Grafted Human Embryonic Progenitors Expressing Neurogenin-2 Stimulate Axonal Sprouting and Improve Motor Recovery after Severe Spinal Cord Injury

7 p.Background: Spinal cord injury (SCI) is a widely spread pathology with currently no effective treatment for any symptom. Regenerative medicine through cell transplantation is a very attractive strategy and may be used in different non-exclusive ways to promote functional recovery. We investigated functional and structural outcomes after grafting human embryonic neural progenitors (hENPs) in spinal cord-lesioned rats.Methods and Principal Findings: With the objective of translation to clinics we have chosen a paradigm of delayed grafting, i.e., one week after lesion, in a severe model of spinal cord compression in adult rats. hENPs were either naive or engineered to express Neurogenin 2 (Ngn2). Moreover, we have compared integrating and non-integrating lentiviral vectors, since the latter present reduced risks of insertional mutagenesis. We show that transplantation of hENPs transduced to express Ngn2 fully restore weight support and improve functional motor recovery after severe spinal cord compression at thoracic level. This was correlated with partial restoration of serotonin innervations at lumbar level, and translocation of 5HT1A receptors to the plasma membrane of motoneurons. Since hENPs were not detectable 4 weeks after grafting, transitory expression of Ngn2 appears sufficient to achieve motor recovery and to permit axonal regeneration. Importantly, we also demonstrate that transplantation of naive hENPs is detrimental to functional recovery.Conclusions and Significance: Transplantation and short-term survival of Ngn2-expressing hENPs restore weight support after SCI and partially restore serotonin fibers density and 5HT1A receptor pattern caudal to the lesion. Moreover, grafting of naive-hENPs was found to worsen the outcome versus injured only animals, thus pointing to the possible detrimental effect of stem cell-based therapy per se in SCI. This is of major importance given the increasing number of clinical trials involving cell grafting developed for SCI patients.This study was supported by the European Union FP6 "RESCUE" STREP; the "Institut pour la Recherche sur la Moelle Epiniere"; the "Academie de Medecine"; the "Societe Francaise de Neurochirurgie"; "Verticale" and the "Association Demain Debout Aquitaine". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Use of the propensity score in observational studies evaluating a surgical procedure

