Search

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.

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

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.

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.

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.

A series of neurological and psychiatric symptoms occur after traumatic brain injury (TBI), with cognitive dysfunction being one of the most prominent sequela. Given that tau hyperphosphorylation is an important cause of cognitive impairment in patients of Alzheimer's disease, our present study detected the presence of hyperphosphorylated tau (p-tau), mainly at Ser404, in multiple brain regions, including the ipsilateral parietal cortex, contralateral hippocampus and prefrontal cortex, immediately after the injury in a mouse TBI model; these changes lasted for at least 4w. All of these brain regions play important roles in working memory. Hyperphosphorylated tau protein was primarily located in neurons and was accompanied by axonal injury and dendritic spine degeneration. Our study demonstrated that p-tau spreads gradually and selectively from the injured cortex to other brain regions after TBI and that all of the affected regions are part of the working memory circuit. These findings provide experimental support for the role of p-tau in cognitive impairment in the early phase after TBI.

Adenosine A2A receptor (A2AR) plays an important regulatory role in the processes of inflammation and fibrosis. However, it is unknown whether A2AR can mediate renal interstitial fibrosis (RIF). To evaluate the effect of genetic A2AR knockout (KO) on the pathological progress of RIF, we applied a unilateral ureteral obstruction (UUO) model of RIF on A2AR KO mice and their wild-type (WT) littermates. Renal pathological assessment was performed at different post-UUO stages using hematoxylin and eosin (HE) and Masson's trichrome staining as well as quantitative morphological analysis. Our data demonstrated that: (i) the extent of RIF was determined by the development of UUO in a time-dependent manner; (ii) A2AR KO exacerbated the pathological progress of RIF in mice at the early post-UUO stage, i.e. day 3 and day 7; (iii) the profibrotic effect of A2AR KO was prominent until the late post-UUO stage, i.e. day 14, at which RIF reached a similar severity level in A2AR KO and WT mice. Our findings revealed that A2AR KO significantly exacerbated the progression of UUO-induced RIF in mice, prominently at the initial stage.

Activation of adenosine A2A receptor aggravates lung damage in a neurogenic mouse model of acute lung injury (ALI) but protects against nonneurogenic ALI., The bone marrow–derived cell (BMDC)–associated inflammatory response plays a key role in the development of acute lung injury (ALI). Activation of adenosine A2A receptor (A2AR) is generally considered to be antiinflammatory, inhibiting BMDC activities to protect against ALI. However, in the present study, we found that in a mouse model of neurogenic ALI induced by severe traumatic brain injury (TBI), BMDC A2AR exerted a proinflammatory effect, aggravating lung damage. This is in contrast to the antiinflammatory effect observed in the mouse oleic acid–induced ALI model (a nonneurogenic ALI model.) Moreover, the A2AR agonist CGS21680 aggravated, whereas the antagonist ZM241385 attenuated, the severe TBI-induced lung inflammatory damage in mice. Further investigation of white blood cells isolated from patients or mouse TBI models and of cultured human or mouse neutrophils demonstrated that elevated plasma glutamate after severe TBI induced interaction between A2AR and the metabotropic glutamate receptor 5 (mGluR5) to increase phospholipase C–protein kinase C signaling, which mediated the proinflammatory effect of A2AR. These results are in striking contrast to the well-known antiinflammatory and protective role of A2AR in nonneurogenic ALI and indicate different therapeutic strategies should be used for nonneurogenic and neurogenic ALI treatment when targeting A2AR.

In addition to spinal fracture and/or dislocation, acute traumatic spinal cord injury (SCI) includes neural tissue and vascular damage at the site of contusion. Vascular damage results in spinal cord ischemia, which is a key factor that contributes to secondary pathogenesis. There is a need to identify spinal cord ischemia secondary to anterior spinal artery (ASA) rupture in acute cervical SCI. After admission, 20 patients with cervical SCI, including 10 cases of central cord syndrome (CCS), four cases of brown-sequard syndrome (BSS) and six cases of tetraplegia (ASIA A), had computed tomographic angiography (CTA) of ASA performed on them. ASA rupture or occlusion was observed. The ASA was visualized in all 20 patients. No ASA rupture was found in CCS and BSS patients, even for sever blunt cervical fracture and dislocation tetraplegia patients, except one stab-wound patient. CTA provides the most detailed, highest resolution imaging of ASA in acute cervical SCI. ASA rupture is not commonly seen in acute blunt cervical SCI.

