DOI: 10.31038/EDMJ.2019335

Abstract

Objective: Hyperglycaemia and diabetic complication during pregnancy interfered with birth defect and traditional usage of phytotherapy take a great attention. The present study searched for illustrate the interference of maternal diabetes on the development of exocrine pancreas during in utero growth and role of ginger extract in improving the disease.

Research design and methods: Sixteen pregnant rats were used and arranged into four groups (n = 4); control, ginger extract group, diabetes, diabetes and ginger supplementation. Diabetes was induced by single i.p. injection of streptozotocin (60 mg/kg in citrate buffer pH 4.5 plus 100mg/kg nicotinamide). Ginger watery extract (200mg/kg body weight) was orally daily supplemented from 5^th day of gestation until 14^th day of gestation. Pregnant were sacrificed at 14^th day of gestation, fetuses were separated and exocrine pancreas were separated and fixed in either 10% phosphate buffered formalin or 2.5 cacodylate buffered glutaraldhyde for transmission electron microscopy. Immunohistochemistry of caspase 3 was carried out. During the gestation period, blood glucose level was measured in the studied groups.

Results: The present results revealed that diabetes developed atrophy and degeneration of the acinar epithelium of the pancreas. At ultrastructural level, there was a considerable missing of the secretory granules and arranged of rough endoplasmic reticulum taking the characteristic feature of fibrosis. Immunohistochemical analysis illustrated over expression of caspase 3 in acina epithelium. However, ginger water extract supplementation to diabetic mother improved the blood glucose level and histo-cytlogical and immunohistochemical picture manifesting marked alleviation of the drastic alterations compared to the control.

Conclusion: The author concluded that ginger extract showed a potent antioxidant activity, decreasing the diabetes associated oxidative stress and improved the fetal exocrine pancreas histo-cytological structure.

Introduction

The pancreas is composed of exocrine and endocrine portion. It is developed from the dorsal and ventral endodermal as a result of the epithelio-mesenchymal interactions. Exocrine acinar cells, are responsible for secretion of zymogens and digestive enzymes through a ductal network leading to the duodenum and facilitated digestion [1, 2]. Acinar cells builds up the exocrine pancreas and secrete up to more than 20 kinds of enzymes, such as proteinase, DNAses and lipases, which are important elements for digestion [3].

The knowledge of type T3c diabetes mellitus (T3cDM, MODY) is still unclear and involved in the impairing of exocrine pancreas, the reservoir of digestive enzymes. Little knowledge about the mark differences between T2DM and T3cDM. T3cDM is developed in early childhood growth [4] and characterized by chronic pancreatitis, pancreatic cancer. Inflammation, fibrosis leading to damage of both endocrine and exocrine function impairing insulin/glucagon secretion and dysfunction of pancreatic enzyme [5]. The prevalence of exocrine dysfunction in type-1 and type-2 diabetic patients reached approximately to 37.7% and 26.2% respectively [6]. Also, the exocrine pancreas dysfunction related to diabetes affects 1.8% of adults with new-onset diabetes. Cell death of the islet of Langerhans alters the secretion of insulin, glucagon, and pancreatic polypeptide resulting fluctuations in glucose levels. Diabetes induced acute or chronic pancreatitis, associated with co-existing cystic fibrosis or hemochromatosis [7,8].

At the same time, there is a great attention to the consumption of traditional medicine. Ginger (Zingiber officinale) is a rhizome popular food spice and showed varieties of bioactive components such as vallinoids, viz. gingerol[9] and paradol, shogaols, zingerone, and galanals A and B [10]. Its administration resulted in improvements of diabetic patients [11–13] and experimental animals [14,15] through managing of hyperglycemia glycemia, oxidative stress, and inflammation.

There was no available work concerned the influence of maternal diabetes on the developing of exocrine pancreas during intrauterine life. The present work searched for illustrated the light and transmission electron microscopically study of fetal exocrine pancreas of diabetic mother and the potential medical phytotherapy of watery ginger extract.

Materials and Methods

1. Applied dose of ginger watery extract

A known weight of dried ginger (Zingiber officinale) was mixed thoroughly with hot water weekly, filtrate and allows standing at room temperature. Each pregnant received 0.5 cc containing 200mg of the extract / kg body weight. Each dose was orally administered daily from 6^th day of gestation till 16^th day of gestation.

2. Induction of diabetes

Experimental type 2 diabetes mellitus was induced in all the rats by a single interperitoneal injection of streptozotocin (60 mg/kg) in citrate buffer (0.05 M) (pH 4.5) and 100mg/kg nicotinamide [16]. Hyperglycemia was verified by measuring the blood glucose within 240- 280 mg/dl were selected for the study.

