Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.

[Spatial Distribution Patterns of Soil Organic Carbon in Karst Forests of the Lijiang River Basin and Its Driving Factors].

The aim of this study was to explore the carbon storage potential of karst forest soils in the Lijiang River Basin, reveal the spatial pattern of soil organic carbon (SOC), investigate the contributions and pathways of each driving factor to the spatial distribution of soil organic carbon, and provide a scientific basis for assessing the carbon cycle function of karst forests in the region. We employed structural equation modeling (SEM) and correlation analysis to investigate the spatial distribution characteristics of forest soil organic carbon in different basin sections (upper, middle, and lower reaches) and soil layers at different depths of the Lijiang River. Additionally, the direct and indirect ratios of each factor were quantified. The results showed that the overall soil layer of karst forest soils in the Lijiang River Basin was shallow, and soil organic carbon was phenoconcentric. The distribution of soil organic carbon content in different watershed sections was upstream > downstream > midstream, and the distribution of readily oxidizable carbon (ROC) and dissolved organic carbon (DOC) was consistent, whereas the distribution of microbial biomass carbon (MBC) was upstream > midstream > downstream. The contribution of various biotic and abiotic factors to the spatial distribution of soil organic carbon in karst forests in the watershed was different, and their contributions were ranked in descending order as:soil physicochemical factors > soil organic carbon active fraction > sample elevation > sample species diversity, with the total effects of 1.148, 0.574, 0.284, and -0.013, respectively. Among them, the sample site elevation had only an indirect effect on soil organic carbon, and the soil organic carbon active fraction had only a direct effect on soil organic carbon. Among the driving factors, total soil nitrogen, soil oxidizable organic carbon, sample site species richness, and soil soluble organic carbon could be used as important predictors of soil organic carbon content in karst forests in the Lijiang River Basin. Therefore, it is necessary to establish an effective eco-environmental protection mechanism covering the whole Lijiang River Basin, to reduce and control the impact of anthropogenic disturbances (especially in the middle urban section of the Lijiang River Basin), and to enhance and protect the species diversity of karst forests in the basin in order to improve soil physicochemical properties, improve and enhance the content of the soil organic carbon active fraction, and enhance the soil organic carbon stocks of karst forests in the Lijiang River Basin through other effective ways, as well as to promote the enhancement of the regional forest carbon sink function.

Genetic ablation of Sarm1 attenuates expression and mislocalization of phosphorylated TDP-43 after mouse repetitive traumatic brain injury.

Traumatic brain injury (TBI), particularly when moderate-to-severe and repetitive, is a strong environmental risk factor for several progressive neurodegenerative disorders. Mislocalization and deposition of transactive response DNA binding protein 43 (TDP-43) has been reported in both TBI and TBI-associated neurodegenerative diseases. It has been hypothesized that axonal pathology, an early event after TBI, may promote TDP-43 dysregulation and serve as a trigger for neurodegenerative processes. We sought to determine whether blocking the prodegenerative Sarm1 (sterile alpha and TIR motif containing 1) axon death pathway attenuates TDP-43 pathology after TBI. We subjected 111 male Sarm1 wild type, hemizygous, and knockout mice to moderate-to-severe repetitive TBI (rTBI) using a previously established injury paradigm. We conducted serial neurological assessments followed by histological analyses (NeuN, MBP, Iba-1, GFAP, pTDP-43, and AT8) at 1 month after rTBI. Genetic ablation of the Sarm1 gene attenuated the expression and mislocalization of phosphorylated TDP-43 (pTDP-43) and accumulation of pTau. In addition, Sarm1 knockout mice had significantly improved cortical neuronal and axonal integrity, functional deficits, and improved overall survival after rTBI. In contrast, removal of one Sarm1 allele delayed, but did not prevent, neurological deficits and neuroaxonal loss. Nevertheless, Sarm1 haploinsufficient mice showed significantly less microgliosis, pTDP-43 pathology, and pTau accumulation when compared to wild type mice. These data indicate that the Sarm1-mediated prodegenerative pathway contributes to pathogenesis in rTBI including the pathological accumulation of pTDP-43. This suggests that anti-Sarm1 therapeutics are a viable approach for preserving neurological function after moderate-to-severe rTBI.

