| Purpose: Retinopathy of prematurity(ROP)is a vasoproliferative retinal disorder that primarily occurs in preterm infants with low birth weight.As the development of perinatal medicine,the survival rates of extremely premature infants and very low birth weight infants are greatly increased,meanwhile the population of ROP is also increasing,especially in those developing countries,such as India or China.At present,Laser photocoagulation?Cryotherapy and anti VEGF therapy are the major treatments of ROP,after Laser photocoagulation or Cryotherapy,It is easy to recurred and get Strabismus?Amblyopia,refractive error or visual field defect in the future.Anti-VEGF monoclonal antibodies play the role in inhibiting abnormal neovascularization in eyes,and at the same time,it also May inhibit the normal vascular development in retinal and is easily recurred or have systemic adverse reactions.At present,damaging or inhibiting angiogenesis directly may prevent progression of the disease,but it still cannot reverse the damage caused by early vascular occlusion and is difficult to solve the problem of ROP fundamentally,ROP still constitutes to be one of the main disorders causing blindness and visual impairment in childhood.Early vascular occlusion and late vascular proliferation caused by the imbalance of promoting / inhibiting angiogenesis factor may be the main pathological mechanism of ROP,Including IGF-1,VEGF,Ang-1,PIGF and so on.Nowadays,these cytokines is becoming hot topics in pathogenesis of ROP.studies have shown that maintaining a certain IGF-1-concentration in the Preterm Infant played a key role in normal retinal vascular development and pathogenesis of ROP.This provides a new idea for ROP prevention and treatment,there were preliminary study in clinical and animal experiments,but the mechanism of the action,intervention route ?dose and time are still not clear,further researches are needed.We established a mouse model of oxygen-induced ROP mice model in the study,and administered a bolus of rh IGF-I on postnatal and applied hyperoxia to screen for the potential role of exogenous IGF-I,to explore the best administration time,dosage and route of rh IGF-1 for the prevention and treatment of ROP,and the influence to the expression of VEGF.Method: postnatal day 7 kun ming mice were randomly divided into air control group,and hyperoxia groups(including hyperoxia saline group,hyperoxia rh IGF-1 group 1,hyperoxia rh IGF-1 group 2,and hyperoxia rh IGF-1 group 3),with 30 mice in each group.Mice in the hyperoxia groups were assessed to expose to(55-65)% Oxygen for 12 days prior to living in room air.At postnatal day 19 the mice were returned to room air.At postnatal day 7 and 8,mice in the hyperoxia rh IGF-1 group 1 received 1mg/kg of rh IGF-1.In the same way and time,mice in the hyperoxia rh IGF-1 group 2 and group 3 respectively received 0.5mg/kg and 0.25mg/kg.Air control group and hyperoxia saline group received the same dose of normal saline.At postnatal day 7?12?19?24,blood samples were collected,and ELISA were used to determine serum levels of IGF-1 and VEGF.At day 19?24,eye Specimens were studied,HE dyeing were used for counting retinal vascular endothelial cells?ADP enzyme for observing retinal blood vessels division,at day 7?12?19?24,we collected eye Specimens for immunohistochemical stain to detect expression of IGF-1 and VEGF protein on retinal.body weight of every group were observed and recorded every day.Results:1.The comparison of body weight between every groups : body weight gradually Increased with advancing posmatal age,the control group growed faster than hyperoxia rh IGF-1 treatment groups,while hyperoxia saline group growed the slowest.at postnatal day 7,there were no statistical significance on Weight(p>0.05),at P12?P19?P24,the comparisons of body weight were statistical significance(p<0.05).body weight of hyperoxia saline group is significantly lower than any other groups(p<0.05).pairwise comparison between the three rh IGF-I treatment groups,the difference was no statistical significance(P>0.05)?Weight of air control group is higher than any of the three group,at P12(P=0.772?0.974?0.949),at P19?P24(P <0.05)?2.The comparison of neovascular endothelial cells: at P24,in hyperoxia saline group,a lot of vascular endothelial cells extending into the inner boundary membrane of retina,compared with every other groups,the difference was statistical significance(P<0.05).pairwise comparison between the three rh IGF-I treatment groups:hyperoxia group received 0.25mg/kg rh IGF-I had more endothelial cells,compared with other two rh IGF-I treatment groups(P<0.05),hyperoxia rh IGF-1 group 1 compared with hyperoxia rh IGF-1 group 2(P=1.000).neovascular endothelial cells of the three rh IGF-I treatment groups is higher than the air control group(P<0.05).3.The serum endogenous IGF-l levels : The serum endogenous IGF-l levels of all groups increased with advancing postnatal age,the control group growed faster than hyperoxia rh IGF-1 treatment groups,while hyperoxia saline group growed the slowest.At P12 ? P19 ? P24,there were statistical significance(p<0.05).with pairwise comparison,hyperoxia saline group was lower than any of other group(P<0.05).With