الفهرس 
AcknowledgmentOnly 14 pages are availabe for public view
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Abstract
Summary The ten-layer albino rat retina is used as a model for studying the development of the nervous system. It is like other areas of the central nervous system derived from the neural tube. The formation of the retina begins in albino rat at day 9 of pregnancy and extends up to about 3 weeks after birth. Zinc is an essential component of the normal metabolism of proteins and nucleic acids and appears to play a key role in maintaining eye function. This component is present in high concentrations in the eye tissue, especially in the retina and choroid. The incidence of congenital malformations in children born to mothers who had zinc deficiency during pregnancy was observed. This study aims to study the effect of zinc deficiency in mothers during pregnancy on retinal prenatal and postnatal development in albino rat. This study was conducted on 48 female rats (3 months old) and 12 adult male rats and rats were mated. The pregnant female rats were divided into two groups (experimental group and control group): • group A (Control): a total number of 24 adult pregnant female rats received single intraperitoneal injection of distilled water on day 9 of gestation. • group B (Experimental): a total number of 24 adult female rats received intraperitoneal injection of 1.10 phenanthroline (purchased from Sigma Company) in a single dose of 30 mg/kg on day 9 of gestation. As for each group, serum zinc level was measured. 72 Male offspring of the treated and control rats were sacrificed at the following prenatal ages: 13th, 15th, 17th and 19th days of gestationand the following postnatal ages: newborn, 7 days,15 days and 21 days. from the offspring of both control and treated mothers, 3 male rats were used in each of the studied age groups for Haematoxylin& Eosin staining technique to study the cytoarchitectural and maturational changes in the retina. Transmission electron microscopic study was done in which 3 male rats were taken from the offspring of both the control & treated mothers at the following ages newborn, 7days, 15 days and 21 days postnatally to show ultrastructure of the different layers of the retina. Immunohistochemical study using anti-Glial fibrillary acidic protein and caspase-3 was used at the newborn, 7 days, 15 days and 21 days postnatal age groups. Morphometric study was done to estimate the thickness of different layers of the retina (photoreceptors layer, outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer) in newborn, 7 days, 15 days and 21 days control and treated postnatal age groups. The results of this study demonstrated that at the gestational age of 13 days old control rat. The optic cup was well formed and it appeared to be consisted of an outer (external) wall and an inner (inverted) wall. By day 15 of gestation in control rat, the pigmented epithelial layer of the retina was present as an uneven, single layer of nearly cuboidal cells, the inverted (neuroblastic) layer was constantly thickened by extensive mitotic activity at its choroidal edge. At the embryonic day 17 of the control group in this study, the neuroblastic layer increased in thickness due to the presence of the mitotic figures. This layer consisted of two parts: an outer part with condensed dark nuclei and inner part of uncondensed paler nuclei. By the day 19 of gestation, the control group in the current study revealed that the inner plexiform layer appeared as a relatively clear zone separating between the darkly stained nuclei of the neuroblastic layer and the round lighter stained ganglion cell nuclei.
Postnatally, by light microscopy, the newborn control rat retina consisted of the pigment epithelium arranged in one row. The neuroblastic layer was formed of two parts, an outer darkly stained compact part and an inner and less compact part with paler nuclei. Moreover, Mitotic figures were common in the outer border of the neural retina with considerable increase in the thickness of the neuroblastic layer. Retina of the control rat’s retina at the 7th postnatal day in this study demonstrated that by the inner segment of the photoreceptors started to appear but the outer segment was not developed. The neuroblastic layer was separated by the outer plexiform layer which appeared as a slight nuclear separation so that the outer part became the outer nuclear layer and the inner part of the neuroblastic layer became the inner nuclear layer. 15 days old control rat retina revealed that all layers of the retina were in the mature condition. Both the outer and inner segments of the photoreceptors could be easily distinguished and were well developed. The outer limiting membrane was seen. Müller cells nuclei could be noticed in the inner nuclear layer. The inner plexiform layer reached its maximal thickness. The ganglion cell layer was formed of one row of cells. The inner limiting membrane was detected. At postnatal day 21, the control rat retina showed that the retina is well formed. The Cone nuclei were observed in the outer half of the outer nuclear layer. Rod nuclei were more densely stained with a unique rosette-shaped dense chromatin and scanty cytoplasm. The nerve fiber layer was considered the axons of the ganglion cells.
