Non-resolving neuroinflammation regulates axon regeneration in chronic spinal cord injury.

Chronic spinal cord injury (SCI) lesions retain increased densities of microglia and macrophages. In acute SCI, macrophages induce growth cone collapse, facilitate axon retraction away from lesion boundaries, as well as play a key role in orchestrating the growth-inhibitory glial scar. Little is known about the role of sustained inflammation in chronic SCI, or whether chronic inflammation affects repair and regeneration. We performed transcriptional analysis using the Nanostring Neuropathology panel to characterize the resolution of inflammation into chronic SCI, to characterize the chronic SCI microenvironment, as well as to identify spinal cord responses to macrophage depletion and repopulation using the CSF1R inhibitor, PLX-5622. We determined the ability for macrophage depletion and repopulation to augment axon growth into chronic lesions both with and without regenerative stimulation using neuronal-specific PTEN knockout (PTEN-KO). PTEN-KO was delivered with spinal injections of retrogradely transported adeno associated viruses (AAVrg's). Both transcriptional analyses and immunohistochemistry revealed the ability for PLX-5622 to significantly deplete inflammation around and within chronic SCI lesions, with a return to pre-depleted inflammatory densities after treatment removal. Neuronal-specific transcripts were significantly elevated in mice after inflammatory repopulation, but no significant effects were observed with macrophage depletion alone. Axon densities significantly increased within the lesion after PLX-5622 treatment with a more consistent effect observed in mice with inflammatory repopulation. PTEN-KO did not further increase axon densities within the lesion beyond effects induced by PLX-5622. We identified that PLX-5622 increased axon densities within the lesion that are histologically identified as 5-HT+and CGRP+, both of which are not robustly transduced by AAVrg's. Our work identified that increased macrophage/microglia densities in the chronic SCI environment may be actively retained by homeostatic mechanisms likely affiliated with a sustained elevated expression of CSF1 and other chemokines. Finally, we identify a novel role of sustained inflammation as a prospective barrier to axon regeneration in chronic SCI.

PTEN knockout using retrogradely transported AAVs transiently restores locomotor abilities in both acute and chronic spinal cord injury.

Restoring function in chronic stages of spinal cord injury (SCI) has often been met with failure or reduced efficacy when regenerative strategies are delayed past the acute or sub-acute stages of injury. Restoring function in the chronically injured spinal cord remains a critical challenge. We found that a single injection of retrogradely transported adeno-associated viruses (AAVrg) to knockout the phosphatase and tensin homolog protein (PTEN) in chronic SCI can effectively target both damaged and spared axons and transiently restore locomotor functions in near-complete injury models. AAVrg's were injected to deliver cre recombinase and/or a red fluorescent protein (RFP) under the human Synapsin 1 promoter (hSyn1) into the spinal cords of C57BL/6 PTENFloxΔ/Δ mice to knockout PTEN (PTEN-KO) in a severe thoracic SCI crush model at both acute and chronic time points. PTEN-KO improved locomotor abilities in both acute and chronic SCI conditions over a 9-week period. Regardless of whether treatment was initiated at the time of injury (acute), or three months after SCI (chronic), mice with limited hindlimb joint movement gained hindlimb weight support after treatment. Interestingly, functional improvements were not sustained beyond 9 weeks coincident with a loss of RFP reporter-gene expression and a near-complete loss of treatment-associated functional recovery by 6 months post-treatment. Treatment effects were also specific to severely injured mice; animals with weight support at the time of treatment lost function over a 6-month period. Retrograde tracing with Fluorogold revealed viable neurons throughout the motor cortex despite a loss of RFP expression at 9 weeks post-PTEN-KO. However, few Fluorogold labeled neurons were detected within the motor cortex at 6 months post-treatment. BDA labeling from the motor cortex revealed a dense corticospinal tract (CST) bundle in all groups except chronically treated PTEN-KO mice, indicating a potential long-term toxic effect of PTEN-KO to neurons in the motor cortex which was corroborated by a loss of ß-tubulin III labeling above the lesion within spinal cords after PTEN-KO. PTEN-KO mice had significantly more ß-tubulin III labeled axons within the lesion when treatment was delivered acutely, but not chronically post-SCI. In conclusion, we have found that using AAVrg's to knockout PTEN is an effective manipulation capable of restoring motor functions in chronic SCI and can enhance axon growth of currently unidentified axon populations when delivered acutely after injury. However, the long-term consequences of PTEN-KO on neuronal health and viability should be further explored.

