Spinal Cord Injury Induces Permanent Reprogramming of Microglia into a Disease-Associated State Which Contributes to Functional Recovery.

Microglia are resident myeloid cells of the CNS. Recently, single-cell RNA sequencing (scRNAseq) has enabled description of a disease-associated microglia (DAM) with a role in neurodegeneration and demyelination. In this study, we use scRNAseq to investigate the temporal dynamics of immune cells harvested from the epicenter of traumatic spinal cord injury (SCI) induced in female mice. We find that as a consequence of SCI, baseline microglia undergo permanent transcriptional reprogramming into a previously uncharacterized subtype of microglia with striking similarities to previously reported DAM as well as a distinct microglial state found during development. Using a microglia depletion model we showed that DAM in SCI are derived from baseline microglia and strongly enhance recovery of hindlimb locomotor function following injury.SIGNIFICANCE STATEMENT Although disease-associated microglia (DAM) have been the subject of strong research interest during recent years (Keren-Shaul, 2017; Jordão, 2019), their cellular origin and their role in "normal" acute injury processes is not well understood. Our work directly addresses the origin and the role of DAM in traumatic injury response. Further, we use a microglia depletion model to prove that DAM in spinal cord injury (SCI) are indeed derived from homeostatic microglia, and that they strongly enhance recovery. Thus, in this work we significantly expand the knowledge of immune response to traumatic injury, demonstrate the applicability to human injury via our unique access to injured human spinal cord tissue, and provide the community with a comprehensive dataset for further exploration.

Interleukin-25 is detrimental for recovery after spinal cord injury in mice.

BACKGROUND: The cytokine, interleukin (IL)-25, is thought to be critically involved in inducing a type 2 immune response which may contribute to regeneration after central nervous system (CNS) trauma. We investigated whether applying recombinant IL-25, locally or systemically, in a mouse model of spinal cord injury (SCI) improves functional and histological recovery. FINDINGS: Repeated systemic administration of IL-25 did not influence functional recovery following SCI. In contrast, a single local administration of IL-25 significantly worsened locomotor outcome, which was evident from a decreased Basso mouse scale (BMS) score compared with phosphate-buffered saline (PBS)-treated controls. This was accompanied by a significant increase in lesion size, demyelination, and T helper cell infiltration. CONCLUSIONS: These data show for the first time that IL-25 is either ineffective when applied systemically or detrimental to spinal cord recovery when applied locally. Our findings question the potential neuroprotective role of IL-25 following CNS trauma.

Complement C5a is detrimental to histological and functional locomotor recovery after spinal cord injury in mice.

Based on the studies on the role of complements C3, C1q and factor B, we hypothesized that complement C5a is detrimental to locomotor recovery at the early stage of secondary injury after spinal cord injury (SCI). To test this hypothesis, we investigated the effect of inhibition of complement C5a receptor (C5aR) by using C5aR antagonist PMX53 (C5aRA) and deficiency of complement C5a receptor (C5aR-/- mice) on histological and locomotor recovery after SCI in mice. We demonstrated that the Basso Mouse Scale scores in the mice injected with C5aRA (C5aRA-mice) at 45min before and 24h after SCI and the C5aR-/- mice were markedly higher than those in the mice treated with saline (Saline-mice) and the C5aR+/+ mice respectively between 7 and 28days after SCI. Also, expression of TNF-α and IL-1ß in C5aRA-mice was significantly lower than that in Saline-mice from 1 to 24h after SCI. In addition, the percentage of microglia/macrophage in C5aRA mice and C5aR-/- mice was significantly lower than those in their corresponding control groups from 1 to 14days after SCI. Furthermore, C5aRA mice and C5aR-/- mice had less GFAP expression in the injured spinal cord epicenter as compared to Saline mice and C5aR+/+ mice at day 28 after SCI. These findings provided evidence that inhibition or deficiency of C5aR could significantly improve histological and functional locomotor recovery after SCI in mice.

Mast cells protect from post-traumatic spinal cord damage in mice by degrading inflammation-associated cytokines via mouse mast cell protease 4.

