The role of gender in cognitive outcomes from stroke.

OBJECTIVE: Stroke can cause cognitive impairment, which can lead to challenges returning to day-to-day activities. Knowing what factors are associated with cognitive impairment post-stroke can be useful for predicting outcomes and guiding rehabilitation. One such factor is gender: previous studies are inconclusive as to whether gender influences cognitive outcomes post-stroke. Accounting for key variables, we examined whether there are gender differences in cognitive outcomes after stroke. METHOD: We analyzed data from neuropsychological assessments of 237 individuals tested in the chronic epoch (≥ 3 months) following ischemic stroke. Using ANCOVA and linear mixed modeling, we examined gender as a predictor of cognition as measured by general cognitive ability (g), Full-Scale IQ, and 18 cognitive tests, controlling for age at stroke onset, education, premorbid intelligence, and lesion volume. RESULTS: There were no significant gender differences in overall cognitive outcomes as measured by g (p = .887) or Full-Scale IQ (p = .801). There were some significant gender differences on specific cognitive tests, with women outperforming men on scores from the Rey Auditory Verbal Learning Test (ps < .01) and men outperforming women on the Wechsler Adult Intelligence Scale Arithmetic and Information subtests (ps < .01). CONCLUSIONS: Our findings indicate that men and women have similar overall cognitive outcomes after stroke, when demographic and lesion factors are accounted for. Although men and women differed in their performance on some individual cognitive tests, neither gender performed systematically better or worse. However, for learning, working memory, and verbal knowledge/comprehension, gender may be an important predictor of outcome post-stroke.

Malonate Promotes Adult Cardiomyocyte Proliferation and Heart Regeneration.

BACKGROUND: Neonatal mouse cardiomyocytes undergo a metabolic switch from glycolysis to oxidative phosphorylation, which results in a significant increase in reactive oxygen species production that induces DNA damage. These cellular changes contribute to cardiomyocyte cell cycle exit and loss of the capacity for cardiac regeneration. The mechanisms that regulate this metabolic switch and the increase in reactive oxygen species production have been relatively unexplored. Current evidence suggests that elevated reactive oxygen species production in ischemic tissues occurs as a result of accumulation of the mitochondrial metabolite succinate during ischemia via succinate dehydrogenase (SDH), and this succinate is rapidly oxidized at reperfusion. Mutations in SDH in familial cancer syndromes have been demonstrated to promote a metabolic shift into glycolytic metabolism, suggesting a potential role for SDH in regulating cellular metabolism. Whether succinate and SDH regulate cardiomyocyte cell cycle activity and the cardiac metabolic state remains unclear. METHODS: Here, we investigated the role of succinate and SDH inhibition in regulation of postnatal cardiomyocyte cell cycle activity and heart regeneration. RESULTS: Our results demonstrate that injection of succinate into neonatal mice results in inhibition of cardiomyocyte proliferation and regeneration. Our evidence also shows that inhibition of SDH by malonate treatment after birth extends the window of cardiomyocyte proliferation and regeneration in juvenile mice. Remarkably, extending malonate treatment to the adult mouse heart after myocardial infarction injury results in a robust regenerative response within 4 weeks after injury via promoting adult cardiomyocyte proliferation and revascularization. Our metabolite analysis after SDH inhibition by malonate induces dynamic changes in adult cardiac metabolism. CONCLUSIONS: Inhibition of SDH by malonate promotes adult cardiomyocyte proliferation, revascularization, and heart regeneration via metabolic reprogramming. These findings support a potentially important new therapeutic approach for human heart failure.

Decreases in the Late Positive Potential to Alcohol Images Among Alcohol Treatment Seekers Following Mindfulness-Based Relapse Prevention.

