White matter (WM) disruption and atrophy is a consequence of traumatic brain injury (TBI) and contributes to persisting cognitive impairment. An increased expression of the myelin-associated axonal outgrowth inhibitor Nogo-A and oligodendrocyte pathology might be negatively associated with postinjury WM changes. Here, we analyzed brain tissue from severe TBI patients, obtained by surgical decompression in the early postinjury phase and postmortem brain tissue of long-term TBI survivors and observed an increased number of Nogo-A+ cells in WM tracts such as the corpus callosum (CC). Likewise, the number of Nogo-A+ cells in the CC was increased from day 7 postinjury to 6 months postinjury (mpi) following central fluid percussion injury (cFPI) in mice. In addition, the number of Olig2+ cells in the CC and capsula externa remained constant, while the numbers of Olig2+/CC1+ and GST-π+ mature oligodendrocytes declined throughout the observation time of 18 months. A significantly lower number of Olig2+/CC1+ cells was found in cFPI mice compared to controls at 18 mpi. Persistent vulnerability of oligodendrocytes in combination with dynamic alterations of Nogo-A expression may have implications for the WM atrophy and insufficient recovery observed after TBI.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality, not least in the elderly. The incidence of aged TBI patients has increased dramatically during the last decades. High age is a highly negative prognostic factor in TBI, and pharmacological treatment options are lacking. We used the controlled cortical impact (CCI) TBI model in 23-month-old male and female mice and analyzed the effect of post-injury treatment with 7,8 dihydroxyflavone (7,8-DHF), a brain-derived neurotrophic factor (BDNF)-mimetic compound, on white matter pathology. Following CCI or sham injury, mice received subcutaneous 7,8-DHF injections (5 ​mg/kg) 30 ​min post-injury and were sacrificed on 2, 7 or 14 days post-injury (dpi) for histological and immunofluorescence analyses. Histological assessment with Luxol Fast Blue (LFB)/Cresyl Violet stain showed that administration of 7,8-DHF resulted in preserved white matter tissue at 2 and 7 dpi with no difference in cortical tissue loss at all investigated time points. Treatment with 7,8-DHF led to reduced axonal swellings at 2 and 7 dpi, as visualized by SMI-31 (Neurofilament Heavy Chain) immunofluorescence, and reduced number of TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labelling)/CC1-positive mature oligodendrocytes at 2 dpi in the perilesional white matter. Post-injury proliferation of Platelet-derived Growth Factor Receptor (PDGFRα)-positive oligodendodrocyte progenitor cells was not altered by 7,8-DHF. Our results suggest that 7,8-DHF can attenuate white matter pathology by mitigating axonal injury and oligodendrocyte death in the aged mouse brain following TBI. These data argue that further exploration of 7,8-DHF towards clinical use is warranted.

Senescence is a negative prognostic factor for outcome and recovery following traumatic brain injury (TBI). TBI-induced white matter injury may be partially due to oligodendrocyte demise. We hypothesized that the regenerative capacity of oligodendrocyte precursor cells (OPCs) declines with age. To test this hypothesis, the regenerative capability of OPCs in young [(10 weeks +/- 2 (SD)] and aged [(62 weeks +/- 10 (SD)] mice was studied in mice subjected to central fluid percussion injury (cFPI), a TBI model causing widespread white matter injury. Proliferating OPCs were assessed by immunohistochemistry for the proliferating cell nuclear antigen (PCNA) marker and labeled by 5-ethynyl-2'-deoxyuridine (EdU) administered daily through intraperitoneal injections (50 mg/kg) from day 2 to day 6 after cFPI. Proliferating OPCs were quantified in the corpus callosum and external capsule on day 2 and 7 post-injury (dpi). The number of PCNA/Olig2-positive and EdU/Olig2-positive cells were increased at 2dpi (p < .01) and 7dpi (p < .01), respectively, in young mice subjected to cFPI, changes not observed in aged mice. Proliferating Olig2+/Nestin+ cells were less common (p < .05) in the white matter of brain-injured aged mice, without difference in proliferating Olig2+/PDGFR alpha+ cells, indicating a diminished proliferation of progenitors with different spatial origin. Following TBI, co-staining for EdU/CC1/Olig2 revealed a reduced number of newly generated mature oligodendrocytes in the white matter of aged mice when compared to the young, brain-injured mice (p < .05). We observed an age-related decline of oligodendrogenesis following experimental TBI that may contribute to the worse outcome of elderly patients following TBI.

