Ablation of the integrin CD11b mac-1 limits deleterious responses to traumatic spinal cord injury and improves functional recovery in mice.

Background: Spinal cord injury (SCI) causes long-term sensorimotor deficits and posttraumatic neuropathic pain, with no effective treatment. In part, this reflects an incomplete understanding of the complex secondary pathobiological mechanisms involved. SCI triggers microglial/macrophage activation with distinct pro-inflammatory or inflammation-resolving phenotypes, which potentiate tissue damage or facilitate functional repair, respectively. The major integrin Mac-1 (CD11b/CD18, αMß2 or CR3), a heterodimer consisting of αM (CD11b) and ß2 (CD18) chains, is generally regarded as a pro-inflammatory receptor in neurotrauma. Multiple immune cells of the myeloid lineage express CD11b, including microglia, macrophages, and neutrophils. In the present study, we examined the effects of CD11b gene ablation on posttraumatic neuroinflammation and functional outcomes after SCI. Methods: Young adult age-matched female CD11b knockout (KO) mice and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation in the injured spinal cord was assessed with qPCR, flow cytometry, NanoString, and RNAseq. Neurological function was evaluated with the Basso Mouse Scale (BMS), gait analysis, thermal hyperesthesia, and mechanical allodynia. Lesion volume was evaluated by GFAP-DAB immunohistochemistry, followed by analysis with unbiased stereology. Results: qPCR analysis showed a rapid and persistent upregulation of CD11b mRNA starting from 1d after injury, which persisted up to 28 days. At 1d post-injury, increased expression levels of genes that regulate inflammation-resolving processes were observed in CD11b KO mice. Flow cytometry analysis of CD45intLy6C-CX3CR1+ microglia, CD45hiLy6C+Ly6G- monocytes, and CD45hiLy6C+Ly6G+ neutrophils revealed significantly reduced cell counts as well as reactive oxygen production in CD11b KO mice at d3 post-injury. Further examination of the injured spinal cord with NanoString Mouse Neuroinflammation Panel and RNAseq showed upregulated expression of pro-inflammatory genes, but downregulated expression of the reactive oxygen species pathway. Importantly, CD11b KO mice exhibited significantly improved locomotor function, reduced cutaneous mechanical/thermal hypersensitivity, and limited tissue damage at 8 weeks post-injury. Conclusion: Collectively, our data suggest an important role for CD11b in regulating tissue inflammation and functional outcome following SCI. Thus, the integrin CD11b represents a potential target that may lead to novel therapeutic strategies for SCI.

Traumatic brain injury-induced inflammatory changes in the olfactory bulb disrupt neuronal networks leading to olfactory dysfunction.

Approximately 20-68% of traumatic brain injury (TBI) patients exhibit trauma-associated olfactory deficits (OD) which can compromise not only the quality of life but also cognitive and neuropsychiatric functions. However, few studies to date have examined the impact of experimental TBI on OD. The present study examined inflammation and neuronal dysfunction in the olfactory bulb (OB) and the underlying mechanisms associated with OD in male mice using a controlled cortical impact (CCI) model. TBI caused a rapid inflammatory response in the OB as early as 24 h post-injury, including elevated mRNA levels of proinflammatory cytokines, increased numbers of microglia and infiltrating myeloid cells, and increased IL1ß and IL6 production in these cells. These changes were sustained for up to 90 days after TBI. Moreover, we observed significant upregulation of the voltage-gated proton channel Hv1 and NOX2 expression levels, which were predominantly localized in microglia/macrophages and accompanied by increased reactive oxygen species production. In vivo OB neuronal firing activities showed early neuronal hyperexcitation and later hypo-neuronal activity in both glomerular layer and mitral cell layer after TBI, which were improved in the absence of Hv1. In a battery of olfactory behavioral tests, WT/TBI mice displayed significant OD. In contrast, neither Hv1 KO/TBI nor NOX2 KO/TBI mice showed robust OD. Finally, seven days of intranasal delivery of a NOX2 inhibitor (NOX2ds-tat) ameliorated post-traumatic OD. Collectively, these findings highlight the importance of OB neuronal networks and its role in TBI-mediated OD. Thus, targeting Hv1/NOX2 may be a potential intervention for improving post-traumatic anosmia.

Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration.

Lipofuscin is an autofluorescent (AF) pigment formed by lipids and misfolded proteins, which accumulates in postmitotic cells with advanced age. Here, we immunophenotyped microglia in the brain of old C57BL/6 mice (>18 months old) and demonstrate that in comparison to young mice, one-third of old microglia are AF, characterized by profound changes in lipid and iron content, phagocytic activity, and oxidative stress. Pharmacological depletion of microglia in old mice eliminated the AF microglia following repopulation and reversed microglial dysfunction. Age-related neurological deficits and neurodegeneration after traumatic brain injury (TBI) were attenuated in old mice lacking AF microglia. Furthermore, increased phagocytic activity, lysosomal burden, and lipid accumulation in microglia persisted for up to 1 year after TBI, were modified by APOE4 genotype, and chronically driven by phagocyte-mediated oxidative stress. Thus, AF may reflect a pathological state in aging microglia associated with increased phagocytosis of neurons and myelin and inflammatory neurodegeneration that can be further accelerated by TBI.

Antibody-Dependent Enhancement Activity of a Plant-Made Vaccine against West Nile Virus.

West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue (DENV) virus. Specifically, a plant-produced virus-like particle (VLP) that displays the WNV Envelope protein domain III (wDIII) elicited both high neutralizing antibody titers and antigen-specific cellular immune responses in mice. Passive transfer of serum from VLP-vaccinated mice protected recipient mice from a lethal challenge of WNV infection. Notably, VLP-induced antibodies did not enhance the infection of Fc gamma receptor-expressing K562 cells by ZIKV or DENV through ADE. Thus, a plant-made wDIII-displaying VLP presents a promising WNV vaccine candidate that induces protective immunity and minimizes the concern of inducing ADE-prone antibodies to predispose vaccinees to severe infection by DENV or ZIKV.

Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice.

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice. Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1+/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI. Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1+/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1+/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1+/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage. Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI.

Elevated soluble death receptor 5 can predict poor prognosis in patients with acute respiratory distress syndrome.

BACKGROUND: The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 5 (DR5), participate in pulmonary cell apoptosis. This study aimed to investigate the clinical value of soluble DR5 and TRAIL for prognosis assessment in acute respiratory distress syndrome (ARDS). RESEARCH DESIGN AND METHODS: Serum and bronchoalveolar lavage fluid (BALF) samples were collected from ARDS patients and controls. Patients were followed-up until death or discharge. Soluble DR5, TRAIL, TNF-α, soluble receptor for advanced glycation end-products (sRAGE), and albumin levels were measured using the Magnetic Luminex or enzyme-linked immunosorbent assays. Data were analyzed according to their distributions and statistical purposes. RESULTS: Serum and BALF DR5 levels were elevated in patients with ARDS; TRAIL elevation and reduction was observed in BALF and serum, respectively. Serum DR5 was higher in non-survivors compared to survivors. Serum DR5 was positively correlated with serum TNF-α and critical illness scores and negatively correlated with serum TRAIL. Serum DR5 exhibited potential for predicting mortality in patients with ARDS. CONCLUSIONS: Serum soluble DR5 elevation, a valuable prognosis predictor in ARDS, may be associated with alveolar epithelial cell apoptosis. TRIAL REGISTRATION: http://www.chictr.org.cn/index.aspx.Uniqueidentifier:ChiCTR-DDD-17013370.

Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice.

Elderly patients with traumatic brain injury (TBI) have greater mortality and poorer outcomes than younger individuals. The extent to which old age alters long-term recovery and chronic microglial activation after TBI is unknown, and evidence for therapeutic efficacy in aged mice is sorely lacking. The present study sought to identify potential inflammatory mechanisms underlying age-related outcomes late after TBI. Controlled cortical impact was used to induce moderate TBI in young and old male C57BL/6 mice. At 12 weeks post-injury, aged mice exhibited higher mortality, poorer functional outcomes, larger lesion volumes, and increased microglial activation. Transcriptomic analysis identified age- and TBI-specific gene changes consistent with a disease-associated microglial signature in the chronically injured brain, including those involved with complement, phagocytosis, and autophagy pathways. Dysregulation of phagocytic and autophagic function in microglia was accompanied by increased neuroinflammation in old mice. As proof-of-principle that these pathways have functional importance, we administered an autophagic enhancer, trehalose, in drinking water continuously for 8 weeks after TBI. Old mice treated with trehalose showed enhanced functional recovery and reduced microglial activation late after TBI compared to the sucrose control group. Our data indicate that microglia undergo chronic changes in autophagic regulation with both normal aging and TBI that are associated with poorer functional outcome. Enhancing autophagy may therefore be a promising clinical therapeutic strategy for TBI, especially in older patients.

