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.

Inhibition of Bruton Tyrosine Kinase Reduces Neuroimmune Cascade and Promotes Recovery after Spinal Cord Injury.

Microglia/astrocyte and B cell neuroimmune responses are major contributors to the neurological deficits after traumatic spinal cord injury (SCI). Bruton tyrosine kinase (BTK) activation mechanistically links these neuroimmune mechanisms. Our objective is to use Ibrutinib, an FDA-approved BTK inhibitor, to inhibit the neuroimmune cascade thereby improving locomotor recovery after SCI. Rat models of contusive SCI, Western blot, immunofluorescence staining imaging, flow cytometry analysis, histological staining, and behavioral assessment were used to evaluate BTK activity, neuroimmune cascades, and functional outcomes. Both BTK expression and phosphorylation were increased at the lesion site at 2, 7, 14, and 28 days after SCI. Ibrutinib treatment (6 mg/kg/day, IP, starting 3 h post-injury for 7 or 14 days) reduced BTK activation and total BTK levels, attenuated the injury-induced elevations in Iba1, GFAP, CD138, and IgG at 7 or 14 days post-injury without reduction in CD45RA B cells, improved locomotor function (BBB scores), and resulted in a significant reduction in lesion volume and significant improvement in tissue-sparing 11 weeks post-injury. These results indicate that Ibrutinib exhibits neuroprotective effects by blocking excessive neuroimmune responses through BTK-mediated microglia/astroglial activation and B cell/antibody response in rat models of SCI. These data identify BTK as a potential therapeutic target for SCI.

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.

Leukemia inhibitory factor modulates the peripheral immune response in a rat model of emergent large vessel occlusion.

BACKGROUND: The migration of peripheral immune cells and splenocytes to the ischemic brain is one of the major causes of delayed neuroinflammation after permanent large vessel stroke. Other groups have demonstrated that leukemia inhibitory factor (LIF), a cytokine that promotes neural cell survival through upregulation of antioxidant enzymes, promotes an anti-inflammatory phenotype in several types of immune cells. The goal of this study was to determine whether LIF treatment modulates the peripheral immune response after stroke. METHODS: Young male (3 month) Sprague-Dawley rats underwent sham surgery or permanent middle cerebral artery occlusion (MCAO). Animals were administered LIF (125 µg/kg) or PBS at 6, 24, and 48 h prior to euthanization at 72 h. Bone marrow-derived macrophages were treated with LIF (20 ng/ml) or PBS after stimulation with interferon gamma + LPS. Western blot was used to measure protein levels of CD11b, IL-12, interferon inducible protein-10, CD3, and the LIF receptor in spleen and brain tissue. ELISA was used to measure IL-10, IL-12, and interferon gamma. Isolectin was used to label activated immune cells in brain tissue sections. Statistical analysis was performed using one-way ANOVA and Student's t test. A Kruskal-Wallis test followed by Bonferroni-corrected Mann-Whitney tests was performed if data did not pass the D'Agostino-Pearson normality test. RESULTS: LIF-treated rats showed significantly lower levels of the LIF receptor and interferon gamma in the spleen and CD11b levels in the brain compared to their PBS-treated counterparts. Fluorescence from isolectin-binding immune cells was more prominent in the ipsilateral cortex and striatum after PBS treatment compared to LIF treatment. MCAO + LIF significantly decreased splenic levels of CD11b and CD3 compared to sham surgery. MCAO + PBS treatment significantly elevated splenic levels of interferon inducible protein-10 at 72 h after MCAO, while LIF treatment after MCAO returned interferon inducible protein 10 to sham levels. LIF administration with interferon gamma + LPS significantly reduced the IL-12/IL-10 production ratio compared to macrophages treated with interferon gamma + LPS alone. CONCLUSIONS: These data demonstrate that LIF promotes anti-inflammatory signaling through alterations of the IL-12/interferon gamma/interferon inducible protein 10 pathway.

