Abel Torres-Espín

Adult Stem Cell Transplants for Spinal Cord Injury Repair: Current State in Preclinical Research

Spinal cord injury (SCI) is a traumatic disorder resulting in a functional deficit that usually leads to severe and permanent paralysis. After the initial insult to the spinal cord, additional structure and function are lost through an active and complex secondary process. Since there is not effective treatment for SCI, several strategies including cellular, pharmacological and rehabilitation therapies have been approached in animal models. Some of them have been proved in clinical trials. In this review we focus on the current state of cell therapies, particularly on cells from adult origin, assayed in preclinical research. Cell types used in SCI therapy include Schwann cells, olfactory ensheathing cells and adult stem cells, such as neural stem cells, umbilical cord blood derived cells, mesenchymal stem cells or induced pluripotent stem cells. There are not yet conclusive evidences on which types of glial or adult stem cells are most effective in SCI treatment. Their ability to incorporate into the damaged spinal cord, to differentiate into neural lineages, to exert neuroprotective effects, to promote regeneration of damaged axons, and to improve functional deficits are still discussed, before translation towards clinical use, as a single therapy or in combination with other strategies.

Bone marrow mesenchymal stromal cells and olfactory ensheathing cells transplantation after spinal cord injury – a morphological and functional comparison in rats

Cell therapy for spinal cord injury (SCI) is a promising strategy for clinical application. Both bone marrow mesenchymal stromal cells (MSCs; also known as bone marrow‐derived ‘mesenchymal stem cells’) and olfactory ensheathing cells (OECs) have demonstrated beneficial effects following transplantation in animal models of SCI. However, due to the large number of affecting parameters that determine the therapy success and the lack of methodological consensus, the comparison of different works is difficult. Therefore, we compared the effects of MSC and OEC transplants at early or delayed time after a spinal cord contusion injury in the rat. Functional outcomes for locomotion, sensory perception and electrophysiological responses were assessed. Moreover, the grafted cells survival and the amount of cavity and spared tissue were studied. The findings indicate that grafted cells survived until 7 days post‐injection, but markedly disappeared in the following 2 weeks. Despite the low survival of the cells, MSC and OEC grafts provided tissue protection after early and delayed transplantation. Nevertheless, only acute MSC grafts improved locomotion recovery in treadmill condition and electrophysiological outcomes with respect to the other injured groups. These results, together with previous works, indicate that the MSC seem a better option than OEC for treatment of contusion injuries.

Beneficial effects of IL-37 after spinal cord injury in mice

Significance Spinal cord injury (SCI) often results in severely impaired locomotor, sensory, and autonomic function. Although inflammation contributes to the physiopathology of SCI, several clinical trials of high doses of dexamethasone or methylprednisolone have not resulted in improved recovery of function. IL-37, a member of the IL-1 family, exerts broad antiinflammatory effects in several mouse models of inflammatory diseases. We report here that mice expressing human IL-37 exhibit reduced inflammation in the central nervous system and demonstrate significantly improved functional disabilities following SCI. The administration of recombinant forms of human IL-37 enhances motor skills after SCI, suggesting that IL-37 could provide a new therapeutic approach to limit the harmful effects of inflammation in neurologic conditions. IL-37, a member of the IL-1 family, broadly reduces innate inflammation as well as acquired immunity. Whether the antiinflammatory properties of IL-37 extend to the central nervous system remains unknown, however. In the present study, we subjected mice transgenic for human IL-37 (hIL-37tg) and wild-type (WT) mice to spinal cord contusion injury and then treated them with recombinant human IL-37 (rIL-37). In the hIL-37tg mice, the expression of IL-37 was barely detectable in the uninjured cords, but was strongly induced at 24 h and 72 h after the spinal cord injury (SCI). Compared with WT mice, hIL-37tg mice exhibited increased myelin and neuronal sparing and protection against locomotor deficits, including 2.5-fold greater speed in a forced treadmill challenge. Reduced levels of cytokines (e.g., an 80% reduction in IL-6) were observed in the injured cords of hIL-37tg mice, along with lower numbers of blood-borne neutrophils, macrophages, and activated microglia. We treated WT mice with a single intraspinal injection of either full-length or processed rIL-37 after the injury and found that the IL-37–treated mice had significantly enhanced locomotor skills in an open field using the Basso Mouse Scale, as well as supported faster speed on a mechanical treadmill. Treatment with both forms of rIL-37 led to similar beneficial effects on locomotor recovery after SCI. This study presents novel data indicating that IL-37 suppresses inflammation in a clinically relevant model of SCI, and suggests that rIL-37 may have therapeutic potential for the treatment of acute SCI.