Brain innate immune response via miRNA-TLR7 sensing in polymicrobial sepsis.

Sepsis-associated encephalopathy (SAE) occurs in sepsis survivors and is associated with breakdown of the blood-brain barrier (BBB), brain inflammation, and neurological dysfunction. We have previously identified a group of extracellular microRNAs (ex-miRNAs), such as miR-146a-5p, that were upregulated in the plasma of septic mice and human, and capable of inducing potent pro-inflammatory cytokines and complements. Here, we established a clinically relevant mouse model of SAE and investigated the role of extracellular miRNAs and their sensor Toll-like receptor 7 (TLR7) in brain inflammation and neurological dysfunction. We observed BBB disruption and a profound neuroinflammatory responses in the brain for up to 14 days post-sepsis; these included increased pro-inflammatory cytokines production, microglial expansion, and peripheral leukocyte accumulation in the CNS. In a battery of neurobehavioral tests, septic mice displayed impairment of motor coordination and neurological function. Sepsis significantly increased plasma RNA and miRNA levels for up to 7 days, such as miR-146a-5p. Exogenously added miR-146a-5p induces innate immune responses in both cultured microglia/astrocytes and the intact brain via a TLR7-dependent manner. Moreover, mice genetically deficient of miR-146a showed reduced accumulation of monocytes and neutrophils in the brain compared to WT after sepsis. Finally, ablation of TLR7 in the TLR7-/- mice preserved BBB integrity, reduced microglial expansion and leukocyte accumulation, and attenuated GSK3ß signaling in the brain, but did not improve neurobehavioral recovery following sepsis. Taken together, these data establish an important role of extracellular miRNA and TLR7 sensing in sepsis-induced brain inflammation.

Sexual dimorphism in neurological function after SCI is associated with disrupted neuroinflammation in both injured spinal cord and brain.

Whereas human spinal cord injury (SCI) is more common in men, the prevalence is growing in women. However, little is known about the effect of biological sex on brain dysfunction and injury mechanisms. To model the highest per capita rate of injury (ages between 16 and 30 years old) in humans, in the present study, young adult or a young/middle-aged male and female C57BL/6 mice were subjected to moderate contusion SCI. When mice were injured at 10-12-week-old, transcriptomic analysis of inflammation-related genes and flow cytometry revealed a more aggressive neuroinflammatory profile in male than females following 3 d SCI, ostensibly driven by sex-specific changes myeloid cell function rather than cell number. Female mice were generally more active at baseline, as evidenced by greater distance traveled in the open field. After SCI, female mice had more favorable locomotor function than male animals. At 13 weeks post-injury, male mice showed poor performance in cognitive and depressive-like behavioral tests, while injured female mice showed fewer deficits in these tasks. However, when injured at 6 months old followed by 8 months post-injury, male mice had considerably less inflammatory activation compared with female animals despite having similar or worse outcomes in affective, cognitive, and motor tasks. Collectively, these findings indicate that sex differences in functional outcome after SCI are associated with the age at onset of injury, as well as disrupted neuroinflammation not only at the site of injury but also in remote brain regions. Thus, biological sex should be considered when designing new therapeutic agents.

Functions and Mechanisms of the Voltage-Gated Proton Channel Hv1 in Brain and Spinal Cord Injury.

