Cortical and Subcortical Alterations and Clinical Correlates after Traumatic Brain Injury.

Background: Traumatic brain injury (TBI) often results in persistent cognitive impairment and psychiatric symptoms, while lesion location and severity are not consistent with its clinical complaints. Previous studies found cognitive deficits and psychiatric disorders following TBI are considered to be associated with prefrontal and medial temporal lobe lesions, however, the location and extent of contusions often cannot fully explain the patient's impairments. Thus, we try to find the structural changes of gray matter (GM) and white matter (WM), clarify their correlation with psychiatric symptoms and memory following TBI, and determine the brain regions that primary correlate with clinical measurements. Methods: Overall, 32 TBI individuals and 23 healthy controls were recruited in the study. Cognitive impairment and psychiatric symptoms were examined by Mini-Mental State Examination (MMSE), Hospital Anxiety and Depression Scale (HADS), and Wechsler Memory Scale-Chinese Revision (WMS-CR). All MRI data were scanned using a Siemens Prisma 3.0 Tesla MRI system. T1 MRI data and diffusion tensor imaging (DTI) data were processed to analyze GM volume and WM microstructure separately. Results: In the present study, TBI patients underwent widespread decrease of GM volume in both cortical and subcortical regions. Among these regions, four brain areas including the left inferior temporal gyrus and medial temporal lobe, supplementary motor area, thalamus, and anterior cingulate cortex (ACC) were highly implicated in the post-traumatic cognitive impairment and psychiatric complaints. TBI patients also underwent changes of WM microstructure, involving decreased fractional anisotropy (FA) value in widespread WM tracts and increased mean diffusivity (MD) value in the forceps minor. The changes of WM microstructure were significantly correlated with the decrease of GM volume. Conclusions: TBI causes widespread cortical and subcortical alterations including a reduction in GM volume and change in WM microstructure related to clinical manifestation. Lesions in temporal lobe may lead to more serious cognitive and emotional dysfunction, which should attract our high clinical attention.

Structural and Functional Alterations of Substantia Nigra and Associations With Anxiety and Depressive Symptoms Following Traumatic Brain Injury.

Backgrounds: Although there are a certain number of studies dedicated to the disturbances of the dopaminergic system induced by traumatic brain injury (TBI), the associations of abnormal dopaminergic systems with post-traumatic anxiety and depressive disorders and their underlying mechanisms have not been clarified yet. In the midbrain, dopaminergic neurons are mainly situated in the substantia nigra (SN) and the ventral tegmental area (VTA). Thus, we selected SN and VTA as regions of interest and performed a seed-based global correlation to evaluate the altered functional connectivity throughout the dopaminergic system post-TBI. Methods: Thirty-three individuals with TBI and 21 healthy controls were recruited in the study. Anxiety and depressive symptoms were examined by the Hospital Anxiety and Depression Scale. All MRI data were collected using a Siemens Prisma 3.0 Tesla MRI system. The volume of SN and the global functional connectivity of the SN and VTA were analyzed. Results: In the present study, patients with TBI reported more anxiety and depressive symptoms. More importantly, some structural and functional alterations, such as smaller SN and reduced functional connectivity in the left SN, were seen in individuals with TBI. Patients with TBI had smaller substantia nigra on both right and left sides, and the left substantia nigra was relatively small in contrast with the right one. Among these findings, functional connectivity between left SN and left angular gyrus was positively associated with post-traumatic anxiety symptoms and negatively associated with depressive symptoms. Conclusions: The TBI causes leftward lateralization of structural and functional alterations in the substantia nigra. An impaired mesocortical functional connectivity might be implicated in post-traumatic anxiety and depression.

Decreased Interhemispheric Functional Connectivity and Its Associations with Clinical Correlates following Traumatic Brain Injury.