IntroductionTraumatic brain injury to thalamo-cortical pathways is associated with posttraumatic morbidity. Diffuse mechanical forces to white matter tracts and deep grey matter regions induce an inflammatory response and vascular damage resulting in progressive neurodegeneration. Pro-inflammatory cytokines, including interleukin-1β (IL-1β), may contribute to the link between nflammation and the injured capillary network after TBI. This study investigates whether IL-1β is a key contributor to capillary alterations and changes in pericyte coverage in the thalamus and cortex after TBI.MethodsAnimals were subjected to central fluid percussion injury (cFPI), a model of TBI causing widespread axonal and vascular pathology, or sham injury and randomized to receive a neutralizing anti-IL-1β or a control, anti-cyclosporin A antibody, at 30 min post-injury. Capillary length and pericyte coverage of cortex and thalamus were analyzed by immunohistochemistry at 2- and 7-days post-injury.Results and ConclusionOur results show that early post-injury attenuation of IL-1β dependent inflammatory signaling prevents capillary damage by increasing pericyte coverage in the thalamus.

Cerebral protein profiling in traumatic brain injury (TBI) is needed to better comprehend secondary injury pathways. Cerebral microdialysis (CMD), in combination with the proximity extension assay (PEA) technique, has great potential in this field. By using PEA, we have previously screened >500 proteins from CMD samples collected from TBI patients. In this study, we customized a PEA panel prototype of 21 selected candidate protein biomarkers, involved in inflammation (13), neuroplasticity/-repair (six), and axonal injury (two). The aim was to study their temporal dynamics and relation to age, structural injury, and clinical outcome. Ten patients with severe TBI and CMD monitoring, who were treated in the Neurointensive Care Unit, Uppsala University Hospital, Sweden, were included. Hourly CMD samples were collected for up to 7 days after trauma and analyzed with the 21-plex PEA panel. Seventeen of the 21 proteins from the CMD sample analyses showed significantly different mean levels between days. Early peaks (within 48?h) were noted with interleukin (IL)-1?, IL-6, IL-8, granulocyte colony-stimulating factor, transforming growth factor alpha, brevican, junctional adhesion molecule B, and neurocan. C-X-C motif chemokine ligand 10 peaked after 3 days. Late peaks (>5 days) were noted with interleukin-1 receptor antagonist (IL-1ra), monocyte chemoattractant protein (MCP)-2, MCP-3, urokinase-type plasminogen activator, Dickkopf-related protein 1, and DRAXIN. IL-8, neurofilament heavy chain, and TAU were biphasic. Age (above/below 22 years) interacted with the temporal dynamics of IL-6, IL-1ra, vascular endothelial growth factor, MCP-3, and TAU. There was no association between radiological injury (Marshall grade) or clinical outcome (Extended Glasgow Outcome Scale) with the protein expression pattern. The PEA method is a highly sensitive molecular tool for protein profiling from cerebral tissue in TBI. The novel TBI dedicated 21-plex panel showed marked regulation of proteins belonging to the inflammation, plasticity/repair, and axonal injury families. The method may enable important insights into complex injury processes on a molecular level that may be of value in future efforts to tailor pharmacological TBI trials to better address specific disease processes and optimize timing of treatments.