3. Experimental animal work

Twenty fertile male and virgin female of albino rats (Rattus norvegicus) (at a ratio of 1 male to 3 females) weighing approximately 200g body weight, obtained from Hellwan Breeding Farm, Ministry of Health, Egypt and used for experimentation. They were housed in good ventilation on a 12-h light and dark cycle. Females were mated (1 male/3 females) overnight and zero dates of gestation were determined the next morning by observing the sperm in vaginal smear. The pregnant were arranged into four groups (n = 4 per each); control, ginger watery extract, diabetes, diabetes and ginger extract. Experimental work was carried out from zero date of gestation till 16^th day intra-uterine growth. Water was allowed ad Libitum. At the end of treatment, they were sacrificed by light diethyl ether anesthesia and dissected at 14^th days prenatal. Maternal blood was collected and serum was separated and kept in refrigerator. Also, the pregnant were dissected and their fetuses were separated and processed for the following investigations:

4. Biochemical investigations:

Serum levels of total cholesterol (TC) [17], Triglycerides (TG) [18] and high density lipoproteins (HDL) [19] were determined. In case of low density lipoproteins (LDL), it was calculated from the total concentrations of cholesterol (TC), HDL-cholesterol and triglycerides according to Friedewald et al [20]. The glucose regularly measured by blood glucometers one touch ultra (Life Scan Milipitas, CA, USA).

5. Histological investigation:

Pancreas of 14^th day-old fetuses of the studied groups were separated and immediately fixed in 10% phosphate buffered formalin (pH 7.4), dehydrated in ascending grades of ethyl alcohol, cleared in xylene and mounted in molten paraplast at 58–62ºC. Five μm histological sections were stained with hematoxylin & eosin and investigated under a bright field light microscope.

6. Transmission electron microscopy

Specimen of pancreas of 16^th day old fetuses of the studied groups were immediately fixed in 2.5 % glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) and post-fixed in 1% osmium tetraoxide, dehydrated in ascending grades of ethyl alcohol, and embedded in epoxy–resin. Ultrathin sections were cut with a diamond knife on a LKB Ultratome IV (LKB Instruments, Bromma, Sweden) and mounted on grids, stained with uranyl acetate and lead citrate, and examined under a Joel 100CX transmission electron microscope (Musashino 3-chome, Akishima, Tokyo 196–8558, Japan).

7. Immunohistochemistry for caspase 3

Deparaffinized histological 5μm thick sections of pancreas of 16 day old fetuses were cut and mounted onto super frost plus glass slides (Fisher Thermo Scientific, Nepean, Ontario, Canada). At a normal room temperature, the tissue sections were processed for antigen retrieval by digestion in 0.05 % trypsin (pH 7.8) for 15 min at 37°C and incubated with the antibodies against caspase 3 (dilution 1:100 Thermo Fisher Scientific, Fremont, CA, USA; Cat. No. A1–70007) for overnight at 4°C, followed by treatment with a horseradish peroxidase streptavidin detection system (Dako), and DAB plus Chromagen for developing the immunoactivity. The immunohistochemical stained slides were counterstained with hematoxylin. Negative control was carried out by incubating slides with 1% non-immune serum phosphate buffer solution (PBS) solution. Specimens were investigated under a Leica BM5000 microscope (Leica Microsystems, Wetzlar, Germany) and photographed.

For assessments of image analysis, slides were photographed using Olympus® digital camera installed on Olympus® microscope with 1/2 X photo adaptor, using 40 X objective. The result images were analyzed on Intel® Core I5® based computer using Video Test morphology® software (Russia) with a specific built-in routine for area, % area measurement and object counting.

Results

Biochemical observations

Ginger-extract supplementation improved the diabetic associated increase of serum glucose level, LDL, triglycerides and total cholesterol levels (Fig.1).

Figure 1. Chart illustrating serum glucose, LDL, TG, HDL and total cholesterol levels. Each result represent the mean ± SD of n = 5. * Significant at P < 0.05. Abbreviations; GL, glucose, HDL, high density lipoprotein; LDL, low density lipoprotein; TC, Total cholesterol.

Exocrine pancreas of 14^th day fetuses

Fetuses of control and ginger-extract-supplemented mother exhibited normal structural pattern of exocrine pancreas. It is composed of tubuloacinar glands containing numerous acinar units. Each composed of pyramidal acinar cells with centrally located nuclei and basophilic cytoplasm surrounding their lumen (Fig. 2 A&B)

Figure 2. Photomicrographs of histological sections of exocrine pancreas of 14th day rat fetuses. A. Control. B. Cinnamon-ginger extract. Note the normal structural pattern of centro-acinar cells and intercalated duct. C. Foetuses of diabetic mother showing disorganized acinus with collapsed duct and damaged acinar lining cells. D. Foetuses of diabetic mother supplemented ginger extract supplementation showing improved cento-acinar cells and intercalated duct. HE. Abbreviations; A, acini, CAC, centro-acinar cell; ICD, intercalated duct ; DA,deformed acinar.

