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Abstract
Diabetes mellitus is a common metabolic disorder associated with degenerative changes in the pancreas. Due to its high prevalence and marked effects on a patient physical and psychological state, diabetes mellitus, which can result in a morbid condition, is a major medical concern.
Moreover, Alloxan is the most widely used drug for induction of diabetes mellitus in laboratory rats .Single intra-peritoneal administration of 180 mg ∕ Kg alloxan leads to a condition closely resembling human type1 diabetes mellitus.
Many alternative medicines were studied to be used in the treatment of diabetes mellitus, beside or instead of the traditional treatment of diabetes with oral hypoglycemic drugs and insulin injections. Green tea and garlic were used nowadays as anti-diabetic agents depending on their powerful hypoglycemic action.
So, the aim of the present work is to study the histological, ultra structural and biochemical effects of green tea and garlic on the pancreatic tissue to asses their use in the treatment of diabetes mellitus.
Seventy two male albino rats weighing 180–200 gm were used in the present work. The rats were divided into six groups, twelve rats each.
Group (I): The rats of this group were used as a control group (non diabetic).
Group (II): Each rat received a single intra-peritoneal dose of ±36 mg of alloxan.
Group (III): Each rat received a single intra-peritoneal dose of ±36 mg of alloxan. Each diabetic rat received a single oral daily dose of green tea (±20mg∕0.5ml) for four weeks.
Group (IV): Each rat received a single intra-peritoneal dose of ± 36 mg of alloxan. Each diabetic rat received a single oral daily dose of garlic extract (±80mg∕0.5ml) for four weeks.
Group (V): Each rat received a single intra-peritoneal dose of ±36 mg alloxan. After four weeks, each diabetic rat received a single oral daily dose of green tea (±20mg∕0.5) for four weeks.
Group (VI): Each rat received a single intra-peritoneal dose of ±36 mg of alloxan. After four weeks, each diabetic rat received a single oral daily dose of garlic extract (±80mg∕0.5ml) for four weeks.
After 24 hours of alloxan injection, blood was collected from the veins of the tail of the rats to confirm occurrence of diabetes mellitus (>200mg∕100ml).
Weight of the rats was measured and then the rats were sacrificed, blood samples were collected and the pancreas was exposed, dissected and specimens were taken. Samples of blood were studied biochemically and specimens of the pancreas were processed to be studied histologically by light and electron microscopes.
Examination of the paraffin sections of the diabetic rats (group II) stained with haematoxylin and eosin revealed remarkable degenerative changes. Acinar cells appeared ill defined with irregular arrangement. Some nuclei were lightly stained and few were dark and small. Some cells appeared to be vacuolated with foamy appearance. Loss of demarcation between the exocrine acini and islet of Langerhans was noticed. The islet cells appeared amalgamated with faint cytoplasm and lightly stained nuclei. Large areas of degeneration were observed. Moreover blood vessels appeared congested.
Semithin sections revealed markedly distorted acini. This was demonstrated by ill defined cell boundaries between the acinar cells. Their cytoplasm was studded with multiple small vacuoles. Some cells contained aggregations of zymogen granules, extending occasionally to the basal part of the cytoplasm. However other acini were almost devoid of these granules. Areas of degeneration appeared in other cells. The nuclei were variable in size and density, and some were irregular. Blood vessels had markedly thickened walls in some sections and were dilated highly congested in other sections.
As regards the islets, it was noticed a decrease in cell density in some sections. Numerous cells exhibited irregular small dark nuclei while few cells had lightly stained vesicular nuclei with prominent nucleoli.
Electron microscopic examination of the pancreas of diabetic rats revealed ill defined cell boundary of the acinar cells. The cytoplasm contained large amount of endoplasmic reticulum which was extensively dilated in some cells together with irregular mitochondria. Some cells had few small rounded basal vacuoles while others contained larger grouped coalased ones. Zymogen granules of variable size were observed in some acinar cells and depleted in others. Nuclei were slightly irregular in shape but some nuclei were bizarre in shape. Congested blood vessel wall was observed.
