الفهرس 
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Abstract
The aim of this study was to evaluate the effects of oral administration of 3 doses of KBrO3 (0.1, 62 and 123 mg/kg) and co-administration of 0.5 ml/kg FO on tongue and parotid salivary gland of adult male albino rat in different durations.
Material and methods:
Animals
210 adult male albino rats with average weight 200 g were used in this study. Animals of each group were caged in separate cages in the animal house at the Faculty of Medicine, Minia University.
Chemical
KBrO3 and FO were purchased from Sigma, St. Louis, MO. USA. through The Egyptian International Center for Import.
Experimental design
The animals were divided into 3 main groups:
1. Control group: composed of 30 rats which received 1ml of distilled water daily through the duration of the experiment (1, 2 and 4 months).
2. Experimental group I: (KBrO3 only)
composed of 90 rats and were divided into 3 subgroups (30 rat in each group), these subgroups were administered KBrO3 orally on a daily basis in the following doses.
• Subgroup 1: 0.1 mg/kg
• Subgroup 2: 62 mg/kg
• Subgroup 3: 123 mg/kg
3. Experimental group II: (KBrO3+ FO)
composed of 90 rats that were divided into 3 subgroups (30 rats in each group), these groups were administered 0.5ml\kg of fish oil (FO) followed by KBrO3 orally on a daily basis in the following doses:
• Subgroup 1: 0.5 ml\kg FO + 0.1 mg/kg KBrO3
• Subgroup 2: 0.5 ml\kg FO + 62 mg/kg KBrO3
• Subgroup 3: 0.5 ml\kg FO +123 mg/kg KBrO3
10 rats from each subgroup of both experimental groups were sacrificed after 1st, 2nd and 4th months and the tongue and parotid glands were prepared for the following stains:
1-Haematoxylin and Eosin stain.
2-Periodic Acid Schiff (PAS) for histochemical staining of parotid gland only.
3-Immunohistochemical staining using Proliferating cell nuclear antigen (PCNA).
Results:
Hematoxylin and eosin results:
Tongue results:
Histopathological examination of tongue in KBrO3 group (gp I) subgroup 1 after the first month revealed histopathological changes. It showed just some atrophic changes in the filiform papilla which appeared slightly shorter with atrophied epithelium and their keratin layer showed thinning & detachment in some areas. In addition to increased spaces between atrophied muscle fibers.
By increasing the dose in subgroup 2 and 3 the atrophy of papillae increased and the fungiform papillae became affected. The epithelium covering the dorsal surface in subgroup 2 exhibited hyperkeratosis while in subgroup 3 it was atrophied with decreased thickness and covered by thin detached keratin layer. On other hand, the basal cell layer showed area of disruption with loss of cohesion and polarity of its cells. Both subgroups also showed atrophy and disorganization of muscle fibers with increased interfibers spaces.
With increasing the duration, the histological changes became more aggravated. In subgroup 1 after the 2nd month, the dorsal epithelium became depressed, thin atrophied and devoid of filiform papillae. While after the 4th month epithelium thickness increased again and contained atrophied filiform papillae together with nuclear changes as nuclear pleomorphism and numerous mitotic figures in higher cell layers. On other hand, muscle atrophy and increased interfiber spaces appeared after both the 2nd and 4th month.
For subgroup 2 and 3 with increasing the duration changes became more aggressive especially in subgroup 3 with the highest dose in which criteria of malignancy were established.
In subgroup 2 after the 2nd month changes increased, filiform papillae lost their regular orientation, moreover the basal cell layer showed proliferation and the muscle layer showed increased spaces between fibers. After the 4th month these changes became more aggravated with nuclear changes like pleomorphism and prominent nucleoli started to appear together with mitotic figures in higher layers, while connective tissue showed increased amount and hyalinization of fibers and blood vessels were dilated and congested with blood. On the other hand, subgroup 3 displayed top to bottom epithelial dysplasia after the 2nd month, nuclear hyperchromatism, numerous normal and abnormal mitotic figures in all epithelial layers, increased connective tissue fibers and congestion of blood vessels were seen. These changes became more exaggerated after the 4th month and tongue sections displayed epithelial cell hyperplasia, pleomorphism of nearly all nuclei in epithelium and connective tissue, fibrosis of connective tissue and eventually, proliferation of epithelium and invasion of connective tissue.
Histopathological examination of FO/KBrO3 group, subgroup 1 after the first month showed that FO nearly maintained normal appearance of epithelium however, the muscle layer appeared to a little extent atrophied. Also in subgroup 2 and 3, FO preserved its protective effect against KBrO3 where filiform papillae retained their regular direction, however there was some atrophic changes in it. On the other hand, muscle fiber atrophy and spacing still exist however they were regularly grouped and organized.
With increasing the duration, KBrO3 effect overcame the FO protective ability. The histological changes were still existing but to a lesser extent in comparison to experimental group I. After the 2nd month, in subgroup 1 dorsal surface retained its covering with papillae however they appeared atrophied and had separated keratin layer. This also was found also in subgroup 2 in addition to disruption of basal cell layer. In sub group 3 there was covering of atrophied papillae but no focal flattening did exist. on other hand, mitotic figures were also restricted to the lower half only.