However, those of diabetic mother exhibited massive damaging of the acinar units and atrophied lumina. The acinar lining cells appeared swollen with pyknotic nuclei and eosinophilic cytoplasm. Interlobular ducts were lined with flattened epithelium (Fig.2C).

On the other hand, fetuses of diabetic mother supplemented ginger-extract revealed improvement of the pancreatic structure but of less developed in comparison with the control (Fig.2D).

At ultrastructural level, the exocrine acinar cells possessed normal cytological structure in control and ginger –treatment. Each exocrine cell possessed centrally located nuclei with peripheral heterochromatin and abundant euchromatin. The cytoplasm contained abundant electron-dense and faint spherical zymogen granules of varying sizes and shape. The granules were membrane-bound. Numerous mitochondria and rough endoplasmic reticulum were detected in-between the secretory granules. Glycogen granules were observed in the cytoplasm of the acinar cells. Blood vessels are detected around the acini and ducts (Figs. 3 A&B).

Figure 3. Transmission electron micrographs of exocrine pancreas of 14th day-old fetuses. A. Control. B. Maternally supplemented ginger extract. Note the presence of normal oval-shaped nuclei with peripheral marginal heterochromatin and central euchromatin. The is rich of electron –dense zymogen granules of different sizes containing in between mitochondria. C&D. Maternally diabetic showing apoptic atrophied nuclei composed electron-dense heterochromatin and surrounded by a network of clumped rough endoplasmic reticulum containing in between atrophied mitochondria (C). Another specimens illustrates nuclei with irregular nuclear envelope, lipid vacuoles and missing of secretory granules (D). E&F. Foetuses of diabetic mother supplemented ginger extract revealing increase secretion of light and electron-dense zymogen granules. Abbreviations; BC, blood capillary; ES, electron-dense-secretory granule; M, mitochondria, N, nuclei; PN, pyknotic nuclei; RER, rough endoplasmic reticulum; V, lipid vacuoles.

In those maternally diabetic, the acinar cells exhibited apoptic nuclei composed of electron-dense heterochromatin and surrounded by a network of clumped rough endoplasmic reticulum enclosed in between atrophied mitochondria manifesting fibrotic structure. In some other cells, the nuclei outlined by irregular nuclear envelope and their cytoplasm contained abundant lipidvacuoles of varying sizes and missing of their inclusion of secretory granules (Figs. 3 C&D).

On the other hand, fetuses of diabetic mother supplemented ginger extract restored the presence of faint secretory granules was comparatively decreased in some cells and increased in other ones. The criteria structure of the acinar cells is improved but less developed compared to the control (Figs. 3 E&F).

Following immunohistochemical staining with caspase 3, the fetuses of diabetic mother exhibited overexpression of dark brown reaction in acinar cells (Fig. 4C) in comparison with the negative staining affinity in control and ginger supplementation (Figs. 4 A&B). Fetuses of diabetic mother supplemented ginger extract revealed marked reduction of the immunohistochemical reaction (Fig. 4D). Image analysis revealed increased regional area of immunohistochemical reaction in acinar cells of diabetic mother compared to the other studied groups (Fig. 5).

Figure 4. Photomicrographs of formalin fixed histological section of exocrine pancreas of 14th day-old fetuses maternally immunohistochemically stained with caspase 3. A. Control. B. Ginger extract-treatment. Note negative immuphistochemically stained with caspase 3. C. Maternally diabetic showing increased immunohistochemical affinity in acinar cells. D. Ginger extract showing marked reduction of the expression of caspase 3.

Figure 5. Chart illustrating image analysis of surface immunohistochemical reactive areas with caspase 3 of acinar cells of fetuses of diabetic mother compared to the other studied groups. Each result represent the mean ± SD of n = 5. Star means significant at P < 0.05.

Discussion

The present findings revealed that 14^th day-old fetuses of diabetic mother exhibited abnormal histological structure of exocrine pancreatic cells including eosinophilia cytoplasm and pyknotic nuclei of acinar cells and almost missing of their lumina. Although there is no detected the presence of inflammatory cells, other authors revealed that inflammation may be occurred during organogenesis of the lymphopoietic-hematopoietic system, based on hematopoietic cell-mesenchymal cell interactions [21].

