RESUMO

Schwann cells (SCs) are essential for the regenerative processes of peripheral nerve injuries. However, their use in cell therapy is limited. In this context, several studies have demonstrated the ability of mesenchymal stem cells (MSCs) to transdifferentiate into Schwann-like cells (SLCs) using chemical protocols or co-culture with SCs. Here, we describe for the first time the in vitro transdifferentiation potential of MSCs derived from equine adipose tissue (AT) and equine bone marrow (BM) into SLCs using a practical method. In this study, the facial nerve of a horse was collected, cut into fragments, and incubated in cell culture medium for 48 h. This medium was used to transdifferentiate the MSCs into SLCs. Equine AT-MSCs and BM-MSCs were incubated with the induction medium for 5 days. After this period, the morphology, cell viability, metabolic activity, gene expression of glial markers glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), p75 and S100ß, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF), and the protein expression of S100 and GFAP were evaluated in undifferentiated and differentiated cells. The MSCs from the two sources incubated with the induction medium exhibited similar morphology to the SCs and maintained cell viability and metabolic activity. There was a significant increase in the gene expression of BDNF, GDNF, GFAP, MBP, p75, and S100ß in equine AT-MSCs and GDNF, GFAP, MBP, p75, and S100ß in equine BM-MSCs post-differentiation. Immunofluorescence analysis revealed GFAP expression in undifferentiated and differentiated cells, with a significant increase in the integrated pixel density in differentiated cells and S100 was only expressed in differentiated cells from both sources. These findings indicate that equine AT-MSCs and BM-MSCs have great transdifferentiation potential into SLCs using this method, and they represent a promising strategy for cell-based therapy for peripheral nerve regeneration in horses.

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RESUMO

Sucrose- and diuretics-based protocols are widely used to induce osmotic diarrhea and dehydration in calves, but they fail to cause metabolic acidosis. In previous studies, calves were fed milk replacers and deprived of water. In this study, we assessed the water, electrolyte, and acid-base imbalances in calves that were fed whole milk and were not completely deprived of water during the induction period. Healthy, male Holstein calves aged 10-12 days were assigned to two groups: free access to water (FWG; n=17) and water deprivation at night (DWG; n=21); and osmotic diarrhea was induced with sucrose added to milk, spironolactone (2mg kg-1) and hydrochlorothiazide (2mg kg-1) orally every 8h for 48h. pH, pCO2, HCO3-, BE, Na+, K+, Cl-, SID3, TPP, AG, Atot, glucose, L-lactate, D-lactate, SIG, and percentage change in plasma volume were measured in venous blood samples taken at 0, 24, and 48h. Data were analyzed using two-way repeated measures ANOVA. Calves showed diarrhea, mild (FWG) to moderate (DWG) dehydration, hyponatremia, and moderate (FWG) to severe (DWG) metabolic acidosis. AG and D-lactate levels were higher and SIG was lower in the DWG, and there was no hyper-L- or D-lactatemia. The magnitude of metabolic acidosis was similar to that observed in natural cases of diarrhea. The protocol for inducing osmotic diarrhea and dehydration should be applied to calves that are fed whole milk and are not completely deprived of water.

Protocolos baseados em sacarose e diuréticos são amplamente usados para induzir diarreia osmótica e desidratação em bezerros, porém não provocam acidose metabólica. Em estudos anteriores, os bezerros foram alimentados com substitutos de leite e privados de água. Neste estudo, avaliaram-se os desequilíbrios hídrico, eletrolítico e ácido base em bezerros que foram alimentados com leite integral e não foram completamente privados de água durante o período de indução. Bezerros HPB machos sadios com 10 a 12 dias de idade foram distribuídos por dois grupos: acesso livre à água (FWG; n=17) e privação de água durante a noite (DWG; n=21); e a diarreia osmótica foi induzida com sacarose adicionada ao leite, espironolactona (2mg kg-1) e hidroclorotiazida (2mg kg-1) por via oral a cada 8h durante 48h. pH, pCO2, HCO3-, BE, Na+, K+, Cl-, SID3, PPT, AG, Atot, glicose, lactato L, lactato D, SIG e alteração percentual no volume plasmático foram medidos em amostras de sangue venoso colhidas em 0, 24 e 48h. Os dados foram analisados usando ANOVA de medidas repetidas bifatorial. Os bezerros apresentaram diarreia, desidratação leve (FWG) a moderada (DWG), hiponatremia e acidose metabólica moderada (FWG) a grave (DWG). Os valores de AG e de lactato D foram maiores e o de SIG foi menor no DWG, e não houve hiperlactatemia L ou D. A magnitude da acidose metabólica foi semelhante à observada em casos naturais de diarreia. O protocolo para induzir diarreia osmótica e desidratação deve ser aplicado a bezerros alimentados com leite integral e não totalmente privados de água.

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