For the last duration FO was still had the ability to protect tissues form KBrO3 effect in all groups as seen in the 2nd duration and dysplastic changes were far away from those seen in experimental group I, no invasion was seen in subgroup 3.
Fibrous content in general appeared to be lesser in FO/KBrO3 compared to KBrO3.
Parotid salivary gland result:
Histopathological examination of parotid gland of experimental group I showed histopathological alternations, subgroup 1 after the first month showed little histological changes in form of cytoplasmic vacuolation in serous acini and striated duct cells, striated ducts appeared dilated together with the blood vessels which appeared congested with blood. In addition to hemorrhage between the acini.
This histological changes became more aggressive with increasing the dose in subgroup 2 and 3, the acini became distended and lost their outlines and circular arrangement especially in subgroup 3. The nuclei showed dysplastic changes in form of hypertrophy, hyperchromatism and pleomorphism in addition to many normal and abnormal mitotic figures, division of nucleus without division of the cytoplasm. On other hand, the striated ducts became more dilated with thinning in their lining epithelium. Blood vessels were more dilated and subgroup 3 showed clotted blood that nearly blocked blood vessel lumen.
With increasing the duration changes became more exaggerated for all subgroups, however the degree of changes was more obvious in subgroup 1 which showed distended acini with more vacuolated cytoplasm with crescent like nuclei, more dilated striated ducts with atrophied lining epithelium and connective tissue hyalinization after the 2nd month while after 4th month vacuolation decreased with deposition of eosinophilic material in serous acini, and their nuclei showed dysplastic changes. In addition to thrombus formation that nearly blocked blood vessel walls.
After the 4th month subgroup 2 showed pleomorphism of fibroblast with inflammatory cell infiltrate appeared in connective tissue stroma and subgroup 3 showed increased spaces between serous acini, degeneration of some acini with karyorrhexis and increased fibrous content in connective tissue stroma and the excretory ducts showed stagnant secretion with cells inside it. While both subgroups shared a common prominent changes like hyperchromatism and prominent nucleoli.
In experimental groups II which was given FO followed by KBrO3, histological examination showed that FO had a strong protective effect in subgroup 1 for the first duration (1 month) only. While in the same subgroup after the 2nd and 3rd durations, subgroups 2 and 3 for the 3 different durations, it alleviated the effect of KBrO3.
In subgroup 1 after the first month, FO co-treatment nearly maintained normal structure of the gland with very few cytoplasmic vacuolation of few acini, slightly dilated striated ducts and blood vessels.
While in subgroups 2 and 3 the protective effect of FO was less obvious especially with increasing the dose in subgroup 3, the cytoplasmic vacuolation appeared in both subgroups but to a lesser extent than their corresponding groups of experimental gp I, nuclear changes were also evident but were very few in subgroup 2 while more evident in subgroup 3 however, they were still lesser than their counterpart of the experimental group I. On the other hand, striated ducts appeared less dilated and blood vessels didn’t show any thrombus formation, just engorged with RBCs which were very few in subgroup 2 while were more in subgroup 3.
By increasing the duration, the protective effect somewhat decreased, disorganized glandular architecture and nuclear changes still occurred but they were lesser in comparison to their corresponding subgroups in group I. however no inflammatory infiltrate had been detected also no fibrosis was seen. In addition, no stagnant secretion was found in any duct in comparison to experimental group I.
PAS histochemical stain results:
PAS staining revealed decrease in PAS stain, experimental group I showed gradual decrease with dose and durations. However, it started to increase again in subgroup 3 after the 4th month.
In experimental group II (FO/KBrO3 group), parotid gland showed improved PAS stain in comparison to experimental group I.
PCNA immunohistochemical:
Tongue results:
Immuohistochemical examination of experimental group I and II, specimens showed increased PCNA labelling index in comparison to the control group. Immunoreactivity was found to gradually increase and appeared in upper epithelial layer, connective tissue cells and muscle cells by increasing the dose and duration, except in subgroup 1 where PCNA immunoreactivity increased after the 1st month and the 2nd month and then slightly decreased after the 4th month.
All experimental group II subgroups showed decrease in PCNA immunoreactivity in comparison to their corresponding subgroups of experimental group I.
Parotid gland results:
Immuohistochemical examination of both experimental group I&II subgroups revealed that there was gradual increase with varying patterns of PCNA staining in acini and intralobular ducts.
Experimental group I showed increase immunoreactivity in comparison experimental group II.
Statistical analysis results:
For the tongue, there was significant increase in the mean values of PCNA area fraction in both experimental groups I & II with the increase of the dose and the durations for all subgroups except subgroup 1 which showed increase then decrease. There was also significant increase in PCNA area fraction mean values of all subgroups of experimental group I when compared to experimental group II subgroups.
For the parotid salivary gland, there was significant increase in the mean values of PCNA area fraction in both experimental groups I &II with the increase of the dose and the durations for all subgroups and there was also significant increase in PCNA area fraction mean values of all subgroups of experimental group I when compared to experimental group II subgroups.