Blockade of interleukin 6 signaling improves the survival rate of transplanted bone marrow stromal cells and increases locomotor function in mice with spinal cord injury

Bone marrow stromal cells (BMSCs) have the potential to improve functional recovery in patients with spinal cord injury (SCI); however, they are limited by low survival rates after transplantation in the injured tissue. Our objective was to clarify the effects of a temporal blockade of interleukin 6 (IL-6)/IL-6 receptor (IL-6R) engagement using an anti-mouse IL-6R monoclonal antibody (MR16-1) on the survival rate of BMSCs after their transplantation in a mouse model of contusion SCI. MR16-1 cotreatment improved the survival rate of transplanted BMSCs, allowing some BMSCs to differentiate into neurons and astrocytes, and improved locomotor function recovery compared with BMSC transplantation or MR16-1 treatment alone. The death of transplanted BMSCs could be mainly related to apoptosis rather than necrosis. Transplantation of BMSC with cotreatment of MR16-1 was associated with a decrease of some proinflammatory cytokines, an increase of neurotrophic factors, decreased apoptosis rates of transplanted BMSCs, and enhanced expression of survival factors Akt and extracellular signal-regulated protein kinases 1/2. We conclude that MR16-1 treatment combined with BMSC transplants helped rescue neuronal cells and axons after contusion SCI better than BMSCs alone by modulating the inflammatory/immune responses and decreasing apoptosis. © 2013 by the American Association of Neuropathologists, Inc.

Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment

Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI). However, the effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in green fluorescent protein (GFP)-positive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of protein kinase C-γ and phosphocyclic AMP response element binding protein expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (p-ERK1/2) in both hematogenous macrophages and resident microglia and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood-spinal cord barrier (BSCB), which was associated with decreased levels of (a) inflammatory cytokines (tumor necrosis factor-α, interleukin-6); (b) mediators of early secondary vascular pathogenesis (matrix metallopeptidase 9); (c) macrophage recruiting factors (CCL2, CCL5, and CXCL10), but increased levels of a microglial stimulating factor (granulocyte-macrophage colony-stimulating factor). These findings support the use of BMSC transplants for SCI treatment. Furthermore, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal-resident microglia and the activity and recruitment of hematogenous macrophages.

Blockade of interleukin-6 effects on cytokine profiles and macrophage activation after spinal cord injury in mice

The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. MR16-1 antibodies versus isotype control antibodies or saline alone was administered immediately after thoracic SCI in mice. MR16-1-treated group samples showed increased neuronal regeneration and locomotor recovery compared with controls. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. MR16-1 treatment promoted arginase-1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site and enhanced positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Development of novel therapeutics and in vitro models for spinal cord injury

