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Traumatic brain injury (TBI) survivors suffer from long-term disability and neuropsychiatric sequelae due to irreparable brain tissue destruction. However, there are still few efficient therapies to promote neurorestoration in damaged brain tissue. This study aimed to investigate whether the pro-oncogenic gene ski can promote neurorestoration after TBI. We established a ski-overexpressing experimental TBI mouse model using adenovirus-mediated overexpression through immediate injection after injury. Hematoxylin-eosin staining, MRI-based 3D lesion volume reconstruction, neurobehavioral tests, and analyses of neuronal regeneration and astrogliosis were used to assess neurorestorative efficiency. The effects of ski overexpression on the proliferation of cultured immature neurons and astrocytes were evaluated using imaging flow cytometry. The Ski protein level increased in the perilesional region at 3 days post injury. ski overexpression further elevated Ski protein levels up to 14 days post injury. Lesion volume was attenuated by approximately 36-55% after ski overexpression, with better neurobehavioral recovery, more newborn immature and mature neurons, and less astrogliosis in the perilesional region. Imaging flow cytometry results showed that ski overexpression elevated the proliferation rate of immature neurons and reduced the proliferation rate of astrocytes. These results show that ski can be considered a novel neurorestoration-related gene that effectively promotes neurorestoration, facilitates neuronal regeneration, and reduces astrogliosis after TBI.

The purpose of the present study was to investigate the effect of glutamate scavenger oxaloacetate (OA) combined with CGS21680, an adenosine A2A receptor (A2AR) agonist, on acute traumatic brain injury (TBI), and to elucidate the underlying mechanisms. C57BL/6J mice were subjected to moderate-level TBI by controlled cortical impact, and then were treated with OA, CGS21680, or OA combined with CGS21680 at acute stage of TBI. At 24 h post TBI, neurological severity score, brain water content, glutamate concentration in cerebrospinal fluid (CSF), mRNA and protein levels of IL-1β and TNF-α, mRNA level and activity of glutamate oxaloacetate aminotransferase (GOT), and ATP level of brain tissue were detected. The results showed that neurological deficit, brain water content, glutamate concentration in CSF, and the inflammatory cytokine IL-1β and TNF-α production were exacerbated in CGS21680 treated mice. Administrating OA suppressed the rise of both glutamate concentration in CSF and brain water content, and elevated the ATP level of cerebral tissue. More interestingly, neurological deficit, brain edema, glutamate concentration, IL-1β and TNF-α levels were ameliorated significantly in mice treated with OA combined with CGS21680. The combined treatment exhibited better therapeutic effects than single OA treatment. We also observed that GOT activity was enhanced in single CGS21680 treatment group, and both the GOT mRNA level and GOT activity were up-regulated in early-stage combined treatment group. These results suggest that A2AR can improve the efficiency of GOT and potentiate the ability of OA to metabolize glutamate. This may be the mechanism that A2AR activation in combination group augmented the neuroprotective effect of OA rather than aggravated the brain damages. Taken together, the present study provides a new insight for the clinical treatment of TBI with A2AR agonists and OA.

Effective treatment for cognitive dysfunction after traumatic brain injury (TBI) is lacking in clinical practice. Increased brain-derived neurotrophic factor (BDNF) expression in cognitive circuits can significantly alleviate cognitive dysfunction in animal models of TBI. Selective 5-hydroxytryptamine receptor 6 (5-HT

In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) have major inhibitory effects on nerve regeneration. They include Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. MAIs possess two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Previous studies have confirmed that the inhibition of NgR only results in a modest increase in regeneration in the CNS; however, the inhibitory effects of PirB with regard to nerve regeneration after binding to MAIs remain controversial. In this study, we demonstrated that PirB is expressed in primary cultures of retinal ganglion cells (RGCs), and the inhibitory effects of the three MAIs on the growth of RGC neurites are not significantly decreased after direct PirB knockdown using adenovirus PirB shRNA. Interestingly, we found that retinal Müller cells expressed PirB and that its knockdown enhanced the regeneration of co-cultured RGC neurites. PirB knockdown also activated the JAK/Stat3 signaling pathway in Müller cells and upregulated ciliary neurotrophic factor levels. These findings indicate that PirB plays a novel role in retinal Müller cells and that its action in these cells may indirectly affect the growth of RGC neurites. The results also reveal that PirB in Müller cells affects RGC neurite regeneration. Our findings provide a novel basis for the use of PirB as a target molecule to promote nerve regeneration. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-020-00510-w) contains supplementary material, which is available to authorized users.

