Closed-head Traumatic Brain Injury Research Articles

Anesthetic Preconditioning of Traumatic Brain Injury Is Ineffective in a Drosophila Model of Obesity.

We tested the hypothesis that obesity influences the pharmacodynamics of volatile general anesthetics (VGAs) by comparing effects of anesthetic exposure on mortality from traumatic brain injury (TBI) in lean and obese Drosophila melanogaster We induced TBI with a high-impact trauma device. Starvation-selection over multiple generations resulted in an obese phenotype (SS flies). Fed flies served as lean controls (FC flies). Adult (1-8-day-old) SS and FC flies were exposed to equianesthetic doses of isoflurane or sevoflurane either before or after TBI. The principal outcome was percent mortality 24 hours after injury, expressed as the Mortality Index at 24 hours (MI24). TBI resulted in a lower MI24 in FC than in SS flies [21 (2.35) and 57.8 (2.14), respectively n = 12, P = 0.0001]. Pre-exposure to isoflurane or sevoflurane preconditioned FC flies to TBI, reducing the risk of death to 0.53 (0.25 to 1.13) and 0.82 (0.43 to 1.58), respectively, but had no preconditioning effect in SS flies. Postexposure to isoflurane or sevoflurane increased the risk of death in SS flies, but only postexposure to isoflurane increased the risk in FC flies [1.39 (0.81 to 2.38)]. Thus, obesity affects the pharmacodynamics of VGAs, thwarting the preconditioning effect of isoflurane and sevoflurane in TBI. SIGNIFICANCE STATEMENT: Inadvertent preconditioning in models of traumatic brain injury (TBI) is a recognized confounder. The findings in a fruit fly (Drosophila melanogaster) model of closed-head TBI indicate that anesthetic pharmacodynamics are profoundly affected by obesity. Specifically, obesity thwarts the brain-protective effect of anesthetic preconditioning. This finding is important for experimental studies of TBI and supports the versatility of the fruit fly as a model for the exploration of anesthetic pharmacodynamics in a wide parameter space.

Translationally Relevant Magnetic Resonance Imaging Markers in a Ferret Model of Closed Head Injury.

Pre-clinical models of traumatic brain injury (TBI) have been the primary experimental tool for understanding the potential mechanisms and cellular alterations that follow brain injury, but the human relevance and translational value of these models are often called into question. Efforts to better recapitulate injury biomechanics and the use of non-rodent species with neuroanatomical similarities to humans may address these concerns and promise to advance experimental studies toward clinical impact. In addition to improving translational aspects of animal models, it is also advantageous to establish pre-clinical outcomes that can be directly compared with the same outcomes in humans. Non-invasive imaging and particularly MRI is promising for this purpose given that MRI is a primary tool for clinical diagnosis and at the same time increasingly available at the pre-clinical level. The objective of this study was to identify which commonly used radiologic markers of TBI outcomes can be found also in a translationally relevant pre-clinical model of TBI. The ferret was selected as a human relevant species for this study with folded cortical geometry and relatively high white matter content and the closed head injury model of engineered rotation and acceleration (CHIMERA) TBI model was selected for biomechanical similarities to human injury. A comprehensive battery of MRI protocols based on common data elements (CDEs) for human TBI was collected longitudinally for the identification of MRI markers and voxelwise analysis of T2, contrast enhancement and diffusion tensor MRI values. The most prominent MRI findings were consistent with focal hemorrhage and edema in the brain stem region following high severity injury as well as vascular and meningeal injury evident by contrast enhancement. While conventional MRI outcomes were not highly conspicuous in less severe cases, quantitative voxelwise analysis indicated diffusivity and anisotropy alterations in the acute and chronic periods after TBI. The main conclusions of this study support the translational relevance of closed head TBI models in intermediate species and identify brain stem and meningeal vulnerability. Additionally, the MRI findings highlight a subset of CDEs with promise to bridge pre-clinical studies with human TBI outcomes.

Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila.

In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.

