Moderate Fluid Percussion Injury Research Articles

Neuronal Membrane Disruption Occurs Late Following Diffuse Brain Trauma in Rats and Involves a Subpopulation of NeuN Negative Cortical Neurons.

The repercussions of traumatic brain injury (TBI) endure years following the initial insult and involve chronic impairments/disabilities. Studies indicate that these morbidities stem from diffuse pathologies, however, knowledge regarding TBI-mediated diffuse pathologies, and in particular, diffuse neuronal membrane disruption, is limited. Membrane disruption has been shown to occur acutely following injury, primarily within neurons, however, the progression of TBI-induced membrane disruption remains undefined. Therefore, the current study investigated this pathology over a longer temporal profile from 6 h to 4 w following diffuse TBI induced using the central fluid percussion injury (CFPI) model in rats. To visualize membrane disruption, animals received an intracerebroventricular infusion of tagged cell-impermeable dextran 2 h prior to experimental endpoints at 6 h, 1 d, 3 d, 1 w, 2 w, or 4 w post-CFPI. The percentage of total neurons demonstrating dextran uptake, indicative of membrane disruption, was quantified within the lateral neocortex layers V and VI from 6 h to 4 w post-injury. We found that membrane disruption displayed a biphasic pattern, where nearly half of the neurons were membrane disrupted sub-acutely, from 6 h to 3 d post-TBI. At 1 w the membrane disrupted population was dramatically reduced to levels indistinguishable from sham controls. However, by 2 and 4 w following CFPI, approximately half of the neurons analyzed displayed membrane disruption. Moreover, our data revealed that a subset of these late membrane disrupted neurons were NeuN negative (NeuN-). Correlative western blot analyses, however, revealed no difference in NeuN protein expression in the lateral neocortex at any time following injury. Furthermore, the NeuN- membrane disrupted neurons did not co-label with traditional markers of astrocytes, microglia, oligodendrocytes, or NG2 cells. Immunohistochemistry against NeuN, paired with a hematoxylin and eosin counter-stain, was performed to quantify the possibility of overall NeuN+ neuronal loss following CFPI. A NeuN- population was observed consistently in both sham and injured animals regardless of time post-injury. These data suggest that there is a consistent subpopulation of NeuN- neurons within the lateral neocortex regardless of injury and that these NeuN- neurons are potentially more vulnerable to late membrane disruption. Better understanding of membrane disruption could provide insight into the mechanisms of diffuse pathology and lead to the discovery of novel treatments for TBI.

Depletion of Microglia Attenuates Dendritic Spine Loss and Neuronal Apoptosis in the Acute Stage of Moderate Traumatic Brain Injury in Mice.

Microglia are the primary immune cells in the central nervous system and undergo significant morphological and transcriptional changes after traumatic brain injury (TBI). However, their exact contribution to the pathogenesis of TBI is still debated and remains to be elucidated. In the present study, thy-1 GFP mice received a colony-stimulating factor 1 receptor inhibitor (PLX3397) for 21 consecutive days, then were subjected to moderate fluid percussion injury (FPI). Brain samples were collected at 1 day and 3 days after FPI for flow cytometry analysis, immunofluorescence, dendrite spine quantification, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and Western blot. We found that PLX3397 treatment significantly attenuated the percentages of resident microglia and infiltrated immune cells. Depletion of microglia promoted neurite outgrowth, preserved dendritic spines and reduced total brain cell and neuronal apoptosis after FPI, which was accompanied by decreased the protein levels of endoplasmic reticulum stress marker proteins, C/EBP-homologous protein and inositol-requiring kinase 1α. Taken together, these findings suggest that microglial depletion may exert beneficial effects in the acute stage of FPI.

Impairment of pericyte-endothelium crosstalk leads to blood-brain barrier dysfunction following traumatic brain injury