Neurons, especially axons, are metabolically demanding and energetically vulnerable during injury. However, the exact energy budget alterations that occur early after axon injury and the effects of these changes on neuronal survival remain unknown. Using a classic mouse model of optic nerve-crush injury, we found that traumatized optic nerves and retinas harbor the potential to mobilize two primary energetic machineries, glycolysis and oxidative phosphorylation, to satisfy the robustly increased adenosine triphosphate (ATP) demand. Further exploration of metabolic activation showed that mitochondrial oxidative phosphorylation was amplified over other pathways, which may lead to decreased retinal ganglion cell (RGC) survival despite its supplement to ATP production. Gene set enrichment analysis of a microarray (GSE32309) identified significant activation of oxidative phosphorylation in injured retinas from wild-type mice compared to those from mice with deletion of phosphatase and tensin homolog (PTEN), while PTEN-/- mice had more robust RGC survival. Therefore, we speculated that the oxidation-favoring metabolic pattern after optic nerve-crush injury could be adverse for RGC survival. After redirecting metabolic flux toward glycolysis (magnifying the Warburg effect) using the drug meclizine, we successfully increased RGC survival. Thus, we provide novel insights into a potential bioenergetics-based strategy for neuroprotection. Electronic supplementary material The online version of this article (10.1007/s12264-020-00490-x) contains supplementary material, which is available to authorized users.

BackgroundCognitive impairment in the late stage of traumatic brain injury (TBI) is associated with the NOD-, LRR and pyrin domain-containing protein 3 (NLRP3) inflammasome, which plays an important role in neuroinflammation. Although classical inflammatory pathways have been well-documented in the late stage of TBI (4–8 weeks post-injury), the mechanism by which the NLRP3 inflammasome impairs cognition is still unclear.MethodsMice lacking the gene encoding for NLRP3 (NLRP3-knockout mice) and their wild-type littermates were used in a controlled cortical impact model of TBI. Levels of NLRP3 inflammasome and inflammatory factors such as IL-1β and HMGB1 were detected in post-injury hippocampal tissue, as well as long-term potentiation. Behaviors were assessed by T-maze test, novel object recognition, and nesting tests. Glycyrrhizin was used to antagonize HMGB1. Calcium imaging were performed on primary neuronal cultures.ResultsBy using the NLRP3-knockout TBI model, we found that the continuous activation of the NLRP3 inflammasome and high mobility group box 1 (HMGB1) release were closely related to cognitive impairment. We also found that inhibition of HMGB1 improved LTP reduction and cognitive function by increasing the phosphorylation level of the NMDAR1 subunit at serine 896 while reducing NLRP3 inflammasome activation.ConclusionNLRP3 inflammasome damages memory in the late stage of TBI primarily through HMGB1 upregulation and provides an explanation for the long-term progression of cognitive dysfunction.

Background In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) exert major inhibitory effects on nerve regeneration: Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp). MAIs have two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Existing studies confirm that inhibiting NgR only exerted a modest disinhibitory effect in CNS. However, the inhibitory effects of PirB on nerve regeneration after binding to MAIs are controversial too. We aimed to further investigate the effect of PirB knockdown on the neuroprotection and axonal regeneration of retinal ganglion cells (RGCs) after optic nerve injury in rats. Methods The differential expression of PirB in the retina was observed via immunofluorescence and western blotting after 1, 3, and 7 days of optic nerve injury (ONI). The retina was locally transfected with adeno-associated virus (AAV) PirB shRNA, then, the distribution of virus in tissues and cells was observed 21 days after AAV transfection to confirm the efficiency of PirB knockdown. Level of P-Stat3 and expressions of ciliary neurotrophic factor (CNTF) were detected via western blotting. RGCs were directly labeled with cholera toxin subunit B (CTB). The new axons of the optic nerve were specifically labeled with growth associated protein-43 (GAP43) via immunofluorescence. Flash visual evoked potential (FVEP) was used to detect the P1 and N1 latency, as well as N1-P1, P1-N2 amplitude to confirm visual function. Results PirB expression in the retina was significantly increased after ONI. PirB knockdown was successful and significantly promoted P-Stat3 level and CNTF expression in the retina. PirB knockdown promoted the regeneration of optic nerve axons and improved the visual function indexes such as N1-P1 and P1-N2 amplitude. Conclusions PirB is one of the key molecules that inhibit the regeneration of the optic nerve, and inhibition of PirB has an excellent effect on promoting nerve regeneration, which allows the use of PirB as a target molecule to promote functional recovery after ONI.