Differential association of baseline body weight and body-weight loss with neurological deficits, histology, and death after repetitive closed head traumatic brain injury

Clinical observations indicate that body weight (BW) extremes are associated with worse outcome after traumatic brain injury (TBI); yet, it is uncertain whether the baseline BW (bBW) may affect outcome after mouse TBI. We retrospectively analyzed 129 similarly aged (9–12 weeks) male C57BL6/J mice that were subjected to repetitive closed head TBI (rTBI) using an established weight drop paradigm as well as 55 sham injured mice. We sought to determine whether the bBW as well as the post-TBI weight relative to baseline (%BW) were associated with a variety of post-rTBI outcomes, including acute model complications (skull fractures and macroscopic hemorrhage), impact seizures, return of the righting reflex (RR), the neurological severity score (NSS), post-rTBI BW-change, and 28-day mortality. In a subset of rTBI mice, we also assessed for potential associations between the bBW and %BW and performance in the novel object recognition (NOR) task and various histological outcomes at 28 days. We found no association between the bBW with acute model complications, impact seizure burden, RR, NSS, and NOR performance at 28 days, as well as cerebral microbleed burden, presence of hyperphosphorylated tau, and TDP-43 pathology after rTBI. However, a higher bBW was associated with a longer time to first impact seizure, a greater microglial activation, astrocytosis, and neuronal loss in the injured cerebral cortex at 28 days. A greater %BW-loss was associated with a shorter impact seizure-free survival, longer time to return of the righting reflex, greater neurological deficit severity as assessed by the NSS and NOR, and worse mortality. On multiple linear regression there was no independent association of the %BW-loss with neuronal loss and neuroinflammation after adjustment for the bBW. These observations indicate that the bBW and %BW-loss may be important biological variables in certain experimental mouse TBI investigations, depending on the outcome measures of interest.

Mouse closed head traumatic brain injury replicates the histological tau pathology pattern of human disease: characterization of a novel model and systematic review of the literature

Traumatic brain injury (TBI) constitutes one of the strongest environmental risk factors for several progressive neurodegenerative disorders of cognitive impairment and dementia that are characterized by the pathological accumulation of hyperphosphorylated tau (p-Tau). It has been questioned whether mouse closed-head TBI models can replicate human TBI-associated tauopathy. We conducted longitudinal histopathological characterization of a mouse closed head TBI model, with a focus on pathological features reported in human TBI-associated tauopathy. Male C57BL/6 J mice were subjected to once daily TBI for 5 consecutive days using a weight drop paradigm. Histological analyses (AT8, TDP-43, pTDP-43, NeuN, GFAP, Iba-1, MBP, SMI-312, Prussian blue, IgG, βAPP, alpha-synuclein) were conducted at 1 week, 4 weeks, and 24 weeks after rTBI and compared to sham operated controls. We conducted a systematic review of the literature for mouse models of closed-head injury focusing on studies referencing tau protein assessment. At 1-week post rTBI, p-Tau accumulation was restricted to the corpus callosum and perivascular spaces adjacent to the superior longitudinal fissure. Progressive p-Tau accumulation was observed in the superficial layers of the cerebral cortex, as well as in mammillary bodies and cortical perivascular, subpial, and periventricular locations at 4 to 24 weeks after rTBI. Associated cortical histopathologies included microvascular injury, neuroaxonal rarefaction, astroglial and microglial activation, and cytoplasmatic localization of TDP-43 and pTDP-43. In our systematic review, less than 1% of mouse studies (25/3756) reported p-Tau using immunostaining, of which only 3 (0.08%) reported perivascular p-Tau, which is considered a defining feature of chronic traumatic encephalopathy. Commonly reported associated pathologies included neuronal loss (23%), axonal loss (43%), microglial activation and astrogliosis (50%, each), and beta amyloid deposition (29%). Our novel model, supported by systematic review of the literature, indicates progressive tau pathology after closed head murine TBI, highlighting the suitability of mouse models to replicate pertinent human histopathology.

Potentiating Hemorrhage in a Periadolescent Rat Model of Closed-Head Traumatic Brain Injury Worsens Hyperexcitability but Not Behavioral Deficits.

