Kevin D. Browne

Multiple proteins implicated in neurodegenerative diseases accumulate in axons after brain trauma in humans

Studies in animal models have shown that traumatic brain injury (TBI) induces the rapid accumulation of many of the same key proteins that form pathologic aggregates in neurodegenerative diseases. Here, we examined whether this rapid process also occurs in humans after TBI. Brain tissue from 18 cases who died after TBI and from 6 control cases was examined using immunohistochemistry. Following TBI, widespread axonal injury was persistently identified by the accumulation of neurofilament protein and amyloid precursor protein (APP) in axonal bulbs and varicosities. Axonal APP was found to co-accumulate with its cleavage enzymes, beta-site APP cleaving enzyme (BACE), presenilin-1 (PS1) and their product, amyloid-beta (Abeta). In addition, extensive accumulation of alpha-synuclein (alpha-syn) was found in swollen axons and tau protein was found to accumulate in both axons and neuronal cell bodies. These data show rapid axonal accumulation of proteins implicated in neurodegenerative diseases including Alzheimers disease and the synucleinopathies. The cause of axonal pathology can be attributed to disruption of axons due to trauma, or as a secondary effect of raised intracranial pressure or hypoxia. Such axonal pathology in humans may provide a unique environment whereby co-accumulation of APP, BACE, and PS1 leads to intra-axonal production of Abeta as well as accumulation of alpha-syn and tau. This process may have important implications for survivors of TBI who have been shown to be at greater risk of developing neurodegenerative diseases.

Thromboembolism and delayed cerebral ischemia after subarachnoid hemorrhage: an autopsy study.

OBJECTIVE:Recent findings have cast doubt on vasospasm as the sole cause of delayed cerebral ischemia after subarachnoid hemorrhage. METHODS:We reviewed the medical records of 29 patients who died after subarachnoid hemorrhage. Brain sections were taken from the insula, cingulate gyrus, and hippocampus. Adjacent sections were stained with hematoxylin-eosin and immunostained for thromboemboli. The density (burden) of the latter was calculated blindly and correlated with evidence for ischemia and with the amount of subarachnoid blood. RESULTS:There is a strong correlation between microclot burden and delayed cerebral ischemia. Patients with clinical or radiological evidence of delayed ischemia had mean microclot burdens of 10.0/cm2 (standard deviation [SD], ±6.6); those without had mean burdens of 2.8 (SD, ±2.6), a highly significant difference (P = 0.002). There is also significant association (P = 0.001) between microclot burden and histological evidence of ischemia, with the mean burdens being 10.9 in sections exhibiting severe ischemia and 4.1 in those in which ischemia was absent. Microclot burden is high in patients who died within 2 days of hemorrhage, decreasing on Days 3 and 4. In delayed ischemia, the numbers rise again late in the first week and remain high until after the second week. In contrast, the average clot burden is low in patients dying without developing delayed ischemia. The amount of blood on an individual slide influenced the microclot burden on that slide to a highly significant extent (P < 0.001). CONCLUSION:Thromboembolism after subarachnoid hemorrhage may contribute to delayed cerebral ischemia, which parallels that caused by vasospasm. The pathogenesis of thromboembolism is discussed.

Neurogenesis and glial proliferation persist for at least one year in the subventricular zone following brain trauma in rats.

In several models of traumatic brain injury in rodents, remarkably progressive tissue loss and neuron death has been observed accompanied by expanding ventricles. Here, we explored potential cell proliferation in the subventricular zone (SVZ) in response to this progressive posttraumatic neurodegeneration. Four-month-old rats (n = 57) were subjected to parasagittal fluid-percussion brain injury or sham treatment (no injury), and their brains were harvested at 2 weeks, 2 months, 6 months, and 1 year (n = 6-8/group) after injury or sham treatment. Brain sections (6 microm) were stained with markers of cell proliferation, Ki-67, and proliferative cell nuclear antigen (PCNA) to detect mitotically active cells. In sham animals, we found a typical age-dependent decrease in Ki-67- and PCNA-labelled cells in the SVZ over the course of 1 year. However, in brain-injured animals, this decrease was reversed culminating in a sixfold increase in the number of cells staining with Ki-67 and a threefold increase in cells staining with PCNA by 1 year following injury compared to age-matched controls. Using double labeling, we also determined that many of the cells staining with Ki-67 or PCNA expressed markers selective for neurons (neurofilament protein) and glia (GFAP). These data suggest that there is a persistent proliferation of neurons and glia in the SVZ following brain trauma that does not diminish during aging as found in non-injured animals.