The presence of zymogen granules in the acinar cells has been reported by Conklin [22] and Laitio et al. [23] in human fetal pancreas. Tadokoro et al. [24] reported the presence of increased number and density of zymogen granules before birth in rats. Inagaki et al. [25] mentioned that the secretory granules are responsible for stored zymogen granucles and secretion of lipase, trypsin, and amylase in fetal acinar cells. Rough endoplasmic reticulum and the Golgi apparatus are involved in synthesis of zymogen granule in the adult [26].

At ultrastructure level, the observed findings reported a detected missing of the secretory granules and increased network of endoplasmic reticulum surrounding the apoptic nuclei forming the fibrotic structure. Lipid vacuoles of varying sizes were distributed in the cytoplasm. Also, atrophied mitochondria with damaging internal structure were observed in comparison with the control.

Similar findings of reduced secretory granules were reported in E2F1/E2F2 compound-mutant mice develop non-autoimmune insulin-deficient diabetes [27].

Diabetes may involvein gene defects leads to pancreatic lipomatosis and exocrine pancreatic insufficiency in childhood, and progressed till 34 years [28]. The observed damage of both mitochondria and rough endoplasmic reticulum facilitated accumulation misfolding protein with intracellular and extracellular aggregation exerting a cytotoxic effect and caused progression of disease of the islets of Langerhans [29].

The apparent reduction and number of mitochondria and deposition of fat vacuoles reflected the depletion of energy level of acinar cells and consequently loss of pancreatic function.

Similar findings were reported by Kaido et al. [30] in a murine model of primary carnitine deficiency.

Also, the present study reported the development of congenital malformation of acute pancreatitis in fetuses of diabetic mother. Damaging of the acinar cells led to depletion of the majority of enzymes needed for digestion.

Pin et al. [31] reported that the acinar cells of the exocrine pancreas is responsible for the development of up >90% of the cells within the pancreas especially β cells. The conversion of acinar cells to either β cells involves alterations in the expression and activity of the transcription factor, and changes in their epigenetic program.

For my opinion in utero poor control of diabetes associated damage of the exocrine pancreas may be increased cell programming and increased the risk susceptibility for the developmental origin of diabetes during adult hood.

At the same time supplementation of ginger to diabetic mother ameliorated the structural pattern of the acinar cells, partially restoring the secretory granules and cell cytoskeletal structure.

Ginger was found to contain several bio components such as gingerol [9] and shogaols and zingerone [10] which improve hyperglycaemia and resoluted oxidative stress and inflammation. This led to decrease the programming of cell death and managing cell growth and differentiation [11,14, 15] .

The authors finally concluded that ginger supplementation during pregnancy may help to improve the antioxidant activity of the pancreatic cells and sustained growth and differentiation against the cytotoxicity of diabetes.

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DOI: 10.31038/EDMJ.2019334

Abstract

Objective: Diabetes is an important public health disease affected different body organs especially eye. The present study aimed to illustrate the cataractous lenses associated complication on ocular organs.

Research design and methods: Following induction of type 2 diabetes for 6 months (streptozotocin 60mg/kg single dose in combination with nicotinamide100 mg./kg body weight), 6 rats of a colony of 47 individuals developed cataract. Control individuals were used of the similar age. The animals groups were anaesthetized and sacrificed. Ocular regions were incised and subjected for histopathology and comet assay. Transmission electron microscopy was carried out for the optic nerve. In case of meibomian gland, beside histopathological investigations, immunohistochemistry of bcl2, caspase 3 and p53 were carried out.

Results: The present findings revealed the development of cataractous lenses, diabetic retinopathy, thickened cornea and damaged stroma and optic neuropathy explained by demyelinated axons. Single gel electrophoresis explained DNA damage of cells of the retina, ciliary organ and optic nerve. Also, there is a marked reduction of the immunohistochemistry of bcl2, caspase 3 and p53 of diabetic meibomian gland, reflecting cell death.

Conclusion: Finally the authors concluded that the cataractous lenses originated through different pathways of diabetic complication.

Introduction

The Diabetes Mellitus (DM) is characterized by hyperglycemia associated with either decreased insulin production in the body or insulin resistance. It is increased among population reached up to 382 million in 2014 [1] and by 2035 more than 592 million may develop with the disease in a ratio of 1 per 10 individual become diabetic [2]. The disease resulted from impairing of β-cell function, hyperglycaemia and impairment of insulin secretion [3].