Spinal cord injury (SCI) is a devastating condition, which results from trauma to the cord, resulting in a primary injury response which leads to a secondary injury cascade, causing damage to both glial and neuronal cells. Following trauma, the central nervous system (CNS) fails to regenerate due to a plethora of both intrinsic and extrinsic factors. Unfortunately, these events lead to loss of both motor and sensory function and lifelong disability and care for sufferers of SCI. There have been tremendous advancements made in our understanding of the mechanisms behind axonal regeneration and remyelination of the damaged cord. These have provided many promising therapeutic targets. However, very few have made it to clinical application, which could potentially be due to inadequate understanding of compound mechanism of action and reliance on poor SCI models. This thesis describes the use of an established neural cell co-culture model of SCI as a medium throughput screen for compounds with potential therapeutic properties. A number of compounds were screened which resulted in a family of compounds, modified heparins, being taken forward for more intense investigation. Modified heparins (mHeps) are made up of the core heparin disaccharide unit with variable sulphation groups on the iduronic acid and glucosamine residues; 2-O-sulphate (C2), 6-O-sulphate (C6) and N-sulphate (N). 2-O-sulphated (mHep6) and N-sulphated (mHep7) heparin isomers were shown to promote both neurite outgrowth and myelination in the SCI model. It was found that both mHeps decreased oligodendrocyte precursor cell (OPC) proliferation and increased oligodendrocyte (OL) number adjacent to the lesion. However, there is a difference in the direct effects on the OL from each of the mHeps; mHep6 increased myelin internode length and mHep7 increased the overall cell size. It was further elucidated that these isoforms interact with and mediate both Wnt and FGF signalling. In OPC monoculture experiments FGF2 treated OPCs displayed increased proliferation but this effect was removed when co-treated with the mHeps. Therefore, suggesting that the mHeps interact with the ligand and inhibit FGF2 signalling. Additionally, it was shown that both mHeps could be partially mediating their effects through the Wnt pathway. mHep effects on both myelination and neurite outgrowth were removed when co-treated with a Wnt signalling inhibitor, suggesting cell signalling mediation by ligand immobilisation and signalling activation as a mechanistic action for the mHeps. However, the initial methods employed in this thesis were not sufficient to provide a more detailed study into the effects the mHeps have on neurite outgrowth. This led to the design and development of a novel microfluidic device (MFD), which provides a platform to study of axonal injury. This novel device is a three chamber device with two chambers converging onto a central open access chamber. This design allows axons from two points of origin to enter a chamber which can be subjected to injury, thus providing a platform in which targeted axonal injury and the regenerative capacity of a compound study can be performed. In conclusion, this thesis contributes to and advances the study of SCI in two ways; 1) identification and investigation of a novel set of compounds with potential therapeutic potential i.e. desulphated modified heparins. These compounds have multiple therapeutic properties and could revolutionise both the understanding of the basic pathological mechanisms underlying SCI but also be a powered therapeutic option. 2) Development of a novel microfluidic device to study in greater detail axonal biology, specifically, targeted axonal injury and treatment, providing a more representative model of SCI than standard in vitro models. Therefore, the MFD could lead to advancements and the identification of factors and compounds relating to axonal regeneration.

Dipsacus asperoides (Xue Duan) inhibits spinal cord injury-induced inflammatory responses in rats

Purpose: To investigate the effect of Dipsacus asperoides (Xue Duan), a traditional Chinese medicine, on rats with spinal cord injury (SCI). Methods: In this study a total of 40 adult rats were used after inducing SCI where Xue Duan was applied on experimental group and phosphate-buffered saline (PBS) was administered in corresponding control groups. Intraperitoneal administration of both compounds for a period of four weeks (28 days) was carried out at a dose of 10 mg/kg/day. Bright field microscopy was performed on the tissues. Results: Bright Field microscopy of tissue sections showed significant reduction in cavity area that resulted from injury, that is from 0.19 ± 0.05 mm2 to 0.09 ± 0.03 mm2 (p < 0.01) in untreated and treated groups respectively. Similarly western blotting results showed a decrease in the expression of NF-kB p65 and I-kBα (p < 0.01). These two compounds are important in increasing secondary pathophysiology in SCI. The results for MPO activity also revealed significantly reduced infiltration of leukocytes to the injury site (p < 0.01). Conclusion: This study reveals the positive effect of the plant material in reducing inflammation in rats with traumatic SCI.

A preclinical study of spinal cord injury focused on cellular and molecular modifications as potential targets for innovative therapies

