Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants. This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed. No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Exploring Adaptive Phenotypes for the Human Calcium-Sensing Receptor Polymorphism R990G.

Rainforest hunter-gatherers from Southeast Asia are characterized by specific morphological features including a particularly dark skin color (D), short stature (S), woolly hair (W), and the presence of steatopygia (S)-fat accumulation localized in the hips (DSWS phenotype). Based on previous evidence in the Andamanese population, we first characterized signatures of adaptive natural selection around the calcium-sensing receptor gene in Southeast Asian rainforest groups presenting the DSWS phenotype and identified the R990G substitution (rs1042636) as a putative adaptive variant for experimental follow-up. Although the calcium-sensing receptor has a critical role in calcium homeostasis by directly regulating the parathyroid hormone secretion, it is expressed in different tissues and has been described to be involved in many biological functions. Previous works have also characterized the R990G substitution as an activating polymorphism of the calcium-sensing receptor associated with hypocalcemia. Therefore, we generated a knock-in mouse for this substitution and investigated organismal phenotypes that could have become adaptive in rainforest hunter-gatherers from Southeast Asia. Interestingly, we found that mouse homozygous for the derived allele show not only lower serum calcium concentration but also greater body weight and fat accumulation, probably because of enhanced preadipocyte differentiation and lipolysis impairment resulting from the calcium-sensing receptor activation mediated by R990G. We speculate that such differential features in humans could have facilitated the survival of hunter-gatherer groups during periods of nutritional stress in the challenging conditions of the Southeast Asian tropical rainforests.

Understanding the neurological implications of acute and long COVID using brain organoids.

As early as in the acute phase of the coronavirus disease 2019 (COVID-19) pandemic, the research community voiced concerns about the long-term implications of infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like many other viruses, can trigger chronic disorders that last months or even years. Long COVID, the chronic and persistent disorder lasting more than 12 weeks after the primary infection with SARS-CoV-2, involves a variable number of neurological manifestations, ranging from mild to severe and even fatal. In vitro and in vivo modeling suggest that SARS-CoV-2 infection drives changes within neurons, glia and the brain vasculature. In this Review, we summarize the current understanding of the neuropathology of acute and long COVID, with particular emphasis on the knowledge derived from brain organoid models. We highlight the advantages and main limitations of brain organoids, leveraging their human-derived origin, their similarity in cellular and tissue architecture to human tissues, and their potential to decipher the pathophysiology of long COVID.

Glycemic status and health-related quality of life (HRQOL) in populations at risk of diabetes in two Latin American cities.

PURPOSE: To estimate the health-related quality of life (HRQOL) according to glycemic status, and its relationship with sociodemographic and clinical factors in a population at risk of developing type 2 diabetes (T2D). METHODS: Cross-sectional study, using cluster sampling. Data were collected from 1135 participants over 30 years of age, at risk of developing T2D from the PREDICOL project. Participants' glycemic status was defined using an oral glucose tolerance test (OGTT). Participants were divided into normoglycemic subjects (NGT), prediabetes and diabetics do not know they have diabetes (UT2D). HRQOL was assessed using the EQ-5D-3L questionnaire of the EuroQol group. Logistic regression and Tobit models were used to examine factors associated with EQ-5D scores for each glycemic group. RESULTS: The mean age of participants was 55.6 ± 12.1 years, 76.4% were female, and one in four participants had prediabetes or unknown diabetes. Participants reported problems most frequently on the dimensions of Pain/Discomfort and Anxiety/Depression in the different glycemic groups. The mean EQ-5D score in NGT was 0.80 (95% CI 0.79-0.81), in prediabetes, 0.81 (95% CI 0.79-0.83), and in participants with UT2D of 0.79 (95% CI 0.76-0.82), respectively. Female sex, older age, city of residence, lower education, receiving treatment for hypertension, and marital status were significantly associated with lower levels of HRQOL in the Tobit regression analysis. CONCLUSIONS: HRQOL of NGT, prediabetes, and UT2D participants was statistically similar. However, factors such as gender, age. and place of residence were found to be significant predictors of HRQOL for each glycemic group.