Sodium Channelopathy Induced by Mild Axonal Trauma Worsens Outcome After a Repeat Injury

There is great concern that one mild traumatic brain injury (mTBI) predisposes individuals to an exacerbated response with a subsequent mTBI. Although no mechanism has been identified, mounting evidence suggests traumatic axonal injury (TAI) plays a role in this process. By using a cell culture system, a threshold of mild TAI was found where dynamic stretch of cortical axons at strains lower than 5% induced no overt pathological changes. However, the axons were found to display an increased expression of sodium channels (NaChs) by 24 hr. After a second, identical mild injury, pathologic increases in [Ca2+]i were observed, leading to axon degeneration. The central role of NaChs in this response was demonstrated by blocking NaChs with tetrodotoxin prior to the second injury, which completely abolished postinjury increases in [Ca2+]i. These data suggest that mild TAI induces a form of sodium channelopathy on axons that greatly exaggerates the pathophysiologic response to subsequent mild injuries.

Chronic ibuprofen administration worsens cognitive outcome following traumatic brain injury in rats

Traumatic brain injury (TBI) can induce progressive neurodegeneration in association with chronic inflammation. Since chronic treatment with the non-steroidal anti-inflammatory drug (NSAID), ibuprofen, improves functional and histopathologic outcome in a mouse model of Alzheimers disease (AD), we investigated whether it would also improve long-term outcome following TBI. Anesthetized adult rats were subjected to fluid percussion brain injury. Over the following 4 months the injured animals received ibuprofen per os (formulated in feed) at the approximate doses of 20 mg/kg body wt/day (n=13), 40 mg/kg body wt/day (n=13), or control (feed only, n=12). Sham animals underwent surgery without injury or ibuprofen treatment (n=9). At 4 months post-injury, a Morris water maze task revealed a profound learning dysfunction in all three injured groups compared to the sham group. Surprisingly, the learning ability of injured animals treated with either chronic ibuprofen regimen was significantly worsened compared to non-treated injured animals. However, there was no difference in the extent of progressive atrophy of the cortex or hippocampus between treated and non-treated injured animals. These data may have important implications for TBI patients who are often prescribed NSAIDs for chronic pain.

Traumatic brain injury induces biphasic upregulation of ApoE and ApoJ protein in rats

Apolipoproteins play an important role in cell repair and have been found to increase shortly after traumatic brain injury (TBI). In addition, apolipoproteins reduce amyloid‐β (Aβ) accumulation in models of Alzheimers disease. Considering that TBI induces progressive neurodegeneration including Aβ accumulation, we explored potential long‐term changes in the gene and protein expression of apolipoproteins E and J (ApoE and J) over 6 months after injury. Anesthetized male Sprague‐Dawley rats were subjected to parasagittal fluid‐percussion brain injury and their brains were evaluated at 2, 4, 7, 14 days, and 1 and 6 months after TBI. In situ hybridization, Western blot, and immunohistochemical analysis demonstrated that although there was a prolonged upregulation in both the gene expression and protein concentration of ApoE and J after injury, these responses were uncoupled. Upregulation of ApoE and J mRNA expression lasted from 4 days to 1 month after injury. In contrast, a biphasic increase in protein concentration and number of immunoreactive cells for ApoE and ApoJ was observed, initially peaking at 2 days (i.e., before increased mRNA expression), returning to baseline by 2 weeks and then gradually increasing through 6 months postinjury. In addition, ApoE and J were found to colocalize with Aβ accumulation in neurons and astrocytes at 1–6 months after injury. Collectively, these data suggest that ApoE and J play a role in the acute sequelae of brain trauma and reemerge long after the initial insult, potentially to modulate progressive neurodegenerative changes.

Long-Term Survival and Integration of Transplanted Engineered Nervous Tissue Constructs Promotes Peripheral Nerve Regeneration

Although peripheral nerve injury is a common consequence of trauma or surgery, there are insufficient means for repair. In particular, there is a critical need for improved methods to facilitate regeneration of axons across major nerve lesions. Here, we engineered transplantable living nervous tissue constructs to provide a labeled pathway to guide host axonal regeneration. These constructs consisted of stretch-grown, longitudinally aligned living axonal tracts inserted into poly(glycolic acid) tubes. The constructs (allogenic) were transplanted to bridge an excised segment of sciatic nerve in the rat, and histological analyses were performed at 6 and 16 weeks posttransplantation to determine graft survival, integration, and host regeneration. At both time points, the transplanted constructs were found to have maintained their pretransplant geometry, with surviving clusters of graft neuronal somata at the extremities of the constructs spanned by tracts of axons. Throughout the transplanted region, there was an intertwining plexus of host and graft axons, suggesting that the transplanted axons mediated host axonal regeneration across the lesion. By 16 weeks posttransplant, extensive myelination of axons was observed throughout the transplant region. Further, graft neurons had extended axons beyond the margins of the transplanted region, penetrating into the host nerve. Notably, this survival and integration of the allogenic constructs occurred in the absence of immunosuppression therapy. These findings demonstrate the promise of living tissue-engineered axonal constructs to bridge major nerve lesions and promote host regeneration, potentially by providing axon-mediated axonal outgrowth and guidance.

Acute treatment with MgSO4 attenuates long-term hippocampal tissue loss after brain trauma in the rat.

