Dissecting Genetic Mechanisms of Differential Locomotion, Depression, and Allodynia after Spinal Cord Injury in Three Mouse Strains.

Strain differences have been reported for motor behaviors, and only a subset of spinal cord injury (SCI) patients develop neuropathic pain, implicating genetic or genomic contribution to this condition. Here, we evaluated neuropsychiatric behaviors in A/J, BALB/c, and C57BL/6 male mice and tested genetic or genomic alterations following SCI. A/J and BALB/c naive mice showed significantly less locomotor activity and greater anxiety-like behavior than C57BL/6 mice. Although SCI elicited locomotor dysfunction, C57BL/6 and A/J mice showed the best and the worst post-traumatic recovery, respectively. Mild (m)-SCI mice showed deficits in gait dynamics. All moderate/severe SCI mice exhibited similar degrees of anxiety/depression. mSCI in BALB/c and A/J mice resulted in depression, whereas C57BL/6 mice did not exhibit depression. mSCI mice had significantly lower mechanical thresholds than their controls, indicating high cutaneous hypersensitivity. C57BL/6, but not A/J and BLAB/c mice, showed significantly lower heat thresholds than their controls. C57BL/6 mice exhibited spontaneous pain. RNAseq showed that genes in immune responses and wound healing were upregulated, although A/J mice showed the largest increase. The cell cycle and the truncated isoform of trkB genes were robustly elevated in SCI mice. Thus, different genomics are associated with post-traumatic recovery, underscoring the likely importance of genetic factors in SCI.

Bi-directional neuro-immune dysfunction after chronic experimental brain injury.

BACKGROUND: It is well established that traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function and that systemic immune changes contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. METHODS: To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham (i.e., 90 days post-surgery) congenic donor mice into otherwise healthy, age-matched, irradiated CD45.2 C57BL/6 (WT) hosts. Immune changes were evaluated by flow cytometry, multiplex ELISA, and NanoString technology. Moderate-to-severe TBI was induced by controlled cortical impact injury and neurological function was measured using a battery of behavioral tests. RESULTS: TBI induced chronic alterations in the transcriptome of BM lineage-c-Kit+Sca1+ (LSK+) cells in C57BL/6 mice, including modified epigenetic and senescence pathways. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI at 8 weeks and 8 months post-reconstitution showed that longer reconstitution periods (i.e., time post-injury) were associated with increased microgliosis and leukocyte infiltration. Pre-treatment with a senolytic agent, ABT-263, significantly improved behavioral performance of aged C57BL/6 mice at baseline, although it did not attenuate neuroinflammation in the acutely injured brain. CONCLUSIONS: TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in hematopoiesis, innate immunity, and neurological function, as well as altered sensitivity to subsequent brain injury.

Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction.

Obesity increases the morbidity and mortality of traumatic brain injury (TBI). Detailed analyses of transcriptomic changes in the brain and adipose tissue were performed to elucidate the interactive effects between high-fat diet-induced obesity (DIO) and TBI. Adult male mice were fed a high-fat diet (HFD) for 12 weeks prior to experimental TBI and continuing after injury. High-throughput transcriptomic analysis using Nanostring panels of the total visceral adipose tissue (VAT) and cellular components in the brain, followed by unsupervised clustering, principal component analysis, and IPA pathway analysis were used to determine shifts in gene expression patterns and molecular pathway activity. Cellular populations in the cortex and hippocampus, as well as in VAT, during the chronic phase after combined TBI-HFD showed amplification of central and peripheral microglia/macrophage responses, including superadditive changes in selected gene expression signatures and pathways. Furthermore, combined TBI and HFD caused additive dysfunction in Y-Maze, Novel Object Recognition (NOR), and Morris water maze (MWM) cognitive function tests. These novel data suggest that HFD-induced obesity and TBI can independently prime and support the development of altered states in brain microglia and VAT, including the disease-associated microglia/macrophage (DAM) phenotype observed in neurodegenerative disorders. The interaction between HFD and TBI promotes a shift toward chronic reactive microglia/macrophage transcriptomic signatures and associated pro-inflammatory disease-altered states that may, in part, underlie the exacerbation of cognitive deficits. Thus, targeting of HFD-induced reactive cellular phenotypes, including in peripheral adipose tissue immune cell populations, may serve to reduce microglial maladaptive states after TBI, attenuating post-traumatic neurodegeneration and neurological dysfunction.