Risk of violence from a current or former partner: Associated factors and classification in a nationwide study in Colombia.

Intimate partner violence (IPV) includes assaults that risk a woman's bodily integrity. Intimate partners commit IPV, people with whom the victim shares (or shared) a close personal or sexual relationship. This phenomenon has a great global and national impact. Thus, it is necessary to establish trends of the risk of physical violence to women by their current or former partner in each department of Colombia and its relationship with sociodemographic and health characteristics. This study uses an ecological approach at the departmental level, with victims of intimate partner violence treated at the National Institute of Legal Medicine and Forensic Sciences (INMLyCF). Potential factors were identified through Bayesian factor analysis and were included in the model to estimate risk. The findings show that the Casanare department had the highest risk of producing victims (SMR: 2.545). In departments where the educational level of women is at or below primary school, there is a high-risk ß = 0.343 (0.285, 0.397) of them being assaulted. For the departments in which the employment of women is in sales and services or office workers, the associated factor presents a higher risk ß = 0.361 (0.201, 0.485), as in the risk related to affiliation with the social security system ß = 0.338 (0.246, 0.498), as well as sexual and reproductive life ß = 0.143 (0.003, 0.322). The following categories were associated with physical gender violence: no education and low participation in making purchases at home ß = 0.106 (0.049, 0.199), low participation in decisions about their health, and visits to family and friends ß = 0.240 (0.170, 0.299). Therefore, public health programs should strengthen women's empowerment in household decisions and increase their educational level to reduce this incidence.

Neumotórax espontáneo bilateral en primigestante adolescente, reporte de un caso / Spontaneous bilateral pneumothorax in a primigestant adolescent, a case report

El neumotórax espontáneo es una patología extremadamente rara durante la gestación. Se define como la presencia de aire dentro de la cavidad pleural que puede generar principalmente dolor torácico y disnea. Esta patología tiene unas bajas incidencia y prevalencia en el embarazo, pero es relevante por una alta tasa de recurrencia, con un buen pronóstico para la madre y el feto si es tempranamente diagnosticada y oportunamente manejada. Se relaciona con factores de riesgo como las maniobras de Valsalva efectuadas durante el trabajo de parto, además de con comorbilidad como el tabaquismo, y con el biotipo longilíneo, entre otros, por lo que son muy importantes una adecuada anamnesis y la evaluación de la exploración física. El obstetra debe sospecharlo ante la clínica de dolor torácico asociado a disnea en gestantes en el trabajo de parto y el parto, y tenerlo en cuenta como diagnóstico diferencial. Es de vital importancia tener un manejo multidisciplinario compuesto por ginecoobstetra, internista, neumólogo y neonatólogo, incluido el apoyo por una unidad de cuidado intensivo para evitar complicaciones materno-perinatales que se puedan asociar al neumotórax espontáneo.

Spontaneous pneumothorax is an extremely rare pathology during pregnancy. It is defined as the presence of air inside the pleural cavity that can mainly generate chest pain and dyspnea. This pathology has a low incidence and prevalence in pregnancy, but a high rate of recurrence with a good prognosis for the mother and the fetus if it is diagnosed early and managed early. It is related to risk factors such as Valsalva maneuvers performed during labor, in addition to comorbidities such as smoking, longilinear biotype, among others, so it is very important to have an adequate history and evaluation of the physical examination. The obstetrician must be attentive to chest pain symptoms associated with dyspnea in pregnant women during labor and delivery, suspect this pathology and take it into account as a differential diagnosis. It is vitally important to have a multidisciplinary management composed of the gynecologist-obstetrician, internist, pulmonologist, neonatologist, including the support of an intensive care unit to avoid maternal-perinatal complications that may be associated with spontaneous pneumothorax.

Health-related quality of life (HRQoL) in a population at risk of type 2 diabetes: a cross-sectional study in two Latin American cities.