Cerebellar dysfunction is commonly observed following traumatic brain injury (TBI). While direct impact to the cerebellum by TBI is rare, cerebellar pathology may be caused by indirect injury via cortico-cerebellar pathways. To address the hypothesis that degeneration of Purkinje cells (PCs), which constitute the sole output from the cerebellum, is linked to long-range axonal injury and demyelination, we used the central fluid percussion injury (cFPI) model of widespread traumatic axonal injury in mice. Compared to controls, TBI resulted in early PC loss accompanied by alterations in the size of pinceau synapses and levels of non-phosphorylated neurofilament in PCs. A combination of vDISCO tissue clearing technique and immunohistochemistry for vesicular glutamate transporter type 2 show that diffuse TBI decreased mossy and climbing fiber synapses on PCs. At 2 days post-injury, numerous axonal varicosities were found in the cerebellum supported by fractional anisotropy measurements using 9.4 T MRI. The disruption and demyelination of the cortico-cerebellar circuits was associated with poor performance of brain-injured mice in the beam-walk test. Despite a lack of direct input from the injury site to the cerebellum, these findings argue for novel long-range mechanisms causing Purkinje cell injury that likely contribute to cerebellar dysfunction after TBI.

Unilateral traumatic brain injury and stroke result in asymmetric postural and motor deficits including contralateral hemiplegia and hemiparesis. In animals, a localized unilateral brain injury recapitulates the human upper motor neuron syndrome in the formation of hindlimb postural asymmetry with contralesional limb flexion and the asymmetry of hindlimb nociceptive withdrawal reflexes. The current view is that these effects are developed due to aberrant activity of motor pathways that descend from the brain into the spinal cord. These pathways and their target spinal circuits may be regulated by local neurohormonal systems that may also mediate effects of brain injury. Here, we evaluate if a unilateral traumatic brain injury induces hindlimb postural asymmetry, a model of postural deficits, and if this asymmetry is spinally encoded and mediated by the endogenous opioid system in rats. A unilateral right-sided controlled cortical impact, a model of clinical focal traumatic brain injury was centred over the sensorimotor cortex and was observed to induce hindlimb postural asymmetry with contralateral limb flexion. The asymmetry persisted after complete spinal cord transection, implicating local neurocircuitry in the development of the deficits. Administration of the general opioid antagonist naloxone and μ-antagonist β-funaltrexamine blocked the formation of postural asymmetry. Surprisingly, κ-antagonists nor-binaltorphimine and LY2444296 did not affect the asymmetry magnitude but reversed the flexion side; instead of contralesional (left) hindlimb flexion the ipsilesional (right) limb was flexed. The postural effects of the right-side cortical injury were mimicked in animals with intact brain via intrathecal administration of the opioid κ-agonist (2)-(trans)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide that induced hindlimb postural asymmetry with left limb flexion. The δ-antagonist naltrindole produced no effect on the contralesional (left) flexion but inhibited the formation of the ipsilesional (right) limb flexion in brain-injured rats that were treated with κ-antagonist. The effects of the antagonists were evident before and after spinal cord transection. We concluded that the focal traumatic brain injury-induced postural asymmetry was encoded at the spinal level, and was blocked or its side was reversed by administration of opioid antagonists. The findings suggest that the balance in activity of the mirror symmetric spinal neural circuits regulating contraction of the left and right hindlimb muscles is controlled by different subtypes of opioid receptors; and that this equilibrium is impaired after unilateral brain trauma through side-specific opioid mechanism.

