Sex differences in the extent of acute axonal pathologies after experimental concussion.

Although human females appear be at a higher risk of concussion and suffer worse outcomes than males, underlying mechanisms remain unclear. With increasing recognition that damage to white matter axons is a key pathologic substrate of concussion, we used a clinically relevant swine model of concussion to explore potential sex differences in the extent of axonal pathologies. At 24 h post-injury, female swine displayed a greater number of swollen axonal profiles and more widespread loss of axonal sodium channels than males. Axon degeneration for both sexes appeared to be related to individual axon architecture, reflected by a selective loss of small caliber axons after concussion. However, female brains had a higher percentage of small caliber axons, leading to more extensive axon loss after injury compared to males. Accordingly, sexual dimorphism in axonal size is associated with more extensive axonal pathology in females after concussion, which may contribute to worse outcomes.

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Early Blood-Brain Barrier Impairment as a Pathological Hallmark in a Novel Model of Closed-Head Concussive Brain Injury (CBI) in Mice.

Concussion, caused by a rotational acceleration/deceleration injury mild enough to avoid structural brain damage, is insufficiently captured in recent preclinical models, hampering the relation of pathophysiological findings on the cellular level to functional and behavioral deficits. We here describe a novel model of unrestrained, single vs. repetitive concussive brain injury (CBI) in male C56Bl/6j mice. Longitudinal behavioral assessments were conducted for up to seven days afterward, alongside the evaluation of structural cerebral integrity by in vivo magnetic resonance imaging (MRI, 9.4 T), and validated ex vivo by histology. Blood-brain barrier (BBB) integrity was analyzed by means of fluorescent dextran- as well as immunoglobulin G (IgG) extravasation, and neuroinflammatory processes were characterized both in vivo by positron emission tomography (PET) using [18F]DPA-714 and ex vivo using immunohistochemistry. While a single CBI resulted in a defined, subacute neuropsychiatric phenotype, longitudinal cognitive testing revealed a marked decrease in spatial cognition, most pronounced in mice subjected to CBI at high frequency (every 48 h). Functional deficits were correlated to a parallel disruption of the BBB, (R2 = 0.29, p < 0.01), even detectable by a significant increase in hippocampal uptake of [18F]DPA-714, which was not due to activation of microglia, as confirmed immunohistochemically. Featuring a mild but widespread disruption of the BBB without evidence of macroscopic damage, this model induces a characteristic neuro-psychiatric phenotype that correlates to the degree of BBB disruption. Based on these findings, the BBB may function as both a biomarker of CBI severity and as a potential treatment target to improve recovery from concussion.

Overexpression of pathogenic tau in astrocytes causes a reduction in AQP4 and GLT1, an immunosuppressed phenotype and unique transcriptional responses to repetitive mild TBI without appreciable changes in tauopathy.

Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, GFAPP301L mice, subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or general tauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing astrocytes in the GFAPP301L model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4) and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal circumstances and following r-mTBI. The identified mechanisms using this GFAPP301L model may suggest targets for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.

Effects of Mild Closed-Head Injury and Subanesthetic Ketamine Infusion on Microglia, Axonal Injury, and Synaptic Density in Sprague-Dawley Rats.

Mild traumatic brain injury (mTBI) affects millions of people in the U.S. Approximately 20-30% of those individuals develop adverse symptoms lasting at least 3 months. In a rat mTBI study, the closed-head impact model of engineered rotational acceleration (CHIMERA) produced significant axonal injury in the optic tract (OT), indicating white-matter damage. Because retinal ganglion cells project to the lateral geniculate nucleus (LGN) in the thalamus through the OT, we hypothesized that synaptic density may be reduced in the LGN of rats following CHIMERA injury. A modified SEQUIN (synaptic evaluation and quantification by imaging nanostructure) method, combined with immunofluorescent double-labeling of pre-synaptic (synapsin) and post-synaptic (PSD-95) markers, was used to quantify synaptic density in the LGN. Microglial activation at the CHIMERA injury site was determined using Iba-1 immunohistochemistry. Additionally, the effects of ketamine, a potential neuroprotective drug, were evaluated in CHIMERA-induced mTBI. A single-session repetitive (ssr-) CHIMERA (3 impacts, 1.5 joule/impact) produced mild effects on microglial activation at the injury site, which was significantly enhanced by post-injury intravenous ketamine (10 mg/kg) infusion. However, ssr-CHIMERA did not alter synaptic density in the LGN, although ketamine produced a trend of reduction in synaptic density at post-injury day 4. Further research is necessary to characterize the effects of ssr-CHIMERA and subanesthetic doses of intravenous ketamine on different brain regions and multiple time points post-injury. The current study demonstrates the utility of the ssr-CHIMERA as a rodent model of mTBI, which researchers can use to identify biological mechanisms of mTBI and to develop improved treatment strategies for individuals suffering from head trauma.