The brain-bone marrow axis and its implications for chronic traumatic brain injury.

Traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function which contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham congenic donor mice into otherwise healthy, age-matched, irradiated hosts. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI showed that longer reconstitution periods were associated with increased microgliosis and leukocyte infiltration. Thus, TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in innate immunity and neurological function, as well as altered sensitivity to subsequent brain injury.

Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction.

Obesity increases the morbidity and mortality of traumatic brain injury (TBI). We performed a detailed analysis of transcriptomic changes in the brain and adipose tissue to examine the interactive effects between high-fat diet-induced obesity (DIO) and TBI in relation to central and peripheral inflammatory pathways, as well as neurological function. Adult male mice were fed a high-fat diet (HFD) for 12 weeks prior to experimental TBI and continuing after injury. Combined TBI and HFD resulted in additive dysfunction in the Y-Maze, novel object recognition (NOR), and Morris water maze (MWM) cognitive function tests. We also performed high-throughput transcriptomic analysis using Nanostring panels of cellular compartments in the brain and total visceral adipose tissue (VAT), followed by unsupervised clustering, principal component analysis, and IPA pathway analysis to determine shifts in gene expression programs and molecular pathway activity. Analysis of cellular populations in the cortex and hippocampus as well as in visceral adipose tissue during the chronic phase after combined TBI-HFD showed amplification of central and peripheral microglia/macrophage responses, including superadditive changes in select gene expression signatures and pathways. These data suggest that HFD-induced obesity and TBI can independently prime and support the development of altered states in brain microglia and visceral adipose tissue macrophages, including the disease-associated microglia/macrophage (DAM) phenotype observed in neurodegenerative disorders. The interaction between HFD and TBI promotes a shift toward chronic reactive microglia/macrophage transcriptomic signatures and associated pro-inflammatory disease-altered states that may, in part, underlie the exacerbation of cognitive deficits. Targeting of HFD-induced reactive cellular phenotypes, including in peripheral adipose tissue macrophages, may serve to reduce microglial maladaptive states after TBI, attenuating post-traumatic neurodegeneration and neurological dysfunction.

Sexually dimorphic extracellular vesicle responses after chronic spinal cord injury are associated with neuroinflammation and neurodegeneration in the aged brain.

BACKGROUND: Medical advances have made it increasingly possible for spinal cord injury (SCI) survivors to survive decades after the insult. But how SCI affects aging changes and aging impacts the injury process have received limited attention. Extracellular vesicles (EVs) are recognized as critical mediators of neuroinflammation after CNS injury, including at a distance from the lesion site. We have previously shown that SCI in young male mice leads to robust changes in plasma EV count and microRNA (miR) content. Here, our goal was to investigate the impact of biological sex and aging on EVs and brain after SCI. METHODS: Young adult age-matched male and female C57BL/6 mice were subjected to SCI. At 19 months post-injury, total plasma EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA). EVs miR cargo was examined using the Fireplex® assay. The transcriptional changes in the brain were assessed by a NanoString nCounter Neuropathology panel and validated by Western blot (WB) and flow cytometry (FC). A battery of behavioral tests was performed for assessment of neurological function. RESULTS: Transcriptomic changes showed a high number of changes between sham and those with SCI. Sex-specific changes were found in transcription networks related to disease association, activated microglia, and vesicle trafficking. FC showed higher microglia and myeloid counts in the injured tissue of SCI/Female compared to their male counterparts, along with higher microglial production of ROS in both injured site and the brain. In the latter, increased levels of TNF and mitochondrial membrane potential were seen in microglia from SCI/Female. WB and NTA revealed that EV markers are elevated in the plasma of SCI/Male. Particle concentration in the cortex increased after injury, with SCI/Female showing higher counts than SCI/Male. EVs cargo analysis revealed changes in miR content related to injury and sex. Behavioral testing confirmed impairment of cognition and depression at chronic time points after SCI in both sexes, without significant differences between males and females. CONCLUSIONS: Our study is the first to show sexually dimorphic changes in brain after very long-term SCI and supports a potential sex-dependent EV-mediated mechanism that contributes to SCI-induced brain changes.