PTEN knockout using retrogradely transported AAVs restores locomotor abilities in both acute and chronic spinal cord injury.

Restoring function in chronic stages of spinal cord injury (SCI) has often been met with failure or reduced efficacy when regenerative strategies are delayed past the acute or sub-acute stages of injury. Restoring function in the chronically injured spinal cord remains a critical challenge. We found that a single injection of retrogradely transported adeno-associated viruses (AAVrg) to knockout the phosphatase and tensin homolog protein (PTEN) in chronic SCI can effectively target both damaged and spared axons and restore locomotor functions in near-complete injury models. AAVrg's were injected to deliver cre recombinase and/or a red fluorescent protein (RFP) under the human Synapsin 1 promoter (hSyn1) into the spinal cords of C57BL/6 PTEN FloxΔ / Δ mice to knockout PTEN (PTEN-KO) in a severe thoracic SCI crush model at both acute and chronic time points. PTEN-KO improved locomotor abilities in both acute and chronic SCI conditions over a 9-week period. Regardless of whether treatment was initiated at the time of injury (acute), or three months after SCI (chronic), mice with limited hindlimb joint movement gained hindlimb weight support after treatment. Interestingly, functional improvements were not sustained beyond 9 weeks coincident with a loss of RFP reporter-gene expression and a near-complete loss of treatment-associated functional recovery by 6 months post-treatment. Treatment effects were also specific to severely injured mice; animals with weight support at the time of treatment lost function over a 6-month period. Retrograde tracing with Fluorogold revealed viable neurons throughout the motor cortex despite a loss of RFP expression at 9 weeks post-PTEN-KO. However, few Fluorogold labeled neurons were detected within the motor cortex at 6 months post-treatment. BDA labeling from the motor cortex revealed a dense corticospinal tract (CST) bundle in all groups except chronically treated PTEN-KO mice indicating a potential long-term toxic effect of PTEN-KO to neurons in the motor cortex. PTEN-KO mice had significantly more ß - tubulin III labeled axons within the lesion when treatment was delivered acutely, but not chronically post-SCI. In conclusion, we have found that using AAVrg's to knockout PTEN is an effective manipulation capable of restoring motor functions in chronic SCI and can enhance axon growth of currently unidentified axon populations when delivered acutely after injury. However, the long-term consequences of PTEN-KO may exert neurotoxic effects.

Advanced Age and Neurotrauma Diminish Glutathione and Impair Antioxidant Defense after Spinal Cord Injury.

Advanced age at the time of spinal cord injury (SCI) exacerbates damage from reactive oxygen species (ROS). Mechanisms underlying this age-dependent response are not well understood and may arise from decreased antioxidant defense. We investigated how spinal cord levels of the antioxidant glutathione (GSH), and its regulation, change with age and SCI. GSH is used by GSH peroxidase to sequester ROS and is recycled by GSH reductase. Male and female, 4- and 14-month-old (MO) mice received a 60 kDyn contusion SCI, and the levels of GSH and its regulatory enzymes were evaluated at one and three days post-injury (dpi). The mice with SCI were treated with N-acetylcysteine-amide (NACA; 150 mg/kg), a cysteine supplement that increases GSH, to determine effects on functional and histological outcomes. GSH was decreased with older age in sham mice, and an SCI-dependent depletion was observed in 4-MO mice by three dpi. Neither age nor injury affected the abundance of proteins regulating GSH synthesis or recycling. GSH peroxidase activity, however, increased after SCI only in 4-MO mice. In contrast, GSH peroxidase activity was increased in 14-MO sham mice, indicating that spinal cords of older mice have an elevated oxidative state. Indeed, 14-MO sham mice had more oxidized protein (3-nitrotyrosine [3-NT]) within their spinal cords compared with 4-MO sham mice. Only 4-MO mice had significant injury-induced increases in 3-NT at three dpi. NACA treatment restored GSH and improved the redox environment in injured 4- and 14-MO mice at one dpi; however, three days of NACA delivery did not improve motor, sensory, or anatomical deficits at 28 dpi in 4-MO mice and trended toward toxicity in all outcomes in 14-MO mice. Our observation suggests that GSH levels at acute stages of SCI play a minimal role in age-dependent outcomes reported after SCI in mice. Collective results implicate elements of injury occurring after three dpi, such as inflammation, as key regulators of age-dependent effects.

