Establishment and validation of a model for brain injury state evaluation and prognosis prediction.

PURPOSE: Traumatic brain injury (TBI) is one of the leading causes of disability and death in modern times, whose evaluation and prognosis prediction have been one of the most critical issues in TBI management. However, the existed models for the abovementioned purposes were defective to varying degrees. This study aims to establish an ideal brain injury state clinical prediction model (BISCPM). METHODS: This study was a retrospective design. The six-month outcomes of patients were selected as the end point event. BISCPM was established by using the split-sample technology, and externally validated via different tests of comparison between the observed and predicted six-month mortality in validating group. TBI patients admitted from July 2006 to June 2012 were recruited and randomly divided into establishing model group and validating model group. Twenty-one scoring indicators were included in BISCPM and divided into three parts, A, B, and C. Part A included movement, pupillary reflex and diameter, CT parameters, and secondary brain insult factors, etc. Part B was age and part C was medical history of the patients. The total score of part A, B and C was final score of BISCPM. RESULTS: Altogether 1156 TBI patients were included with 578 cases in each group. The score of BISCPM from validating group ranged from 2.75 to 31.94, averaging 13.64 ± 5.59. There was not statistical difference between observed and predicted mortality for validating group. The discrimination validation showed that the BISCPM is superior to international mission for prognosis and analysis of clinical trials (IMPACT) lab model. CONCLUSION: BISCPM is an effective model for state evaluation and prognosis prediction of TBI patients. The use of BISCPM could be of great significance for decision-making in management of TBI.

Sirt1-Sirt3 axis regulates human blood-brain barrier permeability in response to ischemia.

Sirtuin1 (Sirt1) and Sirtuin3 (Sirt3) are two well-characterized members of the silent information regulator 2 (Sir2) family of proteins. Both Sirt1 and Sirt3 have been shown to play vital roles in resistance to cellular stress, but the interaction between these two sirtuins has not been fully determined. In this study, we investigated the role of Sirt1-Sirt3 axis in blood-brain barrier (BBB) permeability after ischemia in vitro. Human brain microvascular endothelial cells and astrocytes were co-cultured to model the BBB in vitro and oxygen and glucose deprivation (OGD) was performed to mimic ischemia. The results of transepithelial electrical resistance (TEER) showed that suppression of Sirt1 via siRNA or salermide significantly decreased BBB permeability, whereas Sirt3 knockdown increased BBB permeability. In addition, Sirt1 was shown to regulate Sirt3 expression after OGD through inhibiting the AMPK-PGC1 pathway. Application of the AMPK inhibitor compound C partially prevented the effects of Sirt1-Sirt3 axis on BBB permeability after OGD. The results of flow cytometry and cytochrome c release demonstrated that Sirt1 and Sirt3 exert opposite effects on OGD-induced apoptosis. Furthermore, suppression of Sirt1 was shown to attenuate mitochondrial reactive oxygen species (ROS) generation, which contribute to the Sirt1-Sirt3 axis-induced regulation of BBB permeability and cell damage. In summary, these findings demonstrate that the Sirt1-Sirt3 axis might act as an important modulator in BBB physiology, and could be a therapeutic target for ischemic stroke via regulating mitochondrial ROS generation.

Sirt3 confers protection against neuronal ischemia by inducing autophagy: Involvement of the AMPK-mTOR pathway.

Sirtuin3 (Sirt3) is a member of the silent information regulator 2 (Sir2) family of proteins located in mitochondria that influences almost every major aspect of mitochondrial biology, including ATP generation and reactive oxygen species (ROS) production. Our previous study showed that Sirt3 exerts protective effects against oxidative stress in neuronal cells. In this study, we investigated the role of Sirt3 in neuronal ischemia using an oxygen and glucose deprivation (OGD) model. Sirt3 was up-regulated by OGD and overexpression of Sirt3 through lentivirus transfection significantly reduced OGD-induced lactate dehydrogenase (LDH) release and neuronal apoptosis. These effects were accompanied by reduced hydrogen dioxide (H2O2) production, enhanced ATP generation and preserved mitochondrial membrane potential (MMP). The results of immunocytochemistry and electron microscopy showed that Sirt3 increased autophagy in OGD-injured neurons, which was also confirmed by the increased expression of Beclin-1 as well as LC3-I to LC3-II conversion. In addition, the autophagy inhibitor 3-MA and bafilomycin A1 partially prevented the effects of Sirt3 on LDH release and apoptosis after OGD. The results of western blotting showed that overexpression of Sirt3 in cortical neurons markedly increased the phosphorylation of AMPK, whereas the phosphor-mTOR (p-mTOR) levels decreased both in the presence and absence of OGD insult. Furthermore, pre-treatment with the AMPK inhibitor compound C partially reversed the protective effects of Sirt3. Taken together, these findings demonstrate that Sirt3 protects against OGD insult by inducing autophagy through regulation of the AMPK-mTOR pathway and that Sirt3 may have therapeutic value for protecting neurons from cerebral ischemia.