AIM: Heightened craving among individuals with alcohol use disorder (AUD) has been attributed to a hypersensitivity to alcohol cues in attentional brain networks. Active mindfulness training has been shown to help improve attentional control. Here, we examined alcohol cue-related hypersensitivity among individuals with AUD who received rolling group mindfulness-based relapse prevention (MBRP) in combination with transcranial direct current stimulation (tDCS), over right inferior frontal gyrus. METHODS: Participants (n = 68) viewed a series of emotionally negative, emotionally neutral and alcohol-related images. Following image presentation, participants were asked to rate their level of craving for the alcohol cues, and their level of negative affect evoked by neutral and negative cues. During the task, electroencephalogram (EEG) was recorded to capture an event-related component shown to relate to emotionally salient stimuli: the late positive potential (LPP). Participants who completed a follow-up EEG (n = 37) performed the task a second time after up to eight sessions of MBRP coupled with active or sham tDCS. RESULTS: We found that both craving ratings and the LPP significantly decreased in response to alcohol cues from pre- to post-treatment, but not for other image cues. The magnitude of alcohol image craving reductions was associated with the number of MBRP group sessions attended. Active tDCS was not associated with craving ratings, but it was associated with greater LPP amplitudes across image types. CONCLUSIONS: Taken together, these results suggest that disruption of alcohol-cue hypersensitivity in people with AUD may be a target mechanism of MBRP.

Stem cells: the pursuit of genomic stability.

Stem cells harbor significant potential for regenerative medicine as well as basic and clinical translational research. Prior to harnessing their reparative nature for degenerative diseases, concerns regarding their genetic integrity and mutation acquisition need to be addressed. Here we review pluripotent and multipotent stem cell response to DNA damage including differences in DNA repair kinetics, specific repair pathways (homologous recombination vs. non-homologous end joining), and apoptotic sensitivity. We also describe DNA damage and repair strategies during reprogramming and discuss potential genotoxic agents that can reduce the inherent risk for teratoma formation and mutation accumulation. Ensuring genomic stability in stem cell lines is required to achieve the quality control standards for safe clinical application.

Effects of anodal tDCS on electroencephalography correlates of cognitive control in mild-to-moderate traumatic brain injury.

BACKGROUND: Transcranial direct current stimulation (tDCS) may provide a potential therapy for cognitive deficits caused by traumatic brain injury (TBI), yet its efficacy and mechanisms of action are still uncertain. OBJECTIVE: We hypothesized that anodal tDCS over the left dorsolateral prefrontal cortex (DLPFC) would boost the influence of a cognitive training regimen in a mild-to-moderate TBI (mmTBI) sample. Cognitive enhancement was measured by examining event-related potentials (ERPs) during cognitive control tasks from pre- to post-treatment. METHODS: Thirty-four participants with mmTBI underwent ten sessions of cognitive training with active (n = 17) or sham (n = 17) anodal tDCS to the left DLPFC. ERPs were assessed during performance of an auditory oddball (3AOB), N-back, and dot pattern expectancy (DPX) task before and after treatment. RESULTS: P3b amplitudes significantly decreased from baseline to post-treatment testing, regardless of tDCS condition, in the N-back task. The active tDCS group demonstrated a significantly increased P3a amplitude in the DPX task. No statistically significant stimulation effects were seen during the 3AOB and N-back tasks. CONCLUSION: Active anodal tDCS paired with cognitive training led to increases in P3a amplitudes in the DPX, inferring increased cognitive control. P3b decreased in the N-back task demonstrating the effects of cognitive training. These dissociated P3 findings suggest separate mechanisms invoked by different neuroplasticity-inducing paradigms (stimulation versus training) in brain networks that support executive functioning.

Quantifying Cardiomyocyte Proliferation and Nucleation to Assess Mammalian Cardiac Regeneration.

Neonatal mice display a remarkable ability to regenerate their heart following an injury during the first week of life. A key facet of successful cardiac regeneration is the proliferation of cardiomyocytes to replace the lost cells. Stimulating cardiomyocyte proliferation in the adult heart is a very promising approach to restore cardiac structure and function following injury. Here, we outline our approach to assess cardiomyocyte proliferation following a myocardial injury via the cell cycle markers phospho-histone H3 and Aurora B. We additionally discuss how we assess successful regeneration using wheat germ agglutinin to measure cardiomyocyte size, nuclear staining to quantify cardiomyocyte nucleation, and Trichrome staining to identify myocardial regeneration and scarring in the myocardium.