Objective: To explore the interhemispheric functional coordination following traumatic brain injury (TBI) and its association with posttraumatic anxiety and depressive symptoms. Methods: This was a combination of a retrospective cohort study and a cross-sectional observational study. We investigated the functional coordination between hemispheres by voxel-mirrored homotopic connectivity (VMHC). Grey matter volumes were examined by voxel-based morphometry (VBM), and microstructural integrity of the corpus callosum (CC) was assessed by diffusion tension imaging (DTI). The anxiety and depressive symptoms were evaluated with the Hospital Anxiety and Depression Scale. Results: The VMHC values of the bilateral middle temporal gyrus (MTG) and orbital middle frontal gyrus (MFG) were significantly decreased in TBI patients versus the healthy controls. Weakened homotopic functional connectivity (FC) in the bilateral orbital MFG is moderate positively correlated with anxiety and depressive symptoms. The white matter integrity in the CC was extensively reduced in TBI patients. In the receiver operating characteristic analysis, the VMHC value of the orbital MFG could distinguish TBI from HC with an area under the curve of 0.939 (sensitivity of 1 and specificity of 0.867). Conclusion: TBI disrupts the interhemispheric functional and structural connection, which is correlated with posttraumatic mood disorders. These findings may serve as a clinical indicator for diagnosis.

Association of Anxiety and Depressive Symptoms with Memory Function following Traumatic Brain Injury.

INTRODUCTION: Memory impairment and mood disorders are among the most troubling sequelae following traumatic brain injury (TBI). The relationships between comorbid psychiatric disorders and memory function have not been well illustrated. The aim of the study was to explore the relationships of comorbid anxiety and depressive symptoms with memory function following TBI. METHODS: A total of 46 TBI participants across all levels of injury and 23 healthy controls were enrolled in this case-control study. Wechsler Memory Scale-Chinese Revision (WMS-CR) picture, recognition, associative learning, comprehension memory, and digit span were administered to evaluate several categories of memory capacity. The Hospital Anxiety and Depression Scale (HADS) was employed to evaluate the anxiety and depressive symptoms. Stepwise multiple linear regressions were conducted. RESULTS: Compared to healthy controls, the participants with TBI reported more anxiety and depressive symptoms. In the meanwhile, they performed more poorly on memory tests, showing 1.84 SDs, 1.07 SDs, and 0.68 SDs below healthy participants on visuospatial memory, working memory, and verbal memory, respectively. A variety of variables, including HADS depression, HADS anxiety, age, GCS, and education were associated with posttraumatic memory function in the bivariate models. The stepwise multiple linear regressions demonstrated a negative association between HADS depression and posttraumatic memory function, especially performance on visuospatial and verbal memory and a positive association between education and posttraumatic memory function. CONCLUSION: More depressive symptoms rather than anxiety symptoms and less years of education are significant predictors for posttraumatic memory dysfunction.

Long noncoding RNA NKILA transferred by astrocyte-derived extracellular vesicles protects against neuronal injury by upregulating NLRX1 through binding to mir-195 in traumatic brain injury.

The study aims to investigate the effects of long noncoding RNA (lncRNA) transmitted nuclear factor-κB interacting lncRNA (NKILA)-containing astrocyte-derived small extracellular vesicles (EVs) on traumatic brain injury (TBI). TBI was modeled in vitro by exposing human neurons to mechanical injury and in vivo by controlled cortical impact in a mouse model. The gain- and loss-function approaches were conducted in injured neurons to explore the role of NKILA, microRNA-195 (miR-195) and nucleotide-binding leucine-rich repeat containing family member X1 (NLRX1) in neuronal injury. EVs extracted from NKILA-overexpressing astrocytes were used to treat injured neurons. It was revealed that NKILA was downregulated in injured neurons. Astrocyte co-culture participated in the upregulation of NKILA in injured neurons. Additionally, NKILA could competitively bind to miR-195 that directly targeted NLRX1. Next, the upregulation of NLRX1 or NKILA relived neuronal injury by promoting neuronal proliferation but inhibiting apoptosis. Astrocyte-derived EVs transferred NKILA into neurons, which led to the downregulation of miR-195, upregulation of NLRX1, increased cell proliferation, and decreased cell apoptosis. The in vivo experiments validated that NKILA-containing EVs promoted brain recovery following TBI. Collectively, astrocyte-derived EVs carrying NKILA was found to alleviate neuronal injury in TBI by competitively binding to miR-195 and upregulating NLRX1.