The AMPAR Antagonist Perampanel Attenuates Traumatic Brain Injury Through Anti-Oxidative and Anti-Inflammatory Activity.

Perampanel is a novel α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) antagonist, approved in over 35 countries as an adjunctive therapy for the treatment of seizures. Recently, it was found to exert protective effects against ischemic neuronal injury in vitro. In the present study, we investigated the potential protective effects of perampanel in a traumatic brain injury (TBI) model in rats. Oral administration with perampanel at a dose of 5 mg/kg exerted no major organ-related toxicities. We found that perampanel significantly attenuated TBI-induced brain edema, brain contusion volume, and gross motor dysfunction. The results of Morris water maze test demonstrated that perampanel treatment also improved cognitive function after TBI. These neuroprotective effects were accompanied by reduced neuronal apoptosis, as evidenced by decreased TUNEL-positive cells in brain sections. Moreover, perampanel markedly inhibited lipid peroxidation and obviously preserved the endogenous antioxidant system after TBI. In addition, enzyme-linked immunosorbent assay (ELISA) was performed at 4 and 24 h after TBI to evaluate the expression of inflammatory cytokines. The results showed that perampanel suppressed the expression of pro-inflammatory cytokines TNF-α and IL-1ß, whereas increased the levels of anti-inflammatory cytokines IL-10 and TGF-ß1. These data show that the orally active AMPAR antagonist perampanel affords protection against TBI-induced neuronal damage and neurological dysfunction through anti-oxidative and anti-inflammatory activity.

Homer1a attenuates glutamate-induced oxidative injury in HT-22 cells through regulation of store-operated calcium entry.

Calcium disequilibrium is extensively involved in oxidative stress-induced neuronal injury. Although Homer1a is known to regulate several neuronal calcium pathways, its effects on, or its exact relationship with, oxidative stress-induced neuronal injury has not yet been fully elucidated. We found that Homer1a protected HT-22 cells from glutamate-induced oxidative stress injury by inhibiting final-phase intracellular calcium overload and mitochondrial oxidative stress. In these cells, stromal interactive molecule 1 (STIM1) puncta, but not the protein level, was significantly increased after glutamate treatment. Store-operated calcium entry (SOCE) inhibitors and cells in which a key component of SOCE (STIM1) was knocked out were used as glutamate-induced oxidative stress injury models. Both models demonstrated significant improvement of HT-22 cell survival after glutamate treatment. Additionally, increased Homer1a protein levels significantly inhibited SOCE and decreased the association of STIM1-Orai1 triggered by glutamate. These results suggest that up-regulation of Homer1a can protect HT-22 cells from glutamate-induced oxidative injury by disrupting the STIM1-Oria1 association, and then by inhibiting the SOCE-mediated final-phrase calcium overload. Thus, regulation of Homer1a, either alone or in conjunction with SOCE inhibition, may serve as key therapeutic interventional targets for neurological diseases in which oxidative stress is involved in the etiology or progression of the disease.

Expression of 58-kD Microspherule Protein (MSP58) is Highly Correlated with PET Imaging of Tumor Malignancy and Cell Proliferation in Glioma Patients.

