted and central avascular area increased at P12 . At P14 , neovascularization was seen , peaking at P17 in the Experimental groups 1 , 3 and 5 . In the Experimental Group 4 , the central avascular area increased and neovascularization was seen at P14 , but the central avascular area was reduced and abnormal neovascularization disappeared , with slight constriction of deep vessels at P1 7. Five days later the vascular pattern become almost normal in the Experimental Group 4 . The retinal vascular form in the Experimental group 2 was similar to in the Control group . The average number of neovascular nuclei extending into the vitreous per cross-section in Experimental group 1 ,2,3,4,5 and the Control group was 49.5±1.4 , 5.2±0.7 , 47.7±4.7 , 5.7±2.4 , 29.2±2.5, and 1.2±0.2 respectively . There was significant differences between the Experimental groups 1 , 3 , 5 and the Control group (P<0.001). It was significant between the Experimental groups 1 , 3 and 2,4. The level of VEGF mRNA increased since P7 , peaked at P11 , and declined since P13 to a low level and maintained to P17 in The Control Groups. In Experimental groups 1 , 3 and 5 , it declined since P7 and remained declining during oxygen exposure , while it increased slowly since mice were taken back to room air and increased rapidly since P15 which is significant compared to the Controls, indicating a close relationship with changes of oxygen concentration . The trend of VEGF mRNA in the Experimental group 2 is similar to the Control group. In Experimental groups 4 , it declined since P7, at minimum P11, it increased mildly since P13, but which is no difference compared to the Controls. The level of PEDF mRNA increased since P7, peaked at P13, and declined mildly since P15 in The Control Groups. In Experimental groups 1 , 3and 5 , it increased since P7 and remained increasing during oxygen exposure; while it declined slowly since mice were taken back to room air and declined rapidly since P15 which is significant compared to the Controls , indicating a close relationship with changes of oxygen concentration . The trend of PEDF mRNA in the Experimental group 2 is similar to the Control group. In Experimental groups 4, it increased since P7, at peaked P11, it declined mildly since P13 , but which is no difference compared to the Controls. The level of IGF-1 mRNA decreased since P7, declined a low level at P15, and maintained to P21 in The Control Groups. In Experimental groups 1 , 3 and 5 , it decreased since P7 and remained decreasing during oxygen exposure; while it increased slowly since mice were taken back to room air and peaked respectively at P15, P17, P17, which is significant compared to the Controls , and declined since that time and maintained alow level to P21 . IGF-1 mRNA in the Experimental group 2 declined since 7 , at minimum at P21 . There were no difference between Experiment group 2 and Control group. In Experimental groups 4, it decreased since P7, at minimum P11, it increased mildly since P13, peaked at P15,and declined slowly since P17. Conclusions 1. The effects of different oxygen inhalation modes on retinal vessels development in neonatal mice were different . The obvious fluctuation of inhaled oxygen concentration and abrupt stop of supplemental oxygen after high levels of supplemental oxygen may severely affect the development of retina vascular , forming neovascularization , leading to the pathologic changes similar to retinopathy of prematurity . 2. VEGF , PEDF and IGF-1 were participated in neovascularization , VEGF promoted neovascularization , moreover PEDF is opposite. Hyperxia increased VEGF expression , declined PEDF expression , and leaded to VEGF/PEDF ratio reducing, hypoxia contrarily bringed on the ratio rising, leading to neovascularization . IGF-1 played important role on the development of normal retina vascularization and the pathogenesis of ROP.PART III The effect of supplemental oxygen on neovascularizationin pathogenesis of retinopathy in neonatal miceObjective To study the effect of supplemental oxygen on neovascularization , in order to provide experimental data for proper oxygen therapy for premature infants who formed retinopathy of prematurity .Methods A total of 210 postnatal day (P)7 C57BL/6J mice were randomly assigned into 8 groups according to different supplemental oxygen concentration . Experimental group 1 was exposed to 75% oxygen for 5 days , followed by room air exposure for 5 days . Experimental group 2 , 3 and 4 was exposed to 75% oxygen for 5 days , then exposed respectively to different FiO₂ 30%, 50% and 75% for days 5 , followed by room air exposure for 5 days . Experimental group 5 , 6 and 7 was exposed to 75 % oxygen for 5 days , then to return room for 2 days, and was exposed respectively to different FiO₂ 30%, 50% and 75% for days 5 , followed by room air exposure for 5 days . The Control group was raised in room air for consecutive 17 days . The proliferative neovascular response was appraised by observing the vascularpattern in adenosine diphosphate-ase(ADPase) stained retina flat-mounts and quantitated by counting the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections.Results The peripheral vascular pattern was clear , and a few avascular area were seen in the Control group at P12 . At P14 the avascular area disappeared . Form P17 to P24 , the entire vascular pattern becomne completely normal , deep and shallow two layers vascular net were seen . In the Experimental groups 1, the central vessels become tortuous and constricted and central avascular area increased at P12 . At P14 , neovascularization was seen , peaking at P17 in the Experimental groups 1. In the Experimental groups 2 , the central avascular area increased , neovascularization was seen at P17 , peaking at P19 and P22 in the Experimental groups 2 . In the Experimental Group 3 and 4 , the central avascular area increased and neovascularization was seen at P17, but the central avascular area was reduced and abnormal neovascularization disappeared , with slight constriction of deep vessels at P19 . Five days later the vascular pattern become almost normal . In the Experimental groups 5, the central avascular area increased, neovascularization was seen at P19 , peaking at P21 and P24 . In the Experimental Group 6 and 7,the central avascular area increased and neovascularization was seen at P19 , but the central avascular area was reduced and abnormal neovascularization disappeared , with slight constriction of deep vessels at P21.At P24 the vascular pattern become almost normal .The average number of neovascular nuclei extending into the vitreous per cross-section in the Experimental group 1 ,2,3,4,5,6,7 and the Control group was 47.7±5.2, 24.9±4.0 , 2.6±1.5 , 3.8±2.2 , 31.1±4.8 , 2.0±1.5 , 8.9±3.1 and 1.2±1.1 respectively . There were significant differences between the Experimental groups 1,2,5 and the Control group (P < 0.05) . And there were significant differences between Experimental groups 3,4,6,7 and the Experimental group 1 (P<0.05). Conclusions In the second phase of ROP , the appropriate supplemental oxygen alleviate neovascularization , but it is argue. Abrupt stop of supplemental oxygen after high levels of supplemental oxygen may promote neovascularization .