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S Varon

Publications and source records attributed to S Varon.

At least 181 records · Page 10Linked to original sources
PubMed1982

In vivo regeneration of cut nerves encased in silicone tubes: growth across a six-millimeter gap.

We describe an experimental in vivo system for studying peripheral nerve regeneration, in which the proximal stump of a transected nerve regrows through a transparent silicone chamber toward the distal stump. Physical separation permits examination of the effects of the humoral and/or cellular influences from the distal stump on regenerating fibers before they invade the distal segment itself. A small segment of the rat sciatic nerve was resected, leaving a 6 mm gap which was then encased by a cylindrical silicone chamber. Within the first weeks, a nerve trunk regenerated along the central axis of the chamber bridged the gap between the proximal and distal stumps. When the distal nerve stump was omitted from the distal opening of the chamber, only a thin structure with a few small-caliber fibers extended across the gap. In each instance regenerating nerve appeared as a cord-like structure completely surrounded by clear fluid, a feature which permits easy collection of the extracellular fluid for analysis of its chemical properties and biological activity. This feature also allows in vivo manipulation of the humoral environment in which nerve regeneration occurs.

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PubMed1982

Purified mouse Schwann cells: mitogenic effects of fetal calf serum and fibroblast growth factor.

We present an in vitro bioassay protocol for the detailed examination of mitogenic influences on purified mouse Schwann cells. This involves measurements of (i) increases in Schwann cell number per culture, (ii) incorporation of radiolabeled thymidine per culture, and (iii) the proportion of total Schwann cells that exhibit a labeled nucleus by autoradiography. Using this standard protocol we show that the mitogenic efforts of fetal calf serum must be distinguished from its effects on cell retention to the culture substratum, and preparations of mouse brain or pituitary fibroblast growth factor but not mouse epidermal growth factor are very potent mouse Schwann cell mitogens. These mitogenic effects of serum and fibroblast growth factor have not been reported for rat Schwann cells.

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PubMed1981

Neuronotrophic factors and their antibodies: in vitro microassays for titration and screening.

Using nerve growth factor (NGF), anti-NGF sera and dissociated neonatal mouse dorsal root ganglionic neurons we present a microculture assay methodology for (1) the titration of neurotrophic factor (NTF) activity in monolayer culture, (2) the titration of NTF antibodies which 'block' NTF biological activity, (3) the titration of NTF antibodies that bind and remove (sequester) NTF from culture medium and (4) a large-scale, convenient, and rapid screening for NTF biological activity as well as for NTF 'blocking' or 'sequestering' antibodies. These quantitative and qualitative in vitro microimmunoassays should be applicable to any neuronotrophic factor or its antibody, even when the agent is only available in crude, unpurified form. Since the microculture systems permit the simultaneous screening of one thousand samples per day they should be useful for the detection and quantitation of monoclonal antibodies present in hybridoma-conditioned media.

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PubMed1981

Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons.

We have recently reported the existence within chick embryo heart cell conditioned medium (HCM) of two distinct and independently assayable factors. One agent, ciliary neuronotrophic factor (CNTF), supports the in vitro survival of 8-day chick embryo ciliary ganglionic (CG) neurons. The other factor, polyornithine-attachable neurite promoting factor (PNPF) is required for extensive neuritic growth from these same CNTF-supported CG neurons. In the present study we have examined the occurrence of PNPF activity within nearly 100 different conditioned media using our previously described chick CG bioassay system. From this screening we conclude that: (1) PNPF production is a rather widespread property of cultured neural as well as non-neural cells; and (2) the chick bioassay is sensitive to PNPF activity from all the species examined, including mouse, rat, human and chick cells. We next examined the effects of 3 representative PNPF-containing conditioned media (from chick heart, mouse Schwann and rat Schwannoma) on neurite production from 3 other peripheral ganglionic neuronal cultures (8-day chick dorsal root, 11-day chick sympathetic, and neonatal mouse dorsal root ganglia) as well as 4 central neuronal cultures (8-day chick embryo telencephalon, optic lobe and spinal cord and neonatal mouse cerebellum). The results of these studies indicate: (1) that the peripheral neurons exhibit a dramatic increase in neurite production in response to PNPF which can be easily recognized both qualitatively and quantitatively; whereas (2) the CNS neurons showed essentially no PNPF-induced increase in neurite production. The sole exception to the latter was the appearance within the chick spinal cord cultures of a neuronal population which extended very long neurites in response to PNPF.

