الفهرس 
Materials and methodsOnly 14 pages are availabe for public view
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Abstract
Nicorandil is a member of relatively new class of potassium channel activators used for treatment of angina pectoris. Like any other drug, nicorandil has been reported to exhibit some side effects, drug-induced ulceration is now recognized as a frequent side effect of nicorandil. Propolis is an ancient and important medical remedy that has been used in various clinical situations, with many biological properties, including immunomodulatory, anti-inflammatory, antiulcer, antioxidant, antibacterial, antiviral, antifungal and antiparasite activities. To investigate the effect of nicorandil used separately or coupled with propolis on the gingiva and periodontal ligament of the albino rat, forty adult male albino rats with 120-150 gram body weight were used in this investigation. The animals were divided into 4 groups 10 animals each.
• group I animals served as controls, and supplied with adaily oral dose of 1ml distilled water.
• group II animals were treated with nicorandil in a daily oral dose of 2 mg/kg body weight (Rathod et al., 2011) using curved metallic oro-pharyngeal tube.
• group III animals were treated with propolis in a daily oral dose of 50 mg/kg body weight (ElMasry et al., 2011) using curved metallic oro-pharyngeal tube.
• group IV animals were treated with nicorandil coupled with propolis in the same doses and route of administration as group II and III.
The animals of all the groups were fed natural diet and allowed drinking water adlibitum throughout the whole experimental period which lasted for 3 months. At the end of Summary and Conclusion 219
the experiment the animals of all the groups were sacrificed by cervical dislocation. The upper and lower jaws were dissected out. Specimens from the gingiva were taken from the region of the upper and lower first molars of the left sides of upper and lower jaws, fixed in 10% neutral buffered formalin, processed, embedded in paraffin and sectioned . The right sides of the upper and lower jaws were similarly fixed in 10% neutral buffered formalin, decalcified in 10% EDTA. After complete decalcification they were processed, embedded in paraffin and sectioned. Six micron thick sections of the jaws and gingiva were cut and stained with:
1. Hematoxylin and eosin for histological evaluation.
2. Masson’s trichrome stain for collagen.
Four micron thick sections of the jaws and gingiva were mounted on optiplus positive charged slides to be stained for:
- Immunohistochemical localization of Interleukin 1 beta (IL-1β) using universal kit streptavidin-biotin peroxidas system with Interleukin 1 beta mouse monoclonal antibody.
- Immunohistochemical localization of transforming growth factor beta (TGF-β) in using a ready to use kit of antibody against transforming growth factor β antigen, a supersensitive multilink starter kit that matches the primary antibody and an antigen retrival.
Small sized samples (1mm3 ) from the gingiva of the animals of the different groups were selected and immediately fixed in 3% phosphate buffered glutaraldehyde for one hour, washed in the buffer and post fixed in 1% buffered osmium tetroxide for 1-2 hours at 4οC, washed, dehydrated in ascending grades of ethanol,
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cleared and embedded in epoxy resin. Ultra thin sections were cut and stained with uranyl acetate and lead citrate to be examined with transmission electron microscope.
