Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.     

Traumatic Brain Injury-Induced Excitotoxicity Assessed in a Controlled Cortical Impact Model

Using a controlled cortical impact model of traumatic brain injury (TBI) coupled with tissue microdialysis, interstitial concentrations of aspartate and glutamate (together with serine and glutamine) were assessed in rat frontal cortex. Histological analysis indicated that the severity of injury following severe TBI (depth of deformation = 3.5 mm) was approximately twice that occurring following moderate TBI (depth of deformation = 1.5 mm). Both groups demonstrated significant postinjury maximal increases in excitatory amino acid (EAA) concentration, which were proportional to the severity of injury. The mean ± SEM fold increase in dialysate concentrations of aspartate was 38 ± 13 (n = 5) for moderate TBI and 74 ± 12 (n = 5) for severe TBI. Fold increases in glutamate concentrations were 81 ± 26 and 144 ± 23 for moderate and severe TBI, respectively. Although these increases normalized within 20–30 min following moderate TBI, concentrations of aspartate and glutamate took >60 min to normalize after severe TBI. Changes in levels of nontransmitter amino acids were much smaller. Fold increases for serine concentrations were 4.6 ± 0.6 and 7.6 ± 1.7 in moderate and severe TBI, respectively; glutamine concentrations had similar small fold increases (2.6 ± 0.2 and 4.1 ± 0.6, respectively). Calculation of interstitial concentrations following severe TBI indicated that aspartate and glutamate maximally increased to 123 ± 20 and 414 ± 66 μM, respectively. To determine the extent to which such tissue concentrations of EAAs could contribute to the injury seen in TBI, the EAA receptor agonists N-methyl-d - aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid were slowly injected into rat cortex. Remarkably similar histological injuries were produced by this procedure, supporting the notion that TBI is an excitotoxic injury.     

脑挫裂伤致miR-9 升高促进神经干细胞分化

目的:探讨颅脑损伤后miR-9表达的变化和对神经干细胞分化和增值的影响,为颅脑损伤后神经功能修复治疗提出新的思路。方法:通过RT-PCR技术检测miR-9在挫裂伤脑组织中的表达情况;培养胚胎来源神经干细胞,并通过免疫荧光鉴定神经干细胞及其分化;转染miR-9后,通过MTT测定神经干细胞的增殖情况,和流式细胞仪检测分化神经元所占比例。结果:miR-9在挫裂伤脑组织中表达显著上升。对神经干细胞过表达miR-9可显著促进细胞增殖,并诱导分化成神经元。结论:脑挫裂伤时miR-9显著升高,并具有着促进神经干细胞增值和诱导分化的作用,可为伤后神经功能修复提供新的治疗方法。     

脑挫裂伤致miR-9升高促进神经干细胞分化

目的：探讨颅脑损伤后miR-9表达的变化和对神经干细胞分化和增值的影响,为颅脑损伤后神经功能修复治疗提出新的思路。方法：通过RT-PCR技术检测miR-9在挫裂伤脑组织中的表达情况;培养胚胎来源神经干细胞,并通过免疫荧光鉴定神经干细胞及其分化;转染miR-9后,通过MTT测定神经干细胞的增殖情况,和流式细胞仪检测分化神经元所占比例。结果：miR-9在挫裂伤脑组织中表达显著上升。对神经干细胞过表达miR-9可显著促进细胞增殖,并诱导分化成神经元。结论：脑挫裂伤时miR-9显著升高,并具有着促进神经干细胞增值和诱导分化的作用,可为伤后神经功能修复提供新的治疗方法。     