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PubMed1981

In vitro responses of sympathetic neurons to nerve growth factor and other macromolecular agents.

Cells in the dissociated state from the sympathetic ganglia (SG) of 11-day-old chick embryos, and monolayer cultures of these cells are used to illustrate some of th extrinsic influences that regulate neuronal performance. In culture, the survival of SG neurons can be measured, as an assay for survival-promoting agents. Among the requirements of the SG discussed are: (1) nerve growth factor and other trophic factors that can replace it, (2) serum, and a defined mixture (N1) that can substitute for it, and (3) a minimal presence of non-neuronal cells. Also reviewed are factors that confer neurite-promoting competence on certain culture substrata. Suspensions of SG cells permit analysis of "short-latency" events triggered within minutes of the presentation of nerve growth factors and provide an insight into its possible mode of action. The most striking such event is its control over Na+/K+ pumps, since ionic control is a fundamental feature of living cells and may well mediate their regulation by trophic factors, hormones or mitogens.

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PubMed1981

Trophic activities for dorsal root and sympathetic ganglionic neurons in media conditioned by Schwann and other peripheral cells.

We describe here the use of 3 established nerve growth factor (NGF) targets (cultured neurons from dissociated chick embryo sympathetic ganglia, and chick embryo or neonatal mouse dorsal root ganglia) to investigate neuronotrophic activities in conditioned media (CMs) from: (i) chick embryo heart; (ii) purified mouse Schwann cells; and (iii) clonal rat Schwannoma RN22 cells. In chick sympathetic and mouse dorsal root ganglionic cultures, all 3 CMs supported survival of the same number of neurons as did mouse submaxillary NGF, and in most cases no increased survival resulted from concurrent administration of NGF and any one CM. NGF and CM activities were quantitated in each of the responsive cell systems. No differences were seen when either test population was used for the same agent, or when different CMs were examined on the same test cells. The CM activity, unlike that of NGF, was not blocked by even excess amounts of antiserum against mouse submaxillary NGF. The neuronotrophic activity of CMs appears to reside with macromolecular constituents. None of the CMs displayed trophic activity on chick embryo dorsal root ganglionic neurons. However, at least one of them (RN22 medium) had drastic effects on these ganglionic cells even in the presence of NGF, leaving open the possibility of a 'toxic' factor overriding putative trophic agents.

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PubMed1981

Neurite-promoting factor in conditioned medium from RN22 Schwannoma cultures: bioassay, fractionation, and properties.

On polyornithine (PORN) substrata dissociated 8-day chick embryo ciliary ganglionic neurons will survive if the culture medium is supplemented with Ciliary neuronotrophic Factor. However, neuritic growth will not occur unless the substratum is derivatized with a PORN-bindable Neurite Promoting Factor (PNPF). In this preliminary study we report that soluble PNPF can be (1) assayed by a convenient in vitro system; (2) obtained in relatively large amounts from serum-free media conditioned over RN22 Schwannoma cultures; (3) concentrated by using Amicon XM100 ultrafiltration; and (4) separated from nearly all of the non-active protein by using ion-exchange chromatography. The partially purified PNPF can be concentrated using XM100 and is heat- and protease-sensitive. In the course of these fractionation studies we observed in some cases a concentration-dependent interference with the expression of PNPF activity in the bioassay; we propose graphical methods to permit the simultaneous determination of PNPF and the extent of such interference. Different treatments that affected the interference property did not always affect PNPF activity in a reciprocal manner, leaving open the possibility that the interference with PNPF activity results from reversible alteration of the PNPF molecule, or that there exists a separate interfering agent.

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PubMed1980

Maintenance by nerve growth factor of the intracellular sodium environment in spinal sensory and sympathetic ganglionic cells.