The voltage-gated proton channel Hv1 is a newly discovered ion channel that is highly conserved among species. It is known that Hv1 is not only expressed in peripheral immune cells but also one of the major ion channels expressed in tissue-resident microglia of the central nervous systems (CNS). One key role for Hv1 is its interaction with NADPH oxidase 2 (NOX2) to regulate reactive oxygen species (ROS) and cytosolic pH. Emerging data suggest that excessive ROS production increases and requires proton currents through Hv1 in the injured CNS, and manipulations that ablate Hv1 expression or induce loss of function may provide neuroprotection in CNS injury models including stroke, traumatic brain injury, and spinal cord injury. Recent data demonstrating microglial Hv1-mediated signaling in the pathophysiology of the CNS injury further supports the idea that Hv1 channel may function as a key mechanism in posttraumatic neuroinflammation and neurodegeneration. In this review, we summarize the main findings of Hv1, including its expression pattern, cellular mechanism, role in aging, and animal models of CNS injury and disease pathology. We also discuss the potential of Hv1 as a therapeutic target for CNS injury.

Effects of influenza vaccination on clinical outcomes of chronic obstructive pulmonary disease: A systematic review and meta-analysis.

PURPOSE: Influenza is a threat to patients with chronic obstructive pulmonary disease (COPD), influenza vaccination help to reduce incidence of influenza infection, however, whether it is beneficial to COPD patients in clinical outcomes lacks for evidence due to limited studies and participations. METHODS: We searched PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure (CNKI) and China Science and Technology Journal Database (CSTJ) to retrieve eligible studies regardless of study design published before August 2020, and conducted meta-analysis with odds ratio (OR) and mean difference (MD). The quality of included studies and pooled evidences were assessed. Narrative summaries were provided where data were insufficient for meta-analysis. RESULTS: 2831 COPD patients were included, the pooled results showed that influenza vaccination reduced the exacerbations (P = 0.0001) and trends of hospitalizations (P = 0.09) in COPD patients. Further subgroup analysis showed that the reduction of exacerbations and hospitalizations were significant in patients with FEV1<50 % predicted (P = 0.01 and P < 0.0001 respectively), but not in those with FEV1≥50 % predicted (P = 0.23 and P = 0.76 respectively). No significant effect of influenza vaccination on all-cause mortality was observed. CONCLUSIONS: Our findings support a protective role of influenza vaccination in COPD patients, a yearly influenza vaccination should be strongly recommended for all COPD patients, especially those with severe airflow limitation, to reduce possible influenza infection, and thus associated exacerbations and hospitalizations.

Plant-Produced Antigen Displaying Virus-Like Particles Evokes Potent Antibody Responses against West Nile Virus in Mice.

In this study, we developed a hepatitis B core antigen (HBcAg)-based virus-like particle (VLP) that displays the West Nile virus (WNV) Envelope protein domain III (wDIII) as a vaccine candidate for WNV. The HBcAg-wDIII fusion protein was quickly produced in Nicotiana benthamiana plants and reached a high expression level of approximately 1.2 mg of fusion protein per gram of leaf fresh weight within six days post gene infiltration. Electron microscopy and gradient centrifugation analysis indicated that the introduction of wDIII did not interfere with VLP formation and HBcAg-wDIII successfully assembled into VLPs. HBcAg-wDIII VLPs can be easily purified in large quantities from Nicotiana benthamiana leaves to >95% homogeneity. Further analysis revealed that the wDIII was displayed properly and demonstrated specific binding to an anti-wDIII monoclonal antibody that recognizes a conformational epitope of wDIII. Notably, HBcAg-wDIII VLPs were shown to be highly immunogenic and elicited potent humoral responses in mice with antigen-specific IgG titers equivalent to that of protective wDIII antigens in previous studies. Thus, our wDIII-based VLP vaccine offers an attractive option for developing effective, safe, and low-cost vaccines against WNV.

Spinal cord injury alters microRNA and CD81+ exosome levels in plasma extracellular nanoparticles with neuroinflammatory potential.