Mast cells (MCs) are found abundantly in the central nervous system and play a complex role in neuroinflammatory diseases such as multiple sclerosis and stroke. In the present study, we show that MC-deficient Kit(W-sh/W-sh) mice display significantly increased astrogliosis and T cell infiltration as well as significantly reduced functional recovery after spinal cord injury compared to wildtype mice. In addition, MC-deficient mice show significantly increased levels of MCP-1, TNF-α, IL-10 and IL-13 protein levels in the spinal cord. Mice deficient in mouse mast cell protease 4 (mMCP4), an MC-specific chymase, also showed increased MCP-1, IL-6 and IL-13 protein levels in spinal cord samples and a decreased functional outcome after spinal cord injury. A degradation assay using supernatant from MCs derived from either mMCP4(-/-) mice or controls revealed that mMCP4 cleaves MCP-1, IL-6, and IL-13 suggesting a protective role for MC proteases in neuroinflammation. These data show for the first time that MCs may be protective after spinal cord injury and that they may reduce CNS damage by degrading inflammation-associated cytokines via the MC-specific chymase mMCP4.

Neuroprotective effects of Alda-1 mitigate spinal cord injury in mice: involvement of Alda-1-induced ALDH2 activation-mediated suppression of reactive aldehyde mechanisms.

Spinal cord injury (SCI) is associated with high production and excessive accumulation of pathological 4-hydroxy-trans-2-nonenal (4-HNE), a reactive aldehyde, formed by SCI-induced metabolic dysregulation of membrane lipids. Reactive aldehyde load causes redox alteration, neuroinflammation, neurodegeneration, pain-like behaviors, and locomotion deficits. Pharmacological scavenging of reactive aldehydes results in limited improved motor and sensory functions. In this study, we targeted the activity of mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) to detoxify 4-HNE for accelerated functional recovery and improved pain-like behavior in a male mouse model of contusion SCI. N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1), a selective activator of ALDH2, was used as a therapeutic tool to suppress the 4-HNE load. SCI was induced by an impactor at the T9-10 vertebral level. Injured animals were initially treated with Alda-1 at 2 hours after injury, followed by once-daily treatment with Alda-1 for 30 consecutive days. Locomotor function was evaluated by the Basso Mouse Scale, and pain-like behaviors were assessed by mechanical allodynia and thermal algesia. ALDH2 activity was measured by enzymatic assay. 4-HNE protein adducts and enzyme/protein expression levels were determined by western blot analysis and histology/immunohistochemistry. SCI resulted in a sustained and prolonged overload of 4-HNE, which parallels with the decreased activity of ALDH2 and low functional recovery. Alda-1 treatment of SCI decreased 4-HNE load and enhanced the activity of ALDH2 in both the acute and the chronic phases of SCI. Furthermore, the treatment with Alda-1 reduced neuroinflammation, oxidative stress, and neuronal loss and increased adenosine 5'-triphosphate levels stimulated the neurorepair process and improved locomotor and sensory functions. Conclusively, the results provide evidence that enhancing the ALDH2 activity by Alda-1 treatment of SCI mice suppresses the 4-HNE load that attenuates neuroinflammation and neurodegeneration, promotes the neurorepair process, and improves functional outcomes. Consequently, we suggest that Alda-1 may have therapeutic potential for the treatment of human SCI. Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of MUSC (IACUC-2019-00864) on December 21, 2019.

The Serum SIRT1 Protein is Associated with the Severity of Injury and Neurological Recovery in Mice with Traumatic Spinal Cord Injury.

