gThe Dorothy Russell Memorial Lecture* The molecular and cellular sequelae of experimental traumatic brain injury: pathogenetic mechanisms

The mechanisms underlying secondary or delayed cell death following traumatic brain injury (TBI) are poorly understood. Recent evidence from experimental models of TBI suggest that diffuse and widespread neuronal damage and loss is progressive and prolonged for months to years after the initial insult in selectively vulnerable regions of the cortex, hippocampus, thalamus, striatum, and subcortical nuclei. The development of new neuropathological and molecular techniques has generated new insights into the cellular and molecular sequelae of brain trauma. This paper will review the literature suggesting that alterations in intracellular calcium with resulting changes in gene expression, activation of reactive oxygen species (ROS), activation of intracellular proteases (calpains), expression of neurotrophic factors, and activation of cell death genes (apoptosis) may play a role in mediating delayed cell death after trauma. Recent data suggesting that TBI should be considered as both an inflammatory and/or a neurodegenerative disease is also presented. Further research concerning the complex molecular and neuropathological cascades following brain trauma should be conducted, as novel therapeutic strategies continue to be developed.     

The potential role of mitochondria in pediatric traumatic brain injury

Mitochondria play a central role in cerebral energy metabolism, intracellular calcium homeostasis and reactive oxygen species generation and detoxification. Following traumatic brain injury (TBI), the degree of mitochondrial injury or dysfunction can be an important determinant of cell survival or death. Literature would suggest that brain mitochondria from the developing brain are very different from those from mature animals. Therefore, aspects of developmental differences in the mitochondrial response to TBI can make the immature brain more vulnerable to traumatic injury. This review will focus on four main areas of secondary injury after pediatric TBI, including excitotoxicity, oxidative stress, alterations in energy metabolism and cell death pathways. Specifically, we will describe what is known about developmental differences in mitochondrial function in these areas, in both the normal, physiologic state and the pathologic state after pediatric TBI. The ability to identify and target aspects of mitochondrial dysfunction could lead to novel neuroprotective therapies for infants and children after severe TBI.     

Mechanical trauma induces immediate changes in neuronal network activity

During a traumatic insult to the brain, tissue is subjected to large stresses at high rates which often surpass cellular thresholds leading to cell dysfunction or death. The acute response of neurons to a mechanical trauma, however, is poorly understood. Plasma membrane disruption may be the earliest cellular outcome from a mechanical trauma. The increase in membrane permeability due to such disruptions may therefore play an important role in the initiation of deleterious cascades following brain injury. The immediate consequences of an increase in plasma membrane permeability on the electrophysiological behavior of a neuronal network exposed to the trauma have not been elucidated. We have developed an in vitro model of traumatic brain injury (TBI) that utilizes a novel device capable of applying stress at high rates to neuronal cells cultured on a microelectrode array. The mechanical insult produced by the device caused a transient increase in neuronal plasma membrane permeability, which subsided after 10 min. We were able to monitor acute spontaneous electrophysiological activity of injured cultures for at least 10 min following the insult. Firing frequency, average burst interval and spikes within burst were assessed before and after injury. The electrophysiological responses to the insult were heterogeneous, although an increase in burst intervals and in the variability of the assessed parameters were common. This study provides a multi-faceted approach to elucidate the role of neuronal plasma membrane disruptions in TBI and its functional consequences.     

Mechanical strain injury increases intracellular sodium and reverses Na+/Ca2+ exchange in cortical astrocytes

Traditionally, astrocytes have been considered less susceptible to injury than neurons. Yet, we have recently shown that astrocyte death precedes neuronal death in a rat model of traumatic brain injury (TBI) (Zhao et al.: Glia 44:140-152, 2003). A main mechanism hypothesized to contribute to cellular injury and death after TBI is elevated intracellular calcium ([Ca2+]i). Since calcium regulation is also influenced by regulation of intracellular sodium ([Na+]i), we used an in vitro model of strain-induced traumatic injury and live-cell fluorescent digital imaging to investigate alterations in [Na+]i in cortical astrocytes after injury. Changes in [Na+]i, or [Ca2+]i were monitored after mechanical injury or L-glutamate exposure by ratiometric imaging of sodium-binding benzofuran isophthalate (SBFI-AM), or Fura-2-AM, respectively. Mechanical strain injury or exogenous glutamate application produced increases in [Na+]i that were dependent on the severity of injury or concentration. Injury-induced increases in [Na+]i were significantly reduced, but not completely eliminated, by inhibition of glutamate uptake by DL-threo-beta-benzyloxyaspartate (TBOA). Blockade of sodium-dependent calcium influx through the sodium-calcium exchanger with 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943) reduced [Ca2+]i after injury. KB-R7943 also reduced astrocyte death after injury. These findings suggest that in astrocytes subjected to mechanical injury or glutamate excitotoxicity, increases in intracellular Na+ may be a critical component in the injury cascade and a therapeutic target for reduction of lasting deficits after traumatic brain injury.     