Poor diabetic-treatment resulted in impairment of visual acuity [4] and the development of cataract in child hood [5, 6]. Cataractous lenses were also reported in diabetic experimental animal [7]. Diabetic related cataract was associated with damaging corneal endothelial cells and increase foveal thickness [8]. Also, diabetic was found to induce retinopathy [9, 10] and represent the main cause of impaired visual acuity and blindness associated with elevated serum homocysteine level Hcy [11]. Also, it is involved in reduction of both retinal thickness and retinal blood flow, and increased thickening of the retinal choriocapillaries [12]. The disease increased retinal apoptosis, overexpression of VEGF and increased oxidative stress [13, 14] and altered osmoregulation leading to acidic retina [15]. Middle-aged, obese rhesus monkeys, developed diabetes mellitus characterized by hypertension, high levels of triglycerides and serum cholesterol. The disease associated with intraretinal hemorrhages, damage choriocapillaries, increased basal laminar deposits and hard drusen of Bruch’s membrane and numerical reduction of photoreceptor inner and outer segments [16]. Diabetic retinopathy characterized by up-regulation of pro-inflammatory interleukin-6 and down-regulation of superoxide dismutase and glutathione (GSH)/oxidized glutathione associated with numerical reduction of retinal ganglion cells and increased glial fibrillary acidic protein expression level [17–18].

Concerning cornea which is the transparent organ important for vision. Diabetes caused marked degeneration of corneal secondary nerve fiber branches [19, 20] and enhanced the development of anterior segment disorders such as corneal erosion and ulcer and persistent epithelial defects [21]. There is discrepancy between authors concerning diabetic interference on corneal thickness. Meanwhile Olsen and Busted (1981) [22] reported increased corneal thickness in diabetic patients, Hashemi et al. (2019) [23] reported no variations of corneal thickness between diabetic and non diabetic individuals. Diabetic keratopathy resulted in reduction of corneal sensitivity [24].

The ocular region guards by eye lid containing glands such as the meibomian glands (MGs) which are arranged in parallel strands within the tarsal plates of the eyelids. It secrete oily meibum by meibocytes and transported through the ductal system by ductule and the central duct towards the free lid margin. The gland possesses a characteristic innervations from sympathetic and parasympathetic fibers [25, 26]. The meibum lubricating the ocular surface during blinking and protecting against tear evaporation [27].

Diabetes was found to induced meibomian gland dysfunction [28, 29] leading to depletion of the lipid secretion associated the instability of tear and development of dry eye disease [27, 30].

Little is known about the diabetes related changes on the histopathological structure of the meibomian glands. Recently, a study from Ding et al. demonstrated that insulin stimulated the proliferation of immortalized human meibomian gland epithelial cells (HMGECs), whereas high glucose was found to be toxic for HMGECs [31]. Diabetes was found to reduce the number of glandular structure impairing its functional activity [32]. Also, diabetes was found to be involved in increase of ciliary body thickness [33] and reduction of both retinal nerve fiber layer and vessel density in the optic disc [34].

The present studies searched for illustrating if the opacity associated with other complication of the eye regions or not and the characteristic features of the histopathological alterations.

Materials and Methods

1. Induction of diabetes

Experimental type 2 diabetes mellitus was induced in all the rats by a single interperitoneal injection of streptozotocin (60 mg/kg) in citrate buffer (0.05 M) (pH 4.5) and 100mg/kg ncotinamide [35]. Hyperglycemia was verified by measuring the blood glucose within 240- 280 mg/dl were selected for the study.

2. Experimental animals

Following investigating a colony of 47 individual diabetic rats (Rattusnorvegicus) after 8 months of streptozotocin-induction and weighing approximately 200–250g body weight, 6 rats exhibiting cataractous lenses either unilateral (4/6) or bilateral (2/6). Control healthy individuals (n = 6) were selected. They were housed in good ventilation on a 12-h light and dark cycle. The studied cataractous rats were observed in about 47 individual diabetic rats after 6 months of induction the disease reaching incidence of about 13%. The cataractous group was separated and a similar control non-diabetic group was also selected. Free excess water and food were allowed ad Libitum. The studied groups were sacrificed by light diethyl ether anesthesia and dissected and ocular region, upper eye lid and optic nerves were separated and processed for the following investigations.

3. Histological investigation

Retina, cornea, proximal optic nerve, ciliary organ and upper eye lid of the studied groups were separated and immediately fixed in 10% phosphate buffered formalin (pH 7.4), dehydrated in ascending grades of ethyl alcohol, cleared in xylene and mounted in molten paraplast at 58–62ºC. Five μm histological sections were stained with hematoxylin & eosin and investigated under a bright field light microscope.

