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Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury (DAI) and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal swellings, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals with confirmed DAI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation., (© 2024. The Author(s).)

The postmortem diagnosis of diffuse traumatic axonal injury (dTAI) relies on β-amyloid precursor protein (β-APP) immunohistochemistry. Most reports of factors associating with dTAI are decades old. We compared background characteristics and neuropathology findings of today's Finnish medico-legal autopsy cases with and without β-APP-positive dTAI (dTAI+ and dTAI-, respectively). The cases had suffered a head injury prior to death and underwent a full neuropathological examination including β-APP stain. Background and circumstantial data as well as neuropathology findings were collected from police documents, medical records, and autopsy and neuropathology reports. Prevalence ratios were calculated for each factor to facilitate comparisons between the dTAI+ and dTAI- groups. The dataset comprised 57 cases (66.7% males), with 17 classified as dTAI+ and 40 as dTAI-. Based on prevalence ratios, the factors that had at least two-fold prevalence among dTAI+ cases compared to dTAI- cases were: an unknown injury mechanism; concurrent epidural or subdural haemorrhage; and an accidental manner of death. In contrast, the factors that had at least two-fold prevalence among dTAI- cases compared to dTAI+ cases were: a short postinjury survival (<30 min); concurrent intracerebral/ventricular haemorrhage or contusion; vermal atrophy; and a natural or homicidal manner of death. This study revealed differences in circumstantial features and neuropathology findings between dTAI+ and dTAI- cases in today's medico-legal autopsy material. Data on typical case profiles may help estimate the prior probability of dTAI not only in medico-legal autopsies but also among living patients with head injuries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Diffuse axonal injury (DAI), one of the most common and devastating type of traumatic brain injury, is the result of the shear force on axons due to severe rotational acceleration and deceleration. Neurogranin (NRGN) is a postsynaptic protein secreted by excitatory neurons, and synaptic dysfunction can alter extracellular NRGN levels. In this study, we examined NRGN levels in serum and cerebrospinal fluid (CSF) after experimental DAI in terms of their diagnostic value. Experimental DAI was induced using the Marmarou technique in male Wistar albino rats. Serum and CSF NRGN levels of the sham group, one‑hour, six‑hour, 24‑hour, and 72‑hour post‑DAI groups were measured by ELISA method. DAI was verified by staining with hematoxylin‑eosin and β‑amyloid precursor protein in the rat brain samples. While no histopathological and immunohistochemical changes were observed in the early hours of the post‑DAI groups, the staining of the β‑APP visibly increased over time, with positivity being most frequent and intense in the 72‑hour group. It was found that serum NRGN levels were significantly lower in the 6‑hour group than in the sham group. The serum NRGN levels in the 24‑hour group were significantly higher than those in the sham group. This study showed a dichotomy of post‑DAI serum NRGN levels in consecutive time periods. NRGN levels in CSF were higher in the one‑hour group than in the sham group and returned to baseline by 72 hours, although not significantly. Our study provides an impression of serum and CSF NRGN levels in a rat DAI model in consecutive time periods. Further studies are needed to understand the diagnostic value of NRGN.

Introduction: Diffuse axonal injury (DAI), with high mortality and morbidity both in children and adults, is one of the most severe pathological consequences of traumatic brain injury. Currently, clinical diagnosis, disease assessment, disability identification, and postmortem diagnosis of DAI is mainly limited by the absent of specific molecular biomarkers., Areas Covered: In this review, we first introduce the pathophysiology of DAI, summarized the reported biomarkers in previous animal and human studies, and then the molecular biomarkers such as β-Amyloid precursor protein, neurofilaments, S-100β, myelin basic protein, tau protein, neuron-specific enolase, Peripherin and Hemopexin for DAI diagnosis is summarized. Finally, we put forward valuable views on the future research direction of diagnostic biomarkers of DAI., Expert Opinion: In recent years, the advanced technology has ultimately changed the research of DAI, and the numbers of potential molecular biomarkers was introduced in related studies. We summarized the latest updated information in such studies to provide references for future research and explore the potential pathophysiological mechanism on diffuse axonal injury.

Traumatic axonal injury (TAI) accounts for a large proportion of the mortality of traumatic brain injury (TBI). The diagnosis of TAI is currently of limited use for medicolegal purposes. It is known that axons in TAI are diffusely damaged by secondary processes other than direct head injury. However, the physiopathological mechanism of TAI is still elusive. The present study used RGD peptide, an antagonist of the mechanotransduction protein integrin, to explore the role of integrin-transmitted mechanical signalling in the pathogenesis of rat TAI. The rats were subjected to a linearly accelerating load, and changes in beta-amyloid precursor protein (β-APP) expression, skeleton ultrastructure, skeleton protein neurofilament light (NF-L), and α-tubulin in the brainstem were observed, indicating that RGD could relieve the severity of axonal injury in TAI rats. In addition, the expression of β-integrin was stronger and centralized in the brainstem of the deceased died from TAI compared to other nonviolent causes. This study examined the pathophysiology and biomechanics of TAI and assessed the role of integrin in the injury of microtubules and neurofilaments in TAI. Thus, we propose that integrin-mediated cytoskeletal injury plays an important role in TAI and that integrin has the potential as a biomarker for TAI., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Diffuse axonal injury (DAI) represents a frequent traumatic brain injury (TBI) type, significantly contributing to the dismal neurological prognosis and high mortality in TBI patients. The increase in mortality can be associated with delayed and nonspecific initial symptoms in DAI patients. Additionally, the existing approaches for diagnosis and monitoring are either low sensitivity or high cost. Therefore, novel, reliable, and objective diagnostic markers should be developed to diagnose and monitor DAI prognosis. Urine is an optimal sample to detect biomarkers for DAI noninvasively. Therefore, the DAI rat model was established in this work. Meanwhile, the ultraperformance liquid chromatography quadrupole-time-of-flight hybrid mass spectrometry- (UPLC/Q-TOF MS-) untargeted metabolomics approach was utilized to identify the features of urine metabolomics to diagnose DAI. This work included 57 metabolites with significant alterations and 21 abnormal metabolic pathways from the injury groups. Three metabolites, viz., urea, butyric acid, and taurine, were identified as possible biomarkers to diagnose DAI based on the great fold changes (FCs) and biological functions during DAI. The present study detected several novel biomarkers for noninvasively diagnosing and monitoring DAI and helped understand the DAI-associated metabolic events., Competing Interests: All the authors claim that there exist no competing interests., (Copyright © 2022 Peng Zhang et al.)

Background: Ketogenic diet (KD) has been identified as a potential therapy to enhance recovery after traumatic brain injury (TBI). Diffuse axonal injury (DAI) is a common type of traumatic brain injury that is characterized by delayed axonal disconnection. Previous studies showed that demyelination resulting from oligodendrocyte damage contributes to axonal degeneration in DAI., Aim: The present study tests a hypothesis that ketone bodies from the ketogenic diet confers protection for myelin and attenuates degeneration of demyelinated axon in DAI., Methods: A modified Marmarou's model of DAI was induced in adult rats. The DAI rats were fed with KD and analyzed with western blot, transmission electron microscope, ELISA test and immunohistochemistry. Meanwhile, a co-culture of primary oligodendrocytes and neurons was treated with ketone body β-hydroxybutryate (βHB) to test for its effects on the myelin-axon unit., Results: Here we report that rats fed with KD showed an increased fatty acid metabolism and ketonemia. This dietary intervention significantly reduced demyelination and attenuated axonal damage in rats following DAI, likely through inhibition of DAI-induced excessive mitochondrial fission and promoting mitochondrial fusion. In an in vitro model of myelination, the ketone body βHB increased myelination significantly and reduced axonal degeneration induced by glucose deprivation (GD). βHB robustly increased cell viability, inhibited GD-induced collapse of mitochondrial membrane potential and attenuated death of oligodendrocytes., Conclusion: Ketone bodies protect myelin-forming oligodendrocytes and reduce axonal damage. Ketogenic diet maybe a promising therapy for DAI.