PURPOSE: The purpose of this study was to describe the health-related quality of life (HRQoL) characteristics in a population at risk of developing type 2 diabetes in Barranquilla and Bogotá, Colombia. METHODS: A cross-sectional study with 1135 participants older than 30 years-of-age recruited in Bogotá D.C., and Barranquilla by cluster sampling in 2018 to 2019. The Finnish Diabetes Risk Score (FINDRISC) was used to detect participants at risk of developing type 2 diabetes (T2D). HRQoL was assessed using the EQ-5D-3L questionnaire. Unadjusted and adjusted logistic regression models were used to calculate odds ratios (OR) and their corresponding 95% confidence intervals CI). RESULTS: Moderate or extreme problems appeared more frequently in the dimensions of Pain/Discomfort (60.8%) and Anxiety/Depression (30.8%). The mean score of the EQ-VAS was 74.3 (± 17.3), significantly larger in the state of complete health (11111) compared with those with problems in more than one of the quality-of-life dimensions. Being female and living in Bogota D.C., were associated with greater odds of reporting problems in the Pain (OR 1.6; 95% CI 1.2-2.2) and Discomfort dimensions (OR 1.6; 95% CI 1.2-2.0) respectively and Anxiety/Depression (OR 1.9; 95% CI 1.3-2.7), (OR 9.1; 95% CI 6.6-12.4), respectively. CONCLUSIONS: As living place and sex were associated with dimensions of Pain/Discomfort and Anxiety/Depression in the HRQoL in people at risk of T2D, greater attention should be paid to these determinants of HRQoL to design and reorient strategies with a territorial and gender perspective to achieve better health outcomes. Diabetes is one of the four non-communicable diseases with increasing prevalence in the world, which has made it a serious public health problem. In Colombia, in 2019 diabetes affected 8.4% of the Colombian adult population and more than one million Colombian adults of this age group have hidden or undetected diabetes. This disease is not only characterized by increased premature mortality, loss of productivity, and economic impact, but it also involves a deterioration in the quality of life of people with diabetes with their respective families. However, very Little is known about health-related quality of life (HRQoL) in a population at risk or with prediabetes. This study has evaluated the quality of life in patients at risk of diabetes and their behavior with some variables as sociodemographic, lifestyle, history, and established their difference in two territories of the Colombian Caribbean. The results of this study indicate that the HRQoL of people at risk of type 2 diabetes is affected by factors such as gender, city, dysglycemia, medication for hypertension and education level. Therefore, greater attention should be paid to these determinants of HRQL to design and implement strategies that reduce this risk of developing type 2 diabetes, prevent prediabetes and improve the quality of life in prediabetic or diabetic patients.

Repeated Suicide Attempts Among Service Users of An Emergency Service in Northern Colombia: Characteristics, Associated Factors, And Management / Intento de Suicidio Repetido en Usuarios de un Servicio de Emergencias En Colombia Del Norte: Características, Factores Asociados y Manejo

Abstract Suicide has increased close to 60% in the last four decades worldwide. In Colombia, during the year 2019, 10,9% of violent deaths were due to suicide. This study aimed to identify risk factors predicting repeated suicide attempts. It also aimed to describe the management of suicidal behaviour within an emergency department of Northern Colombia. Dataset comprised 336 medical records of individuals seeking medical assistance for intentional self-harm between 2008-2019; 136 medical records were associated with previously reported suicide attempts. Results from a multivariate logistic regression showed that suicide ideation and having a history of psychiatric disorders significantly predicted repeated suicide attempts. Furthermore, repeated attempts were more likely in underaged individuals and young adults. Management of patients engaging in suicidal behaviour involved hospitalization and outpatient mental health services. However, a few patients were sent home with recommendations or were non-compliant. Findings from this study highlight the importance to develop evidence-based screening and monitoring protocols that prevent repeated suicide attempts.