Age-related changes in plasma extracellular vesicles influence neuroinflammation in the brain and neurological outcome after traumatic spinal cord injury.

Approximately 20% of all spinal cord injuries (SCI) occur in persons aged 65 years or older. Longitudinal, population-based studies showed that SCI is a risk factor for dementia. However, little research has addressed the potential mechanisms of SCI-mediated neurological impairment in the elderly. We compared young adult and aged C57BL/6 male mice subjected to contusion SCI, using a battery of neurobehavioral tests. Locomotor function showed greater impairment in aged mice, which was correlated with reduced, spared spinal cord white matter and increased lesion volume. At 2 months post-injury, aged mice displayed worse performance in cognitive and depressive-like behavioral tests. Transcriptomic analysis identified activated microglia and dysregulated autophagy as the most significantly altered pathways by both age and injury. Flow cytometry demonstrated increased myeloid and lymphocyte infiltration at both the injury site and brain of aged mice. SCI in aged mice was associated with altered microglial function and dysregulated autophagy involving both microglia and brain neurons. Altered plasma extracellular vesicles (EVs) responses were found in aged mice after acute SCI. EV-microRNA cargos were also significantly altered by aging and injury, which were associated with neuroinflammation and autophagy dysfunction. In cultured microglia, astrocytes, and neurons, plasma EVs from aged SCI mice, at a lower concentration comparable to those of young adult SCI mice, induced the secretion of pro-inflammatory cytokines CXCL2 and IL-6, and increased caspase3 expression. Together, these findings suggest that age alters the EVs pro-inflammatory response to SCI, potentially contributing to worse neuropathological and functional outcomes.

Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration.

Lipofuscin is an autofluorescent (AF) pigment formed by lipids and misfolded proteins, which accumulates in postmitotic cells with advanced age. Here, we immunophenotyped microglia in the brain of old C57BL/6 mice (>18 months old) and demonstrate that in comparison to young mice, one-third of old microglia are AF, characterized by profound changes in lipid and iron content, phagocytic activity, and oxidative stress. Pharmacological depletion of microglia in old mice eliminated the AF microglia following repopulation and reversed microglial dysfunction. Age-related neurological deficits and neurodegeneration after traumatic brain injury (TBI) were attenuated in old mice lacking AF microglia. Furthermore, increased phagocytic activity, lysosomal burden, and lipid accumulation in microglia persisted for up to 1 year after TBI, were modified by APOE4 genotype, and chronically driven by phagocyte-mediated oxidative stress. Thus, AF may reflect a pathological state in aging microglia associated with increased phagocytosis of neurons and myelin and inflammatory neurodegeneration that can be further accelerated by TBI.

Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice.

Elderly patients with traumatic brain injury (TBI) have greater mortality and poorer outcomes than younger individuals. The extent to which old age alters long-term recovery and chronic microglial activation after TBI is unknown, and evidence for therapeutic efficacy in aged mice is sorely lacking. The present study sought to identify potential inflammatory mechanisms underlying age-related outcomes late after TBI. Controlled cortical impact was used to induce moderate TBI in young and old male C57BL/6 mice. At 12 weeks post-injury, aged mice exhibited higher mortality, poorer functional outcomes, larger lesion volumes, and increased microglial activation. Transcriptomic analysis identified age- and TBI-specific gene changes consistent with a disease-associated microglial signature in the chronically injured brain, including those involved with complement, phagocytosis, and autophagy pathways. Dysregulation of phagocytic and autophagic function in microglia was accompanied by increased neuroinflammation in old mice. As proof-of-principle that these pathways have functional importance, we administered an autophagic enhancer, trehalose, in drinking water continuously for 8 weeks after TBI. Old mice treated with trehalose showed enhanced functional recovery and reduced microglial activation late after TBI compared to the sucrose control group. Our data indicate that microglia undergo chronic changes in autophagic regulation with both normal aging and TBI that are associated with poorer functional outcome. Enhancing autophagy may therefore be a promising clinical therapeutic strategy for TBI, especially in older patients.