The blood-brain barrier (BBB) constitutes a neurovascular unit formed by microvascular endothelial cells, pericytes, and astrocytes. Brain pericytes are important regulators of BBB integrity, permeability, and blood flow. Pericyte loss has been implicated in injury; however, how the crosstalk among pericytes, endothelial cells, and astrocytes ultimately leads to BBB dysfunction in traumatic brain injury (TBI) remains elusive. In this study, we demonstrate the importance of pericyte-endothelium interaction in maintaining the BBB function. TBI causes the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-β signaling impairment that results in loss of interaction with endothelium and leads to neurovascular dysfunction. Using in vivo mild (7 psi) and moderate (15 psi) fluid percussion injury (FPI) in mice, we demonstrate the expression of various pericyte markers including PDGFR-β, NG2 and CD13 that were significantly reduced with a subsequent reduction in the expression of various integrins; adherent junction protein, N-cadherin; gap junction protein, connexin-43; and tight junction proteins such as occludin, claudin-5, ZO-1, and JAM-a. Impairment of pericyte-endothelium interaction increases the BBB permeability to water that is marked by a significant increase in aquaporin4 expression in injured animals. Similarly, pericyte-endothelium integrity impairment in FPI animals greatly increases the permeability of small-molecular-weight sodium fluorescein and high-molecular-weight-tracer Evans blue across the BBB. In addition, the injury-inflicted animals show significantly higher levels of S100β and NSE in the blood samples compared with controls. In conclusion, our data provide an insight that brain trauma causes an early impairment of pericyte-endothelium integrity and results in BBB dysregulation that initiates pathological consequences associated with TBI.

Wogonin attenuates the deleterious effects of traumatic brain injury in anesthetized Wistar rats

Wogonin, a flavonoid (5,7-dihydroxy-8-methoxyflavone) with reported neuroprotective properties, is widely used in treating inflammatory diseases. The main goal of this study was to explore the role of wogonin in preventing deleterious cardiovascular effects of traumatic brain injury (TBI). Experiments were carried out in adult male urethane-anesthetized, artificially ventilated, Wistar rats, weighing 300–350 gm. TBI was produced by fluid percussion injury (FPI). A significant decrease in blood pressure (BP), heart rate (HR) and greater splanchnic nerve activity (GSNA), which lasted for up to 4 h, was observed after the application of moderate FPI. Intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of wogonin before and after the moderate FPI significantly attenuated the decreases in BP, HR, and GSNA elicited by FPI. Administration of wogonin also prevented the attenuation of baroreflex-induced bradycardia elicited by FPI. Based on these results, it was concluded that administration of wogonin attenuates the deleterious effects of moderate FPI.

Neutralization of Interleukin-1β following Diffuse Traumatic Brain Injury in the Mouse Attenuates the Loss of Mature Oligodendrocytes.

Traumatic brain injury (TBI) commonly results in injury to the components of the white matter tracts, causing post-injury cognitive deficits. The myelin-producing oligodendrocytes (OLs) are vulnerable to TBI, although may potentially be replaced by proliferating oligodendrocyte progenitor cells (OPCs). The cytokine interleukin-1β (IL-1β) is a key mediator of the complex inflammatory response, and when neutralized in experimental TBI, behavioral outcome was improved. To evaluate the role of IL-1β on oligodendrocyte cell death and OPC proliferation, 116 adult male mice subjected to sham injury or the central fluid percussion injury (cFPI) model of traumatic axonal injury, were analyzed at two, seven, and 14 days post-injury. At 30 min post-injury, mice were randomly administered an IL-1β neutralizing or a control antibody. OPC proliferation (5-ethynyl 2′- deoxyuridine (EdU)/Olig2 co-labeling) and mature oligodendrocyte cell loss was evaluated in injured white matter tracts. Microglia/macrophages immunohistochemistry and ramification using Sholl analysis were also evaluated. Neutralizing IL-1β resulted in attenuated cell death, indicated by cleaved caspase-3 expression, and attenuated loss of mature OLs from two to seven days post-injury in brain-injured animals. IL-1β neutralization also attenuated the early, two day post-injury increase of microglia/macrophage immunoreactivity and altered their ramification. The proliferation of OPCs in brain-injured animals was not altered, however. Our data suggest that IL-1β is involved in the TBI-induced loss of OLs and early microglia/macrophage activation, although not the OPC proliferation. Attenuated oligodendrocyte cell loss may contribute to the improved behavioral outcome observed by IL-1β neutralization in this mouse model of diffuse TBI.

The Importance of Inter-Species Variation in Traumatic Brain Injury-Induced Alterations of Microglial-Axonal Interactions.

