Kathryn E. Saatman

Classification of Traumatic Brain Injury for Targeted Therapies

The heterogeneity of traumatic brain injury (TBI) is considered one of the most significant barriers to finding effective therapeutic interventions. In October, 2007, the National Institute of Neurological Disorders and Stroke, with support from the Brain Injury Association of America, the Defense and Veterans Brain Injury Center, and the National Institute of Disability and Rehabilitation Research, convened a workshop to outline the steps needed to develop a reliable, efficient and valid classification system for TBI that could be used to link specific patterns of brain and neurovascular injury with appropriate therapeutic interventions. Currently, the Glasgow Coma Scale (GCS) is the primary selection criterion for inclusion in most TBI clinical trials. While the GCS is extremely useful in the clinical management and prognosis of TBI, it does not provide specific information about the pathophysiologic mechanisms which are responsible for neurological deficits and targeted by interventions. On the premise that brain injuries with similar pathoanatomic features are likely to share common pathophysiologic mechanisms, participants proposed that a new, multidimensional classification system should be developed for TBI clinical trials. It was agreed that preclinical models were vital in establishing pathophysiologic mechanisms relevant to specific pathoanatomic types of TBI and verifying that a given therapeutic approach improves outcome in these targeted TBI types. In a clinical trial, patients with the targeted pathoanatomic injury type would be selected using an initial diagnostic entry criterion, including their severity of injury. Coexisting brain injury types would be identified and multivariate prognostic modeling used for refinement of inclusion/exclusion criteria and patient stratification. Outcome assessment would utilize endpoints relevant to the targeted injury type. Advantages and disadvantages of currently available diagnostic, monitoring, and assessment tools were discussed. Recommendations were made for enhancing the utility of available or emerging tools in order to facilitate implementation of a pathoanatomic classification approach for clinical trials.

Motor and cognitive function evaluation following experimental traumatic brain injury

Traumatic brain injury (TBI) in humans may cause extensive sensorimotor and cognitive dysfunction. As a result, many TBI researchers are beginning to assess behavioral correlates of histologically determined damage in animal models. Although this is an important step in TBI research, there is a need for standardization between laboratories. The ability to reliably test treatments across laboratories and multiple injury models will close the gap between treatment success in the lab and success in the clinic. The goal of this review is to describe and evaluate the tests employed to assess functional outcome after TBI and to overview aspects of cognitive, sensory, and motor function that may be suitable targets for therapeutic intervention.

The 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.

Prolonged calpain-mediated spectrin breakdown occurs regionally following experimental brain injury in the rat.

Calpain, a calcium-activated neutral protease family, has been implicated in the neuropathologic sequelae accompanying various neurological disorders. We have characterized the distribution and time course of calpain activation following brain injury in the rat, using a monoclonal antibody that recognizes calpain-generated breakdown products (BDPs) of spectrin. Adult male Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2–2.4 atm, n = 35) or served as controls (uninjured, n = 12). One group of animals (n = 21) were sacrificed at either 30 minutes (min), 1 day, or 3 days post-injury, and selected brain regions were prepared for Western blot analysis. The remaining animals (n = 26) were sacrificed at 90 min, 4 hours (h), 1 day, or 7 days post-injury, and immunohistochemistry was performed. Spectrin BDPs were found predominantly in the hemisphere ipsilateral to the injury site, located primarily in cortical and hippocampal regions which exhibit neuronal death. Calpain-mediated spectrin breakdown was detected at 90 min in dendrites and axons, and by 4 h in neuronal perikarya. By 1 day post-injury, cortical and hippocampal regions of calpain activation had increased in size. Delayed spectrin breakdown was observed in the thalamus, both at 3 days and 7 days after injury. These results suggest that calpain may play an role in the neurodegenerative process following brain injury.

Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury.

OBJECTIVE Using the neural stem cell (NSC) clone C17.2, we evaluated the ability of transplanted murine NSCs to attenuate cognitive and neurological motor deficits after traumatic brain injury. METHODS Nonimmunosuppressed C57BL/6 mice (n = 65) were anesthetized and subjected to lateral controlled cortical impact brain injury (n = 52) or surgery without injury (sham operation group, n = 13). At 3 days postinjury, all brain-injured animals were reanesthetized and randomized to receive stereotactic injection of NSCs or control cells (human embryonic kidney cells) into the cortex-hippocampus interface in either the ipsilateral or the contralateral hemisphere. One group of animals (n = 7) was killed at either 1 or 3 weeks postinjury to assess NSC survival in the acute posttraumatic period. Motor function was evaluated at weekly intervals for 12 weeks in the remaining animals, and cognitive (i.e., learning) deficits were assessed at 3 and 12 weeks after transplantation. RESULTS Brain-injured animals that received either ipsilateral or contralateral NSC transplants showed significantly improved motor function in selected tests as compared with human embryonic kidney cell-transplanted animals during the 12-week observation period. Cognitive dysfunction was unaffected by transplantation at either 3 or 12 weeks postinjury. Histological analyses showed that NSCs survive for as long as 13 weeks after transplantation and were detected in the hippocampus and/or cortical areas adjacent to the injury cavity. At 13 weeks, the NSCs transplanted ipsilateral to the impact site expressed neuronal (NeuN) or astrocytic (glial fibrillary acidic protein) markers but not markers of oligodendrocytes (2′3′cyclic nucleotide 3′-phosphodiesterase), whereas the contralaterally transplanted NSCs expressed neuronal but not glial markers (double-labeled immunofluorescence and confocal microscopy). CONCLUSION These data suggest that transplanted NSCs can survive in the traumatically injured brain, differentiate into neurons and/or glia, and attenuate motor dysfunction after traumatic brain injury.

