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S Varon

Publications and source records attributed to S Varon.

At least 127 records · Page 7Linked to original sources
PubMed1985

Modification of fibrin matrix formation in situ enhances nerve regeneration in silicone chambers.

The spatial-temporal progress of nerve regeneration was examined in silicone chambers of three different volume capacities: 11, 25, and 75 microliter. In all chambers, the stumps of a transected rat sciatic nerve were sutured into the ends of the chamber leaving a 10 mm gap between the stumps. Chambers were implanted empty (E chambers) or prefilled with saline (PF chambers). A coaxial and continuous fibrin matrix had formed in all chambers by 1 week. In E chambers, the matrices had a proximal-distal taper that was more pronounced in E25 and E75 chambers due to significantly larger matrix diameters in the proximal region. At 3 weeks, vascular and Schwann cell migration and axonal regeneration were less advanced in the E25 and E75 than in the control E11 chambers. The retardation correlated with the presence of an avascular organization of circumferential cells. Saline prefilling affected the caliber and density of fibrin fibers in the 1 week matrices of PF25 and PF75 chambers. The matrices did not have a prominent taper and diameters were progressively larger with increasing chamber volume. Saline prefilling did not affect regeneration progress in 3 week PF11 chambers but did enhance regeneration in the PF25 chambers; a 1.5-fold larger diameter nerve formed at 3 weeks that contained 2.6-fold more axons. Progress in the PF75 chamber was retarded. We conclude that the volume, timing, and nature of the fluid filling a silicone chamber have significant influence on the formation of fibrin matrices. Alterations in matrix formation correlate with substantial changes in the subsequent progress of intrachamber regeneration events.

Animals↗
PubMed1985

Selected nutrients reduce the pyruvate requirement for survival in vitro of chick central nervous system neurons.

Central nervous system neurons, cultured as monolayers at low density, need exogenous pyruvate for their survival. The pyruvate concentrations required by embryonic day 8 (E8) chick forebrain neurons are reduced substantially by other low molecular weight agents present in Dulbecco's modified Eagle's medium (DMEM) and in astroglia-conditioned Eagle's basal medium (EBM). To examine the nature of these pyruvate-sparing molecules, E8 chick forebrain neurons were cultured in EBM containing a concentration of pyruvate too low to support neuronal survival, unless sparing agents were supplied. This strategy permitted the identification of the pyruvate-sparing DMEM constituents, omission of which had led to neuronal loss. Pyruvate sparing required a mixture of (i) serine, (ii) pyridoxal (or riboflavin and biotin), and (iii) six essential amino acids: arginine, cystine, leucine, isoleucine, tyrosine, and tryptophan (each replaceable by corresponding amino acids, except tryptophan). All pyruvate-sparing contributors were optimally needed at or near their DMEM concentrations.

Amino Acids↗
PubMed1985

Developmental changes in the responses of rat chromaffin cells to neuronotrophic and neurite-promoting factors.

This study describes the survival and neurite outgrowth behaviors of cultured adrenal medullary (chromaffin) cells obtained from postnatal rats 1 day (D1) to 100 days (D100) old in response to nerve growth factor (NGF), chick eye ciliary neuronotrophic factor (CNTF), and laminin. In the absence of trophic factors the 4-day survival of cultured chromaffin cells (relative to the number of cells attached at 2 hr) increased from one-third of the cells at D1 to 40% at D8 and 90-100% at D16 and older stages. At saturating concentrations NGF increased cell survival at D8 by 90%, but failed to support all chromaffin cells present at 2 hr. In contrast, CNTF supported the survival of all cells at D8. At D1 NGF and CNTF had only a very small effect on survival during the 4-day culture period, although both factors clearly enhanced the numbers of surviving cells after 8 days. Either NGF or CNTF also elicited neurite outgrowth from rat chromaffin cells, which amounted to approximately 15-20% at D1 and D8 and subsequently decreased to about 5-8% at D30 and virtually zero at D100. At this last age both factors applied together clearly elicited neurites. Such a potentiating effect of NGF and CNTF was also seen at earlier postnatal ages. Laminin did not affect neurite growth at D30 in the absence of trophic factors, as already described for D8 rat chromaffin cells. In the presence of NGF, however, it increased neurite length and branching during a 4-day culture period and even enhanced neurite recruitment at later culture times. These data suggest that rat chromaffin cells undergo age-related changes in their responses to NGF and CNTF and that laminin modulates their neurite outgrowth behaviors in the presence of trophic factors.

Adrenal Glands↗
PubMed1985

GM1 ganglioside accelerates neurite outgrowth from primary peripheral and central neurons under selected culture conditions.

