■ AbstractWe have developed a simple shell-less chick embryo culture system to study glucose-induced malformations. This system involves the culturing of chick embryos from the second day to the fifth day of incubation, with associated yolk and thick and thin albumen outside the egg shell. The system allows the observation of embryonic development of chicks in a glass bowl. Developing embryos at 24 h, 48 h and 72 h incubation, corresponding to the Hamberger Hamilton (HH) stages from 7 to 21, were treated with two concentrations of glucose (50 mM and 100 mM) for 24 h. Glucose treatment resulted in a mortality rate of over 70% in younger embryos. Furthermore, a variety of malformations such as retarded growth, abnormal heart development, macrosomia, exencephaly, etc. were observed in older embryos, which were similar to those reported in mammalian embryos as a consequence of diabetic pregnancy. The glucose-induced malformations were found to be concentration-and stage-dependent, thus emphasizing the roles of the degree of hyperglycemia and the stage of embryonic development in diabetic growth anomalies. Here we demonstrate for the first time that the present system can be used (i) for experiments at early stages of chick embryo development and (ii) for assessing the effects of acute glucose toxicity similar to those reported for mammalian embryos in a hyperglycemic environment.

■ AbstractAnimal models have been used extensively in diabetes research. Studies on animal models have contributed to the discovery and purification of insulin, development of new therapeutic approaches, and progress in fundamental and clinical research. However, conventional rodent and large animal mammalian models face ethical, practical, or technical limitations. Therefore, it would be beneficial developing an alternative model for diabetes research which would overcome these limitations. Amongst other vertebrates, birds are phylogenically closer to mammals, and amongst birds, the chick has been used as one of the favored models in developmental biology, toxicology, cancer research, immunology, and drug testing. Chicken eggs are readily available, have a short incubation period and easily accessible embryos. Based on these inimitable advantages, the present review article aims to discuss the suitability of the chick as a model system to study specific aspects of diabetes. The review focuses on the application of i) chick pancreatic islets for screening of antidiabetic agents and for islet banking, (ii) shell-less chick embryo culture as a model to study hyperglycemia-induced malformations observed in mammalian embryos, and (iii) chick chorioallantoic membrane (CAM) to examine glucose-induced endothelial damage leading to inhibition of angiogenesis.

Species differences in susceptibility of islets to STZ in different mammals have been well documented. Likewise, failure of diabetes induction in birds by streptozotocin has been reported. We hypothesized that the susceptibility of islets to STZ treatment may be related to generation of reactive oxygen species (ROS) and their antioxidant defense mechanisms. To test this hypothesis, we measured the total ROS generated and estimated the damage caused to the chick islets due to STZ treatment, in terms of lipid peroxidation, protein carbonyl formation and DNA strand breaks and compared it with that of mouse islets. We also compared the activities of antioxidant enzymes like catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR) and amount of antioxidant molecules like reduced glutathione (GSH) and uric acid under control and STZ-treated conditions. These studies coupled with viability, functionality and presence of glucose transporter GLUT2 in chick and mouse islets clearly indicated that STZ treatment neither affects viability nor functionality of chick islets whereas those of mouse islets are affected significantly. Here we demonstrate for the first time a correlation between the generation of ROS on STZ treatment and antioxidant status with insensitivity of chick islets to STZ resulting into failure of diabetes induction in chick.

Angiogenesis, the process of new blood vessel formation from pre-existing vessels, is essential for growth and development. Development of drugs that can accelerate or decelerate angiogenesis in the context of various diseases requires appropriate preclinical screening. As angiogenesis involves complex cellular and molecular processes, in vivo studies are superior to in vitro investigations. Conventional in vitro, in vivo, and ex ovo models of angiogenesis are time consuming and tedious, and require sophisticated infrastructure for embryo culture. In the present study, we established an in ovo chick embryo yolk sac membrane (YSM) assay for angiogenesis and tested the angiogenic potential of arginine, conditioned medium (CM) from human adipose tissue and placenta-derived mesenchymal stem cells (ADMSCs-CM and PDMSCs-CM), avastin and vitamin C. The obtained results were confirmed with the routinely employed chick embryo Chorioallantoic Membrane (CAM) assay. Both assays revealed the pro-angiogenic nature of arginine, ADMSCs-CM, and PDMSCs-CM, and the anti-angiogenic effect of avastin and vitamin C. This novel in ovo YSM model is simple, reproducible, and highly economic in terms of the time frame and cost incurred. The proposed model is thus a suitable substitute to the CAM model for pilot screening of potential angiogenic and anti-angiogenic agents.

The therapeutic potential of stem cells has led to renewed interest in regenerative biology. Pancreatic stellate cells have been reported in the mammalian pancreas; however, there are very few reports on stellate cells in the chicken pancreas. The intercalated duct epithelial cells observed in the A and B islets of the chicken pancreas have been claimed to be stellate cells from their morphological appearance. While isolating islets and acinar cells from the chick pancreas, we have found a population of stellate-like cells, which has been successfully propagated in a defined nutrient medium. These cells were immunopositive for vimentin, desmin, and fibronectin and also expressed alkaline phosphatase, indicating their undifferentiated state. On exposure to serum-free medium containing specific nutrients and differentiating agents, these stellate-like-cells gave rise to islet-like cell clusters. Islet-like clusters stained positive for the islet specific stain diphenyl thiocarbazone and were immunopositive for C-peptide indicating de novo insulin synthesis. These clusters secreted insulin in response to glucose challenge, thus suggesting their similarity to islets. Thus stellate cells found in chick pancreatic islets exhibit potential to differentiate into islet-like clusters. Taken together, our study documents for the first time the presence of a stellate-like cell population in chick pancreatic islets providing a source for islet neogenesis.