Advances in Neurorestoratology-Current status and future developments.

Neurorestoratology constitutes a novel discipline aimed at the restoration of damaged neural structures and impaired neurological functions. This area of knowledge integrates and compiles all concepts and strategies dealing with the neurorestoration. Although currently, this discipline has already been well recognized by physicians and scientists throughout the world, this article aimed at broadening its knowledge to the academic circle and the public society. Here we shortly introduced why and how Neurorestoratology was born since the fact that the central nervous system (CNS) can be repaired and the subsequent scientific evidence of the neurorestorative mechanisms behind, such as neurostimulation or neuromodulation, neuroprotection, neuroplasticity, neurogenesis, neuroregeneration or axonal regeneration or sprouting, neuroreplacement, loop reconstruction, remyelination, immunoregulation, angiogenesis or revascularization, and others. The scope of this discipline is the improvement of therapeutic approaches for neurological diseases and the development of neurorestorative strategies through the comprehensive efforts of experts in the different areas and all articulated by the associations of Neurorestoratology and its journals. Strikingly, this article additionally explores the "state of art" of the Neurorestoratology field. This includes the development process of the discipline, the achievements and advances of novel neurorestorative treatments, the most efficient procedures exploring and evaluating outcome after the application of pioneer therapies, all the joining of a multidisciplinary expert associations and the specialized journals being more and more impact. We believe that in a near future, this discipline will evolve fast, leading to a general application of cell-based comprehensive neurorestorative treatments to fulfill functional recovery demands for patients with neurological deficits or dysfunctions.

Repair of central nervous system lesions by transplantation of olfactory ensheathing cells.

Clinical conditions affecting the central nervous system (CNS) fall into two main categories - degenerative conditions in which nerve cells are lost (Alzheimer's, Parkinson's, Huntington's disease, etc.), and traumatic insults which sever nerve fibers but leave their cell bodies and initial parts of the severed axons intact (spinal cord injury, cerebrovascular accidents, or tumors affecting fiber tracts). After injuries of this second type, the survival of the nerve cell bodies and the local sprouting at the severed ends of the proximal stumps of the axons raise the tantalizing possibility of one day learning how to induce these severed fibers to regenerate to their original targets and restore lost functions. This chapter gives an overview of current research into the strategy of transplantation of olfactory ensheathing cells into axotomizing injuries.

Axon regeneration can facilitate or suppress hindlimb function after olfactory ensheathing glia transplantation.

Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.

Further evidence of olfactory ensheathing glia facilitating axonal regeneration after a complete spinal cord transection.

Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT(2A) receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Clinical application of adult olfactory bulb ensheathing glia for nervous system repair.

The ability of adult olfactory bulb ensheathing glia (OB-OEG) to promote histological and functional neural repair has been broadly documented. Pre-clinical studies show that beneficial effects of adult OB-OEG are repeatable in the same type of spinal cord injury initially tested, in other spinal cord and CNS injury models, in different species and after the administration of these cells in different forms (either alone or in combination with other cells, drugs, products or devices). These studies demonstrate the reproducibility, robustness, fundamental nature and relevance of the findings. Therefore, the use of adult OB-OEG for spinal cord injury repair meets the scientific criteria established by the International Campaign for Cures of Spinal Cord Injury Paralysis (ICCP) for the translation to human application. Because there is so much heterogeneity in the way adult OEG is administered, each of these different OEG-based therapies must be individually categorized to determine whether they fulfill the requisites dictated by the consolidated regulatory body to be considered or not as a medicine. In the case they do, in Europe, they shall be subjected to the Regulatory European Framework for Advanced Therapy Medicinal Products and the European Clinical Trials Directive (Directives 2001/20/EC and 2009/120/EC). After a deep analysis of the European Regulation we have concluded that grafts consisting of suspensions of purified adult OEG, to be used for the promotion of axonal regeneration in the CNS, do not comply with the definition of Medicinal Product provided by the European Medicines Agency. In contrast, experimental therapies using OEG in combination with other cell types, drugs, products or devices, or genetically-modified OEG fall under the definitions of Medicinal Product. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Telomerase protects adult rodent olfactory ensheathing glia from early senescence.

Adult olfactory bulb ensheathing glia (OB-OEG) promote the repair of acute, subacute, and chronic spinal cord injuries and autologous transplantation is a feasible approach. There are interspecies differences between adult rodent and primate OB-OEG related to their longevity in culture. Whereas primate OB-OEG exhibit a relatively long life span, under the same culture conditions rodent OB-OEG divide just three to four times, are sensitive to oxidative stress and become senescent after the third week in vitro. Telomerase is a "physiological key regulator" of the life span of normal somatic cells and also has extratelomeric functions such as increased resistance to oxidative stress. To elucidate whether telomerase has a role in the senescence of rodent OB-OEG, we have introduced the catalytic subunit of telomerase mTERT into cultures of these cells by retroviral infection. Native and modified adult rat OB-OEG behaved as telomerase-competent cells as they divided while expressing mTERT but entered senescence once the gene switched off. After ectopic expression of mTERT, OB-OEG resumed division at a nonsenescent rate, expressed p75 and other OEG markers, and exhibited the morphology of nonsenescent OB-OEG. The nonsenescent period of mTERT-OEG lasted 9weeks and then ectopic mTERT switched off and cells entered senescence again. Our results suggest a role of telomerase in early senescence of adult rodent OB-OEG cultures and a protection from oxidative damage. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Chronic spinal injury repair by olfactory bulb ensheathing glia and feasibility for autologous therapy.

