الفهرس 
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Abstract
The tongue ulcers emerge over the eternal skin of mouth and tongue due to infection, brushing the teeth randomly, constipation, heavy drinking and excessive smoking. All the tongue ulcers are recognized by their white colored patches, found over the tongue and some other parts of mouth, causing inflammatory experience to the victim. Though the tongue ulcers can be controlled at initial stage, their exacerbation can’t be ruled out if ignored for long. The tongue ulcers are also known as aphthous ulcers, characterized by their white colored lesions all over the skin of tongue, causing it to swell, bleed and experience excessive inflammation all over its eternal skin. It causes aching and burning sensation to the person while speaking and consuming any edible item.
Diabetes is a multisystem disorder that affects the wound healing process. Chronic wound healing is a troublesome and common complication of diabetes, resulting in significant clinical morbidity, such as nonhealing ulcers, infection, gangrene and amputation. Thus, care for diabetic wounds remains a significant clinical problem and the development of therapies that improve wound healing in diabetic patients is of critical importance.
Spirulina plantensis is a simple, microscopic blue green algal. It grows naturally in fresh, brackish, sewage water and even in saline environment. Spirulina does contain a variety of nutrients, such as protein, a variety of B vitamins and minerals, the antioxidants beta-carotene and vitamin E, and phycocyanin. Phycocyanin, the blue pigment in spirulina, can act as a free radical scavenger and powerful antioxidant. Also it can exert a wide range of anti-inflammatory effects.
So this study is conducted to evaluate the effect of spirulina plantensis in diabetic wound healing in rat tongue mucosa.
Material and methods:
65 albino rats of average weight (150-200gm) were used in this study. The animals were subjected to induction of diabetes and then classified into 4 groups as follow:
Group A: 5 rats did not receive any treatment.
Group B: 20 rats were subjected to tongue ulcer and did not receive any treatment.
Group C: The same as group B but these animals received 1-2 IU/ g/day of insulin NPH.
Group D: The same as group B but these animals received 15 mg/kg body weight Spirulina plantensis extract for 30 days.
Both groups C& D were subjected to ulcer induction in their tongue after one week of starting of diabetic treatment.
Experimental diabetes was induced to all animals by a single intraperitoneal injection of streptozotocin (STZ: 45 mg/kg body weight in 0.1 ml citrate buffer, pH 4.5).The diagnosis of diabetes will be based on hyperglycemia (blood glucose (non-fasting) more than 300 mg/dl) using blood glucose meter (Hie M et al., 2007). The blood glucose level and the body weight of the rats were monitored every week and the results were listed in table. Ulcer was done at the middle of the tongue by using biopsy punch to ensure that all ulcers will have the same size (Jornet PL et al., 2009), the ulcer was circular in shape about 4mm in diameter.
After ulcer induction, specimens were obtained from each animal after 24 hours, 4days, 10days and 21days; tongue from each animal were removed and immediately fixed with 10%formalin solution for histological, and immunohistochemical studies. Specimens were put in glutaraldehyde fixative solution for electron microscope preparation.
(1)Statistical analysis results for the blood glucose level and the body weight of the rats:
It revealed a significant difference between groups B, C&D in different weeks.
(2)The histological examination revealed the following:
Haematoxylin & Eosin Stain:
Group A (Diabetic rats without ulcer induction):
After one week of the diabetic induction the tongue mucosa consisted of normally specialized stratified squamous epithelium with its layers (the basal cell layer, the prickle cell layer, the granular cell layer and the keratinized surface papillae) overlying the lamina propria with fibroblasts and blood vessels. The dorsal surface of the tongue showed normal arrangement and shape of papillae with hyperkeratosis. After 4 weeks of the diabetic induction, the dorsal surface of the tongue appeared with most of the papillae were flattened and with evident hyperkeratosis. Some fungiform papillae appeared elongated with narrow tips and separation of its covering keratin.
Group B (Diabetic rats without treatment):
After 24 hours of ulcer induction, these specimens revealed discontinuity of the epithelium and the underlying connective tissue, the granulation tissue showed inflammatory cell infiltration, after 4 days there was slight proliferation of the covering epithelium and formation of the granulation tissue with dense inflammatory cell infiltration, after 10 days the ulcer was still opened with heavy inflammatory cell infiltration, after 21 days there was prominent proliferation of the covering epithelium to close the ulcer margin as well as the connective tissue stroma.
Group C (Diabetic rats receiving insulin):
After 24 hours of ulcer induction the granulation tissue showed more inflammatory cell infiltration and dilated blood vessels. Mitotic figures appeared in the epithelium. After 4days the sections showed the beginning of healing process via proliferation of the epithelium. The granulation tissue appeared with fibroblasts, collagen fibers, inflammatory cell infiltrations and newly formed blood vessels. After 10 days of ulcer induction the sections showed complete reepithelialization of the ulcerative area with the beginning of reorganization of the epithelium and connective tissue. After 21 days there was complete reorganization of the normal epithelial stratification with normal architecture of the underlying lamina propria.