4. Immunohistochemistry for caspase 3

Histological 5 μm thick sections of upper eye lid were cut and mounted onto super frost plus glass slides (Fisher Thermo Scientific, Nepean, Ontario, Canada). The tissue sections were retained at normal room temperature and processed for antigen retrieval by digestion in 0.05 % trypsin (pH 7.8) for 15 min at 37°C and incubated with antibodies against bcl2, caspase 3 and P53 (dilution 1:100 Thermo Fisher Scientific, Fremont, CA, USA; Cat. No. A1–70007) for overnight at 4°C. Then treated with a horseradish peroxidase streptavidin detection system (Dako), followed with DAB plus Chromagen to detect the immunoactivity by counterstaining with hematoxylin (Sigma). Sections incubated with 1% non-immune serum phosphate buffer solution (PBS) solution served as negative controls. Specimens were observed with a Leica BM5000 microscope (Leica Microsystems, Wetzlar, Germany) and photographed. For assessments of the percentages of positive immunoreactive areas were determined by investigating slides using Olympus® digital camera installed on Olympus® microscope with 1/2 X photo adaptor, using 40 X objective. The result images were analyzed on Intel® Core I5® based computer using Video Test Morphology® software (Russia) with a specific built-in routine for area, % area measurement and object counting.

5. Transmission electron microscopy

Specimen of proximal optic nerve close to the retina of the studied groups were immediately fixed in 0.1M cacodylate buffer (pH 7.4) containing 2.5 % glutaraldehyde) and post-fixed in 1 % osmium tetraoxide, dehydrated in ascending grades of ethyl alcohol, and embedded in epoxy–resin. Ultrathin sections were cut with a diamond knife on a LKB Ultratome IV (LKB Instruments, Bromma, Sweden) and mounted on grids, stained with uranyl acetate and lead citrate, and examined under a Joel 100CX transmission electron microscope (Musashino 3-chome, Akishima, Tokyo 196–8558, Japan).

6. Comet assay

Retina, cornea, optic nerve, ciliary organ specimens of the studied groups were homogenized in chilled homogenizer buffer, pH 7.5, containing 75 mMNaCl and 24 mM Na₂ ethylenediaminetetraacetic acid (EDTA), pH 13. Six µL of the homogenate were suspended on 0.5% low melting agarose and sandwiched between a layer of 0.6% normal-melting agarose and another of 0.5% low melting agarose on fully frosted slides and kept on ice for the polymerization. The slides were the immersed in a lysis solution (1% sodium surcosinate, 2.5 M NaCl, 100 mM Na₂EDTA, 10 mm Tris-HCl, 1% Triton X-100, and 10% DMSO) at 4°C. After 1 h, they were placed in electrophoresis buffer (0.3 M NaOH, 1 mM Na₂EDTA, pH 13) for 10 min at 37 ^oC to allow DNA to unwind. Electrophoresis was carried out for 10 min at 300 mA and 1 V/cm, followed by staining with 20 mg/mL ethidium bromide. Each slide was analyzed using a Leitz Orthoplan (Wetzlar, Germany) epifluorescence microscope. One hundred cells were analyzed on each slide using the Comet assay II automatic digital analysis system. Perspective tail length (mm) is the distance of DNA migration from the center of the body of the nuclear core and is used to evaluate the extent of DNA damage. Tail length was measured automatically by image analysis software [36].

7. Statistical analysis

Data are presented as means ± standard deviation (SD). The statistical analysis was performed with multi-variant analysis of variance (MANOVA) using the SPSS (version 13) software package for Windows, comparing the multivariations between diabetes and control groups. Significance was determined at p < 0.05.

Results

1. Cornea & Retina

The cornea is composed of outer lining epithelium, followed by Bowman’s membrane, stroma, descemet’s membrane and corneal endothelium. Regard to the control (Fig. 1A), the diabetic group revealed that the corneal epithelium was comparatively thickened with vesicular vacuolar degenerated nuclei. The stroma showed patches of hyaline degenerated collagenous fibrils and infiltrated by necrotic regions. Vacuolated keratocytes were detected in between the collagen fibrils of the stroma. The corneal endothelium lining the descemet’s membrane attained considerable thickening (Fig. 1A1).

Figure 1. Photomicrographs of cross histological sections of cornea (A&A1) and retina (B&B1). A. Control showing normal epithelium, basement membrane, stroma and endothelium. B. Diabetic showing thickened epithelium, damaged stroma and thickened endothelium. B. Control retina showing arranged ganglion, inner plexiform, inner nuclear, outer plexiform, outer nuclear and photoreceptor layers. B1. Diabetic retina showing damaged ganglion, thinning inner nuclear (arrow head), damaged outer nuclear (star) and atrophied photoreceptor(arrow) C & A1. Comet assay of control (single star)(C) and cataractous (double star) (C1) retina showing detached retinal cells of diabetic group. Abbreviations; E, endothelium;BM, basement membrane; IPL, inner plexiform layer; ILM, inner limiting membrane; INL, inner nuclear layer; NFL, nerve fiber layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptor; Pg, pigmented cell; St, stroma.