Olfactory bulb ensheathing glia (OB-OEG) promote repair of spinal cord injury (SCI) in rats after transplantation at acute or subacute (up to 45 days) stages. The most relevant clinical scenario in humans, however, is chronic SCI, in which no more major cellular or molecular changes occur at the injury site; this occurs after the third month in rodents. Whether adult OB-OEG grafts promote repair of severe chronic SCI has not been previously addressed. Rats with complete SCI that were transplanted with OB-OEG 4 months after injury exhibited progressive improvement in motor function and axonal regeneration from different brainstem nuclei across and beyond the SCI site. A positive correlation between motor outcome and axonal regeneration suggested a role for brainstem neurons in the recovery. Functional and histological outcomes did not differ after transplantation at subacute or chronic stages. Thus, autologous transplantation is a feasible approach as there is a time frame for patient stabilization and OEG preparation; moreover, the healing effects of OB-OEG on established injuries may offer new therapeutic opportunities for chronic SCI patients.

Comparative gene expression profiling of olfactory ensheathing glia and Schwann cells indicates distinct tissue repair characteristics of olfactory ensheathing glia.

Olfactory ensheathing glia (OEG) are a specialized type of glia that support the growth of primary olfactory axons from the neuroepithelium in the nasal cavity to the brain. Transplantation of OEG in the injured spinal cord promotes sprouting of injured axons and results in reduced cavity formation, enhanced axonal and tissue sparing, remyelination, and angiogenesis. Gene expression analysis may help to identify the molecular mechanisms underlying the ability of OEG to recreate an environment that supports regeneration in the central nervous system. Here, we compared the transcriptome of cultured OEG (cOEG) with the transcriptomes of cultured Schwann cells (cSCs) and of OEG directly obtained from their natural environment (nOEG), the olfactory nerve layer of adult rats. Functional data mining by Gene Ontology (GO)-analysis revealed a number of overrepresented GO-classes associated with tissue repair. These classes include "response to wounding," "blood vessel development," "cell adhesion," and GO-classes related to the extracellular matrix and were overrepresented in the set of differentially expressed genes between both comparisons. The current screening approach combined with GO-analysis has identified distinct molecular properties of OEG that may underlie their efficacy and interaction with host tissue after implantation in the injured spinal cord. These observations can form the basis for studies on the function of novel target molecules for therapeutic intervention after neurotrauma.

Slow- and fast-twitch rat hind limb skeletal muscle phenotypes 8 months after spinal cord transection and olfactory ensheathing glia transplantation.

Paralysed skeletal muscle of rats with spinal cord injury (SCI) undergoes atrophy and a switch in gene expression pattern which leads to faster, more fatigable phenotypes. Olfactory ensheathing glia (OEG) transplants have been reported to promote axonal regeneration and to restore sensory-motor function in animals with SCI. We hypothesized that OEG transplants could attenuate skeletal muscle phenotypic deterioration and that this effect could underlie the functional recovery observed in behavioural tests. A variety of morphological, metabolic and molecular markers were assessed in soleus (SOL) and extensor digitorum longus (EDL) muscles of spinal cord transected (SCT), OEG-transplanted rats 8 months after the intervention and compared with non-transplanted SCT rats and sham-operated (without SCT) controls (C). A multivariate analysis encompassing all the parameters indicated that OEG-transplanted rats displayed skeletal muscle phenotypes intermediate between non-transplanted and sham-operated controls, but different from both. A high correlation was observed between behaviourally tested sensory-motor functional capacity and expression level of slow- and fast-twitch hind limb skeletal muscle phenotypic markers, particularly the histochemical glycerol-3-phosphate dehydrogenase activity (-0.843, P < 0.0001) and the fraction of variant 2s of the slow regulatory myosin light chain isoform (0.848, P < 0.0001) in SOL. Despite the mean overall effect of OEG transplants in patterning skeletal muscle protein expression towards normal, in 6 out of 9 animals they appeared insufficient to overcome fibre type switching and to support a consistent and generalized long-term maintenance of normal skeletal muscle characteristics. The interplay of OEG and exercise-mediated neurotrophic actions is a plausible mechanism underlying OEG transplantation effects on paralysed skeletal muscle.

Adult olfactory bulbs from primates provide reliable ensheathing glia for cell therapy.