Traumatic brain injury (TBI) increases the risk of delayed neurodegenerative processes, including Parkinson’s disease (PD). Interleukin-1beta (IL-1β), a key pro-inflammatory cytokine, may promote secondary injury development after TBI. Conversely, neutralizing IL-1β was found to improve functional recovery following experimental TBI. However, the mechanisms underlying the behavioral improvements observed by IL-1β neutralization are still poorly understood. The present study investigated the role of IL-1β on the microglia response and neuronal changes in the globus pallidus in response to diffuse TBI. Mice were subjected to sham injury or the central fluid percussion injury (cFPI) (a model of traumatic axonal injury), and were randomly administered an IL-1β neutralizing or a control antibody at 30 min post-injury. The animals were analyzed at 2, 7, or 14 days post-injury. When compared to controls, mice subjected to cFPI TBI had increased microglia activation and dopaminergic innervation in the globus pallidus, and a decreased number of parvalbumin (PV) positive interneurons in the globus pallidus. Neutralization of IL-1β attenuated the microglia activation, prevented the loss of PV+ interneurons and normalized dopaminergic fiber density in the globus pallidus of brain-injured animals. These findings argue for an important role for neuro-inflammation in the PD-like pathology observed in TBI.

Alzheimer's disease (AD) is the leading cause of dementia worldwide, affecting over 10% of the elderly population. Epidemiological evidence indicates that traumatic brain injury (TBI) is an important risk factor for developing AD later in life. However, which injury-induced processes that contribute to the disease onset remains unclear. The aim with the present study was to identify cellular processes that could link TBI to AD development, by investigating the chronic impact of two different injury models, controlled cortical impact (CCI) and midline fluid percussion injury (mFPI). The trauma was induced in 3-month-old tg-ArcSwe mice, carrying the Arctic mutation along with the Swedish mutation, and the influence of TBI on AD progression was analyzed at 12- and 24-weeks post-injury. The long-term effect of the TBI on memory deficiency, amyloid-β (Aβ) pathology, neurodegeneration and inflammation was investigated by Morris water maze, PET imaging, immunohistochemistry, and biochemical analyses. Morris water maze analysis demonstrated that mice subjected to CCI or mFPI performed significantly worse than uninjured tg-ArcSwe mice, especially at the later time point. Moreover, the injured mice showed a late upregulation of reactive gliosis, which concurred with a more pronounced Aβ pathology, compared to uninjured AD mice. Our results suggest that the delayed glial activation following TBI may be an important link between the two diseases. However, further studies in both experimental models and human TBI patients will be required to fully elucidate the reasons why TBI increases the risk of neurodegeneration.

Abstract Traumatic brain injury (TBI) is followed by secondary injury mechanisms strongly involving neuroinflammation. To monitor the complex inflammatory cascade in human TBI, we used cerebral microdialysis (MD) and multiplex proximity extension assay (PEA) technology and simultaneously measured levels of 92 protein biomarkers of inflammation in MD samples every three hours for five days in 10 patients with severe TBI under neurointensive care. One μL MD samples were incubated with paired oligonucleotide-conjugated antibodies binding to each protein, allowing quantification by real-time quantitative polymerase chain reaction. Sixty-nine proteins were suitable for statistical analysis. We found five different patterns with either early (<48 h; e.g., CCL20, IL6, LIF, CCL3), mid (48–96 h; e.g., CCL19, CXCL5, CXCL10, MMP1), late (>96 h; e.g., CD40, MCP2, MCP3), biphasic peaks (e.g., CXCL1, CXCL5, IL8) or stable (e.g., CCL4, DNER, VEGFA)/low trends. High protein levels were observed for e.g., CXCL1, CXCL10, MCP1, MCP2, IL8, while e.g., CCL28 and MCP4 were detected at low levels. Several proteins (CCL8, -19, -20, -23, CXCL1, -5, -6, -9, -11, CST5, DNER, Flt3L, and SIRT2) have not been studied previously in human TBI. Cross-correlation analysis revealed that LIF and CXCL5 may play a central role in the inflammatory cascade. This study provides a unique data set with individual temporal trends for potential inflammatory biomarkers in patients with TBI. We conclude that the combination of MD and PEA is a powerful tool to map the complex inflammatory cascade in the injured human brain. The technique offers new possibilities of protein profiling of complex secondary injury pathways.