Interactions between microglia and neuronal components are important for normal CNS function. They are also associated with neuroinflammation and many pathological processes and several studies have explored these interactions in terms of phagocytic engulfment. Much progress has also been made in understanding the consequences of chronic neuroinflammatory changes following trauma. However, little is known about acute alterations to these physical non-phagocytic microglial-neuronal interactions following traumatic brain injury (TBI), and particularly to what degree these post-injury interactions may be influenced by the animal species utilized in pre-clinical models of TBI. To investigate these problems, we evaluated the physical interactions between microglia and injured axons acutely (6 h and 1 day) following central fluid percussion injury (cFPI) in both rats and micro pigs. The physical interactions between Iba-1+ microglia and either normal MBP+ myelinated fibers or APP+ injured axonal swellings in the thalamus were assessed following injury or sham via quantitative image analysis of 3D confocal micrographs. The results indicated that the physical interactions between microglia and injured axonal swellings decreased by nearly half in rats 6 h following cFPI but was consistent with sham control at 1 day post-cFPI. This reduction was also observed in non-injured intact fibers at both timepoints following TBI in the rat. Microglial process interactions with injured axons in the micro pig, however, increased nearly 2-fold compared to interactions with intact axonal segments 1 day post-cFPI. This study shows that the species utilized for in vivo pre-clinical studies influences the manner in which microglial-axonal interactions change following TBI. These species differences can be leveraged to further our understanding of the mechanisms involved in microglial process convergence and how these neuro-immune interactions alter the progression of axonal injury following TBI.

Transient Receptor Potential Melastatin 4 Induces Astrocyte Swelling But Not Death after Diffuse Traumatic Brain Injury.

Traumatic brain injury (TBI) is a prevalent disease with significant costs. Although progress has been made in understanding the complex pathobiology of focal lesions associated with TBI, questions remain regarding the diffuse responses to injury. Expression of the transient receptor potential melastatin 4 (Trpm4) channel is linked to cytotoxic edema during hemorrhagic contusion expansion. However, little is known about Trpm4 following diffuse TBI. To explore Trpm4 expression in diffuse TBI, rats were subjected to a diffuse central fluid percussion injury (CFPI) and survived for 1.5 h to 8 weeks. The total number of Trpm4+ cells, as well as individual cellular intensity/expression of Trpm4, were assessed. Hemotoxylin and eosin (H&E) labeling was performed to evaluate cell damage/death potentially associated with Trpm4 expression following diffuse TBI. Finally, ultrastructural assessments were performed to evaluate the integrity of Trpm4+ cells and the potential for swelling associated with Trpm4 expression. Trpm4 was primarily restricted to astrocytes within the hippocampus and peaked at 6 h post-injury. While the number of Trpm4+ astrocytes returned to sham levels by 8 weeks post-CFPI, cellular intensity occurred in region-specific waves following injury. Correlative H&E assessments demonstrated little evidence of hippocampal damage, suggesting that Trpm4 expression by astrocytes does not precipitate cell death following diffuse TBI. Additionally, ultrastructural assessments showed Trpm4+ astrocytes exhibited twice the soma size compared with Trpm4- astrocytes, indicating that astrocyte swelling is associated with Trpm4 expression. This study provides a foundation for future investigations into the role of Trpm4 in astrocyte swelling and edema following diffuse TBI.

Genetic and Histological Alterations Reveal Key Role of Prostaglandin Synthase and Cyclooxygenase 1 and 2 in Traumatic Brain Injury–Induced Neuroinflammation in the Cerebral Cortex of Rats Exposed to Moderate Fluid Percussion Injury

After the initial insult in traumatic brain injury (TBI), secondary neurodegeneration occurs that is intimately associated with neuroinflammation. Prostaglandin (PG) synthases and cyclooxygenase (COX) 1 and 2 may contribute to inflammation in the brain. Temporal and spatial expression features of PG and COX1 and 2 following trauma may guide the development of antineuroinflammation strategies. Here, we examined PG synthase signaling and COX1 and 2 gene expression levels and COX-1- and 2-positive cell types and their temporal localization in TBI-induced brain in an effort to reveal their participation in the disease’s evolving neuroinflammation. Using brain samples from the cerebral cortex of rats subjected to TBI model of lateral moderate fluid percussion injury (FPI), we sought to characterize the temporal (subacute TBI) and spatial (lateral cortical lesion) brain alterations accompanying the disease progression. Temporal gene expression changes of PG synthase signaling were compared between sham-operated and TBI-treated rats using microarray pathway analysis. Moreover, we examined COX1 and 2 expression patterns and their intracellular distribution in sham-operated and TBI-treated rats by immunohistochemistry. After FPI, COX1 and 2 gene expression levels, and PGE2 synthase increased while PGD2 synthase decreased, suggesting that PGE2 and PGD2 afforded contraindicative effects of inflammation and anti-inflammation, respectively. Immunohistochemical analyses showed that both COX1 and COX2 increased in a time-dependent manner in the brain, specifically in degenerating neurons of the cortex. Interestingly, the expression of COX cell type was cell-specific, in that COX1 was particularly increased in degenerating neurons while COX2 was expressed in macrophages. In view of the dynamic temporal and spatial expression of PG, COX1 and 2 gene expression and localization in the injured brain regulating PG synthase and COX1 and 2 activity will require a careful disease-specific tailoring of treatments to abrogate the neuroinflammation-plagued secondary cell death due to TBI.