Accumulation of Amyloid β and Tau and the Formation of Neurofilament Inclusions Following Diffuse Brain Injury in the Pig

Brain trauma in humans increases the risk for developing Alzheimer disease (AD) and may induce the acute formation of AD-like plaques containing amyloid beta (A beta). To further explore the potential link between brain trauma and neurodegeneration, we conducted neuropathological studies using a pig model of diffuse brain injury. Brain injury was induced in anesthetized animals via nonimpact head rotational acceleration of 110 degrees over 20 ms in the coronal plane (n = 15 injured, n = 3 noninjured). At 1, 3, 7, and 10 days post-trauma, control and injured animals were euthanized and immunohistochemical analysis was performed on brain sections using antibodies specific for A beta, beta-amyloid precursor protein (betaPP), tau, and neurofilament (NF) proteins. In addition to diffuse axonal pathology, we detected accumulation of A beta and tau that colocalized with immunoreactive betaPP and NF in damaged axons throughout the white matter in all injured animals at 3-10 days post-trauma. In a subset of brain injured animals, diffuse A beta-containing plaque-like profiles were found in both the gray and white matter, and accumulations of tau and NF rich inclusions were observed in neuronal perikarya. These results show that this pig model of diffuse brain injury is characterized by accumulations of proteins that also form pathological aggregates in AD and related neurodegenerative diseases.

Temporal window of vulnerability to repetitive experimental concussive brain injury.

OBJECTIVE:Repetitive concussive brain injury (CBI) is associated with cognitive alterations and increased risk of neurodegenerative disease. METHODS:To evaluate the temporal window during which the concussed brain remains vulnerable to a second concussion, anesthetized mice were subjected to either sham injury or single or repetitive CBI (either 3, 5, or 7 days apart) using a clinically relevant model of CBI. Cognitive, vestibular, and sensorimotor function (balance and coordination) were evaluated, and postmortem histological analyses were performed to detect neuronal degeneration, cytoskeletal proteolysis, and axonal injury. RESULTS:No cognitive deficits were observed in sham-injured animals or those concussed once. Mice subjected to a second concussion within 3 or 5 days exhibited significantly impaired cognitive function compared with either sham-injured animals (P < 0.05) or mice receiving a single concussion (P < 0.01). No cognitive deficits were observed when the interconcussion interval was extended to 7 days, suggestive of a transient vulnerability of the brain during the first 5 days after an initial concussion. Although all concussed mice showed transient motor deficits, vestibulomotor dysfunction was more pronounced in the group that sustained two concussions 3 days apart (P < 0.01 compared with all other groups). Although scattered degenerating neurons, evidence of cytoskeletal damage, and axonal injury were detected in selective brain regions between 72 hours and 1 week after injury in all animals sustaining a single concussion, the occurrence of a second concussion 3 days later resulted in significantly greater traumatic axonal injury (P < 0.05) than that resulting from a single CBI. CONCLUSION:These data suggest that a single concussion is associated with behavioral dysfunction and subcellular alterations that may contribute to a transiently vulnerable state during which a second concussion within 3 to 5 days can lead to exacerbated and more prolonged axonal damage and greater behavioral dysfunction.

Insulin-like Growth Factor-1 (IGF-1) Improves both Neurological Motor and Cognitive Outcome Following Experimental Brain Injury

We evaluated the efficacy of insulin-like growth factor-1 (IGF-1) in attenuating neurobehavioral deficits following lateral fluid percussion (FP) brain injury. Male Sprague-Dawley rats (345-425 g, n = 88) were anesthetized and subjected to FP brain injury of moderate severity (2.4-2.9 atm). In Study 1, IGF-1 (1.0 mg/kg, n = 9) or vehicle (n = 14) was administered by subcutaneous injection at 15 min postinjury and similarly at 12-h intervals for 14 days. In animals evaluated daily for 14 days, IGF-1 treatment attenuated motor dysfunction over the 2-week period (P < 0.02). In Study 2, IGF-1 (4 mg/kg/day, n = 8 uninjured, n = 13 injured) or vehicle (n = 8 uninjured, n = 13 injured) was administered for 2 weeks via a subcutaneous pump implanted 15 min postinjury. IGF-1 administration was associated with increased body weight and mild, transient hypoglycemia which was more pronounced in brain-injured animals. At 2 weeks postinjury (P < 0.05), but not at 48 h or 1 week, brain-injured animals receiving IGF-1 showed improved neuromotor function compared with those receiving vehicle. IGF-1 administration also enhanced learning ability (P < 0.03) and memory retention (P < 0.01) in brain-injured animals at 2 weeks postinjury. Taken together, these data suggest that chronic, posttraumatic administration of the trophic factor IGF-1 may be efficacious in ameliorating neurobehavioral dysfunction associated with traumatic brain injury.

Calpain as a Therapeutic Target in Traumatic Brain Injury

SummaryThe 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.

Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models.

Genetic strategies provide new ways to define the molecular cascades that regulate the responses of the mammalian nervous system to injury. Genetic interventions also provide opportunities to manipulate and control key molecular steps in these cascades, so as to modify the outcome of CNS injury. Most current genetic strategies involve the use of mice, an animal that has not heretofore been used extensively for neurotrauma research. Therefore, one purpose of the present review is to consider how mice respond to neural trauma, focusing especially on recent information that reveals important differences between mice and rats, and between different inbred strains of mice. The second aim of this review is to provide a brief introduction to the opportunities, caveats, and potential pitfalls of studies that use genetically modified animals for neurotrauma research.