P. Gauthier

Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.

A membrane permeant nucleic acid stain, ethidium homodimer was used in combination with calcein-AM to document the viability of Schwann cells (SCs) in whole nerves after cold storage assays. Segments of peripheral nerves were, (i) kept intact in buffer (viability controls), (ii) thawed after a cryopreservation, according to a protocol which has been previously shown to maintain the integrity of most nerve components [Ruwe and Trumble, J. Reconstr. Microsurg., 1990, 6: 239-244; Gauthier et al., In 3rd International Symposium on Axonal Regrowth in the Mammalian Spinal Cord and Peripheral Nerve, Deauville, France, 1995, p. 24, abstract], (iii) killed by chemical injury, or (iv) by successive freezing-thawing. Teased preparations of nerve fibers were prepared from the various types of nerve segments and incubated with calcein-AM and ethidium homodimer, which stain, respectively, living and dead cells. In control or cryopreserved nerves, staining with calcein-AM resulted in bright green fluorescence in the cytoplasm of SCs, with no red fluorescence of ethidium homodimer. In contrast, in killed nerve preparations, intense ethidium red fluorescence was observed in SC nuclei, with negligible green calcein cytoplasmic fluorescence. Thus, the combination of calcein-AM/ethidium homodimer appeared as an effective tool for assessing the viability of SCs and determine the quality of cold stored nerve preparations used in graft repair procedures. In addition, the generated fluorescence enabled clear visualization of myelinated fibers by confocal imaging.

Functional reconnections established by central respiratory neurons regenerating axons into a nerve graft bridging the respiratory centers to the cervical spinal cord

The present work investigated, in adult rats, the long‐term functional properties and terminal reconnections of central respiratory neurons regenerating axons within a peripheral nerve autograft bridging two separated central structures. A nerve graft was first inserted into the left medulla oblongata, in which the respiratory centers are located. Three months later, a C3 left hemisection was performed, and the distal tip of the graft was implanted into the C4 left spinal cord at the level of the phrenic nucleus, a natural central inspiratory target. Six to eight months after medullary implantation, the animals (n = 12) were electrophysiologically investigated to test 1) the phrenic target reinnervation by analyzing the phrenic responses elicited by bridge electrical stimulation and 2) the bridge innervation by unitary recordings of the spontaneous activity of regenerated axons within the nerve bridge. In the control group (n = 6), the medullary site of implantation corresponded to the dorsolateral medulla, a region known to be an unsuitable site for inducing respiratory axonal regrowth after nerve grafting. Stimulation of the nerve bridge never elicited phrenic nerve response, and no respiratory units were found within the nerve bridge. In the experimental group (n = 6), the proximal tip of the nerve bridge was implanted within the ventrolateral medulla at the level of the respiratory centers. Electrical stimulation of the nerve bridge induced phrenic nerve responses that reflected a postsynaptic activation of the phrenic target. Subsequent unitary recordings from teased fibers within the bridge revealed the presence of regenerated inspiratory fibers exhibiting discharge patterns typical of medullary inspiratory neurons, which normally make synaptic contacts with the inspiratory phrenic target. These results indicate that, when provided with an appropriate denervated target, central respiratory neurons with regenerated axons along a nerve bridge can remain functional for a long period and can make precise and specific functional reconnections with central homotypic target neurons.

Nasal OEC transplantation promotes respiratory recovery in a subchronic rat model of cervical spinal cord contusion

Engraftment of nasal olfactory ensheathing cells (OEC) is considered as a promising therapeutic strategy for spinal cord repair and one clinical trial has already been initiated. However, while the vast majority of fundamental studies were focused on the recovery of locomotor function, the efficiency of this cellular tool for repairing respiratory motor dysfunction, which affects more than half of paraplegic/tetraplegic patients, remains unknown. Using a rat model that mimics the mechanisms encountered after a cervical contusion that induces a persistent hemi-diaphragmatic paralysis, we assessed the therapeutic efficiency of a delayed transplantation (2 weeks post-contusion) of nasal OECs within the injured spinal cord. Functional recovery was quantified with respiratory behavior tests, diaphragmatic electromyography and neuro-electrophysiological recording of the phrenic motoneurons while axogenesis was evaluated using immunohistochemistry. We show that 3 months post-transplantation, nasal OECs improve i) breathing movements, ii) activities of the ipsilateral diaphragm and corresponding phrenic nerve, and iii) axonal sprouting in the injury site. We also demonstrate that this functional partial recovery is mediated by the restoration of ipsilateral supraspinal command. Our study brings further evidence that olfactory ensheathing cells could have clinical application especially in tetraplegic patients with impaired breathing movements. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Regeneration of Respiratory Pathways within Spinal Peripheral Nerve Grafts

