Cord Injury In Mice Research Articles

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca(2+) signaling using two-photon microscopy.

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

A novel closed-body model of spinal cord injury caused by high-pressure air blasts produces extensive axonal injury and motor impairments

Diffuse axonal injury is thought to be the basis of the functional impairments stemming from mild traumatic brain injury. To examine how axons are damaged by traumatic events, such as motor vehicle accidents, falls, sports activities, or explosive blasts, we have taken advantage of the spinal cord with its extensive white matter tracts. We developed a closed-body model of spinal cord injury in mice whereby high-pressure air blasts targeted to lower thoracic vertebral levels produce tensile, compressive, and shear forces within the parenchyma of the spinal cord and thereby cause extensive axonal injury. Markers of cytoskeletal integrity showed that spinal cord axons exhibited three distinct pathologies: microtubule breakage, neurofilament compaction, and calpain-mediated spectrin breakdown. The dorsally situated axons of the corticospinal tract primarily exhibited microtubule breakage, whereas all three pathologies were common in the lateral and ventral white matter. Individual axons typically demonstrated only one of the three pathologies during the first 24h after blast injury, suggesting that the different perturbations are initiated independently of one another. For the first few days after blast, neurofilament compaction was frequently accompanied by autophagy, and subsequent to that, by the fragmentation of degenerating axons. TuJ1 immunolabeling and mice with YFP-reporter labeling each revealed more extensive microtubule breakage than did βAPP immunolabeling, raising doubts about the sensitivity of this standard approach for assessing axonal injury. Although motor deficits were mild and largely transient, some aspects of motor function gradually worsened over several weeks, suggesting that a low level of axonal degeneration continued past the initial wave. Our model can help provide further insight into how to intervene in the processes by which initial axonal damage culminates in axonal degeneration, to improve outcomes after traumatic injury. Importantly, our findings of extensive axonal injury also caution that repeated trauma is likely to have cumulative adverse consequences for both brain and spinal cord.

Correction: Hesp et al., Chronic Oligodendrogenesis and Remyelination after Spinal Cord Injury in Mice and Rats

Correction: Hesp et al., Chronic Oligodendrogenesis and Remyelination after Spinal Cord Injury in Mice and Rats

Local Injection of Lenti-BDNF at the Lesion Site Promotes M2 Macrophage Polarization and Inhibits Inflammatory Response After Spinal Cord Injury in Mice.

There is much evidence to suggest that brain-derived neurotrophic factor (BDNF) is a prominent candidate in promoting neuroprotection, axonal regeneration, and synaptic plasticity following spinal cord injury (SCI). Although some evidence indicates that BDNF has potent anti-oxidative effects and may be involved in the regulation of the immune response, the effects of BDNF in the inflammatory response during the course of secondary damage after SCI is still unclear. The present study was designed to investigate the effects of BDNF with a special focus on their effect on macrophage polarization after SCI. Adult C57 mice underwent T10 spinal cord clip compression injury and received lenti-BDNF vector injections at the epicenter of the lesion site. Four days later, total BDNF levels were greatly increased in animals that received lenti-BDNF injections. Confocal imaging showed that more than 80% of the lenti-virus infected cells were CD11b-positive macrophages. In addition, the expression of arginase-1 and CD206 (associated with M2 macrophage phenotype) significantly increased in the animals that received lenti-BDNF injections compared with those that received lenti-EGFP injections. On the contrary, the expression of CD16/32 and inducible nitric oxide synthase (M1 phenotype marker) was down-regulated as demonstrated using flow cytometry and immunohistochemistry. Furthermore, the production of interleukin 1β and tumor necrosis factor alpha was significantly reduced whereas the levels of interleukin 10 and interleukin 13 were elevated in subjects that received lenti-BDNF vector injections. The time course of functional recovery revealed that gradual recovery was observed in the subacute phase in lenti-BDNF group, little improvement was observed in lenti-EGFP group. At the axonal level, significant retraction of the CST axons were observed in lenti-EGFP injected animals relative to lenti-BDNF group by biotinylated dextran amine tracing. In addition, compared to lenti-BDNF group markedly demyelination was observed in the lenti-EGFP group using luxol fast blue staining. In conclusion, we found that BDNF could promote the shift of M1 to M2 phenotype and ameliorate the inflammatory microenvironment. Furthermore, the roles of BDNF in immunity modulation may enhance neuroprotective effects and partially contribute to the locomotor functional recovery after SCI.