Post-traumatic epilepsy (PTE) and neurocognitive deficits are devastating sequelae of head injuries that are common in adolescents. Investigating desperately needed treatments is hindered by the difficulties in inducing PTE in rodents and the lack of established immature rat models of pediatric PTE. Hemorrhage is a significant risk factor for PTE, but compared to humans, rats are less prone to bleeding because of their rapid blood coagulation system. In this study, we promoted bleeding in the controlled cortical impact (CCI) closed-head injury model with a 20 min pre-impact 600 IU/kg intraperitoneal heparin injection in postnatal day 35 (P35) periadolescent rats, given the preponderance of such injuries in this age group. Temporo-parietal CCI was performed post-heparin (HTBI group) or post-saline (TBI group). Controls were subjected to sham procedures following heparin or saline administration. Continuous long-term EEG monitoring was performed for 3 months post-CCI. Sensorimotor testing, the Morris water maze, and a modified active avoidance test were conducted between P80 and P100. Glial fibrillary acidic protein (GFAP) levels and neuronal damage were also assessed. Compared to TBI rats, HTBI rats had persistently higher EEG spiking and increased hippocampal GFAP levels (p < 0.05). No sensorimotor deficits were detected in any group. Compared to controls, both HTBI and TBI groups had a long-term hippocampal neuronal loss (p < 0.05), as well as contextual and visuospatial learning deficits (p < 0.05). The hippocampal astrogliosis and EEG spiking detected in all rats subjected to our hemorrhage-promoting procedure suggest the emergence of hyperexcitable networks and pave the way to a periadolescent PTE rat model.

Improved Mild Closed Head Traumatic Brain Injury Outcomes With a Brain-Computer Interface Amplified Cognitive Remediation Training.

This study is a retrospective chart review of 200 clients who participated in a non-verbal restorative cognitive remediation training (rCRT) program between 2012 and 2020. Each client participated in the program for about 16 weeks, and the study as a whole occurred over a five-year period. The program was applied to effect proper neural functional remodeling needed to support resilient, flexible, and adaptable behaviors after encountering a mild closed head traumatic brain injury (mTBI). The rCRT program focused on improving functional performance in executive cognitive control networks as defined by fMRI studies. All rCRT activities were delivered in a semi-game-like manner, incorporating a brain-computer interface (BCI) that provided in-the-moment neural network performance integrity metrics (nPIMs) used to adjust the level of play required to properly engage long-term potentiation (LTP) and long-term depression (LTD) network learning rules.This study reports on t-test and Reliable Change Index (RCI) changes found within individual cognitive abilities’ performance metrics derived from the Woodcock-Johnson Cognitive Abilities III Test. We compared pre- and post-scores from seven cognitive abilities considered dependent on executive cognitive control networks against seven non-executive control abilities. We observed significant improvements (p < 10-4) with large Cohen’s deffect sizes (0.78-1.20) across 13 of 14 cognitive ability domains with a medium effect size (0.49) on the remaining one. The mean percent change for the pooled trained domain was double that observed for the pooled untrained domain, at 17.2% versus 8.3%, respectively. To further adjust for practice effects, practice effect RCI values were computed and further supported the effectiveness of the rCRT (trained RCI 1.4-4.8; untrained RCI 0.­08-0.75).

Sidestream Smoke Affects Dendritic Complexity and Astrocytes After Model Mild Closed Head Traumatic Brain Injury.

Mild traumatic brain injuries can have long-term consequences that interfere with the life of the patient and impose a burden on our health care system. Oxidative stress has been identified as a contributing factor for the progression of neurodegeneration following TBI. A major source of oxidative stress for many veterans is cigarette smoking and second-hand smoke, which has been shown to have an effect on TBI recovery. To examine the potential influences of second-hand smoke during recovery from TBI, we utilized a mouse model of closed head injury, followed by repeated exposure to cigarette smoke and treatment with a neuroprotective antioxidant. We found that neither the mild injuries nor the smoke exposure produced axonal damage detectable with amino cupric silver staining. However, complexity in the dendritic arbors was significantly reduced after mild TBI plus smoke exposure. In the hippocampus, there were astrocytic responses, including Cyp2e1 upregulation, after the injury and tobacco smoke insult. This study provides useful context for the importance of lifestyle changes, such as reducing or eliminating cigarette smoking, during recovery from TBI.

