Longitudinal Magnetic Resonance Imaging Tracking of Transplanted Neural Progenitor Cells in the Spinal Cord Utilizing the Bright-Ferritin Mechanism.

Human neural progenitor cells (hNPCs) hold promise for treating spinal cord injury. Studies to date have focused on improving their regenerative potential and therapeutic effect. Equally important is ensuring successful delivery and engraftment of hNPCs at the injury site. Unfortunately, no current imaging solution for cell tracking is compatible with long-term monitoring in vivo. The objective of this study was to apply a novel bright-ferritin magnetic resonance imaging (MRI) mechanism to track hNPC transplants longitudinally and on demand in the rat spinal cord. We genetically modified hNPCs to stably overexpress human ferritin. Ferritin-overexpressing (FT) hNPCs labeled with 0.2 mM manganese provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, morphology, proliferation, and differentiation. In vivo, 2 M cells were injected into the cervical spinal cord of Rowett nude rats. MRI employed T1-weighted acquisitions and T1 mapping on a 3 T scanner. Conventional short-term cell tracking was performed using exogenous Mn labeling prior to cell transplantation, which displayed transient bright contrast on MRI 1 day after cell transplantation and disappeared after 1 week. In contrast, long-term cell tracking using bright-ferritin allowed on-demand signal recall upon Mn supplementation and precise visualization of the surviving hNPC graft. In fact, this new cell tracking technology identified 7 weeks post-transplantation as the timepoint by which substantial hNPC integration occurred. Spatial distribution of hNPCs on MRI matched that on histology. In summary, bright-ferritin provides the first demonstration of long-term, on-demand, high-resolution, and specific tracking of hNPCs in the rat spinal cord.

Minimizing Blood Loss in Spine Surgery.

STUDY DESIGN: Broad narrative review. OBJECTIVE: To review and summarize the current literature on guidelines, outcomes, techniques and indications surrounding multiple modalities of minimizing blood loss in spine surgery. METHODS: A thorough review of peer-reviewed literature was performed on the guidelines, outcomes, techniques, and indications for multiple modalities of minimizing blood loss in spine surgery. RESULTS: There is a large body of literature that provides a consensus on guidelines regarding the appropriate timing of discontinuation of anticoagulation, aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), and herbal supplements prior to surgery. Additionally, there is a more heterogenous discussion the utility of preoperative autologous blood donation facilitated by erythropoietin and iron supplementation for healthy patients slated for procedures with high anticipated blood loss and for whom allogeneic transfusion is likely. Intraoperative maneuvers available to minimize blood loss include positioning and maintaining normothermia. Tranexamic acid (TXA), bipolar sealer electrocautery, and topical hemostatic agents, and hypotensive anesthesia (mean arterial pressure (MAP) <65 mm Hg) should be strongly considered in cases with larger exposures and higher anticipated blood loss. There is strong level 1 evidence for the use of TXA in spine surgery as it reduces the overall blood loss and transfusion requirements. CONCLUSION: As the volume and complexity of spinal procedures rise, intraoperative blood loss management has become a pivotal topic of research within the field. There are many tools for minimizing blood loss in patients undergoing spine surgery. The current literature supports combining techniques to use a cost- effective multimodal approach to minimize blood loss in the perioperative period.

Traumatic spinal cord injury.

Traumatic spinal cord injury (SCI) has devastating consequences for the physical, social and vocational well-being of patients. The demographic of SCIs is shifting such that an increasing proportion of older individuals are being affected. Pathophysiologically, the initial mechanical trauma (the primary injury) permeabilizes neurons and glia and initiates a secondary injury cascade that leads to progressive cell death and spinal cord damage over the subsequent weeks. Over time, the lesion remodels and is composed of cystic cavitations and a glial scar, both of which potently inhibit regeneration. Several animal models and complementary behavioural tests of SCI have been developed to mimic this pathological process and form the basis for the development of preclinical and translational neuroprotective and neuroregenerative strategies. Diagnosis requires a thorough patient history, standardized neurological physical examination and radiographic imaging of the spinal cord. Following diagnosis, several interventions need to be rapidly applied, including haemodynamic monitoring in the intensive care unit, early surgical decompression, blood pressure augmentation and, potentially, the administration of methylprednisolone. Managing the complications of SCI, such as bowel and bladder dysfunction, the formation of pressure sores and infections, is key to address all facets of the patient's injury experience.