The present study aimed to investigate the association between the serum SIRT1 protein and the severity of spinal cord injury (SCI) as well as the neurological recovery in mice. In this study, the wild-type (WT), Mx1-Cre+ SIRT1loxP/loxP (Mx1), and LCK-Cre+SIRT1loxP/loxP (LCK) mice were subjected to sham surgery, mild, moderate, or severe SCI, respectively. The serum was collected at intervals of 12 h, 1 day (d), 3 d, 5 d, 7 d, 10 d, 14 d, and 21 d after the injury. The locomotor function of all the animals was assessed using the Basso mouse scale (BMS) and the serum SIRT1 proteins were analyzed using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that about 7-10 d after SCI, the levels of SIRT1 protein in the serum correlated significantly with the severity of the injury and at 28 d post-injury, there was a distant neurological recovery (BMS score). The serum SIRT1 concentration in both the Mx1 and LCK mice in the sham group was significantly reduced compared to that in the WT mice, and there was a delayed increase in the serum SIRT1 levels after injury. These findings indicate that the SIRT1 concentrations in the serum of the SCI mice closely correlated with the acute severity and neurological outcome.

Intravenous injection of adult human bone marrow mesenchymal stromal cells attenuates spinal cord ischemia/reperfusion injury in a murine aortic arch crossclamping model.

Objective: We sought to investigate the efficacy of human bone marrow mesenchymal stem/stromal cell (hBM-MSC) in a murine spinal cord ischemia/reperfusion (SCIR) model. Methods: C57BL/6J mice were subjected to SCIR by crossclamping the aortic arch and left subclavian artery for 5.5 minutes. Two hours after reperfusion, hBM-MSCs (hBM-MSC group) or phosphate-buffered saline (control group) were intravenously injected without immunosuppressant. Hindlimb motor function was assessed until day 28 after reperfusion using the Basso Mouse Scale (BMS). The lumbar spinal cord was harvested at hour 24 and day 28, and the histologic number of NeuN-positive motor neurons in 3 cross-sections of each lumbar spinal cord and the gene expression were evaluated. Results: BMS score was 0 throughout the study period in all control mice. BMS score was significantly greater in the hBM-MSC group than the control group from hour 8 (P < .05) to day 28 (P < .01). The numbers of motor neurons at hour 24 (P < .01) and day 28 (P < .05) were significantly preserved in the hBM-MSC group than the control group. mRNA expression levels of proinflammatory cytokines were significantly lower (P < .05), and those of insulin-like growth factor-1 (P < .01) and proangiogenic factors (P < .05) were significantly greater in the hBM-MSC group than the control group at hour 24. Conclusions: hBM-MSC therapy may attenuate SCIR injury by preserving motor neurons, at least in part, through inhibition of proinflammatory cytokines and upregulation of proangiogenic factors in the reperfusion-injured spinal cord.

Matrine promotes neural circuit remodeling to regulate motor function in a mouse model of chronic spinal cord injury.

In chronic phase of spinal cord injury, functional recovery is more untreatable compared with early intervention in acute phase of spinal cord injury. In the last decade, several combination therapies successfully improved motor dysfunction in chronic spinal cord injury. However, their effectiveness is not sufficient. We previously found a new effective compound for spinal cord injury, matrine, which induced axonal growth and functional recovery in acute spinal cord injury mice via direct activation of extracellular heat shock protein 90. Although our previous study clarified that matrine was an activator of extracellular heat shock protein 90, the potential of matrine for spinal cord injury in chronic phase has not been sufficiently evaluated. Thus, this study aimed to investigate whether matrine ameliorates chronic spinal cord injury in mice. Once daily intragastric administration of matrine (100 µmol/kg per day) to spinal cord injury mice were starte at 28 days after injury, and continued for 154 days. Continuous matrine treatment improved hindlimb motor function in chronic spinal cord injury mice. In injured spinal cords of the matrine-treated mice, the density of neurofilament-H-positive axons was increased. Moreover, matrine treatment increased the density of bassoon-positive presynapses in contact with choline acetyltransferase-positive motor neurons in the lumbar spinal cord. These findings suggest that matrine promotes remodeling and reconnection of neural circuits to regulate hindlimb movement. All protocols were approved by the Committee for Animal Care and Use of the Sugitani Campus of the University of Toyama (approval No. A2013INM-1 and A2016INM-3) on May 7, 2013 and May 17, 2016, respectively.

Effects of FABP7 on functional recovery after spinal cord injury in adult mice.