Resumen El suicidio ha aumentado en cerca de un 60% en los últimos cuarenta años. En Colombia, para el año 2019, el 10.9% de las muertes violentas se presentaron por suicidio. Este estudio busca identificar los factores de riesgo asociados con los intentos repetidos de suicidio y además realiza un análisis descriptivo del manejo dado a estos pacientes. Esta investigación utiliza la base de datos de la unidad de emergencias en un hospital del norte de Colombia, y se centra en los pacientes que acudieron al hospital por intento de suicidio entre enero de 2008 y junio de 2019. La base de datos presenta 336 casos de los cuales el 81% corresponden a intentos suicidas y 19% a gestos. Resultados de un análisis de regresión logística multivariada mostraron que la ideación suicida y la historia de desorden psiquiátrico predecían significativamente los intentos suicidas repetidos y que los intentos repetidos eran más probables en el grupo de individuos menores de edad y en adultez temprana. El tratamiento administrado a los pacientes fue hospitalización y servicios ambulatorios; un porcentaje fue enviado a casa con recomendaciones. Los hallazgos de este estudio destacan la importancia del desarrollo de protocolos estandarizados basados en evidencia para prevenir los intentos repetidos.

Chromosome X-wide Analysis of Positive Selection in Human Populations: Common and Private Signals of Selection and its Impact on Inactivated Genes and Enhancers.

The ability of detecting adaptive (positive) selection in the genome has opened the possibility of understanding the genetic basis of population-specific adaptations genome-wide. Here, we present the analysis of recent selective sweeps, specifically in the X chromosome, in human populations from the third phase of the 1,000 Genomes Project using three different haplotype-based statistics. We describe instances of recent positive selection that fit the criteria of hard or soft sweeps, and detect a higher number of events among sub-Saharan Africans than non-Africans (Europe and East Asia). A global enrichment of neural-related processes is observed and numerous genes related to fertility appear among the top candidates, reflecting the importance of reproduction in human evolution. Commonalities with previously reported genes under positive selection are found, while particularly strong new signals are reported in specific populations or shared across different continental groups. We report an enrichment of signals in genes that escape X chromosome inactivation, which may contribute to the differentiation between sexes. We also provide evidence of a widespread presence of soft-sweep-like signatures across the chromosome and a global enrichment of highly scoring regions that overlap potential regulatory elements. Among these, enhancers-like signatures seem to present putative signals of positive selection which might be in concordance with selection in their target genes. Also, particularly strong signals appear in regulatory regions that show differential activities, which might point to population-specific regulatory adaptations.

Comparative Analysis of Mammal Genomes Unveils Key Genomic Variability for Human Life Span.

The enormous mammal's lifespan variation is the result of each species' adaptations to their own biological trade-offs and ecological conditions. Comparative genomics have demonstrated that genomic factors underlying both, species lifespans and longevity of individuals, are in part shared across the tree of life. Here, we compared protein-coding regions across the mammalian phylogeny to detect individual amino acid (AA) changes shared by the most long-lived mammals and genes whose rates of protein evolution correlate with longevity. We discovered a total of 2,737 AA in 2,004 genes that distinguish long- and short-lived mammals, significantly more than expected by chance (P = 0.003). These genes belong to pathways involved in regulating lifespan, such as inflammatory response and hemostasis. Among them, a total 1,157 AA showed a significant association with maximum lifespan in a phylogenetic test. Interestingly, most of the detected AA positions do not vary in extant human populations (81.2%) or have allele frequencies below 1% (99.78%). Consequently, almost none of these putatively important variants could have been detected by genome-wide association studies. Additionally, we identified four more genes whose rate of protein evolution correlated with longevity in mammals. Crucially, SNPs located in the detected genes explain a larger fraction of human lifespan heritability than expected, successfully demonstrating for the first time that comparative genomics can be used to enhance interpretation of human genome-wide association studies. Finally, we show that the human longevity-associated proteins are significantly more stable than the orthologous proteins from short-lived mammals, strongly suggesting that general protein stability is linked to increased lifespan.

