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M Manthorpe

Publications and source records attributed to M Manthorpe.

At least 91 records · Page 5Linked to original sources
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

Fetal calf serum-mediated inhibition of neurite growth from ciliary ganglion neurons in vitro.

Embryonic chick ciliary ganglion (CG) neurons cultured in fetal calf serum-containing medium have been previously reported to extend neurites on polyornithine (PORN) substrata precoated with a neurite-promoting factor (PNPF) from rat schwannoma-conditioned medium. On PORN substrata alone, however, no neuritic growth occurred. This was interpreted as evidence that PORN was an incompetent substratum for ciliary neuritic growth. In this study, we now find that an untreated PORN substratum allows neuritic growth in serum-free defined medium. When PNPF was added to PORN, a more rapid and extensive neuritic response occurred. After 5 hr of culture, a 60% neuritic response occurred on PNPF/PORN, whereas no neurons initiated neurites until 10-12 hr on PORN. The inhibitory effect of fetal calf serum noted above on PORN could be obtained in part by pretreating the substratum with serum for 1 hr. Maximal inhibitory effects in the PORN pretreatment were achieved after 30 min and were not further improved by treatments up to 4 hr. Bovine serum albumin was also found to inhibit neurite growth on PORN to about 60% of the inhibition obtained by an equivalent amount of serum protein. Fetal calf serum was shown to cause a 15% reduction in the percentage of neurons bearing neurites after its addition to 18-hr serum-free PORN cultures and to cause statistically significant reductions in neurite lengths measured 2 hr later.

Animals↗
PubMed1984

Neuronotrophic activities in cerebrospinal fluid of head trauma patients.

Neuronotrophic factors (NTFs) are agents required for neurons to survive in tissue culture. In this study, we investigated the presence of NTFs in cerebrospinal fluid (CSF) of patients with central nervous system (CNS) injury. Cerebrospinal fluid was collected from 15 patients with acute CNS lesions in whom ventricular catheters had been placed to monitor and to facilitate the control of intracranial pressure. Neuronotrophic activity within the CSF was assayed using cultures of neurons derived from fetal rat hippocampus and embryonic chick cerebral cortex. Cerebrospinal fluid from all 15 patients contained NTFs which supported the survival of rat hippocampal neurons. Survival of chick cortex neurons was supported by eight of nine CSF samples. In the 11 patients from whom consecutive CSF samples were available, NTF activity assayed in rat hippocampal cultures tended to decrease during the first several days after CNS injury. In CSF collected from three patients by lumbar puncture for diagnosis of "nontraumatic" conditions, no NTFs were detectable. NTFs supporting hippocampal neurons were also detected in extracts of blood clot obtained from normal volunteers. Neuronotrophic activity in the CSF was heat sensitive, nondialyzable, and macromolecular, suggesting its association with a protein(s). These observations suggested that (i) NTFs are detectable in human CSF after CNS injury, (ii) NTFs appear in response to the injury itself, and (iii) at least some human NTFs can support the survival in culture of nonhuman CNS neurons.

Animals↗
PubMed1984

Use of central neuronal cultures for the detection of neuronotrophic agents.

Neuronotrophic factors, a class of macromolecules thought to be present within the neuronal environment are required to support the survival in vitro of peripheral neurons. In the present study we have established bioassay culture systems suitable for the identification of similar agents for intrinsic neurons of the central nervous system. The striatum, hippocampus and septum of 18 day fetal rats were dissociated and plated in a serum-free medium on a neurite conducive substratum which allows an easy recognition of neurons under phase contrast microscopy. These cultures contain predominantly neurons as assessed by tetanus toxin labelling, a well recognized neuronal marker. Seeding the cell suspensions at decreasing densities yields after 24 h a density dependent survival of the neuronal population. Thus a low seeding density could be chosen where survival of these neurons required an exogenous source of trophic factors. Survival of central neurons was promoted by several conditioned media derived from rodent glial cell cultures, both primary (astroglia, Schwann) and clonal (C6 glioma, Schwannoma). Serial dilutions of these media allowed the titration of their respective neuronotrophic activities. In addition, conditioned media derived from the central neuronal cultures themselves, when seeded at a high density, were also able to support the survival of low density seeded central neurons.