Immunoglobulin G Is Increased in the Injured Spinal Cord in a Sex- and Age-Dependent Manner.

There are limited studies examining age and sex as biological variables in the pathophysiology of spinal cord injury (SCI). The use of older animals and sex-balanced groups in SCI models is increasingly prioritized to better match clinical demographics. Including older animals in SCI studies is technically challenging, and outcomes are unpredictable with respect to biological and treatment responses. Incidental discoveries that are unrelated to the question under investigation often emerge while including age and sex as biological variables. When probing tissue homogenates on Western blots of 4- and 14-month-old (MO) mice, we identified a sex- and age-dependent increase in immunoglobulin G (IgG) within the spinal cords of older, 14-MO mice acutely after SCI, with females having more IgG compared with males. We further probed to determine whether differences in hemorrhage exist between sexes or ages by evaluating hemoglobin within spinal homogenates. Differences in hemoglobin between sexes and ages were not consistently observed. Because IgG was elevated in an age- and sex-dependent manner without of evidence of differences in hemorrhage, our findings point to potential pre-existing differences in IgG within mouse plasma in an age- and sex-dependent manner. This report has identified age- and sex-dependent differences in infiltrating IgG into the injured spinal cord environment that may affect injury and recovery processes. Our findings highlight that systemic contributions to SCI can be sex- and age-dependent and illustrate the value of reporting incidental discoveries.

Acute inflammatory profiles differ with sex and age after spinal cord injury.

BACKGROUND: Sex and age are emerging as influential variables that affect spinal cord injury (SCI) recovery. Despite a changing demographic towards older age at the time of SCI, the effects of sex or age on inflammation remain to be elucidated. This study determined the sex- and age-dependency of the innate immune response acutely after SCI. METHODS: Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the lesion were determined at 3- and 7-day post-injury (DPI) using flow cytometry. Cell counts of microglia and MDMs were obtained using immunohistochemistry to verify flow cytometry results at 3-DPI. Microglia and MDMs were separately isolated using fluorescence-activated cell sorting (FACS) at 3-day post-injury (DPI) to assess RNA expression of 27 genes associated with activation, redox, and debris metabolism/clearance. RESULTS: Flow cytometry revealed that being female and older at the time of injury significantly increased MDMs relative to other phagocytes, specifically increasing the ratio of MDMs to microglia at 3-DPI. Cell counts using immunohistochemistry revealed that male mice have more total microglia within SCI lesions that can account for a lower MDM/microglia ratio. With NanoString analyses of 27 genes, only 1 was differentially expressed between sexes in MDMs; specifically, complement protein C1qa was increased in males. No genes were affected by age in MDMs. Only 2 genes were differentially regulated in microglia between sexes after controlling for false discovery rate, specifically CYBB (NOX2) as a reactive oxygen species (ROS)-associated marker as well as MRC1 (CD206), a gene associated with reparative phenotypes. Both genes were increased in female microglia. No microglial genes were differentially regulated between ages. Differences between microglia and MDMs were found in 26 of 27 genes analyzed, all expressed higher in MDMs with three exceptions. Specifically, C1qa, cPLA2, and CD86 were expressed higher in microglia. CONCLUSIONS: These findings indicate that inflammatory responses to SCI are sex-dependent at both the level of cellular recruitment and gene expression.