7,8-Dihydroxyflavone facilitates the action exercise to restore plasticity and functionality: Implications for early brain trauma recovery

Metabolic dysfunction accompanying traumatic brain injury (TBI) severely impairs the ability of injured neurons to comply with functional demands. This limits the success of rehabilitative strategies by compromising brain plasticity and function, and highlights the need for early interventions to promote energy homeostasis. We sought to examine whether the TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF) normalizes brain energy deficits and reestablishes more normal patterns of functional connectivity, while enhancing the effects of exercise during post-TBI period. Moderate fluid percussion injury (FPI) was performed and 7,8-DHF (5mg/kg, i.p.) was administered in animals subjected to FPI that either had access to voluntary wheel running for 7days after injury or were sedentary. Compared to sham-injured controls, TBI resulted in reduced hippocampal activation of the BDNF receptor TrkB and associated CREB, reduced levels of plasticity markers GAP-43 and Syn I, as well as impaired memory as indicated by the Barnes maze task. While 7,8-DHF treatment and exercise individually mitigated TBI-induced effects, administration of 7,8-DHF concurrently with exercise facilitated memory performance and augmented levels of markers of cell energy metabolism viz., PGC-1α, COII and AMPK. In parallel to these findings, resting-state functional MRI (fMRI) acquired at 2weeks after injury showed that 7,8-DHF with exercise enhanced hippocampal functional connectivity, and suggests 7,8-DHF and exercise to promote increases in functional connectivity. Together, these findings indicate that post-injury 7,8-DHF treatment promotes enhanced levels of cell metabolism, synaptic plasticity in combination with exercise increases in brain circuit function that facilitates greater physical rehabilitation after TBI.

Mild Traumatic Brain Injury Induces Structural and Functional Disconnection of Local Neocortical Inhibitory Networks via Parvalbumin Interneuron Diffuse Axonal Injury.

Diffuse axonal injury (DAI) plays a major role in cortical network dysfunction posited to cause excitatory/inhibitory imbalance after mild traumatic brain injury (mTBI). Current thought holds that white matter (WM) is uniquely vulnerable to DAI. However, clinically diagnosed mTBI is not always associated with WM DAI. This suggests an undetected neocortical pathophysiology, implicating GABAergic interneurons. To evaluate this possibility, we used mild central fluid percussion injury to generate DAI in mice with Cre-driven tdTomato labeling of parvalbumin (PV) interneurons. We followed tdTomato+ profiles using confocal and electron microscopy, together with patch-clamp analysis to probe for DAI-mediated neocortical GABAergic interneuron disruption. Within 3 h post-mTBI tdTomato+ perisomatic axonal injury (PSAI) was found across somatosensory layers 2-6. The DAI marker amyloid precursor protein colocalized with GAD67 immunoreactivity within tdTomato+ PSAI, representing the majority of GABAergic interneuron DAI. At 24 h post-mTBI, we used phospho-c-Jun, a surrogate DAI marker, for retrograde assessments of sustaining somas. Via this approach, we estimated DAI occurs in ~9% of total tdTomato+ interneurons, representing ~14% of pan-neuronal DAI. Patch-clamp recordings of tdTomato+ interneurons revealed decreased inhibitory transmission. Overall, these data show that PV interneuron DAI is a consistent and significant feature of experimental mTBI with important implications for cortical network dysfunction.

Diffuse traumatic brain injury in the mouse induces a transient proliferation of oligodendrocyte progenitor cells in injured white matter tracts.