Translating state-of-the-art spinal cord MRI techniques to clinical use: A systematic review of clinical studies utilizing DTI, MT, MWF, MRS, and fMRI.

BACKGROUND: A recent meeting of international imaging experts sponsored by the International Spinal Research Trust (ISRT) and the Wings for Life Foundation identified 5 state-of-the-art MRI techniques with potential to transform the field of spinal cord imaging by elucidating elements of the microstructure and function: diffusion tensor imaging (DTI), magnetization transfer (MT), myelin water fraction (MWF), MR spectroscopy (MRS), and functional MRI (fMRI). However, the progress toward clinical translation of these techniques has not been established. METHODS: A systematic review of the English literature was conducted using MEDLINE, MEDLINE-in-Progress, Embase, and Cochrane databases to identify all human studies that investigated utility, in terms of diagnosis, correlation with disability, and prediction of outcomes, of these promising techniques in pathologies affecting the spinal cord. Data regarding study design, subject characteristics, MRI methods, clinical measures of impairment, and analysis techniques were extracted and tabulated to identify trends and commonalities. The studies were assessed for risk of bias, and the overall quality of evidence was assessed for each specific finding using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework. RESULTS: A total of 6597 unique citations were identified in the database search, and after full-text review of 274 articles, a total of 104 relevant studies were identified for final inclusion (97% from the initial database search). Among these, 69 studies utilized DTI and 25 used MT, with both techniques showing an increased number of publications in recent years. The review also identified 1 MWF study, 11 MRS studies, and 8 fMRI studies. Most of the studies were exploratory in nature, lacking a priori hypotheses and showing a high (72%) or moderately high (20%) risk of bias, due to issues with study design, acquisition techniques, and analysis methods. The acquisitions for each technique varied widely across studies, rendering direct comparisons of metrics invalid. The DTI metric fractional anisotropy (FA) had the strongest evidence of utility, with moderate quality evidence for its use as a biomarker showing correlation with disability in several clinical pathologies, and a low level of evidence that it identifies tissue injury (in terms of group differences) compared with healthy controls. However, insufficient evidence exists to determine its utility as a sensitive and specific diagnostic test or as a tool to predict clinical outcomes. Very low quality evidence suggests that other metrics also show group differences compared with controls, including DTI metrics mean diffusivity (MD) and radial diffusivity (RD), the diffusional kurtosis imaging (DKI) metric mean kurtosis (MK), MT metrics MT ratio (MTR) and MT cerebrospinal fluid ratio (MTCSF), and the MRS metric of N-acetylaspartate (NAA) concentration, although these results were somewhat inconsistent. CONCLUSIONS: State-of-the-art spinal cord MRI techniques are emerging with great potential to improve the diagnosis and management of various spinal pathologies, but the current body of evidence has only showed limited clinical utility to date. Among these imaging tools DTI is the most mature, but further work is necessary to standardize and validate its use before it will be adopted in the clinical realm. Large, well-designed studies with a priori hypotheses, standardized acquisition methods, detailed clinical data collection, and robust automated analysis techniques are needed to fully demonstrate the potential of these rapidly evolving techniques.

Investigational drugs for the treatment of spinal cord injury: review of preclinical studies and evaluation of clinical trials from Phase I to II.

INTRODUCTION: Efforts in basic research have clarified mechanisms involved in spinal cord injury (SCI), and resulted in positive findings using experimental treatments including cell transplantation and drug administration preclinically. Based on accumulated results, various clinical trials have begun for human SCI. AREAS COVERED: In this review, the authors focus on five investigational drugs: riluzole, minocycline, Rho protein antagonist, magnesium chloride in polyethylene glycol formulation, and basic fibroblast growth factor. All drugs have established safety and tolerability from Phase I clinical trials, and are now in Phase II. They have been proven to have neuroprotective and/or neuroregenerative effects in animal models of SCI. EXPERT OPINION: To date, diverse drugs have been translated into clinical trials, but none have reached clinical application. A key gap was the lack of reliable biomarkers for SCI to fast-track Phase I/II trials. Furthermore, problems were often due to lack of adequate outcome assessments for both animal models and SCI patients. In order to advance clinical trials more quickly and with greater success, more clinically relevant animal models should be used in basic research. Clinically, it is indispensable to use appropriate outcome measurements and to construct a wide network among clinical centers to validate the efficacy of drugs.

Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model of cervical spinal cord injury.

Riluzole, a sodium/glutamate antagonist has shown promise as a neuroprotective agent. It is licensed for amyotrophic lateral sclerosis and is in clinical trial development for spinal cord injury (SCI). This study investigated the therapeutic time-window and pharmacokinetics of riluzole in a rodent model of cervical SCI. Rats were treated with riluzole (8 mg/kg) at 1 hour (P1) and 3 hours (P3) after injury or with vehicle. Afterward, P1 and P3 groups received riluzole (6 (mg/kg) every 12 hours for 7 days. Both P1 and P3 animals had significant improvements in locomotor recovery as measured by open field locomotion (BBB score, BBB subscore). Von Frey stimuli did not reveal an increase in at level or below level mechanical allodynia. Sensory-evoked potential recordings and quantification of axonal cytoskeleton demonstrated a riluzole-mediated improvement in axonal integrity and function. Histopathological and retrograde tracing studies demonstrated that delayed administration leads to tissue preservation and reduces apoptosis and inflammation. High performance liquid chromatography (HPLC) was undertaken to examine the pharmacokinetics of riluzole. Riluzole penetrates the spinal cord in 15 min, and SCI slowed elimination of riluzole from the spinal cord, resulting in a longer half-life and higher drug concentration in spinal cord and plasma. Initiation of riluzole treatment 1 and 3 hours post-SCI led to functional, histological, and molecular benefits. While extrapolation of post-injury time windows from rat to man is challenging, evidence from SCI-related biomarker studies would suggest that the post-injury time window is likely to be at least 12 hours in man.

Translational potential of preclinical trials of neuroprotection through pharmacotherapy for spinal cord injury.

There is a need to enhance the pipeline of discovery and evaluation of neuroprotective pharmacological agents for patients with spinal cord injury (SCI). Although much effort and money has been expended on discovering effective agents for acute and subacute SCI, no agents that produce major benefit have been proven to date. The deficiencies of all aspects of the pipeline, including the basic science input and the clinical testing output, require examination to determine remedial strategies. Where has the neuroprotective/pharmacotherapy preclinical process failed and what needs to be done to achieve success? These are the questions raised in the present review, which has 2 objectives: 1) identification of articles that address issues related to the translational readiness of preclinical SCI pharmacological therapies; and 2) examination of the preclinical studies of 5 selected agents evaluated in animal models of SCI (including blunt force trauma, penetrating trauma, or ischemia). The 5 agents were riluzole, glyburide, magnesium sulfate, nimodipine, and minocycline, and these were selected because of their promise of translational readiness as determined by the North American Clinical Trials Network Consortium. The authors found that there are major deficiencies in the effort that has been extended to coordinate and conduct preclinical neuroprotection/pharmacotherapy trials in the SCI field. Apart from a few notable exceptions such as the NIH effort to replicate promising strategies, this field has been poorly coordinated. Only a small number of articles have even attempted an overall evaluation of the neuroprotective/pharmacotherapy agents used in preclinical SCI trials. There is no consensus about how to select the agents for translation to humans on the basis of their preclinical performance and according to agreed-upon preclinical performance criteria. In the absence of such a system and to select the next agent for translation, the Consortium has developed a Treatment Strategy Selection Committee, and this committee selected the most promising 5 agents for potential translation. The results show that the preclinical work on these 5 agents has left numerous gaps in knowledge about their preclinical performance and confirm the need for significant changes in preclinical neuroprotection/pharmacotherapy trials in SCI. A recommendation is made for the development and validation of a preclinical scoring system involving worldwide experts in preclinical and clinical SCI.

Spinal cord clinical trials and the role for bioengineering.