Myelin as an inflammatory mediator: Myelin interactions with complement, macrophages, and microglia in spinal cord injury.

Spinal cord injury (SCI) triggers chronic intraspinal inflammation consisting of activated resident and infiltrating immune cells (especially microglia/macrophages). The environmental factors contributing to this protracted inflammation are not well understood; however, myelin lipid debris is a hallmark of SCI. Myelin is also a potent macrophage stimulus and target of complement-mediated clearance and inflammation. The downstream effects of these neuroimmune interactions have the potential to contribute to ongoing pathology or facilitate repair. This depends in large part on whether myelin drives pathological or reparative macrophage activation states, commonly referred to as M1 (proinflammatory) or M2 (alternatively) macrophages, respectively. Here we review the processes by which myelin debris may be cleared through macrophage surface receptors and the complement system, how this differentially influences macrophage and microglial activation states, and how the cellular functions of these myelin macrophages and complement proteins contribute to chronic inflammation and secondary injury after SCI.

Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy.

Purpose To examine whether cardiac chemical exchange saturation transfer (CEST) imaging can be serially and noninvasively used to probe cell survival or rejection after intramyocardial implantation in mice. Materials and Methods Experiments were compliant with the National Institutes of Health Guidelines on the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. One million C2C12 cells labeled with either europium (Eu) 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) or saline via the hypotonic swelling technique were implanted into the anterior-lateral left ventricular wall in C57BL/6J (allogeneic model, n = 17) and C3H (syngeneic model, n = 13) mice. Imaging (frequency offsets of ±15 parts per million) was performed 1, 10, and 20 days after implantation, with the asymmetrical magnetization transfer ratio (MTRasym) calculated from image pairs. Histologic examination was performed at the conclusion of imaging. Changes in MTRasym over time and between mice were assessed by using two-way repeated-measures analysis of variance. Results MTRasym was significantly higher in C3H and C57BL/6J mice in grafts of Eu-HP-DO3A-labeled cells (40.2% ± 5.0 vs 37.8% ± 7.0, respectively) compared with surrounding tissue (-0.67% ± 1.7 vs -1.8% ± 5.3, respectively; P < .001) and saline-labeled grafts (-0.4% ± 6.0 vs -1.2% ± 3.6, respectively; P < .001) at day 1. In C3H mice, MTRasym remained increased (31.3% ± 9.2 on day 10, 28.7% ± 5.2 on day 20; P < .001 vs septum) in areas of in Eu-HP-DO3A-labeled cell grafts. In C57BL/6J mice, corresponding MTRasym values (11.3% ± 8.1 on day 10, 5.1% ± 9.4 on day 20; P < .001 vs day 1) were similar to surrounding myocardium by day 20 (P = .409). Histologic findings confirmed cell rejection in C57BL/6J mice. Estimation of graft area was similar with cardiac CEST imaging and histologic examination (R2 = 0.89). Conclusion Cardiac CEST imaging can be used to image cell survival and rejection in preclinical models of cell therapy. © RSNA, 2016 Online supplemental material is available for this article.

Toll-Like Receptors and Dectin-1, a C-Type Lectin Receptor, Trigger Divergent Functions in CNS Macrophages.