Sexual dimorphism in neurological function after SCI is associated with disrupted neuroinflammation in both injured spinal cord and brain.

Whereas human spinal cord injury (SCI) is more common in men, the prevalence is growing in women. However, little is known about the effect of biological sex on brain dysfunction and injury mechanisms. To model the highest per capita rate of injury (ages between 16 and 30 years old) in humans, in the present study, young adult or a young/middle-aged male and female C57BL/6 mice were subjected to moderate contusion SCI. When mice were injured at 10-12-week-old, transcriptomic analysis of inflammation-related genes and flow cytometry revealed a more aggressive neuroinflammatory profile in male than females following 3 d SCI, ostensibly driven by sex-specific changes myeloid cell function rather than cell number. Female mice were generally more active at baseline, as evidenced by greater distance traveled in the open field. After SCI, female mice had more favorable locomotor function than male animals. At 13 weeks post-injury, male mice showed poor performance in cognitive and depressive-like behavioral tests, while injured female mice showed fewer deficits in these tasks. However, when injured at 6 months old followed by 8 months post-injury, male mice had considerably less inflammatory activation compared with female animals despite having similar or worse outcomes in affective, cognitive, and motor tasks. Collectively, these findings indicate that sex differences in functional outcome after SCI are associated with the age at onset of injury, as well as disrupted neuroinflammation not only at the site of injury but also in remote brain regions. Thus, biological sex should be considered when designing new therapeutic agents.

Bidirectional Brain-Systemic Interactions and Outcomes After TBI.

Traumatic brain injury (TBI) is a debilitating disorder associated with chronic progressive neurodegeneration and long-term neurological decline. Importantly, there is now substantial and increasing evidence that TBI can negatively impact systemic organs, including the pulmonary, gastrointestinal (GI), cardiovascular, renal, and immune system. Less well appreciated, until recently, is that such functional changes can affect both the response to subsequent insults or diseases, as well as contribute to chronic neurodegenerative processes and long-term neurological outcomes. In this review, we summarize evidence showing bidirectional interactions between the brain and systemic organs following TBI and critically assess potential underlying mechanisms.

Acute colitis during chronic experimental traumatic brain injury in mice induces dysautonomia and persistent extraintestinal, systemic, and CNS inflammation with exacerbated neurological deficits.

BACKGROUND: Disruptions of brain-gut axis have been implicated in the progression of a variety of gastrointestinal (GI) disorders and central nervous system (CNS) diseases and injuries, including traumatic brain injury (TBI). TBI is a chronic disease process characterized by persistent secondary injury processes which can be exacerbated by subsequent challenges. Enteric pathogen infection during chronic TBI worsened cortical lesion volume; however, the pathophysiological mechanisms underlying the damaging effects of enteric challenge during chronic TBI remain unknown. This preclinical study examined the effect of intestinal inflammation during chronic TBI on associated neurobehavioral and neuropathological outcomes, systemic inflammation, and dysautonomia. METHODS: Dextran sodium sulfate (DSS) was administered to adult male C57BL/6NCrl mice 28 days following craniotomy (Sham) or TBI for 7 days to induce intestinal inflammation, followed by a return to normal drinking water for an additional 7 to 28 days for recovery; uninjured animals (Naïve) served as an additional control group. Behavioral testing was carried out prior to, during, and following DSS administration to assess changes in motor and cognitive function, social behavior, and mood. Electrocardiography was performed to examine autonomic balance. Brains were collected for histological and molecular analyses of injury lesion, neurodegeneration, and neuroinflammation. Blood, colons, spleens, mesenteric lymph nodes (mLNs), and thymus were collected for morphometric analyses and/or immune characterization by flow cytometry. RESULTS: Intestinal inflammation 28 days after craniotomy or TBI persistently induced, or exacerbated, respectively, deficits in fine motor coordination, cognition, social behavior, and anxiety-like behavior. Behavioral changes were associated with an induction, or exacerbation, of hippocampal neuronal cell loss and microglial activation in Sham and TBI mice administered DSS, respectively. Acute DSS administration resulted in a sustained systemic immune response with increases in myeloid cells in blood and spleen, as well as myeloid cells and lymphocytes in mesenteric lymph nodes. Dysautonomia was also induced in Sham and TBI mice administered DSS, with increased sympathetic tone beginning during DSS administration and persisting through the first recovery week. CONCLUSION: Intestinal inflammation during chronic experimental TBI causes a sustained systemic immune response and altered autonomic balance that are associated with microglial activation, increased neurodegeneration, and persistent neurological deficits.