Physiologic progesterone reduces mitochondrial dysfunction and hippocampal cell loss after traumatic brain injury in female rats

Growing literature suggests important sex-based differences in outcome following traumatic brain injury (TBI) in animals and humans. Progesterone has emerged as a key hormone involved in many potential neuroprotective pathways after acute brain injury and may be responsible for some of these differences. Many studies have utilized supraphysiologic levels of post-traumatic progesterone to reverse pathologic processes after TBI, but few studies have focused on the role of endogenous physiologic levels of progesterone in neuroprotection. We hypothesized that progesterone at physiologic serum levels would be neuroprotective in female rats after TBI and that progesterone would reverse early mitochondrial dysfunction seen in this model. Female, Sprague-Dawley rats were ovariectomized and implanted with silastic capsules containing either low or high physiologic range progesterone at 7 days prior to TBI. Control rats received ovariectomy with implants containing no hormone. Rats underwent controlled cortical impact to the left parietotemporal cortex and were evaluated for evidence of early mitochondrial dysfunction (1 h) and delayed hippocampal neuronal injury and cortical tissue loss (7 days) after injury. Progesterone in the low physiologic range reversed the early postinjury alterations seen in mitochondrial respiration and reduced hippocampal neuronal loss in both the CA1 and CA3 subfields. Progesterone in the high physiologic range had a more limited pattern of hippocampal neuronal preservation in the CA3 region only. Neither progesterone dose significantly reduced cortical tissue loss. These findings have implications in understanding the sex-based differences in outcome following acute brain injury.     

From cell death to neuronal regeneration: building a new brain after traumatic brain injury

During the past decade, there has been accumulating evidence of the involvement of passive and active cell death mechanisms in both the clinical setting and in experimental models of traumatic brain injury (TBI). Traditionally, research for a treatment of TBI consists of strategies to prevent cell death using acute pharmacological therapy. However, to date, encouraging experimental work has not been translated into successful clinical trials. The development of cell replacement therapies may offer an alternative or a complementary strategy for the treatment of TBI. Recent experimental studies have identified a variety of candidate cell lines for transplantation into the injured CNS. Additionally, the characterization of the neurogenic potential of specific regions of the adult mammalian brain and the elucidation of the molecular controls underlying regeneration may allow for the development of neuronal replacement therapies that do not require transplantation of exogenous cells. These novel strategies may represent a new opportunity of great interest for delayed intervention in patients with TBI.     

Common patterns of Bcl-2 family gene expression in two traumatic brain injury models

Cell death/survival following traumatic brain injury (TBI) may be a result of alterations in the intracellular ratio of death and survival factors. Bcl-2 family genes mediate both cell survival and the initiation of cell death. Using lysate RNase protection assays, mRNA expression of the anti-cell death genes Bcl-2 and Bcl-xL, and the pro-cell death gene Bax, was evaluated following experimental brain injuries in adult male Sprague-Dawley rats. Both the lateral fluid-percussion (LFP) and the lateral controlled cortical impact (LCI) models of TBI showed similar patterns of gene expression. Anticell death bcl-2 and bcl-xL mRNAs were attenuated early and tended to remain depressed for at least 3 days after injury in the cortex and hippocampus ipsilateral to injury. Pro-cell death bax mRNA was elevated in these areas, usually following the decrease in anti-cell death genes. These common patterns of gene expression suggest an important role for Bcl-2 genes in cell death and survival in the injured brain. Understanding the regulation of these genes may facilitate the development of new therapeutic strategies for a condition that currently has no proven pharmacologic treatments.     