Background/Aims: Although it has been reported that somatostatin (SOM) upregulated the level of 90-kD heat shock protein (Hsp90), which participates in the inflammatory regulation by its client proteins, such as glucocorticoid receptor (GR), it remains unclear if it has a protective role against acute lung injury (ALI). Methods: ALI model was established by the injection of oleic acid (OA) into the tail vein of mice. Lung injury was assessed by histological analysis, lung water content and arterial blood gases. The levels of Hsp90 and GR, the binding capacity and the affinity of GR were examined. Results: It was showed that pretreatment with SOM significantly increased Hsp90 levels and alleviated lung injuries in OA-injected mice. Furthermore, SOM increased the GR expression and improved the affinity of the GR in animals with lung injury. However, little alteration was found in the maximum binding capacity of the GR in mice with or without SOM. Conclusion: The data indicate SOM exerts a protective effect by increasing Hsp90 abundant and further enhancing the affinity of the GR. The beneficial effects of SOM treatment provide a new strategy for modulation of GR efficiency and alleviation of acute lung injury.

OBJECTIVE: To investigate the effects of three different ways of chronic caffeine administration on blast- induced memory dysfunction and to explore the underlying mechanisms. METHODS: Adult male C57BL/6 mice were used and randomly divided into five groups: control: without blast exposure, con-water: administrated with water continuously before and after blast-induced traumatic brain injury (bTBI), con-caffeine: administrated with caffeine continuously for 1 month before and after bTBI, pre-caffeine: chronically administrated with caffeine for 1 month before bTBI and withdrawal after bTBI, post-caffeine: chronically administrated with caffeine after bTBI. After being subjected to moderate intensity of blast injury, mice were recorded for learning and memory performance using Morris water maze (MWM) paradigms at 1, 4, and 8 weeks post-blast injury. Neurological deficit scoring, glutamate concentration, proinflammatory cytokines production, and neuropathological changes at 24 h, 1, 4, and 8 weeks post-bTBI were examined to evaluate the brain injury in early and prolonged stages. Adenosine A1 receptor expression was detected using qPCR. RESULTS: All of the three ways of chronic caffeine exposure ameliorated blast-induced memory deficit, which is correlated with the neuroprotective effects against excitotoxicity, inflammation, astrogliosis and neuronal loss at different stages of injury. Continuous caffeine treatment played positive roles in both early and prolonged stages of bTBI; pre-bTBI and post-bTBI treatment of caffeine tended to exert neu- roprotective effects at early and prolonged stages of bTBI respectively. Up-regulation of adenosine A1 receptor expression might contribute to the favorable effects of chronic caffeine consumption. CONCLUSION: Since caffeinated beverages are widely consumed in both civilian and military personnel and are convenient to get, the results may provide a promising prophylactic strategy for blast-induced neurotrauma and the consequent cognitive impairment. Language: en

Background/Aim: Both peroxisome proliferator-activated receptor (PPAR) δ and Ski are investigate the interaction of PPARδ and Ski and this interaction-associated effect in wound healing. Methods: Effect of PPARδ activation on Ski expression was detected in rat skin fibroblasts by real-time PCR and western blot. Luciferase assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay were performed to identify the binding site of PPARδ in the promoter region of rat Ski gene. And the functional activity of PPARδ regulation to Ski was detected in fibroblast proliferation and rat skin wound healing model. Results: PPARδ agonist GW501516 upregulated Ski expression in a dose-dependent manner. Direct repeat-1 (DR1) response element locating at -865∼-853 in Ski promoter region was identified to mediate PPARδ binding to Ski and associated induction of Ski. Furthermore, PPARδ upregulated Ski to promote fibroblasts proliferation and rat skin wound repair, which could be largely blocked by pre-treated with Ski RNA interference. Conclusion: This study demonstrates that Ski is a novel target gene for PPARδ and upregulation of Ski to promote fibroblast proliferation is implicated in the PPARδ-mediated wound healing.