The results of the electron microscopic examination of the control newborn, 7days, 15 days and 21 days old rat retina in this study indicated that the retinal pigment epithelial cells were flat cells containing numerous mitochondria and abundant free ribosomes. The tightly packed neuroblastic cells in the newborn rat retina had different shapes and sizes of nuclei and dense chromatin. At 7 days old control rat retina, the outer segments of photoreceptors were not apparent at this age but the inner segments were started to be formed of outer ellipsoids that contained mitochondria and glycogen granules. At 15 days old control rat retina, the outer segments of photoreceptors were formed of parallel lamellar structures. The inner nuclear layer contained amacrine cells with large indented nuclei with electron lucent cytoplasm. By 21 days old, control rat retina showed that the inner nuclear layer contained Müller cells which appeared with electron dense cell bodies, dark nuclei and many cytoplasmic processes. The ganglion cells of the newborn, 7 days, 15 days and 21 days old rat retina revealed large nuclei with free ribosomes and mitochondria, Golgi bodies and rough endoplasmic reticulum in the cytoplasm.
Immunohistochemical study using anti glial fibrillary acidic protein (GFAP) showed that immunoreactivity was detected in the nerve fiber and ganglionic layers. GFAP was not detected in any other layer of the control rat retina. Also, there was negative caspase-3 reaction in all the layers of the control rat retina.
In contrast to the control group, the experimental group at gestational day13 in this study showed that apparent shrinkage of the lens vesicle. The optic cup was well formed but it appeared to be shrunken. The external layer of the optic cup contained one cell layer of tall columnar cells with darkly stained nuclei. The inverted layer of the optic cup consisted of several layers of columnar cells with deeply stained pyknotic nuclei. No mitotic figures could be observed. The RPE of zinc-deficient rats of 15th, 17th and 19thdays old embryos consistently contained apoptotic cells with vacuolated cytoplasm and rarified nuclei and the neuroblastic layer showed apparent decrease in thickness and less mitotic features while the ganglion cell layer revealed numerous deeply stained pyknotic nuclei in the 19th gestational day.
By light microscopy in the present study, the retina of the newborn rat of the zinc deficient group showed that the retinal pigment epithelium and the neuroblastic layer contained some vacuolated cells with pyknotic nuclei. In addition to the pyknotic and vacuolated cells in 7days-old zinc deficient albino rat retina; the inner segments of the photoreceptors revealed vacuolations. Moreover, Zinc deficient albino rat at the age of 15 and 21 days old revealed numerous deeply stained pyknotic nuclei and vacuolated cytoplasm in the outer nuclear layer cells. The outer plexiform layer appeared distorted and the inner nuclear layer appeared condensed with most nuclei were deeply stained and pyknotic. This layer showed highly dispersed cells with markedly vacuolated cytoplasm. The ganglion cell layer appeared disorganized with some shrunken deeply stained pyknotic nuclei and vacuolated degenerated cytoplasm. Widening of the spaces between nerve fibers was observed. The inner limiting membrane was disrupted and could be hardly detected.
Electron microscopic examination of the zinc deficient rats in all the studied postnatal age groups in this work, the pigment epithelial cells revealed flat or rounded nuclei surrounded by rarefied cytoplasm containing distorted mitochondria with disrupted cristae, dilated rough endoplasmic reticulum and electron dense inclusion bodies.
In this work, in the treated rats the outer segments of photoreceptors were distorted with marked disorganization and loss of normal orientation of its lamellar disc membranes and some of them showed distended bimembranous discs. Decrease in the density of the outer segments, degeneration in the lamellae and increase in intermembranous disc space were also detected. The inner segments showed disorganization with degenerative vacuoles, distorted swollen mitochondria with disrupted cristae and accumulated glycogen granules.
The inner nuclear layer of the experimental groups contained amacrine cells with dilated nuclear envelop, rarified cytoplasm, dilated rough endoplasmic reticulum and degenerated mitochondria. Müller cells with irregular thin processes were noticed in the zinc deficient rats. The ganglion cells in this study showed an electron dens nucleus with dilated nuclear envelope surrounded by rarified cytoplasm containing swollen mitochondria with disrupted cristae, markedly dilated rough endoplasmic reticulum and many vacuoles.
Immunohistochemical stained experimental albino rat retina sections using GFAP showed strong positive immunoreactivity for GFAP restricted to inner retinal layers. Moreover, there was intense positive caspase-3 reaction in all layers of the treated rat retina.
Morphometric findings in this study showed the presence of non- significant decrease in the total retinal thickness of the retina in all the prenatal studied zinc deficient rats in comparison with their corresponding control. Postnatal, there was statistically highly-significant decrease of the total retinal thickness of the zinc deficient newborn, 7, 15 and 21 days old rats when compared with the same control age group, while there was a significant decrease in both the neuroblastic layer and the ganglion cell layer in zinc deficient newborn rats when compared with the newborn control group. Also there was a significant decrease in the outer nuclear layer, the inner nuclear layer and the ganglion cell layer in zinc deficient7, 15 and 21 days old rats when compared with the same control age group
In conclusion, this study demonstrated that maternal zinc deficiency causes cytoarchitectural and maturational changes in the development of albino rat retina. It is recommended that there should be strict control of zinc level during pregnancy.