Spinal cord injury (SCI) activates macrophages, endowing them with both reparative and pathological functions. The mechanisms responsible for these divergent functions are unknown but are likely controlled through stochastic activation of different macrophage receptor subtypes. Various danger-associated molecular patterns released from dying cells in the injured spinal cord likely activate distinct subtypes of macrophage pattern recognition receptors, including bacterial toll-like receptors (TLRs) and fungal C-type lectin receptors (e.g., dectin-1). To determine the in vivo consequences of activating these receptors, ligands specific for TLR2 or dectin-1 were microinjected, alone or in combination, into intact spinal cord. Both ligands elicit a florid macrophage reaction; however, only dectin-1 activation causes macrophage-mediated demyelination and axonal injury. Coactivating TLR2 reduced the injurious effects of dectin-1 activation. When injected into traumatically injured spinal cord, TLR2 agonists enhance the endogenous macrophage reaction while conferring neuroprotection. Indeed, dieback of axons was reduced, leading to smaller lesion volumes at the peak of the macrophage response. Moreover, the density of NG2+ cells expressing vimentin increased in and near lesions that were enriched with TLR2-activated macrophages. In dectin-1-null mutant (knock-out) mice, dieback of corticospinal tract axons also is reduced after SCI. Collectively, these data support the hypothesis that the ability of macrophages to create an axon growth-permissive microenvironment or cause neurotoxicity is receptor dependent and it may be possible to exploit this functional dichotomy to enhance CNS repair. SIGNIFICANCE STATEMENT: There is a growing appreciation that macrophages exert diverse functions in the injured and diseased CNS. Indeed, both macrophage-mediated repair and macrophage-mediated injury occur, and often these effector functions are elicited simultaneously. Understanding the mechanisms governing the reparative and pathological properties of activated macrophages is at the forefront of neuroscience research. In this report, using in vitro and in vivo models of relevance to traumatic spinal cord injury (SCI), new data indicate that stochastic activation of toll-like and c-type lectin receptors on macrophages causes neuroprotection or neurotoxicity, respectively. Although this manuscript focuses on SCI, these two innate immune receptor subtypes are also involved in developmental processes and become activated in macrophages that respond to various neurological diseases.

IL-4 signaling drives a unique arginase+/IL-1ß+ microglia phenotype and recruits macrophages to the inflammatory CNS: consequences of age-related deficits in IL-4Rα after traumatic spinal cord injury.

Alternative activation of microglia/macrophages (M2a) by interleukin (IL)-4 is purported to support intrinsic growth and repair processes after CNS injury. Nonetheless, alternative activation of microglia is poorly understood in vivo, particularly in the context of inflammation, injury, and aging. Here, we show that aged mice (18-19 months) had reduced functional recovery after spinal cord injury (SCI) associated with impaired induction of IL-4 receptor α (IL-4Rα) on microglia. The failure to successfully promote an IL-4/IL-4Rα response in aged mice resulted in attenuated arginase (M2a associated), IL-1ß, and chemokine ligand 2 (CCL2) expression, and diminished recruitment of IL-4Rα(+) macrophages to the injured spinal cord. Furthermore, the link between reduced IL-4Rα expression and reduced arginase, IL-1ß, and CCL2 expression was confirmed using adult IL-4Rα knock-out (IL-4Rα(KO)) mice. To better understand IL-4Rα-mediated regulation of active microglia, a series of studies was completed in mice that were peripherally injected with lipopolysaccharide and later provided IL-4 by intracerebroventricular infusion. These immune-based studies demonstrate that inflammatory-induced IL-4Rα upregulation on microglia was required for the induction of arginase by IL-4. In addition, IL-4-mediated reprogramming of active microglia enhanced neurite growth ex vivo and increased inflammatory gene expression (i.e., IL-1ß and CCL2) and the corresponding recruitment of CCR2(+)/IL-4Rα(+)/arginase(+) myeloid cells in vivo. IL-4 reprogrammed active microglia to a unique and previously unreported phenotype (arginase(+)/IL-1ß(+)) that augmented neurite growth and enhanced recruitment of peripheral IL-4Rα(+) myeloid cells to the CNS. Moreover, this key signaling cascade was impaired with age corresponding with reduced functional recovery after SCI.

Azithromycin drives alternative macrophage activation and improves recovery and tissue sparing in contusion spinal cord injury.