Traumatic brain injury (TBI) is a condition burdened by an extremely high rate of morbidity and mortality and can result in an overall disability rate as high as 50% in affected individuals. Therefore, the importance of identifying clinical prognostic factors for diffuse axonal injury (DAI) in (TBI) is commonly recognized as critical. The aim of the present review paper is to evaluate the most recent contributions from the relevant literature in order to understand how each single prognostic factor determinates the severity of the clinical syndrome associated with DAI. The main clinical factors with an important impact on prognosis in case of DAI are glycemia, early GCS, the peripheral oxygen saturation, blood pressure, and time to recover consciousness. In addition, the severity of the lesion, classified on the ground of the cerebral anatomical structures involved after the trauma, has a strong correlation with survival after DAI. In conclusion, modern findings concerning the role of reactive oxygen species (ROS) and oxidative stress in DAI suggest that biomarkers such as GFAP, pNF-H, NF-L, microtubule associated protein tau, Aβ42, S-100β, NSE, AQP4, Drp-1, and NCX represent a possible critical target for future pharmaceutical treatments to prevent the damages caused by DAI.

Blood-brain barrier (BBB) disruption exacerbates diffuse axonal injury (DAI), but the underlying mechanisms are not fully understood. Inactivation or deletion of erythropoietin-producing hepatoma (EPH) receptor A2 (EphA2) attenuated BBB damage and promoted tight junction formation. In this study, we aimed to investigate the role of EphA2 in the protection of BBB integrity and the relevant mechanisms involved in a rat model of DAI. Blocking activation of the EphA receptor by EphA2-Fc ameliorated axonal injury, cell apoptosis, and glial activation, protected BBB integrity and increased expression of the tight junction-associated proteins ZO-1, claudin-5 and occludin-1. In vitro BBB models established by human brain microvascular endothelial cells (HBMECs) were subjected to oxygen deprivation (OGD). Treatment with EphrinA1, which activates EphA2, exacerbated the OGD-induced destruction of permeability and integrity of the BBB models by reducing the expression of tight junction-associated proteins. However, inhibition of Rho-associated coiled coil-containing protein kinases 1 and 2 (ROCK1 and 2) abrogated all of the effects of EphrinA1 on the BBB models in vitro. In conclusion, we provide evidence that EphA2 plays an important role in the destruction of BBB integrity by decreasing the expression of tight junction proteins through the ROCK pathway., (Copyright © 2020. Published by Elsevier B.V.)

Objective: We have previously demonstrated that inflammation induced by toll-like receptors (TLRs) 2/4 exert cerebral deleterious effects after diffuse axonal injury (DAI); however, the underlying mechanisms are not fully understood. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine involved in inflammatory responses. The purpose of this study was to investigate the role of MIF in inflammation induced by TLRs in the cortices of DAI rats., Methods: The rat DAI model was established by head rotational acceleration and confirmed by β -APP, HE, and silver staining. MIF protein expression at 3 h, 6 h, 12 h, 1 d, and 3 d after DAI was measured by western blot. The localization of MIF was measured by immunofluorescence. MIF antagonist ISO-1 was intracerebroventricularly injected to inhibit MIF. Neuronal and axonal injury and glial responses were assessed by TUNEL, immunohistochemistry, and TEM. Expression of TLR2, TLR4, ERK, phospho-ERK, NF- κ B, and phospho-NF- κ B was examined by western blot. The level of IL-1 β , IL-6, and TNF- α was measured by ELISA., Results: MIF expression was significantly increased, peaking at 1 day after DAI, and MIF was mainly localized in microglial cells and neurons. ISO-1 suppressed neuronal apoptosis, axonal injury, and glial responses and decreased the expression of downstream signaling molecules related to TLR2/4, including ERK, phospho-ERK, NF- κ B, phospho-NF- κ B, IL-1 β , IL-6, and TNF- α ., Conclusion: MIF was involved in the neuronal and axonal damage through a TLR-related pathway following DAI., Competing Interests: All of the authors involved in the preparation of the manuscript declare no conflict of interest in any form., (Copyright © 2020 Yonglin Zhao et al.)

Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) can alleviate diffuse axonal injury (DAI)-induced apoptosis by regulating expression of heme oxygenase-1 (HO-1), while sulforaphane (SFN) was shown to reduce oxidative stress by increasing the expression of Nrf2. Therefore, we aimed to investigate therapeutic effect of SFN in the treatment of DAI and the ability of SFN to reduce oxidative stress., Methods: The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to observe the effects of H 2 O 2 and SFN on cell viability. Fluorometric assay, Western blot analysis, and flow cytometry were conducted to validate the protective role of SFN in an animal model of DAI. In addition, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were measured in DAI rats treated by SFN, while Western blot, immunohistochemistry assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were carried out to verify the effect of SFN in different animal groups., Results: Cell viability was reduced by H 2 O 2 in a dose-dependent manner, while the treatment by SFN significantly promoted cell growth. Meanwhile the administration of SFN effectively reduced the levels of caspase-3/poly(ADP-ribose) polymerase (PARP) activity increased by the H 2 O 2 treatment, indicating that the protective effect of SFN could be mediated by its ability to suppress caspase-3 activation and PARP cleavage. In addition, the SFN treatment reduced the intracellular reactive oxygen species (ROS) generation induced by H 2 O 2 . Moreover, the MDA levels of SOD/GPx activity in various rat groups showed the protective effects of SFN in DAI rats. It is suspected that the protective effect of SFN was exerted via the activation of the Nrf2/HO-1 signaling pathway. In this study, DAI and DAI + phosphate-buffered saline (PBS) groups also showed the presence of more TUNEL-positive cells compared with the sham-operated group, while the SFN treatment reduced the extent of neuronal apoptosis., Conclusions: By activating the Nrf2/HO-1 signaling pathway and reducing the activity of caspase-3, SFN reduces the apoptosis of neurons in brain trauma-induced DAI., (© 2019 Wiley Periodicals, Inc.)

