Delayed inflammatory mRNA and protein expression after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI. METHODS: Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression. RESULTS: Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma. CONCLUSIONS: These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.

Hyaluronidase reduced edema after experimental traumatic brain injury.

Cerebral edema and the subsequent increased intracranial pressure are associated with mortality and poor outcome following traumatic brain injury. Previous in vitro studies have shown that the Gibbs-Donnan effect, which describes the tendency of a porous, negatively charged matrix to attract positive ions and water, applies to brain tissue and that enzymatic reduction of the fixed charge density can prevent tissue swelling. We tested whether hyaluronidase, an enzyme that degrades the large, negatively charged glycosaminoglycan hyaluronan, could reduce brain edema after traumatic brain injury. In vivo, intracerebroventricular injection of hyaluronidase after controlled cortical impact in mice reduced edema in the ipsilateral hippocampus at 24 h by both the wet-weight/dry-weight method (78.15 ± 0.65% vs. 80.4 ± 0.46%; p < 0.01) and T2-weighted magnetic resonance imaging (13.88 ± 3.09% vs. 29.23 ± 6.14%; p < 0.01). Hyaluronidase did not adversely affect blood-brain-barrier-integrity measured by dynamic contrast-enhanced magnetic resonance imaging, nor did hyaluronidase negatively affect functional recovery after controlled cortical impact measured with the rotarod or Morris water maze tasks. Reduction of fixed charge density by hyaluronidase was confirmed in cortical explants in vitro (5.46 ± 1.15 µg/mg vs. 7.76 ± 1.87 µg/mg; p < 0.05). These data demonstrate that targeting the fixed charge density with hyaluronidase reduced edema in an in vivo mouse model of traumatic brain injury.

Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury.

BACKGROUND: Traumatic Brain Injury (TBI) is a major cause of disability and mortality, to which there is currently no comprehensive treatment. Blood Brain Barrier (BBB) dysfunction is well documented in human TBI patients, yet the molecular mechanisms that underlie this neurovascular unit (NVU) pathology remains unclear. The apolipoprotein-E (apoE) protein has been implicated in controlling BBB integrity in an isoform dependent manner, via suppression of Cyclophilin A (CypA)-Matrix metallopeptidase-9 (MMP-9) signaling cascades, however the contribution of this pathway in TBI-induced BBB permeability is not fully investigated. METHODS: We exposed C57Bl/6 mice to controlled cortical impact and assessed NVU and BBB permeability responses up to 21 days post-injury. We pharmacologically probed the role of the CypA-MMP-9 pathway in BBB permeability after TBI using Cyclosporin A (CsA, 20 mg/kg). Finally, as the apoE4 protein is known to be functionally deficient compared to the apoE3 protein, we used humanized APOE mice as a clinically relevant model to study the role of apoE on BBB injury and repair after TBI. RESULTS: In C57Bl/6 mice there was an inverse relationship between soluble apoE and BBB permeability, such that damaged BBB stabilizes as apoE levels increase in the days following TBI. TBI mice displayed acute pericyte loss, increased MMP-9 production and activity, and reduced tight-junction expression. Treatment with the CypA antagonist CsA in C57Bl/6 mice attenuates MMP-9 responses and enhances BBB repair after injury, demonstrating that MMP-9 plays an important role in the timing of spontaneous BBB repair after TBI. We also show that apoe mRNA is present in both astrocytes and pericytes after TBI. We report that APOE3 and APOE4 mice have similar acute BBB responses to TBI, but APOE3 mice display faster spontaneous BBB repair than APOE4 mice. Isolated microvessel analysis reveals delayed pericyte repopulation, augmented and sustained MMP-9 expression at the NVU, and impaired stabilization of Zonula Occludens-1, Occludin and Claudin-5 expression at tight junctions in APOE4 mice after TBI compared to APOE3 mice. CONCLUSIONS: These data confirm apoE as an important modulator of spontaneous BBB stabilization following TBI, and highlights the APOE4 allele as a risk factor for poor outcome after TBI.

The Effect of the APOE4 Gene on Accumulation of Aß40 After Brain Injury Cannot Be Reversed by Increasing apoE4 Protein.

