Repetitive mild traumatic brain injury in a mouse model produces learning and memory deficits accompanied by histological changes

Concussion or mild traumatic brain injury (mTBI) represents the most common type of brain injury. However, in contrast with moderate or severe injury, there are currently few non-invasive experimental studies that investigate the cumulative effects of repetitive mTBI using rodent models. Here we describe and compare the behavioral and pathological consequences in a mouse model of single (s-mTBI) or repetitive injury (r-mTBI, five injuries given at 48 h intervals) administered by an electromagnetic controlled impactor. Our results reveal that a single mTBI is associated with transient motor and cognitive deficits as demonstrated by rotarod and the Barnes Maze respectively, whereas r-mTBI results in more significant deficits in both paradigms. Histology revealed no overt cell loss in the hippocampus, although a reactive gliosis did emerge in hippocampal sector CA1 and in the deeper cortical layers beneath the injury site in repetitively injured animals, where evidence of focal injury also was observed in the brainstem and cerebellum. Axonal injury, manifest as amyloid precursor protein immunoreactive axonal profiles, was present in the corpus callosum of both injury groups, though more evident in the r-mTBI animals. Our data demonstrate that this mouse model of mTBI is reproducible, simple, and noninvasive, with behavioral impairment after a single injury and increasing deficits after multiple injuries accompanied by increased focal and diffuse pathology. As such, this model may serve as a suitable platform with which to explore repetitive mTBI relevant to human brain injury

Lifelong behavioral and neuropathological consequences of repetitive mild traumatic brain injury

Objective: Exposure to repetitive concussion, or mild traumatic brain injury (mTBI), has been linked with increased risk of long-term neurodegenerative changes, specifically chronic traumatic encephalopathy (CTE). To date, preclinical studies largely have focused on the immediate aftermath of mTBI, with no literature on the lifelong consequences of mTBI in these models. This study provides the first account of lifelong neurobehavioral and histological consequences of repetitive mTBI providing unique insight into the constellation of evolving and ongoing pathologies with late survival. Methods: Male C57BL/6J mice (aged 2–3 months) were exposed to either single or repetitive mild TBI or sham procedure. Thereafter, animals were monitored and assessed at 24 months post last injury for measures of motor coordination, learning deficits, cognitive function, and anxiety-like behavior prior to euthanasia and preparation of the brains for detailed neuropathological and protein biochemical studies. Results: At 24 months survival animals exposed to r-mTBI showed clear evidence of learning and working memory impairment with a lack of spatial memory and vestibule-motor vestibulomotor deficits compared to sham animals. Associated with these late behavioral deficits there was evidence of ongoing axonal degeneration and neuroinflammation in subcortical white matter tracts. Notably, these changes were also observed after a single mTBI, albeit to a lesser degree than repetitive mTBI. Interpretation: In this context, our current data demonstrate, for the first time, that rather than an acute, time limited event, mild TBI can precipitate a lifelong degenerative process. These data therefore suggest that successful treatment strategies should consider both the acute and chronic nature of mTBI

Translational potential of long-term decreases in mitochondrial lipids in a mouse model of Gulf War Illness

Gulf War Illness (GWI) affects 25% of veterans from the 1990–1991 Gulf War (GW) and is accompanied by damage to the brain regions involved in memory processing. After twenty-five years, the chronic pathobiology of GWI is still unexplained. To address this problem, we examined the long-term consequences of GW exposures in an established GWI mouse model to identify biological processes that are relevant to the chronic symptoms of GWI. Three-month old male C57BL6 mice were exposed for 10 days to GW agents (pyridostigmine bromide and permethrin). Barnes Maze testing conducted at 15- and 16-months post-exposure revealed learning and memory impairment. Immunohistochemical analyses showed astroglia and microglia activation in the hippocampi of exposed mice. Proteomic studies identified perturbation of mitochondria function and metabolomics data showed decreases in the Krebs cycle compounds, lactate, β-hydroxybutyrate and glycerol-3 phosphate in the brains of exposed mice. Lipidomics data showed decreases in fatty acids, acylcarnitines and phospholipids, including cardiolipins in the brains of exposed mice. Pilot biomarker studies showed that plasma from exposed mice and veterans with GWI had increases in odd-chain, and decreases in long-chain, acylcarnitines compared to their respective controls. Very long-chain acylcarnitines were decreased in veterans with GWI compared to controls. These studies suggest that mitochondrial lipid disturbances might be associated with GWI and that further investigation is required to determine its role in the pathophysiology of this illness. Targeting mitochondrial function may provide effective therapies for GWI, and that lipid abnormalities could serve as biomarkers of GWI