Diffuse axonal injury (DAI) is one of the most common and severe pathological consequences of traumatic brain injury (TBI). The molecular mechanism of DAI is highly complicated and still elusive, yet a clear understanding is crucial for the diagnosis, treatment, and prognosis of DAI. In our study, we used rats to establish a DAI model and applied isobaric tags for relative and absolute quantitation (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins (DEPs) in the corpus callosum. As a result, a total of 514 proteins showed differential expression between the injury groups and the control. Among these DEPs, 14 common DEPs were present at all seven time points postinjury (1, 3, 6, 12, 24, 48, and 72 h). Next, bioinformatic analysis was performed to elucidate the pathogenesis of DAI, which was found to possibly involve calcium ion-regulatory proteins (e.g., calsenilin and ryanodine receptor 2), cytoskeleton organization (e.g., peripherin, NFL, NFM, and NFH), apoptotic processes (e.g., calsenilin and protein kinase C delta type), and inflammatory response proteins (e.g., complement C3 and C-reactive protein). Moreover, peripherin and calsenilin were successfully confirmed by western blotting to be significantly upregulated during DAI, and immunohistochemical (IHC) analysis revealed that their expression increased and could be observed in axons after injury, thus indicating their potential as DAI biomarkers. Our experiments not only provide insight into the molecular mechanisms of axonal injury in rats during DAI but also give clinicians and pathologists important reference data for the diagnosis of DAI. Our findings may expand the list of DAI biomarkers and improve the postmortem diagnostic rate of DAI., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Diffuse axonal injury (DAI) is an important consequence of traumatic brain injury (TBI). At the moment of trauma, axons rarely disconnect, but undergo cytoskeletal disruption and transport interruption leading to protein accumulation within swellings. The amyloid precursor protein (APP) accumulates rapidly and the standard histological evaluation of axonal pathology relies upon its detection. APP+ swellings first appear as varicosities along intact axons, which can ultimately undergo secondary disconnection to leave a terminal "axon bulb" at the disconnected, proximal end. However, sites of disconnection are difficult to determine with certainty using standard, thin tissue sections, thus limiting the comprehensive evaluation of axon degeneration. The tissue-clearing technique, CLARITY, permits three-dimensional visualization of axons that would otherwise be out of plane in standard tissue sections. Here, we examined the morphology and connection status of APP+ swellings using CLARITY at 6 h, 24 h, 1 week and 1 month following the controlled cortical impact (CCI) model of TBI in mice. Remarkably, many APP+ swellings that appeared as terminal bulbs when viewed in standard 8-µm-thick regions of tissue were instead revealed to be varicose swellings along intact axons when three dimensions were fully visible. Moreover, the percentage of these potentially viable axon swellings differed with survival from injury and may represent the delayed onset of distinct mechanisms of degeneration. Even at 1-month post-CCI, ~10% of apparently terminal bulbs were revealed as connected by CLARITY and are thus potentially salvageable. Intriguingly, the diameter of swellings decreased with survival, including varicosities along intact axons, and may reflect reversal of, or reduced, axonal transport interruption in the chronic setting. These data indicate that APP immunohistochemistry on standard thickness tissue sections overestimates axon disconnection, particularly acutely post-injury. Evaluating cleared tissue demonstrates a surprisingly delayed process of axon disconnection and thus longer window of therapeutic opportunity than previously appreciated. Intriguingly, a subset of axon swellings may also be capable of recovery., (© 2018 International Society of Neuropathology.)

The diagnosis of diffuse axonal injury (DAI) is an important task in forensic pathology and clinical medicine. This study aimed to explore the use of Fourier transform infrared spectroscopy (FTIR) to detect DAI. The DAI area of the rat model was detected point by point by the FTIR-mapping system. Infrared spectral data of DAI were obtained by selecting the amide A band, CH 3 symmetric stretching, collagen triple-helix structure and asymmetric stretching vibrational frequency of nucleic acid and phospholipid PO 2 as the target peak positions. The system can automatically draw infrared spectral color pathological images. In the DAI group, the amide A protein secondary amine N-H stretching vibration and the collagen triple-helix structure of the high-absorption area were consistent with the DAI area confirmed by the silver and β-APP staining. The CH 3 symmetric stretching, nucleic acid and phospholipid PO 2 symmetric stretching vibration absorption spectra showed no significant differences between the experimental and verification groups. The FTIR-mapping technique can visually express the molecular characteristics of DAI, which is expected to be applied to the pathological diagnosis of DAI., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Diffuse axonal injury contributes to the long-term functional morbidity observed after pediatric moderate/severe traumatic brain injury (msTBI). Whole-brain proton magnetic resonance echo-planar spectroscopic imaging was used to measure the neurometabolite levels in the brain to delineate the course of disruption/repair during the first year post-msTBI. The association between metabolite biomarkers and functional measures (cognitive functioning and corpus callosum [CC] function assessed by interhemispheric transfer time [IHTT] using an event related potential paradigm) was also explored. Pediatric patients with msTBI underwent assessments at two times (post-acutely at a mean of three months post-injury, n = 31, and chronically at a mean of 16 months post-injury, n = 24). Healthy controls also underwent two evaluations, approximately 12 months apart. Post-acutely, in patients with msTBI, there were elevations in choline (Cho; marker for inflammation and/or altered membrane metabolism) in all four brain lobes and the CC and decreases in N-acetylaspartate (NAA; marker for neuronal and axonal integrity) in the CC compared with controls, all of which normalized by the chronic time point. Subgroups of TBI showed variable patterns chronically. Patients with slow IHTT had lower lobar Cho chronically than those with normal IHTT; they also did not show normalization in CC NAA whereas those with normal IHTT showed significantly higher levels of CC NAA relative to controls. In the normal IHTT group only, chronic CC Cho and NAA together explained 70% of the variance in long-term cognitive functioning. MR based whole brain metabolic evaluations show different patterns of neurochemistry after msTBI in two subgroups with different outcomes. There is a dynamic relationship between prolonged inflammatory responses to brain damage, reparative processes/remyelination, and subsequent neurobehavioral outcomes. Multimodal studies allow us to test hypotheses about degenerative and reparative processes in patient groups that have divergent functional outcome, with the ultimate goal of developing targeted therapeutic agents.

Diffuse axonal injury (DAI) accounts for more than 50% of all traumatic brain injury. In response to the mechanical damage associated with DAI, the abnormal proteins produced in the neurons and axons, namely, β-APP and p-tau, induce endoplasmic reticulum (ER) stress. Curcumin, a major component extracted from the rhizome of Curcuma longa, has shown potent anti-inflammatory, antioxidant, anti-infection, and antitumor activity in previous studies. Moreover, curcumin is an activator of nuclear factor-erythroid 2-related factor 2 (Nrf2) and promotes its nuclear translocation. In this study, we evaluated the therapeutic potential of curcumin for the treatment of DAI and investigated the mechanisms underlying the protective effects of curcumin against neural cell death and axonal injury after DAI. Rats subjected to a model of DAI by head rotational acceleration were treated with vehicle or curcumin to evaluate the effect of curcumin on neuronal and axonal injury. We observed that curcumin (20 mg/kg intraperitoneal) administered 1 h after DAI induction alleviated the aggregation of p-tau and β-APP in neurons, reduced ER-stress-related cell apoptosis, and ameliorated neurological deficits. Further investigation showed that the protective effect of curcumin in DAI was mediated by the PERK/Nrf2 pathway. Curcumin promoted PERK phosphorylation, and then Nrf2 dissociated from Keap1 and was translocated to the nucleus, which activated ATF4, an important bZIP transcription factor that maintains intracellular homeostasis, but inhibited the CHOP, a hallmark of ER stress and ER-associated programmed cell death. In summary, we demonstrate for the first time that curcumin confers protection against abnormal proteins and neuronal apoptosis after DAI, that the process is mediated by strengthening of the unfolded protein response to overcome ER stress, and that the protective effect of curcumin against DAI is dependent on the activation of Nrf2.