Cortical Endogenic Neural Regeneration of Adult Rat after Traumatic Brain Injury

Focal and diffuse neuronal loss happened after traumatic brain injury (TBI). With little in the way of effective repair, recent interest has focused on endogenic neural progenitor cells (NPCs) as a potential method for regeneration. Whether endogenic neural regeneration happened in the cortex of adult rat after TBI remains to be determined. In this study, rats were divided into a sham group and a TBI group, and the rat model of medium TBI was induced by controlled cortical impact. Rats were injected with BrdU at 1 to 7 days post-injury (dpi) to allow identification of differentiated cells and sacrificed at 1, 3, 7, 14 and 28 dpi for immunofluorescence. Results showed nestin⁺/sox-2⁺ NPCs and GFAP⁺/sox-2⁺ radial glial (RG)-like cells emerged in peri-injured cortex at 1, 3, 7, 14 dpi and peaked at 3 dpi. The number of GFAP⁺/sox-2⁺ cells was less than that of nestin⁺/sox-2⁺ cells. Nestin⁺/sox-2⁺ cells from posterior periventricle (pPV) immigrated into peri-injured cortex through corpus callosum (CC) were found. DCX⁺/BrdU⁺ newborn immature neurons in peri-injured cortex were found only at 3, 7, 14 dpi. A few MAP-2⁺/BrdU⁺ newborn neurons in peri-injured cortex were found only at 7 and 14 dpi. NeuN⁺/BrdU⁺ mature neurons were not found in peri-injured cortex at 1, 3, 7, 14 and 28 dpi. While GFAP⁺/BrdU⁺ astrocytes emerged in peri-injured cortex at 1, 3, 7, 14, 28 dpi and peaked at 7 dpi then kept in a stable state. In the corresponding time point, the percentage of GFAP⁺/BrdU⁺ astrocytes in BrdU⁺ cells was more than that of NPCs or newborn neurons. No CNP⁺/BrdU⁺ oligodendrocytes were found in peri-injured cortex. These findings suggest that NPCs from pPV and reactive RG–like cells emerge in peri-injured cortex of adult rats after TBI. It can differentiate into immature neurons and astrocytes, but the former fail to grow up to mature neurons.     

Berberine Protects against Neuronal Damage via Suppression of Glia-Mediated Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) triggers a series of neuroinflammatory processes that contribute to evolution of neuronal injury. The present study investigated the neuroprotective effects and anti-inflammatory actions of berberine, an isoquinoline alkaloid, in both in vitro and in vivo TBI models. Mice subjected to controlled cortical impact injury were injected with berberine (10 mg·kg⁻¹) or vehicle 10 min after injury. In addition to behavioral studies and histology analysis, blood-brain barrier (BBB) permeability and brain water content were determined. Expression of PI3K/Akt and Erk signaling and inflammatory mediators were also analyzed. The protective effect of berberine was also investigated in cultured neurons either subjected to stretch injury or exposed to conditioned media with activated microglia. Berberine significantly attenuated functional deficits and brain damage associated with TBI up to day 28 post-injury. Berberine also reduced neuronal death, apoptosis, BBB permeability, and brain edema at day 1 post-injury. These changes coincided with a marked reduction in leukocyte infiltration, microglial activation, matrix metalloproteinase-9 activity, and expression of inflammatory mediators. Berberine had no effect on Akt or Erk 1/2 phosphorylation. In mixed glial cultures, berberine reduced TLR4/MyD88/NF-κB signaling. Berberine also attenuated neuronal death induced by microglial conditioned media; however, it did not directly protect cultured neurons subjected to stretch injury. Moreover, administration of berberine at 3 h post-injury also reduced TBI-induced neuronal damage, apoptosis and inflammation in vivo. Berberine reduces TBI-induced brain damage by limiting the production of inflammatory mediators by glial cells, rather than by a direct neuroprotective effect.     

Up-regulation of MCM3 Relates to Neuronal Apoptosis After Traumatic Brain Injury in Adult Rats

Minichromosome maintenance complex component 3, one of the minichromosome maintenance proteins, functions as a part of pre-replication complex to initiate DNA replication in eukaryotes. Minichromosome maintenance complex component 3 (MCM3) was mainly implied in cell proliferation and tumorigenesis. In addition, MCM3 might play an important role in neuronal apoptosis. However, the functions of MCM3 in central nervous system are still with limited acquaintance. In this study, we performed a traumatic brain injury (TBI) model in adult rats. Western blot and immunohistochemistry staining showed up-regulation of MCM3 in the peritrauma brain cortex. The expression patterns of active caspase-3 and Bax, Bcl-2 were parallel with that of MCM3. Immunofluorescent staining and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling suggested that MCM3 was involved in neuronal apoptosis. In conclusion, our data indicated that MCM3 might play an important role in neuronal apoptosis following TBI. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against TBI.     

