Therapeutic effects of in vivo-differentiated stem cell and Matricaria chamomilla L. Oil in diabetic rabbit.

BACKGROUND: The main goal of diabetes therapy is to control blood glucose levels. OBJECTIVES: In this study, the effect of Matricaria chamomilla L. oil as an herbal agent, on therapeutic properties of poly L-lactic acid-based (PLLA) scaffold loaded with differentiated stem cells, is examined in the diabetic rabbit. METHODS: Adipose mesenchymal stem cells (AMSCs) were isolated from male New Zealand White rabbits and after seeding on the PLLA scaffold differentiated in the pancreatic region. In vivo differentiation of AMSCs toward pancreatic progenitor cells was evaluated by quantitative analysis of gene expressions and immunohistochemistry. Then, one normal and five diabetic groups including blank diabetic, scaffold, oil + scaffold, and differentiated cell + scaffold or oil + scaffold were assessed after 21 days of treatment. After the assessment, the diabetic groups were evaluated by clinical parameters and pancreatic histological sections. RESULTS: It was found that AMSCs were differentiated to insulin-producing cells (IPCs) in the pancreatic environment which then used for implantation. Blood glucose in the oil + scaffold, cell + scaffold, and oil + cell + scaffold groups showed a significant decrease after 21 days. In the above mentioned three groups, insulin secretion was increased significantly. Chamomile oil also caused a significant decrease in High-density lipoprotein (HDL), Low-density lipoprotein (LDL), and total cholesterol levels. According to histological sections results, in cell + scaffold and oil + cell + scaffold groups, ß cells were significantly increased compared to blank diabetic group. CONCLUSIONS: Together these data demonstrated chamomile oil along with in vivo-differentiated stem cell is a promising new treatment for diabetes.

Glycine Supplementation Ameliorates Retinal Neuronal Damage in an Experimental Model of Diabetes in Rats: A Light and Electron Microscopic Study.

PURPOSE: To investigate the potential neuroprotective effect of glycine supplementation on the retinal ultrastructure of streptozocin (STZ)-induced diabetic rats. METHODS: Adult male Wistar rats weighing 200-250 g (n = 40) were randomly divided into four groups of 10 each: normal group (C), glycine + normal group (G), STZ group (D), and glycine + STZ group (DG). The G and DG groups received glycine (130 mM and 1% w/v) freely in their drinking water seven days after the induction of diabetes for up to 16 weeks. Retinal samples for histopathology were examined using light and electron microscopy. RESULTS: Diabetes-induced histological changes were attenuated in the retinas of rats in the DG group. The ultrastructural alterations produced by experimental diabetes in the inner nuclear layer, outer nuclear layer, and ganglion cell layer were significantly ameliorated by glycine supplementation. CONCLUSION: Our findings suggest that glycine supplementation effectively attenuates retinal neuronal damage in experimental diabetic rats, and thus may be a potential candidate to protect retinal ultrastructure against diabetes.

Effects on c-Fos expression in the PAG and thalamus by selective input via tetrodotoxin-resistant afferent fibres from muscle and skin.

Nociceptive information from skin and muscle is differently processed at many levels of the central nervous system. However, with regard to nociceptive input from muscle to the thalamus, only few data are available. Here, we investigated the c-Fos expression in the thalamus and the periaqueductal grey matter (PAG) induced by electrical stimulation of tetrodotoxin-resistant (TTX-r), presumably nociceptive, afferent fibres. In addition, a comparison between the effects of TTX-r input from muscle and skin was made. In anaesthetised rats, a skin or a muscle nerve was stimulated electrically for 1h at an intensity supramaximal for unmyelinated fibres. To block TTX-sensitive afferents, TTX was applied to the sciatic nerve. c-Fos was visualized using DAB immunohistochemistry. Here we report for the first time that in the PAG and medial thalamus, the main effect of TTX-r input from muscle was a reduction in c-Fos expression, and that in some thalamic nuclei (e.g. posterior, reuniens, and central medial nuclei), significant differences in the number of c-Fos-positive cells were found after muscle and cutaneous input, respectively. The thalamic regions with the strongest effects of muscle input were the VL bilaterally and the VPL contralaterally (increase in c-Fos expression) as well as the rhomboid nucleus (decrease in c-Fos expression).

In Vivo Differentiation of Mesenchymal Stem Cells into Insulin Producing Cells on Electrospun Poly-L-Lactide Acid Scaffolds Coated with Matricaria chamomilla L. Oil.

OBJECTIVE: This study examined the in vivo differentiation of mesenchymal stem cells (MSCs) into insulin producing cells (IPCs) on electrospun poly-L-lactide acid (PLLA) scaffolds coated with Matricaria chammomila L. (chamomile) oil. MATERIALS AND METHODS: In this interventional, experimental study adipose MSCs (AMSCs) were isolated from 12 adult male New Zealand white rabbits and characterized by flow cytometry. AMSCs were subsequently differentiated into osteogenic and adipogenic lines. Cells were seeded onto either a PLLA scaffold (control) or PLLA scaffold coated with chamomile oil (experimental). A total of 24 scaffolds were inserted into the pancreatic area of each rabbit and placement was confirmed by ultrasound. After 21 days, immunohistochemistry analysis of insulin-producing like cells on protein levels confirmed insulin expression of insulin producing cells (IPSCs). Real-time polymerase chain reaction (PCR) determined the expressions of genes related to pancreatic endocrine development and function. RESULTS: Fourier transform infrared spectroscopy (FTIR) results confirmed the existence of oil on the surface of the PLLA scaffold. The results showed a new peak at 2854 cm(-1) for the aliphatic CH2 bond. Pdx1 expression was 0.051 ± 0.007 in the experimental group and 0.009 ± 0.002 in the control group. There was significantly increased insulin expression in the scaffold coated with chamomile oil (0.09 ± 0.001) compared to control group (0.063 ± 0.009, P≤0.05). Both groups expressed Ngn3 and Pdx1 specific markers and pancreatic tissue was observed at 21 days post transplantation. CONCLUSION: The pancreatic region is an optimal site for differentiation of AMSCs to IPCs. Chamomile oil (as an antioxidant agent) can affect cell adhesion to the scaffold and increase cell differentiation. In addition, the oil may lead to increased blood glucose uptake in pathways in the muscles, liver and fatty tissue of a diabetic animal model by some probable molecular mechanisms.

