The mechanisms of injury-induced apoptosis of neurons within the spinal cord are poorly understood. In this study, we show that spinal cord injury (SCI) induces endoplasmic reticulum stress revealed by the activation of an unbalanced unfolded protein response (UPR). Using a weight-drop contusion model of SCI, the UPR activation was characterized by a quick transient phosphorylation of alpha subunit of eukaryotic initiation factor 2 soon restored by the up-regulation of its regulator Gadd34; an effective cleavage/activation of the ATF6a transcription factor leading to up-regulation of the canonical UPR target genes Chop, Xbp1 and Grp78; the presence of the processing of Xbp1 mRNA indicative of inositol requiring kinase 1 activation, and a gradual accumulation of C/EBP homologous transcription factor protein (CHOP) with concomitant caspase-12 activation. Interestingly, the subcellular distribution of CHOP was found in the nucleus of neurons and oligodendrocytes but in the cytoplasm of astrocytes. Considering the pro-apoptotic action attributed to this transcription factor, this phenomenon might account for the different susceptibility of cell types to dye after SCI.

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We compared the neurological and electrophysiological outcome, glial reactivity, and spared spinal cord connectivity promoted by acute transplantation of olfactory ensheathing cells (group OEC) or Schwann cells (group SC) after a mild injury to the rat spinal cord. Animals were subjected to a photochemical injury of 2.5 min irradiation at the T8 spinal cord segment. After lesion, a suspension containing 180,000 OECs or SCs was injected. A control group (group DM) received the vehicle alone. During 3 months postsurgery, behavioral skills were assessed with open field-BBB scale, inclined plane, and thermal algesimetry tests. Motor (MEPs) and somatosensory evoked potentials (SSEPs) were performed to evaluate the integrity of spinal cord pathways, whereas lumbar spinal reflexes were evaluated by the H reflex responses. Glial fibrillary acidic protein and proteoglycan expressions were quantified immunohistochemically at the injured spinal segments, and the preservation of corticospinal and raphespinal tracts caudal to the lesion was evaluated. Both OEC- and SC-transplanted groups showed significantly better results in all the behavioral tests than the DM group. Furthermore, the OEC group had higher MEP amplitudes and lower H responses than the other two groups. At the injury site, the area of spared parenchyma was greater in transplanted than in control injured rats. OEC-transplanted animals had reduced astrocytic reactivity and proteoglycan expression in comparison with SC-transplanted and DM rats. Taken together, these results indicate that transplantation of both OEC and SC has potential for restoration of injured spinal cords. OEC grafts showed superior ability to reduce glial reactivity and to improve functional recovery.

Disconnection of the axon from the soma of spinal motoneurons (MNs) leads either to a retrograde degenerative process or to a regenerative reaction, depending on the severity and the proximity to the soma of the axonal lesion. The endoplasmic reticulum (ER) is a continuous membranous network that extends from the nucleus to the entire cytoplasm of the neuronal soma, axon and dendrites. We investigated whether axonal injury is sensed by the ER and triggers the activation of protective mechanisms, such as the unfolded protein response (UPR) and autophagy. We found early (at 3 days) accumulation of beclin1, LC3II and Lamp-1, hallmarks of autophagy, in both degenerating MNs after spinal root avulsion and in non-degenerating MNs after distal nerve section, although Lamp-1 disappeared by 5 days only in the former. In contrast, only degenerating MNs presented early activation of IRE1a, revealed by an increase of the spliced isoform of Xbp1 and accumulation of ATF4 in their nucleus, two branches of the UPR, and late BiP downregulation in association with cytoskeletal and organelle disorganization. We conclude that BiP decrease is a signature of the degenerating process, as its overexpression led to an increase in MN survival after root avulsion. Besides, Bcl2 is strongly implicated in the survival pathway activated by BiP overexpression.

Previously, we have shown that transplants of olfactory bulb ensheathing cells promoted regeneration of transected dorsal roots into the spinal cord. In this study, we assessed the ability of regenerating axons to make functional connections in the cord. Dorsal roots L3 to L6 were sectioned close to their entrance into the spinal cord and reapposed after injecting a suspension of ensheathing cells into each dorsal root entry zone (Group G). Afferent regeneration into the cord and recovery of spinal reflexes were compared with animals that received no injection (Group S) or culture medium without cells (Group C). Electrophysiological tests, to measure nerve conduction and spinal reflexes (H response and withdrawal reflex) evoked by stimulation of afferents of the sciatic nerve, were performed. At 14 days after surgery, H response was found in only 1 of 7 rats of Group G, and withdrawal reflexes were absent from all animals. At 60 days, the H response reappeared in 7 of 10 rats of Group G, and 1 of 5 of each of Groups C and S. The withdrawal reflex recovered in 4 of 10 rats of Group G, but in none of Groups C and S. Immunohistochemical labeling for CALCITONIN GENE– RELATED PEPTIDE (CGRP) in rats of Group G showed immunoreactive fibers entering the dorsal horn from sectioned roots, although at lower density than in the contralateral side. In conclusion, transplanted ensheathing cells promoted central regeneration and functional reconnection of regenerating sensory afferents. Ann Neurol 1999;45:207–215