Islet cells (alpha cells) showed few cisterns of endoplasmic reticulum and elongated vesicular mitochondria. Vacuolation of variable size and shape was observed. Secretory granules were dense and multiple in some cells and very few in other cells. Nuclei were regular. While, beta cells showed marked degeneration. The characteristic finding was the assorted appearance of the secretory granules. Frequently, they appeared marginal in their halos. The halos were either; irregular, widened or fused together and some granules showed entire loss of the surrounding halo.
Examination of the paraffin sections stained with haematoxylin and eosin of (group III) showed that most of the acini appeared regular in shape, with well defined boundaries, however the cell boundaries were indistinct . Most of the nuclei were rounded, basal and vesicular. Demarcation between the islets of Langerhans and the acini was well defined. The islets appeared highly cellular. However small rounded multiple areas of degeneration were noticed. The nuclei were variable in size and shape
Examination of the semithin sections showed that most of the acini had a normal appearance with apical zymogen granules having minimal basal extensions. The cells had basal vesicular nuclei with prominent nucleoli. Blood vessels with minimal congestion were observed. Most of islets appeared pale with rounded vesicular nuclei. However, few irregular nuclei were noticed.
Electron microscopic examination revealed that the acinar cells had well defined boundaries, and were pyramidal in shape. Electron dense apical rounded secretory zymogen granules were observed. The cells were studded with parallel cisterns of rough endoplasmic reticulum surrounding almost regular nuclei.
Regarding islets, alpha cells, was studded with small secretory granules of variable density. Moreover, few cisterns of rough endoplasmic reticulum and small oval to rounded mitochondria were observed. However few areas of rarified cytoplasm could be still detected. The nuclei were rounded euchromatic with well defined nuclear membrane. Beta cell showed secretory granules surrounded by a clear halo. However, few granules were small, marginalized and surrounded by a rather wide halo . The cytoplasm contained few scattered small mitochondria and euchromatic rounded nuclei.
Examination of the paraffin sections stained with haematoxylin and eosin of (group IV) revealed variable histopathological changes. Most of the acini had irregular boundaries and appeared as homogenous esinophilic patches devoid of nuclei. Some distorted acini were having pale stained nuclei. Moreover some cells had foamy appearance which was associated either with vesicular nuclei or small shrunken deeply stained nuclei. Some of the islet cells showed degenerated cytoplasm and faint nuclei while others appeared of normal density.
Examination of semithin sections showed mild demarcation between the acinar cells. The later had minute vacuoles in their cytoplasm and apical accumulation of zymogen granules. Nuclei were rounded and vesicular. However, few nuclei were dark and irregular. Islets showed decreased cellularity, with pleomorphic irregular nuclei and few vacuoles in their cytoplasm.
Electron microscopic examination showed demarcation between the acinar cells. Their cytoplasm was rich in parallel cisterns of rough endoplasmic reticulum. Few vacuoles and Large mitochondria were observed .The characteristic finding was that most of the acinar cells exhibited marked depletion of zymogen granules , while other contained few zymogen granules . Nuclei were euchromatic with prominent nucleoli. Blood vessels were noticed to be congested. Esinophil cell infiltration was observed.
Islet (alpha) cells cytoplasm was studded with secretory granules of variable size and density. Some areas of rarified cytoplasm were observed. Nuclei appeared elongated. While, beta cells showed assorted appearance of secretory granules. Some secretory granules were central with its clear halo, others appeared peripheral in its halo. The halos were irregular, widened or fused together and some empty ones were detected. Their cytoplasm contained few cisterns of rough endoplasmic reticulum and multiple rounded mitochondria. Some exhibited distinct cristae while others appeared with faint ill defined matrix. Nuclei were oval with prominent nucleoli.
Examination of the paraffin sections stained with haematoxylin and eosin of (group V) revealed that the acini were still distorted in shape with no distinct cell boundaries Most of the nuclei were basal in position, rounded and vesicular however, few nuclei were dark and shrunken. The islets were highly cellular with some peripheral areas of cell depletion. Nuclei were rounded and vesicular, however, some peripheral nuclei were rather smaller in size, irregular in shape and darkly stained.