The positron emission tomography (PET) radioligand for adenosine A 1 receptor (A1R) [1-methyl- 11 C] 8-dicyclopropylmethyl-1-methyl-3-propylxanthine (MPDX) has recently been developed for human brain imaging. In the present study, we evaluated the alteration of the A1R in patients with diffuse axonal injury (DAI) in chronic stage in vivo. Ten patients with DAI (7 men and 3 women) were included in this study. Three PET examinations were sequentially performed to measure A1R binding with 11 C-MPDX, glucose metabolism with 18 F-fluorodeoxyglucose (FDG), and central benzodiazepine receptor binding with 11 C-flumazenil (FMZ), and decreases of 11 C-FMZ uptake indicate neuronal loss. 11 C- MPDX did not depict any lesion with significantly decreased nondisplaceable binding potential (BP ND ) in comparison to healthy controls (14 men) in region of interest (ROI) analysis. Instead, it showed a significant increase of BP ND in the lower frontal and posterior cingulate cortexes and rolandic area (p < 0.05) in ROI analysis. In 18 F-FDG PET, the standardized uptake values (SUVs) ratio to the whole brain were decreased in anterior and posterior cingulate gyrus compared to controls (14 men and 9 women; p < 0.01). In 11 C-FMZ PET, the SUV ratio to the cerebellum was decreased in anterior cingulate gyrus in ROI analysis (controls, 9 men and 6 women; p < 0.01). The area with significantly increased 11 C-MPDX binding, lower frontal cortex, rolandic area, and posterior cingulate gyrus, did not overlap with the areas of neuronal loss detected by decreased 11 C-FMZ binding and did not completely overlap with area of reduced 18 F-FDG uptake. We obtained the first 11 C-MPDX PET images reflecting the A1R BP ND in human DAI brain in vivo. 11 C-MPDX depicted increased A1R BP ND in the areas surrounding the injured brain, whereas 18 F-FDG demonstrated reduction throughout the brain. The results suggested that A1R might continuously confer neuroprotective or neuromodulatory effects in DAI even in the chronic stage.

Traumatic brain injury (TBI) is one of the world's leading causes of morbidity and mortality among young individuals. TBI applies powerful rotational and translational forces to the brain parenchyma, which results in a traumatic diffuse axonal injury (DAI) responsible for brain swelling and neuronal death. Following TBI, axonal degeneration has been identified as a progressive process that starts with disrupted axonal transport causing axonal swelling, followed by secondary axonal disconnection and Wallerian degeneration. These modifications in the axonal cytoskeleton interrupt the axoplasmic transport mechanisms, causing the gradual gathering of transport products so as to generate axonal swellings and modifications in neuronal homeostasis. Oxidative stress with consequent impairment of endogenous antioxidant defense mechanisms plays a significant role in the secondary events leading to neuronal death. Studies support the role of an altered axonal calcium homeostasis as a mechanism in the secondary damage of axon, and suggest that calcium channel blocker can alleviate the secondary damage, as well as other mechanisms implied in the secondary injury, and could be targeted as a candidate for therapeutic approaches. Reactive oxygen species (ROS)-mediated axonal degeneration is mainly caused by extracellular Ca 2+ . Increases in the defense mechanisms through the use of exogenous antioxidants may be neuroprotective, particularly if they are given within the neuroprotective time window. A promising potential therapeutic target for DAI is to directly address mitochondria-related injury or to modulate energetic axonal energy failure., Competing Interests: The authors declared no conflicts of interest.

Background: Therapeutics specific to neural injury have long been anticipated but remain unavailable. Axons in the central nervous system do not readily regenerate after injury, leading to dysfunction of the nervous system. This failure of regeneration is due to both the low intrinsic capacity of axons for regeneration and the various inhibitors emerging upon injury. After many years of concerted efforts, however, these hurdles to axon regeneration have been partially overcome., Scope of Review: This review summarizes the mechanisms regulating axon regeneration. We highlight proteoglycans, particularly because it has become increasingly clear that these proteins serve as critical regulators for axon regeneration., Major Conclusions: Studies on proteoglycans have revealed that glycans not only assist in the modulation of protein functions but also act as main players-e.g., as functional ligands mediating intracellular signaling through specific receptors on the cell surface. By regulating clustering of the receptors, glycans in the proteoglycan moiety, i.e., glycosaminoglycans, promote or inhibit axon regeneration. In addition, proteoglycans are involved in various types of neural plasticity, ranging from synaptic plasticity to experience-dependent plasticity., General Significance: Although studies on proteins have progressively facilitated our understanding of the nervous system, glycans constitute a new frontier for further research and development in this field. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: To screen for the differential expression proteins in brain tissues of SD rat after diffuse axonal injury （DAI） by isobaric tag for relative and absolute quantification-liquid chromatograph-mass spectrometer/mass spectrometer （iTRAQ-LC-MS/MS）, and to explore potential biomarkers available for the diagnosis of DAI., Methods: Animal models of DAI were established with the Marmarou method as reference, and the subjects were divided into blank control group （ n =4）, sham strike group （ n =4） and fatal strike group （ n =4）, respectively. The proteins in rat brain tissues were detected by iTRAQ-LC-MS/MS, and bioinformatics analysis and verification were performed on the results and screened for the differential expression proteins., Results: A total of 2 016 proteins were identified and quantified. The bioinformatics analysis revealed that the proteins had wide distribution and function, and participated in different biological processes. There were 16 proteins showed differential expression in fatal strike group, including one up-regulated expression protein and 15 down-regulated expression proteins. The results of iTRAQ-LC-MS/MS were confirmed by Western blotting method., Conclusions: Multiple differential expression proteins in rat brain tissues after DAI can be screened by iTRAQ-LC-MS/MS. This not only indicates a research direction for exploring the pathogenesis of DAI, but also provides potential biomarkers available for the diagnosis of DAI., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine)

Diffuse axonal injury (DAI) is the most common and significant pathological features of traumatic brain injury (TBI). However, there are still no effective drugs to combat the formation and progression of DAI in affected individuals. FK506, also known as tacrolimus, is an immunosuppressive drug, which is widely used in transplantation medicine for the reduction of allograft rejection. Previous studies have identified that FK506 may play an important role in the nerve protective effect of the central nervous system. In the present study, apoptosis of neuronal cells was observed following the induction of experimental DAI. The results demonstrated that it was closely related with the upregulation of death‑associated protein kinase 1 (DAPK1). It was hypothesized that FK506 may inhibit the activity of DAPK1 by inhibiting calcineurin activity, which may be primarily involved in anti‑apoptosis following DAI induction. Through researching the expression of nerve regeneration associated proteins (NF‑H and GAP‑43) following DAI, the present study provides novel data to suggest that FK506 promotes axon formation and nerve regeneration following experimental DAI. Therefore, FK506 may be a potent therapeutic for inhibiting nerve injury, as well as promoting the nerve regeneration following DAI.

Treatment of diffuse axonal injury (DAI) remains challenging in clinical practice due to the unclear pathophysiological mechanism. Uncontrolled, excessive inflammation is one of the most recognized mechanisms that contribute to the secondary injury after DAI. Toll like receptor 2 (TLR2) is highlighted for the initiation of a vicious self-propagating inflammatory circle. However, the role and detailed mechanism of TLR2 in secondary injury is yet mostly unknown. In this study, we demonstrated the expression of TLR2 levels in cortex, corpus callosum, and internal capsule and the localization of TLR2 in neurons and glial cells in rat DAI models. Intracerebral knockdown of TLR2 significantly downregulated TLR2 expression, attenuated cortical apoptosis, lessened glial response, and reduced the secondary axonal and neuronal injury in the cortex by inhibiting phosphorylation of mitogen-activated protein kinases (MAPK) including Erk, JNK, and p38, translocation of NF- κ B p65 from the cytoplasm to the nucleus, and decreasing levels of proinflammatory cytokines including interleukin-6, interleukin-1 β , and tumor necrosis factor- α . On the contrary, administration of TLR2 agonist to DAI rats achieved an opposite effect. Collectively, we demonstrated that TLR2 was involved in mediating secondary injury after DAI by inducing inflammation via the MAPK and NF- κ B pathways., Competing Interests: All of the authors involved in preparation of the manuscript declare no conflict of interests in any form.