There is still a lack of pharmacological treatment options for traumatic brain injury (TBI), the dominant cause of death and disability in persons under the age of 40 in the developed part of the world. Clinical TBI is a markedly complex disease, categorized into different subtypes that differ in their pathophysiology, treatment requirements, and long-term consequences. For successful development of novel treatment options, refined preclinical evaluation in rodent TBI models is mandatory. Since persisting cognitive deficits, impaired motor function, depression, and personality changes are common sequelae in TBI patients, preclinical models must produce clinically relevant behavioral deficits. Additionally, clinical TBI is a markedly heterogeneous disease with a severity span from immediately fatal to mild injuries with minor and passing symptoms. Ideally, a rodent TBI model should thus be adjustable in terms of injury severity. One of the most widely used rodent TBI model is the fluid percussion injury (FPI), which meets many of the criteria for a clinically relevant experimental model. The FPI technique relies on a fluid pressure pulse being transmitted into the skull cavity of the animal, allowing for a degree of brain displacement. By placing the craniectomy and the injury site either over the midline of the skull (the central FPI; cFPI) or over one hemisphere (the lateral FPI; lFPI) the injury shows either more diffuse (cFPI) or more focal (lFPI) characteristics. Although FPI has many advantages over other TBI models, including the possibility to vary important injury characteristics, the outcome after TBI may be influenced by other features such as gender, age, species, and even strain which should be considered in the design of the rodent models. In this chapter, we discuss the limitations and advantages, as well as the special considerations necessary when using the FPI model in rodents.

Background/Objectives: Motor impairment induced by traumatic brain injury (TBI) may be mediated through changes in spinal molecular systems regulating neuronal plasticity.We assessed whether a focal controlled cortical impact (CCI) TBI in the rat alters expression of the Tgfb1, c-Fos, Bdnf, and Gap43 neuroplasticity genes in lumbar spinal cord.Approach/Methods: Adult male Sprague-Dawley rats (n = 8) were subjected to a right-side CCI over the anterior sensorimotor hindlimb representation area or sham-injury (n = 8).Absolute expression levels of Tgfb1, c-Fos, Bdnf, and Gapd43 genes were measured by droplet digital PCR in ipsi-and contralesional, dorsal and ventral quadrants of the L4 and L5 spinal cord.The neuronal activity marker c-Fos was analysed by immunohistochemistry in the dorsal L4 and L5 segments.The contravs.ipsilesional expression pattern was examined as the asymmetry index, AI.Results: The Tgfb1 mRNA levels were significantly higher in the CCI vs. sham-injured rats, and in the contravs.ipsilesional dorsal domains in the CCI group.The number of c-Fos-positive cells was elevated in the L4 and L5 segments; and on the contralesional compared to the ipsilesional side in the CCI group.The c-Fos AI in the dorsal laminae was significantly increased by CCI. Conclusions:The results support the hypothesis that focal TBI induces plastic alterations in the lumbar spinal cord that may contribute to either motor recovery or maladaptive motor responses.

Neuroinflammation is a major contributor to the progressive brain injury process induced by traumatic brain injury (TBI), and may play an important role in the pathophysiology of axonal injury. The immediate neuroinflammatory cascade cannot be characterized in the human setting. Therefore, we used the midline fluid percussion injury model of diffuse TBI in rats and a novel microdialysis (MD) method providing stable diffusion-driven biomarker sampling. Immediately post-injury, bilateral amphiphilic tri-block polymer coated MD probes (100kDa cut off membrane) were inserted and perfused with Dextran 500kDa-supplemented artificial cerebrospinal fluid (CSF) to optimize protein capture. Six hourly samples were analyzed for 27 inflammatory biomarkers (9 chemokines, 13 cytokines, and 5 growth factors) using a commercial multiplex biomarker kit. TBI (n=6) resulted in a significant increase compared with sham-injured controls (n=6) for five chemokines (eotaxin/CCL11, fractalkine/CX3CL1, LIX/CXCL5, monocyte chemoattractant protein [MCP]1/CCL2, macrophage inflammatory protein [MIP]1 /CCL3), 10 cytokines (interleukin [IL]-1, IL-1, IL-4, IL-6, IL-10, IL-13, IL-17, IL-18, interferon [IFN]-, tumor necrosis factor [TNF]-), and four growth factors (epidermal growth factor [EGF], granulocyte-macrophage colony-stimulating factor [GM-CSF], leptin, vascular endothelial growth factor [VEGF]). Therefore, diffuse TBI was associated with an increased level of 18 of the 27 inflammatory biomarkers at one through six time points, during the observation period whereas the remaining 9 biomarkers were unaltered. The study shows that diffuse TBI induces an acute increase in a number of inflammatory biomarkers. The novel MD technique provides stable MD sampling suitable for further studies on the early neuroinflammatory cascade in TBI.

