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Abstract
Diabetic retinopathy and keratopathy had attracted major power of both clinical and basic researches because of their clinical importance as a leading cause of blindness. Aminoguanidine proved to have a therapeutic potential for the future prevention of both diabetic keratopathy and retinopathy and it preserves the structure of cornea and retina in perfect form in normal eyes. The present study tried to evaluate the effect of experimentally induced diabetes on the structure of the cornea and retina of albino rats and the effect of aminoguanidine to ameliorate these changes.
Eighty adult male albino rats of weight ranging from 190-220 grams were used in this study. They were classified as follows:
Group I (Control group):-
Consisted of ten animals. It was subdivided into two subgroups.
Subgroup Ia:- Consisted of five animals. They were kept without any treatment throughout the whole period of the experiment.
Subgroup Ib:- Consisted of five animals. Each animal was given a single intraperitoneal injection of 0.5ml of 0.1M/L citrate buffer (Vehicle of streptozotocin).
Group II (Diabetic group):-
Consisted of forty animals. Each animal was given a single intraperitoneal injection of streptozotocin (30 mg/kg body weight) diluted in 0.5 ml of 0.1M/L citrate buffer, pH 4.5.
Group III (Diabetic+Aminoguanidine group):-
Consisted of twenty animals that were confirmed to be diabetic on the third day of experiment followed by administration of aminoguanidine which was given for each rat daily at a dose of (100 mg/kg body weight) for eight weeks.
Group IV (Aminoguanidine group):-
Consisted of ten non diabetic rats. Each rat was given aminoguanidine on the third day from the beginning of the experiment at a dose of (100 mg/kg body weight) daily for eight weeks.
Measurements of body weight and blood glucose were done.
On the last day of the experiment, the rats were sacrificed and both eye balls were enucleated and processed. Rats’ corneas and retinas of different groups were embedded in paraffin, cut and stained with different stains.
1- Haematoxylin and Eosin stain.
2- Histochemical staining (PAS stain).
3- Immunohistochemical staining:-
-For glial fibrillary acidic protein (GFAP) of the astrocytes in retina.
-For caspase-3 to detect apoptosis in both cornea and retina.
Other specimens of rats’ corneas and retinas of different groups were processed for electron microscopic study.
Thicknesses of corneas’ and retinas’ different layers were measured by image analyzer.
Statistical analysis was done using one-way ANOVA test.
Histological examination of corneal sections of the diabetic group showed marked histological affection which appeared focal in distribution. The thickness of all corneal layers was significantly increased as compared to other groups. The corneal epithelium showed degenerative cytoplasmic changes and vacuolations throughout its layers and many apoptotic epithelial cells were noticed. The surface corneal microvilli appeared disrupted or even lost in some areas. There were wide intercellular spaces with interrupted desmosomes in between some epithelial cells. The epithelial basement membrane appeared distorted with unapparent hemidesmosomal junctions. The Bowman’s membrane became irregular with areas of collagen fibrils aggregations. The corneal stroma showed areas of invaded blood capillaries together with inflammatory cellular infiltration. There were areas of deformed, disrupted wide spaced collagen fibrils as well. Stromal cells were distorted and many apoptotic stromal cells were noticed. Descemet’s membrane was irregular. Endothelial cells were distorted with many cytoplasmic vacuolations. Many apoptotic cells were also noticed.
However, diabetic aminoguanidine treated group showed marked restoration of the histological features of the cornea.
Histological examination of retinal sections of diabetic group showed marked histological affection which appeared focal in distribution.
There was a significant decrease in the thickness of outer and inner nuclear and plexiform layers as compared to other groups.
The retinal pigment epithelium showed many cytoplasmic vacuolation of various sizes mostly basal and apparent increase in the dense bodies mostly apical. There was also absence of basal infoldings as compared to control group. The photoreceptor cell outer segments showed extensive disorganization, disruption and marked fragmentation with increased caspase-3 immunoreactivity. As regards the nuclei of rods and cones cells in the outer nuclear layer, they showed marked degeneration. Few apoptotic cells were detected. The outer plexiform layer showed degeneration and vacuolations of some mitochondria in the nerve terminals. The inner nuclear layer showed degenerative changes in the horizontal, bipolar, Muller and amacrine cells. Many apoptotic cells were also noticed. Scattered small darkly stained nuclei were detected in between the cells of inner nuclear layer most probably microglia. Apparent increase in the number of Muller cells was also noticed. Moreover, there was enhanced GFAP immunoreactivity not only in the end feet of Muller cells but it projected towards the inner nuclear layer. The inner plexiform layer showed vacuolated mitochondria in some synaptic nerve terminals. As regards the ganglionic cell layer, the ganglion cells showed marked vacuolation in their cytoplasm. Many apoptotic cells were also noticed. Large elongated blood capillaries with disrupted tight junctions between their lining endothelium were also seen. Optic nerve layer appeared distorted.
- Meanwhile, diabetic aminoguanidine treated group showed marked restoration of the histological features of the retina.
- Moreover, the group which was given aminoguanidine alone showed perfect histological structure of both cornea and retina.