The effects of myelin on macrophage activation are phenotypic specific via cPLA2 in the context of spinal cord injury inflammation.

Spinal cord injury (SCI) produces chronic, pro-inflammatory macrophage activation that impairs recovery. The mechanisms driving this chronic inflammation are not well understood. Here, we detail the effects of myelin debris on macrophage physiology and demonstrate a novel, activation state-dependent role for cytosolic phospholipase-A2 (cPLA2) in myelin-mediated potentiation of pro-inflammatory macrophage activation. We hypothesized that cPLA2 and myelin debris are key mediators of persistent pro-inflammatory macrophage responses after SCI. To test this, we examined spinal cord tissue 28-days after thoracic contusion SCI in 3-month-old female mice and observed both cPLA2 activation and intracellular accumulation of lipid-rich myelin debris in macrophages. In vitro, we utilized bone marrow-derived macrophages to determine myelin's effects across a spectrum of activation states. We observed phenotype-specific responses with myelin potentiating only pro-inflammatory (LPS + INF-γ; M1) macrophage activation, whereas myelin did not induce pro-inflammatory responses in unstimulated or anti-inflammatory (IL-4; M2) macrophages. Specifically, myelin increased levels of pro-inflammatory cytokines, reactive oxygen species, and nitric oxide production in M1 macrophages as well as M1-mediated neurotoxicity. PACOCF3 (cPLA2 inhibitor) blocked myelin's detrimental effects. Collectively, we provide novel spatiotemporal evidence that myelin and cPLA2 play an important role in the pathophysiology of SCI inflammation and the phenotype-specific response to myelin implicate diverse roles of myelin in neuroinflammatory conditions.

Mitochondria exert age-divergent effects on recovery from spinal cord injury.

The extent that age-dependent mitochondrial dysfunction drives neurodegeneration is not well understood. This study tested the hypothesis that mitochondria contribute to spinal cord injury (SCI)-induced neurodegeneration in an age-dependent manner by using 2,4-dinitrophenol (DNP) to uncouple electron transport, thereby increasing cellular respiration and reducing reactive oxygen species (ROS) production. We directly compared the effects of graded DNP doses in 4- and 14-month-old (MO) SCI-mice and found DNP to have increased efficacy in mitochondria isolated from 14-MO animals. In vivo, all DNP doses significantly exacerbated 4-MO SCI neurodegeneration coincident with worsened recovery. In contrast, low DNP doses (1.0-mg/kg/day) improved tissue sparing, reduced ROS-associated 3-nitrotyrosine (3-NT) accumulation, and improved anatomical and functional recovery in 14-MO SCI-mice. By directly comparing the effects of DNP between ages we demonstrate that mitochondrial contributions to neurodegeneration diverge with age after SCI. Collectively, our data indicate an essential role of mitochondria in age-associated neurodegeneration.

Corrigendum: Considerations for Studying Sex as a Biological Variable in Spinal Cord Injury.

[This corrects the article DOI: 10.3389/fneur.2020.00802.].

Considerations for Studying Sex as a Biological Variable in Spinal Cord Injury.

In response to NIH initiatives to investigate sex as a biological variable in preclinical animal studies, researchers have increased their focus on male and female differences in neurotrauma. Inclusion of both sexes when modeling neurotrauma is leading to the identification of novel areas for therapeutic and scientific exploitation. Here, we review the organizational and activational effects of sex hormones on recovery from injury and how these changes impact the long-term health of spinal cord injury (SCI) patients. When determining how sex affects SCI it remains imperative to expand outcomes beyond locomotor recovery and consider other complications plaguing the quality of life of patients with SCI. Interestingly, the SCI field predominately utilizes female rodents for basic science research which contrasts most other male-biased research fields. We discuss the unique caveats this creates to the translatability of preclinical research in the SCI field. We also review current clinical and preclinical data examining sex as biological variable in SCI. Further, we report how technical considerations such as housing, size, care management, and age, confound the interpretation of sex-specific effects in animal studies of SCI. We have uncovered novel findings regarding how age differentially affects mortality and injury-induced anemia in males and females after SCI, and further identified estrus cycle dysfunction in mice after injury. Emerging concepts underlying sexually dimorphic responses to therapy are also discussed. Through a combination of literature review and primary research observations we present a practical guide for considering and incorporating sex as biological variable in preclinical neurotrauma studies.