Although millions of individuals suffer a traumatic brain injury (TBI) worldwide each year, it is only recently that TBI has been recognized as a major public health problem. Beyond the acute clinical manifestations, there is growing recognition that a single severe TBI (sTBI) or repeated mild TBIs (rTBI) can also induce insidious neurodegenerative processes, which may be associated with early dementia, in particular chronic traumatic encephalopathy (CTE). Identified at autopsy examination in individuals with histories of exposure to sTBI or rTBI, CTE is recognized as a complex pathology featuring both macroscopic and microscopic abnormalities. These include cavum septum pellucidum, brain atrophy and ventricular dilation, together with pathologies in tau, TDP-43, and amyloid-β. However, the establishment and characterization of CTE as a distinct disease entity is in its infancy. Moreover, the relative "dose" of TBI, such as the frequency and severity of injury, associated with risk of CTE remains unknown. As such, there is a clear and pressing need to improve the recognition and diagnosis of CTE and to identify mechanistic links between TBI and chronic neurodegeneration.

Background Aims: Cell therapy in neurological disability after traumatic brain injury (TBI) is in its initial clinical stage. We describe our preliminary clinical experience with three patients with diffuse axonal injury (DAI) who were treated with intrathecal administration of autologous mesenchymal stromal cells (MSCs)., Methods: Three patients with established neurological sequelae due to DAI received intrathecally autologous MSCs. The total number of MSCs administered was 60 × 10 6 (one patient), 100 × 10 6 (one patient) and 300 × 10 6 (one patient)., Results: All three patients showed improvement after cell therapy, and subsequent studies with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) showed a diffuse and progressive increase in brain glucose metabolism., Conclusion: Our present results suggest benefit of intrathecal administration of MSCs in patients with DAI, as well as a relationship between this type of treatment and increase in brain glucose metabolism. These preliminary findings raise the question of convenience of assessing the potential benefit of intrathecal administration of MSCs for brain diseases in which a decrease in glucose metabolism represents a crucial pathophysiological finding, such as Alzheimer's disease (AD) and other dementias., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Objectives: To establish the imaging mass spectrometry for analysis of differentially expressed proteins distribution in the rat brains with diffuse axonal injury （DAI） based on matrix assisted laser desorption/ionization-time of flight imaging mass spectrometry （MALDI-TOF-IMS）., Methods: MALDI-TOF-IMS scanning were conducted on the brains of DAI group and control group in the m/z range of 1 000 to 20 000 using AutoflexⅢ MALDI-TOF spectrometer. ClinProTool 2.2 software was used for statistical analysis on the data of two groups, and then the differentially expressed proteins were picked out to conduct imaging. The distribution of the proteins with different m/z in the rat brains was observed., Results: Five proteins with different m/z , including 4 963, 5 634, 6 253, 6 714 and 7 532, differentially expressed in the rat brains with DAI., Conclusions: MALDI-TOF-IMS can be used for studying the differentially expressed proteins in rat brains with DAI and the analysis method is established for exploring the distribution of differentially expressed proteins in the rat brains with DAI using imaging mass spectrometry., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine)

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., (© 2016 American Association of Neuropathologists, Inc. All rights reserved.)

Diffuse axonal injury (DAI) is fairly common during a traumatic brain injury (TBI) and is associated with high mortality. Making an early diagnosis, appropriate therapeutic decisions, and an accurate prognostic evaluation of patients with DAI still pose difficulties for clinicians. The detailed mechanisms of axonal injury after head trauma have yet to be clearly defined and no reliable biomarkers are available for early DAI diagnosis. Therefore, this study employed an established DAI animal model in conjunction with an isobaric tag for relative and absolute quantification (iTRAQ)-based protein identification/quantification approach. Alterations in rat cerebral protein expression were quantified using iTRAQ coupled LC-MS/MS, with differentially expressed proteins between the control groups, sham and sham-injured, and the injury groups, animals that died immediately post-injury and those sacrificed at 1h, 6h, 1d, 3d and 7d post-injury, identified. A total of 1858 proteins were identified and quantified and comparative analysis identified ten candidate proteins that warranted further examination. Of the ten candidate DAI biomarkers, four proteins, citrate synthase (CS), synaptosomal-associated protein 25 (Snap25), microtubule-associated protein 1B (MAP1B) and Rho-associated protein kinase 2 (Rock2), were validated by subsequent Western blot and immunohistochemistry analyses. Our studies not only identified several novel biomarkers that may provide insight into the pathophysiological mechanisms of DAI, but also demonstrated the feasibility of iTRAQ-based quantitative proteomic analysis in cerebral tissue research., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Increasing evidence suggests that secondary injury after diffuse axonal injury (DAI) damages more axons than the initial insult, but the underlying mechanisms of this phenomenon are not fully understood. Recent studies show that toll-like receptor 4 (TLR4) plays a critical role in promoting adaptive immune responses and have been shown to be associated with brain damage. The purpose of this study was to investigate the role of the TLR4 signalling pathway in secondary axonal injury in the cortices of DAI rats. TLR4 was mainly localized in microglial cells and neurons, and the levels of TLR4 downstream signalling molecules, including TLR4, myeloid differentiation primary response gene 88, toll/IR-1-(TIR-) domain-containing adaptor protein inducing interferon-beta, interferon regulatory factor 3, interferon β, nuclear factor κB (NF-κB) p65, and phospho-NF-κB p65, significantly increased and peaked at 1 d after DAI. Inhibition of TLR4 by TAK-242 attenuated apoptosis, neuronal and axonal injury, and glial responses. The neuroprotective effects of TLR4 inhibition were associated with decreases in the levels of TLR4 downstream signalling molecules and inflammatory factors, including interleukin-1β, interleukin-6, and tumour necrosis factor-α. These results suggest that the TLR4 signalling pathway plays an important role in secondary injury and may be an important therapeutic target following DAI.

The exact pathogenesis of multiple sclerosis (MS) is incompletely understood. Although auto-immune responses have an important role in the development of hallmark focal demyelinating lesions, the underlying mechanism of axonal degeneration, the other key player in MS pathology and main determinant of long-term disability, remains unclear and corresponds poorly with inflammatory disease activity. Perfusion-weighted imaging studies have demonstrated that there is a widespread cerebral hypoperfusion in patients with MS, which is present from the early beginning to more advanced disease stages. This reduced cerebral blood flow (CBF) does not seems to be secondary to loss of axonal integrity with decreased metabolic demands but appears to be mediated by elevated levels of the potent vasospastic peptide endothelin-1 in the cerebral circulation. Evidence is evolving that cerebral hypoperfusion in MS is associated with chronic hypoxia, focal lesion formation, diffuse axonal degeneration, cognitive dysfunction, and fatigue. Restoring CBF may therefore emerge as a new therapeutic target in MS.

Evaluating diffuse axonal injury (DAI) remains challenging in clinical sciences since the physiopathologic mechanism of DAI is still unclear. The calcium overload in the axoplasm is considered to be crucial for secondary axonal injury. The present study use calcium channel blocker, nimodipine, to explore the influence of Ca2+ in the pathogenesis of rat DAI. In the DAI group, the expressions of β-APP and NF-L in axons were increased from 12 to 72 h. The ultrastructural observation indicated the axon and vessel injury appeared at 12 h post-injury and severely aggravated from 24 to 72 h. The expression of vWF and brain water content was increased at 12 h after injury and further increased at 24 h. Nimodipine decreased the expression of β-APP, NF-L and vWF, and also attenuated the ultrastructural damage of vascular wall and axons. Furthermore, Ca-dependent enzyme, the calcineurin activity were increased in DAI and nimodipine suppressed the activity of calcineurins (CaN). However, the amount of CaN expression was not changed. Our results showed that disturbances of axonal calcium homeostasis play an important role in the secondary damage of the axon, neuron and capillary vessel which may be related with activating CaN during the acute phase of DAI. Nimodipine can alleviate the secondary damage by suppressing the calcineurin activity.