B Cell‐Targeted Therapy with Anti‐CD20 Monoclonal Antibody Reduced Secondary Tissue Damage and Enhanced Behavioral Recovery Following Experimental Spinal Cord Injury in Mice

Traumatic injury to the spinal cord activates B cells, which culminates in the synthesis of autoantibodies. The functional significance of this immune response is unclear. Antibodies produced after SCI caused pathology, in part by activating intraspinal complement and cells bearing Fc receptors. These data indicate that B cells, through the production of antibodies, affect pathology in SCI. There is increasing appreciation of the important role of B cells in spinal cord trauma and consequently, increasing interest in treating these disorders through B cell‐depletion therapy. The purpose of this study was to investigate the effects of anti‐CD20 mAb B cell depletion therapy within the first 24 hours of SCI.In this study, the T6‐T7 vertebrae in three groups of mice were injured by a 50‐g clip‐induced compression method, and the anti‐murine CD20 IgG2a antibody (clone 18B12, 1 mg/kg) was intravenously administered starting 1 hour and 6 hours postinjury. Animals were sacrified at 24 hours. The anti‐CD20 antibody treatment caused significant attenuation of leukocyte infiltration, reactive oxygen species‐associated enzymes, and secondary tissue damage. Basso‐Beattie‐Bresnahan (BBB) scores were significantly higher in anti‐CD20‐treated mice than controls 10 days postinjury. Our data demonstrate an important role of B cells which could possibly lead to B cell‐based strategies for the treatment of SCI.

Chronic Oligodendrogenesis and Remyelination after Spinal Cord Injury in Mice and Rats

Adult progenitor cells proliferate in the acutely injured spinal cord and their progeny differentiate into new oligodendrocytes (OLs) that remyelinate spared axons. Whether this endogenous repair continues beyond the first week postinjury (wpi), however, is unknown. Identifying the duration of this response is essential for guiding therapies targeting improved recovery from spinal cord injury (SCI) by enhancing OL survival and/or remyelination. Here, we used two PDGFRα-reporter mouse lines and rats injected with a GFP-retrovirus to assess progenitor fate through 80 d after injury. Surprisingly, new OLs were generated as late as 3 months after injury and their processes ensheathed axons near and distal to the lesion, colocalized with MBP, and abutted Caspr+ profiles, suggesting newly formed myelin. Semithin sections confirmed stereotypical thin OL remyelination and few bare axons at 10 wpi, indicating that demyelination is relatively rare. Astrocytes in chronic tissue expressed the pro-OL differentiation and survival factors CNTF and FGF-2. In addition, pSTAT3+ NG2 cells were present through at least 5 wpi, revealing active signaling of the Jak/STAT pathway in these cells. The progenitor cell fate genes Sox11, Hes5, Id2, Id4, BMP2, and BMP4 were dynamically regulated for at least 4 wpi. Collectively, these data verify that the chronically injured spinal cord is highly dynamic. Endogenous repair, including oligodendrogenesis and remyelination, continues for several months after SCI, potentially in response to growth factors and/or transcription factor changes. Identifying and understanding spontaneous repair processes such as these is important so that beneficial plasticity is not inadvertently interrupted and effort is not exerted to needlessly duplicate ongoing spontaneous repair.

All roads go to Salubrinal: endoplasmic reticulum stress, neuroprotection and glial scar formation.

All roads go to Salubrinal: endoplasmic reticulum stress, neuroprotection and glial scar formation.

Transplantation of human bone marrow stromal cell-derived neuroregenrative cells promotes functional recovery after spinal cord injury in mice.