Effect of Sex on Motor Function, Lesion Size, and Neuropathic Pain after Contusion Spinal Cord Injury in Mice.

Spinal cord injury (SCI) causes neurodegeneration, impairs locomotor function, and impacts the quality of life particularly in those individuals in whom neuropathic pain develops. Whether the time course of neurodegeneration, locomotor impairment, or neuropathic pain varies with sex, however, remains understudied. Therefore, the objective of this study in male and female C57BL/6 mice was to evaluate the following outcomes for six weeks after a 75-kdyn thoracic contusion SCI: locomotor function using the Basso Mouse Scale (BMS); spinal cord tissue sparing and rostral-caudal lesion length; and mechanical allodynia and heat hyperalgesia using hindpaw application of Von Frey filaments or radiant heat stimuli, respectively. Although motor function was largely similar between sexes, all of the males, but only half of the females, recovered plantar stepping. Rostral-caudal lesion length was shorter in females than in males. Mechanical allodynia and heat hyperalgesia after SCI developed in all animals, regardless of sex; there were no differences in pain outcomes between sexes. We conclude that contusion SCI yields subtle sex differences in mice depending on the outcome measure but no significant differences in behavioral signs of neuropathic pain.

Delayed Azithromycin Treatment Improves Recovery After Mouse Spinal Cord Injury.

After spinal cord injury (SCI), macrophages infiltrate into the lesion and can adopt a wide spectrum of activation states. However, the pro-inflammatory, pathological macrophage activation state predominates and contributes to progressive neurodegeneration. Azithromycin (AZM), an FDA approved macrolide antibiotic, has been demonstrated to have immunomodulatory properties in a variety of inflammatory conditions. Indeed, we previously observed that post-SCI AZM treatment reduces pro-inflammatory macrophage activation. Further, a combined pre- and post-injury treatment paradigm improved functional recovery from SCI. Therefore, for the current study, we hypothesize that post-injury AZM treatment will improve recovery from SCI. To test this hypothesis, we examined the therapeutic potential of delayed AZM treatment on locomotor, sensory, and anatomical recovery. We administered AZM beginning 30-min, 3-h, or 24-h following contusion SCI in female mice, and then daily for 7 days. AZM administration beginning 30-min and 3-h post-injury improved locomotor recovery with increased stepping function relative to vehicle controls. Further, delaying treatment for 30-min after SCI significantly reduced lesion pathology. Initiating AZM treatment 24-h post-injury was not therapeutically effective. Regardless of the timing of the initial treatment, AZM did not statistically reduce the development of neuropathic pain (mechanical allodynia) nor increase neuron survival. Collectively, these results add to a growing body of evidence supporting AZM's translational potential as a therapeutic agent for SCI and other neuroinflammatory conditions in which patients currently have very few options.

Opportunities for Self-Evaluation Increase Student Calibration in an Introductory Biology Course.

Accurate self-evaluation is critical for learning. Calibration describes the relationship between learners' perception of their performance and their actual performance on a task. Here, we describe two studies aimed at assessing and improving student calibration in a first-semester introductory biology course at a 4-year public institution. Study 1 investigated students' (n = 310) calibration (the difference between estimated and actual exam performance) across one semester. Students were significantly miscalibrated for the first exam: their predicted scores were, on average, significantly higher than their actual scores. The lowest-performing students had the most inaccurate estimates. Calibration improved with each exam. By the final exam, students underestimated their scores. We initiated a second study in the following semester to examine whether explicitly teaching students about self-evaluation strategies would improve their calibration and performance. Instruction in the experimental section (n = 290) focused on students' tendency to overestimate their abilities and provided retrieval-practice opportunities. Students in the experimental section showed better calibration and performance on the first exam compared with students in a control section taught by a different instructor during the same semester (n = 251). These findings suggest that simple instructional strategies can increase students' metacognitive awareness and improve their performance.