There is considerable need for bringing effective therapies for spinal cord injury (SCI) to the clinic. Excellent medical and surgical management has mitigated poor prognoses after SCI; however, few advances have been made to return lost function. Bioengineering approaches have shown great promise in preclinical rodent models, yet there remains a large translational gap to carry these forward in human trials. Herein, we provide a framework of human clinical trials, an overview of past trials for SCI, as well as bioengineered approaches that include: directly applied pharmacologics, cellular transplantation, biomaterials and functional neurorehabilitation. Success of novel therapies will require the correct application of comprehensive preclinical studies with well-designed and expertly conducted human clinical trials. While biologics and bioengineered strategies are widely considered to represent the high potential benefits for those who have sustained a spinal injury, few such therapies have been thoroughly tested with appreciable efficacy for use in human SCI. With these considerations, we propose that bioengineered strategies are poised to enter clinical trials.

Human neuropathological and animal model evidence supporting a role for Fas-mediated apoptosis and inflammation in cervical spondylotic myelopathy.

Although cervical spondylotic myelopathy is a common cause of chronic spinal cord dysfunction in humans, little is known about the molecular mechanisms underlying the progressive neural degeneration characterized by this condition. Based on animal models of cervical spondylotic myelopathy and traumatic spinal cord injury, we hypothesized that Fas-mediated apoptosis and inflammation may play an important role in the pathobiology of human cervical spondylotic myelopathy. We further hypothesized that neutralization of the Fas ligand using a function-blocking antibody would reduce cell death, attenuate inflammation, promote axonal repair and enhance functional neurological outcomes in animal models of cervical spondylotic myelopathy. We examined molecular changes in post-mortem human spinal cord tissue from eight patients with cervical spondylotic myelopathy and four control cases. Complementary studies were conducted using a mouse model of cervical spondylotic myelopathy (twy/twy mice that develop spontaneous cord compression at C2-C3). We observed Fas-mediated apoptosis of neurons and oligodendrocytes and an increase in inflammatory cells in the compressed spinal cords of patients with cervical spondylotic myelopathy. Furthermore, neutralization of Fas ligand with a function-blocking antibody in twy/twy mice reduced neural inflammation at the lesion mediated by macrophages and activated microglia, glial scar formation and caspase-9 activation. It was also associated with increased expression of Bcl-2 and promoted dramatic functional neurological recovery. Our data demonstrate, for the first time in humans, the potential contribution of Fas-mediated cell death and inflammation to the pathobiology of cervical spondylotic myelopathy. Complementary data in a murine model of cervical spondylotic myelopathy further suggest that targeting the Fas death receptor pathway is a viable neuroprotective strategy to attenuate neural degeneration and optimize neurological recovery in cervical spondylotic myelopathy. Our findings highlight the possibility of medical treatments for cervical spondylotic myelopathy that are complementary to surgical decompression.

Spinal cord injury: a systematic review of current treatment options.

BACKGROUND: Spinal cord injury (SCI) is a devastating event often resulting in permanent neurologic deficit. Research has revealed an understanding of mechanisms that occur after the primary injury and contribute to functional loss. By targeting these secondary mechanisms of injury, clinicians may be able to offer improved recovery after SCI. QUESTIONS/PURPOSES: In this review, we highlight advances in the field of SCI by framing three questions: (1) What is the preclinical evidence for the neuroprotective agent riluzole that has allowed this agent to move into clinical trials? (2) What is the preclinical evidence for Rho antagonists that have allowed this group of compounds to move into clinical trials? (3) What is the evidence for early surgical decompression after SCI? METHODS: We conducted a systematic review of MEDLINE and EMBASE-cited articles related to SCI to address these questions. RESULTS: As a result of an improved understanding of the secondary mechanisms of SCI, specific clinical strategies have been established. We highlight three strategies that have made their way from bench to bedside: the sodium-glutamate antagonist riluzole, the Rho inhibitor Cethrin, and early surgical decompression. Each of these modalities is under clinical investigation. We highlight the fundamental science that led to this development. CONCLUSIONS: As our understanding of the fundamental mechanisms of SCI improves, we must keep abreast of these discoveries to translate them into therapies that will hopefully benefit patients. We summarize this process of bench to bedside with regard to SCI.

Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injury.

