الفهرس 
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Abstract
Chlorpyrifos is the insecticide of choice for farmers worldwide. Repeated doses of chlorpyrifos were found to cause significant dysfunction on different organs. It was described as one of the endocrine disruptors, causing long-term changes in endocrine function and it is known to disrupt the thyroid gland and its secretions causing several clinical and subclinical malfunctions. Propolis extract is a well-known antioxidant. In addition, it has anti-inflammatory, immunomodulatory and cytotoxic effects. Therefore, the present study was performed to study the microscopic, morphometric and biochemical alterations in the thyroid gland in adult male albino rats after the administration of chlorpyrifos for successive 3 months and also to assess the possible protective role of propolis extract on these changes. Sixty-five adult male albino rats of two months age and weighing approximately 200-250 gm/rat were used in this study. Rats were divided into two main groups control and experimental groups. All treatments were given by oral gavage. The control group included 15 rats, subdivided into 3 groups (each contained 5 rats). group Ia rats received 0.5 ml of distilled water once daily. group Ib rats received 0.5 ml of corn oil once daily. group Ic rats received Propolis extract dissolved in distilled water (50 mg/kg/day). The experimental group included 50 rats, subdivided into 2 subgroups (each contained 25 rats). group II a rats received Chlorpyrifos (CPF) dissolved in corn oil (6.75mg/Kg/day). group IIb rats received CPF concurrently with Propolis extract in the same mentioned doses. Note, all animals were weighted before the beginning of the experiment. Twenty-four hours after administration of the last dose, each rat was weighted and all animals were fasted overnight. Blood samples were taken from the retro orbital venous plexuses for hormonal assay, then rats were anaesthetized by ether inhalation. An incision was made in the skin of the neck then trachea was exposed and thyroid gland was rapidly obtained from each rat and cut into two lobes. The right lobe was used for the light microscopic study and the left lobe was used for the electron microscopic study. Tissue samples for light microscopic studies were processed to prepare 5µm paraffin sections. Sections were stained with H&E for routine histological examinations, PAS for staining the colloid and Masson’s Trichrome stain for the demonstration of collagenous fibers in the stroma. The left lobe of the thyroid gland was cut into small pieces 1 mm³, fixed in 2.5% glutaraldehyde and processed for electron microscopic study. Semithin sections (1 micron) were stained with toluidine blue stain to confirm histological changes. Ultrathin sections (80-90nm) were stained with urinyl acetate and lead citrate for the examination by TEM. Morphometric study was done to assess the area of thyroid follicles, colloid percentage area, percentage area of fibrosis in addition to the percentage of parafollicular cells. Biochemical study was done to assess the serum levels of T3, T4 and TSH. Examination of H&E stained sections in the thyroid gland of all subgroups of the control group showed the same histological structure. A thin connective tissue capsule was found covering the gland from which septa arise dividing the gland into incomplete lobules. The thyroid parenchyma showed normal organization of variable sized thyroid follicles filled with acidophilic homogenous colloid. Large follicles were present mainly at the periphery, while central follicles had smaller diameter. Central thyroid follicles were lined by a single layer of cuboidal cells with rounded nuclei, while the peripheral ones were lined mainly by flat cells. Parafollicular cells appeared large, pale with vesicular nuclei. Clumps of interfollicular cells were seen in C.T among the thyroid follicles. PAS stained sections of control groups showed PAS positive reaction in the colloid and the basement membrane surrounding the thyroid follicles. Masson’s trichrome stained sections showed fine collagenous fibers in the thin connective tissue capsule and in the connective tissue septa among the thyroid follicles. Semi-thin sections stained with toluidine blue showed the normal histological structure. Few parafollicular cells were noticed with pale stained cytoplasm and vesicular nuclei. The electron microscopic examination of ultrathin sections of the thyroid gland of control group showed that thyroid follicles were lined by a single layer of follicular cells and parafollicular cells. Some follicular cells were active with cuboidal epithelium. They had oval nuclei with clumps of heterochromatin. The apical surface of follicular cells showed long slender microvilli projecting into the luminal colloid and the Lateral borders showed tight junctions. Their cytoplasm exhibited mitochondria, Golgi apparatus, few lysosomes as well as cisternae of rough endoplasmic reticulum. Parafollicular cells had rounded euchromatic nucleus. They were lying on thin basal lamina and did not reach the lumen of the follicle. Their cytoplasm showed numerous electron dense secretory granules, Golgi apparatus, numerous mitochondria and