牛磺酸对脑创伤大鼠的脑保护作用

目的:探讨牛磺酸(Tau)预处理对弥漫性脑创伤(TBI)大鼠脑皮层超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、脑含水量(BWC)和脑皮层水孔通道蛋白4(AQP4)表达的影响。方法:复制大鼠TBI模型,分为假手术组(S组)、TBI组(T组)、低剂量Tau组(L组)和高剂量Tau组(H组),用比色法测定脑皮层匀浆液中SOD活力和MDA含量;干/湿法测定BWC;免疫组织化学检测脑皮层AQP4的表达。结果:T组大鼠脑皮层SOD活力显著低于S组,T组MDA含量、BWC和脑皮层AQP4的表达显著高于S组;H、L组脑皮层SOD活力显著高于T组,H、L组MDA含量、BWC和脑皮层AQP4的表达显著低于T组;H、L组之间差异无显著性。结论:Tau可能通过清除TBI后产生的的氧自由基、下调TBI大鼠脑皮层AQP4的表达减轻脑水肿,发挥其脑保护作用。     

Alpha lipoic acid inhibits oxidative stress‐induced apoptosis by modulating of Nrf2 signalling pathway after traumatic brain injury

Alpha lipoic acid (ALA) is a powerful antioxidant which has been widely used in the treatment of different system diseases, such as cardiovascular and cerebrovascular diseases. But, there are few studies that refer to protective effects and potential mechanisms on traumatic brain injury (TBI). This study was carried out to investigate the neuroprotective effect following TBI and illuminate the underlying mechanism. Weight drop‐injured model in rats was induced by weight‐drop. ALA was administrated via intraperitoneal injection after TBI. Neurologic scores were examined following several tests. Neurological score was performed to measure behavioural outcomes. Nissl staining and TUNEL were performed to evaluate the neuronal apoptosis. Western blotting was engaged to analyse the protein content of the Nuclear factor erythroid 2‐related factor 2 (Nrf2) and its downstream protein factors, including hemeoxygenase‐1 (HO‐1) and quinine oxidoreductase‐1 (NQO1). ALA treatment alleviated TBI‐induced neuron cell apoptosis and improved neurobehavioural function by up‐regulation of Nrf2 expression and its downstream protein factors after TBI. This study presents new perspective of the mechanisms responsible for the neuronal apoptosis of ALA, with possible involvement of Nrf2 pathway.     

Breviscapine provides a neuroprotective effect after traumatic brain injury by modulating the Nrf2 signaling pathway

Breviscapine (BVP) has been widely used in the treatment of several systemic diseases, including those of the cardiovascular and cerebrovascular systems. But, few studies have looked at the neuroprotective effects of BVP and its potential effect in treating traumatic brain injury (TBI). The present study investigated the neuroprotective effect of BVP following TBI and illuminated the underlying mechanism. The weight drop-induced closed diffuse traumatic brain injury method was used to induce TBI in rats. BVP was injected intraperitoneally 30 minutes after TBI. Neurologic scores were performed to measure behavioral outcomes. Nissl staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed on histopathologic tissue sections to evaluate neuronal apoptosis. The nuclear factor erythroid 2-related factor 2 (Nrf2) and its related downstream proteins, including heme oxygenase-1 (HO-1) and quinine oxidoreductase-1 (NQO1) were detected with Western blots. BVP treatment alleviated or attenuated TBI-induced neuron cell apoptosis and improved neurobehavioral functions through the upregulated expression of Nrf2 and its related downstream proteins. This study, using the drug, BVP, we present new mechanisms responsible for neuronal apoptosis in TBI with possible involvement of the Nrf2 pathway.     

Caspase 7: increased expression and activation after traumatic brain injury in rats

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.     

Sirtuin 1 alleviates neuroinflammation-induced apoptosis after traumatic brain injury

Sirtuin 1 (SIRT1) plays a very important role in a wide range of biological responses, such as metabolism, inflammation and cell apoptosis. Changes in the levels of SIRT1 have been detected in the brain after traumatic brain injury (TBI). Further, SIRT1 has shown a neuroprotective effect in some models of neuronal death; however, its role and working mechanisms are not well understood in the model of TBI. This study aimed to address this issue. SIRT1-specific inhibitor (sirtinol) and activator (A3) were introduced to explore the role of SIRT1 in cell apoptosis. Results of the study suggest that SIRT1 plays an important role in neuronal apoptosis after TBI by inhibiting NF-κB, IL-6 and TNF-α deacetylation and the apoptotic pathway sequentially, possibly by alleviating neuroinflammation.     