Extracellular vesicles (EVs) have been implicated mechanistically in the pathobiology of neurodegenerative disorders, including central nervous system injury. However, the role of EVs in spinal cord injury (SCI) has received limited attention to date. Moreover, technical limitations related to EV isolation and characterization methods can lead to misleading or contradictory findings. Here, we examined changes in plasma EVs after mouse SCI at multiple timepoints (1d, 3d, 7d, 14d) using complementary measurement techniques. Plasma EVs isolated by ultracentrifugation (UC) were decreased at 1d post-injury, as shown by nanoparticle tracking analysis (NTA), and paralleled an overall reduction in total plasma extracellular nanoparticles. Western blot (WB) analysis of UC-derived plasma EVs revealed increased expression of the tetraspanin exosome marker, CD81, between 1d and 7d post-injury. To substantiate these findings, we performed interferometric and fluorescence imaging of single, tetraspanin EVs captured directly from plasma with ExoView®. Consistent with WB, we observed significantly increased plasma CD81+ EV count and cargo at 1d post-injury. The majority of these tetraspanin EVs were smaller than 50 nm based on interferometry and were insufficiently resolved by flow cytometry-based detection. At the injury site, there was enhanced expression of EV biogenesis proteins that were also detected in EVs directly isolated from spinal cord tissue by WB. Surface expression of tetraspanins CD9 and CD63 increased in multiple cell types at the injury site; however, astrocyte CD81 expression uniquely decreased, as demonstrated by flow cytometry. UC-isolated plasma EV microRNA cargo was also significantly altered at 1d post-injury with changes similar to that reported in EVs released by astrocytes after inflammatory stimulation. When injected into the lateral ventricle, plasma EVs from SCI mice increased both pro- and anti-inflammatory gene as well as reactive astrocyte gene expression in the brain cortex. These studies provide the first detailed characterization of plasma EV dynamics after SCI and suggest that plasma EVs may be involved in posttraumatic brain inflammation.

Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury.

Acidosis is among the least studied secondary injury mechanisms associated with neurotrauma. Acute decreases in brain pH correlate with poor long-term outcome in patients with traumatic brain injury (TBI), however, the temporal dynamics and underlying mechanisms are unclear. As key drivers of neuroinflammation, we hypothesized that microglia directly regulate acidosis after TBI, and thereby, worsen neurological outcomes. Using a controlled cortical impact model in adult male mice we demonstrate that intracellular pH in microglia and extracellular pH surrounding the lesion site are significantly reduced for weeks after injury. Microglia proliferation and production of reactive oxygen species (ROS) were also increased during the first week, mirroring the increase in extracellular ROS levels seen around the lesion site. Microglia depletion by a colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622, markedly decreased extracellular acidosis, ROS production, and inflammation in the brain after injury. Mechanistically, we identified that the voltage-gated proton channel Hv1 promotes oxidative burst activity and acid extrusion in microglia. Compared to wildtype controls, microglia lacking Hv1 showed reduced ability to generate ROS and extrude protons. Importantly, Hv1-deficient mice exhibited reduced pathological acidosis and inflammation after TBI, leading to long-term neuroprotection and functional recovery. Our data therefore establish the microglial Hv1 proton channel as an important link that integrates inflammation and acidosis within the injury microenvironment during head injury.

The voltage-gated proton channel Hv1 plays a detrimental role in contusion spinal cord injury via extracellular acidosis-mediated neuroinflammation.

Tissue acidosis is an important secondary injury process in the pathophysiology of traumatic spinal cord injury (SCI). To date, no studies have examined the role of proton extrusion as mechanism of pathological acidosis in SCI. In the present study, we hypothesized that the phagocyte-specific proton channel Hv1 mediates hydrogen proton extrusion after SCI, contributing to increased extracellular acidosis and poor long-term outcomes. Using a contusion model of SCI in adult female mice, we demonstrated that tissue pH levels are markedly lower during the first week after SCI. Acidosis was most evident at the injury site, but also extended into proximal regions of the cervical and lumbar cord. Tissue reactive oxygen species (ROS) levels and expression of Hv1 were significantly increased during the week of injury. Hv1 was exclusively expressed in microglia within the CNS, suggesting that microglia contribute to ROS production and proton extrusion during respiratory burst. Depletion of Hv1 significantly attenuated tissue acidosis, NADPH oxidase 2 (NOX2) expression, and ROS production at 3 d post-injury. Nanostring analysis revealed decreased gene expression of neuroinflammatory and cytokine signaling markers in Hv1 knockout (KO) mice. Furthermore, Hv1 deficiency reduced microglia proliferation, leukocyte infiltration, and phagocytic oxidative burst detected by flow cytometry. Importantly, Hv1 KO mice exhibited significantly improved locomotor function and reduced histopathology. Overall, these data suggest an important role for Hv1 in regulating tissue acidosis, NOX2-mediated ROS production, and functional outcome following SCI. Thus, the Hv1 proton channel represents a potential target that may lead to novel therapeutic strategies for SCI.