Neurons from chick embryonic day 8 (E8) ciliary ganglia, E8 and E15 dorsal root ganglia, E8 forebrain, and from rat E18 hippocampus and striatum were cultured as monolayers in the presence or absence of GM1 ganglioside. All of the primary neurons tested were susceptible to an effect of GM1 on their neuritic outgrowth, resulting in a 2- to 3-fold stimulation over control, the recognition of which depended on selecting culture conditions appropriate to each case. The response of E8 ciliary ganglionic neurons required a serum-free medium containing ciliary neuronotrophic factor, and was most pronounced by 8 h at 3 X 10(-8) M GM1. The neuritic response by either E8 or E15 dorsal root ganglionic neurons required serum (greater than or equal to 0.3%), their appropriate neuronotrophic factor, and 100-fold higher GM1 concentrations (presumably reflecting the serum presence), with optimal response times of 12-24 h. For E8 chick forebrain and E18 rat central neurons, GM1 substantially increased the proportion of neurite-bearing neurons in a serum-free pyruvate-containing medium between 7 and 24 h, with an optimal GM1 concentration of 10(-7) M. In all cases, the response to GM1 was a time-related gain, i.e. an earlier onset of neuritic regeneration rather than permanent increase in the number of neurite-bearing neurons.

Animals↗
PubMed1985

Tumor-promoting phorbol diester mimics two distinct neuronotrophic factors.

The two purified neuronotrophic factor proteins, nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF), differ in molecular properties and in their action on certain neuronal cell types. The survival in culture of dissociated chick embryonic day 8 dorsal root ganglionic neurons is supported by NGF and day 8 ciliary ganglionic neurons by CNTF, but neither factor can be a substitute for the other. Here we report that a known tumor-promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), can support the survival of both neuronal types in the absence of either NGF or CNTF and does so with the same efficacy as the corresponding trophic factor. The inactive phorbol derivative, 4-alpha-phorbol-didecanoate, does not support either neuronal type. The combined provision of suboptimal doses of TPA and NGF or CNTF does not reveal synergistic effects. These results suggest that TPA, NGF and CNTF may exert their neuronal survival promoting influences through a common molecular mechanism.

Animals↗
PubMed1985

Parameters of neuritic growth from ciliary ganglion neurons in vitro: influence of laminin, schwannoma polyornithine-binding neurite promoting factor and ciliary neuronotrophic factor.

Ciliary ganglion neurons extend neuritic processes when cultured for 24 h in medium containing ciliary neuronotrophic factor (CNTF) and on a polyornithine substratum precoated with either laminin or a Schwannoma-derived neurite promoting factor (PNPF). We have examined the roles of laminin, PNPF and CNTF for each of four parameters of neuritic growth, including: initiation time, neuronal polarity, neuritic branching and average neurite output (lengths) with time. Increasing laminin and PNPF levels were found to advance the time of neurite initiation as well as shift the majority (70-80%) of the neurons from a unipolar to multipolar neuritic morphology. The polarity imposed by any given concentration of either neurite promoting factor remained constant over the 24 h culture period examined. The average lengths from the longest neurites per neuron over a 10-28 h culture interval were not affected by increasing levels of laminin or PNPF, but total neuritic output per neuron was increased. This increased total neuritic output could be attributed to a combination of earlier neuritic initiation time and an increased neuronal polarity at high laminin or PNPF levels. CNTF at threshold survival levels did not promote initiation time, neuronal polarity or total neuritic output. However, cultures receiving less CNTF than that required for maximal neuronal survival displayed an increased neuronal polarity and a reduced neuritic output before any apparent loss of neurons. Neuritic branching was not affected by either the neurite promoting or trophic factors after 24 h of culture. Laminin and PNPF were found to be indistinguishable in their effects on the ciliary ganglion neurons in each of the four parameters studied.

Animals↗
PubMed1985

Neuronotrophic and neurite-promoting factors: effects on early postnatal chromaffin cells from rat adrenal medulla.