Depression symptoms in neurological patients: A survey of a large cohort of patients with focal brain lesions.

INTRODUCTION: Examining depression following neurological injury is useful for understanding post-lesion depression and depression more generally. The extant literature shows variability in the incidence and severity of depression post-lesion, likely due to heterogeneity in study methodology, patient samples, measures of depression, and time of assessment. Here, we aim to characterize depression symptoms and their demographic correlates in a large sample of individuals in the chronic epoch following a focal brain lesion. METHOD: We sampled 492 individuals who had focal, stable brain lesions and were in the chronic epoch (≥3 months post-onset). Demographic (gender, years of education), temporal (age at lesion onset, time since lesion onset), and lesion (lesion laterality, lesion etiology, lesion volume) factors were used to predict depression symptoms measured by the Beck Depression Inventory (BDI). RESULTS: We found that on average, neurological patients exhibited elevated levels of depression symptoms (although not clinically significant) relative to a community sample, and the neurological patients showed higher rates of mild and moderate depression symptoms than are typical in a community sample. Gender and lesion etiology were predictive of depression symptoms, whereby women and patients with ischemic stroke had higher levels of depression symptoms. CONCLUSIONS: Our results suggest that depression symptom severity may be elevated following a focal brain lesion. Moreover, some individuals may be more likely to develop depression symptoms post-lesion than others. This may be mediated by individual factors such as gender and lesion etiology. The findings have important implications for the diagnosis, prognosis, and treatment of depression in neurological patients.

Autologous Umbilical Cord Blood-Derived Mononuclear Cell Therapy Promotes Cardiac Proliferation and Adaptation in a Porcine Model of Right Ventricle Pressure Overload.

Congenital heart diseases, including single ventricle circulations, are clinically challenging due to chronic pressure overload and the inability of the myocardium to compensate for lifelong physiological demands. To determine the clinical relevance of autologous umbilical cord blood-derived mononuclear cells (UCB-MNCs) as a therapy to augment cardiac adaptation following surgical management of congenital heart disease, a validated model system of right ventricular pressure overload due to pulmonary artery banding (PAB) in juvenile pigs has been employed. PAB in a juvenile porcine model and intramyocardial delivery of UCB-MNCs was evaluated in three distinct 12-week studies utilizing serial cardiac imaging and end-of-study pathology evaluations. PAB reproducibly induced pressure overload leading to chronic right ventricular remodeling including significant myocardial fibrosis and elevation of heart failure biomarkers. High-dose UCB-MNCs (3 million/kg) delivered into the right ventricular myocardium did not cause any detectable safety issues in the context of arrhythmias or abnormal cardiac physiology. In addition, this high-dose treatment compared with placebo controls demonstrated that UCB-MNCs promoted a significant increase in Ki-67-positive cardiomyocytes coupled with an increase in the number of CD31+ endothelium. Furthermore, the incorporation of BrdU-labeled cells within the myocardium confirmed the biological potency of the high-dose UCB-MNC treatment. Finally, the cell-based treatment augmented the physiological adaptation compared with controls with a trend toward increased right ventricular mass within the 12 weeks of the follow-up period. Despite these adaptations, functional changes as measured by echocardiography and magnetic resonance imaging did not demonstrate differences between cohorts in this surgical model system. Therefore, this randomized, double-blinded, placebo-controlled pre-clinical trial establishes the safety of UCB-MNCs delivered via intramyocardial injections in a dysfunctional right ventricle and validates the induction of cardiac proliferation and angiogenesis as transient paracrine mechanisms that may be important to optimize long-term outcomes for surgically repaired congenital heart diseases.

Transcranial direct current stimulation modulates working memory and prefrontal-insula connectivity after mild-moderate traumatic brain injury.