Cytochrome c release and caspase activation after traumatic brain injury

Experimental traumatic brain injury (TBI) results in a rapid and significant necrosis of cortical tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the primary damage resulting in significant neurological dysfunction. The identification of cell death pathways that mediate this secondary traumatic injury have not been elucidated, however recent studies have implicated a role for apoptosis in the neuropathology of traumatic brain injury. The present study utilized a controlled cortical impact model of brain injury to assess the involvement of apoptotic pathways: release of cytochrome c from mitochondria and the activation of caspase-1- and caspase-3-like proteases in the injured cortex at 6, 12 and 24 h post-injury. Collectively, these results demonstrate cytochrome c release from mitochondria and its redistribution into the cytosol occurs in a time-dependent manner following TBI. The release of cytochrome c is accompanied by a time-dependent increase in caspase-3-like protease activity with no apparent increase in caspase-1-like activity. However, pretreatment with a general caspase inhibitor had no significant effect on the amount of cortical damage observed at 7 days post-injury. Our data suggest that several pro-apoptotic events occur following TBI, however the translocation of cytochrome c itself and/or other events upstream of caspase activation/inhibition may be sufficient to induce neuronal cell death.     

Exercise inhibits neuronal apoptosis and improves cerebral function following rat traumatic brain injury

Exercise is reported to inhibit neuronal apoptotic cell death in the hippocampus and improve learning and memory. However, the effect of exercise on inhibition of neuronal apoptosis surrounding the area of damage after traumatic brain injury (TBI) and the improvement of cerebral dysfunction following TBI are unknown. Here, we investigate the effect of exercise on morphology and cerebral function following TBI in rats. Wistar rats received TBI by a pneumatic controlled injury device were randomly divided into two groups: (1) non-exercise group and (2) exercise group. The exercise group ran on a treadmill for 30 min/day at 22 m/min for seven consecutive days. Immunohistochemical and behavioral studies were performed following TBI. The number of single-stranded DNA (ssDNA)-positive cells around the damaged area early after TBI was significantly reduced in the exercise group compared with the non-exercise group (P < 0.05). Furthermore, most ssDNA-positive cells in the non-exercise group co-localized with neuronal cells. However, in the exercise group, a few ssDNA-positive cells co-localized with neurons. In addition, there was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the exercise group compared with the non-exercise group (P < 0.05). These results indicate that exercise following TBI inhibits neuronal degeneration and apoptotic cell death around the damaged area, which results in improvement of cerebral dysfunction. In summary, treadmill running improved cerebral dysfunction following TBI, indicating its potential as an effective clinical therapy. Therefore, exercise therapy (rehabilitation) in the early phase following TBI is important for recuperation from cerebral dysfunction.     

Sexual dimorphism in the inflammatory response to traumatic brain injury

The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate‐to‐severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro‐ (IL‐1β and TNFα) and anti‐inflammatory (Arg1 and TGFβ) cytokines with a single‐phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro‐inflammatory response peaks at 4 h and 7 days, and a delayed anti‐inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post‐TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.     

Calpain as a therapeutic target in traumatic brain injury

The family of calcium-activated neutral proteases, calpains, appears to play a key role in neuropathologic events following traumatic brain injury (TBI). Neuronal calpain activation has been observed within minutes to hours after either contusive or diffuse brain trauma in animals, suggesting that calpains are an early mediator of neuronal damage. Whereas transient calpain activation triggers numerous cell signaling and remodeling events involved in normal physiological processes, the sustained calpain activation produced by trauma is associated with neuron death and axonal degeneration in multiple models of TBI. Nonetheless, the causal relationship between calpain activation and neuronal death is not fully understood. Much remains to be learned regarding the endogenous regulatory mechanisms for controlling calpain activity, the roles of different calpain isoforms, and the in vivo substrates affected by calpain. Detection of stable proteolytic fragments of the submembrane cytoskeletal protein αII-spectrin specific for cleavage by calpains has been the most widely used marker of calpain activation in models of TBI. More recently, these protein fragments have been detected in the cerebrospinal fluid after TBI, driving interest in their potential utility as TBI-associated biomarkers. Post-traumatic inhibition of calpains, either direct or indirect through targets related to intracellular calcium regulation, is associated with attenuation of functional and behavioral deficits, axonal pathology, and cell death in animal models of TBI. This review focuses on the current state of knowledge of the role of calpains in TBI-induced neuropathology and effectiveness of calpain as a therapeutic target in the acute post-traumatic period.     