Sexual Dimorphism of Pain Control: Analgesic Effects of Pioglitazone and Azithromycin in Chronic Spinal Cord Injury.

Central neuropathic pain develops in greater than 75% of individuals suffering a spinal cord injury (SCI). Increasingly, sex is recognized as an important biological variable in the development and treatment of peripheral neuropathic pain, but much less is known about the role of sex in central neuropathic pain and its pharmacological inhibition. To test the hypothesis that efficacy of analgesic therapies differs between males and females in SCI, we used a mouse model of SCI pain to determine the analgesic efficacy of pioglitazone (PIO), U.S. Food and Drug Administration-approved drug for the treatment of diabetes, and azithromycin (AZM), a commonly prescribed macrolide antibiotic with immunomodulatory properties. Male and female mice received moderate-severe T9 contusion SCI (75-kdyn). A robust heat hyperalgesia developed similarly between male and female mice by 4 weeks post-injury and lasted throughout the duration of the study (14 weeks). Three months after SCI, mice were treated with PIO (10 mg/kg, intraperitoneal) or AZM (160 mg/kg, oral). We observed a sex-specific effect of PIO with significant antihyperalgesic effects in females, but not males. In contrast, AZM was effective in both sexes. Our data support the use of PIO and AZM as novel therapies for SCI pain and highlight the importance of considering sex as a biological variable in clinical and experimental SCI pain research.

Reducing age-dependent monocyte-derived macrophage activation contributes to the therapeutic efficacy of NADPH oxidase inhibition in spinal cord injury.

OBJECTIVE: The average age at the time of spinal cord injury (SCI) has increased to 43 years old. Middle-aged mice (14 months old, MO) exhibit impaired recovery after SCI with age-dependent increases in reactive oxygen species (ROS) production through NADPH oxidase (NOX) along with pro-inflammatory macrophage activation. Despite these aging differences, clinical therapies are being examined in individuals regardless of age based upon preclinical data generated primarily using young animals (∼4 MO). Our objective is to test the extent to which age affects SCI treatment efficacy. Specifically, we hypothesize that the effectiveness of apocynin, a NOX inhibitor, is age-dependent in SCI. METHODS: Apocynin treatment (5 mg/kg) or vehicle was administered 1 and 6 h after moderate T9 contusion SCI (50kdyn IH) and then daily for 1 week to 4 and 14 MO mice. Locomotor and anatomical recovery was evaluated for 28 days. Monocyte-derived macrophage (MDM) and microglial activation and ROS production were evaluated at 3 and 28 days post-injury. RESULTS: Apocynin improved functional and anatomical recovery in 14 but not 4 MO SCI mice. Apocynin-mediated recovery was coincident with significant reductions in MDM infiltration and MDM-ROS production in 14 MO SCI mice. Importantly, microglial activation was unaffected by treatment. CONCLUSION: These results indicate that apocynin exhibits age-dependent neuroprotective effects by blocking excessive neuroinflammation through NOX-mediated ROS production in MDMs. Further, these data identify age as a critical regulator for SCI treatment efficacy and indicate that pharmacologically reduced macrophage, but not microglia, activation and ROS production reverses age-associated neurological impairments.

Compression Decreases Anatomical and Functional Recovery and Alters Inflammation after Contusive Spinal Cord Injury.