Activation of the Fas receptor has been recently linked to apoptotic cell death after spinal cord injury (SCI). Although it is generally considered that Fas activation mediates apoptosis predominantly through the extrinsic pathway, we hypothesized that intrinsic mitochondrial signaling could be involved in the underlying mechanism of Fas-induced apoptosis after SCI. In the present study, we utilized the Fejota clip compression model of SCI at T5-6 in C57BL/6 Fas-deficient (lpr) and wild-type mice. Complementary studies were conducted using an in vitro model of trauma or a Fas-activating antibody to induce apoptosis in primary neuronal-glial mixed spinal cord cultures. After in vivo SCI, lpr mice, in comparison with wild-type mice, exhibited reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells at the lesion, reduced expression of truncation of Bid (tBid), apoptosis-inducing factor, activated caspase-9 and activated caspase-3, and increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. After in vitro neurotrauma or the induction of Fas signaling by the Jo2 activating antibody, lpr spinal cord cultures showed an increased proportion of cells retaining mitochondrial membrane integrity and a reduction of tBid expression, caspase-9 and caspase-3 activation, and TUNEL-positive cells as compared to wild-type spinal cord cultures. The neutralization of Fas ligand (FasL) protected against traumatically induced or Fas-mediated caspase-3 activation and the loss of mitochondrial membrane potential and tBid expression in wild-type spinal cord cultures. However, in lpr spinal cord cultures, FasL neutralization had no protective effects. In summary, these data provide direct evidence for the induction of intrinsic mitochondrial signaling pathways following Fas activation after SCI.

Update on the treatment of spinal cord injury.

Acute spinal cord injury (SCI) is a devastating neurological disorder that can affect any individual at a given instance. Current treatment options for SCI include the use of high dose methylprednisolone sodium succinate, a corticosteroid, surgical interventions to stabilize and decompress the spinal cord, intensive multisystem medical management, and rehabilitative care. While utility of these therapeutic options provides modest benefits, there is a critical need to identify novel approaches to treat or repair the injured spinal cord in hope to, at the very least, improve upon the patient's quality of life. Thankfully, several discoveries at the preclinical level are now transitioning into the clinical arena. These include the Surgical Treatment for Acute Spinal Cord Injury Study (STASCIS) Trial to evaluate the role and timing of surgical decompression for acute SCI, neuroprotection with the semisynthetic second generation tetracycline derivative, minocycline; aiding axonal conduction with the potassium channel blockers, neuroregenerative/neuroprotective approaches with the Rho antagonist, Cethrin; the use of anti-NOGO monoclonal antibodies to augment plasticity and regeneration; as well as cell-mediated repair with stem cells, bone marrow stromal cells, and olfactory ensheathing cells. This review overviews the pathobiology of SCI and current treatment choices before focusing the rest of the discussion on the variety of promising neuroprotective and cell-based approaches that have recently moved, or are very close, to clinical testing.

An in vitro model of neurotrauma in organotypic spinal cord cultures from adult mice.

Cellular degeneration after spinal cord injury (SCI) involves numerous pathways. It is essential to use appropriate experimental models in order to understand the complex processes, which evolve after the initial trauma. The purpose of this study was to develop and assess an in vitro model of neurotrauma using organotypic slice culture of adult mice spinal cord. This model will facilitate the investigation of primary and secondary mechanisms of cell death that occurs after SCI. We modified previously described methods for generating organotypic cultures of murine spinal cord. The viability of organotypic cultures was assessed by observing the outgrowth of neurites and by using a mitochondria dependent dye for live cells (tetrazolium dye; MTT). The morphological integrity of cultures was examined histologically by hematoxylin and eosin (H&E) staining for general morphology and with luxol fast blue (LFB) for myelin. Neuronal and glial (GFAP; CNPase) markers were used to identify neurons, astrocytes and oligodendroglia, respectively. Primary injury was achieved by using a weight drop (0.2 g) model of injury. Cell death after primary injury was attenuated by pre-treatment with two known neuroprotective agents: the AMPA/KA blocker CNQX and methylprednisolone. The nuclear markers Propidium iodide and Sytox-green, as well as the TUNEL (in situ terminal deoxytransferase-mediated dUTP nick end labeling) technique, were used as a quantitative indicators of cell death at 24, 48 and 72 h post-injury using a confocal microscope and image analysis software. This novel in vitro model of SCI is easy to reproduce, will facilitate the examination of post-trauma cell death mechanisms and the neuroprotective effects of pharmacological agents and aid in the study of transgenic murine models.