The Effect Of Low-level Lead Exposure On The Retina And Optic Nerve From Rats During The Developmental Stage

Part I Establishment of the developmental animal model of low-level lead exposureObjectiveUsing atomic absorption spectrophotometry to detect retinal and blood lead level of the normal and lead-exposed rats at different ages. The purpose of this study is to understand the change and metabolic pattern of retinal lead from the normal and lead-exposed rats in developmental stage which can be used to provide foundation for further studying lead-induced visual system deficits in children during the developmental period. MethodsTwo groups of healthy female pregnant SD rats were fed with deionized water or0.2% lead acetate solution, respectively, from the third to five days of conception, lasting for the entire gestational and lactational period. The offspring born by the group of rats drinking deionized water were classified as control group, while those born by rats drinking lead acetate were classified as experimental group. All rats at p21 (weaning) began to drink deionized water. The rats of different ages from each group were chosen, weighed and sacrificed at P3, P6, P9, P12, P15, P20,P25, P30, P40, P50. Blood and retina were collected as samples. The atomic absorption spectrophotometry was used to detect retinal and blood lead concentration of normal and lead-exposed rats at different ages. Results1. Blood lead concentration of lead-exposed rats at P6 showed significant increase compared with the controls.2. As development, retinal lead concentration of the lead-exposed rats after born gradually increased, the highest level of retinal lead was observed at P20. There was a significant different in the retinal lead concentration from the lead-exposed rats compared to the control groups.Conclusions1. There is a significant different in the retinal lead concentration from the lead-exposed rats compared to the controls. The highest level of retinal lead is observed at P20. There is no physiological functions which lead is in human body. The high retinal lead level is the foundation to harm retina and visual nervous system.2. The metabolic rule of retinal lead from lead-exposed rats is similar to that of blood lead, However, the half-life of lead in retina is longer than that in blood, and the metabolism may be slower.Part II The effect of low- level lead exposure on the morphology of retina andoptic nerve from rats during the developmental stageObjectiveThe purpose of this study is to establish a developmental framework of retina, understand the change rule in the cross-sectional areas and myelination of the optic nerve from the normal and lead-exposed rats in different developmental stage with a light microscope. The ultrastructural changes of the retina and optic nerve from two groups at p6, pl5, p50 is observed by electron microscope which reveal impact of lead on the morphology of the retina and optic nerve. Methods1. Globes from the normal and lead-exposed rats in different developmental stage were fixed, embeded in paraffin. Sections of the retina through the optic nerve head were stained with HE. Sections of optic nerve were stained HE and luxol fast blue, respectively. All sections were observed with the light microscope.2. Retina in posteror pole of eye and optic nerve from two groups at p6, p15, p50 were fixed, embedded, cut, stained, and observed with the electron microscope.Results1. The morphological pattern of retina from the normal rat during developmental stageCompared with adult rat, the total retinal thickness at neonatal rat was much thicker. On subsequent days, the total retinal thickness was gradually thinner, and reduced approximately 36.48% from P3 to P50. The inner plexiform layer(IPL) and the outer plexiform layer(OPL) of retina were slowly thicker. The IPL had been existed in neonatal rat, and to increase gradually within the developing retina, the thickness of the IPL increased roughly 36.31% from P3 to P50, whereas the OPL was first visible at 9 days postnatal, the thickness of the OPL increased roughly 26.59% from P9 to P50. The thickness of the retinal ganglion cell layer(GCL), the inner nuclear layer(INL), and the outer nuclear layer(ONL) became thinner, especially INL. The thickness of the GCLreduced 39.03% from P3 to P50. The thickness of the INL and the ONL decreased roughly 55.52% and 45.36% from P9 to P50, respectively. A corresponding changes in the number of each nuclear layer was observed as development. It was estimated that 52.28% of RGCs die by apoptsis from P3 to P50. The cell number of the INL and the ONL reduced approximately 40.56% and 20.04% from P9 to P50, respectively. The cell death at three nuclear layer was especially high between P20 to P25.There are only two nuclear layer in the retina from neonata rat, one is the GCL, which RGC has differentiated, another is the neuroblastic layer. The OPL, occuring at