Group D (Diabetic rats receiving spirulina plantensis):
After 24 hours of ulcer induction the granulation tissue showed more inflammatory cell infiltration and dilated blood vessels, after 4 days the epithelium migrated. After 10 days the healing process was evident as the covering stratified epithelium proliferated to close the ulcer margins and covered the underlying connective tissue stroma. After 21 days the section showed complete reepithelialization of the ulcerative area with normal stratification, reorganization of the epithelium and the underlying connective tissue.
The Immunohistochemical examination revealed the following:
The immunohistochemical positive results were detected as brown deposits using alpha smooth muscle actin stain.
Group A(Diabetic rats without ulcer induction):
The brown deposits were detected in the smooth muscle cells of the blood vessel walls in the lamina propria.
In group B,C and D:
After 24 hours of ulcer induction the positive staining was found to be restricted to the smooth muscle cells of the blood vessel walls. At day 4 after ulcer induction in group C&D the expression of the alpha SMA was not only in the walls of blood vessels but also in the fibroblasts of the granulation tissue, while in group B there were negative staining of alpha SMA in the granulation tissue. At 10 days of ulcer induction, the immunoreactive cells had appeared in group B where their expression in group C&D were maximum. After 21 days there was decreasing in the expression of alpha SMA in group B while in group C&D, the only positive reaction was found in the walls of blood vessels.
(3)The Transmission Electron Microscopic examination revealed the following: Group A (Diabetic rats without ulcer induction):
The tongue mucosa showed epithelial cells with degenerated nuclei, swollen mitochondria and widening of the intercellular spaces.
Group B (Diabetic rats without receiving treatment):
After 24 hours of ulcer induction there was little infiltration of the granulation tissue with inflammatory cells with widening of the intercellular spaces between the cells of the mucosa, after 4days of ulcer induction there was heavy infiltration of the granulation tissue with neutrophils, the cells of the mucosa showed signs of inflammation with abnormal configuration of the nucleus, no apparent nucleoli and abnormal chromatin distribution. Mitotic figures appeared. No new blood vessels appeared. After 10 days there was still heavy infiltration of the granulation tissue with neutrophils, mast cell appeared but it contain few dense granules and its plasma membrane disintegrated, the cells of the mucosa showed abnormal configuration of the nucleus with no apparent nucleoli, little mitochondria and widening of the intercellular spaces, fibroblast was irregular in shape and very large, collagen fibers were loosely packed and irregularly arranged. After 21 days the cells of the mucosa showed abnormal configuration of the nucleus, and widening of the intercellular spaces. Fibroblast was irregular in shape. Collagen content was reduced.
Group C (Diabetic rats receiving insulin):
After 24 hours of ulcer induction there was heavy infiltration of the granulation tissue with inflammatory cells (neutrophils) with normal intercellular spaces between the cells of the mucosa. After 4 days the amount of neutrophils decreased and began to be replaced by lymphocyte and macrophage. New blood vessels and mitotic figures appeared. The cells of the mucosa were active with open faced nucleus, clear nucleoli, euchromatin, tonofilaments and many rough endoplasmic reticulums. After 10 days the amount of neutrophils decreased and mast cell appeared; it contained numerous homogeneous electron-dense granules and intact plasma membrane. The cells of the mucosa were active with open faced nucleus, clear nucleoli, normal dispersed chromatin, tonofilaments, rough endoplasmic reticulum and intact desmosomal junctions and intercellular spaces. Fibroblast was intact with regular spindle shape. Collagen fibers appeared more packed and presumably better aligned. After 21 days the cells of the mucosa were intact with intact nucleus, clear nucleoli, euchromatin, intact desmosomal junctions and intercellular spaces. Fibroblast was intact with regular spindle shape. Collagen fibers appeared more packed and presumably better aligned.
Group D (Diabetic rats receiving spirulina):
After 24 hours of ulcer induction there was heavy infiltration of the granulation tissue with inflammatory cells (neutrophils) with normal intercellular spaces between the cells of the mucosa. After 4 days lymphocyte, macrophage, new blood vessels and mitotic figures appeared. The cells of the mucosa were active. After 10 days the amount of neutrophils decreased and mast cell was similar to that of group C. The cells of the mucosa were active. Fibroblast and collagen content was similar to group C. After 21 days the cells of the mucosa were intact with normal configuration of the nucleus, intact intercellular spaces and desmesomal junctions. Fibroblast and collagen content was similar to group C.
Conclusion:
• Diabetes mellitus produce severe alteration and degeneration of the rat tongue mucosa.
• Diabetes mellitus is one such metabolic disorder that impedes normal steps of wound healing process.
• Spirulina plantensis has a beneficial effect in controlling blood glucose levels and is a promising agent for management of diabetes.
• Tongue ulcers in diabetic rats treated with spirulina plantensis showed steps towards healing, recovery, and improvement in their histological, immunohistochemical, and ultrastructural patterns.
Recommendation:
Within the limitations of the study, we suggest that spirulina plantensis may have an important role in the healing of ulcers in diabetic rat tongue.
More investigations should be made to evaluate the benificial effect of spirulina plantensis on wound healing.