Injury to the white matter may lead to impaired neuronal signaling and is commonly observed following traumatic brain injury (TBI). Although endogenous repair of TBI-induced white matter pathology is limited, oligodendrocyte progenitor cells (OPCs) may be stimulated to proliferate and regenerate functionally myelinating oligodendrocytes. Even though OPCs are present throughout the adult brain, little is known about their proliferative activity following axonal injury caused by TBI. We hypothesized that central fluid percussion injury (cFPI) in mice, a TBI model causing wide-spread axonal injury, results in OPC proliferation. Proliferation of OPCs was evaluated in 27 cFPI mice using 5-ethynyl-2'-deoxyuridine (EdU) labeling and a cell proliferation assay at 2 (n = 9), 7 (n = 8) and 21 (n = 10) days post injury (dpi). Sham-injured mice (n = 14) were used as controls. OPC proliferation was quantified by immunohistochemistry using the OPC markers NG2 and Olig2 in several white matter loci including the corpus callosum, external capsule, fimbriae, the internal capsule and cerebral peduncle. The number of EdU/DAPI/Olig2-positive cells were increased in the cFPI group compared to sham-injured animals at 7 days post-injury (dpi; p≤0.05) in the majority of white matter regions. The OPC proliferation had subsided by 21 dpi. The number of EdU/DAPI/NG2 cells was also increase at 7 dpi in the external capsule and fimbriae. These results suggest that traumatic axonal injury in the mouse induces a transient proliferative response of residing OPCs. These proliferating OPCs may replace dead oligodendrocytes and contribute to remyelination, which needs evaluation in future studies.

Adaptive reorganization of retinogeniculate axon terminals in dorsal lateral geniculate nucleus following experimental mild traumatic brain injury

The pathologic process in traumatic brain injury marked by delayed axonal loss, known as diffuse axonal injury (DAI), leads to partial deafferentation of neurons downstream of injured axons. This process is linked to persistent visual dysfunction following mild traumatic brain injury (mTBI), however, examination of deafferentation in humans is impossible with current technology. To investigate potential reorganization in the visual system following mTBI, we utilized the central fluid percussion injury (cFPI) mouse model of mTBI. We report that in the optic nerve of adult male C57BL/6J mice, axonal projections of retinal ganglion cells (RGCs) to their downstream thalamic target, dorsal lateral geniculate nucleus (dLGN), undergo DAI followed by scattered, widespread axon terminals loss within the dLGN at 4days post-injury. However, at 10days post-injury, significant reorganization of RGC axon terminals was found, suggestive of an adaptive neuroplastic response. While these changes persisted at 20days post-injury, the RGC axon terminal distribution did not recovery fully to sham-injury levels. Our studies also revealed that following DAI, the segregation of axon terminals from ipsilateral and contralateral eye projections remained consistent with normal adult mouse distribution. Lastly, our examination of the shell and core of dLGN suggested that different RGC subpopulations may vary in their susceptibility to injury or in their contribution to reorganization following injury. Collectively, these findings support the premise that subcortical axon terminal reorganization may contribute to recovery following mTBI, and that different neural phenotypes may vary in their contribution to this reorganization despite exposure to the same injury.

Aging with Traumatic Brain Injury: Effects of Age at Injury on Behavioral Outcome following Diffuse Brain Injury in Rats

Development and aging are influenced by external factors with the potential to impact health throughout the life span. Traumatic brain injury (TBI) can initiate and sustain a lifetime of physical and mental health symptoms. Over 1.7 million TBIs occur annually in the USA alone, with epidemiology suggesting a higher incidence for young age groups. Additionally, increasing life spans mean more years to age with TBI. While there is ongoing research of experimental pediatric and adult TBI, few studies to date have incorporated animal models of pediatric, adolescent, and adult TBI to understand the role of age at injury across the life span. Here, we explore repeated behavioral performance between rats exposed to diffuse TBI at five different ages. Our aim was to follow neurological morbidities across the rodent life span with respect to age at injury. A single cohort of male Sprague-Dawley rats (n = 69) was received at postnatal day (PND) 10. Subgroups of this cohort (n = 11-12/group) were subjected to a single moderate midline fluid percussion injury at age PND 17, PND 35, 2 months, 4 months, or 6 months. A control group of naïve rats (n = 12) was assembled from this cohort. The entire cohort was assessed for motor function by beam walk at 1.5, 3, 5, and 7 months of age. Anxiety-like behavior was assessed with the open field test at 8 months of age. Cognitive performance was assessed using the novel object location task at 8, 9, and 10 months of age. Depression-like behavior was assessed using the forced swim test at 10 months of age. Age at injury and time since injury differentially influenced motor, cognitive, and affective behavioral outcomes. Motor and cognitive deficits occurred in rats injured at earlier developmental time points, but not in rats injured in adulthood. In contrast, rats injured during adulthood showed increased anxiety-like behavior compared to uninjured control rats. A single diffuse TBI did not result in chronic depression-like behaviors or changes in body weight among any groups. The interplay of age at injury and aging with an injury are translationally important factors that influence behavioral performance as a quality of life metric. More complete understanding of these factors can direct rehabilitative efforts and personalized medicine for TBI survivors.