Experimental models of spinal cord injury (SCI) typically utilize contusion or compression injuries. Clinically, however, SCI is heterogeneous and the primary injury mode may affect secondary injury progression and neuroprotective therapeutic efficacy. Specifically, immunomodulatory agents are of therapeutic interest because the activation state of SCI macrophages may facilitate pathology but also improve repair. It is unknown currently how the primary injury biomechanics affect macrophage activation. Therefore, to determine the effects of compression subsequent to spinal contusion, we examined recovery, secondary injury, and macrophage activation in C57/BL6 mice after SCI with or without a 20 sec compression at two contusion impact forces (50 and 75 kdyn). We observed that regardless of the initial impact force, compression increased tissue damage and worsened functional recovery. Interestingly, compression-dependent damage is not evident until one week after SCI. Further, compression limits functional recovery to the first two weeks post-SCI; in the absence of compression, mice receiving contusion SCI recover for four weeks. To determine whether the recovery plateau is indicative of compression-specific inflammatory responses, we examined macrophage activation with immunohistochemical markers of purportedly pathological (CD86 and macrophage receptor with collagenous structure [MARCO]) and reparative macrophages (arginase [Arg1] and CD206). We detected significant increases in macrophages expression of MARCO and decreases in macrophage Arg1 expression with compression, suggesting a biomechanical-dependent shift in SCI macrophage activation. Collectively, compression-induced alterations in tissue and functional recovery and inflammation highlight the need to consider the primary SCI biomechanics in the design and clinical implementation of immunomodulatory therapies.

Pioglitazone treatment following spinal cord injury maintains acute mitochondrial integrity and increases chronic tissue sparing and functional recovery.

Pioglitazone is an FDA-approved PPAR-γ agonist drug used to treat diabetes, and it has demonstrated neuroprotective effects in multiple models of central nervous system (CNS) injury. Acute treatment after spinal cord injury (SCI) in rats is reported to suppress neuroinflammation, rescue injured tissues, and improve locomotor recovery. In the current study, we additionally assessed the protective efficacy of pioglitazone treatment on acute mitochondrial respiration, as well as functional and anatomical recovery after contusion SCI in adult male C57BL/6 mice. Mice received either vehicle or pioglitazone (10mg/kg) at either 15min or 3h after injury (75kdyn at T9) followed by a booster at 24h post-injury. At 25h, mitochondria were isolated from spinal cord segments centered on the injury epicenters and assessed for their respiratory capacity. Results showed significantly compromised mitochondrial respiration 25h following SCI, but pioglitazone treatment that was initiated either at 15min or 3h post-injury significantly maintained mitochondrial respiration rates near sham levels. A second cohort of injured mice received pioglitazone at 15min post injury, then once a day for 5days post-injury to assess locomotor recovery and tissue sparing over 4weeks. Compared to vehicle, pioglitazone treatment resulted in significantly greater recovery of hind-limb function over time, as determined by serial locomotor BMS assessments and both terminal BMS subscores and gridwalk performance. Such improvements correlated with significantly increased grey and white matter tissue sparing, although pioglitazone treatment did not abrogate long-term injury-induced inflammatory microglia/macrophage responses. In sum, pioglitazone significantly increased functional neuroprotection that was associated with remarkable maintenance of acute mitochondrial bioenergetics after traumatic SCI. This sets the stage for dose-response and delayed administration studies to maximize pioglitazone's efficacy for SCI while elucidating the precise role that mitochondria play in governing its neuroprotection; the ultimate goal to develop novel therapeutics that specifically target mitochondrial dysfunction.

Predictive screening of M1 and M2 macrophages reveals the immunomodulatory effectiveness of post spinal cord injury azithromycin treatment.