Enhancers with tissue-specific activity are enriched in intronic regions.

Tissue function and homeostasis reflect the gene expression signature by which the combination of ubiquitous and tissue-specific genes contribute to the tissue maintenance and stimuli-responsive function. Enhancers are central to control this tissue-specific gene expression pattern. Here, we explore the correlation between the genomic location of enhancers and their role in tissue-specific gene expression. We find that enhancers showing tissue-specific activity are highly enriched in intronic regions and regulate the expression of genes involved in tissue-specific functions, whereas housekeeping genes are more often controlled by intergenic enhancers, common to many tissues. Notably, an intergenic-to-intronic active enhancers continuum is observed in the transition from developmental to adult stages: the most differentiated tissues present higher rates of intronic enhancers, whereas the lowest rates are observed in embryonic stem cells. Altogether, our results suggest that the genomic location of active enhancers is key for the tissue-specific control of gene expression.

Adaptive selection drives TRPP3 loss-of-function in an Ethiopian population.

TRPP3 (also called PKD2L1) is a nonselective, cation-permeable channel activated by multiple stimuli, including extracellular pH changes. TRPP3 had been considered a candidate for sour sensor in humans, due to its high expression in a subset of tongue receptor cells detecting sour, along with its membership to the TRP channel family known to function as sensory receptors. Here, we describe the functional consequences of two non-synonymous genetic variants (R278Q and R378W) found to be under strong positive selection in an Ethiopian population, the Gumuz. Electrophysiological studies and 3D modelling reveal TRPP3 loss-of-functions produced by both substitutions. R278Q impairs TRPP3 activation after alkalinisation by mislocation of H+ binding residues at the extracellular polycystin mucolipin domain. R378W dramatically reduces channel activity by altering conformation of the voltage sensor domain and hampering channel transition from closed to open state. Sour sensitivity tests in R278Q/R378W carriers argue against both any involvement of TRPP3 in sour detection and the role of such physiological process in the reported evolutionary positive selection past event.

Optic vesicle morphogenesis requires primary cilia.

Arl13b is a gene known to regulate ciliogenesis. Functional alterations in this gene's activity have been associated with Joubert syndrome. We found that in Arl13 null mouse embryos the orientation of the optic cup is inverted, such that the lens is abnormally surrounded by an inverted optic cup whose retina pigmented epithelium is oddly facing the surface ectoderm. Loss of Arl13b leads to the disruption of optic vesicle's patterning and expansion of ventral fates. We show that this phenotype is consequence of miss-regulation of Sonic hedgehog (Shh) signaling and demonstrate that the Arl13b-/- eye phenotype can be rescued by deletion of Gli2, a downstream effector of the Shh pathway. This work identified an unexpected role of primary cilia during the morphogenetic movements required for the formation of the eye.

A Gutsy Move for Cell-Based Regenerative Medicine in Parkinson's Disease: Targeting the Gut Microbiome to Sequester Inflammation and Neurotoxicity.

Pharmaceuticals and cell-based regenerative medicine for Parkinson's disease (PD) offer palliative relief but do not arrest the disease progression. Cell therapy has emerged as an experimental treatment, but current cell sources such as human umbilical cord blood (hUCB) stem cells display only partial recapitulation of mature dopaminergic neuron phenotype and function. Nonetheless, stem cell grafts ameliorate PD-associated histological and behavioral deficits likely through stem cell graft-secreted therapeutic substances. We recently demonstrated the potential of hUCB-derived plasma in enhancing motor capabilities and gastrointestinal function, as well as preventing dopaminergic neuronal cell loss, in an 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) rodent model of PD. Recognizing the translational need to test in another PD model, we now examined here the effects of an intravenously transplanted combination of hUCB and plasma into the 6-hydroxydopamine (6-OHDA) lesioned adult rats. Animals received three separate doses of 4 × 106 hUCB cells with plasma beginning at 7 days after stereotaxic 6-OHDA lesion, then behaviorally and immunohistochemically evaluated over 56 days post-lesion. Whereas vehicle-treated lesioned animals exhibited the typical 6-OHDA neurobehavioral symptoms, hUCB and plasma-treated lesioned animals showed significant attenuation of motor function, gut motility, and nigral dopaminergic neuronal survival, combined with diminished pro-inflammatory microbiomes not only in the nigra, but also in the gut. Altogether these data support a regenerative medicine approach for PD by sequestering inflammation and neurotoxicity through correction of gut dysbiosis.