Knockout of Cyclophilin-D Provides Partial Amelioration of Intrinsic and Synaptic Properties Altered by Mild Traumatic Brain Injury.

Mitochondria are central to cell survival and Ca2+ homeostasis due to their intracellular buffering capabilities. Mitochondrial dysfunction resulting in mitochondrial permeability transition pore (mPTP) opening has been reported after mild traumatic brain injury (mTBI). Cyclosporine A provides protection against the mPTP opening through its interaction with cyclophilin-D (CypD). A recent study has found that the extent of axonal injury after mTBI was diminished in neocortex in cyclophilin-D knockout (CypDKO) mice. Here we tested whether this CypDKO could also provide protection from the increased intrinsic and synaptic neuronal excitability previously described after mTBI in a mild central fluid percussion injury mice model. CypDKO mice were crossed with mice expressing yellow fluorescent protein (YFP) in layer V pyramidal neurons in neocortex to create CypDKO/YFP-H mice. Whole cell patch clamp recordings from axotomized (AX) and intact (IN) YFP+ layer V pyramidal neurons were made 1 and 2 days after sham or mTBI in slices from CypDKO/YFP-H mice. Both excitatory post synaptic currents (EPSCs) recorded in voltage clamp and intrinsic cellular properties, including action potential (AP), afterhyperpolarization (AHP), and depolarizing after potential (DAP) characteristics recorded in current clamp were evaluated. There was no significant difference between sham and mTBI for either spontaneous or miniature EPSC frequency, suggesting that CypDKO ameliorates excitatory synaptic abnormalities. There was a partial amelioration of intrinsic properties altered by mTBI. Alleviated were the increased slope of the AP frequency vs. injected current plot, the increased AP, AHP and DAP amplitudes. Other properties that saw a reversal that became significant in the opposite direction include the current rheobase and AP overshoot. The AP threshold remained depolarized and the input resistance remained increased in mTBI compared to sham. Additional altered properties suggest that the CypDKO likely has a direct effect on membrane properties, rather than producing a selective reduction of the effects of mTBI. These results suggest that inhibiting CypD after TBI is an effective strategy to reduce synaptic hyperexcitation, making it a continued target for potential treatment of network abnormalities.

Nicotinamide Treatment in Traumatic Brain Injury: Operation Brain Trauma Therapy.

Nicotinamide (vitamin B3) was the first drug selected for cross-model testing by the Operation Brain Trauma Therapy (OBTT) consortium based on a compelling record of positive results in pre-clinical models of traumatic brain injury (TBI). Adult male Sprague-Dawley rats were exposed to either moderate fluid percussion injury (FPI), controlled cortical impact injury (CCI), or penetrating ballistic-like brain injury (PBBI). Nicotinamide (50 or 500 mg/kg) was delivered intravenously at 15 min and 24 h after injury with subsequent behavioral, biomarker, and histopathological outcome assessments. There was an intermediate effect on balance beam performance with the high (500 mg/kg) dose in the CCI model, but no significant therapeutic benefit was detected on any other motor task across the OBTT TBI models. There was an intermediate benefit on working memory with the high dose in the FPI model. A negative effect of the low (50 mg/kg) dose, however, was observed on cognitive outcome in the CCI model, and no cognitive improvement was observed in the PBBI model. Lesion volume analysis showed no treatment effects after either FPI or PBBI, but the high dose of nicotinamide resulted in significant tissue sparing in the CCI model. Biomarker assessments included measurements of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase-1 (UCH-L1) in blood at 4 or 24 h after injury. Negative effects (both doses) were detected on biomarker levels of GFAP after FPI and on biomarker levels of UCH-L1 after PBBI. The high dose of nicotinamide, however, reduced GFAP levels after both PBBI and CCI. Overall, our results showed a surprising lack of benefit from the low dose nicotinamide. In contrast, and partly in keeping with the literature, some benefit was achieved with the high dose. The marginal benefits achieved with nicotinamide, however, which appeared sporadically across the TBI models, has reduced enthusiasm for further investigation by the OBTT Consortium.

