Enrique Verdú

Influence of aging on peripheral nerve function and regeneration

Abstract   Aging deeply influences several morphologic and functional features of the peripheral nervous system (PNS). Morphologic studies have reported a loss of myelinated and unmyelinated nerve fibers in elderly subjects, and several abnormalities involving myelinated fibers, such as demyelination, remyelination and myelin balloon figures. The deterioration of myelin sheaths during aging may be due to a decrease in the expression of the major myelin proteins (P0, PMP22, MBP). Axonal atrophy, frequently seen in aged nerves, may be explained by a reduction in the expression and axonal transport of cytoskeletal proteins in the peripheral nerve. Aging also affects functional and electrophysiologic properties of the PNS, including a decline in nerve conduction velocity, muscle strength, sensory discrimination, autonomic responses, and endoneurial blood flow. The age‐related decline in nerve regeneration after injury may be attributed to changes in neuronal, axonal, Schwann cell and macrophage responses. After injury, Wallerian degeneration is delayed in aged animals, with myelin remnants accumulated in the macrophages being larger than in young animals. The interaction between Schwann cells and regenerative axons takes longer, and the amount of trophic and tropic factors secreted by reactive Schwann cells and target organs are lower in older subjects than they are in younger subjects. The rate of axonal regeneration becomes slower and the density of regenerating axons decrease in aged animals. Aging also determines a reduction in terminal and collateral sprouting of regenerated fibers, further limiting the capabilities for target reinnervation and functional restitution. These age‐related changes are not linearly progressive with age; the capabilities for axonal regeneration and reinnervation are maintained throughout life, but tend to be delayed and less effective with aging.

Nerve Guides Seeded with Autologous Schwann Cells Improve Nerve Regeneration

This study evaluates the ability of Schwann cells (SCs) transplanted into a nerve guide to improve regeneration and reinnervation after sciatic nerve resection and repair, leaving a 6-mm gap, in the mouse. SCs were isolated from predegenerated adult sciatic nerves and expanded in culture using a chemically defined medium. Syngeneic, isogeneic, and autologous SCs were suspended in Matrigel and seeded in resorbable, permeable poly(l-lactide-co-epsilon-caprolactone) guides at 150,000 cells/tube. Guides containing SCs were compared to guides filled with Matrigel alone and with peroneal nerve autografts. Functional reinnervation was assessed by noninvasive methods to determine recovery of sweating, nociceptive, sensory, and motor functions in the hindpaw during 4 months postoperation. Morphological analysis of the regenerated nerves was performed at the end of follow-up. The group with an autograft achieved faster and higher levels of reinnervation and higher number of regenerated myelinated fibers than groups repaired by tubulization. The immunogenicity of transplanted SCs influenced the outcome of nerve regeneration. Transplants of autologous SCs resulted in slightly lower levels of reinnervation than autografts, but higher recovery and number of regenerated fibers reaching the distal nerve than transplants of isologous and syngeneic SCs, although most of the differences were not statistically significant. Syngeneic SCs did not improve regeneration with respect to acellular guides. Prelabeled transplanted SCs were found to survive into the guide 1-3 months after implantation, to a larger number when they were autologous than syngeneic. Cellular prostheses composed of a resorbable guide seeded with autologous SCs appear as an alternative for repairing long gaps in injured nerves, approaching the success of autografts.

Morphometric and ultrastructural changes with ageing in mouse peripheral nerve.

Qualitative and quantitative information is reported on the morphological changes that occur in nerve fibres and nonneuronal cells of peripheral nerve during the lifetime of the mouse. Tibial nerves of mice aged 6–33 mo were studied. With ageing, collagen accumulates in the perineurium and lipid droplets in the perineurial cells. Macrophages and mast cells increase in number, and onion bulbs and collagen pockets are frequently present. Schwann cells associated with myelinated fibres (MF) slightly decrease in number in parallel with an increase of the internodal length from 6 to 12 mo, but increase in older nerves when demyelination and remyelination are common. The unmyelinated axon to myelinated fibre (UA/MF) ratio was about 2 until 12 mo, decreasing to 1.6 by 27 mo. In older mice, the loss of nerve fibres involves UA (50% loss of 27–33 mo cf. 6 mo) more markedly than MF (35%). In aged nerves wide incisures and infolded or outfolded myelin loops are frequent, resulting in an increased irregularity in the morphology of fibres along the internodes. In the mouse there is an adult time period, 12–20 mo, during which several features of degeneration progressively appear, and an ageing period from 20 mo upwards when the nerve suffers a general disorganisation and marked fibre loss.

Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy

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

Acute transplantation of olfactory ensheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat

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.

Comparison of Regenerative and Reinnervating Capabilities of Different Functional Types of Nerve Fibers

Functional reinnervation of sweat glands (SGs), skin, and muscle in the mouse paw after sciatic nerve lesions was evaluated to allow comparisons of the regeneration efficiency of different functional types of nerve fibers. In four groups of mice the sciatic nerve was crushed, sectioned, and left unrepaired or repaired by suture or tubulization. Reappearance of SG secretion and pinprick responses occurred slightly earlier than recordings of compound muscle and nerve action potentials in all groups. The degree of reinnervation, with respect to preoperative control values, of SGs and skin nociceptors was higher than the amplitude of the action potentials, mainly when the nerve injury was severe. The chances for recovery progressively decreased with the severity of the lesion, affecting the larger nerve fibers most. These results indicate that, after injuries of peripheral nerves, all types of nerve fibers are able to regenerate in the mouse, although small size fibers (sudomotor and nociceptive) allow for a higher degree of functional recovery than large myelinated fibers (skeletomotor and sensory).

Spinal cord injury induces endoplasmic reticulum stress with different cell‐type dependent response

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 ATF6α 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.

Olfactory Ensheathing Cells Transplanted in Lesioned Spinal Cord Prevent Loss of Spinal Cord Parenchyma and Promote Functional Recovery

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 GFAP‐immunolabeled cord sections. The BBB score and the amplitude of motor‐ and somatosensory‐evoked potentials were higher in OECs‐transplanted rats than in DMEM‐injected 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.

Effects of ensheathing cells transplanted into photochemically damaged spinal cord.

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.

Olfactory bulb ensheathing cells enhance peripheral nerve regeneration.

Sciatic nerve resection leaving a 15 mm gap could not be repaired by bridging the stumps with a silicone tube prefilled with a laminin gel. However, when purified olfactory ensheathing cells (EC) were added to the gel filling the tube, successful axonal regeneration was observed in 50% of rats. With 12 mm gaps, regeneration occurred in 79% of rats with transplanted EC compared with 60% of those receiving collagen gel alone. Therefore, ECs help repair severe peripheral nerve injuries, in addition to their ability to promote axonal regeneration within the central nervous system.