BACKGROUND: Macrophages persist indefinitely at sites of spinal cord injury (SCI) and contribute to both pathological and reparative processes. While the alternative, anti-inflammatory (M2) phenotype is believed to promote cell protection, regeneration, and plasticity, pro-inflammatory (M1) macrophages persist after SCI and contribute to protracted cell and tissue loss. Thus, identifying non-invasive, clinically viable, pharmacological therapies for altering macrophage phenotype is a challenging, yet promising, approach for treating SCI. Azithromycin (AZM), a commonly used macrolide antibiotic, drives anti-inflammatory macrophage activation in rodent models of inflammation and in humans with cystic fibrosis. METHODS: We hypothesized that AZM treatment can alter the macrophage response to SCI and reduce progressive tissue pathology. To test this hypothesis, mice (C57BL/6J, 3-month-old) received daily doses of AZM (160 mg/kg) or vehicle treatment via oral gavage for 3 days prior and up to 7 days after a moderate-severe thoracic contusion SCI (75-kdyn force injury). Fluorescent-activated cell sorting was used in combination with real-time PCR (rtPCR) to evaluate the disposition and activation status of microglia, monocytes, and neutrophils, as well as macrophage phenotype in response to AZM treatment. An open-field locomotor rating scale (Basso Mouse Scale) and gridwalk task were used to determine the effects of AZM treatment on SCI recovery. Bone marrow-derived macrophages (BMDMs) were used to determine the effect of AZM treatment on macrophage phenotype in vitro. RESULTS: In accordance with our hypothesis, SCI mice exhibited significantly increased anti-inflammatory and decreased pro-inflammatory macrophage activation in response to AZM treatment. In addition, AZM treatment led to improved tissue sparing and recovery of gross and coordinated locomotor function. Furthermore, AZM treatment altered macrophage phenotype in vitro and lowered the neurotoxic potential of pro-inflammatory, M1 macrophages. CONCLUSIONS: Taken together, these data suggest that pharmacologically intervening with AZM can alter SCI macrophage polarization toward a beneficial phenotype that, in turn, may potentially limit secondary injury processes. Given that pro-inflammatory macrophage activation is a hallmark of many neurological pathologies and that AZM is non-invasive and clinically viable, these data highlight a novel approach for treating SCI and other maladaptive neuroinflammatory conditions.

Stress exacerbates neuron loss and microglia proliferation in a rat model of excitotoxic lower motor neuron injury.

All individuals experience stress and hormones (e.g., glucocorticoids/GCs) released during stressful events can affect the structure and function of neurons. These effects of stress are best characterized for brain neurons; however, the mechanisms controlling the expression and binding affinity of glucocorticoid receptors in the spinal cord are different than those in the brain. Accordingly, whether stress exerts unique effects on spinal cord neurons, especially in the context of pathology, is unknown. Using a controlled model of focal excitotoxic lower motor neuron injury in rats, we examined the effects of acute or chronic variable stress on spinal cord motor neuron survival and glial activation. New data indicate that stress exacerbates excitotoxic spinal cord motor neuron loss and associated activation of microglia. In contrast, hypertrophy and hyperplasia of astrocytes and NG2+ glia were unaffected or were modestly suppressed by stress. Although excitotoxic lesions cause significant motor neuron loss and stress exacerbates this pathology, overt functional impairment did not develop in the relevant forelimb up to one week post-lesion. These data indicate that stress is a disease-modifying factor capable of altering neuron and glial responses to pathological challenges in the spinal cord.

Tissue-resident macrophages specifically express Lactotransferrin and Vegfc during ear pinna regeneration in spiny mice.

The details of how macrophages control different healing trajectories (regeneration vs. scar formation) remain poorly defined. Spiny mice (Acomys spp.) can regenerate external ear pinnae tissue, whereas lab mice (Mus musculus) form scar tissue in response to an identical injury. Here, we used this dual species system to dissect macrophage phenotypes between healing modes. We identified secreted factors from activated Acomys macrophages that induce a pro-regenerative phenotype in fibroblasts from both species. Transcriptional profiling of Acomys macrophages and subsequent in vitro tests identified VEGFC, PDGFA, and Lactotransferrin (LTF) as potential pro-regenerative modulators. Examining macrophages in vivo, we found that Acomys-resident macrophages secreted VEGFC and LTF, whereas Mus macrophages do not. Lastly, we demonstrate the requirement for VEGFC during regeneration and find that interrupting lymphangiogenesis delays blastema and new tissue formation. Together, our results demonstrate that cell-autonomous mechanisms govern how macrophages react to the same stimuli to differentially produce factors that facilitate regeneration.