La chirurgie a évolué dans les dernières décennies. Les techniques ont été rapidement modifiées et sont de plus en plus proposées. L’évaluation des techniques chirurgicales est donc devenue un enjeu de santé publique. Cependant l’évaluation des procédures chirurgicales par des essais contrôlés randomisés est compliquée. Les preuves apportées par des études observationnelles sont donc des alternatives même si celles-ci sont entachées de biais de confusion. L’utilisation du score de propension dans l’analyse statistique des études observationnelles est une méthode qui permet de réduire ce biais et d’augmenter la validité interne de ces études. Le score de propension (SP) est défini pour un patient comme la probabilité de recevoir le traitement évalué, en fonction de ses caractéristiques avant traitement (caractéristiques de base). Ainsi après utilisation du score de propension, les distributions des caractéristiques de base du groupé traité et non traité doivent être similaires. Cette méthode est de plus en plus utilisée en particulier pour évaluer des interventions chirurgicales. Les objectifs de ce travail étaient 1) de comparer l’effet traitement estimé par les études observationnelles utilisant un score de propension et l’effet traitement estimé par des ECR en chirurgie ; 2) de faire une description précise de l’utilisation du score de propension dans les études observationnelles évaluant des interventions chirurgicales. Notre premier travail repose sur une étude méta-épidémiologique. Cette étude incluait 31 questions cliniques qui avaient été traitées dans la littérature par au moins une étude observationnelle utilisant un score de propension et au moins un essai contrôlé randomisé. Après extraction des effets traitement de chacun des 2 types d’études nous avons regroupé les résultats pour l’ensemble des questions cliniques. Nous n’avons pas retrouvé en moyenne de différence statistiquement significative pour l’estimation de l’effet traitement entre les ECR et les études observationnelles avec SP. Cependant, nous avons pu constater des variations dans les 2 sens pouvant parfois être importantes pour certaines questions cliniques. Dans la deuxième partie de cette thèse, nous avons réalisé une revue systématique méthodologique des études observationnelles évaluant une intervention chirurgicale utilisant un score de propension. Sur les 652 articles sélectionnés entre 1980 et 2014, nous avons montré une évolution importante et rapide du nombre d’articles au cours du temps, passant de moins de 10 articles par an avant 2000 à plus de 200 depuis 2013. Dans l’analyse plus fine de 129 articles récents, nous avons pu mettre en avant des limites méthodologiques (analyse avec score de propension mal détaillée, variables incluses dans le score de propension non rapportées, pas ou peu d’information sur les données manquantes, non-respect du principe de l’analyse en intention de traiter). Ainsi, des recommandations pour améliorer la planification, la réalisation, l’analyse et l’écriture du rapport des études observationnelles avec SP ont pu être faite. Les études observationnelles avec score de propension sont de plus en plus fréquentes. Leurs avantages par rapport aux essais contrôlés randomisés dans le domaine de la chirurgie en font une alternative intéressante. En revanche, des limites méthodologiques existent. Certaines limites peuvent être corrigées facilement, d’autres méritent des analyses plus approfondies.Surgery has evolved over the last few decades. Techniques have changed rapidly and are increasingly being proposed. The evaluation of surgical techniques has therefore become a public health issue. However, the evaluation of surgical procedures by randomised controlled trials is complicated. The evidence provided by observational studies is therefore an alternative, even if it is marred by confounding bias. The use of propensity scores in the statistical analysis of observational studies is a method that reduces this bias and increases the internal validity of these studies. The propensity score (PS) is defined for a patient as the probability of receiving the treatment under evaluation, as a function of the patient's pre-treatment characteristics (baseline characteristics). Thus, after using the propensity score, the distributions of the baseline characteristics of the treated and untreated groups should be similar. This method is increasingly used, particularly for evaluating surgical procedures. The objectives of this work were 1) to compare the treatment effect estimated by observational studies using a propensity score and the treatment effect estimated by RCTs in surgery; 2) to provide a precise description of the use of the propensity score in observational studies evaluating surgical interventions. Our first work is based on a meta-epidemiological study. This study included 31 clinical questions that had been addressed in the literature by at least one observational study using a propensity score and at least one randomised controlled trial. After extracting the treatment effects from each of the 2 types of study, we pooled the results for all the clinical questions. On average, we did not find any statistically significant difference for the estimation of the treatment effect between the RCTs and the observational studies with MS. However, we were able to observe variations in both directions, which could sometimes be significant for certain clinical questions. In the second part of this thesis, we conducted a systematic methodological review of observational studies evaluating surgery using a propensity score. Of the 652 articles selected between 1980 and 2014, we showed a significant and rapid change in the number of articles over time, from less than 10 articles per year before 2000 to more than 200 since 2013. In a more detailed analysis of 129 recent articles, we were able to highlight methodological limitations (analysis with propensity score poorly detailed, variables included in the propensity score not reported, no or little information on missing data, failure to comply with the principle of intention-to-treat analysis). As a result, recommendations have been made to improve the planning, conduct, analysis and reporting of observational studies with MS. Observational studies with propensity scores are becoming increasingly common. Their advantages over randomised controlled trials in surgery make them an interesting alternative. However, there are methodological limitations. Some limitations can be easily corrected, while others merit more in-depth analysis

Is hospital information system relevant to detect surgical site infection? Findings from a prospective surveillance study in posterior instrumented spinal surgery

International audienceOBJECT:Spinal instrumentation has a high rate of surgical site infection (SSI), but results greatly vary depending on surveillance methodology, surgical procedures, or quality of follow-up. Our aim was to study true incidence of SSI in spinal surgery by significant data collection, and to compare it with the results obtained through the hospital information system.METHODS:This work is a single center prospective cohort study that included all patients consecutively operated on for spinal instrumentation by posterior approach over a six-month period regardless the etiology. For all patients, a "high definition" prospective method of surveillance was performed by the infection control (IC) department during at least 12 months after surgery. Results were then compared with findings from automatic surveillance though the hospital information system (HIS).RESULTS:One hundred and fifty-four patients were included. We found no hardly difference between "high definition" and automatic surveillance through the HIS, even if HIS tended to under-estimate the infection rate: rate of surgical site infection was 2.60% and gross SSI incidence rate via the hospital information system was 1.95%. Smoking and alcohol consumption were significantly related to a SSI.CONCLUSION:Our SSI rates to reflect the true incidence of infectious complications in posterior instrumented adult spinal surgery in our hospital and these results were consistent with the lower levels of published infection rate. In-house surveillance by surgeons only is insufficiently sensitive. Further studies with more patients and a longer inclusion time are needed to conclude if SSI case detection through the HIS could be a relevant and effective alternative method

Assessment of the Radiation Exposure of Surgeons and Patients During a Lumbar Microdiskectomy and a Cervical Microdiskectomy: A French Prospective Multicenter Study