Animals↗
PubMed1984

Purification of the chick eye ciliary neuronotrophic factor.

Dissociated 8-day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2 1/2-day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion-exchange chromatography, membrane ultrafiltration-concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate-polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10-20 micrograms of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 X 10(6) trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24-h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8-day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.

Animals↗
PubMed1984

Low molecular weight agents support survival of cultured neurons from the central nervous system.

Microcultures of dissociated neurons from various central and peripheral neural tissues were used for quantitative analysis and characterization of trophic agents directed to these test neurons. Media conditioned by a variety of central and peripheral glial as well as muscle cell cultures contain trophic activities for central neurons which are distinct from the more traditional protein factors directed to peripheral neurons, by at least two features: (1) they reside with low molecular weight (Mr less than 1000) agents which are resistant to heat, extremes of pH, and various proteolytic and peptidolytic enzymes; and (2) they are necessary for the short-term survival of a variety of rodent and avian central neurons, but they fail by themselves to support survival of peripheral neurons under the same culture conditions.

Animals↗
PubMed1983

Human laminin isolated in a nearly intact, biologically active form from placenta by limited proteolysis.

A protein with properties of laminin has been isolated from human placental extracts by using monoclonal antibodies. Placental tissue was extracted with 0.5 M NaCl and high molecular weight proteins were isolated from the extract by salt precipitation and gel filtration on Sepharose 6B. The resulting protein fraction which contained material cross-reactive with anti-sera to rat laminin was used as immunogen to prepare hybridomas. Thirteen hybrids produced antibodies which reacted with basement membrane-associated antigens in indirect immunofluorescence of tissues. One of these, 4E10, was characterized in detail. This monoclonal antibody reacted with human laminin as shown by several lines of evidence. Immunoprecipitation from metabolically labeled culture media of a human amniotic epithelial cell line with the 4E10 antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed polypeptides with Mr similar to those of rat laminin. Immunochromatography of placental extracts obtained by limited pepsin digestion yielded material with main polypeptides at 160 and 130 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. These peptic fragments cross-reacted with rat laminin in immunodiffusion and enzyme immunoassay, and a polyclonal antiserum against the fragments reacted with basement membranes in tissues in a manner identical with the 4E10 antibody. Electron microscopic images of the human peptic fragments showed structures similar to the cross-shaped images of murine laminins, although the short arms were truncated to various degrees or even absent. The isolated peptic fragments also displayed biological activity similar to that of murine laminins in that the outgrowth of neurites by neuronal cells was promoted on plates coated with the fragments.

Antibodies, Monoclonal↗
PubMed1983

Neuronotrophic activity in brain wounds of the developing rat. Correlation with implant survival in the wound cavity.

Neuronotrophic activity accumulates in a wound cavity created in the entorhinal/occipital cortex of developing rats. These trophic factors support the survival of neurons in monolayer cultures of chick embryo spinal cord, ciliary ganglion, sympathetic ganglion and dorsal root ganglion, as well as of mouse dorsal root ganglion. Trophic activity was very low both in non-injured brain tissue and in the wound cavity 1 day post-lesion, but it increased 15- to 300-fold during the subsequent 2-5 days. Together with the trophic activity in the wound fluid were other substances which interfered with the survival of spinal cord neurons. The neuronotrophic factors appeared to be proteins immunologically distinct from mouse submaxillary nerve growth factor. Fragments of rat embryo corpus striatum placed in the cortical wound cavity immediately after its formation showed very poor subsequent survival and no innervation of the host hippocampus. However, if implantation was delayed by 3 or 6 days with respect to the time at which the receiving cavity was made, the survival was greatly improved and innervation of the host took place. The time course for the accumulation of the trophic factors in the cavity paralleled the delay leading to increased survival of brain grafts. It is suggested that the neuronotrophic activity accumulating in the wound cavity during the delay period may be responsible for the increased survival of the implants.

Animals↗
PubMed1983

Endogenous and exogenous factors support neuronal survival and choline acetyltransferase activity in embryonic spinal cord cultures.