Do Achievement Goals Differently Orient Students' Academic Engagement Through Learning Strategy and Academic Self-Efficacy and Vary by Grade.

Background: Previous cross-sectional studies were on the basis of three categories of achievement goal orientation; therefore, it is not yet possible to fully examine whether achievement goal orientation affects academic engagement through learning strategies and self-efficacy and whether those effects vary by grade. Then, it is necessary to further explore whether different achievement goal orientations affect academic engagement through the mediation of learning strategies and academic self-efficacy from the perspective of integration of achievement goal orientation theory and social cognitive theory under the premise of four classifications of achievement goal orientation, if so, whether there is consistent-path structure between grades. Methods: Participants were 1429 high school students (647 male,782 female) were token as subjects through cluster sampling. The Achievement Goal Orientation Scale, Learning Strategies Scale, Academic self-efficacy Scale,and Academic Engagement Scale were used to measure achievement goal orientations, learning strategies, academic self-efficacy and academic engagement. Results: The mastery approach, performance approach, and performance avoidance indirectly predicted academic engagement through the chained mediated effects of learning strategies and academic self-efficacy, respectively. There were no direct or indirect predictive effects of mastery avoidance on students' academic engagement. The path structure constructs were consistent across grades, except for grade differences in the predictive relationships between mastery approach on learning strategies and mastery avoidance on learning strategies. Conclusion: As external achievement goals originate from others, regardless of valence approach or avoidance, performance goals indirectly orient academic engagement through chain multiple mediators of learning strategies and academic self-efficacy. As internal achievement goal originates from the individual itself, the mastery approach not only directly but also indirectly orients academic engagement through chain multiple mediators of learning strategies and academic self-efficacy. The path structure remains consistent but local relations vary across school years in China. Finally, the possible psychological mechanisms of goal orientations are discussed.

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.

Molecular Docking Insight into the Label-Free Fluorescence Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA) is the most common mycotoxin and can be found in wheat, corn and other grain products. As OTA pollution in these grain products is gaining prominence as a global issue, the demand to develop OTA detection technology has attracted increasing attention. Recently, a variety of label-free fluorescence biosensors based on aptamer have been established. However, the binding mechanisms of some aptasensors are still unclear. Herein, a label-free fluorescent aptasensor employing Thioflavin T (ThT) as donor for OTA detection was constructed based on the G-quadruplex aptamer of the OTA aptamer itself. The key binding region of aptamer was revealed by using molecular docking technology. In the absence of the OTA target, ThT fluorescent dye binds with the OTA aptamer to form an aptamer/ThT complex, and results in the fluorescence intensity being obviously enhanced. In the presence of OTA, the OTA aptamer binds to OTA because of its high affinity and specificity to form an aptamer/OTA complex, and the ThT fluorescent dye is released from the OTA aptamer into the solution. Therefore, the fluorescence intensity is significantly decreased. Molecular docking results revealed that OTA is binding to the pocket-like structure and surrounded by the A29-T3 base pair and C4, T30, G6 and G7 of the aptamer. Meanwhile, this aptasensor shows good selectivity, sensitivity and an excellent recovery rate of the wheat flour spiked experiment.

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

A Dual-Mode Method Based on Aptamer Recognition and Time-Resolved Fluorescence Resonance Energy Transfer for Histamine Detection in Fish.

Histamine produced via the secretion of histidine decarboxylase by the bacteria in fish muscles is a toxic biogenic amine and of significant concern in food hygiene, since a high intake can cause poisoning in humans. This study proposed a fluorometric and colorimetric dual-mode specific method for the detection of histamine in fish, based on the fluorescence labeling of a histamine specific aptamer via the quenching and optical properties of gold nanoparticles (AuNPs). Due to the fluorescence resonance energy transfer phenomenon caused by the proximity of AuNPs and NaYF4:Ce/Tb, resulting in the quenching of the fluorescence signal in the detection system, the presence of histamine will compete with AuNPs to capture the aptamer and release it from the AuNP surface, inducing fluorescence recovery. Meanwhile, the combined detection of the two modes showed good linearity with histamine concentration, the linear detection range of the dual-mode synthesis was 0.2-1.0 µmol/L, with a detection limit of 4.57 nmol/L. Thus, this method has good selectivity and was successfully applied to the detection of histamine in fish foodstuffs with the recoveries of 83.39~102.027% and 82.19~105.94% for Trichiurus haumela and Thamnaconus septentrionalis, respectively. In addition, this method was shown to be simple, rapid, and easy to conduct. Through the mutual verification and combined use of the two modes, a highly sensitive, rapid, and accurate dual-mode detection method for the analysis of histamine content in food was established, thereby providing a reference for the monitoring of food freshness.

