Functional Connectivity Increases in Response to High-Definition Transcranial Direct Current Stimulation in Patients with Chronic Disorder of Consciousness.

High-definition transcranial direct current stimulation (HD-tDCS) has been shown to play an important role in improving consciousness in patients with disorders of consciousness (DOCs), but its neuroelectrophysiological evidence is still lacking. To better explain the electrophysiological mechanisms of the effects of HD-tDCS on patients with DOCs, 22 DOC patients underwent 10 anodal HD-tDCS sessions of the left dorsolateral prefrontal cortex (DLPFC). This study used the Coma Recovery Scale-Revised (CRS-R) to assess the level of consciousness in DOC patients. According to whether the CRS-R score increased before and after stimulation, DOC patients were divided into a responsive group and a non-responsive group. By comparing the differences in resting-state EEG functional connectivity between different frequency bands and brain regions, as well as the relationship between functional connectivity values and clinical scores, the electrophysiological mechanism of the clinical effects of HD-tDCS was further explored. The change of the phase locking value (PLV) on the theta frequency band in the left frontal-parietooccipital region was positively correlated with the change in the CRS-R scores. As the number of interventions increased, we observed that in the responsive group, the change in PLV showed an upward trend, and the increase in the PLV appeared in the left frontal-parietooccipital region at 4-8 Hz and in the intra-bifrontal region at 8-13 Hz. In the non-responsive group, although the CRS-R scores did not change after stimulation, the PLV showed a downward trend, and the decrease in the PLV appeared in the intra-bifrontal region at 8-13 Hz. In addition, at the three-month follow-up, patients with increased PLV in the intra-bifrontal region at 8-13 Hz after repeated HD-tDCS stimulation had better outcomes than those without. Repeated anodal stimulation of the left DLPFC with HD-tDCS resulted in improved consciousness in some patients with DOCs. The increase in functional connectivity in the brain regions may be associated with the improvement of related awareness after HD-tDCS and may be a predictor of better long-term outcomes.

Dynamic Changes of Brain Activity in Different Responsive Groups of Patients with Prolonged Disorders of Consciousness.

As medical technology continues to improve, many patients diagnosed with brain injury survive after treatments but are still in a coma. Further, multiple clinical studies have demonstrated recovery of consciousness after transcranial direct current stimulation. To identify possible neurophysiological mechanisms underlying disorders of consciousness (DOCs) improvement, we examined the changes in multiple resting-state EEG microstate parameters after high-definition transcranial direct current stimulation (HD-tDCS). Because the left dorsolateral prefrontal cortex is closely related to consciousness, it is often chosen as a stimulation target for tDCS treatment of DOCs. A total of 21 patients diagnosed with prolonged DOCs were included in this study, and EEG microstate analysis of resting state EEG datasets was performed on all patients before and after interventions. Each of them underwent 10 anodal tDCS sessions of the left dorsolateral prefrontal cortex over 5 consecutive working days. According to whether the clinical manifestations improved, DOCs patients were divided into the responsive (RE) group and the non-responsive (N-RE) group. The dynamic changes of resting state EEG microstate parameters were also analyzed. After multiple HD-tDCS interventions, the duration and coverage of class C microstates in the RE group were significantly increased. This study also found that the transition between microstates A and C increased, while the transition between microstates B and D decreased in the responsive group. However, these changes in EEG microstate parameters in the N-RE group have not been reported. Our findings suggest that EEG neural signatures have the potential to assess consciousness states and that improvement in the dynamics of brain activity was associated with the recovery of DOCs. This study extends our understanding of the neural mechanism of DOCs patients in consciousness recovery.

Exendin-4 promotes proliferation of adipose-derived stem cells through ERK and JNK signaling pathways.

Adipose-derived stem cell (ADSC) transplantation has emerged as a potential tool for the treatment of cardiovascular disease. However, with a limited renewal capacity and the need for mass cells during the engraftment, strategies are needed to enhance ADSC proliferative capacity. In this study, we explored the effects of exendin-4 (Ex-4), a glucagon-like peptide-1 analog, on the growth of ADSCs, focusing in particular on c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Firstly, ADSCs were isolated and cultured in vitro. Then, flow cytometry demonstrated that ADSCs were positive for CD90 and CD29 but negative for CD31, CD34, and CD45. Ex-4 (0-50 nM) treatment increased ADSC proliferation in a dose-dependent manner but had no effects on stem cell markers of ADSCs. Moreover, we found that Ex-4 treatment elevated the phosphorylation levels of the JNK and ERK signaling pathways. Furthermore, utilization of Ex-4 also promoted cyclin D1 and cyclin E protein expression, which was accompanied by more Edu(+) cells and a higher percentage of cells in the S-phase of the cell cycle after Ex-4 treatment. In parallel, the application of inhibitors SP600125 and PD98059, inhibitors of the JNK and ERK signaling pathways, respectively, not only reversed such effects of Ex-4 on JNK and ERK but also resulted in lower percentages of S-phase cells and fewer numbers of Edu(+) cells. In summary, Ex-4 has no effects on stem cell markers in ADSCs but promotes ADSC growth via JNK and ERK signaling pathways.