Olfactory bulb ensheathing glia (OB-OEG) from adult rodents promote functional and morphological repair after grafting into injured spinal cords. To provide insight into the feasibility of using OB-OEG in human therapy, we studied OB-OEG in primates to determine their suitability for spinal cord transplantation. Here, we show that OEG can be obtained from olfactory bulbs of adult macaca mulatta and nemestrina monkeys and compare their characteristics to those obtained from rats. In contrast to rodent OB-OEG, primate OB-OEG are nonsenescent, exhibit a longer lifespan, are less sensitive to high oxygen culture environment, and maintain a phenotype suitable for grafting for up to 2.5 months in vitro. Three-week cultures (short term) derived from a single macaca olfactory bulb provide enough OEG for autologous transplantation at the acute stage of injury, and after long-term cultures (2.5 months) may yield an additional 20 billion. OEG can be frozen for later use. Therefore, primate adult olfactory bulbs constitute a reliable source of OEG for cell therapy, and successful culture of these cells make autologous transplantation feasible.

OEG implantation and step training enhance hindlimb-stepping ability in adult spinal transected rats.

Numerous treatment strategies for spinal cord injury seek to maximize recovery of function and two strategies that show substantial promise are olfactory bulb-derived olfactory ensheathing glia (OEG) transplantation and treadmill step training. In this study we re-examined the issue of the effectiveness of OEG implantation but used objective, quantitative measures of motor performance to test if there is a complementary effect of long-term step training and olfactory bulb-derived OEG implantation. We studied complete mid-thoracic spinal cord transected adult female rats and compared four experimental groups: media-untrained, media-trained, OEG-untrained and OEG-trained. To assess the extent of hindlimb locomotor recovery at 4 and 7 months post-transection we used three quantitative measures of stepping ability: plantar stepping performance until failure, joint movement shape and movement frequency compared to sham controls. OEG transplantation alone significantly increased the number of plantar steps performed at 7 months post-transection, while training alone had no effect at either time point. Only OEG-injected rats plantar placed their hindpaws for more than two steps by the 7-month endpoint of the study. OEG transplantation combined with training resulted in the highest percentage of spinal rats per group that plantar stepped, and was the only group to significantly improve its stepping abilities between the 4- and 7-month evaluations. Additionally, OEG transplantation promoted tissue sparing at the transection site, regeneration of noradrenergic axons and serotonergic axons spanning the injury site. Interestingly, the caudal stump of media- and OEG-injected rats contained a similar density of serotonergic axons and occasional serotonin-labelled interneurons. These data demonstrate that olfactory bulb-derived OEG transplantation improves hindlimb stepping in paraplegic rats and further suggest that task-specific training may enhance this OEG effect.

Long-term care of paraplegic laboratory mammals.

Repair of spinal cord injuries (SCIs) is still a major clinical challenge. Several attempts have been made to find a cure for this condition in experimental animals that could be extrapolated to humans. A key for success seems the availability of optimum animal models for testing different therapies. Complete spinal cord lesion in mammals is considered the most accurate injury model. In addition, long-term survival of animals seems more appropriate, as this increases the efficacy of the repair strategies. However, paraplegic animals require special care and treatment for proper longterm maintenance, and to date, there are no published protocols. This lack of available information has discouraged scientists from working with this injury model. Over the past 7 years, we have tested the repair efficacy of olfactory ensheathing glia in paraplegic rats for survival periods of more than 8 months. To keep these animals healthy for this long time, we adapted and administered treatments used in people with paraplegia. These same protocols (developed for rodents in our group) are being applied to paraplegic monkeys. In this review, we provide an overview of the proper handling and care of paraplegic adult laboratory mammals for long periods. This information might help other groups to optimize the outcome obtained and to better evaluate the prospect of a given experimental repair strategy. In addition, the use of human treatments in paraplegic animals provides a more realistic model for a later transfer to the clinical arena.

Olfactory ensheathing glia transplantation: a therapy to promote repair in the mammalian central nervous system.

A therapy to treat injuries to the central nervous system (CNS) is, to date, a major clinical challenge. The devastating functional consequences they cause in human patients have encouraged many scientists to search, in animal models, for a repair strategy that could, in the future, be applied to humans. However, although several experimental approaches have obtained some degree of success, very few have been translated into clinical trials. Traumatic and demyelinating lesions of the spinal cord have attracted several groups with the same aim: to find a way to promote axonal regeneration, remyelination, and functional recovery, by using a simple, safe, effective, and viable procedure. During the past decade, olfactory ensheathing glia (OEG) transplantation has emerged as a very promising experimental therapy to promote repair of spinal cords, after different types of injuries. Transplants of these cells promoted axonal regeneration and functional recovery after partial and complete spinal cord lesions. Moreover, olfactory ensheathing glia were able to form myelin sheaths around demyelinated axons. In this article, we review these recent advances and discuss to what extent olfactory ensheathing glia transplantation might have a future as a therapy for different spinal cord affections in humans.