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.

Azithromycin reduces hemoglobin-induced innate neuroimmune activation.

Neonatal intraventricular hemorrhage (IVH) releases blood products into the lateral ventricles and brain parenchyma. There are currently no medical treatments for IVH and surgery is used to treat a delayed effect of IVH, post-hemorrhagic hydrocephalus. However, surgery is not a cure for intrinsic brain injury from IVH, and is performed in a subacute time frame. Like many neurological diseases and injuries, innate immune activation is implicated in the pathogenesis of IVH. Innate immune activation is a pharmaceutically targetable mechanism to reduce brain injury and post-hemorrhagic hydrocephalus after IVH. Here, we tested the macrolide antibiotic azithromycin, which has immunomodulatory properties, to reduce innate immune activation in an in vitro model of microglial activation using the blood product hemoglobin (Hgb). We then utilized azithromycin in our in vivo model of IVH, using intraventricular blood injection into the lateral ventricle of post-natal day 5 rat pups. In both models, azithromycin modulated innate immune activation by several outcome measures including mitochondrial bioenergetic analysis, cytokine expression and flow cytometric analysis. This suggests that azithromycin, which is safe for neonates, could hold promise for modulating innate immune activation after IVH.

Microglia as drivers of neurodegeneration: The role of innate-adaptive immune signaling.

Microglia contribute to neurodegeneration through numerous mechanisms. In this issue of Neuron, Shi et al.1 identify a maladaptive innate-adaptive immune axis with CD8+ T cells, mediated through microglial CCL2/8 and CCR2/5, in radiation-induced brain injury and stroke. Their findings across species and injuries suggest broader implications for neurodegenerative conditions.

Challenges in Translating Regenerative Therapies for Spinal Cord Injury.

Regenerating the injured spinal cord is a substantial challenge with many obstacles that need to be overcome to achieve robust functional benefits. This abundance of hurdles can partly explain the limited success when applying regenerative intervention treatments in animal models and/or people. In this article, we elaborate on a few of these obstacles, starting with the applicability of animal models and how they compare to the clinical setting. We then discuss the requirement for combinatorial interventions and the associated problems in experimental design, including the addition of rehabilitative training. The article expands on differences in lesion sizes and locations between humans and common animal models, and how this difference can determine the success or failure of an intervention. An additional and frequently overlooked problem in the translation of interventions that applies beyond the field of neuroregeneration is the reporting bias and the lack of transparency in reporting findings. New data mandates are tackling this problem and will eventually result in a more balanced view of the field. Finally, we will discuss strategies to negotiate the challenging course of successful translation to facilitate successful translation of regeneration promoting interventions.

Effects of Acute Ethanol Intoxication on Spinal Cord Injury Outcomes in Female Mice.