Noninvasive oral cancer screening based on local residual adaptation network using optical coherence tomography.

Oral cancer is known as one of the relatively common malignancy types worldwide. Despite the easy access of the oral cavity to examination, the invasive biopsy is still essential for final diagnosis, which requires laborious operation and complicated trained specialists. With the development of deep learning, the artificial intelligence (AI) technique is applied for oral cancer examinations and alleviates the workload of manual screening on biopsy. However, existing computer-aided oral cancer diagnostic methods focus on oral cavity environment photos and histology images, which require complicated operations for doctors and are invasive and painful for patients. As a noninvasive, real-time imaging technique, optical coherence tomography (OCT) can express sufficient identical information for oral cancer screening, but it has not been effectively explored for automatic oral cancer diagnosis. This paper proposes a novel deep learning method named Local Residual Adaptation Network (LRAN) for noninvasive oral cancer screening on OCT images, collected from 25 patients in Beijing Stomatological Hospital. Our proposed LRAN consists of a Residual Feature Representation (RFR) module and a Local Distribution Adaptation (LDA) module. Specifically, RFR firstly adopts stacked residual blocks as the backbone network to learn feature representations for training data, optimized by the Cross-Entropy loss, and then deploy Euclidean distance to measure the distribution distance between training and testing OCT images. Finally, LRAN achieves distribution-gap bridging by the LDA module, which integrates local maximum mean discrepancy constraint to estimate and minimize the distribution discrepancy between training and testing sets within the same category. We also collected an OCT-based oral cancer image dataset to evaluate the effectiveness of the proposed method, and it achieves an accuracy of 91.62%, a sensitivity of 91.66%, and a specificity of 92.58% on this self-collected dataset. Furthermore, we conduct a quantitative and qualitative analysis, and the results demonstrate LRAN model has excellent capability to solve the noninvasive oral cancer screening task.

The lncRNA-AK046375 Upregulates Metallothionein-2 by Sequestering miR-491-5p to Relieve the Brain Oxidative Stress Burden after Traumatic Brain Injury.

We previously discovered that traumatic brain injury (TBI) induces significant perturbations in long noncoding RNA (lncRNA) levels in the mouse cerebral cortex, and lncRNA-AK046375 is one of the most significantly changed lncRNAs after TBI. lncRNA-AK046375 overexpression and knockdown models were successfully constructed both in vitro and in vivo. In cultured primary cortical neurons and astrocytes, lncRNA-AK046375 sequestered miR-491-5p, thereby enhancing the expression of metallothionein-2 (MT2), which ameliorated oxidative-induced cell injury. In addition, upregulated lncRNA-AK046375 promoted the recovery of motor, learning, and memory functions after TBI in C57BL/6 mice, and the underlying mechanism may be related to ameliorated apoptosis, inhibited oxidative stress, reduced brain edema, and relieved loss of tight junction proteins at the blood-brain barrier in the mouse brain. Therefore, we conclude that lncRNA-AK046375 enhances MT2 expression by sequestering miR-491-5p, ultimately strengthening antioxidant activity, which ameliorates neurological deficits post-TBI.

Essential role of MALAT1 in reducing traumatic brain injury.