Regarding to the normal structure of the retina (Fig, 1B ), the diabetic group showed comparative reduction of the retinal thickness. There was comparative numerical reduction of the inner and outer nuclear cells. The nuclei of these cell layer were electron-dese manifesting clumping of the nuclear chromatin. The photoreceptor layer was comparatively atrophied. The ganglion cells were markedly missing and the nerve fiber become thin, fragile and vacuolated (Fig.1B1). Diabetic retina showed detached retinal cells manifesting damaged DNa (Fig. 1C1) compared to non-changed in control (Fig.1C). The whole retina, inner and outer nuclear layer and photoreceptors attained considerable reduction in diabetic group in comparison with the control (Fig. 2).

Figure 2. Chart illustrated retinal layer thickness of diabetic group. Abbreviations; GL, gamglion layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptor; R, retina. each result represent mean ±SD (n-5). Diabetic retina and photoreceptor thickness are significant in diabetic group at P < 0.05.

2. Ciliary organ and optic nerve

The ciliary body occupied the interface between the iris and the choroids. It is made up of two ring-shaped components: the pars plicata and the pars plana. The parsplicata detected in the anterior region of the ciliary body, at the scleral spur. The ciliary body composed of branched ciliary processes. It is lined by two layers of columnar cells. The outer one is pigmented, mean while the inner lining cells lack pigmentation. The lumen is enclosed with fine collagenous network (Fig. 3 A). Following application of comet assay the control ciliary cells are round (Fig.3A1).

Figure 3A-B1. Photomicrographs of histological sections of ciliary organ. A. Control showing branched ciliary organ. A1. Comet assay of control showing normal round cells (single star). B&B1. Diabetic rat showing atrophied ciliary organ (B) with detached damaged cells post comet assay (double star) (B1).

Figure 2C-C1. Photomicrographs of histological sections of control optic nerve showing attachement with retina in optic disc and compacted axons and contents of neuronal cells(dark arrow head).C2. Transmission electron micrographs of control showing compacted myelinated axons. C3. Comet assay of control showing round cells (Single star). D-D2 Diabetic group showing fragile histological section and vacuolated neurons (white arrow head) (D) and demyelinated electron micrograph (D1) and detached neuronal cells of optic nerve after comet assay (D2). Abbreviations; C, connective tissue; CE, ciliary epithelium; DCE, damaged ciliary epithelium; DMA, degenerated mitochondrial axons.LI, lymphocytic infiltration; M, mitochondria; MA, mitochondrial axons;

Experimental diabetic group revealed missing of the ciliary processes and comparative thinning of their lining epithelium. Congestion of their blood capillaries, widened lumen and distortion of their collagenous network were observed (Fig. 3B). Following single gel electrophoresis (comet assay), revealed DNA segregation assessed by formation of detached tail region manifested DNA damage (Fig. 3B1).

Optic nerve

Concerning the optic nerve, light microscopically, these are formed of densely aggregated and enclosed in-between cells with basophilic nuclei suspected the oligodendrocytes and astrocytes (Figs. 3 C&C1). Ultrastructurally, the nerve axons are densely packed and composed of myelinated and non-myelinated axons of varying small size. The axon enclosed by several layers of myelin sheath. Abundant mitochondria are distributed the cytoplasm of the inner compartment of the axons (Fig.3 C2).

Diabetic–treatment revealed widespread of vacuoles and fragility of the nerve fibers, and comparative reduction of the oligodendrocytes. Many of them possessed vacuolated axoplasm (Fig. 3D). Ultrastructurally, vacuolation and demyelination of the theaxonal sheath were detected (Fig. 3 D1). Following application comet assay, there was apparent increase of apoptic cells with detached tail region (Fig.3D2) compared to non-changed in control (Fig.3C3).

3. Meibomian gland

In control, the meibomian gland consisted of numerous branched acini that opened into a central duct through short ductules. Each acinus consisted of a basal layer of flattened cells. The central duct was lined by stratified squamous epithelium (Figs.4A1-A3).

Figure 4. A, Photomacrograph of normal (A) and diabetic cataract (B).A1-A3. Photomicroph of histological sections of control meibomian gland showing normal branched acinar gland. B1-B3. Diabetic rat showing atrophied and degenerated meibomian gland (B1&B2) and degenerated germinativum of epidermis (B3) illustrated by arrow head. Abbreviations; DHf, degenerated hair follicle ; DMG, damaged meibomian gland; E, epidermis; HF, hair follicle ; MG, meibomian gland.HE

In diabetic group, the gland exhibiteda considerable atrophy and degeneration of the acinar cells (Figs. 4B1-B3).