Exogenous insulin-like growth factor 1 attenuates acute ischemic stroke-induced spatial memory impairment via modulating inflammatory response and tau phosphorylation.

Acute ischemic stroke is one of the main causes of mortality and morbidity worldwide. The present study aimed to explore the effects of exogenous insulin-like growth factor 1 (IGF-1) on the cognitive injuries induced by acute ischemic stroke and the underlying mechanisms. Acute ischemic stroke rat model was established via transient occlusion of the left middle cerebral artery to male Sprague-Dawley rats. IGF-1 was administered intravenously every other day 24 h after surgery for 14 days. Cognitive functions were determined by Morris water maze assay. Cerebral infarction and edema were determined by riphenyltetrazolium chloride staining and cerebral water content measurement. ELISA and Western blot were performed to detect concentrations of target proteins. Ischemic stroke rats exhibited reduced plasma IGF-1 level and impaired cognitive functions. Intravenous IGF-1 delivery increased the IGF-1 levels in plasma, ischemic amygdala, hippocampus and cortex, improved the neurological dysfunction, cognitive deficits, cerebral infarction and brain edema. Furthermore, IGF-1 relieved the systemic and cerebral inflammatory response by inhibiting the secretion of pro-inflammatory cytokines, interleukin (IL)-6, IL-1ß, and tumor necrosis factor alpha (TNF-α), in serum and ischemic hippocampus of ischemic rats. Additionally, IGF-1 attenuated tau phosphorylation in ischemic hippocampus. In short, intravenous IGF-1 administration attenuates acute ischemic stroke-induced cognitive injuries in the experimental rat model possibly via modulating inflammatory response and tau phosphorylation, and might be of promising therapeutic value to ischemic stroke in the future.

Associations between serum tau, neurological outcome, and cognition following traumatic brain injury.

OBJECTIVE: To investigate the dynamic change in the serum Tau protein early after acute traumatic brain injury (TBI) and its association with neurological outcome and cognitive function. SUBJECTS AND METHODS: Around 229 patients with acute TBI and 30 healthy subjects were evaluated for the serum levels of Tau protein on 1, 3, 5, 7, and 14 days after TBI. The relationships of the serum levels of Tau protein and initial GCS and GOS at 6 months post-injury were also analyzed. Further, 95 TBI patients were assessed with their cognitive function with Montreal cognitive assessment (MoCA) score. RESULTS: Serum Tau was significantly higher in patients with TBI at 1, 3, 5, 7, and 14 days. The serum Tau at each point was significantly lower respectively in the patients with mild TBI than that in medium and severe TBI. The serum Tau was significantly lower in patients with good outcome compared to the poor outcome group. The early serum Tau was negatively correlated with both GCS and GOS. In the TBI group, 39 (41%) out of 95 patients developed cognitive dysfunction assessed by MoCA. Tau protein at day 1, 3, and 5 after TBI was significantly correlated with cognitive dysfunction at 6 months after TBI. CONCLUSIONS: Acute Tau associations with neurological outcomes and cognition may implicate white matter damage and neuronal degeneration. Serum Tau may be used as a reliable biological marker for early diagnosis and cognitive recovery following TBI.