Fluid percussion injury (FPI) is a widely used experimental model for studying traumatic brain injury (TBI). However, little is known about how the brain mechanically responds to fluid impacts and how the mechanical pressures/strains of the brain correlate to subsequent brain damage for rodents during FPI. Hence, we developed a numerical approach to simulate FPI experiments on rats and characterize rat brain pressure/strain responses at a high resolution. A previous rat brain model was improved with a new hexahedral elements-based skull model and a new cerebrospinal fluid (CSF) layer. We validated the numerical model against experimentally measured pressures from FPI. Our results indicated that brain tissues under FPI experienced high pressures, which were slightly lower (10–20%) than input saline pressure. Interestingly, FPI was a mixed focus- and diffuse-type injury model with highest strains (12%) being concentrated in the ipsilateral cortex under the fluid-impact site and diffuse strains (5–10%) being spread to the entire brain, which was different from controlled cortical impact in which high strains decreased gradually away from the impact site.

Asymmetric Hindlimb Motor Response To Focal Traumatic Brain Injury Is Controlled By Side-Specific Opioid Mechanism

Abstract 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.

Reduced Intracranial Pressure After Treatment With Anti-Secretory Factor 16 In A Rat Model Of Traumatic Brain Injury

A Dedicated 21-Plex Pea Panel For High-Sensitive Protein Biomarker Detection Using Micro-Dialysis In Traumatic Brain Injury

A synthetic peptide with antisecretory activity, antisecretory factor (AF)-16, improves injury-related deficits in water and ion transport and decreases intracranial pressure after experimental cold lesion injury and encephalitis although its role in traumatic brain injury (TBI) is unknown. AF-16 or an inactive reference peptide was administrated intranasally 30 min following midline fluid percussion injury (mFPI; n = 52), a model of diffuse mild-moderate TBI in rats. Sham-injured (n = 14) or naïve (n = 24) animals were used as controls. The rats survived for either 48 h or 15 days post-injury. At 48 h, the animals were tested in the Morris water maze (MWM) for memory function and their brains analyzed for cerebral edema. Here, mFPI-induced brain edema compared to sham or naïve controls that was significantly reduced by AF-16 treatment (p < 0.05) although MWM performance was not altered. In the 15-day survival groups, the MWM learning and memory abilities as well as histological changes were analyzed. AF-16-treated brain-injured animals shortened both MWM latency and swim path in the learning trials (p < 0.05) and improved probe trial performance compared to brain-injured controls treated with the inactive reference peptide. A modest decrease by AF-16 on TBI-induced changes in hippocampal glial acidic fibrillary protein (GFAP) staining (p = 0.11) was observed. AF-16 treatment did not alter any other immunohistochemical analyses (degenerating neurons, beta-amyloid precursor protein (β-APP), and Olig2). In conclusion, intranasal AF-16-attenuated brain edema and enhanced visuospatial learning and memory following diffuse TBI in the rat. Intranasal administration early post-injury of a promising neuroprotective substance offers a novel treatment approach for TBI.