Unusual combinations of neurodegenerative pathologies with chronic traumatic encephalopathy (CTE) complicates clinical prediction of CTE.

BACKGROUND AND PURPOSE: Chronic traumatic encephalopathy (CTE) has gained widespread attention due to its association with multiple concussions and contact sports. However, CTE remains a postmortem diagnosis, and the link between clinical symptoms and CTE pathology is poorly understood. This study aimed to investigate the presence of copathologies and their impact on symptoms in former contact sports athletes. METHODS: This was a retrospective case series design of 12 consecutive cases of former contact sports athletes referred for autopsy. Analyses are descriptive and include clinical history as well as the pathological findings of the autopsied brains. RESULTS: All participants had a history of multiple concussions, and all but one had documented progressive cognitive, psychiatric, and/or motor symptoms. The results showed that 11 of the 12 participants had evidence of CTE in the brain, but also other copathologies, including different combinations of tauopathies, and other rare entities. CONCLUSIONS: The heterogeneity of symptoms after repetitive head injuries and the diverse pathological combinations accompanying CTE complicate the prediction of CTE in clinical practice. It is prudent to consider the possibility of multiple copathologies when clinically assessing patients with repetitive head injuries, especially as they age, and attributing neurological or cognitive symptoms solely to presumptive CTE in elderly patients should be discouraged.

Preclinical characterization of macrophage-adhering gadolinium micropatches for MRI contrast after traumatic brain injury in pigs.

The choroid plexus (ChP) of the brain plays a central role in orchestrating the recruitment of peripheral leukocytes into the central nervous system (CNS) through the blood-cerebrospinal fluid (BCSF) barrier in pathological conditions, thus offering a unique niche to diagnose CNS disorders. We explored whether magnetic resonance imaging of the ChP could be optimized for mild traumatic brain injury (mTBI). mTBI induces subtle, yet influential, changes in the brain and is currently severely underdiagnosed. We hypothesized that mTBI induces sufficient alterations in the ChP to cause infiltration of circulating leukocytes through the BCSF barrier and developed macrophage-adhering gadolinium [Gd(III)]-loaded anisotropic micropatches (GLAMs), specifically designed to image infiltrating immune cells. GLAMs are hydrogel-based discoidal microparticles that adhere to macrophages without phagocytosis. We present a fabrication process to prepare GLAMs at scale and demonstrate their loading with Gd(III) at high relaxivities, a key indicator of their effectiveness in enhancing image contrast and clarity in medical imaging. In vitro experiments with primary murine and porcine macrophages demonstrated that GLAMs adhere to macrophages also under shear stress and did not affect macrophage viability or functions. Studies in a porcine mTBI model confirmed that intravenously administered macrophage-adhering GLAMs provide a differential signal in the ChP and lateral ventricles at Gd(III) doses 500- to 1000-fold lower than those used in the current clinical standard Gadavist. Under the same mTBI conditions, Gadavist did not offer a differential signal at clinically used doses. Our results suggest that macrophage-adhering GLAMs could facilitate mTBI diagnosis.

Acute Changes in the Resting Brain Networks in Concussion Patients: Small-World Topology Perspective.