Enhanced Akt/GSK-3ß/CREB signaling mediates the anti-inflammatory actions of mGluR5 positive allosteric modulators in microglia and following traumatic brain injury in male mice.

We have previously shown that treatment with a mGluR5 positive allosteric modulator (PAM) is neuroprotective after experimental traumatic brain injury (TBI), limiting post-traumatic neuroinflammation by reducing pro-inflammatory microglial activation and promoting anti-inflammatory and neuroprotective responses. However, the specific molecular mechanisms governing this anti-inflammatory shift in microglia remain unknown. Here we show that the mGluR5 PAM, VU0360172 (VuPAM), regulates microglial inflammatory responses through activation of Akt, resulting in the inhibition of GSK-3ß. GSK-3ß regulates the phosphorylation of CREB, thereby controlling the expression of inflammation-related genes and microglial plasticity. The anti-inflammatory action of VuPAM in microglia is reversed by inhibiting Akt/GSK-3ß/CREB signaling. Using a well-characterized TBI model and CX3CR1gfp/+ mice to visualize microglia in vivo, we demonstrate that VuPAM enhances Akt/GSK-3ß/CREB signaling in the injured cortex, as well as anti-inflammatory microglial markers. Furthermore, in situ analysis revealed that GFP + microglia in the cortex of VuPAM-treated TBI mice co-express pCREB and the anti-inflammatory microglial phenotype marker YM1. Taken together, our data show that VuPAM decreases pro-inflammatory microglial activation by modulating Akt/GSK-3ß/CREB signaling. These findings serve to clarify the potential neuroprotective mechanisms of mGluR5 PAM treatment after TBI, and suggest novel therapeutic targets for post-traumatic neuroinflammation. Cover Image for this issue: https://doi.org/10.1111/jnc.15048.

Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury.

Acidosis is among the least studied secondary injury mechanisms associated with neurotrauma. Acute decreases in brain pH correlate with poor long-term outcome in patients with traumatic brain injury (TBI), however, the temporal dynamics and underlying mechanisms are unclear. As key drivers of neuroinflammation, we hypothesized that microglia directly regulate acidosis after TBI, and thereby, worsen neurological outcomes. Using a controlled cortical impact model in adult male mice we demonstrate that intracellular pH in microglia and extracellular pH surrounding the lesion site are significantly reduced for weeks after injury. Microglia proliferation and production of reactive oxygen species (ROS) were also increased during the first week, mirroring the increase in extracellular ROS levels seen around the lesion site. Microglia depletion by a colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622, markedly decreased extracellular acidosis, ROS production, and inflammation in the brain after injury. Mechanistically, we identified that the voltage-gated proton channel Hv1 promotes oxidative burst activity and acid extrusion in microglia. Compared to wildtype controls, microglia lacking Hv1 showed reduced ability to generate ROS and extrude protons. Importantly, Hv1-deficient mice exhibited reduced pathological acidosis and inflammation after TBI, leading to long-term neuroprotection and functional recovery. Our data therefore establish the microglial Hv1 proton channel as an important link that integrates inflammation and acidosis within the injury microenvironment during head injury.

Spinal cord injury alters microRNA and CD81+ exosome levels in plasma extracellular nanoparticles with neuroinflammatory potential.