Cell death mechanisms and modulation in traumatic brain injury

Cell death after traumatic brain injury (TBI) is a major cause of neurological deficits and mortality. Understanding the mechanisms of delayed post-traumatic cell loss may lead to new therapies that improve outcome. Although TBI induces changes in multiple cell types, mechanisms of neuronal cell death have been the predominant focus. Recent work has emphasized the diversity of neuronal death phenotypes, which have generally been defined by either morphological or molecular changes. This diversity has led to confusing and at times contradictory nomenclature. Here we review the historical basis of proposed definitions of neuronal cell death, with the goal of clarifying critical research questions and implications for therapy in TBI. We believe that both morphological and molecular features must be used to clarify post-traumatic cell death and related therapeutic targets. Further, we underscore that the most effective neuroprotective strategies will need to target multiple pathways to reflect the regional and temporal changes underlying diverse neuronal cell death phenotypes.     

VEGF protects rat cortical neurons from mechanical trauma injury induced apoptosis via the MEK/ERK pathway

Traumatic brain injury (TBI) is a serious insult that frequently leads to neurological dysfunction or death. Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and vascular permeability. Recently, VEGF has been identified as a neurotrophic factor and has been implicated in the pathogenic mechanisms of TBI. However, the possible mechanisms of VEGF in primary or secondary injuries after TBI are largely unknown. The present study attempted to determine whether VEGF has a protective effect on primary cortical neurons against mechanical trauma injury, which is an in vitro insult mimicking traumatic brain injury. We found that pretreatment of primary cortical neurons in culture with VEGF decreased neuronal death in a concentration-dependent manner, and VEGF counteracted the mechanical trauma mediated apoptotic death of cultured cortical neurons. VEGF up-regulates the activity of ERK (extracellular signal-regulated kinase) in cultured cortical neurons and U0126 (a mitogen activated protein kinase kinase (MEK) inhibitor) suppressed VEGF induced activity of ERK. Furthermore, incubation of cells with U0126 attenuated the ability of VEGF to protect neurons against mechanical trauma-induced apoptosis. Therefore, the present study supports the notion that MEK/ERK pathway is involved in VEGF mediated neuroprotection against mechanical trauma injury.     

Neuropathological and biochemical features of traumatic injury in the developing brain

Trauma to the developing brain constitutes a poorly explored field. Some recent studies attempting to model and study pediatric head trauma, the leading cause of death and disability in the pediatric population, revealed interesting aspects and potential targets for future research. Trauma triggers both excitotoxic and apoptotic neurodegeneration in the developing rat brain. Excitotoxic neurodegeneration develops and subsides rapidly (within hours) whereas apoptotic cell death occurs in a delayed fashion over several days following the initial traumatic insult. Apoptotic neurodegeneration contributes in an age-dependent fashion to neuronal injury following head trauma, with the immature brain being exceedingly sensitive. In the most vulnerable ages the apoptosis contribution to the extent of traumatic brain damage far outweighs that of the excitotoxic component.     

Selective CDK inhibitor limits neuroinflammation and progressive neurodegeneration after brain trauma

Traumatic brain injury (TBI) induces secondary injury mechanisms, including cell-cycle activation (CCA), which lead to neuronal cell death, microglial activation, and neurologic dysfunction. Here, we show progressive neurodegeneration associated with microglial activation after TBI induced by controlled cortical impact (CCI), and also show that delayed treatment with the selective cyclin-dependent kinase inhibitor roscovitine attenuates posttraumatic neurodegeneration and neuroinflammation. CCI resulted in increased cyclin A and D1 expressions and fodrin cleavage in the injured cortex at 6 hours after injury and significant neurodegeneration by 24 hours after injury. Progressive neuronal loss occurred in the injured hippocampus through 21 days after injury and correlated with a decline in cognitive function. Microglial activation associated with a reactive microglial phenotype peaked at 7 days after injury with sustained increases at 21 days. Central administration of roscovitine at 3 hours after CCI reduced subsequent cyclin A and D1 expressions and fodrin cleavage, improved functional recovery, decreased lesion volume, and attenuated hippocampal and cortical neuronal cell loss and cortical microglial activation. Furthermore, delayed systemic administration of roscovitine improved motor recovery and attenuated microglial activation after CCI. These findings suggest that CCA contributes to progressive neurodegeneration and related neurologic dysfunction after TBI, likely in part related to its induction of microglial activation.     

