Cryopreserved astrocytes maintain biological properties: Support of neuronal survival and differentiation.

BACKGROUND: Astrocytes, one of the main glial cell types, play critical roles in the central nervous system (CNS) development and function, including support of neuronal survival and differentiation, blood brain barrier formation, synapse homeostasis and injury response. Cell isolation and culture techniques have been proved to be a powerful tool to study astrocyte physiology and function. Due to financial constraints and rigid biosafety and ethics rules to use animal models, freezing techniques and the creation of cell banks emerged as alternatives to optimize the use of experimental animals. One of the main challenges, however, of these techniques is to guarantee that conserved cells keep their biological properties. NEW METHOD: In this work, we characterized morphologically and functionally murine secondary astrocyte cultures that have been submitted to freezing/thawing procedures. RESULTS: Morphological characterization of SAC (secondary astrocyte culture) and SFAC (secondary frozen-astrocyte culture) did not reveal significant differences on astrocyte morphology, confluence time and cell number along culture period. Functionally, SAC and SFAC did not reveal differences in their potential to support neuronal survival, maturation, neuritogenesis and synapse formation. CONCLUSIONS: Our results suggest that murine astrocytes that are submitted to freezing/thawing procedure maintain morphological and functional characteristics when compared with non-frozen astrocytes. Thus, this methodological approach is a valuable tool for in vitro research and might allow experimental optimization and reduction of animal use.

Transforming Growth Factor ß1/SMAD Signaling Pathway Activation Protects the Intestinal Epithelium from Clostridium difficile Toxin A-Induced Damage.

Clostridium difficile, the main cause of diarrhea in hospitalized patients, produces toxins A (TcdA) and B (TcdB), which affect intestinal epithelial cell survival, proliferation, and migration and induce an intense inflammatory response. Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine affecting enterocyte and immune/inflammatory responses. However, it has been shown that exposure of intestinal epithelium to a low concentration of TcdA induces the release of TGF-ß1, which has a protective effect on epithelial resistance and a TcdA/TGF-ß signaling pathway interaction. The activation of this pathway in vivo has not been elucidated. The aim of this study was to investigate the role of the TGF-ß1 pathway in TcdA-induced damage in a rat intestinal epithelial cell line (IEC-6) and in a mouse model of an ileal loop. TcdA increased the expression of TGF-ß1 and its receptor, TßRII, in vitro and in vivo TcdA induced nuclear translocation of the transcription factors SMAD2/3, a hallmark of TGF-ß1 pathway activation, both in IEC cells and in mouse ileal tissue. The addition of recombinant TGF-ß1 (rTGF-ß) prevented TcdA-induced apoptosis/necrosis and restored proliferation and repair activity in IEC-6 cells in the presence of TcdA. Together, these data show that TcdA induces TGF-ß1 signaling pathway activation and suggest that this pathway might play a protective role against the effect of C. difficile-toxin.