BACKGROUND: The acute changes that occur in the small-world topology of the brain in concussion patients remain unclear. Here, we investigated acute changes in the small-world organization of brain networks in concussion patients and their influence on persistent post-concussion symptoms. METHODS: Eighteen concussion patients and eighteen age-matched controls were enrolled in this study. All participants underwent computed tomography, magnetic resonance imaging (MRI), susceptibility weighted imaging, and blood oxygen level-dependent functional MRI. A complex network analysis method based on graph theory was used to calculate the parameters of small-world networks under different degrees of network sparsity. All subjects were evaluated using the Glasgow Coma Scale and Rivermead Postconcussion Symptom Questionnaire. RESULTS: Compared with the controls, the normalized cluster coefficient (γ) of whole brain networks in patients and the "small-world" index (σ) was slightly enhanced, whereas the standardized minimum path (λ) was slightly shorter. Whole brain effect (Eglobal) and local effect (Elocal) changes were not pronounced. Under the condition of minimum network sparsity (Dmin = 0.13), the numbers of nodes in the "right intraorbital superior frontal gyrus" (Anatomical Automatic Labeling, AAL26), right globus pallidus (AAL76), and bilateral temporal transverse gyrus (AAL79,80) in brain concussion patients were significantly lower. The numbers of nodes in the left subcapital lobe (AAL61) and left occipital gyrus (AAL51) were significantly higher, and the normalized cluster coefficients of the right intraorbital supraphalus (AAL26) and left posterior cingulate gyrus (AAL35) were significantly increased. The normalized clustering coefficients of the right triangular subfrontal gyrus (AAL55) (based on the normalized clustering coefficients of nodes in AAL14) and left sub-parietal lobes (AAL61) were significantly reduced. The mean local effects of nodes in the right intraorbital upper frontal gyrus (AAL26), left posterior cingulate gyrus (AAL35), and bilateral auxiliary motor cortex (AAL19, 20) were enhanced, whereas the mean local effects of the bilateral triangular inferior frontal gyrus (AAL13,14) and left insular cap (AAL11) were reduced (p < 0.05). CONCLUSIONS: The overall trend of network topology abnormalities in patients was random, and generalized and local functional abnormalities were seen. Changes in the function and affective circuitry of the resting default network were particularly pronounced in these patients, which we speculate may be one of the main drivers of the cognitive dysfunction and mood changes seen in concussion patients.

Dynamic Dysregulation of the Triple Network of the Brain in Mild Traumatic Brain Injury and Its Relationship With Cognitive Performance.

A triple network model consisting of a default network, a salience network, and a central executive network has recently been used to understand connectivity patterns in cognitively normal versus dysfunctional brains. This study aimed to explore changes in the dynamic connectivity of triplet network in mild traumatic brain injury (mTBI) and its relationship to cognitive performance. In this work, we acquired resting-state functional magnetic resonance imaging (fMRI) data from 30 mTBI patients and 30 healthy controls (HCs). Independent component analysis, sliding time window correlation, and k-means clustering were applied to resting-state fMRI data. Further, we analyzed the relationship between changes in dynamic functional connectivity (FC) parameters and clinical variables in mTBI patients. The results showed that the dynamic functional connectivity of the brain triple network was clustered into five states. Compared with HC, mTBI patients spent longer in state 1, which is characterized by weakened dorsal default mode network (DMN) and anterior salience network (SN) connectivity, and state 3, which is characterized by a positive correlation between DMN and SN internal connectivity. Mild TBI patients had fewer metastases in different states than HC patients. In addition, the mean residence time in state 1 correlated with Montreal Cognitive Assessment scores in mTBI patients; the number of transitions between states correlated with Glasgow Coma Score in mTBI patients. Taken together, our findings suggest that the dynamic properties of FC in the triple network of mTBI patients are abnormal, and provide a new perspective on the pathophysiological mechanism of cognitive impairment from the perspective of dynamic FC.

Tau Imaging in Late Traumatic Brain Injury: A [18F]MK-6240 Positron Emission Tomography Study.

