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S D Skaper

Publications and source records attributed to S D Skaper.

At least 145 records · Page 8Linked to original sources
PubMed1982

Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site.

A cavity was made in the brain (entorhinal cortex) of developing or adult rats, and a small piece of Gelfoam was emplaced to collect fluid secreted into the wound. The neuronotrophic activity of the fluid was assayed with sympathetic and parasympathetic neurons in culture. The results show that wounds in the brain of developing or adult rats stimulate the accumulation of neuronotrophic factors and that the activity of these factors increases over the first few days after infliction of the damage.

Adrenergic Fibers↗
PubMed1982

Regulation of Na+,K+ pump activity by nerve growth factor in chick embryo dorsal root ganglion cells.

Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+,K+-pump-mediated) K+ influx over a period of several hours. Addition of NGF to NGF-deprived cells caused 1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by 2) a very rapid, but transient overstimulation of the Na+,K+-pump-mediated K+ influx. Restoration of normal Na+,K+-pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1-100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF-deprived cells and could not be elicited in NGF-supported steady-state cells or in cells having already received delayed NGF once. This transient Na+,K+-pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+,K+-pump was specific for the NGF molecule and could not be mimicked by other proteins.

Animals↗
PubMed1982

Molecular requirements for survival of cultured avian and rodent dorsal root ganglionic neurons responding to different trophic factors.

We have previously demonstrated that both peripheral and central neurons from 8 day embryonic chick and newborn mouse can be maintained in a serum-free medium using the N1 supplement consisting of insulin, transferrin, putrescine, progesterone, and selenite. In the present studies we show that dorsal root ganglionic (DRG) neurons from embryonic chick (E7-E15) and neonatal mouse can be cultured in a serum-free environment with only the addition of insulin and transferrin, plus Nerve Growth Factor (NGF). Chick DRG from E10-E15 contain a population of neurons sensitive to a chick embryo eye-derived ganglionic neuronotrophic factor (GNTF), which is distinct from the neuronal subset dependent upon NGF. The GNTF-dependent chick neurons can also be maintained in culture with insulin and transferrin supplements. Neonatal mouse DRG neurons, whether supported by NGF or eye-derived GNTF, likewise survive in serum-free medium with only insulin and transferrin. Limited numbers of neurons survive for the first 24 hours in a serum-free medium lacking insulin or transferrin, but failed to display neurite outgrowth even in the presence of added trophic factor.

Animals↗
PubMed1982

Ionic behaviors and nerve growth factor dependence in developing embryonic chick ganglia. I. Studies with intact dorsal root ganglia.

We have recently shown that intact and dissociated 8-day embryonic (E8) chick dorsal root ganglia (DRG) lose the ability to regulate their intracellular Na+, K+ levels when deprived of nerve growth factor (NGF) for 6 h; recovery occurs within minutes of NGF presentation. These ganglionic neurons are believed to depend on NGF for survival and neurite production over a defined period of embryonic life--between about E6 and E15 in the chick. Using intact DRG from E6-E16 chick embryos we determined developmental changes in: (i) 22Na+ accumulation in the presence and absence of NGF, or in the presence of ouabain; and (ii) intra- and extracellular fluid spaces. Sodium accumulation, in the presence of NGF, increases from E6 to E10. It parallels the total fluid space under ouabain but then decreases conspicuously between E10 and E16, despite little change in the latter. NGF thus prevents Na+ accumulation during the early period, and becomes increasingly irrelevant for this behavior in later (after E10) development. These data are interpreted as indicating that: (i) NGF is required for ionic control by DRG neurons up to E10; and (ii) indigenous behaviors for the control of ion pump mechanism(s) are progressively acquired by these cells from E10 to E16, in parallel with the decreasing ionic relevance of NGF. These findings are consistent with the view that the ionic responses to NGF correlate closely with the survival and neurite-promoting effects of this factor.

Age Factors↗
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.

Animals↗
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.

Animals↗
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.

Animals↗
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.

Animals↗
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.

Animals↗
PubMed1980

Survival, proliferation and morphological specialization of mouse Schwann cells in a serum-free, fully defined medium.

Neonatal mouse dorsal root ganglionic (DRG) cell dissociates were cultured in a synthetic medium with horse serum or the serum-free supplement N1 (insulin, transferrin, progesterone, putrescine, selenium). Serum-supplemented cultures with added nerve growth factor (NGF) yielded neurons, small flat and spindle cells (Schwann) and large flat cells (fibroblastic elements). However, in serum-free, N1-supplemented medium plus exogenous NGF, neurons and Schwann cells predominated, with very few large flat cells. In the N1 medium most Schwann cells assumed a typical spindle shape and were associated with neuritic processes when neurons were present. Upon addition of serum, virtually all of the Schwann cells appeared to abandon physical contact with the neurites and develop a more flattened morphology. In N1 medium without NGF (no neurites), most Schwann cells still assumed a spindle shape and formed characteristic chain-like associations. Autoradiographic techniques, as well as numerical analyses, demonstrated that in N1 medium Schwann cells were able to proliferate when associated with neurites but only slightly so in their absence. These Schwann cells showed a marked increase in proliferation when serum was added regardless of the presence or absence of neurites. The above observations may provide a basis for the preparation of purified Schwann cells, alone or in combination with their neurons.

Animals↗