Extracellular vesicles (EVs) have been implicated mechanistically in the pathobiology of neurodegenerative disorders, including central nervous system injury. However, the role of EVs in spinal cord injury (SCI) has received limited attention to date. Moreover, technical limitations related to EV isolation and characterization methods can lead to misleading or contradictory findings. Here, we examined changes in plasma EVs after mouse SCI at multiple timepoints (1d, 3d, 7d, 14d) using complementary measurement techniques. Plasma EVs isolated by ultracentrifugation (UC) were decreased at 1d post-injury, as shown by nanoparticle tracking analysis (NTA), and paralleled an overall reduction in total plasma extracellular nanoparticles. Western blot (WB) analysis of UC-derived plasma EVs revealed increased expression of the tetraspanin exosome marker, CD81, between 1d and 7d post-injury. To substantiate these findings, we performed interferometric and fluorescence imaging of single, tetraspanin EVs captured directly from plasma with ExoView®. Consistent with WB, we observed significantly increased plasma CD81+ EV count and cargo at 1d post-injury. The majority of these tetraspanin EVs were smaller than 50 nm based on interferometry and were insufficiently resolved by flow cytometry-based detection. At the injury site, there was enhanced expression of EV biogenesis proteins that were also detected in EVs directly isolated from spinal cord tissue by WB. Surface expression of tetraspanins CD9 and CD63 increased in multiple cell types at the injury site; however, astrocyte CD81 expression uniquely decreased, as demonstrated by flow cytometry. UC-isolated plasma EV microRNA cargo was also significantly altered at 1d post-injury with changes similar to that reported in EVs released by astrocytes after inflammatory stimulation. When injected into the lateral ventricle, plasma EVs from SCI mice increased both pro- and anti-inflammatory gene as well as reactive astrocyte gene expression in the brain cortex. These studies provide the first detailed characterization of plasma EV dynamics after SCI and suggest that plasma EVs may be involved in posttraumatic brain inflammation.

Delayed microglial depletion after spinal cord injury reduces chronic inflammation and neurodegeneration in the brain and improves neurological recovery in male mice.

Neuropsychological deficits, including impairments in learning and memory, occur after spinal cord injury (SCI). In experimental SCI models, we and others have reported that such changes reflect sustained microglia activation in the brain that is associated with progressive neurodegeneration. In the present study, we examined the effect of pharmacological depletion of microglia on posttraumatic cognition, depressive-like behavior, and brain pathology after SCI in mice. Methods: Young adult male C57BL/6 mice were subjected to moderate/severe thoracic spinal cord contusion. Microglial depletion was induced with the colony-stimulating factor 1 receptor (CSF1R) antagonist PLX5622 administered starting either 3 weeks before injury or one day post-injury and continuing through 6 weeks after SCI. Neuroinflammation in the injured spinal cord and brain was assessed using flow cytometry and NanoString technology. Neurological function was evaluated using a battery of neurobehavioral tests including motor function, cognition, and depression. Lesion volume and neuronal counts were quantified by unbiased stereology. Results: Flow cytometry analysis demonstrated that PLX5622 pre-treatment significantly reduced the number of microglia, as well as infiltrating monocytes and neutrophils, and decreased reactive oxygen species production in these cells from injured spinal cord at 2-days post-injury. Post-injury PLX5622 treatment reduced both CD45int microglia and CD45hi myeloid counts at 7-days. Following six weeks of PLX5622 treatment, there were substantial changes in the spinal cord and brain transcriptomes, including those involved in neuroinflammation. These alterations were associated with improved neuronal survival in the brain and neurological recovery. Conclusion: These findings indicate that pharmacological microglia-deletion reduces neuroinflammation in the injured spinal cord and brain, improving recovery of cognition, depressive-like behavior, and motor function.

Mithramycin selectively attenuates DNA-damage-induced neuronal cell death.