As a highly evolutionary conserved long non-coding RNA, metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was first demonstrated to be related to lung tumor metastasis by promoting angiogenesis. To investigate the role of MALAT1 in traumatic brain injury, we established mouse models of controlled cortical impact and cell models of oxygen-glucose deprivation to mimic traumatic brain injury in vitro and in vivo. The results revealed that MALAT1 silencing in vitro inhibited endothelial cell viability and tube formation but increased migration. In MALAT1-deficient mice, endothelial cell proliferation in the injured cortex, functional vessel density and cerebral blood flow were reduced. Bioinformatic analyses and RNA pull-down assays validated enhancer of zeste homolog 2 (EZH2) as a downstream factor of MALAT1 in endothelial cells. Jagged-1, the Notch homolog 1 (NOTCH1) agonist, reversed the MALAT1 deficiency-mediated impairment of angiogenesis. Taken together, our results suggest that MALAT1 controls the key processes of angiogenesis following traumatic brain injury in an EZH2/NOTCH1-dependent manner.

Downregulation of miR-491-5p promotes neovascularization after traumatic brain injury.

MicroRNA-491-5p (miR-491-5p) plays an important role in regulating cell proliferation and migration; however, the effect of miR-491-5p on neovascularization after traumatic brain injury remains poorly understood. In this study, a controlled cortical injury model in C57BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro, respectively. In the in vivo model, quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5p increased or decreased following the intracerebroventricular injection of an miR-491-5p agomir or antagomir, respectively, and the expression of miR-491-5p decreased slightly after traumatic brain injury. To detect the neuroprotective effects of miR-491-p, neurological severity scores, Morris water maze test, laser speckle techniques, and immunofluorescence staining were assessed, and the results revealed that miR-491-5p downregulation alleviated neurological dysfunction, promoted the recovery of regional cerebral blood flow, increased the number of lectin-stained microvessels, and increased the survival of neurons after traumatic brain injury. During the in vitro experiments, the potential mechanism of miR-491-5p on neovascularization was explored through quantitative real-time-polymerase chain reaction, which showed that miR-491-5p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5p mimic or inhibitor, respectively. Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5p. Cell counting kit 8 (CCK-8) assay, flow cytometry, and 2?,7?-dichlorofluorescein diacetate (DCFH-DA) assay results confirmed that the downregulation of miR-491-5p increased brain microvascular endothelial cell viability, reduced cell apoptosis, and alleviated oxidative stress under oxygen-glucose deprivation conditions. Cell scratch assay, Transwell assay, tube formation assay, and western blot assay results demonstrated that miR-491-5p downregulation promoted the migration, proliferation, and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway. These findings confirmed that miR-491-5p downregulation promotes neovascularization, restores cerebral blood flow, and improves the recovery of neurological function after traumatic brain injury. The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress. All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China (approval No. 2020-304) on June 22, 2020.

Neat1 decreases neuronal apoptosis after oxygen and glucose deprivation.

Studies have shown that downregulation of nuclear-enriched autosomal transcript 1 (Neat1) may adversely affect the recovery of nerve function and the increased loss of hippocampal neurons in mice. Whether Neat1 has protective or inhibitory effects on neuronal cell apoptosis after secondary brain injury remains unclear. Therefore, the effects of Neat1 on neuronal apoptosis were observed. C57BL/6 primary neurons were obtained from the cortices of newborn mice and cultured in vitro, and an oxygen and glucose deprivation cell model was established to simulate the secondary brain injury that occurs after traumatic brain injury in vitro. The level of Neat1 expression in neuronal cells was regulated by constructing a recombinant adenovirus to infect neurons, and the effects of Neat1 expression on neuronal apoptosis after oxygen and glucose deprivation were observed. The experiment was divided into four groups: the control group, without any treatment, received normal culture; the oxygen and glucose deprivation group were subjected to the oxygen and glucose deprivation model protocol; the Neat1 overexpression and Neat1 downregulation groups were treated with Neat1 expression intervention techniques and were subjected to the in oxygen and glucose deprivation protocol. The protein expression levels of neurons p53-induced death domain protein 1 (PIDD1, a pro-apoptotic protein), caspase-2 (an apoptotic priming protein), cytochrome C (a pro-apoptotic protein), and cleaved caspase-3 (an apoptotic executive protein) were measured in each group using the western blot assay. To observe changes in the intracellular distribution of cytochrome C, the expression levels of cytochrome C in the cytoplasm and mitochondria of neurons from each group were detected by western blot assay. Differences in the cell viability and apoptosis rate between groups were detected by cell-counting kit 8 assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, respectively. The results showed that the apoptosis rate, PIDD1, caspase-2, and cleaved caspase-3 expression levels significantly decreased, and cell viability significantly improved in the Neat1 overexpression group compared with the oxygen and glucose deprivation group; however, Neat1 downregulation reversed these changes. Compared with the Neat1 downregulation group, the cytosolic cytochrome C level in the Neat1 overexpression group significantly decreased, and the mitochondrial cytochrome C level significantly increased. These data indicate that Neat1 upregulation can reduce the release of cytochrome C from the mitochondria to the cytoplasm by inhibiting the PIDD1-caspase-2 pathway, reducing the activation of caspase-3, and preventing neuronal apoptosis after oxygen and glucose deprivation, which might reduce secondary brain injury after traumatic brain injury. All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China, on December 19, 2020 (approval No. 2020-895).