Axonal injury contributes greatly to neurological dysfunction following traumatic brain injury (TBI), but current histological diagnostic methods are limited in identifying the pathological profiles of injured axons and unable to provide an objective and accurate quantification. Fourier transform infrared microspectroscopy (FTIRM) has the ability to offer macromolecular bioinformatics of the tissues including biochemical composition and structure by calculating band absorption intensity. In this study, axonal injury in the brainstem of rats with traumatic brain injury at 72 h post-trauma, which was confirmed with beta-amyloid precursor protein (β-APP) immunostaining, was detected with FTIRM technique. The lower intensity of infrared absorbance under the amide I band corresponds strongly to the area of axonal injury, and further analysis of amide I band shows significant differences in protein conformation between injured and normal axons. The findings indicate that using FTIRM technique, the amide I band has potentials to be a infrared spectral marker of axonal injury., (© 2015 American Academy of Forensic Sciences.)

This study investigated the correlation between AQP4 expression and DTI in rat brainstems after diffuse axonal injuries (DAI). Forty rats were imaged before injury and reimaged at 3, 6, 12, 24 and 72 h post-injury. A control group of 8 rats was imaged and sacrificed for histology but not injured. After brain injury, AQP4 expression and ADC values in the brainstems increased gradually, reaching peak values at 24 h and 12 h, respectively. FA values decreased within 72 h. There was a negative correlation between ADC values and brainstem AQP4 expression at 12 h, and a positive correlation at 24 h or 72 h (P<0.01), respectively. Changes in the ADC and FA values in the brainstems indicated brain edema and severe axonal injuries. The correlations between AQP4 expression and time-dependent ADC values aid in understanding brain edema development after DAI.

Introduction: Blast injury to brain, a hundred-year old problem with poorly characterized neuropathology, has resurfaced as health concern in recent deployments in Iraq and Afghanistan. To characterize the neuropathology of blast injury, we examined the brains of veterans for the presence of amyloid precursor protein (APP)-positive axonal swellings typical of diffuse axonal injury (DAI) and compared them to healthy controls as well as controls with opiate overdose, anoxic-ischemic encephalopathy, and non-blast TBI (falls and motor vehicle crashes)., Results: In cases with blast history, we found APP (+) axonal abnormalities in several brain sites, especially the medial dorsal frontal white matter. In white matter, these abnormalities were featured primarily by clusters of axonal spheroids or varicosities in a honeycomb pattern with perivascular distribution. Axonal abnormalities colocalized with IBA1 (+) reactive microglia and had an appearance that was distinct from classical DAI encountered in TBI due to motor vehicle crashes. Opiate overdose cases also showed APP (+) axonal abnormalities, but the intensity of these lesions was lower compared to cases with blast histories and there was no clear association of such lesions with microglial activation., Conclusions: Our findings demonstrate that many cases with history of blast exposure are featured by APP (+) axonopathy that may be related to blast exposure, but an important role for opiate overdose, antemortem anoxia, and concurrent blunt TBI events in war theater or elsewhere cannot be discounted.

Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. Here, we show glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, we find injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. We provide evidence for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. We propose that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury., Competing Interests: The authors have declared that no competing interests exist.

Amyloid-β (Aβ) protein and its precursor, amyloid-β precursor protein (β-APP), have traditionally been used in the diagnosis of Alzheimer disease. Their use in diagnosis of traumatic brain injury by forensic analysis is becoming more widespread. However, to date, no reliable small animal model exists to evaluate these brain injury indicators. To address this, we have studied primary brain-stem injury in rats to assess the appearance of diffuse axonal injury in brain sections and correlate these findings with appearance of Aβ and relative β-APP mRNA levels. Using an EnVision 2-step immunohistochemical staining method to measure axon diameter, we found that there was significant difference in axon diameters within the medulla oblongata and several time points after brain injury, ranging from 3 to 24 hours. In addition, mRNA expression levels of β-APP increased following brain injury, peaking 3 hours following injury and decreasing back to baseline levels by 24 hours after injury. These results suggest that using immunohistochemistry and reverse transcription-polymerase chain reaction to detect changes in Aβ-associated axonal changes and β-APP mRNA levels, respectively, can be useful for the diagnosis of diffuse axonal injury during autopsy at early time points following fatal brain injury.

Significant attention has recently been drawn to the potential link between head trauma and the development of neurodegenerative disease, namely chronic traumatic encephalopathy (CTE). The acute neurotrauma associated with sports-related concussions in athletes and blast-induced traumatic brain injury in soldiers elevates the risk for future development of chronic neurodegenerative diseases such as CTE. CTE is a progressive disease distinguished by characteristic tau neurofibrillary tangles (NFTs) and, occasionally, transactive response DNA binding protein 43 (TDP43) oligomers, both of which have a predilection for perivascular and subcortical areas near reactive astrocytes and microglia. The disease is currently only diagnosed postmortem by neuropathological identification of NFTs. A recent workshop sponsored by National Institute of Neurological Disorders and Stroke emphasized the need for premortem diagnosis, to better understand disease pathophysiology and to develop targeted treatments. In order to accomplish this objective, it is necessary to discover the mechanistic link between acute neurotrauma and the development of chronic neurodegenerative and neuropsychiatric disorders such as CTE. In this review, we briefly summarize what is currently known about CTE development and pathophysiology, and subsequently discuss injury-induced pathways that warrant further investigation. Understanding the mechanistic link between acute brain injury and chronic neurodegeneration will facilitate the development of appropriate diagnostic and therapeutic options for CTE and other related disorders.

Aims: We investigated the axonal morphological changes and expression of both tau protein and β-APP following concussion to the medulla oblongata, in a rat model of chronic alcoholism., Methods: Fifty-nine male Sprague-Dawley rats were randomly divided into EtOH, EtOH-TBI and control groups (water group, water-TBI group). To establish chronic alcoholic rats, rats were intragastrically given edible spirituous liquor twice daily. Rats also received a blow on the occipital tuberosity with an iron pendulum. Morphological changes and expression of tau and β-APP proteins in the medulla oblongata were examined., Results: (a) Nerve fibre thickening and twisting were observed in alcoholic rats, with nerve fibre changes becoming more significant following a concussion blow, which leads to some nerve fibres fracturing. (b) Transmission electron microscopy revealed that the nerve fibre myelin became loosened and displayed lamellar separation, which became more significant following concussion. (c) The integral optical density (IOD) sum value of β-APP of the EtOH-TBI group was lower than that in the EtOH group (P < 0.05); the Tau IOD sum value of the EtOH-TBI group was higher than that in the EtOH group (P < 0.05)., Conclusion: (a) Chronic alcoholism caused nerve fibre and neuronal morphology damage in the rat medulla oblongata, with structural damage becoming more significant following concussion. (b) Concussion changed the expression of β-APP and tau protein in chronic alcoholic rat medulla oblongata, suggesting that chronic alcoholism can lead to severe axonal injury following a concussion blow. (c) The effect of chronic alcoholism may be synergistic the concussion blow to promote animal injury and death.