Effects of Exendin-4 on bone marrow mesenchymal stem cell proliferation, migration and apoptosis in vitro.

Mesenchymal stem cells (MSC) are regarded as an attractive source of therapeutic stem cells for myocardial infarction. However, their limited self-renewal capacity, low migration capacity and poor viability after transplantation hamper the clinical use of MSC; thus, a strategy to enhance the biological functions of MSC is required. Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, exerts cell-protective effects on many types of cells. However, little information is available regarding the influence of Ex-4 on MSC. In our study, MSC were isolated from bone marrow and cultured in vitro. After treatment with Ex-4, MSC displayed a higher proliferative capacity, increased C-X-C motif receptor 4 (CXCR4) expression and an enhanced migration response. Moreover, in H2O2-induced apoptosis, Ex-4 preserved mitochondrial function through scavenging ROS and balancing the expression of anti- and pro-apoptotic proteins, leading to the inhibition of the mitochondria-dependent cell death pathways and increased cell survival. Moreover, higher phospho-Akt (p-Akt) expression was observed after Ex-4 intervention. However, blockade of the PI3K/Akt pathway with inhibitors suppressed the above cytoprotective effects of Ex-4, suggesting that the PI3K/Akt pathway is partly responsible for Ex-4-mediated MSC growth, mobilization and survival. These findings provide an attractive method of maximizing the effectiveness of MSC-based therapies in clinical applications.

Exendin-4 enhances the migration of adipose-derived stem cells to neonatal rat ventricular cardiomyocyte-derived conditioned medium via the phosphoinositide 3-kinase/Akt-stromal cell-derived factor-1α/CXC chemokine receptor 4 pathway.

Adipose­derived stem cells (ADSCs) are considered a suitable source of cells for the repair of tissue following acute myocardial infarction (AMI); however, the transplantation efficiency of ADSCs remains low. Therefore, identification of an efficient method to enhance the migration of engrafted cells to the target site is required. The present study used exendin­4 (Ex­4), a glucagon­like peptide­1 receptor agonist, to optimize the migratory capacity of ADSCs. The aim was to determine the effect and mechanisms of Ex­4 on the migration of ADSCs to neonatal rat ventricular cardiomyocyte­derived conditioned medium (NRVC­CM). The ADSCs and cardiomyocytes were cultured in vitro. Following incubation of the ADSCs with Ex­4, cell proliferation was measured using an MTT assay and the expression levels of CXC chemokine receptor 4 (CXCR4) were investigated by reverse transctiption quantitative polymerase chain reaction (RT­qPCR), western blot analysis and flow cytometry. In addition, the expression levels of stromal cell­derived factor­1α (SDF­1α) were evaluated in the NRVC­CM treated with Ex­4 by ELISA, RT­qPCR and western blot analysis. The migration of the ADSCs to the NRVC­CM was examined using a Transwell assay. Changes in the protein expression levels of phosphorylated (p­)Akt were examined in the two types of cell by western blot analysis. The results suggested that Ex­4 promoted the proliferation and expression of CXCR4 in the ADSCs, increased the secretion of SDF­1α in the cardiomyocytes and increased the expression levels of p­Akt in both cells. However, the alterations to the SDF­1α/CXCR4 cascade in the cells were abrogated following pretreatment with LY­294002, a phosphoinositide 3­kinase(PI3K) inhibitor. Furthermore, a Transwell migration assay revealed marked translocation of the ADSCs through the membranes, towards the NRVC­CM, following treatment with Ex­4. However, these effects were reduced significantly by pretreatment of the cells with the SDF­1α/CXCR4 cascade antagonist, AMD3100, and the PI3K inhibitor, LY­294002. These results indicated that Ex­4 augmented the SDF­1α/CXCR4 cascade by activating the PI3K/Akt pathways in the ADSCs and NRVCs. Furthermore, enhancement of the PI3K/Akt-SDF-1α/CXCR4 pathway may be important in the migratory response of ADSCs to NRVC­CM in vitro.

Exendin-4 protects adipose-derived mesenchymal stem cells from apoptosis induced by hydrogen peroxide through the PI3K/Akt-Sfrp2 pathways.