OBJECTIVEElucidating the mechanisms of neuronal injury is crucial for the development of spinal cord injury (SCI) treatments. Brain-type fatty acid-binding protein 7 (FABP7) is expressed in the adult rodent brain, especially in astrocytes, and has been reported to play a role in astrocyte function in various types of brain damage; however, its role after SCI has not been well studied. In this study, the authors evaluated the expression change of FABP7 after SCI using a mouse spinal cord compression model and observed the effect of FABP7 gene knockout on neuronal damage and functional recovery after SCI.METHODSFemale FABP7 knockout (KO) mice with a C57BL/6 background and their respective wild-type littermates were subjected to SCI with a vascular clip. The expression of FABP7, neuronal injury, and functional recovery after SCI were analyzed in both groups of mice.RESULTSWestern blot analysis revealed upregulation of FABP7 in the wild-type mice, which reached its peak 14 days after SCI, with a significant difference in comparison to the control mice. Immunohistochemistry also showed upregulation of FABP7 at the same time points, mainly in proliferative astrocytes. The number of surviving ventral neurons in the FABP7-KO mice at 28 days after SCI was significantly lower than that observed in the wild-type mice. In addition, motor functional recovery in the FABP7-KO mice was significantly worse than that of the wild-type mice.CONCLUSIONSThe findings of this study indicate that FABP7 could have a neuroprotective role that might be associated with modulation of astrocytes after SCI. FABP7 could potentially be a therapeutic target in the treatment of SCI.

A combination of mesenchymal stem cells and scaffolds promotes motor functional recovery in spinal cord injury: a systematic review and meta-analysis.

OBJECTIVE: There is controversy about the role of scaffolds as an adjunctive therapy to mesenchymal stem cell (MSC) transplantation in spinal cord injury (SCI). Thus, the authors aimed to design a meta-analysis on preclinical evidence to evaluate the effectiveness of combination therapy of scaffold + MSC transplantation in comparison with scaffolds alone and MSCs alone in improving motor dysfunction in SCI. METHODS: Electronic databases including Medline, Embase, Scopus, and Web of Science were searched from inception until the end of August 2018. Two independent reviewers screened related experimental studies. Animal studies that evaluated the effectiveness of scaffolds and/or MSCs on motor function recovery following experimental SCI were included. The findings were reported as standardized mean difference (SMD) and 95% confidence interval (CI). RESULTS: A total of 34 articles were included in the meta-analysis. Analyses show that combination therapy in comparison with the scaffold group alone (SMD 2.00, 95% CI 1.53-2.46, p < 0.0001), the MSCs alone (SMD 1.58, 95% CI 0.84-2.31, p < 0.0001), and the nontreated group (SMD 3.52, 95% CI 2.84-4.20, p < 0.0001) significantly improved motor function recovery. Co-administration of MSCs + scaffolds only in the acute phase of injury (during the first 3 days after injury) leads to a significant recovery compared to scaffold alone (SMD 2.18, p < 0.0001). In addition, the cotransplantation of scaffolds with bone marrow-derived MSCs (SMD 1.99, p < 0.0001) and umbilical cord-derived MSCs (SMD 1.50, p = 0.001) also improved motor function following SCI. CONCLUSIONS: The findings showed that scaffolds + MSCs is more effective than scaffolds and MSCs alone in improving motor function following SCI in animal models, when used in the acute phase of injury.

Lentivirus-mediated silencing of the CTGF gene suppresses the formation of glial scar tissue in a rat model of spinal cord injury.