BACKGROUND/AIMS: The nucleolar 58-kDa microspherule protein (MSP58) has important transcriptional regulation functions and plays a crucial role in the tumorigenesis and progression of cancers. 3'-deoxy-3'-[18F]fluorothymidine (FLT) has emerged as a promising positron emission tomography (PET) tracer for evaluating tumor malignancy and cell proliferation. METHODS: In the present study, the expression of MSP58 was evaluated by immunohistochemistry and the corresponding PET image was examined using FLT-PET in 55 patients with various grades of gliomas. RESULTS: The immunoreactivity score (IRS) of MSP58 increased with tumor grade with grade IV gliomas exhibiting the highest expression and showed a highly significant positive correlation with the Ki-67 index (r = 0.65, P < 0.001). The IRS of MSP58 in the tumor showed a highly significant positive correlation with corresponding FLT uptake value (r = 0.61, P < 0.001). The correlation between MSP58 expression and glioma malignancy was also confirmed by immunofluorescence, RT-PCR and western blot analysis. FLT uptake value also exhibited a highly significant positive correlation with the Ki-67 index (r = 0.59, P < 0.001). Kaplan-Meier analysis revealed that MSP58 expression has a significant prognostic ability for the overall survival time similar to that found in the uptake value of FLT-PET. CONCLUSION: These results indicate that MSP58 plays an important role in cell proliferation and will be one of the potential candidates of molecular therapy targeting proliferation. FLT-PET might be used as an early measure of treatment response in the proliferation-targeted therapy.

Downregulation of STIM2 improves neuronal survival after traumatic brain injury by alleviating calcium overload and mitochondrial dysfunction.

Although store-operated calcium entry (SOCE) has been implicated in several neurological disorders, the exact mechanism for its role in traumatic brain injury (TBI) has not been elucidated. In this study, we found that TBI upregulated the expression of a calcium sensor protein called stromal interactive molecule 2 (STIM2); however, the levels of its homologue, STIM1, were unaffected. Both STIM1 and STIM2 are crucial components of SOCE, both in vivo and in vitro. Using shRNA, we discovered that downregulation of STIM2, but not STIM1, significantly improved neuronal survival in both an in vitro and in vivo model of TBI, decreasing neuronal apoptosis, and preserving neurological function. This neuroprotection was associated with alleviating TBI-induced calcium overload and preserving mitochondrial function. Additionally, downregulation of STIM2 not only inhibited Ca(2+) release from the endoplasmic reticulum (ER), but also reduced SOCE-mediated Ca(2+) influx, decreased mitochondrial Ca(2+), restored mitochondrial morphology and improved mitochondrial function, including MMP maintenance, ROS production and ATP synthesis. These results indicate that inhibition of STIM2 can protect neurons from TBI by inhibiting calcium overload and preserving mitochondrial function. This suggests that STIM2 might be an effective interventional target for TBI.

Sirt3 protects cortical neurons against oxidative stress via regulating mitochondrial Ca2+ and mitochondrial biogenesis.

Oxidative stress is a well-established event in the pathology of several neurobiological diseases. Sirt3 is a nicotinamide adenine nucleotide (NAD+)-dependent protein deacetylase that regulates mitochondrial function and metabolism in response to caloric restriction and stress. This study aims to investigate the role of Sirt3 in H2O2 induced oxidative neuronal injury in primary cultured rat cortical neurons. We found that H2O2 treatment significantly increased the expression of Sirt3 in a time-dependent manner at both mRNA and protein levels. Knockdown of Sirt3 with a specific small interfering RNA (siRNA) exacerbated H2O2-induced neuronal injury, whereas overexpression of Sirt3 by lentivirus transfection inhibited H2O2-induced neuronal damage reduced the generation of reactive oxygen species (ROS), and increased the activities of endogenous antioxidant enzymes. In addition, the intra-mitochondrial Ca2+ overload, but not cytosolic Ca2+ increase after H2O2 treatment, was strongly attenuated after Sirt3 overexpression. Overexpression of Sirt3 also increased the content of mitochondrial DNA (mtDNA) and the expression of mitochondrial biogenesis related transcription factors. All these results suggest that Sirt3 acts as a prosurvival factor playing an essential role to protect cortical neurons under H2O2 induced oxidative stress, possibly through regulating mitochondrial Ca2+ homeostasis and mitochondrial biogenesis.

Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells.