Background: Persistent posttraumatic symptoms (PPS) may manifest after a mild-moderate traumatic brain injury (mmTBI) even when standard brain imaging appears normal. Transcranial direct current stimulation (tDCS) represents a promising treatment that may ameliorate pathophysiological processes contributing to PPS. Objective/Hypothesis: We hypothesized that in a mmTBI population, active tDCS combined with training would result in greater improvement in executive functions and post-TBI cognitive symptoms and increased resting state connectivity of the stimulated region, i.e., left dorsolateral prefrontal cortex (DLPFC) compared to control tDCS. Methods: Thirty-four subjects with mmTBI underwent baseline assessments of demographics, symptoms, and cognitive function as well as resting state functional magnetic resonance imaging (rsfMRI) in a subset of patients (n = 24). Primary outcome measures included NIH EXAMINER composite scores, and the Neurobehavioral Symptom Inventory (NSI). All participants received 10 daily sessions of 30 min of executive function training coupled with active or control tDCS (2 mA, anode F3, cathode right deltoid). Imaging and assessments were re-obtained after the final training session, and assessments were repeated after 1 month. Mixed-models linear regression and repeated measures analyses of variance were calculated for main effects and interactions. Results: Both active and control groups demonstrated improvements in executive function (EXAMINER composite: p < 0.001) and posttraumatic symptoms (NSI cognitive: p = 0.01) from baseline to 1 month. Active anodal tDCS was associated with greater improvements in working memory reaction time compared to control (p = 0.007). Reaction time improvement correlated significantly with the degree of connectivity change between the right DLPFC and the left anterior insula (p = 0.02). Conclusion: Anodal tDCS improved reaction time on an online working memory task in a mmTBI population, and decreased connectivity between executive network and salience network nodes. These findings generate important hypotheses for the mechanism of recovery from PPS after mild-moderate TBI.

Activation of P53 Via Nutlin-3a Reveals Role for P53 In ROS Signaling During Cardiac Differentiation of hiPSCs.

Activation of the transcription factor P53 within cancer cells is a well-characterized pathway, whereas the effects of P53 activation during development remain largely unexplored. Previous research has indicated that increased levels of P53 protein during key murine developmental stages cause defects in multiple embryonic tissues, including the heart. These findings were confirmed in several different mouse models of congenital heart defects, but P53 activation in a human system of cardiovascular development is not available. Utilizing human induced pluripotent stem cells (hiPSCs), we characterized the normal levels of P53 during cardiac differentiation and showed that levels of P53 are high in hiPSCs and decrease upon cardiac lineage commitment. We also observed P53 localization changed from mainly cytoplasmic in iPS colonies to the nucleus in the Nkx2-5 + cardiac progenitor stage. Pharmacological-mediated increase of P53 protein levels with the Mdm2 inhibitor Nutlin-3a during early (mesoderm to cardiac mesoderm) stages of cardiogenesis resulted in a sizeable loss of cardiomyocytes due to increased apoptosis and cell cycle arrest. Interestingly, increasing P53 levels did not result in apoptosis at later (cardiac progenitor to beating cardiomyocytes) stages of the cardiac differentiation. These results illustrate the temporal sensitivity to increased P53 levels during cardiogenesis. We conducted RNA-Seq on these cells with or without Nutlin-3a to ascertain transcriptional differences due to increased P53 at the different stages during the differentiation. Our results from the RNA-Seq revealed up-regulation of Sestrins after Nutlin-3a treatment suggesting a new role for P53 in the metabolism of cardiac regeneration.

Ventromedial Prefrontal-Anterior Cingulate Hyperconnectivity and Resilience to Apathy in Traumatic Brain Injury.