This review summarizes recent developments that have contributed to our understanding of how adenosine 2A receptors (A2ARs) modulate brain damage in various animal models of acute neurological injuries, including brain ischemia, traumatic brain injury, spinal cord injury and hemorrhage stroke. The main conclusions are: (1) pharmacological, neurochemical and molecular/genetic approaches to the complex actions of A2AR in different cellular elements suggest that A2AR activation exerts bidirectional effect (detrimental or protective) after brain insults; (2) modulation of glutamate excitotoxicity and neuroinflammation are involved in the protection of A2AR agonists or antagonists, but the bidirectional effect of A2AR is largely due to the bidirectional regulation of neuroinflammation (anti-inflammation or proinflammation) by A2AR on immune cells such as microglia cells and peripheral bone marrow cells; and (3) the bidirectional effect of A2AR on neuroinflammation and brain injury depends on the distinct and sometimes opposite actions of A2AR in various cellular elements and on different injury models and associated pathological conditions. The local glutamate level in the brain injury is one of the crucial factors that contribute to the direction of A2AR effect on neuroinflammation and brain injury outcome. These developments presented here clearly highlight the complexity of using A2AR agents therapeutically in acute neuronal injuries and confirm that A2AR ligands have many promising characteristics that encourage the pursuit of their full therapeutic potential.

We recently demonstrated that Ski is a novel wound healing-related factor that promotes fibroblast proliferation and inhibits collagen secretion. Here, we show that increasing local Ski expression by gene transfer not only significantly accelerated wound healing by relieving inflammation, accelerating re-epithelialization and increasing formation of granulation tissue, but also reduced scar formation by decreasing collagen production in rat dermal wounds. Similarly, ski gene transfer accelerated wound healing, reduced the protuberant height and volume of scars and increased collagen maturity in a hypertrophic scar model in the rabbit ear. Conversely, reducing Ski expression in the wound by RNA interference resulted in significantly slower wound healing and increased scar area in rat dermal wounds. We demonstrated that these effects of Ski are associated with transforming growth factor-β-mediated signalling pathways through both Smad2/3-dependent and Smad-independent pathways. Together, our results define a dual role for Ski in promoting wound healing and alleviating scar formation, identifying a new target for therapeutic approaches to preventing scar hyperplasia and accelerating wound healing.

During brain injury, extracellular adenosine and glutamate levels increase rapidly and dramatically. We hypothesized that local glutamate levels in the brain dictates the adenosine–adenosine A2Areceptor (A2AR) effects on neuroinflammation and brain damage outcome. Here, we showed that, in the presence of low concentrations of glutamate, the A2AR agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) inhibited lipopolysaccharide (LPS)-induced nitric oxide synthase (NOS) activity of cultured microglial cells, an effect that was dependent on the protein kinase A (PKA) pathway. However, in high concentrations of glutamate, CGS21680 increased LPS-induced NOS activity in a protein kinase C (PKC)-dependent manner. Thus, increasing the local level of glutamate redirects A2AR signaling from the PKA to the PKC pathway, resulting in a switch in A2AR effects from antiinflammatory to proinflammatory. In a cortical impact model of traumatic brain injury (TBI) in mice, brain water contents, behavioral deficits, and expression of tumor necrosis factor-α, interleukin-1 mRNAs, and inducible NOS were attenuated by administering CGS21680 at post-TBI time when brain glutamate levels were low, or by administering the A2AR antagonist ZM241385 [4-(2-{[5-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-yl]amino}ethyl)phenol] at post-TBI time when brain glutamate levels were elevated. Furthermore, pre-TBI treatment with the glutamate release inhibitor (S)-4C3HPG [(S)-4-carboxy-3-hydroxyphenylglycine] converted the debilitating effect of CGS21680 administered at post-TBI time with high glutamate level to a neuroprotective effect. This further indicates that the switch in the effect of A2AR activation in intact animals from antiinflammatory to proinflammatory is dependent on glutamate concentration. These findings identify a novel role for glutamate in modulation of neuroinflammation and brain injury via the adenosine–A2AR system.