Endothelial Regulation by Exogenous Annexin A1 in Inflammatory Response and BBB Integrity Following Traumatic Brain Injury.

BACKGROUND AND TARGET: Following brain trauma, blood-brain barrier (BBB) disruption and inflammatory response are critical pathological steps contributing to secondary injury, leading to high mortality and morbidity. Both pathologies are closely associated with endothelial remodeling. In the present study, we concentrated on annexin A1 (ANXA1) as a novel regulator of endothelial function after traumatic brain injury. METHODS: After establishing controlled cortical impact (CCI) model in male mice, human recombinant ANXA1 (rANXA1) was administered intravenously, followed by assessments of BBB integrity, brain edema, inflammatory response, and neurological deficits. RESULT: Animals treated with rANXA1 (1 µg/kg) at 1 h after CCI exhibited optimal BBB protection including alleviated BBB disruption and brain edema, as well as endothelial junction proteins loss. The infiltrated neutrophils and inflammatory cytokines were suppressed by rANXA1, consistent with decreased adhesive and transmigrating molecules from isolated microvessels. Moreover, rANXA1 attenuated the neurological deficits induced by CCI. We further found that the Ras homolog gene family member A (RhoA) inhibition has similar effect as rANXA1 in ameliorating brain injuries after CCI, whereas rANXA1 suppressed CCI-induced RhoA activation. CONCLUSION: Our findings suggest that the endothelial remodeling by exogenous rANXA1 corrects BBB disruption and inflammatory response through RhoA inhibition, hence improving functional outcomes in CCI mice.

Fecal Microbiota Transplantation Is a Promising Method to Restore Gut Microbiota Dysbiosis and Relieve Neurological Deficits after Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) can induce persistent fluctuation in the gut microbiota makeup and abundance. The present study is aimed at determining whether fecal microbiota transplantation (FMT) can rescue microbiota changes and ameliorate neurological deficits after TBI in rats. METHODS: A controlled cortical impact (CCI) model was used to simulate TBI in male Sprague-Dawley rats, and FMT was performed for 7 consecutive days. 16S ribosomal RNA (rRNA) sequencing of fecal samples was performed to analyze the effects of FMT on gut microbiota. Modified neurological severity score and Morris water maze were used to evaluate neurobehavioral functions. Metabolomics was used to screen differential metabolites from the rat serum and ipsilateral brains. The oxidative stress indices were measured in the brain. RESULTS: TBI induced significance changes in the gut microbiome, including the alpha- and beta-bacterial diversity, as well as the microbiome composition at 8 days after TBI. On the other hand, FMT could rescue these changes and relieve neurological deficits after TBI. Metabolomics results showed that the level of trimethylamine (TMA) in feces and the level of trimethylamine N-oxide (TMAO) in the ipsilateral brain and serum was increased after TBI, while FMT decreased TMA levels in the feces, and TMAO levels in the ipsilateral brain and serum. Antioxidant enzyme methionine sulfoxide reductase A (MsrA) in the ipsilateral hippocampus was decreased after TBI but increased after FMT. In addition, FMT elevated SOD and CAT activities and GSH/GSSG ratio and diminished ROS, GSSG, and MDA levels in the ipsilateral hippocampus after TBI. CONCLUSIONS: FMT can restore gut microbiota dysbiosis and relieve neurological deficits possibly through the TMA-TMAO-MsrA signaling pathway after TBI.