Apathy is a common and impairing sequela of traumatic brain injury (TBI). Yet, little is known about the neural mechanisms determining in which patients apathy does or does not develop post-TBI. We aimed to elucidate the impact of TBI on motivational neural circuits and how this shapes apathy over the course of TBI recovery. Resting-state functional magnetic resonance imaging data were collected in patients with subacute mild TBI (n = 44), chronic mild-to-moderate TBI (n = 26), and nonbrain-injured control participants (CTRL; n = 28). We measured ventromedial prefrontal cortex (vmPFC) functional connectivity (FC) as a function of apathy, using an a priori vmPFC seed adopted from a motivated decision-making study in an independent TBI study cohort. Patients reported apathy using a well-validated tool for assaying apathy in TBI. The vmPFC-to-wholebrain FC was contrasted between groups, and we fit regression models with apathy predicting vmPFC FC. Subacute and chronic TBI caused increased apathy relative to CTRL, replicating previous work suggesting that apathy has an enduring impact in TBI. The vmPFC was functionally connected to the canonical default network, and this architecture did not differ between subacute TBI, chronic TBI, and CTRL groups. Critically, in TBI, increased apathy scores predicted decreased vmPFC-dorsal anterior cingulate cortex (dACC) FC. Last, we subdivided the TBI group based on patients above versus below the threshold for "clinically significant apathy," finding that TBI patients with clinically significant apathy demonstrated comparable vmPFC-dACC FC to CTRLs, whereas TBI patients with subthreshold apathy scores demonstrated vmPFC-dACC hyperconnectivity relative to both CTRLs and patients with clinically significant apathy. Post-TBI vmPFC-dACC hyperconnectivity may represent an adaptive compensatory response, helping to maintain motivation and enabling resilience to the development of apathy after neurotrauma. Given the role of vmPFC-dACC circuits in value-based decision making, rehabilitation strategies designed to improve this ability may help to reduce apathy and improve functional outcomes in TBI.

Joint analysis of frontal theta synchrony and white matter following mild traumatic brain injury.

Some of the most disabling aspects of mild traumatic brain injury (mTBI) include lingering deficits in executive functioning. It is known that mTBI can damage white matter tracts, but it remains unknown how this structural brain damage translates into cognitive deficits. This experiment utilized theta band phase synchrony to identify the dysfunctional neural operations that contribute to cognitive problems following mTBI. Sub-acute stage (< 2 weeks) mTBI patients (N = 52) and healthy matched controls (N = 32) completed a control-demanding task with concurrent EEG. Structural MRI was also collected. While there were no performance-specific behavioral differences between groups in the dot probe expectancy task, the degree of theta band phase synchrony immediately following injury predicted the degree of symptom recovery two months later. Although there were no differences in fractional anisotropy (FA) between groups, joint independent components analysis revealed that a smaller network of lower FA-valued voxels contributed to a diminished frontal theta phase synchrony network in the mTBI group. This finding suggests that frontal theta band markers of cognitive control are sensitive to sub-threshold structural aberrations following mTBI.

Cerebral Perfusion Effects of Cognitive Training and Transcranial Direct Current Stimulation in Mild-Moderate TBI.

Background: Persistent post-traumatic symptoms (PPS) after traumatic brain injury (TBI) can lead to significant chronic functional impairment. Pseudocontinuous arterial spin labeling (pCASL) has been used in multiple studies to explore changes in cerebral blood flow (CBF) that may result in acute and chronic TBI, and is a promising neuroimaging modality for assessing response to therapies. Methods: Twenty-four subjects with chronic mild-moderate TBI (mmTBI) were enrolled in a pilot study of 10 days of computerized executive function training combined with active or sham anodal transcranial direct current stimulation (tDCS) for treatment of cognitive PPS. Behavioral surveys, neuropsychological testing, and magnetic resonance imaging (MRI) with pCASL sequences to assess global and regional CBF were obtained before and after the training protocol. Results: Robust improvements in depression, anxiety, complex attention, and executive function were seen in both active and sham groups between the baseline and post-treatment visits. Global CBF decreased over time, with differences in regional CBF noted in the right inferior frontal gyrus (IFG). Active stimulation was associated with static or increased CBF in the right IFG, whereas sham was associated with reduced CBF. Neuropsychological performance and behavioral symptoms were not associated with changes in CBF. Discussion: The current study suggests a complex picture between mmTBI, cerebral perfusion, and recovery. Changes in CBF may result from physiologic effect of the intervention, compensatory neural mechanisms, or confounding factors. Limitations include a small sample size and heterogenous injury sample, but these findings suggest promising directions for future studies of cognitive training paradigms in mmTBI.