Transplantation of bone marrow stromal cells (BMSCs) for spinal cord injury (SCI) has been shown to improve functional outcome. BMSCs can be easily obtained from bone marrow aspirate and have fewer problems in the clinical application for human SCI from the ethical and legal points of view. Recently, we produced cells with neural stem and/or progenitor cell property and neural regeneration supporting capacity from human bone marrow stromal cells (human bone marrow stromal cell-derived neuroregenerative cells: hBMSC-NRs). The aim of the present study was to clarify the effectiveness of transplantation of hBMSC-NRs to injured spinal cord of severe combined immunodeficiency (NOD/SCID) mice. Neurite outgrowth assay of PC-12 cells was performed. One week after a T9-level contusion SCI, hBMSCs or hBMSC-NRs were transplanted into the spinal cord. After the transplantation, functional and histological examinations were performed. Conditioned media of hBMSC-NRs significantly promoted the neurite outgrowth of PC-12 cells in vitro. Transplanted hBMSC-NRs survived in the injured spinal cord 8 weeks after SCI. Immunohistochemistry revealed that the density of serotonin-positive fibers of the transplanted group was significantly higher than that of the control group at the epicenter and caudal segment to the injured site. The recovery of hind limb function of the hBMSC-NRs group was significantly better than that of the control group. In conclusion, hBMSC-NRs can be one of the realistic candidates for cell transplantation therapy for human SCI.

The neuron regrowth is associated with the proliferation of neural precursor cells after leukemia inhibitory factor administration following spinal cord injury in mice.

ObjectivesTo explore whether LIF could promote the proliferation of neural precursor cells (NPCs) and to analyze the correlation between increased NPCs and FluoroGold (FG) labeled neurons in mice after spinal cord injury (SCI).MethodsMotor behavior was assessed using Rotarod and Platform Hang tests; neurons in the corticospinal and rubrospinal systems were labeled with FG, NPCs were immustained with nestin-FITC conjugate. The numbers of FG-labeled neurons and NPCs were estimated, and the correlation between FG-labeled neurons and NPCs was assessed.ResultsMice in the SCI group showed negligible recovery of locomotor behavior; in contrast, mice in the LIF group showed a statically significant improvement. Both FG-labeled neurons and NPCs were significantly increased in the LIF group compared to the SCI group, and this increase in FG-labeled neurons and NPCs showed a clear association above the lesion level.ConclusionsLIF could promote locomotive behaviors in mice post-SCI by encouraging the proliferation of NPCs; LIF may in fact be a potential cytokine for the induction of NPCs post-SCI.

Aldose Reductase Regulates Microglia/Macrophages Polarization Through the cAMP Response Element-Binding Protein After Spinal Cord Injury in Mice.

Inflammatory reactions are the most critical pathological processes occurring after spinal cord injury (SCI). Activated microglia/macrophages have either detrimental or beneficial effects on neural regeneration based on their functional polarized M1/M2 subsets. However, the mechanism of microglia/macrophage polarization to M1/M2 at the injured spinal cord environment remains unknown. In this study, wild-type (WT) or aldose reductase (AR)-knockout (KO) mice were subjected to SCI by a spinal crush injury model. The expression pattern of AR, behavior tests for locomotor activity, and lesion size were assessed at between 4h and 28days after SCI. We found that the expression of AR is upregulated in microglia/macrophages after SCI in WT mice. In AR KO mice, SCI led to smaller injury lesion areas compared to WT. AR deficiency-induced microglia/macrophages induce the M2 rather than the M1 response and promote locomotion recovery after SCI in mice. In the in vitro experiments, microglia cell lines (N9 or BV2) were treated with the AR inhibitor (ARI) fidarestat. AR inhibition caused 4-hydroxynonenal (HNE) accumulation, which induced the phosphorylation of the cAMP response element-binding protein (CREB) to promote Arg1 expression. KG501, the specific inhibitor of phosphorylated CREB, could cancel the upregulation of Arg1 by ARI or HNE stimulation. Our results suggest that AR works as a switch which can regulate microglia by polarizing cells to either the M1 or the M2 phenotype under M1 stimulation based on its states of activity. We suggest that inhibiting AR may be a promising therapeutic method for SCI in the future.

Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice

Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n=22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.