Downregulation of microRNA-9-5p promotes synaptic remodeling in the chronic phase after traumatic brain injury.

The level of microRNA-9-5p (miRNA-9-5p) in brain tissues is significantly changed in the chronic phase after traumatic brain injury (TBI). However, the effect of miRNA-9-5p on brain function after TBI has not been elucidated. In this study, we used a controlled cortical impact (CCI) model to induce TBI in Sprague-Dawley rats. Brain microvascular endothelial cells (BMECs), astrocytes, and neurons were extracted from immature Sprague-Dawley rats and cocultured to reconstruct the neurovascular unit (NVU) in vitro. The results showed that downregulation of miRNA-9-5p in the chronic phase contributed to neurological function recovery by promoting astrocyte proliferation and increasing the release of astrocyte-derived neurotrophic factors around injured brain tissues after TBI. A dual-luciferase reporter assay validated that miRNA-9-5p was a post-transcriptional modulator of thrombospondin 2 (Thbs-2), and downregulation of miRNA-9-5p promoted Thbs-2 expression in astrocytes. Furthermore, we verified that Thbs-2 can promote Notch pathway activation by directly binding to Jagged and Notch. Through in vitro experiments, we found that the expression of synaptic proteins and the number of synaptic bodies were increased in neurons in the NVU, which was constructed using astrocytes pretreated with miRNA-9-5p inhibitor. Moreover, we also found that downregulation of miRNA-9-5p promoted Thbs-2 expression in astrocytes, which activated the Notch/cylindromatosis/transforming growth factor-ß-activated kinase 1 pathway in neurons and promoted the expression of synaptic proteins, including post-synaptic density protein 95 and synaptotagmin. Based on these results, miRNA-9-5p may be a new promising prognostic marker and treatment target for TBI.

Bexarotene promotes microglia/macrophages - Specific brain - Derived Neurotrophic factor expression and axon sprouting after traumatic brain injury.

Traumatic brain injury (TBI) has been regarded as one of the leading cause of injury-related death and disability. White matter injury after TBI is characterized by axon damage and demyelination, resulting in neural network impairment and neurological deficit. Brain-derived neurotrophic factor (BDNF) can promote white matter repair. The activation of peroxisome proliferator-activated receptor gamma (PPARγ) has been reported to promote microglia/macrophages towards anti-inflammatory state and therefore to promote axon regeneration. Bexarotene, an agonist of retinoid X receptor (RXR), can activate RXR/PPARγ heterodimers. The aim of the present study was to identify the effect of bexarotene on BDNF in microglia/macrophages and axon sprouting after TBI in mice. Bexarotene was administered intraperitoneally in C57BL/6 mice undergoing controlled cortical impact (CCI). PPARγ dependency was determined by intraperitoneal administration of a PPARγ antagonist T0070907. We found that bexarotene promoted axon regeneration indicated by increased growth associated protein 43 (GAP43) expression, myelin basic protein (MBP) expression, and biotinylated dextran amine (BDA)+ axon sprouting. Bexarotene also increased microglia/macrophages-specific brain derived neurotrophic factor (BDNF) expression after TBI. In addition, bexarotene reduced the number of pro-inflammatory microglia/macrophages while increased the number of anti-inflammatory microglia/macrophages after TBI. Moreover, bexaortene inhibited pro-inflammatory cytokine secretion. In addition, bexarotene treatment improved neurological scores and cognitive function of CCI-injured mice. These effects of bexarotene were partially abolished by T0070907. In conclusion, bexarotene promotes axon sprouting, increases microglia/macrophages-specific BDNF expression, and induces microglia/macrophages from a pro-inflammatory state towards an anti-inflammatory one after TBI at least partially in a PPARγ-dependent manner.

Pentraxin 3 contributes to neurogenesis after traumatic brain injury in mice.