International audienceOBJECTIVE:Cervical and lumbar disk herniations are the most frequently carried out procedures in spinal surgery. Often, a few snapshots during the procedure are necessary to validate the level or to position the implant. The objective of this study is to quantitatively estimate the radiation received by a spine surgeon and patient during a low-dose radiation procedure.METHODS:We conducted a prospective multicenter study in France from November 2014 to April 2015. Four spine centers were monitored for radiation received by surgeons during interventions for lumbar disk herniation and cervical disk herniation.RESULTS:A total of 134 patients were included. For lumbar disk herniation, the average exposure for the surgeon was 0.584 μSv on the chest, 5.291 μSv on the lens, and 9.295 μSv on the hands per procedure. For these procedures, the dose area product (DAP) was 94.2 ± 198.4 cGy·cm(2), and the fluoroscopic time was 10.2 ± 16.9 seconds. For a herniated cervical disk, the average exposure for the surgeon was 0.122 μSv on the chest, 3.106 μSv on the lens, and 7.143 μSv on the hands per procedure. For these procedures, the DAP was 35.7 ± 72.1 cGy·cm(2), and the fluoroscopic time was 19.7 ± 13.7 seconds.CONCLUSIONS:Exposure to x-rays for surgeons and patients during surgery for lumbar disk herniation is higher than during surgery for cervical herniation disk. Our results show that radiation exposure to the spine surgeon is still far below the annual dose limits

Potential adverse effects of cyclosporin A on kidneys after spinal cord injury

Study design:Cell transplantation strategies are gaining increasing interest for spinal cord injury (SCI) with the objective of promoting spinal cord repair. To avoid allogenic graft rejection, an adequate immune suppression is required, and one of the most potent and commonly used immunosuppressives is cyclosporin A (CsA). In SCI, permanent sensory motor loss is combined with modifications of drug absorption, distribution and elimination.Objectives:The objectives of this study were to thoroughly explore histological and functional outcomes of CsA treatment in a rat model of spinal cord compression.Setting: Experiments were carried out at the Institute for Neurosciences of Montpellier (France), the Integrative Biology of Neurodegeneration Laboratory (Spain) and in the Novartis Institutes for BioMedical Research (Switzerland) for CsA blood concentration determination.Methods:We first evaluated histological outcomes of CsA treatment on kidneys and spinal cord after SCI. We then investigated whether SCI modified CsA blood concentration. Finally, using behavioral analysis, we assessed the potential CsA impact on functional recovery.Results:When spinal-cord-injured rats were treated with a CsA dose of 10 mg kg -1 per day, we observed deleterious effects on kidneys, associated with modifications of CsA blood concentration. Adding an antibiotic treatment reduced kidney alteration without modifying CsA blood concentration. Finally, we showed that CsA treatment per se modified neither functional recovery nor lesion extension.Conclusion:This study pinpoints the absolute requirement of careful CsA monitoring in the clinical setting for patients with SCI to minimize potential unexpected effects and avoid therapeutic failure.Peer reviewe

Surgical-site infection in spinal injury: incidence and risk factors in a prospective cohort of 518 patients

International audiencePURPOSE:To investigate the incidence of surgical-site infection (SSI) and determinate the risk factors of SSI in the context of spinal injury.METHODS:From February 1, 2011 to July 31, 2011, for a multicentre cohort of patients with acute spinal injury, we prospectively censored those with SSI for at least 12 months. We recorded epidemiologic characteristics and details of surgical procedure and postoperative care for each patient. We calculated the incidence of SSI at 1, 3 and 12 months after surgery. Univariate and multivariate analysis were used to establish the association of risk factors and SSI. We studied clinical outcomes by a visual analog scale for pain and physical and mental component summaries (PCS and MCS) of the Medical Outcomes Survey 36-Item Short Form (SF-36).RESULTS:At 1 year, among 518 patients, we recorded 25 SSI events, with median occurrence at 16 days (25-75 % quartile: 13-44 days). Incidence of SSI was 3.2 % (95 % confidence interval [1.9-5.3 %]) at 1 month, 3.7 % (95 % [2.2-5.8 %]) at 3 months and 4.6 % (95 % CI [3-6.9 %]) at 12 months. On multivariate analysis, age, presence of diabetes and surgical duration were predictors of SSI (p = 0.009, p = 0.047, and p = 0.015 respectively). At 12 months, infected and non-infected patients did not differ in pain (p = 0.58) or SF-36 PCS (p = 0.8) or MCS (p = 0.68).CONCLUSIONS:In this large prospective multicentre study in the context of spinal injury, we obtained an equivalent incidence rate and risk factors of SSI as found in the literature for elective spinal surgery

A Combination of Ex vivo Diffusion MRI and Multiphoton to Study Microglia/Monocytes Alterations after Spinal Cord Injury