The apolipoprotein E (apoE) protein is involved in clearance of ß-amyloid (Aß) from the brain; and the APOE4 gene is associated with Aß plaque formation in humans following traumatic brain injury (TBI). Here, we examined the association between apoE and Aß40 after experimental TBI and the effects of APOE alleles on this relationship. We report a biphasic response of soluble apoE protein after TBI with an acute reduction at 1 day postinjury followed by an increase at 7 days postinjury. TBI-induced Aß40 levels decreased as soluble apoE levels increased. In APOE4 mice there was a diminished apoE response to TBI that corresponded to prolonged accumulation of TBI-induced Aß40 versus that in APOE3 mice. Amyloid precursor protein processing was similar in APOE3 and APOE4 mice suggesting that impaired clearance was responsible for the abnormal accumulation of Aß40 in the latter. Treatment of APOE4 mice with bexarotene for 7 days increased apoE4 protein levels but was not sufficient to reduce TBI-induced Aß40 Thus, rapid clearance of TBI-induced Aß40 occurs in mice but these pathways are impaired in APOE4 carriers. These data may help explain the deposition of Aß in APOE4 carriers and the increased incidence of brain Aß plaques following TBI.

Injury-Induced Neurogenesis: Mechanisms and Relevance.

Partial recovery from brain injury due to trauma, hypoxia, or stroke, is ubiquitous and occurs largely through unknown mechanisms. It is now well accepted that injury enhances proliferation of quiescent stem and progenitor cells in specialized niches within the brain. However, whether this injury-induced neurogenesis contributes to recovery after brain injury remains controversial. Recent evidence suggests that hippocampal neural stem/precursor cell activation and subsequent neurogenesis are responsible for at least some aspects of spontaneous recovery following brain injury from a variety of causes. However, other aspects of injury-induced neurogenesis, including its contribution to adverse sequelae such as seizures, are still being investigated. The purpose of this review is to provide an overview of adult hippocampal neurogenesis and how it relates to injury and explain how current mouse technology is allowing for better understanding of whether manipulating this natural process might eventually help inform therapy following brain injury.

Polypathology and dementia after brain trauma: Does brain injury trigger distinct neurodegenerative diseases, or should they be classified together as traumatic encephalopathy?

Neuropathological studies of human traumatic brain injury (TBI) cases have described amyloid plaques acutely after a single severe TBI, and tau pathology after repeat mild TBI (mTBI). This has helped drive the hypothesis that a single moderate to severe TBI increases the risk of developing late-onset Alzheimer's disease (AD), while repeat mTBI increases the risk of developing chronic traumatic encephalopathy (CTE). In this review we critically assess this position-examining epidemiological and case control human studies, neuropathological evidence, and preclinical data. Epidemiological studies emphasize that TBI is associated with the increased risk of developing multiple types of dementia, not just AD-type dementia, and that TBI can also trigger other neurodegenerative conditions such as Parkinson's disease. Further, human post-mortem studies on both single TBI and repeat mTBI can show combinations of amyloid, tau, TDP-43, and Lewy body pathology indicating that the neuropathology of TBI is best described as a 'polypathology'. Preclinical studies confirm that multiple proteins associated with the development of neurodegenerative disease accumulate in the brain after TBI. The chronic sequelae of both single TBI and repeat mTBI share common neuropathological features and clinical symptoms of classically defined neurodegenerative disorders. However, while the spectrum of chronic cognitive and neurobehavioral disorders that occur following repeat mTBI is viewed as the symptoms of CTE, the spectrum of chronic cognitive and neurobehavioral symptoms that occur after a single TBI is considered to represent distinct neurodegenerative diseases such as AD. These data support the suggestion that the multiple manifestations of TBI-induced neurodegenerative disorders be classified together as traumatic encephalopathy or trauma-induced neurodegeneration, regardless of the nature or frequency of the precipitating TBI.

Apolipoprotein E Regulates Injury-Induced Activation of Hippocampal Neural Stem and Progenitor Cells.