Epidemiological studies have identified prior traumatic brain injury (TBI) as a risk factor for developing Alzheimer's disease (AD). Neurofibrillary tangles (NFTs) are common to AD and chronic traumatic encephalopathy following repetitive mild TBI. However, it is unclear if a single TBI is sufficient to cause accumulation of NFTs. We performed a [18F]MK-6240 positron emission tomography (PET) imaging study to assess NFTs in patients who had sustained a single TBI at least 2 years prior to study inclusion. Fourteen TBI patients (49 ± 20 years; 5 M/9 F; 8 moderate-severe, 1 mild-probable, 5 symptomatic-possible TBI) and 40 demographically similar controls (57 ± 19 years; 19 M/21 F) underwent simultaneous [18F]MK-6240 PET and magnetic resonance imaging (MRI) as well as neuropsychological assessment including the Cambridge Neuropsychological Test Automated Battery (CANTAB). A region-based voxelwise partial volume correction was applied, using parcels obtained by FreeSurfer v6.0, and standardized uptake value ratios (SUVR) were calculated relative to the cerebellar gray matter. Group differences were assessed on both a voxel- and a volume-of-interest-based level and correlations of [18F]MK-6240 SUVR with time since injury as well as with clinical outcomes were calculated. Visual assessment of TBI images did not show global or focal increases in tracer uptake in any subject. On a group level, [18F]MK-6240 SUVR was not significantly different in patients versus controls or between subgroups of moderate-severe TBI versus less severe TBI. Within the TBI group, One Touch Stockings problem solving and spatial working memory (executive function), reaction time (attention), and Mini-Mental State Examination (MMSE) (global cognition) were associated with [18F]MK-6240 SUVR. We found no group-based increase of [18F]MK-6240 brain uptake in patients scanned at least 2 years after a single TBI compared with healthy volunteers, which suggests that no NFTs are building up in the first years after a single TBI. Nonetheless, correlations with cognitive outcomes were found that warrant further investigation.

Mild traumatic brain injury in Drosophila melanogaster alters reactive oxygen and nitrogen species in a sex-dependent manner.

Traumatic brain injury (TBI) is an outside force causing a modification in brain function and/or structural brain pathology that upregulates brain inducible nitric oxide synthase (iNOS), instigating increased levels of nitric oxide activity which is implicated in secondary pathology leading to behavioral deficits (Hall et al., 2012; Garry et al., 2015; Kozlov et al., 2017). In mammals, TBI-induced NO production activates an immune response and potentiates metabolic crisis through mitochondrial dysfunction coupled with vascular dysregulation; however, the direct influence on pathology is complicated by the activation of numerous secondary cascades and activation of other reactive oxygen species. Drosophila TBI models have demonstrated key features of mammalian TBI, including temporary incapacitation, disorientation, motor deficits, activation of innate immunity (inflammation), and autophagy responses observed immediately after injury (Katzenberger et al., 2013; Barekat et al., 2016; Simon et al., 2017; Anderson et al., 2018; Buhlman et al., 2021b). We hypothesized that acute behavioral phenotypes would be associated with deficits in climbing behavior and increased oxidative stress. Because flies lack mammalian-like cardiovascular and adaptive immune systems, we were able to make our observations in the absence of vascular disruption and adaptive immune system interference in a system where highly targeted interventions can be rapidly evaluated. To demonstrate the induction of injury, ten-day-old transgenic flies received an injury of increasing angles from a modified high impact trauma (HIT) device where angle-dependent increases occurred for acute neurological behavior assessments and twenty-four-hour mortality, and survival was significantly decreased. Injury caused sex-dependent effects on climbing activity and measures of oxidative stress. Specifically, after a single 60-degree HIT, female flies exhibited significant impairments in climbing activity beyond that observed in male flies. We also found that several measures of oxidative stress, including Drosophila NOS (dNOS) expression, protein nitration, and hydrogen peroxide production were significantly decreased in female flies. Interestingly, protein nitration was also decreased in males, but surpassed sham levels with a more severe injury. We also observed decreased autophagy demand in vulnerable dopaminergic neurons in female, but not male flies. In addition, mitophagy initiation was decreased in females. Collectively, our data suggest that TBI in flies induces acute behavioral phenotypes and climbing deficits that are analogous to mammalian TBI. We also observed that various indices of oxidative stress, including dNOS expression, protein tyrosine nitration, and hydrogen peroxide levels, as well as basal levels of autophagy, are altered in response to injury, an effect that is more pronounced in female flies.

Multifaceted neural and vascular pathologies after pediatric mild traumatic brain injury.