Spinal Cord Injury (SCI) is a devastating condition for human and animal health. In SCI particularly, neurons, oligodendrocytes precursor cells, and mature oligodendrocytes are highly vulnerable to the toxic microenvironment after the lesion and susceptible to the elevated levels of noxious stimuli. Thus the regenerative response of the organism in case of SCI is significantly reduced, and only little spontaneous amelioration is observed in lesioned patients during the early phases. This work mainly focuses on studying and characterizing the modification induced by the SCI in a preclinical animal model. We investigated the ECM composition in the spinal cord segments surrounding the primary lesion site at a gene expression level. We found Timp1 and CD44 as a crucial hub in the secondary cascade of SCI in both spinal cord segments surrounding the lesion site. Interestingly, a temporal and anatomical difference in gene expression, indicating a complex regulation of ECM genes after SCI that could be used as a tool for regenerative medicine. We also investigated the modification in synaptic plasticity-related gene expression in spinal and supraspinal areas involved in motor control. We confirmed the anatomical and temporal difference in gene expression in spinal cord tissue. This analysis suggests that a molecular mapping of the lesion-induced modification could be a useful tool for regenerative medicine. In the last part, we evaluated the efficacy of an implantable biopolymer loaded with an anti-inflammatory drug and a pro-myelinating agent on the acute phase of SCI in our preclinical model. We found a consistent reduction of the inflammatory state in the spinal lesion site and the cord's surrounding segments. Moreover, we found increased preservation of the spinal cord tissue with a related upregulation of neuronal and oligodendroglial markers after lesion. Our treatment showed effective ameliorating functional outcome and reducing the lesion extension in the chronic phase.

Increased EMG response following electromyographic biofeedback treatment of rectus femoris muscle after spinal cord injury

Objective To study increases in electromyographic (EMG) response from the right and left rectus femoris muscles of individuals with long-term cervical spinal cord injuries after EMG biofeedback treatment. Design Repeated measure trials compared EMG responses before and after biofeedback treatment in patients with spinal cord injuries. Main outcome measures The Neuroeducator was used to analyse and provide feedback of the EMG signal and to measure EMG response. Setting Department of Traumatic Orthopaedics, School of Medicine, University of Sao Paulo, Brazil. Participants Twenty subjects (three men and 17 women), between 21 and 49 years of age, with incomplete spinal cord injury at level C6 or higher (range C2 to C6). Of these subjects, 10 received their spinal cord injuries from motor vehicle accidents, one from a gunshot, five from diving, three from falls and one from spinal disc herniation. Results Significant differences were found in the EMG response of the right rectus femoris muscle between pre-initial (T1), post-initial (T2) and additional (T3) biofeedback treatment with the subjects in a sitting position [mean (standard deviation) T1: 26 mu V (29); T2: 67 mu V (50); T3: 77 mu V (62)]. The mean differences and 95% confidence intervals for these comparisons were as follows: T1 to T2, -40.7 (-53.1 to -29.4); T2 to T3, -9.6 (-26.1 to 2.3). Similar differences were found for the left leg in a sitting position and for both legs in the sit-to-stand condition. Conclusions The EMG responses obtained in this study showed that treatment involving EMG biofeedback significantly increased voluntary EMG responses from right and left rectus femoris muscles in individuals with spinal cord injuries. (C) 2010 Chartered Society of Physiotherapy. Published by Elsevier Ltd. All rights reserved.

Spinal Cord T-Cell Infiltration in the Rat Spared Nerve Injury Model: A Time Course Study.

The immune system is involved in the development of neuropathic pain. In particular, the infiltration of T-lymphocytes into the spinal cord following peripheral nerve injury has been described as a contributor to sensory hypersensitivity. We used the spared nerve injury (SNI) model of neuropathic pain in Sprague Dawley adult male rats to assess proliferation, and/or protein/gene expression levels for microglia (Iba1), T-lymphocytes (CD2) and cytotoxic T-lymphocytes (CD8). In the dorsal horn ipsilateral to SNI, Iba1 and BrdU stainings revealed microglial reactivity and proliferation, respectively, with different durations. Iba1 expression peaked at D4 and D7 at the mRNA and protein level, respectively, and was long-lasting. Proliferation occurred almost exclusively in Iba1 positive cells and peaked at D2. Gene expression observation by RT-qPCR array suggested that T-lymphocytes attracting chemokines were upregulated after SNI in rat spinal cord but only a few CD2/CD8 positive cells were found. A pronounced infiltration of CD2/CD8 positive T-cells was seen in the spinal cord injury (SCI) model used as a positive control for lymphocyte infiltration. Under these experimental conditions, we show early and long-lasting microglia reactivity in the spinal cord after SNI, but no lymphocyte infiltration was found.