Assessment of the Effects of Altered Amyloid-Beta Clearance on Behavior following Repeat Closed-Head Brain Injury in Amyloid-Beta Precursor Protein Humanized Mice.

Traumatic brain injury (TBI) increases the risk for dementias including Alzheimer's disease (AD) and chronic traumatic encephalopathy. Further, both human and animal model data indicate that amyloid-beta (Aβ) peptide accumulation and its production machinery are upregulated by TBI. Considering the clear link between chronic Aβ elevation and AD as well as tau pathology, the role(s) of Aβ in TBI is of high importance. Endopeptidases, including the neprilysin (NEP)-like enzymes, are key mediators of Aβ clearance and may affect susceptibility to pathology post-TBI. Here, we use a "humanized" mouse model of Aβ production, which expresses normal human amyloid-beta precursor protein (APP) under its natural transcriptional regulation and exposed them to a more clinically relevant repeated closed-head TBI paradigm. These transgenic mice also were crossed with mice deficient for the Aβ degrading enzymes NEP or NEP2 to assess models of reduced cerebral Aβ clearance in our TBI model. Our results show that the presence of the human form of Aβ did not exacerbate motor (Rotarod) and spatial learning/memory deficits (Morris water maze) post-injuries, while potentially reduced anxiety (Open Field) was observed. NEP and NEP2 deficiency also did not exacerbate these deficits post-injuries and was associated with protection from motor (NEP and NEP2) and spatial learning/memory deficits (NEP only). These data suggest that normally regulated expression of wild-type human APP/Aβ does not contribute to deficits acutely after TBI and may be protective at this stage of injury.

Differential Association of Baseline Body Weight and Body-Weight Loss with Neurological Deficits, Histology, and Death after Repetitive Closed Head Traumatic Brain Injury

Differential Association of Baseline Body Weight and Body-Weight Loss with Neurological Deficits, Histology, and Death after Repetitive Closed Head Traumatic Brain Injury

Association of Baseline Body Weight with Neurological Deficits, Histology, and Death after Repetitive Closed Head Traumatic Brain Injury in the Mouse

Association of Baseline Body Weight with Neurological Deficits, Histology, and Death after Repetitive Closed Head Traumatic Brain Injury in the Mouse

Acute damage to the blood–brain barrier and perineuronal net integrity in a clinically-relevant rat model of traumatic brain injury.

Closed-head, frontal impacts in which the brain undergoes both lateral and rotational acceleration comprise the majority of human traumatic brain injury (TBI). Here, we utilize a clinically relevant model to examine the effects of a single concussion on aspects of brain integrity: the blood-brain barrier, the perineuronal nets (PNNs), and diffuse axonal injury. Adult, male Sprague-Dawley rats received either a frontal, closed-head concussive TBI, or no injury and were evaluated at 1- or 7-day post-injury. Using immunolabeling for albumin, we observed a significant increase in the permeability of the blood-brain barrier at 1-, but not 7-day post-injury. Breakdown of the PNN, as measured by the binding of wisteria floribunda, was observed at 1-day post-injury in the dorsal, lateral, and ventral cortices. This difference was resolved at 7-day. Finally, axonal injury was identified at both 1- and 7-day post-injury. This preclinical model of closed-head, frontal TBI presents a useful tool with which to understand better the acute pathophysiology of a single, frontal TBI.

Interactions among Genetic Background, Anesthetic Agent, and Oxygen Concentration Shape Blunt Traumatic Brain Injury Outcomes in Drosophila melanogaster.