Up-Regulation of CCT8 Related to Neuronal Apoptosis after Traumatic Brain Injury in Adult Rats

Neurochemical Research - Traumatic brain injury (TBI) initiates a series of neurochemical and signaling changes that could eventually lead to neuronal apoptosis. Recent studies indicated that...     

Mitochondrial Dysfunction Contributes to Cell Death Following Traumatic Brain Injury in Adult and Immature Animals

Secondary injury following traumatic brain injury (TBI) is characterized by a variety of pathophysiologic cascades. Many of these cascades can have significant detrimental effects on cerebral mitochondria. These include exposure of neurons to excitotoxic levels of excitatory neurotransmitters with intracellular calcium influx, generation of reactive oxygen species, and production of peptides that participate in apoptotic cell death. Both experimental and clinical TBI studies have documented mitochondrial dysfunction, and animal studies suggest this dysfunction begins early and may persist for days following injury. Furthermore, interventions targeting mitochondrial mechanisms have shown neuroprotection after TBI. Continued evaluation and understanding of mitochondrial mechanisms contributing to neuronal cell death and survival after TBI is indicated. In addition, important underlying factors, such as brain maturation, that influence mitochondrial function should be studied. The ability to identify, target, and manipulate mitochondrial dysfunction may lead to the development of novel therapies for the treatment of adult and pediatric TBI.     

Influence of Post-Traumatic Stress Disorder on Neuroinflammation and Cell Proliferation in a Rat Model of Traumatic Brain Injury

Long-term consequences of traumatic brain injury (TBI) are closely associated with the development of severe psychiatric disorders, such as post-traumatic stress disorder (PTSD), yet preclinical studies on pathological changes after combined TBI with PTSD are lacking. In the present in vivo study, we assessed chronic neuroinflammation, neuronal cell loss, cell proliferation and neuronal differentiation in specific brain regions of adult Sprague-Dawley male rats following controlled cortical impact model of moderate TBI with or without exposure to PTSD. Eight weeks post-TBI, stereology-based histological analyses revealed no significant differences between sham and PTSD alone treatment across all brain regions examined, whereas significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle, but not cerebellum, in animals that received TBI alone and combined TBI-PTSD compared with PTSD alone and sham treatment. Additional immunohistochemical results revealed a significant loss of CA3 pyramidal neurons in the hippocampus of TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Further examination of neurogenic niches revealed a significant downregulation of Ki67-positive proliferating cells, but not DCX-positive neuronally migrating cells in the neurogenic subgranular zone and subventricular zone for both TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Comparisons of levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD revealed that PTSD did not exacerbate the neuropathological hallmarks of TBI. These results indicate a progressive deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized TBI patients.     

吸氢对大鼠颅脑损伤引起的急性炎症反应的抑制作用

为了探讨吸氢对大鼠创伤性颅脑损伤（traumatic brain injury,TBI）急性期炎症反应的影响,将6周龄雄性SD大鼠随机分为假手术组、TBI组和吸氢治疗组。采用悬浮芯片技术检测TBI后2、6和24 h的血清细胞因子水平;TBI后24 h采用改良的神经功能缺失评分法（modified neurological severity score,mNss）评估吸氢的神经保护作用,同时取脑组织进行尼氏染色分析并对血清生化指标进行检测。神经功能评分表明,TBI大鼠吸氢后24 h内神经功能就有显著改善,尼氏染色进一步验证了吸氢对神经元的保护作用;血清细胞因子的检测表明,吸氢对TBI引起的急性炎症反应具有很好的抑制作用,表现为7种促炎因子的血清水平在TBI后2 h明显降低。此外,吸氢还可明显降低血清中心脏和肝脏标志物水平,提示吸氢对TBI急性期心脏和肝脏功能损伤具有保护作用。研究提示吸氢可能通过抑制TBI急性期的炎症反应发挥其神经保护作用。     

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.     

