Almudena Ramón-Cueto

Functional Recovery of Paraplegic Rats and Motor Axon Regeneration in Their Spinal Cords by Olfactory Ensheathing Glia

Axonal regeneration in the lesioned mammalian central nervous system is abortive, and this causes permanent disabilities in individuals with spinal cord injuries. In adult rats, olfactory ensheathing glia (OEG) transplants successfully led to functional and structural recovery after complete spinal cord transection. From 3 to 7 months post surgery, all OEG-transplanted animals recovered locomotor functions and sensorimotor reflexes. They presented voluntary hindlimb movements, they supported their body weight, and their hindlimbs responded to light skin contact and proprioceptive stimuli. In addition, relevant motor axons (corticospinal, raphespinal, and coeruleospinal) regenerated for long distances within caudal cord stumps. Therefore, OEG transplantation provides a useful repair strategy in adult mammals with traumatic spinal cord injuries. Our results with these cells could lead to new therapies for the treatment of spinal cord lesions in humans.

Regeneration into the spinal cord of transected dorsal root axons is promoted by ensheathing glia transplants.

The permissivity of adult olfactory bulb to the ingrowth of olfactory axons could be due to the unique properties of ensheathing glia. To test whether these glial cells could be used to promote axonal regeneration in a spontaneously nonregenerating system, we transplanted suspensions of pure ensheathing cells into a rhizotomized spinal cord segment. Ensheathing cells were purified away from other cell types by immunoaffinity, using anti-p75 nerve growth factor receptor. After laminectomy at the lower thoracic level, the spinal cord was exposed and one dorsal root (T10) was completely transected at the cord entry point. The root stump was microsurgically anastomosed to the cord and a suspension of ensheathing cells was transplanted in the spinal cord at the dorsal root entry zone. Three weeks after transplantation, numerous regenerating dorsal root axons were observed reentering the spinal cord. Ingrowth of dorsal root axons was observed using DiI and antibodies against calcitonin gene-related peptide and growth-associated protein. Primary sensory afferents invaded laminae 1, 2, and 3, grew through laminae 4 and 5, and reached the dorsal grey commissure and lamina 4 of the contralateral side. We did not observe regenerating axons within the ipsilateral ventral horn and dorsal column. Transplanted ensheathing cells reached the same laminae as axons. Neither ensheathing cells nor regenerating axons invaded those laminae they did not innervate under normal circumstances. In conclusion, the regeneration of injured dorsal root axons into the adult spinal cord was possible after ensheathing glia transplantation. The use of ensheathing cells as stimulators of axonal growth might be generalized to other central nervous system injuries.

Olfactory ensheathing glia: properties and function.

The failure of regenerating axons to grow within the adult mammalian central nervous system (CNS) does not apply to the olfactory bulb (OB). In this structure, normal and transected olfactory axons are able to enter, regenerate, and reestablish lost synaptic contacts with their targets, throughout the lifetime of the organism. A remarkable difference between an axonal growth-permissive structure such as the OB and the remaining CNS resides in the presence of ensheathing glia in the former. These cells exhibit phenotypic and functional properties known to be involved in the process of axonal elongation that may explain the permissibility of the OB to axonal growth. In addition, transplants of ensheathing glia were successfully used to promote axonal regeneration within the injured adult CNS. The axonal growth-promoting properties of ensheathing glia make the study of this cell type interesting to provide an insight into the mechanisms underlying the process of axonal regeneration. Therefore, in this article we review the developmental, morphologic, immunocytochemical, and functional properties presented by this unique glial cell type, and correlate them with the axonal growth-promoting ability of ensheathing glia. In addition, we provide some evidence of the potentiality that ensheathing glia might have as a promoter of axonal regeneration within the injured nervous system.

Can experiments in nonhuman primates expedite the translation of treatments for spinal cord injury in humans

Can experiments in nonhuman primates expedite the translation of treatments for spinal cord injury in humans?

In vitro enfolding of olfactory neurites by p75 NGF receptor positive ensheathing cells from adult rat olfactory bulb

Secondary cultures of adult rat olfactory bulb (OB) contained three different types of cell: (i) process‐bearing cells; (ii) macrophage‐like cells and (iii) fusiform cells. The immunohistochemical properties of process‐bearing cells closely corresponded to those described for ensheathing glia in vivo. The most distinctive feature of these cells was their immunoreactivity for low affinity nerve growth factor receptor (NGFR). Process‐bearing cells also shared the ultrastructural properties of ensheathing glia in vivo, as well as the ability to ensheath olfactory axons. In contrast, macrophage‐like cells had the immunostaining properties of microglia, and fusiform cells were likely capillary endothelial cells.

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.

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.

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.

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.

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.