Intracerebroventricular Administration of hNSCs Improves Neurological Recovery after Cardiac Arrest in Rats.

Irreversible brain injury and neurological dysfunction induced by cardiac arrest (CA) have long been a clinical challenge due to lack of effective therapeutic interventions to reverse neuronal loss and prevent secondary reperfusion injury. The neuronal regenerative potential of neural stem cells (NSCs) provides a possible solution to this clinical deficit. We investigated the neuronal recovery potential of human neural stem cells (hNSCs) via intracerebroventricular (ICV) xenotransplantation after CA in rats and the effects of transplanted NSCs on the proliferation and migration of endogenous NSCs. Outcome measures included neurological functional recovery measured by neurological deficit score (NDS), electrophysiologic analysis of EEG, and assessment of proliferation and migration at the cellular level and the Wnt/ß-catenin pathway at the molecular level. Neurological functional assessment based on aggregate neurological deficit score (NDS) showed better recovery of function after hNSCs therapy (P < 0.05). Tracking of stem cells' proliferation with Ki67 antibody suggested that the NSCs group had more prominent proliferation compared to control group (number of Ki67+ cells, Control VS. NSC: 89.0 ± 31.6 VS. 352.7 ± 97.3, P < 0.05). In addition, cell migration tracked by Dcx antibody showed more Dcx + cells migrated to the far distance zone from SVZ in the treatment group (P < 0.05). Further immunofluorescence staining confirmed that the expression of the Wnt signaling pathway protein (ß-catenin) was upregulated in the NSC group (P < 0.05). ICV delivery of hNSCs promotes endogenous NSC proliferation and migration and ultimately enhances neuronal survival and neurological functional recovery. Wnt/ß-catenin pathway may be involved in the initiation and maintenance of this enhancement.Graphical abstract.

Delayed microglial depletion after spinal cord injury reduces chronic inflammation and neurodegeneration in the brain and improves neurological recovery in male mice.

Neuropsychological deficits, including impairments in learning and memory, occur after spinal cord injury (SCI). In experimental SCI models, we and others have reported that such changes reflect sustained microglia activation in the brain that is associated with progressive neurodegeneration. In the present study, we examined the effect of pharmacological depletion of microglia on posttraumatic cognition, depressive-like behavior, and brain pathology after SCI in mice. Methods: Young adult male C57BL/6 mice were subjected to moderate/severe thoracic spinal cord contusion. Microglial depletion was induced with the colony-stimulating factor 1 receptor (CSF1R) antagonist PLX5622 administered starting either 3 weeks before injury or one day post-injury and continuing through 6 weeks after SCI. Neuroinflammation in the injured spinal cord and brain was assessed using flow cytometry and NanoString technology. Neurological function was evaluated using a battery of neurobehavioral tests including motor function, cognition, and depression. Lesion volume and neuronal counts were quantified by unbiased stereology. Results: Flow cytometry analysis demonstrated that PLX5622 pre-treatment significantly reduced the number of microglia, as well as infiltrating monocytes and neutrophils, and decreased reactive oxygen species production in these cells from injured spinal cord at 2-days post-injury. Post-injury PLX5622 treatment reduced both CD45int microglia and CD45hi myeloid counts at 7-days. Following six weeks of PLX5622 treatment, there were substantial changes in the spinal cord and brain transcriptomes, including those involved in neuroinflammation. These alterations were associated with improved neuronal survival in the brain and neurological recovery. Conclusion: These findings indicate that pharmacological microglia-deletion reduces neuroinflammation in the injured spinal cord and brain, improving recovery of cognition, depressive-like behavior, and motor function.

Dementia, Depression, and Associated Brain Inflammatory Mechanisms after Spinal Cord Injury.