Dynamic changes in neurodevelopment and cognitive functioning occur during adolescence, including a switch from reactive to more proactive forms of cognitive control, including response inhibition. Pediatric mild traumatic brain injury (pmTBI) affects these cognitions immediately post-injury, but the role of vascular versus neural injury in cognitive dysfunction remains debated. This study consecutively recruited 214 sub-acute pmTBI (8-18 years) and age/sex-matched healthy controls (HC; N = 186), with high retention rates (>80%) at four months post-injury. Multimodal imaging (functional MRI during response inhibition, cerebral blood flow and cerebrovascular reactivity) assessed for pathologies within the neurovascular unit. Patients exhibited increased errors of commission and hypoactivation of motor circuitry during processing of probes. Evidence of increased/delayed cerebrovascular reactivity within motor circuitry during hypercapnia was present along with normal perfusion. Neither age-at-injury nor post-concussive symptom load were strongly associated with imaging abnormalities. Collectively, mild cognitive impairments and clinical symptoms may continue up to four months post-injury. Prolonged dysfunction within the neurovascular unit was observed during proactive response inhibition, with preliminary evidence that neural and pure vascular trauma are statistically independent. These findings suggest pmTBI is characterized by multifaceted pathologies during the sub-acute injury stage that persist several months post-injury.

Associations of prior concussion severity with brain microstructure using mean apparent propagator magnetic resonance imaging.

Magnetic resonance imaging (MRI) diffusion studies have shown chronic microstructural tissue abnormalities in athletes with history of concussion, but with inconsistent findings. Concussions with post-traumatic amnesia (PTA) and/or loss of consciousness (LOC) have been connected to greater physiological injury. The novel mean apparent propagator (MAP) MRI is expected to be more sensitive to such tissue injury than the conventional diffusion tensor imaging. This study examined effects of prior concussion severity on microstructure with MAP-MRI. Collegiate-aged athletes (N = 111, 38 females; ≥6 months since most recent concussion, if present) completed semistructured interviews to determine the presence of prior concussion and associated injury characteristics, including PTA and LOC. MAP-MRI metrics (mean non-Gaussian diffusion [NG Mean], return-to-origin probability [RTOP], and mean square displacement [MSD]) were calculated from multi-shell diffusion data, then evaluated for associations with concussion severity through group comparisons in a primary model (athletes with/without prior concussion) and two secondary models (athletes with/without prior concussion with PTA and/or LOC, and athletes with/without prior concussion with LOC only). Bayesian multilevel modeling estimated models in regions of interest (ROI) in white matter and subcortical gray matter, separately. In gray matter, the primary model showed decreased NG Mean and RTOP in the bilateral pallidum and decreased NG Mean in the left putamen with prior concussion. In white matter, lower NG Mean with prior concussion was present in all ROI across all models and was further decreased with LOC. However, only prior concussion with LOC was associated with decreased RTOP and increased MSD across ROI. Exploratory analyses conducted separately in male and female athletes indicate associations in the primary model may differ by sex. Results suggest microstructural measures in gray matter are associated with a general history of concussion, while a severity-dependent association of prior concussion may exist in white matter.

Co-administration of dl-3-n-butylphthalide and neprilysin is neuroprotective in Alzheimer disease associated with mild traumatic brain injury.

dl-3-n-Butylphthalide is a potent synthetic Chinese celery extract that is highly efficient in inducing neuroprotection in concussive head injury (CHI), Parkinson's disease, Alzheimer's disease, stroke as well as depression, dementia, anxiety and other neurological diseases. Thus, there are reasons to believe that dl-3-n-butylphthalide could effectively prevent Alzheimer's disease brain pathology. Military personnel during combat operation or veterans are often the victims of brain injury that is a major risk factor for developing Alzheimer's disease in their later lives. In our laboratory we have shown that CHI exacerbates Alzheimer's disease brain pathology and reduces the amyloid beta peptide (AßP) inactivating enzyme neprilysin. We have used TiO2 nanowired-dl-3-n-butylphthalide in attenuating Parkinson's disease brain pathology exacerbated by CHI. Nanodelivery of dl-3-n-butylphthalide appears to be more potent as compared to the conventional delivery of the compound. Thus, it would be interesting to examine the effects of nanowired dl-3-n-butylphthalide together with nanowired delivery of neprilysin in Alzheimer's disease model on brain pathology. In this investigation we found that nanowired delivery of dl-3-n-butylphthalide together with nanowired neprilysin significantly attenuated brain pathology in Alzheimer's disease model with CHI, not reported earlier. The possible mechanism and clinical significance is discussed based on the current literature.