Acute hyperglycaemia exacerbates spinal cord injury in mice, and predicts poor recovery in human patients

Acute hyperglycaemia exacerbates spinal cord injury in mice, and predicts poor recovery in human patients

Central but not peripheral administration of XPro1595 is therapeutic following moderate spinal cord injury in mice

Central but not peripheral administration of XPro1595 is therapeutic following moderate spinal cord injury in mice

Acute hyperglycemia impairs functional improvement after spinal cord injury in mice and humans.

Spinal cord injury (SCI) is a devastating disorder for which the identification of exacerbating factors is urgently needed. We demonstrate that transient hyperglycemia during acute SCI is a detrimental factor that impairs functional improvement in mice and human patients after acute SCI. Under hyperglycemic conditions, both in vivo and in vitro, inflammation was enhanced through promotion of the nuclear translocation of the nuclear factor κB (NF-κB) transcription factor in microglial cells. During acute SCI, hyperglycemic mice exhibited progressive neural damage, with more severe motor deficits than those observed in normoglycemic mice. Consistent with the animal study findings, a Pearson χ(2) analysis of data for 528 patients with SCI indicated that hyperglycemia on admission (glucose concentration ≥126 mg/dl) was a significant risk predictor of poor functional outcome. Moreover, a multiple linear regression analysis showed hyperglycemia at admission to be a powerful independent risk factor for a poor motor outcome, even after excluding patients with diabetes mellitus with chronic hyperglycemia (regression coefficient, -1.37; 95% confidence interval, -2.65 to -0.10; P < 0.05). Manipulating blood glucose during acute SCI in hyperglycemic mice rescued the exacerbation of pathophysiology and improved motor functional outcomes. Our findings suggest that hyperglycemia during acute SCI may be a useful prognostic factor with a negative impact on motor function, highlighting the importance of achieving tight glycemic control after central nervous system injury.

Excitability changes in the sciatic nerve and triceps surae muscle after spinal cord injury in mice.

BackgroundFrom the onset to the chronic phase of spinal cord injury (SCI), peripheral axons and muscles are subjected to abnormal states of activity. This starts with very intense spasms during the first instant of SCI, through a no activity flaccidity phase, to a chronic hyperactivity phase. It remains unclear how the nature of this sequence may affect the peripheral axons and muscles.MethodsWe set out to investigate the changes in excitability of the sciatic nerve and to characterize the properties of muscle contractility after contusive injury of the mouse thoracic spinal cord.ResultsThe following changes were observed in animals after SCI: 1) The sciatic nerve compound action potential was of higher amplitudes and lower threshold, with the longer strength-duration time constant and faster conduction velocity; 2) The latency of the onset of muscle contraction of the triceps surae muscle was significantly shorter in animals with SCI; 3) The muscle twitches expressed slower rising and falling slopes, which were accompanied by prolonged contraction duration in SCI animals compared to controls.ConclusionThese findings suggest that in peripheral nerves SCI promotes hyperexcitability, which might contribute to mechanisms of spastic syndrome.

CCR5 blockade promotes M2 macrophage activation and improves locomotor recovery after spinal cord injury in mice.

Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Chemokine Receptor 5 (CCR5), a major co-receptor for macrophage-tropic human immunodeficiency viruses, is expressed on the surface of monocytes/macrophages, dendritic cells, activated T cells, and NK cells. Recent papers have indicated the important role of CCR5 in SCI, but the mechanism is still unknown. In our current study, CCR5 blockade displayed increased myelin sparring and enhanced SC repair process. The number of CD4(+) T cells, CD8(+) T cells, Ly6G(+) neutrophils and CD11b(+) macrophages were all significantly lower in the anti-CCR5 group than that in the control group after SCI. The IL-4 and IL-13 levels in anti-CCR5 group were markedly higher than that in control group after SCI. Correspondingly, the anti-CCR5-treated group showed increased numbers of Arg1- or CD206-expressing macrophages compared with the control IgG group. Furthermore, CCR5 blockade promoted PPARγ activation, and the increased numbers of M2 macrophages induced by CCR5 blockade were both reversed with additional PPARγ antagonist treatment. In conclusion, our present work provides evidence to support the concept that CCR5 blockade promotes M2 macrophage activation and improves locomotor recovery after SCI in mice.

Central but not systemic administration of XPro1595 is therapeutic following moderate spinal cord injury in mice.