Pathogens such as bacterial lipopolysaccharide (LPS) play an important role in promoting the production of the inflammatory cytokines interleukin-1 beta (IL-1β) and tumour necrosis factor-α (TNF-α) in response to infection or damage in microglia. However, whether different signalling pathways regulate these two inflammatory factors remains unclear. The protein kinase C (PKC) family is involved in the regulation of inflammation, and our previous research showed that the activation of the PKC pathway played a key role in the LPS-induced transformation of the adenosine A2A receptor (A2AR) from anti-inflammatory activity to pro-inflammatory activity under high glutamate concentrations. Therefore, in the current study, we investigated the role of PKC in the LPS-induced production of these inflammatory cytokines in mouse primary microglia. GF109203X, a specific PKC inhibitor, inhibited the LPS-induced expression of IL-1β messenger ribonucleic acid and intracellular protein in a dose-dependent manner. Moreover, 5 µM GF109203X prevented LPS-induced IL-1β expression but did not significantly affect LPS-induced TNF-α expression. PKC promoted IL-1β expression by regulating the activity of NF-κB but did not significantly impact the activity of ERK1/2. A2AR activation by CGS21680, an A2AR agonist, facilitated LPS-induced IL-1β expression through the PKC pathway at high glutamate concentrations but did not significantly affect LPS-induced TNF-α expression. Taken together, these results suggest a new direction for specific intervention with LPS-induced inflammatory factors in response to specific signalling pathways and provide a mechanism for A2AR targeting, especially after brain injury, to influence inflammation by interfering with A2AR.

Traumatic brain injury-induced acute lung injury (TBI-ALI) is a serious complication of traumatic brain injury (TBI). Our previous clinical study found that high levels of blood glutamate after TBI were closely related to the occurrence and severity of TBI-ALI, while it remains unknown whether a high concentration of blood glutamate directly causes or aggravates TBI-ALI. We found that inhibition of the adenosine A2A receptor (A2AR) after brain injury alleviated the TBI-ALI; however, it is unknown whether lowering blood glutamate levels in combination with inhibiting the A2AR would lead to better effects. Using mouse models of moderate and severe TBI, we found that intravenous administration of L-glutamate greatly increased the lung water content, lung-body index, level of inflammatory markers in bronchoalveolar lavage fluid and acute lung injury score and significantly decreased the PaO2/FiO2 ratio. Moreover, the incidence of TBI-ALI and the mortality rate were significantly increased, and the combined administration of A2AR activator and exogenous glutamate further exacerbated the above damaging effects. Conversely, lowering the blood glutamate level through peritoneal dialysis or intravenous administration of oxaloacetate notably improved the above parameters, and a further improvement was seen with concurrent A2AR genetic inactivation. These data suggest that A2AR activation aggravates the damaging effect of high blood glutamate concentrations on the lung and that combined treatment targeting both A2AR and blood glutamate may be an effective way to prevent and treat TBI-ALI.

Caffeine is a substance that is consumed worldwide, and it may exert neuroprotective effects against various cerebral insults, including neurotrauma, which is the most prevalent injury among military personnel. To investigate the effects of caffeine on high-intensity blast wave-induced severe blast injury in mice, three different paradigms of caffeine were applied to male C57BL/6 mice with severe whole body blast injury (WBBI). The results demonstrated that chronic caffeine treatment alleviated blast-induced traumatic brain injury (bTBI); however, both chronic and acute caffeine treatments exacerbated blast-induced lung injuries and, more importantly, increased both the cumulative and time-segmented mortalities postinjury. Interestingly, withdrawing caffeine intake preinjury resulted in favorable outcomes in mortality and lung injury, similar to the findings in water-treated mice, and had the trend to attenuate brain injury. These findings demonstrated that although drinking coffee or caffeine preparations attenuated blast-induced brain trauma, these beverages may place personnel in the battlefield at high risk of casualties, which will help us re-evaluate the therapeutic strategy of caffeine application, particularly in multiple-organ-trauma settings. Furthermore, these findings provided possible strategies for reducing the risk of casualties with caffeine consumption, which may help to change the coffee-drinking habits of military personnel.