Partial recovery from even severe traumatic brain injury (TBI) is ubiquitous and occurs largely through unknown mechanisms. Recent evidence suggests that hippocampal neural stem/progenitor cell (NSPC) activation and subsequent neurogenesis are responsible for at least some aspects of spontaneous recovery following TBI. Apolipoprotein E (ApoE) regulates postnatal neurogenesis in the hippocampus and is therefore a putative mediator of injury-induced neurogenesis. Further, ApoE isoforms in humans are associated with different cognitive outcomes following TBI. To investigate the role of ApoE in injury-induced neurogenesis, we exposed wild-type, ApoE-deficient, and human ApoE isoform-specific (ApoE3 and ApoE4) transgenic mice crossed with nestin-green fluorescent protein (GFP) reporter mice to controlled cortical impact (CCI) and assessed progenitor activation at 2 d post-injury using unbiased stereology. GFP+ progenitor cells were increased by approximately 120% in the ipsilateral hippocampus in injured wild-type mice, compared with sham mice (p<0.01). Co-localization of GFP+ cells with bromodeoxyrudine (BrdU) to label dividing cells indicated increased proliferation of progenitors in the injured hippocampus (p<0.001). This proliferative injury response was absent in ApoE-deficient mice, as no increase in GFP+ cells was observed in the injured hippocampus, compared with sham mice, despite an overall increase in proliferation indicated by increased BrdU+ cells (86%; p<0.05). CCI-induced proliferation of GFP+ cells in both ApoE3 and ApoE4 mice but the overall response was attenuated in ApoE4 mice due to fewer GFP+ cells at baseline. We demonstrate that ApoE is required for injury-induced proliferation of NSPCs after experimental TBI, and that this response is influenced by human APOE genotype.

Experimental traumatic brain injury induces rapid aggregation and oligomerization of amyloid-beta in an Alzheimer's disease mouse model.

Soluble amyloid-beta (Aß) oligomers are hypothesized to be the pathogenic species in Alzheimer's disease (AD), and increased levels of oligomers in the brain subsequent to traumatic brain injury (TBI) may exacerbate secondary injury pathways and underlie increased risk of developing AD in later life. To determine whether TBI causes Aß aggregation and oligomerization in the brain, we exposed triple transgenic AD model mice to controlled cortical impact injury and measured levels of soluble, insoluble, and oligomeric Aß by enzyme-linked immunosorbent assay (ELISA) at 1, 3, and 7 days postinjury. TBI rapidly increased levels of both soluble and insoluble Aß40 and Aß42 in the injured cortex at 1 day postinjury. We confirmed previous findings that identified damaged axons as a major site of Aß accumulation using both immunohistochemistry and biochemistry. We also report that soluble Aß oligomers were significantly increased in the injured cortex, as demonstrated by both ELISA and Western blot. Interestingly, the mouse brain is able to rapidly clear trauma-induced Aß, with both soluble and insoluble Aß species returning to sham levels by 7 days postinjury. In conclusion, we demonstrate that TBI causes acute accumulation and aggregation of Aß in the brain, including the formation of low- and high-molecular-weight Aß oligomers. The formation and aggregation of Aß into toxic species acutely after injury may play a role in secondary injury cascades after trauma and, chronically, may contribute to increased risk of developing AD in later life.

It's money! Real-world grant experience through a student-run, peer-reviewed program.

Grantsmanship is an integral component of surviving and thriving in academic science, especially in the current funding climate. Therefore, any additional opportunities to write, read, and review grants during graduate school may have lasting benefits on one's career. We present here our experience with a small, student-run grant program at Georgetown University Medical Center. Founded in 2010, this program has several goals: 1) to give graduate students an opportunity to conduct small, independent research projects; 2) to encourage graduate students to write grants early and often; and 3) to give graduate students an opportunity to review grants. In the 3 yr since the program's start, 28 applications have been submitted, 13 of which were funded for a total of $40,000. From funded grants, students have produced abstracts and manuscripts, generated data to support subsequent grant proposals, and made new professional contacts with collaborators. Above and beyond financial support, this program provided both applicants and reviewers an opportunity to improve their writing skills, professional development, and understanding of the grants process, as reflected in the outcome measures presented. With a small commitment of time and funding, other institutions could implement a program like this to the benefit of their graduate students.

Controlled cortical impact results in an extensive loss of dendritic spines that is not mediated by injury-induced amyloid-beta accumulation.