Chronic repetitive mild traumatic brain injury results in reduced cerebral blood flow, axonal injury, gliosis, and increased T-Tau and Tau oligomers

Exposure to repetitive mild traumatic brain injury (mTBI) is a risk factor for chronic traumatic encephalopathy, which is characterized by patchy deposition of hyperphosphorylated tau aggregates in neurons and astrocytes at the depths of cortical sulci. We developed an mTBI paradigm to explore effects of repetitive concussive-type injury over several months in mice with a human tau genetic background (hTau). Two injuries were induced in the hTau mice weekly over a period of 3 or 4 months and the effects were compared with those in noninjured sham animals. Behavioral and in vivo measures and detailed neuropathological assessments were conducted 6 months after the first injury. Our data confirm impairment in cerebral blood flow and white matter damage. This was accompanied by a 2-fold increase in total tau levels and mild increases in tau oligomers/conformers and pTau (Thr231) species in brain gray matter. There was no evidence of neurofibrillary/astroglial tangles, neuropil threads, or perivascular foci of tau immunoreactivity. There were neurobehavioral deficits (ie, disinhibition and impaired cognitive performance) in the mTBI animals. These data support the relevance of this new mTBI injury model for studying the consequences of chronic repetitive mTBI in humans, and the role of tau in TBI

Reduction of β-amyloid pathology by celastrol in a transgenic mouse model of Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Aβ deposits represent a neuropathological hallmark of Alzheimer's disease (AD). Both soluble and insoluble Aβ species are considered to be responsible for initiating the pathological cascade that eventually leads to AD. Therefore, the identification of therapeutic approaches that can lower Aβ production or accumulation remains a priority. NFκB has been shown to regulate BACE-1 expression level, the rate limiting enzyme responsible for the production of Aβ. We therefore explored whether the known NFκB inhibitor celastrol could represent a suitable compound for decreasing Aβ production and accumulation <it>in vivo</it>.</p> <p>Methods</p> <p>The effect of celastrol on amyloid precursor protein (APP) processing, Aβ production and NFκB activation was investigated by western blotting and ELISAs using a cell line overexpressing APP. The impact of celastrol on brain Aβ accumulation was tested in a transgenic mouse model of AD overexpressing the human APP695sw mutation and the presenilin-1 mutation M146L (Tg PS1/APPsw) by immunostaining and ELISAs. An acute treatment with celastrol was investigated by administering celastrol intraperitoneally at a dosage of 1 mg/Kg in 35 week-old Tg PS1/APPsw for 4 consecutive days. In addition, a chronic treatment (32 days) with celastrol was tested using a matrix-driven delivery pellet system implanted subcutaneously in 5 month-old Tg PS1/APPsw to ensure a continuous daily release of 2.5 mg/Kg of celastrol.</p> <p>Results</p> <p><it>In vitro</it>, celastrol dose dependently prevented NFκB activation and inhibited BACE-1 expression. Celastrol potently inhibited Aβ_1-40and Aβ_1-42production by reducing the β-cleavage of APP, leading to decreased levels of APP-CTFβ and APPsβ. <it>In vivo</it>, celastrol appeared to reduce the levels of both soluble and insoluble Aβ_1-38, Aβ_1-40and Aβ_1-42. In addition, a reduction in Aβ plaque burden and microglial activation was observed in the brains of Tg PS1/APPsw following a chronic administration of celastrol.</p> <p>Conclusions</p> <p>Overall our data suggest that celastrol is a potent Aβ lowering compound that acts as an indirect BACE-1 inhibitor possibly by regulating BACE-1 expression level via an NFκB dependent mechanism. Additional work is required to determine whether chronic administration of celastrol can be safely achieved with cognitive benefits in a transgenic mouse model of AD.</p