The heteromeric complexes of adenosine 2A receptor (A2AR) and N-methyl-D-aspartate receptor (NMDAR) have recently been confirmed in cell experiments, while its in situ detection at the subcellular level of brain tissue has not yet been achieved. Proximity Ligation Assay (PLA) enables the detection of low-abundance proteins and their interactions at the cellular level with high specificity and sensitivity, while Transmission electron microscope (TEM) is an excellent tool for observing subcellular structures. To develop a highly efficient and reproducible technique for in situ detection of protein interactions at subcellular levels, in this study, we modified the standard PLA sample preparation method to make the samples suitable for analysis by transmission electron microscopy. Using this technique, we successfully detected the heteromers of A2AR and NMDAR1, the essential subunit of NMDA receptor on the hippocampal synaptic structure in mice. Our results show that the distribution of this heteromer is different in different hippocampal subregions. This technique holds the potential for being a reliable method to detect protein interactions at the subcellular level and unravel their unknown functions.

Objectives The present study clarified the role and signalling pathway of Ski in regulating proliferation and apoptosis in fibroblasts under high‐glucose (HG) conditions. Materials and Methods The proliferation and apoptosis of rat primary fibroblasts were assessed using EdU incorporation and TUNEL assays. The protein and phosphorylation levels of the corresponding factors were measured using immunofluorescence staining and Western blotting. Immunoprecipitation was used to determine the interactions between Ski and FoxO1 or Ski and HDAC1. The Ski protein was overexpressed via recombinant adenovirus transfection, and FoxO1 and HDAC1 were knocked down using targeted small‐interfering RNA. Results The present study found that HG inhibited fibroblast proliferation, increased apoptosis and reduced Ski levels in rat primary fibroblasts. Conversely, increasing Ski protein levels alleviated HG‐induced proliferation inhibition and apoptosis promotion. Increasing Ski protein levels also increased Ski binding to FoxO1 to decrease FoxO1 acetylation, and interfering with FoxO1 caused loss of the regulatory effect of Ski in fibroblasts under HG. Increasing Ski protein levels decreased FoxO1 acetylation via HDAC1‐mediated deacetylation. Conclusions Therefore, these findings confirmed for the first time that Ski regulated fibroblast proliferation and apoptosis under HG conditions via the FoxO1 pathway., Under HG conditions, FoxO1 acetylation level was increased. As a transcription factor, FoxO1 involved in the induction of a special subset of genes that regulate cellular proliferation or apoptosis, etc Increased Ski protein increased the binding of Ski to FoxO1, which mediated deacetylation through Ski binding with HDAC1 and resulted in a reduction in FoxO1 acetylation level. Therefore, the role of FoxO1 transcription factor was inhibited.

Background: Adenosine 2A receptor (A2AR) is involved in many physiological and pathological functions and serves as an important drug target. Inhibition of A2AR may alleviate symptoms associated wi...

Gastrointestinal stromal tumors (GISTs) are the most commonly diagnosed primary mesenchymal tumors of the gastrointestinal tract and 30% of GISTs are associated with a high recurrence risk or metastasis. The current risk classification criteria of the National Comprehensive Cancer Network are based on tumor size, mitotic activity and localization. Investigating additional biomarkers associated with clinical risk may aid in the diagnosis of GIST and improves prediction of patient prognosis. In the present study, the value of using the expression levels of the oncoprotein ski as a prognostic predictor for GISTs was investigated. The results demonstrated that high ski expression levels were correlated with high risk and recurrence rates and indicated poor prognosis regarding median disease-free survival. Overall, the present study suggests that ski expression levels may serve as a predictor for clinical risk and prognosis of patients with GISTs.

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A

BackgroundVacuolar sorting protein 35 (VPS35), a critical component of retromer, is essential for selective endosome-to-Golgi retrieval of membrane proteins. It is highly expressed in microglial cells, in addition to neurons. We have previously demonstrated microglial VPS35’s functions in preventing hippocampal, but not cortical, microglial activation, and in promoting adult hippocampal neurogenesis. However, microglial VPS35’s role in the cortex in response to ischemic stroke remains largely unclear.MethodsWe used mice with VPS35 cKO (conditional knockout) in microglial cells and examined and compared their responses to ischemic stroke with control mice. The brain damage, cell death, changes in glial cells and gene expression, and sensorimotor deficits were assessed by a combination of immunohistochemical and immunofluorescence staining, RT-PCR, Western blot, and neurological functional behavior tests.ResultsWe found that microglial VPS35 loss results in an increase of anti-inflammatory microglia in mouse cortex after ischemic stroke. The ischemic stroke-induced brain injury phenotypes, including brain damage, neuronal death, and sensorimotor deficits, were all attenuated by microglial VPS35-deficiency. Further analysis of protein expression changes revealed a reduction in CX3CR1 (CX3C chemokine receptor 1) in microglial VPS35-deficient cortex after ischemic stroke, implicating CX3CR1 as a potential cargo of VPS35 in this event.ConclusionTogether, these results reveal an unrecognized function of microglial VPS35 in enhancing ischemic brain injury-induced inflammatory microglia, but suppressing the injury-induced anti-inflammatory microglia. Consequently, microglial VPS35 cKO mice exhibit attenuation of ischemic brain injury response.