BackgroundGlial cell activation and overproduction of inflammatory mediators in the central nervous system (CNS) have been implicated in acute traumatic injuries to the CNS, including spinal cord injury (SCI). Elevated levels of the proinflammatory cytokine tumor necrosis factor (TNF), which exists in both a soluble (sol) and a transmembrane (tm) form, have been found in the lesioned cord early after injury. The contribution of solTNF versus tmTNF to the development of the lesion is, however, still unclear.MethodsWe tested the effect of systemically or centrally blocking solTNF alone, using XPro1595, versus using the drug etanercept to block both solTNF and tmTNF compared to a placebo vehicle following moderate SCI in mice. Functional outcomes were evaluated using the Basso Mouse Scale, rung walk test, and thermal hyperalgesia analysis. The inflammatory response in the lesioned cord was investigated using immunohistochemistry and western blotting analyses.ResultsWe found that peripheral administration of anti-TNF therapies had no discernable effect on locomotor performances after SCI. In contrast, central administration of XPro1595 resulted in improved locomotor function, decreased anxiety-related behavior, and reduced damage to the lesioned spinal cord, whereas central administration of etanercept had no therapeutic effects. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI.ConclusionsThese studies suggest that, by selectively blocking solTNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. This protection may be mediated via alteration of the inflammatory environment without suppression of the neuroprotective effects of tmTNF signaling through TNFR2.

Tegaserod, a small compound mimetic of polysialic acid, promotes functional recovery after spinal cord injury in mice

In a previous study, we have shown that the small organic compound tegaserod, a drug approved for clinical application in an unrelated condition, is a mimic of the regeneration-beneficial glycan polysialic acid (PSA) in a mouse model of femoral nerve injury. Several independent observations have shown positive effects of PSA and its mimetic peptides in different paradigms of injury of the central and peripheral mammalian nervous systems. Since small organic compounds generally have advantages over metabolically rapidly degraded glycans and the proteolytically vulnerable mimetic peptides, a screen for a small PSA mimetic compound was successfully carried out, and the identified molecule proved to be beneficial in neurite outgrowth in vitro, independent of its originally described function as a 5-HT4 receptor agonist. In the present study, a mouse spinal cord compression device was used to elicit severe compression injury. We show that tegaserod promotes hindlimb motor function at 6weeks after spinal cord injury compared to the control group receiving vehicle only. Immunohistology of the spinal cord rostral and caudal to the lesion site showed increased numbers of neurons, and a reduced area and intensity of glial fibrillary acidic protein immunoreactivity. Quantification of regrowth/sprouting of axons immunoreactive for tyrosine hydroxylase and serotonin showed increased axonal density rostral and caudal to the injury site in the ventral horns of mice treated with tegaserod. The combined observations suggest that tegaserod has the potential for treatment of spinal cord injuries in higher vertebrates.

New reliable scoring system, Toyama mouse score, to evaluate locomotor function following spinal cord injury in mice.

BackgroundAmong the variety of methods used to evaluate locomotor function following a spinal cord injury (SCI), the Basso Mouse Scale score (BMS) has been widely used for mice. However, the BMS mainly focuses on hindlimb movement rather than on graded changes in body support ability. In addition, some of the scoring methods include double or triple criteria within a single score, which likely leads to an increase in the deviation within the data. Therefore we aimed to establish a new scoring method reliable and easy to perform in mice with SCI.FindingsOur Toyama Mouse Score (TMS) was established by rearranging and simplifying the BMS score and combining it with the Body Support Scale score (BSS). The TMS reflects changes in both body support ability and hindlimb movement. The definition of single score is made by combing multiple criteria in the BMS. The ambiguity was improved in the TMS. Using contusive SCI mice, hindlimb function was measured using the TMS, BMS and BSS systems. The TMS could distinguish changes in hindlimb movements that were evaluated as the same score by the BMS. An analysis of the coefficient of variation (CV) of score points recorded for 11 days revealed that the CV for the TMS was significantly lower than the CV obtained using the BMS. A variation in intra evaluators was lower in the TMS than in the BMS.ConclusionThese results suggest that the TMS may be useful as a new reliable method for scoring locomotor function for SCI models.