Evaluation of the chronic effects of spinal cord injury (SCI) has long focused on sensorimotor deficits, neuropathic pain, bladder/bowel dysfunction, loss of sexual function, and emotional distress. Although not well appreciated clinically, SCI can cause cognitive impairment including deficits in learning and memory, executive function, attention, and processing speed; it also commonly leads to depression. Recent large-scale longitudinal population-based studies indicate that patients with isolated SCI (without concurrent brain injury) are at a high risk of dementia associated with substantial cognitive impairments. Yet, little basic research has addressed potential mechanisms for cognitive impairment and depression after injury. In addition to contributing to disability in their own right, these changes can adversely affect rehabilitation and recovery and reduce quality of life. Here, we review clinical and experimental work on the complex and varied responses in the brain following SCI. We also discuss potential mechanisms responsible for these less well-examined, important SCI consequences. In addition, we outline the existing and developing therapeutic options aimed at reducing SCI-induced brain neuroinflammation and post-injury cognitive and emotional impairments.

Serum Endostatin Is a Novel Marker for COPD Associated with Lower Lung Function, Exacerbation and Systemic Inflammation.

Backgrounds and Aims: It is well known that angiogenesis contributes to the progression of chronic obstructive pulmonary disease (COPD) by initiating the remodeling of bronchial vasculature. However, the specific molecular mechanisms are incompletely understood. This research aimed to explore whether endostatin, a member of endogenous antiangiogenic proteins, is a biomarker in COPD and plays a role in the angiogenesis of COPD. Methods: 100 stable COPD patients, 130 patients with acute exacerbation (AECOPD) and 68 healthy volunteers were recruited in this research. Lung function test was conducted in the healthy people and stable COPD patients. Serum endostatin, C-reactive protein (CRP) and vascular endothelial growth factor (VEGF) of all the subjects were measured by Human Magnetic Luminex Screening Assay. Results: Serum endostatin level was significantly higher in stable COPD compared with healthy control and even more in AECOPD patients (P<0.001). Besides, stable COPD patients with frequent exacerbation (≥2 exacerbations per year) in the last 1 year had a higher concentration of endostatin in the circulation compared to the patients with less exacerbation (P=0.037). Furthermore, circulatory endostatin was negatively associated with forced expiratory volume in 1 s % predicted (FEV1%pre), an index of lung function in the stable COPD group (P=0.009). Finally, endostatin was positively correlated to serum CRP in COPD group (including stable and AECOPD) (P=0.005) and all the subjects (P<0.001), but only associated with VEGF in the total participants (P=0.002), not in the COPD group. Conclusion: These results suggested that endostatin is a biomarker for COPD and associated with lower lung function, exacerbation, and systemic inflammation. Endostatin potentially contributes to the pathogenesis of COPD.

Sustained neuronal and microglial alterations are associated with diverse neurobehavioral dysfunction long after experimental brain injury.

Traumatic brain injury (TBI) can cause progressive neurodegeneration, sustained neuroinflammation and chronic neurological dysfunction. Few experimental studies have explored the long-term neurobehavioral and functional cellular changes beyond several months. The present study examined the effects of a single moderate-level TBI on functional outcome 8 months after injury. Male C57BL/6 mice were subjected to controlled cortical impact injury and followed for changes in motor performance, learning and memory, as well as depressive-like and social behavior. We also used a novel flow cytometry approach to assess cellular functions in freshly isolated neurons and microglia from the injured tissue. There were marked and diverse, sustained neurobehavioral changes in injured mice. Compared to sham controls, chronic TBI mice showed long-term deficits in gait dynamics, nest building, spatial working memory and recognition memory. The tail suspension, forced swim, and sucrose consumption tests showed a marked depressive-like phenotype that was associated with impaired sociability. At the cellular level, there were lower numbers of Thy1+Tuj1+ neurons and higher numbers of activated CD45loCD11b+ microglia. Functionally, both neurons and microglia exhibited significantly higher levels of oxidative stress after injury. Microglia exhibited chronic deficits in phagocytosis of E. coli bacteria, and increased uptake of myelin and dying neurons. Living neurons showed decreased expression of synaptophysin and postsynaptic density (PSD)-95, along with greater numbers of microtubule-associated protein light chain 3 (LC3)-positive autophagosomes and increased mitochondrial mass that suggest dysregulation of autophagy. In summary, the late neurobehavioral changes found after murine TBI are similar to those found chronically after moderate-severe human head injury. Importantly, such changes are associated with microglial dysfunction and changes in neuronal activity.