The Effect of Functional Electrically Stimulated Ambulation Training on Locomotor Function and Quality of Life in Individuals With Incomplete Spinal Cord Injury

The purpose of this study was to investigate the effects of a 12-week FES-ambulation program on locomotor function and quality of life after incomplete spinal cord injury. Six individuals with incomplete SCI participated in the study. Over-ground walking endurance (6MWT), speed (10MWT), independence (WISCI II) and body-weight support were assessed. Quality of life was assessed via the SF-36, WHOQOL-BREF, Perceived Stress Scale, Center of Epidemiological Studies for Depression scale, and task self-efficacy. Participants experienced significant improvements in walking endurance (223.6±141.5m to 297.3±164.5m; p=0.03), body-weight support (55.3±12.6% to 14.7±23.2%; p= 0.005) and four of the six participants showed improvements on the WISCI II scale (1-4 points). In addition, there was a significant reduction in reported bodily pain (6.5±1.2 to 5.0±1.7; p=0.04). Therefore, FES-ambulation is an effective means for enhancing over-ground locomotor function in individuals with incomplete SCI. It may also be an effective method for reducing pain in individuals with SCI.

A prediction model for establishing the disability degree in adults with spinal cord injury: results based on WHO-DAS II perspective

Objective: To establish a prediction model of the degree of disability in adults with Spinal CordInjury (SCI ) based on the use of the WHO-DAS II . Methods: The disability degree was correlatedwith three variable groups: clinical, sociodemographic and those related with rehabilitation services.A model of multiple linear regression was built to predict disability. 45 people with sci exhibitingdiverse etiology, neurological level and completeness participated. Patients were older than 18 andthey had more than a six-month post-injury. The WHO-DAS II and the ASIA impairment scale(AIS ) were used. Results: Variables that evidenced a significant relationship with disability were thefollowing: occupational situation, type of affiliation to the public health care system, injury evolutiontime, neurological level, partial preservation zone, ais motor and sensory scores and number ofclinical complications during the last year. Complications significantly associated to disability werejoint pain, urinary infections, intestinal problems and autonomic disreflexia. None of the variablesrelated to rehabilitation services showed significant association with disability. The disability degreeexhibited significant differences in favor of the groups that received the following services: assistivedevices supply and vocational, job or educational counseling. Conclusions: The best predictiondisability model in adults with sci with more than six months post-injury was built with variablesof injury evolution time, AIS sensory score and injury-related unemployment.

Temporal modifications in bone following spinal cord injury in rats

Introduction: The aim of this study was to investigate the temporal modifications in bone mass, bone biomechanical properties and bone morphology in spinal cord injured rats 2, 4 and 6 weeks after a transection. Material and methods: Control animals were randomly distributed into four groups (n = 10 each group): control group (CG) - control animals sacrificed immediately after surgery; spinal cord-injured 2 weeks (2W) - spinal cord-injured animals sacrificed 2 weeks after surgery; spinal cord-injured 4 weeks (4W) - spinal cord-injured animals sacrificed 4 weeks after surgery; spinal cord-injured 6 weeks (6W) - spinal cord-injured animals sacrificed 6 weeks after surgery. Results: Biomechanical properties of the right tibia were determined by a threepoint bending test and injured animals showed a statistically significant decrease in maximal load compared to control animals. The right femur was used for densitometric analysis and bone mineral content of the animals sacrificed 4 and 6 weeks after surgery was significantly higher compared to the control animals and animals sacrificed 2 weeks after surgery. Histopathological and morphological analysis of tibiae revealed intense resorptive areas in the group 2 weeks after injury only. Conclusions: The results of this study show that this rat model is a valuable tool to investigate bone remodeling processes specifically associated with SCI. Taken together, our results suggest that spinal cord injury induced bone loss within 2 weeks after injury in rats.