Repeated mild traumatic brain injury triggers pathology in asymptomatic C9ORF72 transgenic mice.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative diseases that represent ends of the spectrum of a single disease. The most common genetic cause of FTD and ALS is a hexanucleotide repeat expansion in the C9orf72 gene. Although epidemiological data suggest that traumatic brain injury (TBI) represents a risk factor for FTD and ALS, its role in exacerbating disease onset and course remains unclear. To explore the interplay between traumatic brain injury and genetic risk in the induction of FTD/ALS pathology we combined a mild repetitive traumatic brain injury paradigm with an established bacterial artificial chromosome transgenic C9orf72 (C9BAC) mouse model without an overt motor phenotype or neurodegeneration. We assessed 8-10 week-old littermate C9BACtg/tg (n = 21), C9BACtg/- (n = 20) and non-transgenic (n = 21) mice of both sexes for the presence of behavioural deficits and cerebral histopathology at 12 months after repetitive TBI. Repetitive TBI did not affect body weight gain, general neurological deficit severity, nor survival over the 12-month observation period and there was no difference in rotarod performance, object recognition, social interaction and acoustic characteristics of ultrasonic vocalizations of C9BAC mice subjected to repetitive TBI versus sham injury. However, we found that repetitive TBI increased the time to the return of the righting reflex, reduced grip force, altered sociability behaviours and attenuated ultrasonic call emissions during social interactions in C9BAC mice. Strikingly, we found that repetitive TBI caused widespread microglial activation and reduced neuronal density that was associated with loss of histological markers of axonal and synaptic integrity as well as profound neuronal transactive response DNA binding protein 43 kDa mislocalization in the cerebral cortex of C9BAC mice at 12 months; this was not observed in non-transgenic repetitive TBI and C9BAC sham mice. Our data indicate that repetitive TBI can be an environmental risk factor that is sufficient to trigger FTD/ALS-associated neuropathology and behavioural deficits, but not paralysis, in mice carrying a C9orf72 hexanucleotide repeat expansion.

Repeated Mild Traumatic Brain Injury and JZL184 Produce Sex-Specific Increases in Anxiety-Like Behavior and Alcohol Consumption in Wistar Rats.

Alcohol use disorder (AUD) is highly comorbid with traumatic brain injury (TBI). Previously, using a lateral fluid percussion model (LFP) (an open-head injury model) to generate a single mild to moderate traumatic brain injury (TBI) we showed that TBI produces escalation in alcohol drinking, that alcohol exposure negatively impacts TBI outcomes, and that the endocannabinoid degradation inhibitor (JZL184) confers significant protection from behavioral and neuropathological outcomes in male rodents. In the present study, we used a weight drop model (a closed-head injury model) to produce repeated mild TBI (rmTBI; three TBIs separated by 24 hours) in male and female rats to examine the sex-specific effects on anxiety-like behavior and alcohol consumption, and whether systemic treatment with JZL184 would reverse TBI effects on those behaviors. In two separate studies, adult male and female Wistar rats were subjected to rmTBI or sham procedure using the weight drop model. Physiological measures of injury severity were collected from all animals. Animals in both studies were allowed to consume alcohol using an intermittent 2-bottle choice procedure (12 pre-TBI sessions and 12 post-TBI sessions). Neurological severity and neurobehavioral scores (NSS and NBS, respectively) were tested 24 hours after the final injury. Anxiety-like behavior was tested at 37-38 days post-injury in Study 1-, and 6-8-days post-injury in Study 2. Our results show that females exhibited reduced respiratory rates relative to males with no significant differences between Sham and rmTBI, no effect of rmTBI or sex on righting reflex, and increased neurological deficits in rmTBI groups in both studies. In Study 1, rmTBI increased alcohol consumption in female but not male rats. Male rats consistently exhibited higher levels of anxiety-like behavior than females. The rmTBI did not affect anxiety-like behavior 37-38 days post-injury. In Study 2, rmTBI once again increased alcohol consumption in female but not male rats, and repeated systemic treatment with JZL184 did not affect alcohol consumption. Also in Study 2, rmTBI increased anxiety-like behavior in males but not females and repeated systemic treatment with JZL184 produced an unexpected increase in anxiety-like behavior 6-8 days post-injury. In summary, rmTBI increased alcohol consumption in female rats, systemic JZL184 treatment did not alter alcohol consumption, and both rmTBI and systemic JZL184 treatment increased anxiety-like behavior 6-8 days post-injury in males but not females, highlighting robust sex differences in rmTBI effects.