Stimulating ideas for heart regeneration: the future of nerve-directed heart therapy.

Ischemic heart disease is the leading cause of death worldwide. The blockade of coronary arteries limits oxygen-rich blood to the heart and consequently there is cardiomyocyte (CM) cell death, inflammation, fibrotic scarring, and myocardial remodeling. Unfortunately, current therapeutics fail to effectively replace the lost cardiomyocytes or prevent fibrotic scarring, which results in reduced cardiac function and the development of heart failure (HF) in the adult mammalian heart. In contrast, neonatal mice are capable of regenerating their hearts following injury. However, this regenerative response is restricted to the first week of post-natal development. Recently, we identified that cholinergic nerve signaling is necessary for the neonatal mouse cardiac regenerative response. This demonstrates that cholinergic nerve stimulation holds significant potential as a bioelectronic therapeutic tool for heart disease. However, the mechanisms of nerve directed regeneration in the heart remain undetermined. In this review, we will describe the historical evidence of nerve function during regeneration across species. Specifically, we will focus on the emerging role of cholinergic innervation in modulating cardiomyocyte proliferation and inflammation during heart regeneration. Understanding the role of nerves in mammalian heart regeneration and adult cardiac remodeling can provide us with innovative bioelectronic-based therapeutic approaches for treatment of human heart disease.

Single-Cell RNA-Sequencing and Optical Electrophysiology of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Discordance Between Cardiac Subtype-Associated Gene Expression Patterns and Electrophysiological Phenotypes.

The ability to accurately phenotype cells differentiated from human induced pluripotent stem cells (hiPSCs) is essential for their application in modeling developmental and disease processes, yet also poses a particular challenge without the context of anatomical location. Our specific objective was to determine if single-cell gene expression was sufficient to predict the electrophysiology of iPSC-derived cardiac lineages, to evaluate the concordance between molecular and functional surrogate markers. To this end, we used the genetically encoded voltage indicator ArcLight to profile hundreds of hiPSC-derived cardiomyocytes (hiPSC-CMs), thus identifying patterns of electrophysiological maturation and increased prevalence of cells with atrial-like action potentials (APs) between days 11 and 42 of differentiation. To profile expression patterns of cardiomyocyte subtype-associated genes, single-cell RNA-seq was performed at days 12 and 40 after the populations were fully characterized with the high-throughput ArcLight platform. Although we could detect global gene expression changes supporting progressive differentiation, individual cellular expression patterns alone were not able to delineate the individual cardiomyocytes into atrial, ventricular, or nodal subtypes as functionally documented by electrophysiology measurements. Furthermore, our efforts to understand the distinct electrophysiological properties associated with day 12 versus day 40 hiPSC-CMs revealed that ion channel regulators SLMAP, FGF12, and FHL1 were the most significantly increased genes at day 40, categorized by electrophysiology-related gene functions. Notably, FHL1 knockdown during differentiation was sufficient to significantly modulate APs toward ventricular-like electrophysiology. Thus, our results establish the inability of subtype-associated gene expression patterns to specifically categorize hiPSC-derived cells according to their functional electrophysiology, and yet, altered FHL1 expression is able to redirect electrophysiological maturation of these developing cells. Therefore, noncanonical gene expression patterns of cardiac maturation may be sufficient to direct functional maturation of cardiomyocytes, with canonical gene expression patterns being insufficient to temporally define cardiac subtypes of in vitro differentiation.

Quantification of Etoposide Hypersensitivity: A Sensitive, Functional Method for Assessing Pluripotent Stem Cell Quality.

Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells' documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells. Stem Cells Translational Medicine 2017;6:1829-1839.