MicroRNA-326 decreases tau phosphorylation and neuron apoptosis through inhibition of the JNK signaling pathway by targeting VAV1 in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and age-related neurological dysfunction. Abundant data have profiled microRNA (miR) patterns in healthy, aging brain, and in the moderate and late-stages of AD. Herein, this study aimed to explore whether miR-326 could influence neuron apoptosis in AD mice and how miR-326 functions in this process. The candidate differentially expressed gene VAV1 was obtained by microarray analysis, and miRNAs that could regulate VAV1 candidate gene were predicted. Luciferase activity determination confirmed VAV1 as a target gene of miR-326. AD mice models were established for investigating the effect of miR-326 on AD mice. The overexpression of miR-326 contributed to decreased time of the mice to find the platform and the escape latency and increased residence time on the target area. Besides, elevation of miR-326 decreased Aß deposition and contents of Aß1-40 and Aß1-42 . Moreover, miR-326 overexpression increased neuron cell ability, mediated cell entry, and inhibited neuron apoptosis via JNK signaling pathway. Of crucial importance, miR-326 negatively regulated the expression of VAV1, inhibited tau phosphorylation, and blocked the activation of the JNK signaling pathway. Taken together these observations, we demonstrate that miR-326 improves cognitive function of AD mice and inhibits neuron apoptosis in AD mice through inactivation of the JNK signaling pathway by targeting VAV1. Based on those findings, miR-326 might exert promise as target for the treatment of AD.

Astrocytic Insulin-Like Growth Factor-1 Protects Neurons Against Excitotoxicity.

BACKGROUND: Exogenous insulin like growth factor-1 (IGF-1) is known to be neuroprotective in animal models with brain insults, while it can also cause hyperexcitability in rodents. In this regard, the role of endogenous IGF-1 in brain responses to brain insults like excitotoxicity, a common pathology in brain injuries, remains to be elucidated. Here, we investigated the potential role of cell-specific endogenous IGF-1 in the kainic acid (KA) -induced degeneration of the neurons. METHODS: Kainic acid was given to primary cultured cortical neurons and co-cultured astrocytes were added as a supportive system. We evaluated the cell proliferation rate, IGF-1 level in different groups and applied the PCR-Chip assay to explore the downstream of IGF-1. In addition, we applied the viral transfer of astrocytic IGF-1 to rodents treated with KA and assessed the associated molecular marker and behavioral outcomes in these rodents. RESULTS: We found KA induced increased cell death and hyperphosphorylated tau in neurons; co-cultured astrocytes could prevent these pathologies, and this rescuing effect was abrogated with blockade of the astrocytic IGF-1 with AG1024 (IGF-1R inhibitor). PCR-Chip assay identified that astrocytic IGF-1 could decrease the p-GSK-3 at Tyr 216 in neurons treated with KA and this effect was abrogated with AG1024 as well. In addition, in vivo study showed that gene transfer of astrocytic IGF-1 decreased p-tau and cognitive dysfunction in KA mice. CONCLUSION: Our results show astrocytic IGF-1 exhibits neuroprotective properties in neurodegenerative processes in the CNS.

Upregulation of Connexin-43 is Critical for Irradiation-induced Neuroinflammation.

BACKGROUND: Radiation therapy is widely used for the treatment of pituitary adenomas. Unfortunately, it might raise the risk of ischemic stroke, with neuroinflammation being a major pathological process. Astrocytes are the most abundant cell type in the central nervous system and have been reported for playing important roles in ischemic stroke. OBJECTIVE: Here we studied how γ-radiation would introduce astrocytes into a detrimental state for neuroinflammation and provide new theory evidence and target for the clinical management of inflammation- related neural damage after radiation-induced ischemic stroke. METHOD: HA-1800 cells were treated with γ-radiation and then the protein and mRNA levels of Connexin (Cx)-43 were evaluated by western and q-PCR. The culture supernatant was collected and the concentrations of the inflammatory factors were determined by ELISA. MiRNA complementary to Cx-43 was designed through the online tools. RESULTS: Cx-43 is upregulated in the treatment of γ-radiation in astrocytes and γ-radiation introduced the detrimental function of astrocytes: cell viability was reduced while the apoptotic cells were increased. Inflammatory factors like tumor necrosis factor alpha, interferon gamma, interleukin-6, interleukin 1-beta were dramatically up-regulated by the irradiation. MiR-374a rescued irradiation induced Cx-43 up-regulation of astrocytes and eliminated detrimental function triggered by γ-radiation. CONCLUSION: Cx-43 expression level may play an important role in the inflammation-related neural damage after irradiation-induced ischemic stroke.

Phosphorylation of connexin 43 induced by traumatic brain injury promotes exosome release.