Adrenal chromaffin cells from early postnatal rats maintained in culture have previously been shown to grow neuritic processes and survive better in the presence of nerve growth factor (NGF). In the present study we have quantitated the effects on chromaffin cell (postnatal day (D) 8) survival and neurite outgrowth of: NGF, ciliary neuronotrophic factor (CNTF), activities contained in various types of conditioned media (CM), and various substrata (laminin, fibronectin and polyornithine-binding neurite-promoting factor from RN 22 Schwannoma cells - PNPF). At saturating concentrations CNTF (50 ng/ml) and C6 glioma cell CM, (50-fold concentrated) supported survival over the 4-day culture period of all the chromaffin cells present in culture 2 h after seeding. NGF (50 ng/ml) and the non-concentrated CMs from primary Schwann cell and astrocytes as well as Schwannoma and C6 glioma cell cultures, achieved the maintenance of only about half the number of cells above the baseline survival as compared to CNTF and the concentrated C6-CM. These results are compatible with two subsets of D8 chromaffin cells, one only supported by CNTF and the concentrated CM and the other supported by either NGF or CNTF. Either NGF or CNTF elicited neurite outgrowth from 15-20% of the surviving cells. Combination of maximal doses of NGF and CNTF caused a small increase in neurite recruitment beyond that elicited by either factor alone. Low doses of CNTF added to the effect of NGF, shifting the NGF titration curve by about 4-fold. Neurite outgrowth was also induced by the concentrated, but not the unconcentrated C6-CM. Laminin, fibronectin and PNPF did not affect the fibronectin and PNPF did not affect the recruitment of neurites as compared to a polyornithine substratum unless the cultures were supplemented with a neuronotrophic factor and carried for 7 days. However, even before showing effects on neurite recruitment these substrata affected various neuritic performances, such as length, neurite numbers and endings per cell.

Adrenal Medulla↗
PubMed1985

Comparison of the effects of laminin and the polyornithine-binding neurite promoting factor from RN22 Schwannoma cells on neurite regeneration from cultured newborn and adult rat dorsal root ganglion neurons.

We have investigated the effects of two neurite promoting factors (NPFs)--laminin and the semipurified polyornithine-binding neurite promoting factor (PNPF-1) from RN 22 Schwannoma cells--on neurite regeneration from dissociated newborn and adult rat dorsal root ganglion (DRG) neurons during 24 and 48 h culture periods in the absence of exogenous neuronotrophic factors. Both laminin and PNPF, when used to pretreat the polyornithine substratum, significantly enhanced neurite recruitment from surviving newborn and adult DRG neurons as compared to an untreated polyornithine substratum. However, the responses of newborn neurons at saturating concentrations of laminin and PNPF were consistently greater (46% neurite-bearing cells at 24 h, 81% at 48 h) than those of adult neurons (14 and 45%, respectively). The responsive neurons of both newborn and adult DRG displayed extensive neuritic networks at 48 h. The ED50 of laminin, or PNPF was 0.15-0.2 micrograms/ml for both newborn and adult neurons. The similarities in the responses of newborn and adult DRG neurons to NPFs validate the use of neurons from embryonic and newborn animals for the in vitro assays of NPFs that can be collected from injured and regenerating adult peripheral nervous tissues.

Age Factors↗
PubMed1985

The output of neuronotrophic and neurite-promoting agents from rat brain astroglial cells: a microculture method for screening potential regulatory molecules.

Throughout embryonic development, as well as in response to injury of the central nervous system, astroglial cells may present neurons with a critical supply of neuronotrophic and neurite-promoting factors which control, respectively, neuronal survival and axonal growth. The identification of such astroglial cell-derived factors, as well as of specific extrinsic agents regulating their production, will require the use of in vitro techniques. We define here a new microculture system in which added agents can be screened for their ability to enhance or inhibit the output of trophic and neurite-promoting factors from purified neonatal rat brain astroglial cells. With such a procedure, thousands of replicate secondary astroglial cultures can be set-up and maintained in chemically defined medium, on a defined substratum and in a viable, low proliferative stable state. These cultured astroglial cells release into their medium at least three distinct and separable types of agents addressing nerve cells in vitro: (i) high molecular weight trophic factors (Mr greater than 10,000) which support the survival of embryonic peripheral neurons; (ii) low molecular weight trophic agents (Mr less than 10,000) supporting embryonic central neurons; and (iii) polyornithine-binding neurite-promoting factors which enhance neuritic regeneration for both peripheral and central neurons. The temporal release patterns of these three agents from astroglial cultures are quite distinct suggesting that their output is independently regulated.

Animals↗
PubMed1985

Specific replacements of pyruvate for trophic support of central and peripheral nervous system neurons.