Objective determination of the time of brain contusion is of key importance in medicolegal neurotraumatology. Currently, the progress of immunohistochemistry allows the study of structural elements of cells including neurofilaments, i.e. neuronal cytoskeletal proteins possessing properties that could be used for determining the age of brain injury in forensic medicine. The purpose of this study was to review recently published literature with a focus on studies investigating changes which occur in neurofilaments after brain trauma, both in animal models and in human biological material. The review has shown a lack of data on temporal changes in neurofilament expression after human brain trauma which could be used for determining the age of injuries in forensic medicine.

Epidemiological evidence links severe or repeated traumatic brain injury (TBI) to the development of Alzheimer's disease (AD). Accumulation of amyloid precursor protein (APP) occurs with high frequency after TBI, particularly in injured axons, and APP may be cleaved to amyloid-β (Aβ) peptides playing key pathophysiological roles in AD. We used cerebral microdialysis (MD) to test the hypothesis that interstitial Aβ levels are altered following TBI and are related to the injury type, cerebral energy metabolism, age of the patient, and level of consciousness. In the present report, we evaluated 10 mechanically ventilated patients (7 male, 3 female, ages 18-76 years) with a severe TBI, who had intracranial pressure and MD monitoring. Each MD sample was analyzed for hourly routine energy metabolic biomarkers (MD-lactate, MD-pyruvate, MD-glucose, and MD-lactate/pyruvate ratio), cellular distress biomarkers (MD-glutamate, MD-glycerol), and MD-urea. The remaining MD samples were analyzed for Aβ1-40 (Aβ40; n=765 samples) and Aβ1-42 (Aβ42; n=765 samples) in pooled 2 h fractions up to 14 days post-injury, using the Luminex xMAP technique, allowing detection with high temporal resolution of the key Aβ peptides Aβ40 and Aβ42. Data are presented using medians and 25th and 75th percentiles. Both Aβ40 and Aβ42 were consistently higher in patients with predominately diffuse axonal injury compared with patients with focal TBI at days 1-6 post- injury, Aβ42 being significantly increased at 113-116 h post-injury (p<0.05). The Aβ levels did not correlate with the interstitial energy metabolic situation, age of the patient, or the level of consciousness. These results support that interstitial generation of potentially toxic Aβ species may occur following human TBI, particularly related to axonal injury.

Moderate to severe traumatic brain injury (TBI) in humans and rats induces measurable metabolic changes, including a sustained depression in cerebral glucose uptake. However, the effect of a mild TBI on brain glucose uptake is unclear, particularly in rodent models. This study aimed to determine the glucose uptake pattern in the brain after a mild lateral fluid percussion (LFP) TBI. Briefly, adult male rats were subjected to a mild LFP and positron emission tomography (PET) imaging with (18)F-fluorodeoxyglucose ((18)FDG), which was performed prior to injury and at 3 and 24 h and 5, 9, and 16 days post-injury. Locomotor function was assessed prior to injury and at 1, 3, 7, 14, and 21 days after injury using modified beam walk tasks to confirm injury severity. Histology was performed at either 10 or 21 days post-injury. Analysis of function revealed a transient impairment in locomotor ability, which corresponds to a mild TBI. Using reference region normalization, PET imaging revealed that mild LFP-induced TBI depresses glucose uptake in both the ipsilateral and contralateral hemispheres in comparison with sham-injured and naïve controls from 3 h to 5 days post-injury. Further, areas of depressed glucose uptake were associated with regions of glial activation and axonal damage, but no measurable change in neuronal loss or gross tissue damage was observed. In conclusion, we show that mild TBI, which is characterized by transient impairments in function, axonal damage, and glial activation, results in an observable depression in overall brain glucose uptake using (18)FDG-PET.

Inflammation can be prevented in most inflammatory brain diseases, while tissue repair of the lesioned central nervous system (CNS) is still a major challenge. The CNS is difficult to access for protein therapeutics due to the blood-brain barrier. Here, we show that genetically engineered embryonic stem cell-derived microglia (ESdM) are a suitable therapeutic vehicle for neurotrophin-3 (NT3) in experimental autoimmune encephalomyelitis (EAE). The intravenously transplanted ESdM migrated into the inflammatory CNS lesions and engrafted there as microglial cells. EAE afflicted mice treated with ESdM that were genetically modified to express NT3 showed stable recovery from disease symptoms. The NT3-transduced ESdM created an anti-inflammatory cytokine milieu in the spinal cord and promoted neuronal sprouting. Furthermore, mice treated with NT3-transduced ESdM showed less axonal injury and reduced demyelination. Thus, genetically modified ESdM represent a suitable tool to introduce therapeutic neuroprotective and repair-promoting proteins into the CNS in neuroinflammatory diseases.

Over the past 70years, diffuse axonal injury (DAI) has emerged as one of the most common and important pathological features of traumatic brain injury (TBI). Axons in the white matter appear to be especially vulnerable to injury due to the mechanical loading of the brain during TBI. As such, DAI has been found in all severities of TBI and may represent a key pathologic substrate of mild TBI (concussion). Pathologically, DAI encompasses a spectrum of abnormalities from primary mechanical breaking of the axonal cytoskeleton, to transport interruption, swelling and proteolysis, through secondary physiological changes. Depending on the severity and extent of injury, these changes can manifest acutely as immediate loss of consciousness or confusion and persist as coma and/or cognitive dysfunction. In addition, recent evidence suggests that TBI may induce long-term neurodegenerative processes, such as insidiously progressive axonal pathology. Indeed, axonal degeneration has been found to continue even years after injury in humans, and appears to play a role in the development of Alzheimer's disease-like pathological changes. Here we review the current understanding of DAI as a uniquely mechanical injury, its histopathological identification, and its acute and chronic pathogenesis following TBI., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There are no established biomarkers for mild traumatic brain injury (mTBI), in part because post-concussive symptoms (PCS) are subjective and conventional imaging is typically unremarkable. To test whether diffuse axonal abnormalities quantified with three-dimensional (3D) proton magnetic resonance spectroscopic imaging (¹H-MRSI) correlated with patients' PCS, we retrospectively studied 26 mTBI patients (mean Glasgow Coma Scale [GCS] score of 14.7), 18- to 56-year-olds and 13 controls three to 55 days post-injury. All were scanned at 3 Tesla with T1- and T2-weighted MRI and 3D ¹H-MRSI (480 voxels over 360 cm³, ∼30% of the brain). On scan day, patients completed a symptom questionnaire, and those who indicated at least one of the most common subacute mTBI symptoms (headache, dizziness, sleep disturbance, memory deficits, blurred vision) were grouped as PCS-positive. Global gray matter and white matter (GM/WM) absolute concentrations of N-acetylaspartate (NAA), choline (Cho), creatine (Cr) and myo-inositol (mI) in PCS-positive and PCS-negative patients were compared to age- and gender-matched controls using two-way analysis of variance. The results showed that the PCS-negative group (n=11) and controls (n=8) did not differ in any GM or WM metabolite level. The PCS-positive patients (n=15) had lower WM NAA than the controls (n=12; 7.0 ± 0.6 versus 7.9 ± 0.5mM; p=0.0007). Global WM NAA, therefore, showed sensitivity to the TBI sequelae associated with common PCS in patients with mostly normal neuroimaging, as well as GCS scores. This suggests a potential biomarker role in a patient population in which objective measures of injury and symptomatology are currently lacking.