Heat shock protein 90 (Hsp90), encoded by the murine hsp84 and hsp86 genes in mice, is a pivotal regulator of glucocorticoid receptor (GR) function in the hypothalamus-pituitary-adrenal axis and affords stress protection. To explore the underlying molecular mechanisms of strain susceptibility to traumatic stress, we investigated the alteration by Hsp90 of the function of the glucocorticoid-glucocorticoid receptor (GC-GR) pathway in attenuating stress responses in C57BL/6 and BALB/c mice using the whole-body blast injury (WBBI) model. We found that C57BL/6 mice had a lower WBBI-induced mortality, higher nuclear GR level, and higher glucocorticoid-response element (GRE) binding activity than BALB/c mice. This study is the first report identifying four genetic variations of the murine hsp84 gene: 226AC, 996GC, 1483GC, and 2000GT. These nucleotide changes occur in the functional domains associated with the nuclear/cytosolic translocation of GR, GR-Hsp90 interaction, ATP binding, and self-dimerization of Hsp90, respectively. Further, we used a specific Hsp90 inhibitor, geldanamycin (GA), to assess the role of Hsp90 in the discriminative traumatic response in C57BL/6 mice. Pretreatment with GA reduced nuclear GR levels and GRE binding activity, and enhanced WBBI-induced mortality. These findings suggest that Hsp90 may underlie the strain-selective (C57BL/6 versus BALB/c) susceptibility to WBBI by mediating the nuclear translocation of GRs and GRE binding. Thus, pharmacological manipulation of Hsp90 may represent a therapeutic strategy to modify the function of the GC-GR pathway and traumatic stress response.

Caffeine, the most consumed psychoactive drug and non-specific adenosine receptor antagonist, has recently been shown to exert a neuroprotective effect against brain injury in animal models of Parkinson’s disease (PD) and stroke. However, the effects of caffeine on traumatic brain injury (TBI) are not known. In this study, we investigated the effects of acute and chronic caffeine treatment on brain injury in a cortical-impact model of TBI in mice. Following TBI, neurological deficits, cerebral edema, as well as inflammatory cell infiltration were all significantly attenuated in mice pretreated chronically (for 3 weeks) with caffeine in drinking water compared with the mice pretreated with saline. Furthermore, we found that chronic caffeine treatment attenuated glutamate release and inflammatory cytokine production, effects that were correlated with an upregulation of brain A 1 receptor mRNA. By contrast, acute treatment with caffeine (i.p. injection, 30 min before TBI) was not effective in protecting against TBI-induced brain injury. These results suggest that chronic (but not acute) caffeine treatment attenuates brain injury, possibly by A 1 receptor-mediated suppression of glutamate release and inhibition of excessive inflammatory cytokine production. These results highlight the potential benefit of chronic caffeine intake for preventing TBI and provide a rationale for the epidemiological investigation of the potential association between TBI and human caffeine intake.