Following immunohistochemical investigations, bcl2 revealed marked depletion of the immunostaining reaction in the gland of diabetic rats compared to the increased expression in the control (Fig.5A&B). On the other hand both caspase 3 and P53 were over expressed in those of diabetic mother manifesting cell death (Figs. 5) in comparison with the control (Figs. 5A1, A2, B1 &B2). Image analysis revealed decreased bcl2 and increased both caspase 3 and p53 active area of immunohistochemical reaction those of diabetic mothers in comparison with the control (Fig.6).

Figure 5. Photomicrographs of formalin fixed histological sections of meibomian glands immunohistochemical stained with Bcl2(A&B), Caspase 3 (A1 &B1) and P53 (A2 &B2). Note decreased expression of Bcl2 (B), increased expression of caspase 3( B1) and overexpression of p53 (B2) in diabetic group compared to control (A-A2). Arrow head pointed to the immunostaining activity.

Figure 6. Chart illustrating of the image analysis of percentages immunoreactive area of diabetic group in comparison with the control. Each result represent the mean SD (n = 5). Diabetic is significant at P < 0.05.

Discussion

The diabetes mellitus (DM) is a public health problem widely increased among population. The present findings revealed induction of cataractous lenses of 6/47 individuals with about 13%.

The present findings agree with the work of El-Sayyad et al. [37–39], Becker et al. [5] and Wu et al. [6] in human and El-Sayyad et al. [37] and Miller and Brines [7] in experimental animals.

Development of cataractous lenses in diabetic patients was found to result from damaging of lens epithelium [37, 40] associated with releasing of free radicals and reduction of the capacity of both antioxidant enzymes and aldose reductase [39, 41] altering glucose metabolism and accumulation of sorbitol enhanced cell damage and development of cataract [39, 42, 43].

Also, diabetic cataract was found to be associated with corneal damage assessed by thickened epithelial lining layer, necrotic stromal patches and thickened endothelium.

The present findings supported the work of Olsen and Busted [22] and Hashemi et al. [23] that reported increased corneal thickness in diabetic patients. Diabetic keratopathy was also, mentioned by Bikbova et al. [24] which impaired the corneal sensitivity. Alterations of diabetic cornea may contribute to damage of the corneal nerve fibers [20, 44] and progress the development of anterior segment disorders [21] resulting in impairing the vision.

Also, the diabetic retina attained a comparatively reduced thickness; numerical reduction of inner and outer nuclear cells and atrophied photoreceptors coincides with increased detached retinal cells manifested DNA damage.

The observed diabetic retinopathy supported the work of El-Sayyad et al. [37], Kim and Yu [33], Yao et al. [12] and Li et al [10]. The authors mentioned that the retinopathy impaired the visual acuity and the development of blindness. Diabetic retinopathy related to blindness affected about 800 cases each year [45].

At the same time, the detected atrophy and damaged photoreceptors supported the work of Johnson et al. [16] and El-Sayyad et al. [37].

Diabetic retinopathy was found to exhibit over expression of inflammatory markers, down-regulation of antioxidant enzymes associated with increased retinal cell death [13, 14, 17].

The present data revealed that diabetes caused abnormal non-branched biliary processes and reduced thickness of their lining epithelium parallel with single strand DNA damage. The present findings agree with [33] following investigating diabetic patients.

Ciliary organ is important for contraction and relaxation of lens allowing it for accommodation. Damage of ciliary epithelium and defect of collagenous fibrils of the ciliary lumina interfered with retinal dystrophies [46].

The present findings revealed neuropathy of optic nerve of diabetic rat assessed by fragility and degeneration of nerve fibers, demyelinated axons and decrease of neuronal cells coincides with single strand DNA damage of astrocytes. Similar findings of optic neuropathy were detected in aging rats which exhibited similar diabetic deterioration of the neuronal cells [47].

Diabetes associated microvascular abnormalities may facilitate the progress of ischemia of the anterior optic nerve [48].

On the other hand the meibomian glands are important for ocular function through secretion of oily meibum which lubricate the ocular surfaces and protecting against tear evaporation [49].

The present findings revealed that diabetes induced atrophy and degeneration of meibomian glands. Following immunohistochemical staining, decreased expression of bcl2 and overexpression of caspase3 and p53, the markers of cell death were detected.

Similar findings of diabetes associated meibomian gland dysfunction were reported [28, 31, 32]. Decreased lipid secretion was found to be associated with the instability of tear and progress of dry eye disease [27, 30].

Finally the authors concluded that the cataractous lenses originated through different pathways of diabetic complication.

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