BACKGROUND CONTEXT: One of the many reactive changes following a spinal cord injury (SCI) is the formation of a glial scar, a reactive cellular process whereby glial cells accumulate and surround the central nervous system injury sites to seal in the wound. Thus, the inhibition of glial scar is of great importance for SCI recovery. PURPOSE: This study aimed to explore the effect of lentivirus-mediated silencing of the CTGF gene on the formation of glial scar tissue in a rat model of SCI. STUDY DESIGN: This is a prospective study. STUDY SAMPLE: A total of 56 Wistar female rats aged 8 weeks were randomly selected for this study. OUTCOME MEASURES: The motor function of the rats was assessed using the Basso, Beattie, and Bresnahan (BBB) functional scale, footprint analysis of gait, and the Basso Mouse Scale (BMS). Quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry were performed to detect the mRNA and protein expressions of glial fibrillary acidic protein (GFAP), vimentin, fibronectin, and laminin in the spinal cord tissues. METHODS: A rat model of SCI was successfully established. Fifty-six male Wistar rats were randomly selected and assigned into four groups (14 rats in each group): the sham operation group, the SCI model group, the negative control (NC) group (SCI rats transfected with empty vector plasmids), and the siRNA-CTGF group (SCI rats transfected with lentivirus CTGF siRNA). RESULTS: The SCI rats showed decreased activity and were dragging their bodies while moving. Compared with the sham operation group, the BBB and BMS scores in the SCI model, NC, and siRNA-CTGF groups significantly decreased. However, the BBB and BMS scores in the siRNA-CTGF group were higher than those in the SCI model and NC groups. The mRNA and protein expressions of GFAP, vimentin, fibronectin, and laminin significantly increased in the SCI model, NC, and siRNA-CTGF groups in comparison with those in the sham operation group. Furthermore, the mRNA and protein expressions of GFAP, vimentin, fibronectin, and laminin in the siRNA-CTGF group were lower than those in the SCI model and NC groups 28 days after transfection. CONCLUSIONS: These findings indicate that lentivirus-mediated silencing of the CTGF gene can suppress the formation of glial scar tissue after SCI.

A progressive compression model of thoracic spinal cord injury in mice: function assessment and pathological changes in spinal cord.

Non-traumatic injury accounts for approximately half of clinical spinal cord injury, including chronic spinal cord compression. However, previous rodent spinal cord compression models are mainly designed for rats, few are available for mice. Our aim is to develop a thoracic progressive compression mice model of spinal cord injury. In this study, adult wild-type C57BL/6 mice were divided into two groups: in the surgery group, a screw was inserted at T9 lamina to compress the spinal cord, and the compression was increased by turning it further into the canal (0.2 mm) post-surgery every 2 weeks up to 8 weeks. In the control group, a hole was drilled into the lamina without inserting a screw. The results showed that Basso Mouse Scale scores were lower and gait worsened. In addition, the degree of hindlimb dysfunction in mice was consistent with the degree of spinal cord compression. The number of motor neurons in the anterior horn of the spinal cord was reduced in all groups of mice, whereas astrocytes and microglia were gradually activated and proliferated. In conclusion, this progressive compression of thoracic spinal cord injury in mice is a preferable model for chronic progressive spinal cord compression injury.

Basophils are dispensable for the recovery of gross locomotion after spinal cord hemisection injury.

Basophils are the smallest population of granulocytes found in the circulation. They have crucial and nonredundant roles in allergic disorders, in protection from parasite infections, in autoimmunity, and in the regulation of type 2 immunity. They share phenotypic and functional properties with mast cells, which exert substantial protective effects after traumatic brain injury and spinal cord injury, although they are considered one of the most proinflammatory cell types in the body. In contrast, the in vivo functions of basophils in central nervous system trauma are still obscure and not well studied. In this study, we show that by comparing spinal cord injury in wild type vs. basophil-deficient Mcpt8Cre transgenic mice, the locomotor recovery is not affected in mice depleted in basophils. In addition, no substantial differences were observed in the lesion size and in the astrocytic and macrophage/microglia reaction between both mouse strains. Hence, despite the multiple properties shared with mast cells, these data show, for the first time, to our knowledge, that basophils are dispensable for the functional recovery process after hemisection injury to the spinal cord in mice.

Fenbendazole improves pathological and functional recovery following traumatic spinal cord injury.