Examination of the semithin sections revealed regular acini studded with apical zymogen granules and basal vesicular nuclei. The cytoplasm of some acini appeared rarified and depleted from granules. Blood vessels were markedly congested. Loss of demarcation between acini and islets was noticed. Most islet cells appeared pale with vesicular rounded but some cells had dark and degenerated nuclei
Electron microscopic examination showed that acinar cells had slightly dilated cisterns of rough endoplasmic reticulum , zymogen granules were prominent, variable in size and density . Few areas of rarified cytoplasm were observed particularly in between zymogen granules.
Islet (alpha cells) appeared studded with secretory granules of variable size and density. On the other hand the cytoplasm exhibited small, dispersed cisterns of rough endoplasmic reticulum, few mitochondria. Nuclei were oval in shape with patchy chromatin condensation. Nearby cells were studded with dilated cisterns of endoplasmic reticulum and swollen disrupted mitochondria. On the other hand, beta cells cytoplasm showed multiple secretory granules having dense core surrounded by a clear halo. Some empty halos, or halos with small faint granules were seen. Less frequently coalesced halos were observed. Parallel cisterns of rough endoplasmic reticulum were also observed in between the granules. Nuclei were euchromatic with prominent nucleoli.
Examination of the paraffin sections stained with haematoxylin and eosin of (group VI) showed variable changes. The acini still have marked distorted appearance, They were amalgamated with ill defined borders. Some acinar cells showed foamy cytoplasm and irregular arrangement of nuclei, irregular esinophilic patches devoid of nuclei were noticed. Islets showed slight decrease in their cellularity, some cells had moderatly stained nuclei while other cells had faintly stained nuclei.
Examination of the semithin sections confirmed the presence of distorted acini. The presence of multiple vacuoles in the acinar cells was a prominent feature. Wide areas of degeneration were encountered. Some acini were studded with dispersed zymogen granules while other cells were devoid completely from these granules. The cells devoid of zymogen granules showed variation in their nuclear appearance, some manifested slightly irregular vesicular nuclei others, exhibited dark nuclei either large in size or shrunken. Islet cells appeared pale, some cells had vesicular rounded nuclei while others had dark irregular nuclei.
Electron microscopic examination showed that acinar cells contained groups of zymogen granules. Some cells had extensive cisterns of rough endoplasmic reticulum which was markedly dilated in other cells. Cytoplasm showed variable degrees of degeneration, some cells manifested localized rarified areas of cytoplasm in between zymogen granules. Occasionally, wide areas of degenerated cytoplasm were noticed. Some nuclei had distinct nuclear membrane and peripheral chromatin condensation, other nuclei appeared small, pyknotic and electron dense with blurred nuclear membrane.
Islet (alpha) cells had pale cytoplasm, studded with secretory granules variable in size and density. Cytoplasm contained small rounded mitochondria and interrupted cisterns of rough endoplasmic reticulum. However, areas of rarified cytoplasm were noticed. In addition, vacuolated areas with whorly appearance could be detected. Nuclei were dark and elongated in some cells. Beta cells appeared degenerated. Few secretory granules preserved their central position in the clear halo but most of the secretory granules appeared marginalized or pale. Many empty halos were detected either single or fused. Nuclei appeared slightly shrunken with indistinct nuclear membrane and peripheral chromatin condensation.
Biochemical studies were performed, blood glucose, serum insulin and serum amylase were measured then statistical analysis was done to show that green tea when given as a protective agent showed best results in improving body weight , blood glucose and serum amylase .While garlic as a protective agent showed better results regarding serum insulin. On the other hand, both of green tea and garlic when used as a treatment, showed less significant results.
Therefore, we can suggest that green tea can protect and delay diabetic complications and may be beneficial in treatment of diabetes, however, this might need further studies about its combined effect with anti-diabetic drugs. While garlic may be considered less beneficial than green tea as a protective agent. However, it is proved to potentiate the anti-diabetic effect of insulin and certain anti-diabetic medications.