Sirtuins (Sirt) are a family of phylogenetically conserved nicotinamide adenine nucleotide (NAD(+))-dependent protein deacetylases, among which Sirt3 resides primarily in the mitochondria and serves as a stress responsive deacetylase, playing a role in protecting cells from damage under stress conditions. The present study aimed to investigate the role of Sirt3 in hydrogen peroxide (H(2)O(2))-induced oxidative neuronal injury in HT22 mouse hippocampal cells. Treatment with H(2)O(2) increased the expression of Sirt3 in a dose- and time-dependent manner, and the knockdown of Sirt3 using specific small interfering RNA (siRNA) exacerbated the H(2)O(2)-induced neuronal injury. The overexpression of Sirt3 induced by lentiviral transfection significantly reduced the generation of reactive oxygen species (ROS) and lipid peroxidation following injury, whereas the activities of endogenous antioxidant enzymes were not affected. Further experiments revealed that the H(2)O(2)-induced inhibition of mitochondrial complex activity and adenosine triphosphate (ATP) synthesis, the decrease in mitochondrial Ca(2+) buffering capacity and mitochondrial swelling were all partly reversed by Sirt3. Furthermore, the overexpression of Sirt3 attenuated the release of cytochrome c, the increase in the Bax/Bcl-2 ratio, as well as caspase-9/caspase-3 activity induced by H(2)O(2), and eventually inhibited apoptotic neuronal cell death. These results suggest that Sirt3 acts as a prosurvival factor, playing an essential role in protecting HT22 cells under H(2)O(2)-induced oxidative stress, possibly by inhibiting ROS accumulation and the activation of the mitochondrial apoptotic pathway.

Activation of mGluR5 attenuates NMDA-induced neurotoxicity through disruption of the NMDAR-PSD-95 complex and preservation of mitochondrial function in differentiated PC12 cells.

Glutamate-mediated toxicity is implicated in various neuropathologic conditions, and activation of ionotropic and metabotropic glutamate receptors is considered to be the most important mechanism. It has been reported that pharmacological saturation of metabotropic glutamate receptors (mGluRs) can facilitate N-methyl-D-aspartate receptor (NMDAR) related signaling cascades, but the mechanism leading to mGluR-NMDAR interactions in excitotoxic neuronal injury has remained unidentified. In the present study, we investigated the role of mGluR5 in the regulation of N-methyl-D-aspartate (NMDA)-induced excitotoxicity in differentiated PC12 cells. We found that activation of mGluR5 with the specific agonist R,S-2-chloro-5-hydroxyphenylglycine (CHPG) increased cell viability and inhibited lactate dehydrogenase (LDH) release in a dose-dependent manner. CHPG also inhibited an increase in the Bax/Bcl-2 ratio, attenuated cleavage of caspase-9 and caspase-3, and reduced apoptotic cell death after NMDA treatment. The NMDA-induced mitochondrial dysfunction, as indicated by mitochondrial reactive oxygen species (ROS) generation, collapse of mitochondrial membrane potential (MMP), and cytochrome c release, was also partly prevented by CHPG treatment. Furthermore, CHPG blocked the NMDA-induced interaction of NMDAR with postsynaptic density protein-95 (PSD-95), but had no effects on intracellular calcium concentrations. All these results indicated that activation of mGluR5 protects differentiated PC12 cells from NMDA-induced neuronal excitotoxicity by disrupting NMDAR-PSD-95 interaction, which might be an ideal target for investigating therapeutic strategies in various neurological diseases where excitotoxicity may contribute to their pathology.

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease.

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP(+) induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP(+) injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca(2+) concentration ([Ca(2+)]cyt) and Ca(2+) in endoplasmic reticulum (ER) ([Ca(2+)]ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca(2+) release up to 120min after MPP(+) injury. Furthermore, decrease of [Ca(2+)]cyt induced by Homer1 knockdown in MPP(+) treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP(+) insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.

Salvianolic acid B induces apoptosis in human glioma U87 cells through p38-mediated ROS generation.

Salvianolic acid B (SalB), the main water-soluble bioactive compounds isolated from the traditional Chinese medical herb Danshen, has been shown to exert anti-cancer effect in several cancer cell lines. The aim of our study was to investigate the potential anti-cancer effect of SalB in human glioma U87 cells. We found that treatment with SalB significantly decreased cell viability of U87 cells in a dose- and time-dependent manner. SalB also enhanced the intracellular ROS generation and induced apoptotic cell death in U87 cells. Western blot analysis suggested that SalB increased the phosphorylation of p38 MAPK and p53 in a dose-dependent manner. Moreover, blocking p38 activation by specific inhibitor SB203580 or p38 specific siRNA partly reversed the anti-proliferative and pro-apoptotic effects, and ROS production induced by SalB treatment. The anti-tumor activity of SalB in vivo was also demonstrated in U87 xenograft glioma model. All of these findings extended the anti-cancer effect of SalB in human glioma cell lines, and suggested that these inhibitory effects of SalB on U87 glioma cell growth might be associated with p38 activation mediated ROS generation. Thus, SalB might be concerned as an effective and safe natural anticancer agent for glioma prevention and treatment.