cisternae of rough endoplasmic reticulum. The administration of chlorpyrifos daily for successive 3 months resulted in a significant histopathological and ultrastructural alterations in the thyroid gland. The capsule of the gland was thickened. Many follicles were small in size. Some of the follicles appeared with irregular outline, others appeared with exfoliated desquamated cells in the lumen. Some follicles revealed disrupted basal lamina and others appeared with multiple layers of follicular cells with pyknotic and degenerated nuclei that disappeared at some follicles. Congested blood capillaries were noticed. Some follicles showed hemorrhage inside their lumen. Parafollicular cells were hypertrophied with dark pyknotic nuclei, and showed hyperplasia and neoplasia at some areas. Interfollicular tissue was expanded by the proliferating interfollicular cells and interfollicular hemorrhage. PAS reaction in most of the follicles of rats’ thyroid gland of chlorpyrifos treated group showed a weak reaction in the colloid and basement membrane which was lost at some areas. In addition, there was a statistically significant decrease in the percentage area of colloid in this group as compared to control group. Masson’s trichrome stained sections revealed excess blue stained collagen fibers in the thickened capsule as well as the interlobular septa with an increase in the collagen fibers among the thyroid follicles in the interfollicular tissue and around blood vessels. There was a highly significant increase in the percentage area of fibrosis as compared with those of control groups. Semi-thin sections stained with toluidine blue showed disturbed normal architecture of thyroid follicles. Parafollicular cells were increased in number and size. There was a statistically significant increase in the percentage of parafollicular cells as compared with control groups. These light microscopic results were accompanied with a significant decrease in the serum levels of T3 and T4 and a significant increase in the serum level of TSH. The electron microscopic examination of ultrathin sections of chlorpyrifos treated group rats’ thyroid gland showed hyperplastic thyroid follicle lined by more than one layer of follicular cells. The apical surface showed partial loss of microvilli. The cytoplasm contained markedly dilated cisternae of rough endoplasmic reticulum and Golgi apparatus, multiple dense lysosomal granules, small scattered vacuoles and degenerated mitochondria. Some follicles were surrounded by thick collagen bundles. The follicular cells had irregular pyknotic nuclei. Parafollicular cells contained fewer secretory granules with lower electron density as compared to those of control group. Light microscopic examination of H&E stained sections of chlorpyrifos and propolis treated group showed improvement in the architecture of thyroid gland. Most of the follicles were more or less similar to the control group. However, some follicles showed follicular hyperplasia, desquamated cells in the lumen and congested blood vessels. In addition, there was a statistically significant increase in the size of thyroid follicles in this group as compared to chlorpyrifos group but still significantly decreased as compared to control group. PAS reaction of nearly all the follicles was more or less similar to that of the control group. The basement membranes of most of the follicles were intact while that of others were thin and weakly stained. In addition, there was a statistically significant increase in the percentage area of colloid compared to chlorpyrifos group. Masson’s trichrome stained sections of prophylactic group showed normal number of collagenous fibers among the thyroid follicles. There was a significant decrease in the percentage area of fibrosis as compared with those of chlorpyrifos group. Semi-thin sections stained with toluidine blue showed restoration of normal architecture of most of thyroid follicles. There was a statistically significant decrease in the percentage of parafollicular cells as compared with those of chlorpyrifos group. These light microscopic results were accompanied with a significant increase in the serum levels of T3 and T4 as compared to chlorpyrifos group and a significant decrease when compared to control group. Electron microscopic examination of ultrathin sections of chlorpyrifos and propolis treated group rats’ thyroid gland showed that most of the follicles were more or less similar to that of control group. The apical surface of the follicular cells exhibited normal arrangement of microvilli projecting into luminal colloid. Rough endoplasmic reticulum had slightly dilated cisternae in some follicles, while cisternae were regular in most of the follicles. Nuclei in most of follicular cells were regular with prominent nucleolus. Parafollicular cells restored its numerous specific secretory electron dense granules. A significant increase in the mortality rate of rats after chlorpyrifos administration was noticed, which was significantly decreased when propolis was used concurrently with chlorpyrifos. In addition, body weight of rats of chlorpyrifos group showed significant decrease. While animals’ weight was improved with propolis administration.