Following traumatic brain injury (TBI), the time window during which secondary injuries develop provides a window for therapeutic interventions. During this time, many TBI victims undergo exposure to hyperoxia and anesthetics. We investigated the effects of genetic background on the interaction of oxygen and volatile general anesthetics with brain pathophysiology after closed-head TBI in the fruit fly Drosophila melanogaster. To test whether sevoflurane shares genetic risk factors for mortality with isoflurane and whether locomotion is affected similarly to mortality, we used a device that generates acceleration–deceleration forces to induce TBI in ten inbred fly lines. After TBI, we exposed flies to hyperoxia alone or in combination with isoflurane or sevoflurane and quantified mortality and locomotion 24 and 48 h after TBI. Modulation of TBI–induced mortality and locomotor impairment by hyperoxia with or without anesthetics varied among fly strains and among combinations of agents. Resistance to increased mortality from hyperoxic isoflurane predicted resistance to increased mortality from hyperoxic sevoflurane but did not predict the degree of locomotion impairment under any condition. These findings are important because they demonstrate that, in the context of TBI, genetic background determines the latent toxic potentials of oxygen and anesthetics.

Argon Inhalation for 24h After Closed-Head Injury Does not Improve Recovery, Neuroinflammation, or Neurologic Outcome in Mice.

In recent years, the noble gas argon (Ar) has been extensively studied for its organ protection properties. While mounting in vitro and in vivo evidence indicates that argon provides neuroprotection in ischemic brain injury, its neuroprotective potential in traumatic brain injury (TBI) has not been evaluated in vivo. We tested the hypothesis that prolonged inhalation of 70% or 79% argon for 24h after closed-head injury (CHI) improves neurologic outcome and overall recovery at 36days post-injury. We also compared effects of the 30% or 21% residual oxygen on argon's potential neuroprotective capacity. Adult male C57/black mice (n = 240) were subjected to closed-head traumatic brain injury, followed by inhalation of 70% argon or nitrogen (30% oxygen), or 79% argon or nitrogen (21% oxygen) for 24h. Neurologic outcome (rotarod, neuroscore, and Morris water maze) was evaluated for up to 36days post-injury. Histologic parameters of neurologic degeneration (Fluoro-Jade staining) and inflammation (F4/80 microglia immunostaining) were assessed in subgroups at 24h and on post-injury day 7. Our CHI protocol consistently resulted in significant brain injury. After argon inhalation for 24h at either concentration, mice did not show significant improvement with regard to neuroscores, rotarod performance, Morris water maze performance, or overall recovery (body weight), compared to nitrogen controls, up to 36days. At 7days post-injury, histologic markers of neurodegeneration and inflammation, particularly in the hippocampus, consistently demonstrated significant injury. Notably, recovery was reduced in mice treated with the higher oxygen concentration (30%) after CHI compared to 21%. Prolonged argon treatment did not improve neurologic outcome, overall recovery (weight), nor markers of neurodegeneration or neuroinflammation after significant CHI compared to nitrogen. While neuroprotective in predominately ischemic injury, argon did not provide protection after TBI in this model, highlighting the crucial importance of assessing argon's strengths and weaknesses in preclinical models to fully understand its organ protective potential in different pathologies and gas mixtures.

Loss of the Antimicrobial Peptide Metchnikowin Protects Against Traumatic Brain Injury Outcomes in Drosophila melanogaster.

Neuroinflammation is a major pathophysiological feature of traumatic brain injury (TBI). Early and persistent activation of innate immune response signaling pathways by primary injuries is associated with secondary cellular injuries that cause TBI outcomes to change over time. We used a Drosophila melanogaster model to investigate the role of antimicrobial peptides (AMPs) in acute and chronic outcomes of closed-head TBI. AMPs are effectors of pathogen and stress defense mechanisms mediated by the evolutionarily conserved Toll and Immune-deficiency (Imd) innate immune response pathways that activate Nuclear Factor kappa B (NF-κB) transcription factors. Here, we analyzed the effect of null mutations in 10 of the 14 known Drosophila AMP genes on TBI outcomes. We found that mutation of Metchnikowin (Mtk) was unique in protecting flies from mortality within the 24 h following TBI under two diet conditions that produce different levels of mortality. In addition, Mtk mutants had reduced behavioral deficits at 24 h following TBI and increased lifespan either in the absence or presence of TBI. Using a transcriptional reporter of gene expression, we found that TBI increased Mtk expression in the brain. Quantitative analysis of mRNA in whole flies revealed that expression of other AMPs in the Toll and Imd pathways as well as NF-κB transcription factors were not altered in Mtk mutants. Overall, these results demonstrate that Mtk plays an infection-independent role in the fly nervous system, and TBI-induced expression of Mtk in the brain activates acute and chronic secondary injury pathways that are also activated during normal aging.