C-ski is a complicated regulating factor for fibroblast proliferation and an important co-repressor of Smad3. Although inhibiting Smad3 activity can markedly promote wound healing because Smad3 mediates the role of transforming growth factor-beta in inhibiting cell proliferation and inducing cell apoptosis; there has been no report on whether c-ski is expressed during wound healing and the relationship between its expression and wound healing. By establishing animal models of normal and radiation-impaired wound healing and using immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction, we found that c-ski was expressed after wounding and reached its peak on day 9 and then significantly decreased. C-ski was present in all repair cells, and was especially prominent in fibroblasts. Compared with the control side, the irradiated side showed a lower expression of c-ski on postwound days 3-9, but higher on day 15, and not significantly different after the wound was healed. The expression of Smad3 was in contrast to the c-ski and cellular proliferation was similar to that of c-ski expression. The apoptosis index was significantly higher on the irradiated side on days 3-9 compared with the control side. In vitro, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide results showed that c-ski could reverse the inhibitory role of Smad3 on fibroblast proliferation. Flow cytometry analysis found that c-ski also diminished fibroblast apoptosis induced by Smad3 transfection. These results suggest that there is not only obvious expression of this regulatory protein but there is also a significant change in the levels of c-ski during wound healing. Its in vivo expression pattern and experiments in vitro suggest that c-ski may be involved in tissue repair by repressing Smad3 activity. Radiation can reduce c-ski and increase Smad3 expression, resulting in elevated Smad3 activity, resulting in diminished cell proliferation, cell apoptosis, and wound-healing delays.

Tau is a microtubule-associated protein, and the oligomeric and hyperphosphorylated forms of tau are increased significantly after neurotrauma and considered important factors in mediating cognitive dysfunction. Blockade of adenosine A2A receptors, either by caffeine or gene knockout (KO), alleviates cognitive dysfunction after traumatic brain injury (TBI). We postulated that A2AR activation exacerbates cognitive impairment via promoting tau hyperphosphorylation. Using a mouse model of moderate controlled cortical impact, we showed that TBI induced hyperphosphorylated tau (p-tau) in the hippocampal dentate gyrus and spatial memory deficiency in the Morris water maze test at 7 days and 4 weeks after TBI. Importantly, pharmacological blockade (A2AR antagonist ZM241385 or non-selective adenosine receptor antagonist caffeine) or genetic inactivation of A2ARs reduced the level of tau phosphorylation at Ser404 and alleviated spatial memory dysfunction. The A2AR control of p-tau is further supported by the observations that a KO of A2AR decreased the activity of the tau phosphorylation kinases, glycogen synthase kinase-3β (GSK-3β) and protein kinase A (PKA) after TBI, and by that CGS21680 (A2AR agonist) exacerbated okadaic acid-induced tau hyperphosphorylation in cultured primary hippocampal neurons. Lastly, CGS21680-induced neuronal tau hyperphosphorylation and axonal injury were effectively alleviated by individual treatments with ZM241385 (A2AR antagonist), H89 (PKA antagonist) and SB216763 (GSK-3β antagonist), or by the combined treatment with H89 and SB216763. Our findings suggest a novel mechanism whereby A2AR activation triggers cognitive dysfunction by increasing the phosphorylation level of tau protein after TBI and suggest a promising therapeutic and prophylactic strategy by targeting aberrant A2AR signaling via tau phosphorylation.

Traumatic brain injury (TBI), particularly explosive blast-induced TBI (bTBI), has become the most prevalent injury among military personnel. The disruption of cognitive function is one of the most serious consequences of bTBI because its long-lasting effects prevent survivors fulfilling their active duty and resuming normal civilian life. However, the mechanisms are poorly understood and there is no treatment available. This study investigated the effects of adenosine A2A receptor (A2AR) on bTBI-induced cognitive deficit, and explored the underlying mechanisms. After being subjected to moderate whole-body blast injury, mice lacking the A2AR (A2AR knockout (KO)) showed less severity and shorter duration of impaired spatial reference memory and working memory than wild-type mice did. In addition, bTBI-induced cortical and hippocampal lesions, as well as proinflammatory cytokine expression, glutamate release, edema, cell loss, and gliosis in both early and prolonged phases of the injury, were significantly attenuated in A2AR KO mice. The results suggest that early injury and chronic neuropathological damages are important mechanisms of bTBI-induced cognitive impairment, and that the impairment can be attenuated by preventing A2AR activation. These findings suggest that A2AR antagonism is a potential therapeutic strategy for mild-to-moderate bTBI and consequent cognitive impairment.