P9, which divides the neuroblastic layer into the INL and the ONL. The thickness of the neuroblastic layer reduced 5.73% from P3 to P6, the cell number lessen 3.41%.2. the morphological pattern of retina from lead-exposed rats during developmental stageWe found decreased the total retinal thickness at the lead-exposed rats compared with the control groups(P<0.05), which decreased 12.73%-24.15% from P3 to P50, average 21.45%. A signicant reduction in the thickness of the ONL from the lead-exposed rats was observed compared with the control groups (P<0.05), the thickness decreased 8.13%-28.44% from P9 to P50, average 15.61%. The decrease in cell was (P<0.05) 12.26%-28.47%, average 14.48%. Decreased thickness of the neuroblastic layer from the lead-exposed rats , which decreased (P<0.05)11.46%-19.91% from P3 to P6, average 15.69%, was consistent with decreased cell populations of the neuroblastic layer, which decreased (P<0.05) 16.19%-18.26%, average 17.23% compared with the control groups. No difference were found in the thickness of the GCL, INL, IPL, OPL, and the cell number of the GCL, INL from two groups (P>0.05). The RGC death at controls was high before roughly P25, whereas that from the lead-exposed rat was high before at P30. Subsequently, a slow decrease in the incidence of cell death occurred. The higher level of the cell death in the INL and the ONL from the control groups was before P25 and P30, respectively, whereas that from the lead-exposed rat was before P40. These findings suggested that the cell apoptosis period in the lead-exposed rat obviously lengthened.3. The morphological pattern of the optic nerve from the normal rats during developmental stageThe optic nerve cross-sectional area gradually increased, which the maximum postnatal growth of the optic nerve occurred between P3 and P15. The number of the glial cell reached a peak at P15-P20. Myelination of optic nerve began at 6 days postnatal. Over the ensuing 4 weeks, myelination increased rapidly, and finished roughly at P30.4. The morphological pattern of the optic nerve from the lead-exposed rats during developmental stageThe cross-sectional area of the optic nerve was significantly reduced in the lead-exposed groups compared to the control nerves(P<0.05), which decreased 11.07%-29.15% fromP3 to P50, average 19.10%. The percentage of myelin was also significantly reduced in the lead-exposed groups compared to the controls (P<0.05), which decreased 24.30%-75.81% fromP6 to P50, average 41.54%. There were no significant differences between experimental animals and control animals in the glial cell populations of the optic nerve. This findings indicated that lead may do not harm to the number of glial cell of the optic nerve, mainly caused myelin deficits.5. the ultrastructure changes of retina and optic nerve from the normal and lead-exposed rat in different developmental stageThe ultrastructure changes of retina in rat at P6, P15, P50: It was difficult to discern the difference between the cell of the INL and the ONL in normal retina at P6 with a light microscope, so we merely called them the cell of the neuroblastic layer. However, under electron microscop the cells from two nuclear layer in retina were different in size and tinction. The cell body of the INL was bigger, longer, undertint in color, whereas the cell body of the ONL was smaller, round, deep in color. The arrangement of the cell of two nuclear layer was sparse. The structure of mitochondria and Golgi complex was clear. The outer segment membranous disc of the photoreceptor cell lined up in order, the mitochondrial cristae in the inner segment was successive, round. The cell nucleus of the ONL was regular in shape, dyeing evenly, a small quantity ofkaryopyknosis. The cell of the INL and the ONL in retina at P15 and P50 arranged uniformly and compact. However, the ONL of retina from the lead-exposed rats at P6, P15, P50 was characterized at the ultrastructural level by the condensation of nuclear chromatin ,shrinkage of the cell soma, blebbing of the plasma membrane ,fragmentation of the cell into pieces and cellular membrane introcession. The outer segment membranous disc of the photoreceptor cell was swelling and blur, the stack partly disassociated, arranged irregularly ,the membrane gaps expanded. The mitochondria in the inner segment was swelling, a smaller proportion of crista was breakage and vacuolization.this phenomenon was less observed in the INL. The results indicated that lead more damaged the cell of the ONL.The ultrastructure changes of the optic nerve in rat at P6, P15, P50: the optic fibers in the control animals at P6 were divided into bundles, each bundle contained the different amounts of nervous fibers. Myelin occurred in the majority of samples, showing two to three turns of loosely wrapped myelin membrane, and became more compact at P15, P50. The axions were relatively integrity and the structure of the