increasing doses of rh IGF-1 intervention,the serum IGF-l levels were increasing,hyperoxia rh IGF-1 group 1 and hyperoxia rh IGF-1 group 2 compared with each other(P>0.05),all the two groups were higher than hyperoxia rh IGF-1 group 3(P<0.05),but were lower than the air control group(P>0.05).hyperoxia rh IGF-1 group 3 was lower than the air control group(P<0.05).4.The serum VEGF levels: at p7,there were no statistical significance on serum VEGF levels between all the groups(F=0.134,P=0.968),at P12?P19?P24,there were statistical significance(p<0.05).at P12?P19,the serum VEGF levels of hyperoxia saline group is lower than any other group(p<0.05),but at P24,it was the highest(p<0.05).At P12?P19,the serum VEGF levels of air control group was higher than any other group(p<0.001),while at P24,compared with hyperoxia rh IGF-1 group 1?hyperoxia rh IGF-1 group 2 respectively,there were no statistical significance(P= 0.991?0.979).hyperoxia rh IGF-1 group 3 was higher than air control group(P=0.005).pairwise comparison between three rh IGF-I treatment group,at P12?P19,hyperoxia rh IGF-1 group 3 was lower than the other two groups(P <0.05),at P24,it was hight than the other two groups(P <0.05).there were no statistical significance between hyperoxia rh IGF-1 group 1and hyperoxia rh IGF-1 group 2 at P12?P19?P24(P>0.05).5.The retinal blood vessel division: ADPase-stained of retinal blood vessels was assessed at P19?P24 to observe vascular morphology.air control group :at P19?P24,retinal vessels were basically mature,large blood vessels Walk naturally,and has branch rules,while distributed radially to peripheral of the retina.The retinal blood vessels had no occlusion or dilation;hyperoxia saline group: at P19 caliber of retinal vascular growed thinner significantly,runed stiff,and had no small blood vessels branch.at the central of retina,there were a range of avascular area.The retinal capillary network occluded widely.at P24,a lot of neovascularization could be seen.hyperoxia rh IGF-1 group 1 and hyperoxia rh IGF-1 group 2:at P19,large blood vessels were more nature,and Vascular caliber were thicker,there were a smaller range of avascular area.The blocking of retinal capillary network was not clear.at P24,neovascularization on retina was not obvious.hyperoxia rh IGF-1 group 3: at P19,caliber of retinal vascular growed a little thin,the range of avascular area grower bigger,while still can be seen few small vessel branches in the central of the retina,there was a small range of capillary network blocked in the peripheral of retina.at P24,vessels had slightly circuity in the Central,some abnormal vascular proliferation can be seen in the peripheral and central of retina.6.Expression level of the IGF-1 target gene on retinal: it gradually increased with advancing posmatal age,the control group growed faster than hyperoxia rh IGF-1 treatment groups,while hyperoxia saline group growed the slowest.at P12?P19?P24,there were statistical significance(p<0.05).hyperoxia saline group was lower than all other groups(P<0.05).pairwise comparison between three rh IGF-I treatment groups:there were no statistical significance between hyperoxia rh IGF-1 group 1 and hyperoxia rh IGF-1 group 2(P>0.05),all the two groups were higher than hyperoxia rh IGF-1 group 3(P<0.05),but were lower than the air control group(P>0.05).hyperoxia rh IGF-1 group 3 was also lower than the air control group too(P<0.05).7.Expression level of the VEGF target gene on retinal: the levels gradually decreased with advancing posmatal age,the air control group decreased fewer than hyperoxia rh IGF-1 treatment groups,while hyperoxia saline group decreased the most.After P19,the level began to rise rapidly in the hyperoxia saline group,and the hyperoxia rh IGF-1 Group 3 take the second,while the air control group continued to show a slow downward trend,hyperoxia rh IGF-1 group 1 and hyperoxia rh IGF-1 Group 2 were slightly increased.at P12?P19?P24,there were statistical significance(p<0.05).at P12 ? P19,its levels of hyperoxia saline group was lower than any other group(p<0.05),but at P24,it was the highest(p<0.05).at P12?P19,the levels of air control group were higher than any other group(p<0.001),while compared with hyperoxia rh IGF-1 group 1?hyperoxia rh IGF-1 group 2 at P24,it showed no statistical significance(P= 0.692?0.639).hyperoxia rh IGF-1 group 3 was higher than air control group(P=0.017).pairwise comparison between three rh IGF-I treatment group,at P12?P19,hyperoxia rh IGF-1 group 3 was lower than the other two groups(P <0.05),at P24,hyperoxia rh IGF-1 group 3 was higher than the other two groups(P <0.05).there were no statistical significance between hyperoxia rh IGF-1 group 1 and hyperoxia rh IGF-1 group 2 at P12?P19?P24(P>0.05).Conclusions:1.The Improved method can successfully induce ROP model in neonatal mice.2.Exogenous complementing of rh IGF-1 early can be effective in reducing the proliferation of retinal blood vessels,and reducing the severity of ROP.3.In the study,a large,medium and small doses(1mg / kg,0.5mg / kg,0.25 mg / kg)of rh IGF-I were assigned to oxygen-induced ROP mice model,while 1mg / kg,0.5mg / kg had the better effect. |
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