Optimization and validation of a modified radial-arm water maze protocol using a murine model of mild closed head traumatic brain injury.

Cognitive impairments can be a significant problem after a traumatic brain injury (TBI), which affects millions worldwide each year. There is a need for establish reproducible cognitive assays in rodents to better understand disease mechanisms and to develop therapeutic interventions towards treating TBI-induced impairments. Our goal was to validate and standardize the radial arm water maze (RAWM) test as an assay to screen for cognitive impairments caused by TBI. RAWM is a visuo-spatial learning test, originally designed for use with rats, and later adapted for mice. The present study investigates whether test procedures, such us the presence of extra-maze cues influences learning and memory performance. C57BL/6 mice were tested in an 8-arm RAWM using a four-day protocol. We demonstrated that two days of training, exposing the mice to extra-maze cues and a visible platform, influenced learning and memory performance. Mice that did not receive training performed poorer compared to mice trained. To further validate our RAWM protocol, we used scopolamine. We, also, demonstrated that a single mild closed head injury (CHI) caused deficits in this task at two weeks post-CHI. Our data supported the use of 7 trials per day and a spaced training protocol as key factor to unmask memory impairment following CHI. Here, we provide a detailed standard operating procedure for RAWM test, which can be applied to a variety of mouse models including neurodegenerative diseases and pathology, as well as when pharmacological approaches are used.

Zebrafish model of posttraumatic epilepsy

Posttraumatic epilepsy (PTE) is defined as recurrent and unprovoked seizures occurring >1week after traumatic brain injury (TBI). Animal studies of PTE are lengthy and expensive. In this study, we developed a cost-effective PTE animal model using zebrafish to bridge the gap between in vitro studies and low-throughput animal studies. We used two different sets of parameters (G1 and G2) to induce closed-head TBI in adult zebrafish using pulsed high-intensity focused ultrasound. Injured fish and naive controls were evaluated for behavioral deficits and spontaneous behavioral seizure activity up to 21 days postinjury (DPI). We also assessed behavioral seizure susceptibility to a subconvulsive dose of pentylenetetrazole (PTZ; 2.5mmol·L-1 ) and recorded electrophysiological signals to confirm seizure activity up to 40 DPI. In addition, we investigated injury-related changes in the blood-brain barrier and expression levels of various proteins altered in rodent and human TBI. The G2 parameters resulted in a more severe TBI, with a mortality rate of 25%, as well as motor dysfunction and heightened anxiety persisting at 21 DPI. One hundred percent of the G2 group showed spontaneous myocloniclike behavior, and 80% demonstrated tonic-clonic-like behavioral seizures by 21 DPI. Such activities were not detected in the naive group. After the application of 2.5mmol·L-1 PTZ, 100% of injured zebrafish had cloniclike seizures at 21 DPI, versus 30% of the naive group. We also demonstrated electrographic seizure activity at 40 DPI, which was not detected in the naive controls. Lastly, we observed acute blood-brain barrier dysfunction and increased levels of HMGB1 and ratios of phosphorylated/total Akt and tau through 21 DPI. Together, the results indicate that severe TBI in the adult zebrafish leads to similar behavioral and physiological changes to those of more traditional models, including the development of PTE, and suggest this may be a useful model that can accelerate research in TBI/PTE.

Automated Quantification of Immunohistochemical Staining of Large Animal Brain Tissue Using QuPath Software

Large scale unbiased quantification of immunohistochemistry (IHC) is time consuming, expensive, and/or limited in scope. Heterogeneous tissue types such as brain tissue have presented a further challenge to the development of automated analysis, as differing cellular morphologies result in either limited applicability or require large amounts of training tissue for machine-learning methods. Here we present the use of QuPath, a free and open source software, to quantify whole-brain sections stained with the immunohistochemical markers IBA1 and AT8, for microglia and phosphorylated tau respectively. The pixel-based method of analysis herein allows for statistical comparison of global protein expression between brains and generates heat-maps of stain intensity, visualizing stain signal across whole sections and permitting more specific investigation of regions of interest. This method is fast, automated, unbiased, and easily replicable. We compared swine brains that had undergone a closed head traumatic brain injury with brains of sham animals, and found a global increase in both microglial signal expression and phosphorylated tau. We discuss the IHC methods necessary to utilize this analysis and provide detailed instruction on the use of QuPath in the pixel-based analysis of whole-slide images.