Human parthenogenetic neural stem cell grafts promote multiple regenerative processes in a traumatic brain injury model.

International Stem Cell Corporation human parthenogenetic neural stem cells (ISC-hpNSC) have potential therapeutic value for patients suffering from traumatic brain injury (TBI). Here, we demonstrate the behavioral and histological effects of transplanting ISC-hpNSC intracerebrally in an animal model of TBI. Methods: Sprague-Dawley rats underwent a moderate controlled cortical impact TBI surgery. Transplantation occurred at 72 h post-TBI with functional readouts of behavioral and histological deficits conducted during the subsequent 3-month period after TBI. We characterized locomotor, neurological, and cognitive performance at baseline (before TBI), then on days 0, 1, 7, 14, 30, 60, and 90 (locomotor and neurological), and on days 28-30, 58-60, and 88-90 (cognitive) after TBI. Following completion of behavioral testing at 3 months post-TBI, animals were euthanized by transcardial perfusion and brains harvested to histologically characterize the extent of brain damage. Neuronal survival was revealed by Nissl staining, and stem cell engraftment and host tissue repair mechanisms such as the anti-inflammatory response in peri-TBI lesion areas were examined by immunohistochemical analyses. Results: We observed that TBI groups given high and moderate doses of ISC-hpNSC had an improved swing bias on an elevated body swing test for motor function, increased scores on forelimb akinesia and paw grasp neurological tests, and committed significantly fewer errors on a radial arm water maze test for cognition. Furthermore, histological analyses indicated that high and moderate doses of stem cells increased the expression of phenotypic markers related to the neural lineage and myelination and decreased reactive gliosis and inflammation in the brain, increased neuronal survival in the peri-impact area of the cortex, and decreased inflammation in the spleen at 90 days post-TBI. Conclusion: These results provide evidence that high and moderate doses of ISC-hpNSC ameliorate TBI-associated histological alterations and motor, neurological, and cognitive deficits.

Endothelial Progenitor Cells Modulate Inflammation-Associated Stroke Vasculome.

Stroke remains a major unmet clinical need that warrants novel therapies. Following an ischemic insult, the cerebral vasculature secretes inflammatory molecules, creating the stroke vasculome profile. The present study evaluated the therapeutic effects of endothelial cells on the inflammation-associated stroke vasculome. qRT-PCR analysis revealed that specific inflammation-related vasculome genes BRM, IκB, Foxf1, and ITIH-5 significantly upregulated by oxygen glucose deprivation (OGD. Interestingly, co-culture of human endothelial cells (HEN6) with human endothelial cells (EPCs) during OGD significantly blocked the elevations of BRM, IκB, and Foxf1, but not ITIH-5. Next, employing the knockdown/antisense technology, silencing the inflammation-associated stroke vasculome gene, IκB, as opposed to scrambled knockdown, blocked the EPC-mediated protection of HEN6 against OGD. In vivo, stroke animals transplanted with intracerebral human EPCs (300,000 cells) into the striatum and cortex 4 h post ischemic stroke displayed significant behavioral recovery up to 30 days post-transplantation compared to vehicle-treated stroke animals. At 7 days post-transplantation, quantification of the fluorescent staining intensity in the cortex and striatum revealed significant upregulation of the endothelial marker RECA1 and a downregulation of the stroke-associated vasculome BRM, IKB, Foxf1, ITIH-5 and PMCA2 in the ipsilateral side of cortex and striatum of EPC-transplanted stroke animals relative to vehicle-treated stroke animals. Altogether, these results demonstrate that EPCs exert therapeutic effects in experimental stroke possibly by modulating the inflammation-plagued vasculome.