The overlooked aspect of excitotoxicity: Glutamate‐independent excitotoxicity in traumatic brain injuries

Traumatic brain injury (TBI) is a leading major cause of morbidity and mortality in youth and individuals under 45 year age. A wide variety of cellular and molecular mechanisms have been identified contributing to the pathogenesis of TBI. A better understanding of the pathophysiology behind TBI is essential for providing more effective treatment. Excitotoxicity as one of the secondary molecular events is a major contributing factor in apoptosis and neuronal death following the initial injury in TBI. Excitotoxicity is the rapid overload and influx of calcium into the cell cytoplasm, activating a series of deleterious signaling cascades causing the cell to undergo apoptosis. Conventional understanding is that the rapid influx of calcium is initiated through glutamate release. However, there are overlooked glutamate‐independent mechanisms that cause the rapid calcium influx into the neuronal cytoplasm, evoking or contributing to excitotoxicity. Therefore, the focus of this review will be on the role of the glutamate‐independent excitotoxic mechanisms of the mechanosensitive response of NMDA receptors, mechanoporation of the cell membrane, ischemia, and the release of calcium from intracellular stores. In conclusion, the shear and stretch forces during a TBI event may result in the mechanosensitive activation of NMDA receptors which contribute to glutamate‐independent excitotoxicity.     

Post-traumatic stress disorder vs traumatic brain injury

Post-traumatic stress disorder (PTSD) and traumatic brain injury (TBI) often coexist because brain injuries are often sustained in traumatic experiences. This review outlines the significant overlap between PTSD and TBI by commencing with a critical outline of the overlapping symptoms and problems of differential diagnosis. The impact of TBI on PTSD is then described, with increasing evidence suggesting that mild TBI can increase risk for PTSD. Several explanations are offered for this enhanced risk. Recent evidence suggests that impairment secondary to mild TBI is largely attributable to stress reactions after TBI, which challenges the long-held belief that postconcussive symptoms are a function of neurological insult This recent evidence is pointing to new directions for treatment of postconcussive symptoms that acknowledge that treating stress factors following TBI may be the optimal means to manage the effects of many TBIs.     

In vitro mechanical strain trauma alters neuronal calcium responses: Implications for posttraumatic epilepsy

Traumatic brain injury (TBI) is known to initiate a series of chemical cascades resulting in neuronal dysfunction and death. Epidemiology studies have found that a prior incidence of TBI is the most important cause of remote symptomatic epilepsy in young adults and children. TBI-induced changes in neuronal sensitivity to stimulation may contribute to acute seizures and the eventual generation of epilepsy. This study examined TBI-induced changes in neuronal sensitivity to stimulation by measuring intracellular calcium ([Ca(++) ](i) ) responses in neurons during glutamate application in vitro. Initial experiments examined neuronal and glial cell death and determined that a 31% mechanical strain trauma to mixed neuronal-astrocyte rat cortical cultures produced a trend, but no significant cell death at 48?h after injury. Subsequent experiments utilized this magnitude of trauma to examine the sensitivity of cortical neurons to changes in [Ca(++) ](i) in response to 100-μm glutamate at five time points postinjury (1, 6, 24, 48, and 72?h). Traumatically strain-injured neurons responded with a dynamic change in the accumulation of [Ca(++) ](i) , with a significant increase at 48?h and a significant decrease at 72?h as compared to uninjured cultures. These data highlight that TBI leads to abnormal responsiveness to stimulation, an indicator of neuronal dysfunction in surviving cells. Such changes in sensitivity to stimulation may also be associated with changes in excitability in the first hours to days after TBI, and may play a role in early posttraumatic seizures observed in patients with TBI. In addition, this study provides an in vitro paradigm for testing the function of surviving cells following treatment interventions targeted at reducing cell death and dysfunction.