Purification and Culture of Adult Rat Dorsal Root Ganglia Neurons

To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns. Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS

Neurotransmitter Phenotype Plasticity in Cultured Dissociated Adult Rat Dorsal Root Ganglia: An Immunocytochemical Study

Culturing sympathetic ganglion neurons in vitro may modify phenotypic expression of some neurotransmitters. For dorsal root ganglia (DRG), contradictory results have been reported; most studies have used immature material. We have therefore performed a detailed immunocytochemical analysis of the transmitter content of cultured adult rat DRG neurons. To demonstrate possible modifications of neurotransmitter phenotypes, we have compared the results obtained with the same techniques on neurons cultured for 3 days and on freshly dissociated DRG cells. Also, the transmitter profile of cultured neurons was compared with that known from in situ studies. Out of 22 antigens studied, 20 were detected in cultured DRG neurons. All of them were expressed in small and/or intermediate-sized cells. Large neurons only contained CGRP, VIP, NPY, beta-END, ENK, and GABA. The percentage of immunostained neurons varied for the various antisera: less than 10% of cultured neurons were positive for ENK, beta-LPH, beta-END, DYN, VASO, and OXY; 10-30% for SOM, CCK, CAT, and SP; and greater than 30% for NPY, CRF, GLU, NT, VIP, GABA, GRP, CGRP, 5-HT, and TRH. In the latter two groups of transmitters (except CGRP), the proportion of immunoreactive neurons was by far larger in cultured than in freshly dissociated DRG. The most pronounced (greater than 25%) increase in the proportion of positively stained neurons after culturing was observed for the GRP, CRF, TRH, and 5-HT antisera. Serotonin was the only transmitter identified in cultured but not in freshly dissociated cells. These data indicate, on one hand, that various antigens, for example, CAT, GABA, NT, TRH, NPY, beta-LPH, and beta-END, which up to now have not been described in DRG in situ, can be detected immunocytochemically a few hours after dissociation of adult rat DRG. On the other hand, several transmitters, for example, VIP, NPY, SP, GABA, GLU, NT, GRP, CRF, TRH, and 5-HT, are expressed in a significantly higher proportion of cells in cultured than in freshly dissociated preparations. This might reflect a change in the phenotypic expression of transmitters due to the new environment generated by the culture conditions, a hypothesis that can be tested by measuring specific mRNA levels. Moreover, considering the plasticity and multipotentiality of their transmitter phenotype, cultured adult DRG neurons might represent an interesting material for autografts into the injured central nervous system

Mechanism of Cell Communication in the Regenerating Peripheral Nervous System

peer reviewe

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

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

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)2cAMP-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

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

Potassium-Induced Release of an Endogenous Toxic Activity for Outer Hair Cells and Auditory Neurons in the Cochlea: A New Pathophysiological Mechanism in Meniere's Disease?

In Meniere's disease, the increase of extracellular potassium concentration in the perilymph is thought to play a key role in determining the progressive loss of cochlear hair cells. In this paper, we describe a serum-free culture preparation of hair cells from 5 day-old rat and report the release by the cochlea, in response to an increase of extracellular potassium concentration, of a cytotoxic activity active on hair cells and auditory neurons. The toxic activity is associated with low molecular weight (less than 10,000 Dalton) molecule(s) as revealed by ultrafiltration. Morphological studies performed on the organ of Corti incubated during 24 h in the presence of the cochlea-derived toxic activity (CTA), show that this factor is toxic for hair cells and not for supporting or surrounding cells. The release of CTA occurs both in the spiral ganglion and in the organ of Corti. We suggest that this cochlea-derived toxic activity may play an important role in the pathophysiology of the hearing loss that occurs during the progression of Meniere's disease

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 corticospinal tract as assessed after anterograde tracing or of descending aminergic fibers could be demonstrated