In vivo nonlinear imaging of corneal structures with special focus on BALB/c and streptozotocin-diabetic Thy1-YFP mice
EXPERIMENTAL EYE RESEARCH
Authors: Ehmke, Tobias; Leckelt, Janine; Reichard, Maria; Weiss, Heike; Hovakimyan, Marina; Heisterkamp, Alexander; Stachs, Oliver; Baltrusch, Simone
Abstract
Two-photon microscopy (TPM) allows high contrast imaging at a subcellular resolution scale. In this work, the microscopy technique was applied to visualize corneal structures in two mouse models (BALE/c and B6.Cg-Tg(Thy1-YFP)16Jrs/J) in vivo. In particular, the transgenic Thy1-YFP mice expressing the yellow fluorescent protein (YFP) in all motor and sensory neurons had been used for investigating the nerve fiber density in healthy and streptozotocin-diabetic mice. This model is clinically relevant since patients suffering from diabetes mellitus have a high risk to develop small fiber neuropathy. Nonlinear laser scanning microscopy displayed a reduction of nerve fiber density in streptozotocin-diabetic versus healthy mice and confirmed data obtained by confocal laser scanning microscopy (CLSM). In recent years, corneal CLSM was proved to be an appropriate non-invasive tool for an early diagnosis of diabetic neuropathy. Nevertheless, validation of the CLSM method for the clinical routine is currently a matter of investigation and requires confirmation by further studies and complementary techniques. Thus, the present study provides further evidence of corneal confocal microscopy as a promising technique for non-invasive detection of diabetic neuropathy. Information derived from these experiments may become clinically relevant and help to develop new drugs for treatment of diabetic neuropathy. (c) 2015 Elsevier Ltd. All rights reserved.
Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions
CYTOTECHNOLOGY
Authors: Sharma, Ankur; Shah, Syed Mohmad; Tiwari, Manish; Roshan, Mayank; Singh, Manoj Kumar; Singla, Suresh Kumar; Palta, Prabhat; Manik, Radhay Sham; Chauhan, Manmohan Singh
Abstract
In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.