Dissociated 4-day (stage 23) chick embryo lumbar cord cells were cultured at low or high cell densities for 1 or 5 days in the presence or absence of added spinal neuronotrophic factor (supplied as RN22 Schwannoma conditioned medium, RCM). In low density, 1-day cultures neuronal survival was dependent on added RCM whereas by 5 days no neurons survived, even in the presence of RCM. In high density 1-day cultures a substantial neuronal population could survive even without added RCM and a large proportion of this neuronal population would survive for 5 days. When conditioned media from high density lumbar cord cultures was supplied to low density unsupplemented cultures, a similar level of 5-day neuronal survival resulted. However, no neurons survived in RCM-supplemented 5-day high density cultures, indicating the presence in RCM of a material toxic for the neurons. Both the RCM and the high density lumbar culture-conditioned medium supported considerable choline acetyltransferase activity indicating the presence within these cultures of motoneurons.

Animals↗
PubMed1983

Temporal changes of neuronotrophic activities accumulating in vivo within nerve regeneration chambers.

The presence of neuronotrophic factors (NTFs) in noninjured sciatic nerve extract and the course of their accumulation from 3 h to 30 days after nerve transection was examined. Rat sciatic nerves were transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers leaving a 10-mm interstump gap. Previous studies had shown that regeneration occurs in chambers containing both stumps but is absent in chambers lacking the distal stump. Chambers became completely filled with fluid 10 to 12 h after implantation. Fluid from chambers without nerve stumps (open-ended) implanted adjacent to nerve-containing chambers had markedly lower trophic activities than those containing one or both stumps. In fluid collected from chambers containing both proximal and distal nerve stumps, the highest titers of NTFs directed to sensory neurons were measured at 3 h posttransection whereas the highest titers of NTFs directed to sympathetic and spinal cord neurons were detected at 1 and 3 days, respectively. Chambers containing only the proximal or only the distal stumps showed similar temporal dynamics for sensory and sympathetic NTFs. Sensory and sympathetic neuronotrophic activity in extracts of proximal and distal stumps followed a similar temporal course to those in chamber fluid. Extracts of nonlesion nerve segments 5 mm from the transection site contained higher sensory and lower sympathetic trophic activity than extracts including the transection site. Spinal cord activity was undetectable in all extracts. Antiserum to nerve growth factor had no effect on fluid or extracts containing high sensory or sympathetic activities. These observations suggested that (i) some NTFs may be present in normal nerves and others may be synthesized or accumulated in response to nerve injury, (ii) sensory, sympathetic, and spinal cord NTFs are separate agents and immunochemically distinct from nerve growth factor, (iii) NTFs predominantly originate from nerve stumps rather than from surrounding fluid, and (iv) proximal and distal nerve stumps accumulate and release NTFs at similar rates.

Animals↗
PubMed1983

Functional receptors for vasoactive intestinal polypeptide in cultured astroglia from neonatal rat brain.

The effects of vasoactive intestinal polypeptide (VIP) were assessed on astroglia cultured from rat CNS. In these cultures VIP (500 nM) promoted the hydrolysis of [3H]glycogen newly synthesized from [3H]glucose. This effect on [3H]glycogen levels was also observed with the structurally related peptide PHI-27 and with other substances which had been demonstrated to promote glycogenolysis in rodent CNS in vitro such as: norepinephrine (NE), serotonin, histamine, adenosine, K+ and dibutyryl cyclic-AMP (dbcAMP). Furthermore, VIP (500 nM) and PHI 27 (500 nM), when applied to astroglial cultures in serum-free medium, displayed marked effects on the morphological appearance of the cell population: they converted the flat cells present in the cultures into cells with typical astrocytic morphology. As previously reported, this effect on the cellular morphology of the cultures was also observed, under identical experimental conditions, after NE and dbcAMP application. These studies demonstrate that cultured rat neonatal astroglia possess receptors for VIP, and suggest that a cyclic AMP accumulation may mediate both the metabolic and morphologic components of this response.

Animals↗
PubMed1983

Laminin promotes neuritic regeneration from cultured peripheral and central neurons.

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co-purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.

Animals↗
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

Isoelectric focusing of the chick eye ciliary neuronotrophic factor.