Adipose-derived mesenchymal stem cells (ADMSCs)-based therapy is a promising modality for the treatment of myocardial infarction in the future. However, the majority of transplanted cells are readily lost after transplantation because of hypoxia and oxidative stress. An efficient means to enhance the ability of ADMSCs to survive under pathologic conditions is required. In our study, we explored the effects of exendin-4 (Ex-4) on ADMSCs apoptosis in vitro induced by hydrogen peroxide, focusing in particular on mitochondrial apoptotic pathways and PI3K/Akt-secreted frizzled-related protein 2 (Sfrp2) survival signaling. We demonstrated that ADMSCs subjected to H2O2 for 12h exhibited impaired mitochondrial function and higher apoptotic rate. However, Ex-4 (1-20 nM) preconditioning for 12h could protect ADMSCs against H2O2-mediated apoptosis in a dose-dependent manner. Furthermore, Ex-4 pretreatment upregulated the levels of superoxide dismutase and glutathione as well as downregulating the production of reactive oxygen species and malondialdehyde. Western blots revealed that increased antiapoptotic proteins Bcl-2 and c-IAP1/2 as well as decreased proapoptotic proteins Bax and cytochrome c appeared in ADMSCs with Ex-4 incubation, which inhibited the caspase-9-involved mitochondrial apoptosis pathways with evidence showing inactivation of caspase-9/3 and preservation of mitochondrial membrane potential. Furthermore, we illustrated that Ex-4 enhanced Akt phosphorylation, which increased the expression of Sfrp2. Notably, blockade of the PI3K/Akt pathway or knockdown of Sfrp2 with siRNA obviously abolished the protective effects of Ex-4 on mitochondrial function and ADMSCs apoptosis under H2O2. In summary, this study confirmed that H2O2 induced ADMSCs apoptosis through mitochondria-dependent cell death pathways, and Ex-4 preconditioning may reduce such apoptosis of ADMSCs through the PI3K/Akt-Sfrp2 pathways.

Real-time tracking of adipose tissue-derived stem cells with injectable scaffolds in the infarcted heart.

Adipose tissue-derived stem cells (ADSCs) has shown promise in the emerging field of regenerative medicine. Many studies have highlighted the importance of coadministering a "scaffold" for increasing intramyocardial retention of stem cells. In this work, an optimized method was developed for efficient transduction of ADSCs with a lentiviral vector carrying a triple-fusion reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). The transduced ADSCs were assessed on biological performance and transplanted into infarcted heart with fibrin scaffolds. In vivo cell retention was tracked by bioluminescence imaging (BLI) and micro positron emission tomography/computed tomography (PET/CT) imaging. Histological assessment was performed for regeneration potentials. The results showed that lentiviral transduction did not influence cell functions. In vitro imaging analysis showed a robust linear correlation between cell numbers and BLI signals (R (2) = 0.99) as well as between cell numbers and radiotracer uptakes (R (2) = 0.98). Transduced ADSCs were visualized in the heart under both BLI and PET/CT imaging, contributing to cardiomyocyte regeneration and angiogenesis in the implanted areas. Compared with BLI monitoring, PET/CT data provided precise localization for cell retention. Thus, a combination of imaging modalities can assist in reliable and efficient monitoring of transplanted cells, holding great potential for the transplantation of injectable scaffolds encapsulating stem cells in treating heart disease.

Transplantation of adipose tissue-derived stem cells overexpressing heme oxygenase-1 improves functions and remodeling of infarcted myocardium in rabbits.

Adipose tissue-derived stem cells (ADSCs) are a promising source of autologous stem cells that are used for regeneration and repair of infracted heart. However, the efficiency of their transplantation is under debate. One of the possible reasons for marginal improvement in ADSCs transplantation is the significant cell death rate of implanted cells after being grafted into injured heart. Therefore, overcoming the poor survival rate of implanted cells may improve stem cell therapy. Due to limited improvement concerning direct stem cell therapy, gene-transfer methods are used to enhance cellular cardiomyoplasty efficacy. Heme oxygenase-1 (HO-1) can provide various types of cells with protection against oxidative injury and apoptosis. However, exact effects of autologous ADSCs combined with HO-1 on cardiac performance remains unknown. In this study, rabbits were treated with ADSCs transduced with HO-1 (HO-1-ADSCs), treated with non-transduced ADSCs, or injected with phosphate buffered saline 14 days after experimental myocardial infarction was induced, when autologous ADSCs were obtained simultaneously. Four weeks after injection, echocardiography showed significant improvements for cardiac functions and left ventricular dimensions in HO-1-ADSCs-treated animals. Structural consequences of transplantation were determined by detailed histological analysis, which showed differentiation of HO-1-ADSCs to cardiomyocyte-like tissues and lumen-like structure organizations. Apart from improvement in angiogenesis and scar areas, more connexin 43-positive gap junction and greater tyrosine hydroxylase-positive cardiac sympathetic nerves sprouting were observed in the HO-1-ADSCs-treated group compared with ADSCs group. These data suggest that the transplantation of autologous ADSCs combined with HO-1 transduction is a feasible and efficacious method for improving infarcted myocardium.