Influence of traumatic brain injury on extracellular tau elimination at the blood–brain barrier

Repetitive head trauma has been associated with the accumulation of tau species in the brain. Our prior work showed brain vascular mural cells contribute to tau processing in the brain, and that these cells progressively degenerate following repetitive mild traumatic brain injury (r-mTBI). The current studies investigated the role of the cerebrovasculature in the elimination of extracellular tau from the brain, and the influence of r-mTBI on these processes. Following intracranial injection of biotin-labeled tau, the levels of exogenous labeled tau residing in the brain were elevated in a mouse model of r-mTBI at 12 months post-injury compared to r-sham mice, indicating reduced tau elimination from the brain following head trauma. This may be the result of decreased caveolin-1 mediated tau efflux at the blood–brain barrier (BBB), as the caveolin inhibitor, methyl-β-cyclodextrin, significantly reduced tau uptake in isolated cerebrovessels and significantly decreased the basolateral-to-apical transit of tau across an in vitro model of the BBB. Moreover, we found that the upstream regulator of endothelial caveolin-1, Mfsd2a, was elevated in r-mTBI cerebrovessels compared to r-sham, which coincided with a decreased expression of cerebrovascular caveolin-1 in the chronic phase following r-mTBI (> 3 months post-injury). Lastly, angiopoietin-1, a mural cell-derived protein governing endothelial Mfsd2a expression, was secreted from r-mTBI cerebrovessels to a greater extent than r-sham animals. Altogether, in the chronic phase post-injury, release of angiopoietin-1 from degenerating mural cells downregulates caveolin-1 expression in brain endothelia, resulting in decreased tau elimination across the BBB, which may describe the accumulation of tau species in the brain following head trauma

MMP9 modulation improves specific neurobehavioral deficits in a mouse model of Alzheimer’s disease

Background Matrix metallopeptidase 9 (MMP9) has been implicated in a variety of neurological disorders, including Alzheimer’s disease (AD), where MMP9 levels are elevated in the brain and cerebrovasculature. Previously our group demonstrated apolipoprotein E4 (apoE4) was less efficient in regulating MMP9 activity in the brain than other apoE isoforms, and that MMP9 inhibition facilitated beta-amyloid (Aβ) elimination across the blood–brain barrier (BBB) Methods In the current studies, we evaluated the impact of MMP9 modulation on Aβ disposition and neurobehavior in AD using two approaches, (1) pharmacological inhibition of MMP9 with SB-3CT in apoE4 x AD (E4FAD) mice, and (2) gene deletion of MMP9 in AD mice (MMP9KO/5xFAD) Results Treatment with the MMP9 inhibitor SB-3CT in E4FAD mice led to reduced anxiety compared to placebo using the elevated plus maze. Deletion of the MMP9 gene in 5xFAD mice also reduced anxiety using the open field test, in addition to improving sociability and social recognition memory, particularly in male mice, as assessed through the three-chamber task, indicating certain behavioral alterations in AD may be mediated by MMP9. However, neither pharmacological inhibition of MMP9 or gene deletion of MMP9 affected spatial learning or memory in the AD animals, as determined through the radial arm water maze. Moreover, the effect of MMP9 modulation on AD neurobehavior was not due to changes in Aβ disposition, as both brain and plasma Aβ levels were unchanged in the SB-3CT-treated E4FAD animals and MMP9KO/AD mice compared to their respective controls. Conclusions In total, while MMP9 inhibition did improve specific neurobehavioral deficits associated with AD, such as anxiety and social recognition memory, modulation of MMP9 did not alter spatial learning and memory or Aβ tissue levels in AD animals. While targeting MMP9 may represent a therapeutic strategy to mitigate aspects of neurobehavioral decline in AD, further work is necessary to understand the nature of the relationship between MMP9 activity and neurological dysfunction

Mural cell dysfunction leads to altered cerebrovascular tau uptake following repetitive head trauma