Excitatory amino acid transporters (EAATs) on cerebral vascular endothelial cells play an important role in maintaining glutamate homeostasis in the brain. The dysfunction of endothelial EAATs is an important reason for the dramatically elevated brain glutamate levels after brain injury, such as traumatic brain injury (TBI). The adenosine A2A receptor (A2AR) plays an important role in regulating the brain glutamate level after brain injury; however, researchers have not clearly determined whether this role was related to its ability to regulate endothelial EAATs. Activation of A2AR in vitro not only decreased the PKA- and glutamate level-dependent strengthening of the interaction between NKA-α1 and the FXYD1 subunit and the subsequent decrease in the activity of Na+/K+-ATPases (NKAs) but also enhanced its interaction with EAATs and ultimately aggravated the reverse transport function of endothelial EAATs under oxygen-glucose deprivation (OGD) conditions. Conversely, inhibition of A2AR restored the normal transport of EAAT. Moreover, A2AR inhibition increased NKA activity and decreased its interaction with EAATs in isolated brain capillaries after TBI, further confirming its role in endothelial EAATs in vivo. Based on our results, A2AR played an important role in regulating endothelial EAAT function, and strategies that restore the normal transport of endothelial EAATs through the inhibition of A2AR might serve as an effective treatment for brain injury.

Recent studies have shown that after traumatic brain injury (TBI), the number of autophagosomes is markedly increased in brain cells surrounding the wound; however, whether autophagy is enhanced or suppressed by TBI remains controversial. In our study, we used a controlled cortical impact system to establish models of mild, moderate and severe TBI. In the mild TBI model, the levels of autophagy-related protein 6 (Beclin1) and autophagy-related protein 12 (ATG12)-autophagy-related protein 5 (ATG5) conjugates were increased, indicating the enhanced initiation of autophagy. Furthermore, the level of the autophagic substrate sequestosome 1 (SQSTM1) was decreased in the ipsilateral cortex. This result, together with the results observed in tandem mRFP-GFP-LC3 adeno-associated virus (AAV)-infected mice, indicates that autophagosome clearance was also increased after mild TBI. Conversely, following moderate and severe TBI, there was no change in the initiation of autophagy, and autophagosome accumulation was observed. Next, we used chloroquine (CQ) to artificially impair autophagic flux in the injured cortex of the mild TBI model and found that the severity of trauma was obviously exacerbated. In addition, autophagic flux and trauma severity were significantly improved in adenosine A2A receptor (A2AR) knockout (KO) mice subjected to moderate TBI. Thus, A2AR may be involved in regulating the impairment of autophagic flux in response to brain injury. Our findings suggest that whether autophagy is increased after TBI is associated with whether autophagic flux is impaired, and the impairment of autophagic flux exacerbates the severity of trauma. Furthermore, A2AR may be a target for alleviating the impairment in autophagic flux after TBI.

Traumatic brain injury-induced acute lung injury (TBI-ALI) is a serious complication after brain injury for which predictive factors are lacking. In this study, we found significantly elevated blood glutamate concentrations in patients with TBI or multiple peripheral trauma (MPT), and patients with more severe injuries showed higher blood glutamate concentrations and longer durations of elevated levels. Although the increase in amplitude was similar between the two groups, the duration was longer in the patients with TBI. There were no significant differences in blood glutamate concentrations in the patients with MPT with regard to ALI status, but the blood glutamate levels were significantly higher in the patients with TBI-ALI than in those without ALI. Moreover, compared to patients without ALI, patients with TBI showed a clearly enhanced inflammatory response that was closely correlated with the blood glutamate levels. The blood glutamate concentration was also found to be a risk factor (adjusted odds ratio, 2.229; 95% CI, 1.082–2.634) and was a better predictor of TBI-ALI than the Glasgow Coma Scale (GCS) score. These results indicated that dramatically increased blood glutamate concentrations were closely related to the occurrence of TBI-ALI and could be used as a predictive marker for “at-risk” patients.