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Alterations of microRNAs expression profiles in small extracellular vesicle after traumatic brain injury in mice

Traumatic brain injury (TBI) is one of the leading causes of mortality and morbidity worldwide. Tools available for diagnosis and therapy are limited. Small extracellular vesicle (sEV) microRNAs (miRNAs) play an important role in TBI disease progression. This study aimed to investigate the alterations in sEV miRNAs expression in the mouse brain extracellular space after TBI. Twenty-four C57BL/6J mice were randomly divided into two groups (12/group). The TBI group was subjected to all surgical procedures and fluid percussion injury (FPI). The sham group only underwent surgery. Brain specimens were collected 3 h after TBI/sham. The brain sEV were isolated. Differentially expressed miRNAs were identified. A total of 50 miRNAs were observed to be differentially expressed (fold change ≥1.5 and P<0.05) after TBI, including 5 upregulated and 45 downregulated. The major enriched Gene Ontology terms were metabolic processes, cell, intracellular, organelle, cytoplasm, axon, binding, protein kinase activity, protein binding, and protein dimerization activity. The KEGG pathway analysis predicted that the pathways affected by the variation of miRNAs in sEVs after TBI included the Wnt signaling pathway and NF-κB signaling pathway. The changes in five miRNAs were confirmed by qRT-PCR. In conclusion, this study demonstrated the differential expression of a series of miRNAs in brain sEV after TBI, which might be correlated with post-TBI physiological and pathological processes. The findings might also provide novel targets for further investigating the molecular mechanisms underlying TBI and potential therapeutic interventions.     

Differential modulation of carbachol and trans-ACPD-stimulated phosphoinositide turnover following traumatic brain injury

In the fluid percussion model of traumatic brain injury (TBI), we examined muscarinic and metabotropic glutamate receptor-stimulated polyphosphoinositide (PPI) turnover in rat hippocampus. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device. Carbachol and (±)-1-Aminocyclopentane-trans-1,3.-dicarboxylic acid (trans-ACPD)-stimulated PPI hydrolysis was assayed in hippocampus from injured and sham-injured controls at both 1 hour and 15 days following injury. At 1 hour after TBI, the response to carbachol was enhanced in injured rats by up to 200% but the response to trans-ACPD was diminished by as much as 28%. By contrast, at 15 days after TBI, the response to carbachol was enhanced by 25% and the response to trans-ACPD was enhanced by 73%. The ionotropic glutamate agonists N-methyl-D-aspartate (NMDA), and -amino-3 hydroxy-5-methyl-4-isoxazolepropionate (AMPA), did not increase PPI hydrolysis in either sham or injured rats and injury did not alter basal hydrolysis. Thus, hippocampal muscarinic and metabotropic receptors linked to phospholipase C are differentially altered by TBI.Abbreviations used TBI traumatic brain injury - EAA excitatory amino acids - PPI polyphosphoinositides - IP inositol phosphates - NMDA N-methyl-D-aspartate - AMPA -amino-3-hydroxy-5-methylisoxazole-4-propionate - trans-ACPD (±)-1-Aminocyclopentanetrans-1,3-dicarboxylic acid - LTP long term potentiation     

Clinical characteristics and pathophysiological mechanisms of focal and diffuse traumatic brain injury

Traumatic brain injury (TBI) is a frequent and clinically highly heterogeneous neurological disorder with large socioeconomic consequences. TBI severity classification, based on the hospital admission Glasgow Coma Scale (GCS) score, ranges from mild (GCS 13–15) and moderate (GCS 9–12) to severe (GCS ≤ 8). The GCS reflects the risk of dying from TBI, which is low after mild (∼1%), intermediate after moderate (up to 15%) and high (up to 40%) after severe TBI. Intracranial damage can be focal, such as epidural and subdural haematomas and parenchymal contusions, or diffuse, for example traumatic axonal injury and diffuse cerebral oedema, although this distinction is somewhat arbitrary. Study of the cellular and molecular post-traumatic processes is essential for the understanding of TBI pathophysiology but even more to find therapeutic targets for the development of neuroprotective drugs to be eventually used in human beings. To date, studies in vitro and in vivo, mainly in animals but also in human beings, are unravelling the pathological TBI mechanisms at high pace. Nevertheless, TBI pathophysiology is all but completely elucidated. Neuroprotective treatment studies in human beings have been disappointing thus far and have not resulted in commonly accepted drugs. This review presents an overview on the clinical aspects and the pathophysiology of focal and diffuse TBI, and it highlights several acknowledged important events that occur on molecular and cellular level after TBI.     