Central nervous system (CNS) injury has been observed to lead to microglia activation and monocytes infiltration at the lesion site. Ex vivo diffusion magnetic resonance imaging (diffusion MRI or DWI) allows detailed examination of CNS tissues, and recent advances in clearing procedures allow detailed imaging of fluorescent-labeled cells at high resolution. No study has yet combined ex vivo diffusion MRI and clearing procedures to establish a possible link between microglia/monocytes response and diffusion coefficient in the context of spinal cord injury (SCI). We carried out ex vivo MRI of the spinal cord at different time-points after spinal cord transection followed by tetrahydrofuran based clearing and examined the density and morphology of microglia/monocytes using two-photon microscopy. Quantitative analysis revealed an early marked increase in microglial/monocytes density that is associated with an increase in the extension of the lesion measured using diffusion MRI. Morphological examination of microglia/monocytes somata at the lesion site revealed a significant increase in their surface area and volume as early as 72 hours post-injury. Time-course analysis showed differential microglial/monocytes response rostral and caudal to the lesion site. Microglia/monocytes showed a decrease in reactivity over time caudal to the lesion site, but an increase was observed rostrally. Direct comparison of microglia/monocytes morphology, obtained through multiphoton, and the longitudinal apparent diffusion coefficient (ADC), measured with diffusion MRI, highlighted that axonal integrity does not correlate with the density of microglia/monocytes or their somata morphology. We emphasize that differential microglial/monocytes reactivity rostral and caudal to the lesion site may thus coincide, at least partially, with reported temporal differences in debris clearance. Our study demonstrates that the combination of ex vivo diffusion MRI and two-photon microscopy may be used to follow structural tissue alteration. Lesion extension coincides with microglia/monocytes density; however, a direct relationship between ADC and microglia/monocytes density and morphology was not observed. We highlighted a differential rostro-caudal microglia/monocytes reactivity that may correspond to a temporal difference in debris clearance and axonal integrity. Thus, potential therapeutic strategies targeting microglia/monocytes after SCI may need to be adjusted not only with the time after injury but also relative to the location to the lesion site

A Novel Translational Model of Spinal Cord Injury in Nonhuman Primate

International audienceSpinal cord injuries (SCI) lead to major disabilities affecting > 2.5 million people worldwide. Major shortcomings in clinical translation result from multiple factors, including species differences, development of moderately predictive animal models, and differences in methodologies between preclinical and clinical studies. To overcome these obstacles, we first conducted a comparative neuroanatomical analysis of the spinal cord between mice, Microcebus murinus (a nonhuman primate), and humans. Next, we developed and characterized a new model of lateral spinal cord hemisection in M. murinus. Over a 3-month period after SCI, we carried out a detailed, longitudinal, behavioral follow-up associated with in vivo magnetic resonance imaging (1H-MRI) monitoring. Then, we compared lesion extension and tissue alteration using 3 methods: in vivo 1H-MRI, ex vivo 1H-MRI, and classical histology. The general organization and glial cell distribution/morphology in the spinal cord of M. murinus closely resembles that of humans. Animals assessed at different stages following lateral hemisection of the spinal cord presented specific motor deficits and spinal cord tissue alterations. We also found a close correlation between 1H-MRI signal and microglia reactivity and/or associated post-trauma phenomena. Spinal cord hemisection in M. murinus provides a reliable new nonhuman primate model that can be used to promote translational research on SCI and represents a novel and more affordable alternative to larger primates

Correlation of in vivo and ex vivo H-1-MRI with histology in two severities of mouse spinal cord injury

Spinal cord injury (SCI) is a debilitating neuropathology with no effective treatment. Magnetic resonance imaging (MRI) technology is the only method used to assess the impact of an injury on the structure and function of the human spinal cord. Moreover, in pre-clinical SCI research, MRI is a non-invasive method with great translational potential since it provides relevant longitudinal assessment of anatomical and structural alterations induced by an injury. It is only recently that MRI techniques have been effectively used for the follow-up of SCI in rodents. However, the vast majority of these studies have been carried out on rats and when conducted in mice, the contusion injury model was predominantly chosen. Due to the remarkable potential of transgenic mice for studying the pathophysiology of SCI, we examined the use of both in and ex vivo 1H-MRI (9.4 T) in two severities of the mouse SCI (hemisection and over-hemisection) and documented their correlation with histological assessments. We demonstrated that a clear distinction between the two injury severities is possible using in and ex vivo 1H-MRI and that ex vivo MR images closely correlate with histology. Moreover, tissue modifications at a remote location from the lesion epicenter were identified by conventional ex vivo MRI analysis. Therefore, in vivo MRI has the potential to accurately identify in mice the progression of tissue alterations induced by SCI and is successfully implemented by ex vivo MRI examination. This combination of in and ex vivo MRI follow-up associated with histopathological assessment provides a valuable approach for further studies intended to evaluate therapeutic strategies on SCI