Third eyelid in the small Indian mongoose (Herpestes javanicus): a morphological and histological study.

The Indian mongoose (Herpestes javanicus) is native to parts of Asia, Iran. The purpose of this study was to describe the gross anatomy of the cartilage and histology of the superficial gland of the third eyelid of two adult mongooses. The animals, in terminal stages of disease and near death due to aging or unknown reasons, were referred from Park Zoo (Shiraz, Iran) to our center. By using a modified maceration technique, the morphological characteristics of the cartilage were examined. For histological examinations of the superficial gland of the third eyelid, the samples were stained with haematoxylin and eosin. Also, to detect the elastic fibers in the cartilage sections were stained with orcein and Weigert's resorcin-fuchsin. The cartilage consisted of an ovoid appendix and a mild reverse sigmoid crossbar. Elastic fibers were scattered throughout the cartilage but were more concentrated in the center. The superficial gland of the third eyelid was compound tubuloacinar with serous acini.

Evaluation of offspring sex ratio, sex hormones and antioxidant enzymes following exposure to methyl tertiary butyl ether in adult male Sprague-Dawley rats.

Methyl tertiary butyl ether (MTBE) is an oxygenated fuel additive which has been used widely in many parts of the world. This experiment was performed to determine the effect of MTBE on offspring sex ratio, sex hormones and antioxidant enzymes. A total of 20 adult Sprague-Dawley male rats were divided into four groups and received 0, 400, 800 and 1600 mg/kg/day MTBE by gavages for 30 consecutive days. At the end of the experiment, blood samples were taken for determination of sex hormones and antioxidant enzymes. Then, male rats were mated with healthy unexposed female rats and sex of offspring was determined after birth. Sex ratio was 0.48, 0.50, 0.43 and 0.50 in 0, 400, 800 and 1600 mg/kg/day MTBE groups, respectively (P = 0.91). There was significant decreasing trend for luteinizing hormone (LH) and testosterone in experimental groups (rs = -0.50, P = 0.030 and rs = -0.67, P = 0.002, respectively). No changes were observed for superoxide dismutase. However, decrease in glutathione peroxidase (GPX) was observed in all treatment groups compared with control which was significant in 400 mg/kg/day MTBE group (P = 0.016). The present study showed that paternal exposure to oral MTBE has no effect on offspring sex ratio; while, MTBE exposure could exert dose-dependent changes in serum testosterone and LH in treatment groups. The results of the present study, need to be clarified in the future studies.

Developmental Competence and Pluripotency Gene Expression of Cattle Cloned Embryos Derived from Donor Cells Treated with 5-aza-2'-deoxycytidine.

BACKGROUND: Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells. MATERIALS AND METHODS: Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine (5-aza-dc; 0-0.3 µM), a DNA methyl transferase inhibitor, improved development and expression of Oct-4. RESULTS: In comparison with untreated cells, 0.01 and 0.08 µM 5-aza-dc treated cells insignificantly decreased the blastocyst rate (32.1% vs. 28.6% and 27.2%, respectively) while it was significant for 0.3 µM treated cells (6.5%). Embryo quality as measured by the total cell number (TCN) decreased in a dose-related fashion, which was significant at 0.08 and 0.3 µM 5-aza-dc treated cells when compared with 0 and 0.01 µM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 µM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 µM 5-aza-dc remained unchanged. CONCLUSION: These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryos.

Somatic cell-induced hyperacetylation, but not hypomethylation, positively and reversibly affects the efficiency of in vitro cloned blastocyst production in cattle.

5-Aza-2'-deoxycytidine (AzC), trichostatin A (TSA), and its natural mimetic, sodium butyrate (NaB), are antineoplastic drugs that can modify the epigenetic status of donor cells prior to somatic cell nuclear transfer (SCNT). In this study, we used fibroblast cells treated with these drugs to investigate the direct and indirect effects of induced changes in DNA methylation and acetylation of the lysine 9 residue of histone H3 (H3K9). Additionally, we assayed cellular characteristics (cell growth, cell proliferation, cell cycle progression, and apoptosis) and SCNT efficiency in response to these drugs as well as monitoring these effects 24 h after removing the drugs. We observed the following: (1) AzC, TSA, and NaB all showed dose-dependent effects on different cellular characteristics; (2) TSA and NaB induced H3K9 hyperacetylation accompanied by DNA hypermethylation, whereas AzC induced DNA hypomethylation with no effect on H3K9 hyperacetylation; (3) TSA and NaB improved cloning efficiency, whereas AzC reduced it; and (4) unlike AzC, the effects of TSA and NaB on cellular characteristics and SCNT efficiency were reversed following drug removal. Our results indicate that somatic cells treated with TSA and NaB show better survival and recovery rates following the removal of these drugs. Moreover, H3K9 hyperacetylation (induced with TSA and NaB), but not DNA hypomethylation (induced with AzC), favors cloning efficiency.