A pathological characteristic of repetitive traumatic brain injury (TBI) is the deposition of hyperphosphorylated and aggregated tau species in the brain and increased levels of extracellular monomeric tau are believed to play a role in the pathogenesis of neurodegenerative tauopathies. The pathways by which extracellular tau is eliminated from the brain, however, remains elusive. The purpose of this study was to examine tau uptake by cerebrovascular cells and the effect of TBI on these processes. We found monomeric tau interacts with brain vascular mural cells (pericytes and smooth muscle cells) to a greater extent than other cerebrovascular cells, indicating mural cells may contribute to the elimination of extracellular tau, as previously described for other solutes such as beta-amyloid. Consistent with other neurodegenerative disorders, we observed a progressive decline in cerebrovascular mural cell markers up to 12 months post-injury in a mouse model of repetitive mild TBI (r-mTBI) and human TBI brain specimens, when compared to control. These changes appear to reflect mural cell degeneration and not cellular loss as no difference in the mural cell population was observed between r-mTBI and r-sham animals as determined through flow cytometry. Moreover, freshly isolated r-mTBI cerebrovessels showed reduced tau uptake at 6 and 12 months post-injury compared to r-sham animals, which may be the result of diminished cerebrovascular endocytosis, as caveolin-1 levels were significantly decreased in mouse r-mTBI and human TBI cerebrovessels compared to their respective controls. Further emphasizing the interaction between mural cells and tau, similar reductions in mural cell markers, tau uptake, and caveolin-1 were observed in cerebrovessels from transgenic mural cell-depleted animals. In conclusion, our studies indicate repeated injuries to the brain causes chronic mural cell degeneration, reducing the caveolar-mediated uptake of tau by these cells. Alterations in tau uptake by vascular mural cells may contribute to tau deposition in the brain following head trauma and could represent a novel therapeutic target for TBI or other neurodegenerative disorders. [Abstract copyright: Published by Elsevier Inc.

Molecular Pathobiology of the Cerebrovasculature in Aging and in Alzheimers Disease Cases With Cerebral Amyloid Angiopathy

Cerebrovascular dysfunction and cerebral amyloid angiopathy (CAA) are hallmark features of Alzheimer's disease (AD). Molecular damage to cerebrovessels in AD may result in alterations in vascular clearance mechanisms leading to amyloid deposition around blood vessels and diminished neurovascular-coupling. The sequelae of molecular events leading to these early pathogenic changes remains elusive. To address this, we conducted a comprehensive in-depth molecular characterization of the proteomic changes in enriched cerebrovessel fractions isolated from the inferior frontal gyrus of autopsy AD cases with low (85.5 ± 2.9 yrs) vs. high (81 ± 4.4 yrs) CAA score, aged-matched control (87.4 ± 1.5 yrs) and young healthy control (47 ± 3.3 yrs) cases. We employed a 10-plex tandem isobaric mass tag approach in combination with our ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method. Enriched cerebrovascular fractions showed very high expression levels of proteins specific to endothelial cells, mural cells (pericytes and smooth muscle cells), and astrocytes. We observed 150 significantly regulated proteins in young vs. aged control cerebrovessels. The top pathways significantly modulated with aging included chemokine, reelin, HIF1α and synaptogenesis signaling pathways. There were 213 proteins significantly regulated in aged-matched control vs. high CAA cerebrovessels. The top three pathways significantly altered from this comparison were oxidative phosphorylation, Sirtuin signaling pathway and TCA cycle II. Comparison between low vs. high CAA cerebrovessels identified 84 significantly regulated proteins. Top three pathways significantly altered between low vs. high CAA cerebrovessels included TCA Cycle II, Oxidative phosphorylation and mitochondrial dysfunction. Notably, high CAA cases included more advanced AD pathology thus cerebrovascular effects may be driven by the severity of amyloid and Tangle pathology. These descriptive proteomic changes provide novel insights to explain the age-related and AD-related cerebrovascular changes contributing to AD pathogenesis. Particularly, disturbances in energy bioenergetics and mitochondrial biology rank among the top AD pathways altered in cerebrovessels. Targeting these failed mechanisms in endothelia and mural cells may provide novel disease modifying targets for developing therapeutic strategies against cerebrovascular deterioration and promoting cerebral perfusion in AD. Our future work will focus on interrogating and validating these novel targets and pathways and their functional significance