The Postnatal Development Study Of Neuroglobin Expression In Rat's Retina And Intra-retinal Ultrastructural Distribution Of Neuroglobin In Normal Adult Rats

A large proportion of blindness diseases are associated with circulatory disorder of retinas,which is mainly caused by ischemia and the induced hypoxia. Thus the molecular mechanism of retinal oxygen metabolism,including the distribution,transport,and consumption of oxygen,is the key point to prevent and treat this kind of disease.Neuroglobin(Ngb),discovered by Burmester et al.in 2000,is now considered to exert its protective effect in hypoxia-induced neuronal injury as a neuron-specific respiratory protein.Researches have shown that the retina is the top one in all tissues on oxygen consumption rates,and at the same time,also on the concentration of Ngb.Taken together it is suggestive that Ngb may play an important role in retinal oxygen metabolism.In the present study,we observed the expression of Ngb in rat's retina in physiological state,including its expression in the postnatal development and the ultrastructural distribution in normal adult rats,in order to explore its possible roles and provide experimental evidence for the mechanism of its protection for retinal neurons in hypoxia-induced injury.Purpose:1,To observe the postnatal development of rat's retina and the expression of Ngb in order to identify the development-related changes of Ngb;2,To investigate the intra-retinal expression of Ngb in normal adult rats. Methods and Contents:Part 1:The Postnatal Development Study of Ngb Expression in Rat's Retina1,Eyes of 6 groups of SD rats(aged from 0 day to 2～3 months after birth,4 rats each) were enucleated and made into slides to prepare for following examinations.H-E staining was used for histological observation,and the immunohistochemical staining was used to analyze the position of Ngb;2,Collected the retinas of the 6 groups' rats as mentioned above(4 rats each), and detected the amount of Ngb expression of each group by Western Blot;Part 2:The Ultrastructural Distribution of Ngb in Normal Adult Rats Four normal female adult Sprague-Dawdey rats were used,and the ultrastructural location of expressed Ngb in the retina was determined by immunogold-silver staining.Results:1,H-E staining showed that the retina of newborn rats was immature.On the first day after birth,we can only identify the RPE layer,neuroblastic layer,IPL and GCL of the retina.The similar structure was observed on the third day. After one week,INL and ONL were independent together with OPL and photoreceptor IS appearing.GCL at this time point was thinner than before. Two weeks after birth,about the 14^th day,the retina structure of 10 distinctive layers had already showed up;2,The Ngb was observed to express in the cytoplasm of the retinal cells by the immunohistochemical staining.On the first and the third day after birth,it was obviously seen in the GCL and RPE layer and there was still some weak positive staining in the interior of the neuroblastic layer.One week later,Ngb was strongly detected in GCL and photoreceptor IS.Some weak positive staining was found in IPL,INL(especially the interior side),OPL and RPE layer.The position of Ngb expression on the 14^th day was similar to the 7^th day,but the intensity changed a little.The strongest expression were seen in GCL and photoreceptor IS,the stronger in IPL,INL and OPL,and the expression was weak in RPE layer.The similar expression was seen in P₂₈ and P_A;3,The Ngb protein bands of the 6 groups mentioned above were all detected by WB.There were significant difference in Ngb abundance between the 6 groups(p＜0.05).The expressed Ngb on the 14^th day was the most plentiful;4,Stained Ngb proteins were detected in GCL,IPL,INL,OPL,photoreceptor IS and RPE layer in normal adult rats by electron microscope.In the cells,Ngb immunoreactivity presented in the cytoplasm,mainly around the mitochondria and outside the cisterna of rough endoplasmic reticulum and Golgi complex.It was also found between the synaptic vesicles.Conclusion:1,The differentiation of all retinal layers is completed at postnatal 2 weeks;2,Ngb is expressed during the whole postnatal development of rat's retina.Its position and intensity has corresponded to the retinal structure of that time point,and above all the expression of Ngb shows a close relationship with the distribution of mitochondria in retinas,which suggests that Ngb may participate the oxygen metabolism in postnatal development of rat's retina;3,The expression of Ngb is significantly enhanced on the 14^th day after birth, when the rats just opened their eyes,which further suggests Ngb may play an important role in the retinal oxygen metabolism;4,The special ultrastructural location of expressed Ngb in the retina suggests a close relationship between Ngb and oxygen supply in neurons.