Abstract Approximately one in three traumatic spinal cord injuries (SCIs) occurs during or shortly after the consumption of alcohol. A small number of retrospective clinical studies report variable effects of alcohol intoxication on mortality, neurological recovery, and complications after SCI. Some of these studies demonstrate a protective effect of alcohol intoxication on SCI outcomes, whereas others show an increased complication risk. Pre-clinical studies in rat, ferret, and feline SCI models report a detrimental effect of ethanol intoxication on hemorrhage, motor recovery, and biochemical markers of tissue injury. However, no studies to date have investigated the neuropathological consequences of ethanol intoxication at the time of SCI or the reciprocal effect of SCI on ethanol metabolism. Therefore, we combined a pre-clinical mouse model of acute ethanol intoxication and experimental vertebral level T9 contusion SCI to investigate their interactive effects in female mice. We first investigated the effect of SCI on ethanol metabolism and found that T9 SCI does not alter ethanol metabolism. However, we did find that isoflurane anesthesia significantly slowed ethanol metabolism independent of SCI. We also determined how acute ethanol intoxication at the time of SCI alters locomotor recovery and lesion pathology. Using the Basso Mouse Scale (BMS) and CatWalk XT Gait Analysis System, we assessed locomotor recovery for 6 weeks after injury and observed that acute ethanol intoxication at the time of injury did not alter locomotor recovery. We also found no effect of ethanol intoxication on heat hyperalgesia development. There was, however, a detrimental effect of ethanol on tissue sparing after SCI. Therefore, we conclude that acute alcohol intoxication at the time of injury may contribute to the neuropathological consequences of SCI.

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.

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.

Transforming growth factor α transforms astrocytes to a growth-supportive phenotype after spinal cord injury.

Astrocytes are both detrimental and beneficial for repair and recovery after spinal cord injury (SCI). These dynamic cells are primary contributors to the growth-inhibitory glial scar, yet they are also neuroprotective and can form growth-supportive bridges on which axons traverse. We have shown that intrathecal administration of transforming growth factor α (TGFα) to the contused mouse spinal cord can enhance astrocyte infiltration and axonal growth within the injury site, but the mechanisms of these effects are not well understood. The present studies demonstrate that the epidermal growth factor receptor (EGFR) is upregulated primarily by astrocytes and glial progenitors early after SCI. TGFα directly activates the EGFR on these cells in vitro, inducing their proliferation, migration, and transformation to a phenotype that supports robust neurite outgrowth. Overexpression of TGFα in vivo by intraparenchymal adeno-associated virus injection adjacent to the injury site enhances cell proliferation, alters astrocyte distribution, and facilitates increased axonal penetration at the rostral lesion border. To determine whether endogenous EGFR activation is required after injury, SCI was also performed on Velvet (C57BL/6J-Egfr(Vel)/J) mice, a mutant strain with defective EGFR activity. The affected mice exhibited malformed glial borders, larger lesions, and impaired recovery of function, indicating that intrinsic EGFR activation is necessary for neuroprotection and normal glial scar formation after SCI. By further stimulating precursor proliferation and modifying glial activation to promote a growth-permissive environment, controlled stimulation of EGFR at the lesion border may be considered in the context of future strategies to enhance endogenous cellular repair after injury.

Achieving CNS axon regeneration by manipulating convergent neuro-immune signaling.

After central nervous system (CNS) trauma, axons have a low capacity for regeneration. Regeneration failure is associated with a muted regenerative response of the neuron itself, combined with a growth-inhibitory and cytotoxic post-injury environment. After spinal cord injury (SCI), resident and infiltrating immune cells (especially microglia/macrophages) contribute significantly to the growth-refractory milieu near the lesion. By targeting both the regenerative potential of the axon and the cytotoxic phenotype of microglia/macrophages, we may be able to improve CNS repair after SCI. In this review, we discuss molecules shown to impact CNS repair by affecting both immune cells and neurons. Specifically, we provide examples of pattern recognition receptors, integrins, cytokines/chemokines, nuclear receptors and galectins that could improve CNS repair. In many cases, signaling by these molecules is complex and may have contradictory effects on recovery depending on the cell types involved or the model studied. Despite this caveat, deciphering convergent signaling pathways on immune cells (which affect axon growth indirectly) and neurons (direct effects on axon growth) could improve repair and recovery after SCI. Future studies must continue to consider how regenerative therapies targeting neurons impact other cells in the pathological CNS. By identifying molecules that simultaneously improve axon regenerative capacity and drive the protective, growth-promoting phenotype of immune cells, we may discover SCI therapies that act synergistically to improve CNS repair and functional recovery.