P. Delrée

Effects of Schwann Cell Transplantation in a Contusion Model of Rat Spinal Cord Injury

Cultured Schwann cells were transplanted at various delays into a spinal cord contusion injury performed at low thoracic level in adult female rats. The Schwann cells were purified from the dorsal root ganglia of adult syngeneic animals. The transplants were well tolerated, and the transplanted Schwann cells invaded the injured spinal cord. As quantified using video image analysis, the survival and growth of the transplanted cells were poor when the grafting procedure was performed 3–4 days after injury and very good when performed immediately or 10 days after injury, in which cases post‐traumatic micro‐ and macrocavitation were strongly reduced. In animals grafted immediately after injury but not in animals grafted after 10 days, post‐traumatic astrogliosis was much reduced. The Schwann cells transplanted area was invaded by numerous regenerating axons, the vast majority of which were, based on the neurotransmitter (CGRP and SP) profile, originating from dorsal root ganglion. No regeneration of the cortico‐spinal tract as assessed after anterograde tracing or of descending aminergic fibers could be demonstrated.

Neuronotrophic Effect of Developing Otic Vesicle on Cochleo-Vestibular Neurons: Evidence for Nerve Growth Factor Involvement

In the developing inner ear, the existence of a neuronal death and of a peripheral target-derived trophic effect on cochleovestibular neurons has been documented. Using cultures of rat cochleovestibular neurons, we show that the E12 otic vesicle releases a factor promoting the survival and the neuritogenesis of these neurons, and that this effect is mimicked by NGF. The effect of the optic vesicle conditioned medium (OVCM) on cochleovestibular neurons is suppressed by anti-NGF antibodies. OVCM is neuronotrophic for NGF-sensitive sympathetic neurons, an effect that is also suppressed by anti-NGF antibodies, further demonstrating the presence of biologically active nerve growth factor.

Sciatic Nerve Regeneration through Venous or Nervous Grafts in the Rat

This study analyses the interest of isologous venous grafts filled with saline or with Schwann cells versus nerve grafts as guides for regeneration of the sciatic nerve in 35 Wistar rats. Electrophysiological parameters (conduction velocities and distal latencies of motor responses) and the functional index of De Medinacelli were measured several times from 1 month to 1 year after surgery. An histological analysis was performed on 2 control rats and on 3 rats killed 6 or 12 months after surgery: the total number of fibers was counted on a montage photoprint of the whole nerve, and the diameters of axons and the thickness of the myelin sheath were measured on digitized images. With a portion of nerve as guide, the regeneration is faster than with a vein. However, regeneration after 6 months is at least as good with a venous graft filled with Schwann cells, as assessed by electrophysiological, functional, and histological analysis. The addition of Schwann cells in grafted veins allows the nerve to regenerate through longer gaps than previously described (25 vs 15 mm). In order to assess the quality of nerve regeneration, functional, electrophysiological, and histological analysis are complementary.

Grafts of syngenic cultured, adult dorsal root ganglion-derived Schwann cells to the injured spinal cord of adult rats: preliminary morphological studies

Highly enriched cultures of Schwann cells were obtained from adult rat dorsal root ganglia and implanted (5 x 10(5) -9 x 10(5) cells) in the spinal cord of syngenic adult rats at the site of an acute compression lesion produced by a subdural inflatable microballoon. These autografts survived and invaded the host tissue, reducing central cavitation and astrocytic gliosis. They dramatically promoted ingrowth of axons, the majority of which appeared to come from the dorsal roots as judged by their neuropeptide content. Invasion of the transplants by descending, e.g. aminergic fibers, was negligible at survival times of up to 4 months. Nonetheless, autologous Schwann cells, which are readily available in the host, represent a promising material for grafts into the injured spinal cord.

Potassium-induced release of neuronotoxic activity by astrocytes

Medium conditioned by newborn rat cerebral cortex microexplants contains neuronotoxic activity for cerebellar granule cells and hippocampal neurons. The neuronotoxic activity is associated with low-molecular weight molecule(s) (less than 1000 Da) and resists to heating and to freezing and thawing. Using nearly homogenous cultures of neurons or astrocytes, we show that the neuronotoxic activity is released by the latter cell type. This release is enhanced by increasing extracellular K+-concentration. Astrocytes also secrete neuronotrophic activity whose release is not affected by external K+. Neurons can be desensitized against the neuronotoxic activity.

Kainate and NMDA toxicity for cultured developing and adult rat spiral ganglion neurons: further evidence for a glutamatergic excitatory neurotransmission at the inner hair cell synapse

In the inner ear, the excitatory amino acid glutamate is a proposed neurotransmitter acting at the synapse between hair cells and afferent auditory neurons. Using cultures of 5-day-old rat auditory neurons, we show that the afferent auditory neuronal population can be divided, on the basis of its sensitivity to the neuronotoxic effect of glutamate and its analogs, in at least 3 subpopulations, one responding to N-methyl-D-aspartate (NMDA), one responding to kainate and a third minor one unresponsive to NMDA, kainic acid and glutamate. No toxic effect of quisqualate is observed. The use of specific antagonists (kynurenate and 2-amino-5-phosphonovalerate (DAP-5) demonstrates the specificity of the receptors to the excitatory amino acids on the afferent auditory neurons. Afferent auditory neurons from adult rats can also be cultured and in these preparations only the large neurons are sensitive to glutamate, kainate and NMDA while the small neurons are not responsive, suggesting that a glutamatergic neurotransmission occurs only at this synapse between the inner hair cells and the large radial afferent auditory neurons. We also show that, in vitro, the organ of Corti releases, in response to an increased potassium concentration and in the presence of calcium, a toxic activity for the afferent auditory neurons that is antagonized by kynurenate and DAP-5. Pathophysiological implications are discussed.