Pathogens such as bacterial lipopolysaccharide (LPS) play an important role in promoting the production of the inflammatory cytokines interleukin-1 beta (IL-1β) and tumour necrosis factor-α (TNF-α) in response to infection or damage in microglia. However, whether different signalling pathways regulate these two inflammatory factors remains unclear. The protein kinase C (PKC) family is involved in the regulation of inflammation, and our previous research showed that the activation of the PKC pathway played a key role in the LPS-induced transformation of the adenosine A

Trauma is an important health problem in modern society. Its serious consequences, such as sepsis and multiple organ dysfunction, are the leading causes of high morbidity and mortality. The heat shock proteins (HSP) increase rapidly under a wide variety of stressful conditions to protect cell from damage. Overexpression of HSP is associated with apoptosis inhibition, inflammatory response and immunomodulation in severe trauma. Therefore, the changes in expression levels of various heat shock proteins or the differences in genetic background will affect the processes and medical outcomes of patients or animal models of diseases. Most importantly, HSP play differential and complex roles in the regulation of inflammation and immunity at different pathological stages and locations after severe trauma. In addition to the traditional therapeutic methods, strategies by the control of HSP expression and consideration of individual genetic background will pave the way for the better treatment and prediction of serious complications caused by severe trauma. In this chapter, the recent progresses of HSP in severe trauma and its complications and the possibilities of HSP as therapeutic targets will be discussed.

We previously demonstrated that cellular Sloan-Kettering Institute (c-Ski) played a dual role, both promoting wound healing and alleviating scar formation. However, its mechanism and therapeutic effects are not clear, especially compared with widely used treatments, such as basic fibroblast growth factor (bFGF) administration. However, Ski treatment led to an even shorter healing time and a more significant reduction in scar area than bFGF treatment. The mechanism underlying this difference was related to a reduced inflammatory response, more rapid re-epithelialization, less collagen after healing and a greater reduction in the proportion of alpha-smooth muscle actin and SMemb-positive cells after Ski treatment. These results not only confirm that Ski plays a dual role in promoting healing and reducing scarring but also suggest that Ski yields better treatment effects than bFGF, indicating better potential therapeutic effects in wound repair.

Purpose Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. Methods The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. Results The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. Conclusions These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.

Electromagnetic methods have been applied in many advanced applications, such as circuit design, geophysical exploration, medical imaging, and environmental perception. Since it can represent a high-fidelity virtual experiment quite close to the real world, these methods are more reliable than approximate methods. However, as working frequency gets smaller and smaller, the discretized linear system become poorly conditioned and thus difficult to solve. Typical examples include Airborne Transient Electromagnetics (ATEM) systems and Control-Source Electromagnetics (CSEM) Systems, either in time domain or in frequency domain. The purpose of this study is to develop an effective method for solving any desired frequency system especially including these low-frequency electromagnetic models. Here we propose a spectral element method (SEM) with the divergence free constraint and apply it to solve subsurface electromagnetic exploration problem. Because Gauss’ law is now explicitly enforce, making system matrix well-conditioned even at extremely low frequency, we have capability to solve the linear system which cannot be solved correctly before. Several numerical experiments show that the proposed method is accuracy and efficiency.

Aim To explore the more suitable concentration of glutamate or N-methyl-D-aspartic acid (NMDA) for intravitreal injection to establish a rat model of retinal neurodegeneration. Methods We injected different doses of glutamate (20 or 50 nmol) or NMDA (40 nmol) into the vitreous chambers of rats, then measured the concentration of glutamate and retinal thickness, quantified apoptotic cells and determined the degree of tau hyperphosphorylation at different time points. T-test was used for comparison of two groups. One-way ANOVA and Turkey's multiple comparisons test were used for comparisons of different groups, and P values below 0.05 were considered statistically significant. Results The glutamate level in the rats treated with 50 nmol of glutamate was twice that of the control group and persisted two weeks. Seven days after intravitreal injection of 50 nmol of glutamate, three parameters [inner retinal thickness (IRT), retinal thickness (RT) and ganglion cell layer (GCL) cell number] were reduced significantly. Furthermore, numerous TUNEL-positive cells were observed in the GCL one day after intravitreal injection of 50 nmol of glutamate, the expression of the apoptosis-related factor cleaved casepase-3 was markedly increased compared with the expression levels in the other treatment groups, and the expression levels of tau s396 and tau s404 were significantly increased compared with those in the control group. Conclusion This study demonstrates that the intravitreal injection of 50 nmol of glutamate can establish the more effective retinal neurodegeneration animal model relative to other treatment groups.

Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-β1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-β1 concentrations, but the mechanism by which low TGF-β1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-β1 concentrations not only promoted c-ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-β1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-β1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-β1/Smad pathway.

Excitatory amino acid transporters (EAATs) on cerebral vascular endothelial cells play an important role in maintaining glutamate homeostasis in the brain. The dysfunction of endothelial EAATs is an important reason for the dramatically elevated brain glutamate levels after brain injury, such as traumatic brain injury (TBI). The adenosine A

BackgroundAcute lung injury (ALI) is a serious complication of stroke that occurs with a high incidence. Our preclinical results indicated that ALI might be related to blood glutamate levels after brain injury. The purpose of this study was to assess dynamic changes in blood glutamate levels in patients with stroke and to determine the correlation between blood glutamate levels, ALI, and long-term prognosis after stroke.MethodsVenous blood samples were collected from controls and patients with stroke at admission and on the third and seventh day after the onset of stroke. Patients were followed for 3 months. The correlations among blood glutamate levels, severities of stroke and ALI, and long-term outcomes were analyzed, and the predictive values of blood glutamate levels and severity scores for ALI were assessed.ResultsIn this study, a total of 384 patients with stroke were enrolled, with a median age of 59 years. Patients showed significantly increased blood glutamate levels within 7 days of stroke onset (p

It is widely accepted that glutamate is the most important excitatory neurotransmitter in the central nervous system (CNS). However, there is also a large amount of glutamate in the blood. Generally, the concentration gradient of glutamate between intraparenchymal and blood environments is stable. However, this gradient is dramatically disrupted under a variety of pathological conditions, resulting in an amplifying cascade that causes a series of pathological reactions in the CNS and peripheral organs. This eventually seriously worsens a patient’s prognosis. These two “isolated” systems are rarely considered as a whole even though they mutually influence each other. In this review, we summarize what is currently known regarding the maintenance, imbalance and regulatory mechanisms that control the intraparenchymal-blood glutamate concentration gradient, discuss the interrelationships between these systems and further explore their significance in clinical practice.

Explosive blast injury has become the most prevalent injury in recent military conflicts and terrorist attacks. The magnitude of this kind of polytrauma is complex due to the basic physics of blast and the surrounding environments. Therefore, development of stable, reproducible and controllable animal model using an ideal blast simulation device is the key of blast injury research. The present review addresses the modeling of blast injury and applications of shock tubes.

With the increasing incidence of blast injury, the research on its mechanisms and protective measures draws more and more attention. Blast injury has many characteristics different from general war injuries or trauma. For example, soldiers often have various degrees of visceral injury without significant surface damage, combined injuries and arterial air embolism. Researchers in China began to investigate blast injury later than the United States and Sweden, but the development is so fast that lots of achievements have been gained, including the development of biological shock tube, the mechanisms and characteristics of blast injury in various organs, as well as protective measures under special environments. This article reviews the past and current situation of blast injury research in China.

Cognitive impairments in Huntington's disease (HD) are attributed to a dysfunction of the cortico-striatal pathway and significantly affect the quality of life of the patients, but this has not been a therapeutic focus in HD to date. We postulated that adenosine A(2A) receptors (A(2A)R), located at pre- and post-synaptic elements of the cortico-striatal pathways, modulate striatal neurotransmission and synaptic plasticity and cognitive behaviors. To critically evaluate the ability of A(2A)R inactivation to prevent cognitive deficits in early HD, we cross-bred A(2A)R knockout (KO) mice with two R6/2 transgenic lines of HD (CAG120 and CAG240) to generate two double transgenic R6/2-CAG120-A(2A)R KO and R6/2-CAG240-A(2A)R KO mice and their corresponding wild-type (WT) littermates. Genetic inactivation of A(2A)R prevented working memory deficits induced by R6/2-CAG120 at post-natal week 6 and by R6/2-CAG240 at post-natal month 2 and post-natal month 3, without modifying motor deficits. Similarly the A2(A)R antagonist KW6002 selectively reverted working memory deficits in R6/2-CAG240 mice at post-natal month 3. The search for possible mechanisms indicated that the genetic inactivation of A(2A)R did not affect ubiquitin-positive neuronal inclusions, astrogliosis or Thr-75 phosphorylation of DARPP-32 in the striatum. Importantly, A(2A)R blockade preferentially controlled long-term depression at cortico-striatal synapses in R6/2-CAG240 at post-natal week 6. The reported reversal of working memory deficits in R6/2 mice by the genetic and pharmacological inactivation of A(2A)R provides a proof-of-principle for A(2A)R as novel targets to reverse cognitive deficits in HD, likely by controlling LTD deregulation.