Traumatic brain injury (TBI) caused by the external force leads to the neuronal dysfunction and even death. TBI has been reported to significantly increase the phosphorylation of glial gap junction protein connexin 43 (Cx43), which in turn propagates damages into surrounding brain tissues. However, the neuroprotective and anti-apoptosis effects of glia-derived exosomes have also been implicated in recent studies. Therefore, we detected whether TBI-induced phosphorylation of Cx43 would promote exosome release in rat brain. To generate TBI model, adult male Sprague-Dawley rats were subjected to lateral fluid percussion injury. Phosphorylated Cx43 protein levels and exosome activities were quantified using Western blot analysis following TBI. Long-term potentiation (LTP) was also tested in rat hippocampal slices. TBI significantly increased the phosphorylated Cx43 and exosome markers expression in rat ipsilateral hippocampus, but not cortex. Blocking the activity of Cx43 or ERK, but not JNK, significantly suppressed TBI-induced exosome release in hippocampus. Furthermore, TBI significantly inhibited the induction of LTP in hippocampal slices, which could be partially but significantly restored by pretreatment with exosomes. The results imply that TBI-activated Cx43 could mediate a nociceptive effect by propagating the brain damages, as well as a neuroprotective effect by promoting exosome release. NEW & NOTEWORTHY We have demonstrated in rat traumatic brain injury (TBI) models that both phosphorylated connexin 43 (p-Cx43) expression and exosome release were elevated in the hippocampus following TBI. The promoted exosome release depends on the phosphorylation of Cx43 and requires ERK signaling activation. Exosome treatment could partially restore the attenuated long-term potentiation. Our results provide new insight for future therapeutic direction on the functional recovery of TBI by promoting p-Cx43-dependent exosome release but limiting the gap junction-mediated bystander effect.

Exosomal lncRNA GAS5 regulates the apoptosis of macrophages and vascular endothelial cells in atherosclerosis.

Atherosclerosis is universally recognized as a chronic lipid-induced inflammation of the vessel wall. Oxidized low density lipoprotein (oxLDL) drives the onset of atherogenesis involving macrophages and endothelial cells (ECs). Our earlier work showed that expression of long noncoding RNA-growth arrest-specific 5 (lncRNA GAS5) was significantly increased in the plaque of atherosclerosis collected from patients and animal models. In this study, we found that knockdown of lncRNA GAS5 reduced the apoptosis of THP-1 cells treated with oxLDL. On the contrary, overexpression of lncRNA GAS5 significantly elevated the apoptosis of THP-1 cells after oxLDL stimulation. The expressions of apoptotic factors including Caspases were changed with lncRNA GAS5 levels. Moreover, lncRNA GAS5 was found in THP-1 derived-exosomes after oxLDL stimulation. Exosomes derived from lncRNA GAS5-overexpressing THP-1 cells enhanced the apoptosis of vascular endothelial cells after taking up these exosomes. However, exosomes shed by lncRNA GAS5 knocked-down THP-1 cells inhibited the apoptosis of endothelial cells. These findings reveal the function of lncRNA GAS5 in atherogenesis which regulates the apoptosis of macrophages and endothelial cells via exosomes and suggest that suppressing the lncRNA GAS5 might be an effective way for the therapy of atherosclerosis.

Phosphorylation of astrocytic connexin43 by ERK1/2 impairs blood-brain barrier in acute cerebral ischemia.