Similarly, and immediately after jaws dissection, half the number of the separate jaws were fixed in 3% phosphate buffered glutaraldehyde for one hour, washed in the buffer and post fixed in 1% buffered osmium tetroxide for 1-2 hours at 4οC, then the specimens’ were decalcified in 10% EDTA. After complete decalcification small sized jaw specimens (1mm3) were cut from the region of periodontal ligament of the decalcified jaws, washed, dehydrated in ascending grades of ethanol, cleared and embedded in epoxy resin. Ultra thin sections were cut and stained with uranyl acetate and lead citrate to study the periodontal ligament by transmission electron microscope. Results: Histological Results: The histological results revealed that nicorandil affected the gingiva and periodontal ligaments as it produced variable degrees of atrophic changes to the extent of formation of ulcerative lesions in the gingival epithelium. Edematous and swollen epithelial cells were frequently seen. There was loss of the characteristic pattern of the epithelial ridges, sometimes they were partially or completely absent. Also attachment epithelium showed marked atrophy/or ulceration in addition to folding of its basement membrane. Collagen fibers in the subepithelial lamina propria of the gingiva and in the periodontal ligaments suffered dissociation and degeneration with apparent decrease in the number of fibroblasts in addition to a lot of inflammatory cell infiltrate were seen. Summary and Conclusion 221
Masson’s trichrome stained sections confirmed the marked degeneration and dissociation of the collagen fibers in the tissues under investigation. Histological findings in the gingiva and periodontal ligaments of group III that received propolis only, appeared normal with nearly the same histological features of normal tissues, however it showed apparent increase in number of cells of the surface epithelium leading to slight acanthosis, mild increase in granular cell layer and its keratohyaline granules in addition to slight hyperkeratosis. Numerous clear cells were observed not only among the basal cells but also among upper spinous cell layer. Slight increase in the collagen fibers of the periodontal ligaments and in subepithelial lamina propria of the gingiva were found, and this is confirmed by Masson’s trichrome stained sections in the tissues under investigation. In case of group IV animals that received combined nicorandil with propolis there were many histological signs indicating the effect of propolis in improvement the of structure of the gingiva and PDL, where the epithelium of gingiva, and dentogingival junction was no more atrophic since it partially regained its thickness, ulcerative areas were rarely encountered, Also, Collagen fibers of periodontal ligaments and in the subepithelial lamina of gingiva revealed decrease in the degree of degeneration with increase in the number of fibroblasts and this was more proven in Masson’s trichrome stained sections. Ultrasturctural results:
The ultrasturctural changes of the gingiva confirmed and added to the structural findings, manifested as degrees of cytoplasmic vacuolizations of the cells of the surface epithelium, wide intercellular spaces, nuclear alterations, mitochondrial enlargement and decrease in keratohyaline granules, in addition to a number of inflammatory cells that Summary and Conclusion 222
appeared between epithelial cells. Degeneration of fibroblasts and collagen fibers in the subepithelial lamina propria of the gingiva and periodontal ligament sections were found, where collagen bundles appeared fragmentated or lysed, a lot of inflammatory cells infiltrate were also observed. The gingiva of the animals of group III that received propolis showed almost normal ultrasturctural features of normal surface epithelium, moreover there were narrowing in the intercellular spaces, increased tonofilaments, increase in keratohyaline granules of granular layer, Langerhans cells were detected in the spinous layer between epithelial cells Normal appearance of the collagen fibers of lamina propria of the gingiva and PDL, however there were mild increase in the density of collagen fibers and increase number of fibroblasts. The gingiva of the animals of group IV that received nicorandil and propolis combined revealed improvement in the ultrasturctural features, where cells of different strata showed less signs of degeneration in comparison with those in group II that received nicorandil only. Intercellular spaces appeared narrower, marked decrease in the cytoplasmic vacuolizations. There was also improvement in ultrasturctural appearance of the collgen fibers and fibroblasts in the PDL and in the lamina propria of the gingiva which appeared more organized, fibroblasts appeared more or less normal in addition to some inflammatory cells were detected. lmmunohistochemical results:
• Interleukin 1 beta ( IL-1β):
In group I&III animals, immunohistochemical localization of IL-1β showed negative staining reaction in the epithelial cells of the different strata of the gingiva, negative staining reaction was revealed in the fibers and cells of PDL and lamina propria of the gingiva, while blood vessels showed negative to weakly positive reaction. Summary and Conclusion 223
The intensity of the staining reactivity was increased in group II animals. However improvement in the staining pattern was observed in group IV animals treated with combined nicorandil and propolis.
• Transforming growth factor beta ( TG-F-β):
In group I&III animals, immunohistochemical localization of TGF-β showed weakly positive staining reaction in the epithelial cells of the different strata of the gingiva, weakly positive staining reactivity in the fibers and cells and moderately positive staining reaction in the blood vessels of PDL and lamina propria of the gingiva. The intensity of the staining reactivity was almost negative in group II animals. However an increase in the staining pattern was observed in group IV animals treated with combined nicorandil and propolis. Conclusion Depending on the results of the present study, the following conclusions could be reached:
1- Administration of nicorandil produces alteration and degeneration in the gingival and PDL tissues of the rats.
2- Propolis offers a promising therapeutic value in prevention and overcoming the side effects of nicorandil on gingiva and PDL.
3- Administration of propolis alone enhanced the barrier function of the gingival epithelium and the integrity of the PDL.
4- It is comprehensible that we should now switch on to “back to nature” approach where propolis seems a promising alternative medicine for the control of diseases with good response and for counteracting the side effects of certain medicaments, and more attention should pave towards
propolis and its plausible use in different diseases