Background: Nogo-A, a major myelin-associated inhibitor, can inhibit injured optic nerve regeneration. However, whether Amino-Nogo is the most important functional domain of Nogo-A remains unknown. This study aimed to identify the role of Amino-Nogo following optic nerve injury, and the mechanism of the Amino-Nogo-integrin αv signaling pathway in vivo. Methods: Sprague-Dawley rats with optic nerve crush injury were injected with Nogo-A siRNA (Nogo-A-siRNA), the Nogo-66 functional domain antagonist peptide of Nogo-A (Nep1-40) or a recombinant rat Amino-Nogo-A protein (∆20) into the vitreous cavity to knock down Nogo-A, inhibit Nogo-66 or activate the Amino-Nogo, resparately. Retinal ganglion cell (RGC) density, axon regeneration and the pattern of NPN of visual electrophysiology (flash visual evoked potentials [F-VEP]) at different times post-injury were investigated. Results: Our study revealed a lower RGC survival rate; shorter axonal outgrowth; longer N1, P1 and N2 waves latencies; and lower N1-P1 and P1-N2 amplitudes in the Δ20 group, and Δ20 treatment significantly attenuated integrin αv expression and phosphorylated focal adhesion kinase (p-FAK) levels. In the Nep1-40 and Nogo-A siRNA groups, there were higher RGC survival rates, longer axonal outgrowth, shorter N1 and P1 wave latencies, and higher N1-P1 and P1-N2amplitudes. Nogo-A siRNA treatment significantly increased integrin αv expression and p-FAK levels. Nepl-40 treatment did not alter integrin αv expression. In addition, there was no significant change in integrin α5 in any group. Conclusion: These results suggest that the integrin signaling pathway is regulated by Amino-Nogo, which inhibits optic nerve regeneration and functional recovery, and that the integrin subunit involved might be integrin αv but not integrin α5.

Glucocorticoids are commonly used for the treatment of pancreatitis and complicated acute lung injury and help to reduce the mortality rates of both. The effect of gene variants in heat shock protein 90 (Hsp90), a key chaperone molecule of the glucocorticoid receptor (GR), on the therapeutic effect of glucocorticoids is unclear. Our study aims to investigate the different susceptibility to glucocorticoid treatment in BALB/c and C57BL/6 mice carrying different Hsp90 genotypes in an animal model of pancreatitis-induced lung injury. Compared with BALB/c mice, C57BL/6 mice have lower mortality rates, decreased water content in their lungs, and a lower level of IL-1 beta in an animal model of acute pancreatitis. C57BL/6 mice show a greater therapeutic effect and increased GR binding activities with glucocorticoid responsive element compared to BALB/c mice after a 0.4 mg/kg dexamethasone (DEX) treatment. Treatment with a higher dose of DEX (4 mg/kg) significantly reduced mortality rates and increased GR-GRE binding activity in both strains of mice, and there was no significant difference between the two strains. DEX did not exert a protective role after geldanamycin, a specific inhibitor of Hsp90, was administered in both strains of mice. Our study revealed that Hsp90 gene variants are responsible for the greater therapeutic effect of DEX in C57BL/6 mice compared to BALB/c mice, which implies that combining DEX treatment with Hsp90 regulation would promote the efficiency of DEX and would be an effective way to alleviate the side effects of hormone therapy.

Traumatic brain injury (TBI) can induce cognitive dysfunction due to the regional accumulation of hyperphosphorylated tau protein (p-tau). However, the factors that cause p-tau to concentrate in specific brain regions remain unclear. Here, we show that AQP4 polarization in the perivascular astrocytic end feet was impaired after TBI, which was most prominent in the ipsilateral brain tissue surrounding the directly impacted region and the contralateral hippocampal CA1 area and was accompanied by increased local p-tau, changes in dendritic spine density and morphology, and upregulation of the adenosine A2A receptor (A2AR). The critical role of the A2AR signaling in these pathological changes was confirmed by alleviation of the impairment of AQP4 polarity and accumulation of p-tau in the contralateral CA1 area in A2AR knockout mice. Given that p-tau can be released to the extracellular space and that the astroglial water transport via AQP4 is involved in tau clearance from the brain interstitium, our results suggest that regional disruption of AQP4 polarity following TBI may reduce the clearance of the toxic interstitial solutes such as p-tau and lead to changes in dendritic spine density and morphology. This may explain why TBI patients are more vulnerable to cognitive dysfunction.