Identification of Injury Specific Proteins in a Cell Culture Model of Traumatic Brain Injury

The complicated secondary molecular and cellular mechanisms following traumatic brain injury (TBI) are still not fully understood. In the present study, we have used mass spectrometry to identify injury specific proteins in an in vitro model of TBI. A standardized injury was induced by scalpel cuts through a mixed cell culture of astrocytes, oligodendrocytes and neurons. Twenty-four hours after the injury, cell culture medium and whole-cell fractions were collected for analysis. We found 53 medium proteins and 46 cell fraction proteins that were specifically expressed after injury and the known function of these proteins was elucidated by an extensive literature survey. By using time-lapse microscopy and immunostainings we could link a large proportion of the proteins to specific cellular processes that occur in response to trauma; including cell death, proliferation, lamellipodia formation, axonal regeneration, actin remodeling, migration and inflammation. A high percentage of the proteins uniquely expressed in the medium after injury were actin-related proteins, which normally are situated intracellularly. We show that two of these, ezrin and moesin, are expressed by astrocytes both in the cell culture model and in mouse brain subjected to experimental TBI. Interestingly, we found many inflammation-related proteins, despite the fact that cells were present in the culture. This study contributes with important knowledge about the cellular responses after trauma and identifies several potential cell-specific biomarkers.     

Cell Proliferation and Neurogenesis in Adult Mouse Brain

Neurogenesis, the formation of new neurons, can be observed in the adult brain of many mammalian species, including humans. Despite significant progress in our understanding of adult neurogenesis, we are still missing data about the extent and location of production of neural precursors in the adult mammalian brain. We used 5-ethynyl-2''-deoxyuridine (EdU) to map the location of proliferating cells throughout the entire adult mouse brain and found that neurogenesis occurs at two locations in the mouse brain. The larger one we define as the main proliferative zone (MPZ), and the smaller one corresponds to the subgranular zone of the hippocampus. The MPZ can be divided into three parts. The caudate migratory stream (CMS) occupies the middle part of the MPZ. The cable of proliferating cells emanating from the most anterior part of the CMS toward the olfactory bulbs forms the rostral migratory stream. The thin layer of proliferating cells extending posteriorly from the CMS forms the midlayer. We have not found any additional aggregations of proliferating cells in the adult mouse brain that could suggest the existence of other major neurogenic zones in the adult mouse brain.     

Inflammation in Traumatic Brain Injury: Role of Cytokines and Chemokines

A traumatic injury to the adult mammalian central nervous system (CNS), such as a stab wound lesion, results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. The roles of cytokines and growth factors released locally by the damaged endogenous cells are recognized in controlling the cellular changes that occur following CNS injury. However, the role of chemokines, a novel class of chemoattractant cytokines, is only recently being studied in regulating inflammatory cell invasion in the injured/diseased CNS (1). The mRNAs for several chemokines have been shown to be upregulated in experimental allergic encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, but chemokine expression in traumatic brain injury has not been studied in detail. Astrocytes have been demonstrated to participate in numerous processes that occur following injury to the CNS. In particular, astrocytic expression of cytokines and growth factors in the injured CNS has been well reviewed (2). Recently a few studies have detected the presence of chemokines in astrocytes following traumatic brain injury (3,4). These studies have suggested that chemokines may represent a promising target for future therapy of inflammatory conditions. This review summarizes the events that occur in traumatic brain injury and discusses the roles of resident and non-resident cells in the expression of growth factors, cytokines and chemokines in the injured CNS.     