Activity dependent therapies modulate the spinal changes that motoneurons suffer after a peripheral nerve injury.

Injury of a peripheral nerve not only leads to target denervation, but also induces massive stripping of spinal synapses on axotomized motoneurons, with disruption of spinal circuits. Even when regeneration is successful, unspecific reinnervation and the limited reconnection of the spinal circuits impair functional recovery. The aim of this study was to describe the changes that axotomized motoneurons suffer after peripheral nerve injury and how activity-dependent therapies and neurotrophic factors can modulate these events. We observed a marked decrease in glutamatergic synapses, with a maximum peak at two weeks post-axotomy, which was only partially reversed with time. This decrease was accompanied by an increase in gephyrin immunoreactivity and a disintegration of perineuronal nets (PNNs) surrounding the motoneurons. Direct application of neurotrophins at the proximal stump was not able to reverse these effects. In contrast, activity-dependent treatment, in the form of treadmill running, reduced the observed destructuring of perineuronal nets and the loss of glutamatergic synapses two weeks after injury. These changes were proportional to the intensity of the exercise protocol. Blockade of sensory inputs from the homolateral hindlimb also reduced PNN immunoreactivity around intact motoneurons, and in that case treadmill running did not reverse that loss, suggesting that the effects of exercise on motoneuron PNN depend on increased sensory activity. Preservation of motoneuron PNN and reduction of synaptic stripping by exercise could facilitate the maintenance of the spinal circuitry and benefit functional recovery after peripheral nerve injury.

Neurite-J: an image-J plug-in for axonal growth analysis in organotypic cultures.

BACKGROUND Previous studies in our lab proposed a method of dorsal root ganglia (DRG) and spinal cord slice (SC) organotypic 3D cultures to study motor and sensory axonal regeneration. Although these models are useful to test how different factors affect axonal growth, manual sample analysis can be inaccurate and time-consuming. Thus, we designed and set-up a plug-in to quantify axonal growth in 3D organotypic cultures. NEW METHOD DRG and SC were cultured in a 3D collagen matrix. Explants were maintained in culture medium (control condition) or in culture medium supplemented with neurotrophins. Neurites were immunolabeled against RT-97 and pictures were obtained using an epifluorescence microscope. To quantify axonal growth we adapted the Sholl method of concentric rings to our cultures and the algorithm was implemented as an ImageJ plug-in. COMPARISON WITH EXISTING METHOD(S) Our method and plug-in was compared with standard Sholl method demonstrating better accuracy. In comparison with Neurite-J, manual measures of axonal growth in organotypic cultures require more time and provide fewer data than our proposed method. RESULTS Neurite-J gives a reliable quantitative analysis of neurite growth, providing counts of neurite number and neurite area at different distances from the explant. Moreover, this plug-in follows lineal and semi-logarithmic analysis of the Sholl method, yielding a numerical value of neurite outgrowth useful for comparing different experimental conditions. CONCLUSION Neurite-J provides a quantification method of neurite arbors in 3D organotypic cultures that gives the researcher an easy, fast and reliable tool to study axonal growth.

Gene Expression Changes in the Injured Spinal Cord Following Transplantation of Mesenchymal Stem Cells or Olfactory Ensheathing Cells

Transplantation of bone marrow derived mesenchymal stromal cells (MSC) or olfactory ensheathing cells (OEC) have demonstrated beneficial effects after spinal cord injury (SCI), providing tissue protection and improving the functional recovery. However, the changes induced by these cells after their transplantation into the injured spinal cord remain largely unknown. We analyzed the changes in the spinal cord transcriptome after a contusion injury and MSC or OEC transplantation. The cells were injected immediately or 7 days after the injury. The mRNA of the spinal cord injured segment was extracted and analyzed by microarray at 2 and 7 days after cell grafting. The gene profiles were analyzed by clustering and functional enrichment analysis based on the Gene Ontology database. We found that both MSC and OEC transplanted acutely after injury induce an early up-regulation of genes related to tissue protection and regeneration. In contrast, cells transplanted at 7 days after injury down-regulate genes related to tissue regeneration. The most important change after MSC or OEC transplant was a marked increase in expression of genes associated with foreign body response and adaptive immune response. These data suggest a regulatory effect of MSC and OEC transplantation after SCI regarding tissue repair processes, but a fast rejection response to the grafted cells. Our results provide an initial step to determine the mechanisms of action and to optimize cell therapy for SCI.