Diffuse traumatic axonal injury in mice induces complex behavioural alterations that are normalized by neutralization of interleukin-1β.

Widespread traumatic axonal injury (TAI) results in brain network dysfunction, which commonly leads to persisting cognitive and behavioural impairments following traumatic brain injury (TBI). TBI induces a complex neuroinflammatory response, frequently located at sites of axonal pathology. The role of the pro-inflammatory cytokine interleukin (IL)-1β has not been established in TAI. An IL-1β-neutralizing or a control antibody was administered intraperitoneally at 30 min following central fluid percussion injury (cFPI), a mouse model of widespread TAI. Mice subjected to moderate cFPI (n = 41) were compared with sham-injured controls (n = 20) and untreated, naive mice (n = 9). The anti-IL-1β antibody reached the target brain regions in adequate therapeutic concentrations (up to ~30 μg/brain tissue) at 24 h post-injury in both cFPI (n = 5) and sham-injured (n = 3) mice, with lower concentrations at 72 h post-injury (up to ~18 μg/g brain tissue in three cFPI mice). Functional outcome was analysed with the multivariate concentric square field (MCSF) test at 2 and 9 days post-injury, and the Morris water maze (MWM) at 14-21 days post-injury. Following TAI, the IL-1β-neutralizing antibody resulted in an improved behavioural outcome, including normalized behavioural profiles in the MCSF test. The performance in the MWM probe (memory) trial was improved, although not in the learning trials. The IL-1β-neutralizing treatment did not influence cerebral ventricle size or the number of microglia/macrophages. These findings support the hypothesis that IL-1β is an important contributor to the processes causing complex cognitive and behavioural disturbances following TAI.

Autophagy Modulation by Lanthionine Ketimine Ethyl Ester Improves Long-Term Outcome after Central Fluid Percussion Injury in the Mouse.

Diffuse axonal injury is recognized as a progressive and long-term consequence of traumatic brain injury. Axonal injury can have sustained negative consequences on neuronal functions such as anterograde and retrograde transport and cellular processes such as autophagy that depend on cytoarchitecture and axon integrity. These changes can lead to somatic atrophy and an inability to repair and promote plasticity. Obstruction of the autophagic process has been noted after brain injury, and rapamycin, a drug used to stimulate autophagy, has demonstrated positive effects in brain injury models. The optimization of drugs to promote beneficial autophagy without negative side effects could be used to attenuate traumatic brain injury and promote improved outcome. Lanthionine ketimine ethyl ester, a bioavailable derivative of a natural sulfur amino acid metabolite, has demonstrated effects on autophagy both in vitro and in vivo. Thirty minutes after a moderate central fluid percussion injury and throughout the survival period, lanthionine ketimine ethyl ester was administered, and mice were subsequently evaluated for learning and memory impairments and biochemical and histological changes over a 5-week period. Lanthionine ketimine ethyl ester, which we have shown previously to modulate autophagy markers and alleviate pathology and slow cognitive decline in the 3 × TgAD mouse model, spared cognition and pathology after central fluid percussion injury through a mechanism involving autophagy modulation.

Simvastatin Treatment in Traumatic Brain Injury: Operation Brain Trauma Therapy.

Simvastatin, the fourth drug selected for testing by Operation Brain Trauma Therapy (OBTT), is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor used clinically to reduce serum cholesterol. In addition, simvastatin has demonstrated potent antineuroinflammatory and brain edema reducing effects and has shown promise in promoting functional recovery in pre-clinical models of traumatic brain injury (TBI). The purpose of this study was to assess the potential neuroprotective effects of oral administration of simvastatin on neurobehavioral, biomarker, and histopathological outcome measures compared across three pre-clinical TBI animal models. Adult male Sprague-Dawley rats were exposed to either moderate fluid percussion injury (FPI), controlled cortical impact injury (CCI), or penetrating ballistic-like brain injury (PBBI). Simvastatin (1 or 5 mg/kg) was delivered via oral gavage at 3 h post-injury and continued once daily out to 14 days post-injury. Results indicated an intermediate beneficial effect of simvastatin on motor performance on the gridwalk (FPI), balance beam (CCI), and rotarod tasks (PBBI). No significant therapeutic benefit was detected, however, on cognitive outcome across the OBTT TBI models. In fact, Morris water maze (MWM) performance was actually worsened by treatment in the FPI model and scored full negative points for low dose in the MWM latency and swim distance to locate the hidden platform. A detrimental effect on cortical tissue loss was also seen in the FPI model, and there were no benefits on histology across the other models. Simvastatin also produced negative effects on circulating glial fibrillary acidic protein biomarker outcomes that were evident in the FPI and PBBI models. Overall, the current findings do not support the beneficial effects of simvastatin administration over 2 weeks post-TBI using the oral route of administration and, as such, it will not be further pursued by OBTT.