microfilament and microtubule was clear andthe glial cells pigmentated evenly. However, the optic nerve in the lead-exposed rats was smaller than that in the controls, few myelination was observed at P6. a small quantity of axions was wrapped one to two turns of filiform myelin membrane. This demonstrated that the myelination of the optic nerve following the lead-exposed rats was delayed. The myelin of the optic nerve in experimental animals at P15, P50 was few and thin, which accompanied with myelin sheath solution, amotic, disaggregation and disappear. However, there was no disruption of the microfilament and microtubule were not seen. Conclusions1. The retinal thickness in the normal rats was thinner with age. The physiological apoptosis was observed in the cell of all nuclear layer. The optic nerve in size gradually increased, which the maximum postnatal growth of the optic nerve occurred between P3 and P15. The myelination started at P6 and completed roughly at P30.2. Lead exposure caused delayed maturation and hypoplasty of the retina and opticnerve from the lead-exposed rats during the developmental stage.3. Lead's toxic effects could result in the pathological apoptosis of retinal cell ,specially the cell of the ONL.4. Low-level Lead caused demyelination at the developing optic nerve. Few influenceof axion, the microfilament and microtubule of the optic nerve was seen. Part III The effect of low-level lead exposure on the gene expression of NMDAreceptor subunit in retina at developmentObjectiveTo demonstrate the pattern of the NMDA receptor subunits mRNA expression in retina from the normal and lead-exposed rats during the different developmental stage by using a semiquantitative polymerase chain reaction method, understand effect of lead on development and plasticity of visual system, and thus provide the molecular mechanisms of lead-induced visual impairment. MethodsRats were sacrificed at P3, P6, P9, P12, P15, P20,P25, P30, P40, P50. The retina was harvested. Total RNA was extracted from the retina with Trizol reagent and reverse transcribed to cDNA. The amplification was performed with a thermocycler. Each polymerase chain reaction (PCR) product was separated by gel electrophoresis on 2% agarose and then photographed under ultraviolet light. The intensity of the products was analyzed using Quantity One. Results1. the pattern of NMDA receptor subunit mRNA expression in retina from the controls during the developmental stageNMDA-NR1 mRNA expression in retina started increasing on P3 and reached its peak on P9-P12, from then on .declined gradually with age; NMDA-2A mRNAexpression in retina at relatively low levels at birth increased by degrees with growth, whereas, NMDA-2B mRNA in retina at relatively high expression levels at birth declined by degrees. This findings suggested that maturation process of the retina from the normal rats was also the course which shifted NMDA-2B( early developmental stage) to NMDA-2A(maturation phase).2. Effect of lead on the pattern of NMDA receptor subunit mRNA expression in the retina during the developmental stageNMDA- NR1 mRNA expression in retina at relatively low levels at birth slowly increased with development and reached its peak at P25, thus declined gradually. The peak of NMDA- NR1 mRNA expression in the retina from the lead-exposed rats delayed for 13 days compared with that from the controls. NMDA-NR1 mRNA expression in the retina from lead-exposed rats was upregulated from P3 to P50 compared with that from the controls. NMDA-2A mRNA expression in the retina at relatively low levels at birth mildly increased, with development, the maximal expression of NMDA-2A mRNA was on day 25, thus declined. NMDA-2A mRNA expression in the retina from the lead-exposed rats was downregulated at P25, P30, P40 and P50 compared with that from the controls. the higher NMDA-2B mRNA expression in retina from the lead-exposed rats at birth remained at relatively high expression level with age. The upregulation of NMDA-2B mRNA expression in the retina from the lead-exposed rats at P40 and P50 was seen. with development of the retina from the lead-exposed rats, NMDA- 2A mRNA expression declined, whereas NMDA- 2B mRNA expression increased. The ratio of NMDA-2A to NMDA-2B was low, so transconformation of NMDA receptor subunit mRNA expression in retina from lead-exposed rats in the process of maturation lagged behind. This indicated that the development in retina was delayed. Conclusions1. In terms of NMDA receptor in retina, the maturation process of the retina from the normal rats was also the course which shifted NMDA-2B to NMDA-2A.2. Transconformation of NMDA receptor subunit mRNA expression in the retinafrom the lead-exposed rats in the process of maturation lagged behind. This indicatedthat the development in the retina was delayed.3. Lead caused abnormal NMDA receptor subunit mRNA expression in the retina atdevelopmental stage.