Proteomic identification of biomarkers of traumatic brain injury

Traumatic brain injury (TBI) is a major national health problem without a US Food and Drug Administration-approved therapy. This review summarizes the importance of discovering relevant TBI protein biomarkers and presents logical rationale that neuroproteomic technologies are uniquely suited for the discovery of otherwise unnoticed TBI biomarkers. It highlights that one must make careful decisions when choosing which paradigm (human vs. animal models) and which biologic samples to use for such proteomic studies. It further outlines some of the desirable attributes of an ideal TBI biomarker and discusses how biomarkers discovered proteomically are complementary to those identified by traditional approaches. Lastly, the most important sequela of any proteomically identified TBI biomarker is validation in preclinical or clinical samples.     

Modulation of autophagy in traumatic brain injury

Traumatic brain injury (TBI) is defined as a traumatically induced structural injury or physiological disruption of brain function as a result of external forces, leading to adult disability and death. A growing body of evidence reveals that alterations in autophagy-related proteins exist extensively in both experimentally and clinically after TBI. Of note, the autophagy pathway plays an essential role in pathophysiological processes, such as oxidative stress, inflammatory response, and apoptosis, thus contributing to neurological properties of TBI. With this in mind, this review summarizes a comprehensive overview on the beneficial and detrimental effects of autophagy in pathophysiological conditions and how these activities are linked to the pathogenesis of TBI. Moreover, the relationship between oxidative stress, inflammation, apoptosis, and autophagy occur TBI. Ultimately, multiple compounds and various drugs targeting the autophagy pathway are well described in TBI. Therefore, autophagy flux represents a potential clinical therapeutic value for the treatment of TBI and its complications.     

Minocycline attenuates neuronal apoptosis and improves motor function after traumatic brain injury in rats

Minocycline is a type of tetracycline antibiotic with broad-spectrum antibacterial activity that has been demonstrated to protect the brain against a series of central nervous system diseases. However, the precise mechanisms of these neuroprotective actions remain unknown. In the present study, we found that minocycline treatment significantly reduced HT22 cell apoptosis in a mechanical cell injury model. In addition, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining confirmed the neuroprotective effects of minocycline in vivo through the inhibition of apoptosis in a rat model of controlled cortical impact (CCI) brain injury. The western blotting analysis revealed that minocycline treatment significantly downregulated the pro-apoptotic proteins BAX and cleaved caspase-3 and upregulated the anti-apoptotic protein BCL-2. Furthermore, the beam-walking test showed that the administration of minocycline ameliorated traumatic brain injury (TBI)-induced deficits in motor function. Taken together, these findings suggested that minocycline attenuated neuronal apoptosis and improved motor function following TBI.     

颅脑创伤后大鼠脑组织脑红蛋白表达变化及其与神经元凋亡的关系研究

目的：研究大鼠弥漫性颅脑创伤后脑组织中脑红蛋白的表达变化情况,探究创伤后脑红蛋白表达变化及其与神经元凋亡的关系。方法：采用雄性SD大鼠50只,随机分为10组（n=5）空白对照组、伤后30min、1h、2h、6h、12h、24h、48h、72h和5d组。以Marmarou’s自由落体打击装置复制颅脑创伤模型,采用免疫组化技术检测伤后不同时间脑组织中脑红蛋白的表达情况及神经元凋亡相关基因Bax、Bcl-2表达情况,并对所得数据进行统计学分析。结果：致伤区皮层神经元脑红蛋白表达分别于伤后2h、72h呈现出两次高峰表达;伤后30min～1h、48～72h期间大脑皮层区脑红蛋白表达的上调均伴随着Bax/Bcl-2比值上升趋势减缓甚至呈现下降趋势。结论：弥漫性颅脑创伤后脑组织中脑红蛋白的高表达在一定程度上可以拮抗创伤应激及伤后继发缺血、缺氧性损伤所导致的神经元凋亡,在颅脑创伤的超早期（〈3h）、急性期（〈72h）可能具有一定的神经保护作用。