Quantitative assessment of locomotion and interlimb coordination in rats after different spinal cord injuries

Animal models of spinal cord injury (SCI) are intended to mimic the main features of human spinal cord lesions, although sometimes it becomes a difficult task to find the right technique to discriminate the severity of the lesion as well as to assess different aspects of functional recovery. For this reason, we have used several functional methods to assess gross and fine locomotion deficits, as well as electrophysiological data to study the dysfunctions underlying the behavioral changes. Moreover, an extensive study based on the quantification of alternation and coordination parameters during gait has been done. Spinal cord injuries of varying severity (mild contusion, moderate contusion and hemisection) were performed at the thoracic level in adult rats that were followed-up for 6 weeks. Lesions resulting in similar scores in the open field test (i.e. mild contusion and hemisection) caused more marked differences in fine coordination when assessed by quantitative coordination analysis based on a digitized walking treadmill. In conclusion, gross and fine deficits can be detected using a battery of tests based on the performance of the animals during tasks of different difficulty. When used appropriately, they become useful tools to study functional recovery due to spontaneous plastic changes or to therapeutic interventions after SCI, as well as to test the effects of new therapies.

Increased migration of olfactory ensheathing cells secreting the Nogo receptor ectodomain over inhibitory substrates and lesioned spinal cord

Olfactory ensheathing cell (OEC) transplantation emerged some years ago as a promising therapeutic strategy to repair injured spinal cord. However, inhibitory molecules are present for long periods of time in lesioned spinal cord, inhibiting both OEC migration and axonal regrowth. Two families of these molecules, chondroitin sulphate proteoglycans (CSPG) and myelin-derived inhibitors (MAIs), are able to trigger inhibitory responses in lesioned axons. Mounting evidence suggests that OEC migration is inhibited by myelin. Here we demonstrate that OEC migration is largely inhibited by CSPGs and that inhibition can be overcome by the bacterial enzyme Chondroitinase ABC. In parallel, we have generated a stable OEC cell line overexpressing the Nogo receptor (NgR) ectodomain to reduce MAI-associated inhibition in vitro and in vivo. Results indicate that engineered cells migrate longer distances than unmodified OECs over myelin or oligodendrocyte-myelin glycoprotein (OMgp)-coated substrates. In addition, they also show improved migration in lesioned spinal cord. Our results provide new insights toward the improvement of the mechanisms of action and optimization of OEC-based cell therapy for spinal cord lesion.

Single pellet grasping following cervical spinal cord injury in adult rat using an automated full-time training robot

Task specific motor training is a common form of rehabilitation therapy in individuals with spinal cord injury (SCI). The single pellet grasping (SPG) task is a skilled forelimb motor task used to evaluate recovery of forelimb function in rodent models of SCI. The task requires animals to obtain food pellets located on a shelf beyond a slit at the front of an enclosure. Manually training and testing rats in the SPG task requires extensive time and often yields results with high outcome variability and small therapeutic windows (i.e., the difference between pre- and post-SCI success rates). Recent advances in automated SPG training using automated pellet presentation (APP) systems allow rats to train ad libitum 24h a day, 7 days a week. APP trained rats have improved success rates, require less researcher time, and have lower outcome variability compared to manually trained rats. However, it is unclear whether APP trained rats can perform the SPG task using the APP system after SCI. Here we show that rats with cervical SCI can successfully perform the SPG task using the APP system. We found that SCI rats with APP training performed significantly more attempts, had slightly lower and less variable final score success rates, and larger therapeutic windows than SCI rats with manual training. These results demonstrate that APP training has clear advantages over manual training for evaluating reaching performance of SCI rats and represents a new tool for investigating rehabilitative motor training following CNS injury.

Functional involvement of the lumbar spinal cord after contusion to T8 spinal segment of the rat.

PURPOSE To evaluate the effects on locomotion and lumbar motoneuron function after a contusion to the midthoracic spinal cord of the rat. METHODS Five animals received a moderate contusion on T8, and over 28 days postoperation (dpo) locomotion and motor electrophysiological outcome were compared with five sham-operated animals. RESULTS At 28 dpo, the contused animals supported their body weight (BBB score =11.5 ± 0.5) and stepped uncoordinatedly. Motor evoked potentials recorded in the tibialis anterior (TA) and plantar muscles (PL), and longitudinal interlimb reflexes recorded in the TA muscles were abolished. The M wave recorded in the TA showed a decrease in amplitude by 7 dpo, which remained invariable until the end of the evaluation (88 ± 3% of {preoperative} values), whereas in the PL muscle it was not affected. Injured animals presented hyperreflexia, as shown by an increased H/M ratio. Histological analysis showed similar number of retrogradely traced TA motoneurons between groups, and that contused animals presented hypertrophied astrocytes in the most rostral but not caudal segments of the lumbar enlargement. CONCLUSION These results indicate that after contusion to the thoracic spinal cord, the lumbar segments undergo structural and functional changes, following a rostro-caudal gradient extension.