Cognitive deficits develop 1 month after diffuse brain injury and are exaggerated by microglia-associated reactivity to peripheral immune challenge

Traumatic brain injury (TBI) elicits immediate neuroinflammatory events that contribute to acute cognitive, motor, and affective disturbance. Despite resolution of these acute complications, significant neuropsychiatric and cognitive issues can develop and progress after TBI. We and others have provided novel evidence that these complications are potentiated by repeated injuries, immune challenges and stressors. A key component to this may be increased sensitization or priming of glia after TBI. Therefore, our objectives were to determine the degree to which cognitive deterioration occurred after diffuse TBI (moderate midline fluid percussion injury) and ascertain if glial reactivity induced by an acute immune challenge potentiated cognitive decline 30days post injury (dpi). In post-recovery assessments, hippocampal-dependent learning and memory recall were normal 7dpi, but anterograde learning was impaired by 30dpi. Examination of mRNA and morphological profiles of glia 30dpi indicated a low but persistent level of inflammation with elevated expression of GFAP and IL-1β in astrocytes and MHCII and IL-1β in microglia. Moreover, an acute immune challenge 30dpi robustly interrupted memory consolidation specifically in TBI mice. These deficits were associated with exaggerated microglia-mediated inflammation with amplified (IL-1β, CCL2, TNFα) and prolonged (TNFα) cytokine/chemokine expression, and a marked reactive morphological profile of microglia in the CA3 of the hippocampus. Collectively, these data indicate that microglia remain sensitized 30dpi after moderate TBI and a secondary inflammatory challenge elicits robust microglial reactivity that augments cognitive decline. Statement of SignificanceTraumatic brain injury (TBI) is a major risk factor in development of neuropsychiatric problems long after injury, negatively affecting quality of life. Mounting evidence indicates that inflammatory processes worsen with time after a brain injury and are likely mediated by glia. Here, we show that primed microglia and astrocytes developed in mice 1month following moderate diffuse TBI, coinciding with cognitive deficits that were not initially evident after injury. Additionally, TBI-induced glial priming may adversely affect the ability of glia to appropriately respond to immune challenges, which occur regularly across the lifespan. Indeed, we show that an acute immune challenge augmented microglial reactivity and cognitive deficits. This idea may provide new avenues of clinical assessments and treatments following TBI.

Rodent Models of Traumatic Brain Injury: Methods and Challenges.

Traumatic brain injury (TBI) has been named the most complex disease in the most complex organ of the body. It is the most common cause of death and disability in the Western world in people <40 years old and survivors commonly suffer from persisting cognitive deficits, impaired motor function, depression and personality changes. TBI may vary in severity from uniformly fatal to mild injuries with rapidly resolving symptoms and without doubt, it is a markedly heterogeneous disease. Its different subtypes differs in their pathophysiology, treatment options and long-term consequences and to date, there are no pharmacological treatments with proven clinical benefit available to TBI patients. To enable development of novel treatment options for TBI, clinically relevant animal models are needed. Due to their availability and low costs, numerous rodent models have been developed which have substantially contributed to our current understanding of the pathophysiology of TBI. The most common animal models used in laboratories worldwide are likely the controlled cortical impact (CCI) model, the central and lateral fluid percussion injury (FPI) models, and weight drop/impact acceleration (I/A) models. Each of these models has inherent advantages and disadvantages; these need to be thoroughly considered when selecting the rodent TBI model according to the hypothesis and design of the study. Since TBI is not one disease, refined animal models must take into account the clinical features and complexity of human TBI. To enhance the possibility of establishing preclinical efficacy of a novel treatment, the preclinical use of several different experimental models is encouraged as well as varying the species, gender, and age of the animal. In this chapter, the methods, limitations, and challenges of the CCI and FPI models of TBI used in rodents are described.