Evaluating spatiotemporal microstructural alterations following diffuse traumatic brain injury.

BackgroundDiffuse traumatic brain injury (TBI) is known to lead to microstructural changes within both white and grey matter detected in vivo with diffusion tensor imaging (DTI). Numerous studies have shown alterations in fractional anisotropy (FA) and mean diffusivity (MD) within prominent white matter tracts, but few have linked these to changes within the grey matter with confirmation via histological assessment. This is especially important as alterations in the grey matter may be predictive of long-term functional deficits.MethodsA total of 33 male Sprague Dawley rats underwent severe closed-head TBI. Eight animals underwent tensor-based morphometry (TBM) and DTI at baseline (pre-TBI), 24 hours (24 h), 7, 14, and 30 days post-TBI. Immunohistochemical analysis for the detection of ionised calcium-binding adaptor molecule 1 (IBA1) to assess microglia number and percentage of activated cells, β-amyloid precursor protein (APP) as a marker of axonal injury, and myelin basic protein (MBP) to investigate myelination was performed at each time-point.ResultsDTI showed significant alterations in FA and RD in numerous white matter tracts including the corpus callosum, internal and external capsule, and optic tract and in the grey-matter in the cortex, thalamus, and hippocampus, with the most significant effects observed at 14 D post-TBI. TBM confirmed volumetric changes within the hippocampus and thalamus. Changes in DTI were in line with significant axonal injury noted at 24 h post-injury via immunohistochemical analysis of APP, with widespread microglial activation seen within prominent white matter tracts and the grey matter, which persisted to 30 D within the hippocampus and thalamus. Microstructural alterations in MBP+ve fibres were also noted within the hippocampus and thalamus, as well as the cortex.ConclusionThis study confirms the widespread effects of diffuse TBI on white matter tracts which could be detected via DTI and extends these findings to key grey matter regions, with a comprehensive investigation of the whole brain. In particular, the hippocampus and thalamus appear to be vulnerable to ongoing pathology post-TBI, with DTI able to detect these alterations supporting the clinical utility in evaluating these regions post-TBI.

Greater neurodegeneration and behavioral deficits after single closed head traumatic brain injury in adolescent versus adult male mice.

Traumatic brain injury (TBI) is a major public health concern affecting 2.8million people per year in the United States, of whom about 1million are children under 19years old. Animal models of TBI have been developed and used in multiple ages of animals, but direct comparisons of adult and adolescent populations are rare. The current studies were undertaken to directly compare outcomes between adult and adolescent male mice, using a closed head, single-impact model of TBI. Six-week-old adolescent and 9-week-old adult male mice were subjected to mild-moderate TBI. Histological measures for neurodegeneration, gliosis, and microglial neuroinflammation, and behavioral tests of locomotion and memory were performed. Adolescent TBI mice have increased mortality (Χ2 =20.72, p<0.001) compared to adults. There is also evidence of hippocampal neurodegeneration in adolescents that is not present in adults. Hippocampal neurodegeneration correlates with histologic activation of microglia, but not with increased astrogliosis. Adults and adolescents have similar locomotion deficits after TBI that recover by 16days postinjury. Adolescents have memory deficits as evidenced by impaired novel object recognition between 3-4 and 4-16days postinjury (F1,26 =5.23, p=0.031) while adults do not. In conclusion, adults and adolescents within a close age range (6-9weeks) respond to TBI differently. Adolescents are more severely affected by mortality, neurodegeneration, and inflammation in the hippocampus compared to adults. Adolescents, but not adults, have worse memory performance after TBI that lasts at least 16days postinjury.