Regulatory T-cells within bone marrow-derived stem cells actively confer immunomodulatory and neuroprotective effects against stroke.

Regulatory T-cells (Tregs) may exert a neuroprotective effect on ischemic stroke by inhibiting both inflammation and effector T-cell activation. Transplantation of human bone marrow-derived stem cells (BMSCs) in ischemic stroke affords neuroprotection that results in part from the cells' anti-inflammatory property. However, the relationship between Tregs and BMSCs in treatment of ischemic stroke has not been fully elucidated. Here, we tested the hypothesis that Tregs within the BMSCs represent active mediators of immunomodulation and neuroprotection in experimental stroke. Primary rat neuronal cells were subjected to an oxygen-glucose deprivation and reperfusion (OGD/R) condition. The cells were re-perfused and co-cultured with Tregs and/or BMSCs. We detected a minority population of Tregs within BMSCs with both immunocytochemistry (ICC) and flow cytometry identifying cells expressing phenotypic markers of CD4, CD25, and FoxP3 protein. BMSCs with the native population of Tregs conferred maximal neuroprotection compared to the treatment conditions containing 0%, 10%, and 100% relative ratio Tregs. Increasing the Treg population resulted in increased IL6 secretion and decreased FGF-ß secretion by BMSCs. This study shows that a minority population of Tregs exists within the therapeutic BMSC population, which serves as robust mediators of the immunomodulatory and neuroprotective effect provided by BMSC transplantation.

Chronic Upregulation of Cleaved-Caspase-3 Associated with Chronic Myelin Pathology and Microvascular Reorganization in the Thalamus after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is associated with long-term disabilities and devastating chronic neurological complications including problems with cognition, motor function, sensory processing, as well as behavioral deficits and mental health problems such as anxiety, depression, personality change and social unsuitability. Clinical data suggest that disruption of the thalamo-cortical system including anatomical and metabolic changes in the thalamus following TBI might be responsible for some chronic neurological deficits following brain trauma. Detailed mechanisms of these pathological processes are not completely understood. The goal of this study was to evaluate changes in the thalamus following TBI focusing on cleaved-caspase-3, a specific effector of caspase pathway activation and myelin and microvascular pathologies using immuno- and histochemistry at different time points from 24 h to 3 months after controlled cortical impact (CCI) in adult Sprague-Dawley rats. Significant increases in cleaved-caspase-3 immunoreactivity in the thalamus were observed starting one month and persisting for at least three months following experimental TBI. Further, the study demonstrated an association of cleaved-caspase-3 with the demyelination of neuronal processes and tissue degeneration in the gray matter in the thalamus, as reflected in alterations of myelinated fiber integrity (luxol fast blue) and decreases in myelin basic protein (MBP) immunoreactivity. The immunofluorescent counterstaining of cleaved-caspase-3 with endothelial barrier antigen (EBA), a marker of blood-brain barrier, revealed limited direct and indirect associations of cleaved caspase-3 with blood-brain barrier damage. These results demonstrate for the first time a significant chronic upregulation of cleaved-caspase-3 in selected thalamic regions associated with cortical regions directly affected by CCI injury. Further, our study is also the first to report that significant upregulation of cleaved-caspase-3 in selected ipsilateral thalamic regions is associated with microvascular reorganization reflected in the significant increases in the number of microvessels with blood-brain barrier alterations detected by EBA staining. These findings provide new insights into potential mechanisms of TBI cell death involving chronic activation of caspase-3 associated with disrupted cortico-thalamic and thalamo-cortical connectivity. Moreover, this study offers the initial evidence that this upregulation of activated caspase-3, delayed degeneration of myelinated nerve fibers and microvascular reorganization with impaired blood-brain barrier integrity in the thalamus might represent reciprocal pathological processes affecting neuronal networks and brain function at the chronic stages of TBI.