The clinical manifestations that occur after traumatic brain injury (TBI) include a wide range of cognitive, emotional, and behavioral deficits. The loss of excitatory synapses could potentially explain why such diverse symptoms occur after TBI, and a recent preclinical study has demonstrated a loss of dendritic spines, the postsynaptic site of the excitatory synapse, after fluid percussion injury. The objective of this study was to determine if controlled cortical impact (CCI) also resulted in dendritic spine retraction and to probe the underlying mechanisms of this spine loss. We used a unilateral CCI and visualized neurons and dendtritic spines at 24 h post-injury using Golgi stain. We found that TBI caused a 32% reduction of dendritic spines in layer II/III of the ipsilateral cortex and a 20% reduction in the dendritic spines of the ipsilateral dentate gyrus. Spine loss was not restricted to the ipsilateral hemisphere, however, with similar reductions in spine numbers recorded in the contralateral cortex (25% reduction) and hippocampus (23% reduction). Amyloid-ß (Aß), a neurotoxic peptide commonly associated with Alzheimer disease, accumulates rapidly after TBI and is also known to cause synaptic loss. To determine if Aß contributes to spine loss after brain injury, we administered a γ-secretase inhibitor LY450139 after TBI. We found that while LY450139 administration could attenuate the TBI-induced increase in Aß, it had no effect on dendritic spine loss after TBI. We conclude that the acute, global loss of dendritic spines after TBI is independent of γ-secretase activity or TBI-induced Aß accumulation.

The effect of injury severity on behavior: a phenotypic study of cognitive and emotional deficits after mild, moderate, and severe controlled cortical impact injury in mice.

Traumatic brain injury (TBI) can cause a broad array of behavioral problems including cognitive and emotional deficits. Human studies comparing neurobehavioral outcomes after TBI suggest that cognitive impairments increase with injury severity, but emotional problems such as anxiety and depression do not. To determine whether cognitive and emotional impairments increase as a function of injury severity we exposed mice to sham, mild, moderate, or severe controlled cortical impact (CCI) and evaluated performance on a variety of neurobehavioral tests in the same animals before assessing lesion volume as a histological measure of injury severity. Increasing cortical impact depth successfully produced lesions of increasing severity in our model. We found that cognitive impairments in the Morris water maze increased with injury severity, as did the degree of contralateral torso flexion, a measure of unilateral striatal damage. TBI also caused deficits in emotional behavior as quantified in the forced swim test, elevated-plus maze, and prepulse inhibition of acoustic startle, but these deficits were not dependent on injury severity. Stepwise regression analyses revealed that Morris water maze performance and torso flexion predicted the majority of the variability in lesion volume. In summary, we find that cognitive deficits increase in relation to injury severity, but emotional deficits do not. Our data suggest that the threshold for emotional changes after experimental TBI is low, with no variation in behavioral deficits seen between mild and severe brain injury.

Modulation of ABCA1 by an LXR agonist reduces ß-amyloid levels and improves outcome after traumatic brain injury.

Traumatic brain injury (TBI) increases brain beta-amyloid (Aß) in humans and animals. Although the role of Aß in the injury cascade is unknown, multiple preclinical studies have demonstrated a correlation between reduced Aß and improved outcome. Therefore, therapeutic strategies that enhance Aß clearance may be beneficial after TBI. Increased levels of ATP-binding cassette A1 (ABCA1) transporters can enhance Aß clearance through an apolipoprotein E (apoE)-mediated pathway. By measuring Aß and ABCA1 after experimental TBI in C57BL/6J mice, we found that Aß peaked early after injury (1-3 days), whereas ABCA1 had a delayed response (beginning at 3 days). As ABCA1 levels increased, Aß levels returned to baseline levels-consistent with the known role of ABCA1 in Aß clearance. To test if enhancing ABCA1 levels could block TBI-induced Aß, we treated TBI mice with the liver X-receptor (LXR) agonist T0901317. Pre- and post-injury treatment increased ABCA1 levels at 24 h post-injury, and reduced the TBI-induced increase in Aß. This reduction in Aß was not due to decreased amyloid precursor protein processing, or a shift in the solubility of Aß, indicating enhanced clearance. T0901317 also limited motor coordination deficits in injured mice and reduced brain lesion volume. These data indicate that activation of LXR can reduce Aß accumulation after TBI, and is accompanied by improved functional recovery.