We studied the effects of olfactory ensheathing cells (OECs) transplanted in a photochemical spinal cord injury in adult rats. After dorsal laminectomy at T8 vertebra, subjacent spinal cord was bathed with rose Bengal for 10 min and illuminated with visible light by means of an optic fiber connected to a halogen lamp for 2.5 min at maximal intensity of 8 kLux. Eight injured rats received a suspension of OECs in DMEM, and another eight rats received DMEM alone. Locomotor ability scored by the BBB scale, pain sensibility by the plantar algesimetry test, and motor-and somatosensory-evoked potentials by electrophysiological techniques were evaluated for 3 months postsurgery. Finally, all rats were perfused with paraformaldehyde and transverse sections from the spinal cord segment at the lesion site were immunostained against GFAP. Area of the preserved spinal cord parenchyma was measured from the GFAPimmunolabeled cord sections. The BBB score and the amplitude of motor-and somatosensory-evoked potentials were higher in OECs-transplanted rats than in DMEMinjected animals throughout follow-up, whereas the withdrawal response to heat noxious stimulus was lower in OEC-than in DMEM-injected rats. The area of preserved spinal cord was significantly larger in OECs-transplanted rats than in DMEM-injected animals. These results indicate that OECs promote functional and morphological preservation of the spinal cord after photochemical injury. GLIA 42:275-286, 2003.

Transplantation of olfactory ensheathing cells (OECs) into photochemically damaged rat spinal cord diminished astrocyte reactivity and parenchyma cavitation. The photochemical lesion performed at T12--L1 resulted in severe damage to the spinal cord, so that during the first 15 days postoperation all rats dragged their hindlimbs and did not respond to pinprick. The maximal area and volume of the cystic cavities were lower in transplanted than in non-transplanted rats, not significantly at the T12--L1 lesion site, but significantly at T9--T10 and L4--L6 cord levels. The density of astrocytes in the grey matter was similar at T12--L1 and L4--L6 in non-transplanted and trans- planted rats, but lower in the latter at T9--T10 level. However, in non-transplanted rats all astrocytes showed a hypertrophied appearance, with long and robust processes heavily GFAP-positive, and overexpression of proteoglycan inhibitor of neuritogenesis, whereas in transplanted rats only a few astrocytes showed hypertrophy and the majority had short, thin processes. These results indicate that OECs transplanted into damaged adult rat spinal cord exert a neuroprotective role by reducing astrocytic gliosis and cystic cavitation.

Spinal root avulsion leads to a progressive loss of axotomized motoneurons (MNs). Nowadays, there is no effective treatment to prolong MN survival that could permit recovery as a result of delayed surgical repair. Administration of Sigma-1 receptor (Sig-1R) ligands has been reported to promote beneficial effects after several types of neural injury. In order to shed light of whether Sig-1R ligands could promote MN survival after root avulsion, L4-L5 spinal roots were unilaterally avulsed in adult rats and the Sig-1R agonist Pre084 was administered at different doses. The ventral spinal cords of the animals were studied from 3 to 21 days post-operation (DPO) by using histological, immunohistochemical, and Western blot techniques. Daily treatment with 0.25 mg/kg Pre084 significantly promoted MN survival (68% vs 43% in untreated rats) at 21 DPO, an effect that was antagonized by coadministration of BD1063, an antagonist of Sig-1R. There was a reduction in astroglial- associated immunoreactivity in rats treated with Pre084. Moreover, Pre084 produced an increase in the Sig-1R co-chaperone BiP within MNs, and an increase of GDNF expression by astrocytes in the ventral horn early after injury. Although the mechanisms promoting MN survival by Pre084 remain unclear, we hypothesize that it is mediated at least in part through the increase in these cytoprotective factors. Therefore, early application of Sig-1R agonist appears to be a promising therapy to improve MN survival after root avulsion.