The treatment of spasticity with Delta9-tetrahydrocannabinol in persons with spinal cord injury

STUDY DESIGN: Open label study to determine drug dose for a randomized double-blind placebo-controlled parallel study. OBJECTIVES: To assess the efficacy and side effects of oral Delta(9)-tetrahydrocannabinol (THC) and rectal THC-hemisuccinate (THC-HS) in SCI patients. SETTING: REHAB Basel, Switzerland. METHOD: Twenty-five patients with SCI were included in this three-phase study with individual dose adjustment, each consisting of 6 weeks. Twenty-two participants received oral THC open label starting with a single dose of 10 mg (Phase 1, completed by 15 patients). Eight subjects received rectal THC-HS (Phase 2, completed by seven patients). In Phase 3, six patients were treated with oral THC and seven with placebo. Major outcome parameters were the spasticity sum score (SSS) using the Modified Ashworth Scale (MAS) and self-ratings of spasticity. RESULTS: Mean daily doses were 31 mg with THC and 43 mg with THC-HS. Mean SSS for THC decreased significantly from 16.72 (+/-7.60) at baseline to 8.92 (+/-7.14) on day 43. Similar improvement was seen with THC-HS. We observed a significant improvement of SSS with active drug (P=0.001) in the seven subjects who received oral THC in Phase 1 and placebo in Phase 3. Major reasons for drop out were increase of pain and psychological side effects. CONCLUSION: THC is an effective and safe drug in the treatment of spasticity. At least 15-20 mg per day were needed to achieve a therapeutic effect.

External validation study of a clinical prediction rule for ambulation outcomes after traumatic spinal cord injury

The main objective of this study was to determine the external validity of a clinical prediction rule developed by the European Multicenter Study on Human Spinal Cord Injury (EM-SCI) to predict the ambulation outcomes 12 months after traumatic spinal cord injury. Data from the North American Clinical Trials Network (NACTN) data registry with approximately 500 SCI cases were used for this validity study. The predictive accuracy of the EM-SCI prognostic model was evaluated using calibration and discrimination based on 231 NACTN cases. The area under the receiver-operating-characteristics curve (ROC) curve was 0.927 (95% CI 0.894 – 0.959) for the EM-SCI model when applied to NACTN population. This is lower than the AUC of 0.956 (95% CI 0.936 – 0.976) reported for the EM-SCI population, but suggests that the EM-SCI clinical prediction rule distinguished well between those patients in the NACTN population who were able to achieve independent ambulation and those who did not achieve independent ambulation. The calibration curve suggests that higher the prediction score is, the better the probability of walking with the best prediction for AIS D patients. In conclusion, the EM-SCI clinical prediction rule was determined to be generalizable to the adult NACTN SCI population.^

Matrix Metalloproteinases And Left Ventricular Function And Structure In Spinal Cord Injured Subjects.

Subjects with spinal cord injury (SCI) exhibit impaired left ventricular (LV) diastolic function, which has been reported to be attenuated by regular physical activity. This study investigated the relationship between circulating matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and echocardiographic parameters in SCI subjects and the role of physical activity in this regard. Forty-two men with SCI [19 sedentary (S-SCI) and 23 physically-active (PA-SCI)] were evaluated by clinical, anthropometric, laboratory, and echocardiographic analysis. Plasmatic pro-MMP-2, MMP-2, MMP-8, pro-MMP-9, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay and zymography. PA-SCI subjects presented lower pro-MMP-2 and pro-MMP-2/TIMP-2 levels and improved markers of LV diastolic function (lower E/Em and higher Em and E/A values) than S-SCI ones. Bivariate analysis showed that pro-MMP-2 correlated inversely with Em and directly with E/Em, while MMP-9 correlated directly with LV mass index and LV end-diastolic diameter in the whole sample. Following multiple regression analysis, pro-MMP-2, but not physical activity, remained associated with Em, while MMP-9 was associated with LV mass index in the whole sample. These findings suggest differing roles for MMPs in LV structure and function regulation and an interaction among pro-MMP-2, diastolic function and physical activity in SCI subjects.

Surgical treatment of patients with cervical spinal cord injury by anterior approach using bone graft

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