Emerging evidence indicates that pentraxin 3 is an acute-phase protein that is linked with the immune response to inflammation. It is also a newly discovered marker of anti-inflammatory A2 reactive astrocytes, and potentially has multiple protective effects in stroke; however, its role in the adult brain after traumatic brain injury is unknown. In the present study, a moderate model of traumatic brain injury in mice was established using controlled cortical impact. The models were intraventricularly injected with recombinant pentraxin 3 (the recombinant pentraxin 3 group) or an equal volume of vehicle (the control group). The sham-operated mice underwent craniotomy, but did not undergo the controlled cortical impact. The potential neuroprotective and neuroregenerative roles of pentraxin 3 were investigated on days 14 and 21 after traumatic brain injury. Western blot assay showed that the expression of endogenous pentraxin 3 was increased after traumatic brain injury in mice. Furthermore, the neurological severity test and wire grip test revealed that recombinant pentraxin 3 treatment reduced the neurological severity score and increased the wire grip score, suggesting an improved recovery of sensory-motor functions. The Morris water maze results demonstrated that recombinant pentraxin 3 treatment reduced the latency to the platform, increased the time spent in the correct quadrant, and increased the number of times traveled across the platform, thus suggesting an improved recovery of cognitive function. In addition, to investigate the effects of pentraxin 3 on astrocytes, specific markers of A2 astrocytes were detected in primary astrocyte cultures in vitro using western blot assay. The results demonstrated that pentraxin 3 administration activates A2 astrocytes. To explore the protective mechanisms of pentraxin 3, immunofluorescence staining was used. Intraventricular injection of recombinant pentraxin 3 increased neuronal maintenance in the peri-injured cortex and ipsilateral hippocampus, increased the number of doublecortin-positive neural progenitor cells in the subventricular and subgranular zones, and increased the number of bromodeoxyuridine (proliferation) and neuronal nuclear antigen (mature neuron) double-labeled cells in the hippocampus and peri-injured cortex. Pentraxin 3 administration also increased the number of neurospheres and the number of bromodeoxyuridine and doublecortin double-labeled cells in neurospheres, and enhanced the proliferation of neural progenitor cells in primary neural progenitor cell cultures in vitro. In conclusion, recombinant pentraxin 3 administration activated A2 astrocytes, and consequently improved the recovery of neural function by increasing neuronal survival and enhancing neurogenesis. All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China on March 1, 2016.

Apolipoprotein E promotes white matter remodeling via the Dab1-dependent pathway after traumatic brain injury.

INTRODUCTION: Axonal injury results in long-term neurological deficits in traumatic brain injury (TBI) patients. Apolipoprotein E (ApoE) has been reported to activate intracellular adaptor protein Disabled-1 (Dab1) phosphorylation via its interaction with ApoE receptors. The Dab1 pathway acts as a regulator of axonal outgrowth and growth cone formation in the brain. AIMS: We hypothesized that ApoE may alleviate axonal injury and regulate axonal regeneration via the Dab1 pathway after TBI. RESULTS: In this study, we established a model of controlled cortical impact (CCI) to mimic TBI in vivo. Using diffusion tensor imaging to detect white matter integrity, we demonstrated that APOE-deficient mice exhibited lower fractional anisotropy (FA) values than APOE+/+ mice at 28 days after injury. The expression levels of axonal regeneration and synapse plasticity biomarkers, including growth-associated protein 43 (GAP43), postsynaptic density protein 95 (PSD-95), and synaptophysin, were also lower in APOE-deficient mice. In contrast, APOE deficiency exerted no effects on the levels of myelin basic protein (MBP) expression, oligodendrocyte number, or oligodendrocyte precursor cell number. Neurological severity score (NSS) and behavioral measurements in the rotarod, Morris water maze, and Y maze tests revealed that APOE deficiency caused worse neurological deficits in CCI mice. Furthermore, Dab1 activation downregulation by the ApoE receptor inhibitor receptor-associated protein (RAP) or Dab1 shRNA lentivirus attenuated the beneficial effects of ApoE on FA values, GAP43, PSD-95, and synaptophysin expression, and neurological function tests. Additionally, the effects of ApoE on axonal regeneration were further validated in vitro. In a mechanical scratch injury model of primary cultured neurons, recombinant ApoE protein treatment enhanced axonal outgrowth and growth cone formation in injured neurons; however, these effects were attenuated by Dab1 shRNA, consistent with the in vivo results. CONCLUSION: Collectively, these data suggest that ApoE promotes axonal regeneration partially through the Dab1 pathway, thereby contributing to functional recovery following TBI.