Central respiratory neurons exhibit normal activity after axonal regeneration within blind-ended peripheral nerve grafts (PNGs) inserted near the corresponding cell bodies in the medullary respiratory centers. Part of these medullary respiratory neurons project toward the spinal cord and contribute to descending respiratory pathways that control respiratory motoneurons. The present work investigates to what extent cervical respiratory pathways could be directed out of the central nervous system within PNGs inserted distant to the medullary respiratory nuclei. In adult rats (n = 13), autologous segments of the peroneal nerve were implanted into the ventrolateral part of the C2 spinal cord at the level of the descending respiratory pathways. Two to four months after grafting, electrophysiological recording of teased graft filaments (n = 562) revealed the presence of regenerated nerve fibers with unitary impulse traffic (n = 164) in all tested PNGs (n = 6). Respiratory discharges (n = 52) corresponded to efferent and afferent activity. Efferent respiratory discharges (n = 32) originated from central respiratory neurons which remained functional and preserved afferent connections. Retrograde horseradish peroxidase labeling applied to the distal cut end of PNGs (n = 7) revealed stained (42/1997) neurons in areas where respiratory cells have been described. Afferent respiratory discharges (n = 20) were synchronized with lung inflation but their origin (stretch pulmonary receptors and/or respiratory muscle receptors) was not determined. On the basis of additional data from light and electron microscopy of PNGs, comparison was made between anatomical, retrograde labeling, and electrophysiological data. The main conclusion is that spinal PNGs appear to be able to promote axonal regeneration of functional respiratory efferent and afferent pathways.

Alteration of Forebrain Neurogenesis after Cervical Spinal Cord Injury in the Adult Rat

Spinal cord injury (SCI) triggers a complex cellular response at the injury site, leading to the formation of a dense scar tissue. Despite this local tissue remodeling, the consequences of SCI at the cellular level in distant rostral sites (i.e., brain), remain unknown. In this study, we asked whether cervical SCI could alter cell dynamics in neurogenic areas of the adult rat forebrain. To this aim, we quantified BrdU incorporation and determined the phenotypes of newly generated cells (neurons, astrocytes, or microglia) during the subchronic and chronic phases of injury. We find that subchronic SCI leads to a reduction of BrdU incorporation and neurogenesis in the olfactory bulb and in the hippocampal dentate gyrus. By contrast, subchronic SCI triggers an increased BrdU incorporation in the dorsal vagal complex of the hindbrain, where most of the newly generated cells are identified as microglia. In chronic condition 90 days after SCI, BrdU incorporation returns to control levels in all regions examined, except in the hippocampus, where SCI produces a long-term reduction of neurogenesis, indicating that this structure is particularly sensitive to SCI. Finally, we observe that SCI triggers an acute inflammatory response in all brain regions examined, as well as a hippocampal-specific decline in BDNF levels. This study provides the first demonstration that forebrain neurogenesis is vulnerable to a distal SCI.

Axonal regeneration from central respiratory neurons of the adult rat into peripheral nerve autografts: effects of graft location within the medulla.

In adult rats, autologous segments of the peroneal nerve were implanted into the medulla oblongata at the level of the obex either on the midline (midline grafts, n = 4), where respiratory axons decussate, or on the left side (lateral grafts, n = 5) in the area of the respiratory cell bodies. Several months after the graft implantation, spontaneous unitary activities (n = 225) were recorded within the grafts and were found to arise from both central respiratory (R, n = 72) and non-respiratory (NR, n = 153) neurons which were giving off regenerated axons along the nerve grafts. The graft reinnervation by respiratory axons was found to be significantly more abundant within the medullary lateral grafts than within the midline grafts. This finding offers further support of the conclusion that the reinnervation of grafts by axons from central neurons is enhanced when the graft is placed proximally to the cell bodies.