Upregulation of 3-MST Relates to Neuronal Autophagy After Traumatic Brain Injury in Mice

3-mercaptopyruvate sulfurtransferase (3-MST) was a novel hydrogen sulfide (H₂S)-synthesizing enzyme that may be involved in cyanide degradation and in thiosulfate biosynthesis. Over recent years, considerable attention has been focused on the biochemistry and molecular biology of H₂S-synthesizing enzyme. In contrast, there have been few concerted attempts to investigate the changes in the expression of the H₂S-synthesizing enzymes with disease states. To investigate the changes of 3-MST after traumatic brain injury (TBI) and its possible role, mice TBI model was established by controlled cortical impact system, and the expression and cellular localization of 3-MST after TBI was investigated in the present study. Western blot analysis revealed that 3-MST was present in normal mice brain cortex. It gradually increased, reached a peak on the first day after TBI, and then reached a valley on the third day. Importantly, 3-MST was colocalized with neuron. In addition, Western blot detection showed that the first day post injury was also the autophagic peak indicated by the elevated expression of LC3. Importantly, immunohistochemistry analysis revealed that injury-induced expression of 3-MST was partly colabeled by LC3. However, there was no colocalization of 3-MST with propidium iodide (cell death marker) and LC3 positive cells were partly colocalized with propidium iodide. These data suggested that 3-MST was mainly located in living neurons and may be implicated in the autophagy of neuron and involved in the pathophysiology of brain after TBI.     

Environmental Circadian Disruption Worsens Neurologic Impairment and Inhibits Hippocampal Neurogenesis in Adult Rats After Traumatic Brain Injury

Circadian rhythms modulate many physiologic processes and behaviors. Therefore, their disruption causes a variety of potential adverse effects in humans and animals. Circadian disruption induced by constant light exposure has been discovered to produce pathophysiologic consequences after brain injury. However, the underlying mechanisms that lead to more severe impairment and disruption of neurophysiologic processes are not well understood. Here, we evaluated the effect of constant light exposure on the neurobehavioral impairment and survival of neurons in rats after traumatic brain injury (TBI). Sixty adult male Sprague–Dawley rats were subjected to a weight-drop model of TBI and then exposed to either a standard 12-/12-h light/dark cycle or a constant 24-h light/light cycle for 14 days. Our results showed that 14 days of constant light exposure after TBI significantly worsened the sensorimotor and cognitive deficits, which were associated with decreased body weight, impaired water and food intake, increased cortical lesion volume, and decreased neuronal survival. Furthermore, environmental circadian disruption inhibited cell proliferation and newborn cell survival and decreased immature cell production in rats subjected to the TBI model. We conclude that circadian disruption induced by constant light exposure worsens histologic and neurobehavioral impairment and inhibits neurogenesis in adult TBI rats. Our novel findings suggest that light exposure should be decreased and circadian rhythm reestablished in hospitalized TBI patients and that drugs and strategies that maintain circadian rhythm would offer a novel therapeutic option.     

Oligodendrocyte Birth and Death following Traumatic Brain Injury in Adult Mice

Oligodendrocytes are responsible for producing and maintaining myelin throughout the CNS. One of the pathological features observed following traumatic brain injury (TBI) is the progressive demyelination and degeneration of axons within white matter tracts. While the effect of TBI on axonal health has been well documented, there is limited information regarding the response of oligodendrocytes within these areas. The aim of this study was to characterize the response of both mature oligodendrocytes and immature proliferative oligodendrocyte lineage cells across a 3 month timecourse following TBI. A computer-controlled cortical impact model was used to produce a focal lesion in the left motor cortex of adult mice. Immunohistochemical analyses were performed at 48 hours, 7 days, 2 weeks, 5 weeks and 3 months following injury to assess the prevalence of mature CC-1⁺ oligodendrocyte cell death, immature Olig2⁺ cell proliferation and longer term survival in the corpus callosum and external capsule. Decreased CC-1 immunoreactivity was observed in white matter adjacent to the site of injury from 2 days to 2 weeks post TBI, with ongoing mature oligodendrocyte apoptosis after this time. Conversely, proliferation of Olig2⁺ cells was observed as early as 48 hours post TBI and significant numbers of these cells and their progeny survived and remained in the external capsule within the injured hemisphere until at least 3 months post injury. These findings demonstrate that immature oligodendrocyte lineage cells respond to TBI by replacing oligodendrocytes lost due to damage and that this process occurs for months after injury.