Cardiac arrest-induced global cerebral ischemia injury (CA-GCII) usually leads to a

Cardiac arrest-induced global cerebral ischemia injury (CA-GCII) usually leads to a poor neurological outcome without an effective treatment. ameliorated brain pathology and improved neurofunctional performance in CA-GCII rats after transplantation. These findings provide a proof of concept for the further validation of engineered BMMSCs for the treatment of CA-GCII patients in clinical practice in the future. Cardiac arrest (CA) is usually associated with both high morbidity and mortality rates and poses the most serious threat to human life.1 In recent years, with the continuous improvement in the emergency network and broad application Ostarine enzyme inhibitor of cardiopulmonary Ostarine enzyme inhibitor resuscitation and defibrillation technology, there is an increase in the success rate of resuscitation of out-of-hospital CA patients with restoration of spontaneous circulation (ROSC).2 However, many of these patients usually suffer from complicated neural dysfunction or even death as a sequel to brain damage due to global cerebral ischemia following CA.3 Despite the use of neuroprotective medicines and hypothermic Ostarine enzyme inhibitor treatments, which to some extent may ameliorate brain injury in clinic,4 there is still an urgent demand for new treatments to improve the prognosis of CA-induced brain injury. In this regard, stem cells offer exciting promise for rebuilding the nervous system to treat devastating brain disorders including global cerebral ischemia resulting from CA. Because of the multipotency to divide and to differentiate into functional neural cells,5 bone marrow-derived mesenchymal stem cells (BMMSCs) have been widely tested for the treatment of a broad spectrum of degenerative disorders, such as cardiovascular diseases and neurologic complications.6, 7, 8, 9 We and others previously showed the therapeutic benefit of BMMSCs in CA-induced global cerebral ischemic injury (CA-GCII).10, 11 Transplantation of BMMSCs TGFBR1 significantly improved neural functional recovery of rats from CA-GCII. Mechanistic analysis suggests that the therapeutic efficacy of stem cells transplanted might be mediated by secreted vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF).12, 13 Therefore, to optimize the efficacy further, in this study we employed lentiviral vectors to induce overexpression of both BDNF and VEGF in BMMSCs, thereafter examined the neuroprotective efficacy of the virus-transduced BMMSCs for treatments of brain lesions and neurofunctional deficits after CA-GCII in rats. The results indicate markedly enhanced neuroprotective potency of BMMSCs for CA-induced global cerebral ischemia. Results Lentivirus-mediated overexpression of BDNF and VEGF in rat BMMSCs Once MSCs were prepared from rat bone marrow and grown in flasks, the identity of these cells was identified by both morphology and specific molecular markers. Phase-contrast microscopy exhibited their adherence onto the surface of plastic substrates with a fibroblast-like appearance (Physique 1a), whereas flow cytometry analysis revealed that both CD44 and CD90, two specific surface markers for MSC, were positive in the majority (61.8 and 91.2%, respectively) of cells after three passages, and only a small portion of cells showed expression of blood cell-related markers, CD34 and C45 (Determine 1b). Cells were co-transduced with the lentivirus constructs Ostarine enzyme inhibitor carrying an expression cassette for rat BDNF or VEGF along with ZsGreen1 or tdTomato fluorescent protein, respectively. Fluorescence microscopy detected expression of both fluorescent proteins in most cells, indicating the success of transduction at a high efficiency (Physique 1c), as ZsGreen1 and tdTomato open reading frames were directed by an internal ribosome entry site (IRES) within either BDNF or VEGF cassette. It is notable that both ZsGreen1 and tdTomato proteins exhibit stronger fluorescence in the nucleus or perinuclear area than the cytoplasm as reported.14, 15 RT-qPCR using specific primers for BDNF or VEGF confirmed a dramatic increase in expression of both BDNF and VEGF transcripts in the cells co-transduced by the lentiviruses carrying both genes compared with cells.