BACKGROUND: Connexins are a family of transmembrane proteins that form gap junctions, which are important for diffusion of cytosolic factors such as ions and second messenger signaling molecules. Our previous study has shown that Connexin40 (Cx40), one dominant connexin expressed in brain, was involved in brain injury. In this study, Cx43, another dominant connexin in brain, was investigated. Using bilateral common carotid artery occlusion-induced ischemia rat model, we tested the expression and phosphorylation level of Cx43 as well as heteromeric Cx40/Cx43 complex formation in brain after ischemia induction. We screened total 16 kinase inhibitors to identify the kinase for Cx43 phosphorylation and confirmed the result using siRNA targeting the specific kinase. Finally, we explored the role of the identified kinase in brain damage using in vivo rat model. RESULTS: We discovered that phosphorylation of Cx43 increased after ischemia. The formation of Cx40/Cx43 heteromeric complex on membrane also increased. Inhibition of ERK activity resulted in inhibition of Cx43 phosphorylation on astrocytes. In in vivo model, application of ERK inhibitor and siRNA prevented brain damage and protected blood-brain barrier integrity in rat. CONCLUSION: Our study provides evidence that Cx43 phosphorylation by ERK is implicated in ischemia induced brain damage.

Involvement of autophagy in connexin 40 reduction in the late phase of traumatic brain injury in rats.

Brain trauma can activate an attenuation of connexin gap junction that is implicated in neuronal injury, but the underlying cellular mechanisms remain incompletely understood. Here, we aimed to study whether autophagy, a stress-response process for recycling of intracellular proteins and organelles, is involved in the reduction of connexin 40 (Cx40) during the late phase of traumatic brain injury (TBI). In a rat model of TBI induced by controlled cortical impact (CCI), we found that Cx40 protein in the brain started to decline at post-surgery day 2 and the decrease continued for up to day 6. Such a relatively late response of Cx40 following TBI was found to be coincident with the substantial induction of neuron degeneration and autophagy, elevated autophagic vacuole numbers, and induced LC3-II and p62 levels. At day 4 post-injury, the extent of co-localization between LC3 and Cx40 was greatly enhanced, and the reduction of Cx40 was rescued by the administration of an autophagy inhibitor chloroquine. Thus, autophagy stimulated in the injured brains may act as a suppressing mechanism to decrease gap junction protein Cx40 in the late phase of TBI.

Connexin40 correlates with oxidative stress in brains of traumatic brain injury rats.

BACKGROUND: Oxidative stress is an important factor in the pathophysiologic changes after traumatic brain injury (TBI). Connexin43 (Cx43) was reported to contribute to cerebral damage. However, the impacts of Cx40 have not been investigated in detail. OBJECTIVE: In the present study, we hypothesized that Cx40 was involved in oxidative stress-induced brain injury after TBI. METHODS: The controlled cortical impact (CCI) model was introduced to Wistar rats as a TBI model. Neurological deficits, oxidative stress and Cx40 were evaluated in TBI rats and N-acetylcysteine (NAC)-treated TBI rats. Neurological severity score (NSS) was used to assess neurological deficits. Brain infarction was measured by histo-staining. Brain edema was evaluated by measuring the brain water content. Cortex samples were collected to measure the tissue levels of malonyldialdehyde (MDA), nitric oxide (NO) and glutathione (GSH) and NADPH oxidase activity. Cx40 expression was determined by Western-blot. RESULTS: TBI-induced brain injuries gradually increased from 6 h to 24 h post CCI, and the severity remained till 72 h. The level of oxidative stress was consistent with the extent of neurological deficits. Cx40 was upregulated after TBI in a linear correlated manner with increased oxidative stress. With NAC intervention, both neurological deficits and oxidative stress were significantly attenuated. Meanwhile, elevated Cx40 expression in cortex was also prevented by NAC treatment. CONCLUSION: These studies revealed the relationship between levels of Cx40 and oxidative stress after TBI. The cortex Cx40 expression was positively correlated with the cerebral oxidative stress, indicating the involvement of Cx40 in the progress of brain damage.

IGF-1: an endogenous link between traumatic brain injury and Alzheimer disease?