During a study of spinal cord injury (SCI), mice in our colony were treated with the anthelmintic fenbendazole to treat pinworms detected in other mice not involved in the study. As this was not part of the original experimental design, we subsequently compared pathological and functional outcomes of SCI in female C57BL/6 mice who received fenbendazole (150 ppm, 8 mg/kg body weight/day) for 4 weeks prior to moderate contusive SCI (50 kdyn force) as compared to mice on the same diet without added fenbendazole. The fenbendazole-treated mice exhibited improved locomotor function, determined using the Basso mouse scale, as well as improved tissue sparing following contusive SCI. Fenbendazole may exert protective effects through multiple possible mechanisms, one of which is inhibition of the proliferation of B lymphocytes, thereby reducing antibody responses. Autoantibodies produced following SCI contribute to the axon damage and locomotor deficits. Fenbendazole pretreatment reduced the injury-induced CD45R-positive B cell signal intensity and IgG immunoreactivity at the lesion epicenter 6 weeks after contusive SCI in mice, consistent with a possible effect on the immune response to the injury. Fenbendazole and related benzimadole antihelmintics are FDA approved, exhibit minimal toxicity, and represent a novel group of potential therapeutics targeting secondary mechanisms following SCI.

Compression injury in the mouse spinal cord elicits a specific proliferative response and distinct cell fate acquisition along rostro-caudal and dorso-ventral axes.

Harnessing the regenerative capabilities of endogenous precursor cells in the spinal cord may be a useful tool for clinical treatments aimed at replacing cells lost as a consequence of disease or trauma. To better understand the proliferative properties and differentiation potential of the adult spinal cord after injury, we used a mouse model of compression spinal cord injury (SCI). After injury, adult mice were administered BrdU to label mitotic cells and sacrificed at different time-points for immunohistochemical analysis. Our data show that the rate of proliferation increased in all regions of the spinal cord 1day after injury, peaked after 3days, and remained elevated for at least 14days after injury. Proliferation was greater at the injury epicenter than in rostral and caudal adjacent spinal segments. The number of proliferative cells and rate of proliferation varied between dorsal and ventral regions of the spinal cord and between the gray and white matter. Newly generated cells expressed markers for progenitor cells (Nestin and Olig2), oligodendrocytes (Sox10), astrocytes (S100b and glial fibrillary acidic protein), and microglia (Iba1), but not neuronal markers (Map2 and NeuN). Marker expression varied with regard to the dorso-ventral region, rostro-caudal proximity to the injury epicenter, and time after injury. At early time-points after injury, BrdU(+) cells mainly expressed microglial/macrophage and astrocytic markers, while at these same time-points in the control spinal cord the mitotic cells predominately expressed oligodendrocyte and oligodendrocyte progenitor cell markers. The profile of proliferation and cell fate marker expression indicates that after moderate compression, the spinal cord has the capacity to generate a variety of glial cells but not neurons, and that this pattern is space and time specific. Future studies should focus on ways to control proliferation and cell fate after injury to aid the development of cell replacement treatments for SCI.

Functional consequences of ethidium bromide demyelination of the mouse ventral spinal cord.

Ethidium bromide (EB) has been extensively used in the rat as a model of spinal cord demyelination. However, this lesion has not been addressed in the adult mouse, a model with unlimited genetic potential. Here we characterize behavioral function, inflammation, myelin status and axonal viability following bilateral injection of 0.20 mg/mL ethidium bromide or saline into the ventral white matter (VWM) of female C57Bl/6 mice. EB-induced VWM demyelination significantly reduced spared VWM and Basso Mouse Scale (BMS) scores persisting out to 2 months. Chronic hindlimb dysfunction was accompanied by a persistent inflammatory response (demonstrated by CD45(+) immunofluorescence) and axonal loss (demonstrated by NF-M immunofluorescence and electron microscopy; EM). These cellular responses differ from the rat where inflammation resolves by 3-4 weeks and axon loss is minimal following EB demyelination. As these data suggest that EB-injection in the mouse spinal cord is a non-remyelinating lesion, we sought to ask whether wheel running could promote recovery by enhancing plasticity of local lumbar circuitry independent of remyelination. This did not occur as BMS and Treadscan assessment revealed no significant effect of wheel running on recovery. However, this study defines the importance of descending ventral motor pathways to locomotor function in the mouse as VWM loss results in a chronic hindlimb deficit.