A procedure is presented in which a crude extract from selected chick embryo intraocular tissues is submitted to analytical polyacrylamide slab gel isoelectric focusing. The extract contains a protein, ciliary neuronotrophic factor (CNTF) which can be eluted in active form from focused gels in a region occupied by only two protein bands. A "slot" technique is presented in which we demonstrate that the eluted CNTF activity focuses in the very restricted region between the two visible bands and is not associated with either band. Silver stain-densitometry is used to correlate staining intensity with protein concentration and from such an analysis it is concluded that the CNTF protein represents an extremely low proportion of total extract protein and that the minimum CNTF specific activity eluted from gel slices is 10(6) trophic units per mg protein. This one-step procedure will be used in the future to prepare highly purified CNTF for antibody generation.

Animals↗
PubMed1982

Comparative features of spinal neuronotrophic factors in fluids collected in vitro and in vivo.

Survival in monolayer culture of 4-day (stage 23) chick embryo lumbar spinal cord neurons can be regulated by two opposing activities. One, spinal neuronotrophic activity, promotes neuronal survival; and the other, spinal neuronotoxic activity, eliminates the neurons from the culture even when the trophic support is present at an optimal concentration. Quantitative microbioassays for each activity are presented and used to measure the relative amounts of each agent within different sources including glial, muscle, and spinal cord cell-conditioned media and fluid collected from peripheral and central nervous tissue lesions. Although both activities were present in all of the sources tested, their concentrations in the wound fluids were orders of magnitude greater than in the conditioned media. The fluid-derived trophic activities were inactivated by heat and trypsin and nondialyzable, whereas all of the conditioned media-derived trophic activities were heat- and trypsin-resistant and dialyzable.

Animals↗
PubMed1982

Spinal cord neuronotrophic factors (SCNTFs): I. Bioassay of schwannoma and other conditioned media.

We present a procedure for the dissociation and growth in serum-free defined culture medium of 4-day chick embryo lumbar spinal cord (LC4) neurons. LC4 neurons will not survive for even 24 h without the addition of trophic supplements (putative spinal cord neuronotrophic factors, SCNTFs). Serum-free medium conditioned over chick embryo heart and skeletal muscle, mouse Schwann and rat RN22 Schwannoma cell cultures were found to contain SCNTF activity which could be quantitated using a convenient neuronal survival bioassay. RN22 conditioned medium also contains polyornithine-binding neurite promoting factors (PNPFs) which can be physically separated from SCNTF. When SCNTF and PNPF were presented to LC4 neurons individually or in combination (i) SCNTF, but not PNPF, supported neuronal survival whereas (ii) PNPF, but not SCNTF, induced neurite production. When LC4 neurons were grown in SCNTF alone, nearly all of them exhibited a flattened, circular, 'fried-egg' morphology. The subsequent addition of PNPF caused these cells to extend long neurites with characteristic terminal growth-cone-like structures.

Animals↗
PubMed1982

Lectin reactivity of PNPF, a polyornithine-binding neurite-promoting factor.

The fate of dissociated neurons from 8-day chick embryo ciliary ganglia, cultured in serum-containing media on polyornithine substrata, is influenced by two different macromolecular factors. The neurons will die within 24 h in the absence of CNTF, the eye-derived ciliary neuronotrophic factor. Even when supported by CNTF, however, ciliary neurons do not grow neurites unless the polyornithine substratum is coated with PNPF, a polyornithine-binding neurite-promoting factor. PNPF activity present in rat Schwannoma-conditioned medium has been shown to behave as a large, acidic, trypsin-sensitive molecule. In the experiments reported here the lectin reactivity of PNPF has been investigated. Using lectin affinity chromatography PNPF was found to bind to concanavalin A and wheat germ agglutinin from which it could be respectively eluted with the specific sugars alpha-methyl-D-mannoside and N-acetyl-D-glucosamine. PNPF did not bind to Ulex europaeus or Dolichus biflorus agglutinins. Pretreatment of polyornithine-bound PNPF with concanavalin A before cell seeding prevented neurite outgrowth from ciliary neurons in a dose-dependent manner, without affecting neuronal survival. This inhibitory effect of concanavalin A could be removed with alpha-methyl-D-mannoside. Wheat germ agglutinin failed to inhibit the neurite-promoting effects of polyornithine-bound PNPF.

Animals↗