Increased intracellular calcium ([Ca(2+)](i)) is a key pathological mechanism involved in secondary neuronal injury and cell death due to diffuse axonal injury (DAI). To date, this increased [Ca(2+)](i) is believed to be mainly caused by dysfunction of voltage-gated sodium channels and mechanoporation of the plasma membrane. Store-operated calcium entry (SOCE) is another source of Ca(2+) influx, and stromal interaction molecule 1 (STIM1) is considered as a sensor and a regulator of SOCE. In this study, we established a DAI in vivo model in rats by lateral head rotation. Using immunohistochemistry, real-time RT-PCR and Western blot, we investigated STIM1 expression levels in the cerebral cortex of rats after lateral head rotational injury. Results revealed notably high STIM1 expression in neurons in the early stages (within 24 h) of DAI. STIM1 began to increase at 6 h post-injury (PI) peaked at 12 h PI, and then gradually decreased. At 2 days PI, STIM1 expression in the injury group showed no significant difference compared with that of the control group. These results indicate that abnormal SOCE may participate in Ca(2+) overload of neurons in the early stages after DAI via enhanced STIM1 expression., (Copyright © 2012 Elsevier B.V. All rights reserved.)

DAI is a leading cause of the patient's death or lasting vegetable state following severe TBI, and up to now the detailed mechanism of axonal injury after head trauma is still unclear. Inflammatory responses have been proved to be an important mechanism of neural injury after TBI. However, most of these studies are concerned with focal cerebral injury following head trauma. In contrast to focal injury, studies on the inflammatory reaction following DAI are only beginning. And in this article, we aimed to review such studies. From the studies reviewed, immune response cells would become reactive around the sites of axonal injury after DAI. Besides, the concentrations of several important inflammatory factors, such as IL-1 family, IL-6 and TNF-ɑ, increased after DAI as well, which implies the participation of inflammatory responses. It can be concluded that inflammatory responses probably participate in the neural injury in DAI, but at present the study of inflammatory responses following DAI is still limited and the clear effects of inflammatory response on axonal injury remain to be more explored.

The aim of this study is to assess the effects of alcohol on traumatic brain injury by using diffusion tensor imaging (DTI) and evaluate aquaporin-4(AQP4) expression changes in rat brainstems following acute alcohol intoxication with diffuse axonal injury (DAI). We further investigated the correlation between the AQP4 expression and DTI in the brain edema. Eighty-five rats were imaged before and after injury at various stages. DTI was used to measure brainstem apparent diffusion coefficient (ADC) and fractional anisotropy (FA), with immunostaining being used to determine AQP4 expression. After acute alcoholism with DAI, ADC values of the brainstem first decreased within 6 h and then elevated. FA values began to decline by 1 h, reaching a minimum at 24 h after trauma. There was a negative correlation between ADC values and brainstem AQP4 expression at 6 h and positive correlation at 6 h to 24 h. Changes of ADC and FA values in DAI with acute alcoholism indicate the effects of ethanol on brain edema and the severity of axonal injury. The correlations between ADC values and the brainstem AQP4 expression at different time points suggest that AQP4 expression follows an adaptative profile to the severity of brain edema.

Traumatic axonal injury (TAI) plays a major role in the development of neurological impairments after traumatic brain injury (TBI), but it is commonly difficult to evaluate it precisely and early with conventional histological biomarkers, especially when the patients experience short-term survival after TBI. Diffusion tensor imaging (DTI) has shown some promise in detecting TAI, but longitudinal studies on the compromised white matter with DTI at early time points (≤72 h) following impact acceleration TBI are still absent. In the present study, rats were subjected to the Marmarou model and imaged with DTI at 3, 12, 24, and 72 h (n = 5 each) post-injury. Using a region-of-interest-based approach, the regions of interest including the corpus callosum, bilateral external capsule, internal capsule, and pyramidal tract were studied. Two DTI parameters, fraction anisotropy and axial diffusivity, were significantly reduced from 3 to 72 h in each region after trauma, corresponding to the gradient of axonal damage demonstrated by immunohistochemical staining of β-amyloid precursor protein and neurofilament light chain. Remarkably, DTI changes predicted the approximate time in the acute phase following TBI. These results indicate that the temporal profiles of diffusion parameters in DTI may be able to provide a tool for early diagnosis of TAI following impact acceleration TBI.

Since mild traumatic brain injury (mTBI) often leads to neurological symptoms even without clinical MRI findings, our goal was to test whether diffuse axonal injury is quantifiable with multivoxel proton MR spectroscopic imaging ((1)H-MRSI). T1- and T2-weighted MRI images and three-dimensional (1)H-MRSI (480 voxels over 360 cm(3), about 30 % of the brain) were acquired at 3 T from 26 mTBI patients (mean Glasgow Coma Scale score 14.7, 18-56 years old, 3-55 days after injury) and 13 healthy matched contemporaries as controls. The N-acetylaspartate (NAA), choline (Cho), creatine (Cr) and myo-inositol (mI) concentrations and gray-matter/white-matter (GM/WM) and cerebrospinal fluid fractions were obtained in each voxel. Global GM and WM absolute metabolic concentrations were estimated using linear regression, and patients were compared with controls using two-way analysis of variance. In patients, mean NAA, Cr, Cho and mI concentrations in GM (8.4 ± 0.7, 6.9 ± 0.6, 1.3 ± 0.2, 5.5 ± 0.6 mM) and Cr, Cho and mI in WM (4.8 ± 0.5, 1.4 ± 0.2, 4.6 ± 0.7 mM) were not different from the values in controls. The NAA concentrations in WM, however, were significantly lower in patients than in controls (7.2 ± 0.8 vs. 7.7 ± 0.6 mM, p = 0.0125). The Cho and Cr levels in WM of patients were positively correlated with time since mTBI. This (1)H-MRSI approach allowed us to ascertain that early mTBI sequelae are (1) diffuse (not merely local), (2) neuronal (not glial), and (3) in the global WM (not GM). These findings support the hypothesis that, similar to more severe head trauma, mTBI also results in diffuse axonal injury, but that dysfunction rather than cell death dominates shortly after injury.

A single traumatic brain injury is associated with an increased risk of dementia and, in a proportion of patients surviving a year or more from injury, the development of hallmark Alzheimer's disease-like pathologies. However, the pathological processes linking traumatic brain injury and neurodegenerative disease remain poorly understood. Growing evidence supports a role for neuroinflammation in the development of Alzheimer's disease. In contrast, little is known about the neuroinflammatory response to brain injury and, in particular, its temporal dynamics and any potential role in neurodegeneration. Cases of traumatic brain injury with survivals ranging from 10 h to 47 years post injury (n = 52) and age-matched, uninjured control subjects (n = 44) were selected from the Glasgow Traumatic Brain Injury archive. From these, sections of the corpus callosum and adjacent parasaggital cortex were examined for microglial density and morphology, and for indices of white matter pathology and integrity. With survival of ≥3 months from injury, cases with traumatic brain injury frequently displayed extensive, densely packed, reactive microglia (CR3/43- and/or CD68-immunoreactive), a pathology not seen in control subjects or acutely injured cases. Of particular note, these reactive microglia were present in 28% of cases with survival of >1 year and up to 18 years post-trauma. In cases displaying this inflammatory pathology, evidence of ongoing white matter degradation could also be observed. Moreover, there was a 25% reduction in the corpus callosum thickness with survival >1 year post-injury. These data present striking evidence of persistent inflammation and ongoing white matter degeneration for many years after just a single traumatic brain injury in humans. Future studies to determine whether inflammation occurs in response to or, conversely, promotes white matter degeneration will be important. These findings may provide parallels for studying neurodegenerative disease, with traumatic brain injury patients serving as a model for longitudinal investigations, in particular with a view to identifying potential therapeutic interventions.