Alterations in iron content, iron-regulatory proteins and behaviour without tau pathology at one year following repetitive mild traumatic brain injury.

Repetitive mild traumatic brain injury (r-mTBI) has increasingly become recognised as a risk factor for the development of neurodegenerative diseases, many of which are characterised by tau pathology, metal dyshomeostasis and behavioural impairments. We aimed to characterise the status of tau and the involvement of iron dyshomeostasis in repetitive controlled cortical impact injury (5 impacts, 48 h apart) in 3-month-old C57Bl6 mice at the chronic (12-month) time point. We performed a battery of behavioural tests, characterised the status of neurodegeneration-associated proteins (tau and tau-regulatory proteins, amyloid precursor protein and iron-regulatory proteins) via western blot; and metal levels using bulk inductively coupled plasma-mass spectrometry (ICP-MS). We report significant changes in various ipsilateral iron-regulatory proteins following five but not a single injury, and significant increases in contralateral iron, zinc and copper levels following five impacts. There was no evidence of tau pathology or changes in tau-regulatory proteins following five impacts, although some changes were observed following a single injury. Five impacts resulted in significant gait deficits, mild anhedonia and mild cognitive deficits at 9-12 months post-injury, effects not seen following a single injury. To the best of our knowledge, we are the first to describe chronic changes in metals and iron-regulatory proteins in a mouse model of r-mTBI, providing a strong indication towards an overall increase in brain iron levels (and other metals) in the chronic phase following r-mTBI. These results bring to question the relevance of tau and highlight the involvement of iron dysregulation in the development and/or progression of neurodegeneration following injury, which may lead to new therapeutic approaches in the future.

Tau Positron Emission Tomography and Neurocognitive Function Among Former Professional American-Style Football Players.

American-style football (ASF) players experience repetitive head impacts that may result in chronic traumatic encephalopathy neuropathological change (CTE-NC). At present, a definitive diagnosis of CTE-NC requires the identification of localized hyperphosphorylated Tau (p-Tau) after death via immunohistochemistry. Some studies suggest that positron emission tomography (PET) with the radiotracer [18F]-Flortaucipir (FTP) may be capable of detecting p-Tau and thus establishing a diagnosis of CTE-NC among living former ASF players. To assess associations between FTP, football exposure, and objective neuropsychological measures among former professional ASF players, we conducted a study that compared former professional ASF players with age-matched male control participants without repetitive head impact exposure. Former ASF players and male controls underwent structural magnetic resonance imaging and PET using FTP for p-Tau and [11C]-PiB for amyloid-ß. Former players underwent neuropsychological testing. The ASF exposure was quantified as age at first exposure, professional career duration, concussion signs and symptoms burden, and total years of any football play. Neuropsychological testing included measures of memory, executive functioning, and depression symptom severity. P-Tau was quantified as FTP standardized uptake value ratios (SUVR) and [11C]-PiB by distribution volume ratios (DVR) using cerebellar grey matter as the reference region. There were no significant differences in [18F]-FTP uptake among former ASF players (n = 27, age = 50 ± 7 years) compared with control participants (n = 11, age = 55 ± 4 years), nor did any participant have significant amyloid-ß burden. Among ASF participants, there were no associations between objective measures of neurocognitive functioning and [18F]-FTP uptake. There was a marginally significant difference, however, between [18F]-FTP uptake isolated to the entorhinal cortex among players in age-, position-, and race-adjusted models (p = 0.05) that may represent an area of future investigation. The absence of increased [18F]-FTP uptake in brain regions previously implicated in CTE among former professional ASF players compared with controls questions the utility of [18F]-FTP PET for clinical evaluation in this population.