Plasminogen activators in developing peripheral nervous system, cellular origin and mitogenic effect.

Newborn rat dorsal root ganglia release two different plasminogen activators (PAs): the urokinase (UK) and the tissue (tPA) type. The former is secreted by neurons while the latter is secreted by Schwann cells. tPA release by Schwann cells is modulated by choleratoxin, a known mitogen for these cells. UK but not tPA stimulates in a dose-dependent fashion the proliferation of Schwann cells. This effect is observed in the absence of plasminogen, suggesting that the substrate for PAs in the developing nervous system is not plasminogen. Since UK is secreted by neurons, our data suggest a new mechanism for neuronal control of Schwann cell proliferation.

Modulation of proteolytic activity during neuritogenesis in the PC12 nerve cell: differential control of plasminogen activator and plasminogen activator inhibitor activities by nerve growth factor and dibutyryl-cyclic AMP.

Extracellular proteolysis is considered to be required during neuritic outgrowth to control the adhesiveness between the growing neurite membrane and extracellular matrix proteins. In this work, PC12 nerve cells were used to study the modulation of proteolytic activity during neuronal differentiation. PC12 cells were found to contain and release a 70–75‐kDa tissue‐type plasminogen activator (tPA) and a much less abundant 48‐kDa urokinase‐type plasminogen activator. A plasminogen activator inhibitor (PAI) activity with molecular sizes of 54 and 58 kDa was also detected in PC12 cell conditioned medium and formed high‐molecular‐mass complexes with released tPA. Release of PAI activity was dependent on treatment with nerve growth factor (NGF), whereas tPA synthesis and release were under control of a cyclic AMP‐ dependent mechanism and increased on treatment with dibutyryl‐cyclic AMP [(But)2cAMP] or cholera toxin. Simultaneous treatment with NGF and (But)2cAMP resulted in increases of both tPA and PAI release and enhancement of tPA‐PAI complex formation. The resulting plasminogen activator activity in conditioned medium was high in (But)2cAMP‐treated cultures with short neuritic outgrowth but remained low in NGF‐ or NGF plus (But)2 cAMP‐treated cultures, where neurite extension was, respectively, large and very large. These results suggest that excess proteolytic activity may be detrimental to neuritic outgrowth and that not only PAI release but also tPA‐PAI complex formation is associated with production of large and stable neuritic outgrowth. This can be understood as an involvement of PAI in the protection against neurite‐destabilizing proteolytic activity.

Neurono-Glial Interactions and Neural Plasticity

Publisher Summary This chapter summarizes recent data from laboratory on intercellular messages that are exchanged between neurons and astroglia. Although in several instances, these data have been obtained using embryonic or neonatal material, it is believed that they are relevant to the understanding of brain reaction to injury and neuronal plasticity. Three aspects have been considered in the chapter: (1) neuronal regulation of astroglia proliferation; (2) astroglial regulation of neuronal survival and death; and (3) glial modulation of neuronal neurotransmitter phenotype. Some neurodegenerative disorders could result from a primary astroglial deficit. The chapter points out possible involvement of glia neuronal interactions at some steps of the pathophysiology of these disorders. The essential role of glial cells during development is now a widely accepted concept. There is no reason to believe that glial cells should not play similarly important roles during aging.

Cultured Astroglia Release a Neuronotoxic Activity That Is Not Related to the Excitotoxins

Neuronal death after brain injury is thought to be in part the result of the activity of the excitotoxins, a family of excitatory amino acids which are released by neurones. We have also described an astroglial cell-derived neuronotoxic activity of low molecular weight whose release can be induced by depolarizing events such as an increase in extracellular potassium concentration. We study here the relationship between this astroglia-derived neuronotoxic activity present in astroglia-conditioned medium (ACM) and the excitotoxins. Using a colorimetric assay of neuronal survival, we show that the ACM neuronotoxic activity, is able to induce the death of all types of neurones tested, including those which are insensitive to excitotoxins. Furthermore, the ACM neuronotoxic activity does not require for its action the extracellular ionic composition which is needed for the activity of excitotoxins. Finally, the ACM neuronotoxic activity is not blocked by competitive or non-competitive antagonists of the various classes of excitotoxin receptors. Those data demonstrate that the astroglia-derived neuronotoxic activity is not related to the excitotoxins. Still, because astrocytes can also be depolarized by members of the excitotoxin family, the possibility exists that the release of astroglia-derived neuronotoxic activity would follow the rise in extracellular excitatory amino acid concentration during nervous system injury.