Previous studies have shown that magnesium salts and the noncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist, NPS 1506, attenuated short‐term cognitive deficits and histopathological changes associated with traumatic brain injury (TBI). We evaluated the long‐term effects of both therapies after brain trauma. Young adult rats were subjected to parasagittal fluid‐percussion brain injury and received either MgSO4 (125 μmol/400 g rat; n = 12) 15 min post‐injury, NPS 1506 (1.15 mg/kg; n = 12) 15 min and 4 hr post‐injury, or vehicle (n = 9) 15 min post‐injury. Uninjured animals (sham) received vehicle (n = 10). Learning function in these animals was evaluated using a water maze paradigm 8 months after injury or sham treatment, and the brains were examined for cortical and hippocampal tissue loss. Compared to sham animals, injured vehicle‐treated animals displayed a substantial learning dysfunction, indicated by an increased latency to find a hidden platform in the water maze (P < 0.001). No improvements in learning, however, were found for injured animals treated with NPS 1506 or MgSO4. Injury induced >30% loss of tissue in the ipsilateral cortex in vehicle‐treated animals that was not reduced in animals treated with either NPS 1506 or MgSO4. Treatment with MgSO4 significantly reduced progressive tissue loss in the hippocampus (P < 0.001). These findings are the first to demonstrate long‐term neuroprotection of hippocampal tissue by an acute treatment in a TBI model. These data also show that the previously reported broad efficacy of MgSO4 or NPS 1506 observed shortly after brain trauma could not be detected 8 months post‐injury.

Hemostatic and neuroprotective effects of human recombinant activated factor VII therapy after traumatic brain injury in pigs

Human recombinant activated factor-VII (rFVIIa) has been used successfully in the treatment of spontaneous intracerebral hemorrhage. In addition, there is increasing interest in its use to treat uncontrolled bleeding of other origins, including trauma. The aim of this study was to evaluate the safety and potential effectiveness of rFVIIa to mitigate bleeding using a clinically relevant model of traumatic brain injury (TBI) in the pig. A double injury model was chosen consisting of (1) an expanding cerebral contusion induced by the application of negative pressure to the exposed cortical surface and (2) a rapid rotational acceleration of the head to induce diffuse axonal injury (DAI). Injuries were performed on 10 anesthetized pigs. Five minutes after injury, 720 microg/kg rFVIIa (n=5) or vehicle control (n=5) was administered intravenously. Magnetic resonance imaging (MRI) studies were performed within 30 min and at 3 days post-TBI to determine the temporal expansion of the cerebral contusion. Euthanasia and histopathologic analysis were performed at day 3. This included observations for hippocampal neuronal degeneration, axonal pathology and microclot formation. The expansion of contusion volume over the 3 days post-injury period was reduced significantly in animals treated with rFVIIa compared to vehicle controls. Surprisingly, immunohistochemical analysis demonstrated that the number of dead/dying hippocampal neurons and axonal pathology was reduced substantially by rFVIIa treatment compared to vehicle. In addition, there was no difference in the extent of microthrombi between groups. rFVIIa treatment after TBI in the pig reduced expansion of hemorrhagic cerebral contusion volume without exacerbating the severity of microclot formation. Finally, rFVIIa treatment provided a surprising neuroprotective effect by reducing hippocampal neuron degeneration as well as the extent of DAI.

A Porcine Model of Traumatic Brain Injury via Head Rotational Acceleration

Unique from other brain disorders, traumatic brain injury (TBI) generally results from a discrete biomechanical event that induces rapid head movement. The large size and high organization of the human brain makes it particularly vulnerable to traumatic injury from rotational accelerations that can cause dynamic deformation of the brain tissue. Therefore, replicating the injury biomechanics of human TBI in animal models presents a substantial challenge, particularly with regard to addressing brain size and injury parameters. Here we present the historical development and use of a porcine model of head rotational acceleration. By scaling up the rotational forces to account for difference in brain mass between swine and humans, this model has been shown to produce the same tissue deformations and identical neuropathologies found in human TBI. The parameters of scaled rapid angular accelerations applied for the model reproduce inertial forces generated when the human head suddenly accelerates or decelerates in falls, collisions, or blunt impacts. The model uses custom-built linkage assemblies and a powerful linear actuator designed to produce purely impulsive non-impact head rotation in different angular planes at controlled rotational acceleration levels. Through a range of head rotational kinematics, this model can produce functional and neuropathological changes across the spectrum from concussion to severe TBI. Notably, however, the model is very difficult to employ, requiring a highly skilled team for medical management, biomechanics, neurological recovery, and specialized outcome measures including neuromonitoring, neurophysiology, neuroimaging, and neuropathology. Nonetheless, while challenging, this clinically relevant model has proven valuable for identifying mechanisms of acute and progressive neuropathologies as well as for the evaluation of noninvasive diagnostic techniques and potential neuroprotective treatments following TBI.