Modified mouse model of repeated mild traumatic brain injury through a thinned-skull window and fluid percussion.

Mild traumatic brain injury (mTBI) is a clinically highly heterogeneous neurological disorder, none of the existing animal models can replicate the entire sequelae. This study aimed to develop a modified closed head injury (CHI) model of repeated mTBI (rmTBI) for investigating Ca2+ fluctuations of the affected neural network, the alternations of electrophysiology, and behavioral dysfunctions. The transcranial Ca2+ study protocol includes AAV-GCaMP6s infection in the right motor cortex, thinned-skull preparation, and two-photon laser scanning microscopy (TPLSM) imaging. The CHI rmTBI model is fabricated using the thinned-skull site and applying 2.0 atm fluid percussion with 48-h interval. The neurological dysfunction, minor motor performance, evident mood, spatial working, and reference deficits we found in this study mimic the clinically relevant syndromes after mTBI. Besides, our study revealed that there was a trend of transition from Ca2+ singlepeak to multipeak and plateau, and the total Ca2+ activities of multipeaks and plateaus (p < .001 vs. pre-rmTBI value) were significantly increased in ipsilateral layer 2/3 motor neurons after rm TBI. In parallel, there is a low-frequency power shift from delta to theta band (p < .01 vs. control) in the ipsilateral layer 2/3 of motor cortex of the rmTBI mice, and the overall firing rates significantly increased (p < .01 vs. control). Moreover, rmTBI causes slight cortical and hippocampal neuron damage and possibly induces neurogenesis in the dentate gyrus (DG). The alternations of Ca2+ and electrophysiological characteristics in layer 2/3 neuronal network, histopathological changes, and possible neurogenesis may play concertedly and partially contribute to the functional outcome post-rmTBI.

Therapeutic effect of a hydrogel-based neural stem cell delivery sheet for mild traumatic brain injury.

OBJECTIVE: There are no effective clinically applicable treatments for neuronal dysfunction after mild traumatic brain injury (TBI). Here, we evaluated the therapeutic effect of a new delivery method of mouse neural stem cell (mNSC) spheroids using a hydrogel, in terms of improvement in damaged cortical lesions and cognitive impairment after mild TBI. METHODS: mNSCs were isolated from the subventricular zone and subgranular zone by a hydrogel-based culture system. GFP-transduced mNSCs were generated into spheroids and wrapped into a sheet for transplantation. Male C57BL/6J mice were randomly divided into four groups: sham operation, TBI, TBI with mNSC spheroids, and TBI with mNSC spheroid sheet transplantation covering the damaged cortex. Histopathological and immunohistochemical features and cognitive function were evaluated 7, 14, and 28 days after transplantation following TBI. RESULTS: Hydrogel-based culture systems and mNSC isolation were successfully established from the adult mice. Essential transcription factors for NSCs, such as SOX2, PAX6, Olig2, nestin, and doublecortin (DCX), were highly expressed in the mNSCs. A transplanted hydrogel-based mNSC spheroid sheet showed good engraftment and survival ability, differentiated into TUJ1-positive neurons, promoted angiogenesis, and reduced neuronal degeneration. Also, TBI mice treated with mNSC spheroid sheet transplantation exhibited a significantly increased preference for a new object, suggesting improved cognitive function compared to the mNSC spheroids or no treatment groups. CONCLUSION: Transplantation with a hydrogel-based mNSC spheroid sheet showed engraftment, migration, and stability of delivered cells in a hostile microenvironment after TBI, resulting in improved cognitive function via reconstruction of the damaged cortex. STATEMENT OF SIGNIFICANCE: This study presents the therapeutic effect of a new delivery method of mouse neural stem cells spheroids using a hydrogel, in terms of improvement in damaged cortical lesions and cognitive impairment after traumatic brain injury. Collagen/fibrin hydrogel allowed long-term survival and migratory ability of NSCs spheroids. Furthermore, transplanted hydrogel-based mNSCs spheroids sheet showed good engraftment, migration, and stability of delivered cells in a hostile microenvironment, resulting in reconstruction of the damaged cortex and improved cognitive function after TBI. Therefore, we suggest that a hydrogel-based mNSCs spheroids sheet could help to improve cognitive impairment after TBI.