There is a growing body of evidence that the insulin-like growth factor-1 (IGF-1) is dynamically involved in the regulation of body homeostasis and glucose regulation. Traumatic brain injury (TBI) is considered to be a risk factor for Alzheimer's disease (AD). As alterations of IGF-1 have been implicated in both TBI and AD and the IGF-1 signaling also mediates the neuronal excitability and synaptic plasticity in both diseases, we propose that IGF-1 may act as the endogenous connection between TBI and AD. Growing evidence suggests that dysfunction of this pathway contributes to the progressive loss of neurons in Alzheimer's disease (AD), one of the most frequent neurodegenerative disorders. These findings have led to numerous studies in preclinical models of neurodegenerative disorders targeting IGF-1 signaling with currently available antidiabetics. These studies have shown that exogenous administration of IGF-1 reverses signaling abnormalities and has neuroprotective effects. In the first part of this review, we discuss physiological functions of IGF-1 signaling pathway including its distribution within the brain and its relationship with TBI and AD. In the second part, we undertake a comprehensive overview of IGF-1 signaling in TBI and AD, respectively. We then detail targeted IGF-1 in preclinical models of neurodegeneration and the design of clinical trials that have used anti-diabetics for treating AD patients. We close with future considerations that treat relevant issues for successful translation of these encouraging preclinical results into clinical sessions.

Involvement of Connexin40 in the Protective Effects of Ginsenoside Rb1 Against Traumatic Brain Injury.

Ginsenosides are the major active components of ginseng, which have been proven to be effective in therapies for neurodegenerative diseases. Ginsenoside Rb1 (GS-Rb1) is the most abundant among all the identified ginsenosides and has been shown to exert neuroprotective effects, although the underlying molecular mechanisms remain unclear. Connexins are a family of transmembrane proteins that form gap junctions, which are important for diffusion of cytosolic factors such as ions and second messenger signaling molecules. Previous studies have shown that a subset of connexin proteins is involved in neuroprotection. We investigated the protective effects of GS-Rb1 against traumatic brain injury (TBI) and the potential mechanism using TBI mouse model. We discovered that TBI-induced brain injury and up-regulation of connexin40 (Cx40) protein expression as early as 6 h post-TBI, which was reversed by administration of GS-Rb1. In addition, we found that the protective effects of GS-Rb1 are dose and time dependent and are partially mediated through phosphorylation of ERK1/2 signaling pathway, as evidenced by the abolishment of GS-Rb1-mediated elevation of p-ERK1/2 expression and inhibition of Cx40 expressions when ERK inhibitor U0126 was used. Our study provides evidence that Cx40 is implicated in TBI-induced brain injuries, and GS-Rb1 exerts neuroprotective activity against TBI involving down-regulation of Cx40 expression.

Protective effect of ginsenoside Rb1 on integrity of blood-brain barrier following cerebral ischemia.

Ginsenosides, the major bioactive compounds in ginseng root, have been found to have antioxidant, immunomodulatory, and anti-inflammatory activities. In the present study, we sought to investigate whether and how ginsenoside Rb1 (GS-Rb1), the most abundant ginsenoside, can protect blood-brain barrier (BBB) integrity following cerebral ischemia in middle cerebral artery occlusion (MCAO) animal model. ICR mice underwent MCAO and received GS-Rb1 by intraperitoneal injection at 3 h after reperfusion. We evaluated infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression at 48 h after MCAO. We further examined whether GS-Rb1 protected BBB integrity by suppressing post-ischemic inflammation-induced activity of matrix metalloproteinase-9 (MMP-9) and nicotinamide adenine dinucleotide phosphate oxidase (NOX). First, GS-Rb1 decreased infarction and improved neurological deficits in MCAO animals. In addition, GS-Rb1 reduced EB extravasation and brain edema and preserved expression of tight junction proteins in the ischemic brain. Moreover, GS-Rb1 inhibited expression of pro-inflammatory factors including nitric oxide synthase and IL-1ß, but increased expression of anti-inflammatory markers arginase 1 and IL-10 in the ischemic brain. Consistently, GS-Rb1 attenuated ischemia-induced expression and activity of MMP9. Finally, GS-Rb1 reduced NOX-4 mRNA expression and NOX activity in ischemic brain. These results suggest that GS-Rb1 protects loss of BBB integrity in ischemic stroke by suppressing neuroinflammation induction of MMP-9 and NOX4-derived free radicals, and indicate its potential for treating brain injuries, such as ischemia and stroke.