DNA damage triggers cell death mechanisms contributing to neuronal loss and cognitive decline in neurological disorders, including traumatic brain injury (TBI), and as a side effect of chemotherapy. Mithramycin, which competitively targets chromatin-binding sites of specificity protein 1 (Sp1), was used to examine previously unexplored neuronal cell death regulatory mechanisms via rat primary neurons in vitro and after TBI in mice (males). In primary neurons exposed to DNA-damage-inducing chemotherapy drugs in vitro we showed that DNA breaks sequentially initiate DNA-damage responses, including phosphorylation of ATM, H2AX and tumor protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent intrinsic apoptosis. Mithramycin was highly neuroprotective in DNA-damage-dependent neuronal cell death, inhibiting chemotherapeutic-induced cell death cascades downstream of ATM and p53 phosphorylation/activation but upstream of p53-induced expression of pro-apoptotic molecules. Mithramycin reduced neuronal upregulation of BH3-only proteins and mitochondrial dysfunction, attenuated caspase-3/7 activation and caspase substrates' cleavage, and limited c-Jun activation. Chromatin immunoprecipitation indicated that mithramycin attenuates Sp1 binding to pro-apoptotic gene promoters without altering p53 binding suggesting it acts by removing cofactors required for p53 transactivation. In contrast, the DNA-damage-independent neuronal death models displayed caspase initiation in the absence of p53/BH3 activation and were not protected even when mithramycin reduced caspase activation. Interestingly, experimental TBI triggers a multiplicity of neuronal death mechanisms. Although markers of DNA-damage/p53-dependent intrinsic apoptosis are detected acutely in the injured cortex and are attenuated by mithramycin, these processes may play a reduced role in early neuronal death after TBI, as caspase-dependent mechanisms are repressed in mature neurons while other, mithramycin-resistant mechanisms are active. Our data suggest that Sp1 is required for p53-mediated transactivation of neuronal pro-apoptotic molecules and that mithramycin may attenuate neuronal cell death in conditions predominantly involving DNA-damage-induced p53-dependent intrinsic apoptosis.

Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.

Dementia, Depression, and Associated Brain Inflammatory Mechanisms after Spinal Cord Injury.

Evaluation of the chronic effects of spinal cord injury (SCI) has long focused on sensorimotor deficits, neuropathic pain, bladder/bowel dysfunction, loss of sexual function, and emotional distress. Although not well appreciated clinically, SCI can cause cognitive impairment including deficits in learning and memory, executive function, attention, and processing speed; it also commonly leads to depression. Recent large-scale longitudinal population-based studies indicate that patients with isolated SCI (without concurrent brain injury) are at a high risk of dementia associated with substantial cognitive impairments. Yet, little basic research has addressed potential mechanisms for cognitive impairment and depression after injury. In addition to contributing to disability in their own right, these changes can adversely affect rehabilitation and recovery and reduce quality of life. Here, we review clinical and experimental work on the complex and varied responses in the brain following SCI. We also discuss potential mechanisms responsible for these less well-examined, important SCI consequences. In addition, we outline the existing and developing therapeutic options aimed at reducing SCI-induced brain neuroinflammation and post-injury cognitive and emotional impairments.

Longitudinal Assessment of Sensorimotor Function after Controlled Cortical Impact in Mice: Comparison of Beamwalk, Rotarod, and Automated Gait Analysis Tests.

Traumatic brain injury (TBI) patients are reported to experience long-term sensorimotor dysfunction, with gait deficits evident up to 2 years after the initial brain trauma. Experimental TBI including rodent models of penetrating ballistic-like brain injury and severe controlled cortical impact (CCI) can induce impairments in static and dynamic gait parameters. It is reported that the majority of deficits in gait-related parameters occur during the acute phase post-injury, as functional outcomes return toward baseline levels at chronic time points. In the present study, we carried out a longitudinal analysis of static, temporal and dynamic gait patterns following moderate-level CCI in adult male C57Bl/6J mice using the automated gait analysis apparatus, CatWalk. For comparison, we also performed longitudinal assessment of fine-motor coordination and function in CCI mice using more traditional sensorimotor behavioral tasks such as the beamwalk and accelerating rotarod tasks. We determined that longitudinal CatWalk analysis did not detect TBI-induced deficits in static, temporal, or dynamic gait parameters at acute or chronic time points. In contrast, the rotarod and beamwalk tasks showed that CCI mice had significant motor function impairments as demonstrated by deficits in balance and fine-motor coordination through 28 days post-injury. Stereological analysis confirmed that CCI produced a significant lesion in the parietal cortex at 28 days post-injury. Overall, these findings demonstrate that CatWalk analysis of gait parameters is not useful for assessment of long-term sensorimotor dysfunction after CCI, and that more traditional neurobehavioral tests should be used to quantify acute and chronic deficits in sensorimotor function.