In vitro pre-degenerated nerve autografts support CNS axonal regeneration

In the present study we compared, in adult rats, the axonal regeneration of central respiratory neurons within autologous fresh (f-; grafted immediately after removal) and pre-degenerated (pd-; grafted after being stored during 3 days in saline at +8 degrees C) peripheral nerve grafts (PNGs) implanted within the C2 cervical spinal cord. The proximal end of the left peroneal nerve was implanted in the site of projection of medullary respiratory neurons (ventro-lateral quadrant) and the distal part of each nerve graft was left unconnected (blind-ended graft). PNGs were examined 2 to 4 months after grafting. Central neurons regenerating axons within the PNGs were studied by recording spontaneous unit activity from small strands teased from the grafts. In control f-PNGs (n = 9), 248 filaments had spontaneous activities, 58 of these were respiratory-related, i.e. had discharge patterns identical to those of normal respiratory (inspiratory and expiratory) neurons. The presence of regenerated nerve fibers with spontaneous unitary impulse traffic (n = 216) was found in all pd-PNGs (n = 5). Thirty-four had respiratory patterns identical to those found within f-PNGs and corresponded to efferent activity. No statistically significant differences in axonal regrowth were found between f- and pd-PNGs. In conclusion, f- and pd-PNGs were equally capable of promoting axonal regeneration of central neurons. The neural components (Schwann cells and others) required for axonal regeneration of adult central neurons are still effective following 3 days of in vitro peripheral nerve degeneration without special storage conditions (oxygenation, medium inducing ATP synthesis). These results have clinical implications for nerve graft surgery when time is required for typing the tissues of both donor and recipient (post-mortem allografts) or transportation of graft material.

Central respiratory neurons of the adult rat regrow axons preferentially into peripheral nerve autografts implanted within ventral rather than within dorsal parts of the medulla oblongata

A great number of severed central nervous system (CNS) neurons of the adult rat have the capillary to regrow axons into peripheral nerve autografts. In the present experiment, autologous segments of the peroneal nerve were inserted perpendicularly to the dorsal surface of the medulla oblongata more or less laterally within either the ventral respiratory group or the so-called dorsal respiratory group (ventrolateral grafts, n = 5; dorsolateral grafts, n = 5). From 2 to 4.5 months after the graft implantation, spontaneous unitary activities (n = 197) were recorded within all the grafted nerves: they were found to arise from both central respiratory (R, n = 60) and non-respiratory (NR, n = 137) neurons which were giving off regenerated axons along the nerve grafts. The graft reinnervation by respiratory axons was found to be significantly more abundant within the medullary ventrolateral grafts than within the dorsolateral ones. The low rate of axonal regeneration from respiratory neurons observed within the dorsolateral grafts provides further evidence that the number of the respiratory neurons in the dorsal respiratory group, if present at all, is much smaller than that of the ventral respiratory group in the rat.

Central respiratory neuronal activity after axonal regeneration within blind-ended peripheral nerve grafts: time course of recovery and loss of functional neurons

Autologous segments of peroneal nerve were implanted into the medulla of adult rats to induce axonal regeneration of central neurons axotomised during the grafting procedure. Grafts were inserted in the midline where respiratory axons decussate or laterally, either in the nucleus tractus solitarius or in the nucleus ambiguus, close to respiratory cell bodies. The distal part of each graft was left unconnected (blind-ended graft). Between 2 and 30 months post-implantation, unit recordings from single fibres were made from small strands teased from the grafts to investigate activity of neurons regenerating axons. Spontaneous respiratory and non-respiratory activity was present only in grafts examined between 2 and 6 months post-implantation. Respiratory units had discharge patterns identical to those of normal inspiratory or expiratory neurons; their responses to lung inflation and asphyxia were also similar to those of central respiratory neurons. No spontaneous activity was present in the grafts examined 7–30 months post-implantation. Moreover, asphyxia, which normally enhances the activity of central respiratory neurons, failed to elicit activity. These results were similar in all grafts, regardless of the site of implantation. The presence of spontaneous activity only between 2 and 6 months post-implantation indicates that once axonal growth of respiratory neurons is stopped within blind-ended grafts, those neurons still exhibited normal functional properties for 3 months. The absence of activity 6 months after grafting suggests that loss of functional regenerating respiratory neurons does not occur progressively and follows an “all or nothing” rule within blind-ended grafts.