When embryonic central nervous system neurons are seeded at low densities with Eagle's basal medium supplemented with the serum substitute N1, glucose, and glutamine, neuronal survival for even 24 h requires the additional supply of exogenous pyruvate--and so does the survival of many peripheral nervous system neurons. Pyruvate can be replaced by alpha-ketoglutarate or oxaloacetate, but not by Krebs cycle substrates that are not keto acids. Most other alpha-keto acids tested (though not beta- or gamma-keto acids) also mimic pyruvate. The apparent equivalence to pyruvate of all these compounds includes identical ED50 values (300 microM for embryonic avian fore-brain neurons, 30-40 microM for rat hippocampal neurons), and also identical susceptibilities to the pyruvate-sparing effects of other low-molecular-weight agents present in Dulbecco's modified Eagle's medium or in astroglia conditioned medium. The substitute alpha-keto acids, however--unlike pyruvate, alpha-ketoglutarate, or oxaloacetate--support cell survival only in the presence of alpha-amino acids that transaminate to alpha-ketoglutarate, oxaloacetate, or pyruvate. The alpha-keto acids, therefore, operate as acceptors of amino groups from appropriate donors to generate Krebs cycle-relevant substrates. Consistent with this view, [14C]glutamate did not generate appreciable 14CO2 unless accompanied by a suitable alpha-keto acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Purified proteins acting on cultured chick embryo ciliary ganglion neurons.

Chick embryo ciliary ganglion neurons in dissociated monolayer culture have been used to examine molecular requirements for neuronal survival and neurite growth. These neurons will rapidly die in vitro unless supplied with an adequate level of ciliary neuronotrophic factor (CNTF), and even in the presence of CNTF they will not vigorously extend neurites on polyornithine substrata unless supplied with appropriate amounts of polyornithine-binding neurite-promoting factors (PNPFs). Recent work on the purification and partial characterization of embryonic chick eye CNTF and rat schwannoma PNPF is reviewed, and in vitro responses of ciliary ganglion neurons to other purified proteins such as laminin, fibronectin, insulin, and nerve growth factor are mentioned.

Animals↗
PubMed1985

Isolation and characterization of rat schwannoma neurite-promoting factor: evidence that the factor contains laminin.

Rat RN22 schwannoma cells in vitro release into their growth medium a macromolecular factor that, when bound to polyornithine-coated culture substrata, will stimulate neuritic regeneration from axotomized peripheral and central neurons. During the purification of this factor, the neurite-promoting activity co-purifies with laminin immunoreactivity as measured by an enzyme-linked immunoadsorbent assay. The purified factor has an immunoreactivity per milligram of protein similar to that of purified rat yolk sac tumor laminin. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, the purified factor exhibits a major band at 200 kilodaltons (kD) and two minor ones at about 130 and 35 kD. The 200-kD band comigrates with the 200-kD band of purified rat laminin. After SDS-PAGE under non-reducing conditions, the rat schwannoma factor and rat laminin both exhibit a band in the 900-kD range with the schwannoma factor band migrating slightly faster than the laminin one. The 200-kD (reducing conditions) and 900-kD (non-reducing conditions) bands of both the schwannoma factor and laminin are stained by immunoblotting with antisera raised against rat and human laminin and against a partially purified preparation of the schwannoma factor. On immunoblots the 400-kD band of laminin (a band not seen in the schwannoma factor preparation) also stains with all three antisera. When the antibodies from each of the three antisera are immobilized on protein A-agarose beads, the beads will completely remove from solution the neurite-promoting activities of both the schwannoma factor and laminin. Antibodies raised against rat laminin fail to block the neurite-promoting activity of the purified schwannoma factor but totally block that of rat laminin. In contrast, antibodies raised against the schwannoma factor will block the neurite-promoting activities of both the schwannoma factor and laminin. By rotary shadowing electron microscopy the schwannoma factor preparation exhibits cross-shaped images similar but not identical to those previously reported for rat and mouse laminin. In addition, the schwannoma factor preparation contains images resembling proteoglycans.

Animals↗
PubMed1985

Pyruvate participation in the low molecular weight trophic activity for central nervous system neurons in glia-conditioned media.

Conditioned media from glial cell cultures contain low molecular weight agents which can support survival of CNS neurons in the absence of recognized protein neuronotrophic factors. A similar support is provided to CNS neurons by selected basal media, and pyruvate is the critical medium constituent responsible for their trophic competence. Eagle's basal medium, which contains no pyruvate, acquires pyruvate when conditioned over astroglial cell cultures. Enzymatic degradation of the pyruvate in the astroglia-conditioned medium leads to corresponding losses in its low molecular weight trophic activity for CNS neurons. Quantitative correlations between pyruvate content and CNS trophic activity demonstrate that pyruvate is the main trophic ingredient of the glia-conditioned medium, and other low molecular weight substances, acquired during conditioning, reduce the pyruvate concentration required for its trophic effect. The "pyruvate-sparing" substances, as yet unidentified, are not the serine and Fe3+ which have pyruvate-sparing competence for peripheral, ciliary ganglionic neurons. These findings, together with previous observations, propose that prenatal neurons fail to generate or retain endogenous pyruvate at the levels for their survival-sustaining activities.