Spinal cord injury (SCI) triggers a heterogeneous macrophage response that when experimentally polarized toward alternative forms of activation (M2 macrophages) promotes tissue and functional recovery. There are limited pharmacological therapies that can drive this reparative inflammatory state. In the current study, we used in vitro systems to comprehensively defined markers of macrophages with known pathological (M1) and reparative (M2) properties in SCI. We then used these markers to objectively define the macrophage activation states after SCI in response to delayed azithromycin treatment. Mice were subjected to moderate-severe thoracic contusion SCI. Azithromycin or vehicle was administered beginning 30 minutes post-SCI and then daily for 3 or 7 days post injury (dpi). We detected a dose-dependent polarization toward purportedly protective M2 macrophages with daily AZM treatment. Specifically, AZM doses of 10, 40, or 160 mg/kg decreased M1 macrophage gene expression at 3 dpi while the lowest (10 mg/kg) and highest (160 mg/kg) doses increased M2 macrophage gene expression at 7 dpi. Azithromycin has documented immunomodulatory properties and is commonly prescribed to treat infections in SCI individuals. This work demonstrates the utility of objective, comprehensive macrophage gene profiling for evaluating immunomodulatory SCI therapies and highlights azithromycin as a promising agent for SCI treatment.

Age increases reactive oxygen species production in macrophages and potentiates oxidative damage after spinal cord injury.

Age potentiates neurodegeneration and impairs recovery from spinal cord injury (SCI). Previously, we observed that age alters the balance of destructive (M1) and protective (M2) macrophages; however, the age-related pathophysiology in SCI is poorly understood. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) contributes to reactive oxygen species (ROS)-mediated damage and macrophage activation in neurotrauma. Further, NOX and ROS increase with central nervous system age. Here, we found significantly higher ROS generation in 14 versus 4-month-old (MO) mice after contusion SCI. Notably, NOX2 increased in 14 MO ROS-producing macrophages suggesting that macrophages and NOX contribute to SCI oxidative stress. Indicators of lipid peroxidation, a downstream cytotoxic effect of ROS accumulation, were significantly higher in 14 versus 4 MO SCI mice. We also detected a higher percentage of ROS-producing M2 (Arginase-1-positive) macrophages in 14 versus 4 MO mice, a previously unreported SCI phenotype, and increased M1 (CD16/32-positive) macrophages with age. Thus, NOX and ROS are age-related mediators of SCI pathophysiology and normally protective M2 macrophages may potentiate secondary injury through ROS generation in the aged injured spinal cord.

Clinical safety of the ProMRI pacemaker system in patients subjected to thoracic spine and cardiac 1.5-T magnetic resonance imaging scanning conditions.

BACKGROUND: Permanent cardiac pacemakers have historically been considered a contraindication to magnetic resonance imaging (MRI). OBJECTIVE: The purpose of the ProMRI Phase B Study, a multicenter, prospective, single-arm, nonrandomized study, was to evaluate the clinical safety of the Biotronik ProMRI pacemaker system in patients undergoing thoracic spine and cardiac MRI. METHODS: The ProMRI Phase B study enrolled 245 patients with stable baseline pacing indices implanted with an Entovis pacemaker (DR-T or SR-T) and Setrox 53-cm and/or 60-cm lead(s). Device interrogation was performed at enrollment, pre- and post-MRI scan, and 1 and 3 months post-MRI. End-points were (1) freedom from MRI- and pacing system-related serious adverse device effects through 1 month post-MRI; (2) freedom from atrial and ventricular MRI-induced pacing threshold increase (>0.5 V); and (3) freedom from P- and R-wave amplitude attenuation (<50%), or P wave <1.5 mV, or R wave <5.0 mV at 1 month post-MRI. RESULTS: In total, 216 patients completed the MRI and 1-month post-MRI follow-up. One adverse event possibly related to the implanted system and the MRI procedure occurred, resulting in a serious adverse device effect-free rate of 99.6% (220/221; P < .0001. Freedom from atrial and ventricular pacing threshold increase was 100% (194/194, P < .001) and 100% (206/206, P < .001) respectively. Freedom from P- and R-wave amplitude attenuation was 98.2% (167/170, P < .001) and 100% (188/188, P < .001) respectively. CONCLUSION: The results of the ProMRI Phase B study demonstrate the clinical safety and efficacy of the ProMRI pacemaker system in patients subjected to thoracic spine and cardiac MRI conditions.