A promising advance in our understanding of the mode of action of nerve growth factor (NGF) has been provided by our recent finding that dissociated cells from 8-day embryonic chick dorsal root ganglia (DRG) lose their competence to maintain a low intracellular Na+ when incubated without NGF for 6 h, and promptly recover it on delayed presentation of NGF. To ascertain whether control of intracellular Na+ is a general feature of the NGF action on its target neurons, we have now tested several NGF-sensitive tissues, both as intact ganglia and dissociates, for their ability to (i) accumulate large amounts of 22Na+ over a period of hours in the absence of NGF, and (ii) rapidly extrude the accumulated radioactivity upon delayed presentation of NGF. Intact and dissociated chick embryo DRG and sympathetic ganglia, as well as dissociated mouse DRG, displayed the expected Na+ responses to the lack or the administration of NGF. No such responses were observed with intact mouse DRG, which do not require NGF in explant cultures for neuritic outgrowth, or with intact and dissociated chick embryo ciliary ganglia, which are not sensitive to NGF in either explant or dissociated cell cultures. Thus, in all ganglionic preparations examined, the occurrence of Na+ responses to exogenous NGF correlated with traditional responses to NGF in culture. This together with other recent data, reinforces the view that ionic control may be a critical element in the mechanism of action of NGF.

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PubMed1980

Purification of mouse Schwann cells using neurite-induced proliferation in serum-free monolayer culture.

We have recently reported that neonatal mouse dorsal root ganglionic Schwann cells will (i) survive and assume characteristic morphologies in a serum-free, fully defined cultured medium (N1 medium), (ii) proliferate extensively in the same N1 medium if neurons are also present and maintained by nerve growth factor, and (iii) display a strong proliferative response to serum even in the absence of neuronal elements, while also undergoing marked changes in their morphology and their associative behavior toward neurites. In this report, we present a detailed procedure, based upon these earlier observations, which yields purified cultures of either neurons plus associated Schwann cells or Schwann cells in the absence of neurons. The procedure utilizes the neuritic mitogen for selective expansion of Schwann cell numbers in serum-free primary cultures, and a secondary culture step involving neuronal removal and additional Schwann cell expansion using the serum mitogen. The procedure requires 9 days for the generation of 3-4 X 10(6) Schwann cells from 12 newborn mice (with a Schwann cell to neuron ration of 10) and an additional 6-7 days for the generation of a neuron-free secondary population of 40 X 10(6) Schwann cells with less than 3% contamination by identifiable ganglionic fibroblasts.

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PubMed1980

Cholinergic neuronotrophic factors: V. Segregation of survival- and neurite-promoting activities in heart-conditioned media.

Chick embryo ciliary ganglionic (CG) neurons will not survive in monolayer culture unless special supplements are provided in the medium. We have previously reported that two such supplements, chick embryo extract and medium conditioned over chick heart cell cultures (HCM) share the capacity to support survival of CG neurons but differ in their neurite-promoting effects. Thus, embryo extract elicited neuritic outgrowth only on collagen and HCM did so only on polyornithine (PORN), although both agents supported neuronal survival on both substrata. We report here the separation and quantitation of two different HCM components. One is a trophic agent which supports survival of CG neurons on either collagen or PORN, but does not seem to adsorb to either substratum. The other is a neurite-promoting factor (NPF) which adsorbs to PORN but not to collagen. Overnight incubation of HCM on PORN yields two products: (i) an NPF-deprived HCM, that has no neurite-promoting activity and (ii) an NPF-coated PORN, that promotes neuritic development of CG neurons trophically supported by either embryo extract or NPF-deprived HCM. CG requirements for neuritic outgrowth were also examined in explant cultures. No neurites were present after 24 h when explants were cultured in plain medium on PORN. Very extensive radial neuritic outgrowth was observed when explants were cultured in HCM on fresh PORN, or in NPF-deprived HCM on NPF-derivatized PORN. In contrast to what happens with dissociated cells, neuritic outgrowth was also present when ganglia were cultured in NPF-deprived HCM on fresh PORN. However, neurites grew radially only to a limited extent, after which they adopted a circular pattern grossly concentric to the ganglionic explant. It is proposed that explanted ciliary ganglia produce a neurite-promoting factor that coats the PORN substratum in widening circles.

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