Animals↗
PubMed1985

Localized survival of ciliary ganglionic neurons identifies neuronotrophic factor bands on nitrocellulose blots.

A novel and sensitive method has been developed to identify ciliary neuronotrophic factors (CNTFs) from tissue extracts after blotting to nitrocellulose paper. The CNTF proteins are required for the in vitro survival of embryonic chick ciliary ganglionic neurons. Tissue extracts containing such CNTFs are electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Purified ciliary ganglionic neurons are seeded on the surface of the nitrocellulose blot, and the culture is incubated for 24 hr in medium lacking CNTF. CNTF can be localized on the blot because it retains its ability to support the survival of the neurons cultured on the nitrocellulose. A band of viable neurons, easily visualized by staining with a vital dye, is supported by the blotted CNTF polypeptide. The number of neurons surviving on the blotted CNTF is related to the amount of CNTF originally loaded on the electrophoretic gel. As little as 2 ng (16 trophic units) of CNTF protein contained in crude tissue extracts can be loaded on the sodium dodecyl sulfate gel and still be recognized by the cultured neurons. This method was used to identify CNTF polypeptides from extracts of adult rat nerve (24,000 and 19,000 daltons) and from tissue found near experimentally induced adult rat brain lesions (24,000 daltons). The electrophoretic mobilities of these peptides are distinct from the previously purified chick eye CNTF polypeptide (20,400 daltons).

Animals↗
PubMed1984

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers.

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Chemically defined requirements for the survival of cultured 8-day chick embryo ciliary ganglion neurons.

We have previously demonstrated that both peripheral and central neurons from embryonic chick and newborn mouse can be maintained in a serum-free defined culture medium containing the appropriate neuronotrophic agent and the N1 supplement consisting of insulin, transferrin, putrescine, progesterone and selenite. In the present studies we have examined the short-term survival requirements of 8-day embryonic chick ciliary ganglion (CG) neurons. By comparing CG neuronal survival in our standard culture medium, Eagle's Basal Medium (EBM), with several other commercially available basal media, we have established that CG neurons also have specific requirements for pyruvate, serine and iron (Fe3+), in addition to their trophic factor (Ciliary Neuronotrophic Factor, CNTF) and the N1 supplement. The data suggest the existence of 3 subsets of CG neurons differing in their essential needs, namely: (1) those supported by glucose in the absence of pyruvate, (2) those requiring exogenous pyruvate but not serine or Fe3+, and (3) those which need pyruvate, serine and Fe3+. The minimal effective concentration of pyruvate could be decreased by a factor of 50 in the concurrent presence of serine and Fe3+. Serine was also a limiting element in the survival of some of these CG neurons. The Fe3+ concentration required by the same neurons was considerably diminished with the availability of transferrin, perhaps reflecting an increased Fe3+ transmembrane transport efficiency. Insulin was found to be the only N1 ingredient required for the survival of CG neurons. Insulin was a constant requirement for all 3 subsets of CG neurons, even when cultured in the total absence of glucose (but presence of pyruvate).

Animals↗
PubMed1984

Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber.

A new peripheral nerve forms across a 10 mm gap within a silicone chamber regeneration model when the distal segment of a transected sciatic nerve, connected to its end organs, is sutured into the distal end of the chamber. We have tested the ability of other tissue inserts to support axonal regeneration in the chamber. When an isolated 2 mm piece of sciatic nerve was sutured into the distal end, fibrin matrix formation, cell immigration and axonal regeneration were identical to those occurring in the control. When the distal nerve insert was replaced with a 2 mm piece of skin or a ligation, a matrix did not form and subsequent cell immigration and axonal regeneration did not occur. When a 2 mm piece of tendon was inserted, a matrix did form at 1 week, but a structure across the gap was observed at later time periods in only 2 out of 7 chambers. The matrix either dissolved before cells could enter the chamber or did not promote cellular immigration and subsequent axonal regeneration. When the distal end was left open, a matrix formed and cells from the reactive tissue outside the chamber entered the matrix and formed a granulation tissue bridge across the gap. This tissue failed to support axonal regeneration; at 3 weeks, axons stopped 1 mm beyond the proximal stump at the interface with the granulation tissue. Thus, matrix formation and a cellular bridge are necessary but not sufficient to ensure regeneration. Successful regeneration across the silicone chamber gap requires humoral and/or cellular contributions available from peripheral nervous tissue and not from the other tested tissues.

Animals↗