Human amnion membrane as a substratum for axonal elongation in vitro and in vivo.
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Amnion↗
Biomedical subjects
S Varon
Publications and source records attributed to S Varon.
Humoral and surface-anchored factors in development and repair of the nervous system.
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Animals↗
Peripheral nerve regeneration in Gore-tex chambers.
Gore-tex chambers were used to bridge a 6 mm gap between the proximal and distal nerve stumps of a rat sciatic nerve. The wall structure of these chambers is characterized by "nodes" interconnected by smaller fibrils. Chambers with internodal distances of 5, 10 and 30 microns were used. Some 30 microns chambers were coated from the outside with Gore-tex (0.2 micron internodal distance) and others were coated from the inside. Regeneration after 12 weeks, as evidenced by muscle action potential recordings and light microscopy, was successful regardless of what type of chamber had been used. The organization of the nerve structure varied among different chamber types. A well organized coaxial nerve structure with myelinated axons was observed if inner-coated chambers were used. In chambers that were not coated or in outer-coated chambers tissue completely filled the chambers, and myelinated axons were arranged in mini-fascicles surrounded by loose connective tissue.
Animals↗
Biological activities of nerve growth factor bound to nitrocellulose paper by Western blotting.
We have previously developed a cell-blot technique to visualize directly in tissue extracts molecules that display the biological activity of ciliary neuronotrophic factors (CNTFs). This technique involves SDS-PAGE of the tissue extract, Western blotting on nitrocellulose paper, neuronal cell culture on the paper, and, using a vital dye, visualization of the neurons that selectively survive on the trophic factor band. In this report, we show that (1) NGF, either purified or in a crude extract from submaxillary glands, can also be successfully recognized using a slightly modified cell-blot technique; (2) a variety of ganglionic neurons can respond to distinct nitrocellulose-anchored trophic factors; (3) while CNTF and NGF can both support the survival of their common target cells, only NGF also promotes neuritic extension; and (4) both the dimeric and the monomeric forms of immobilized beta-NGF are active.
Animals↗
Exogenous matrix precursors promote functional nerve regeneration across a 15-mm gap within a silicone chamber in the rat.
When silicone regeneration chambers are implanted empty, axonal regeneration fails if the interstump gap length is greater than 10 mm. Previous experiments using the 10-mm gap model demonstrated that regeneration success correlated with the dimension and/or consistency of the naturally formed acellular fibrin matrix. Both spatial and temporal parameters of regeneration could be stimulated through modifications of the fibrin matrix by prefilling the chambers at the time of implantation either with phosphate-buffered saline or plasma dialyzed against phosphate-buffered saline. In the present experiments, similar modification of matrix formation was found to promote successful regeneration across 15-mm and 20-mm interstump gap lengths. In addition, prefilling 15-mm-gap chambers with dialyzed plasma resulted in a 3.5-fold increase in the incidence of functional restitution detected at 8 weeks after implantation over the outcome with chambers prefilled with phosphate-buffered saline.
Animals↗
Nerve regeneration chamber: evaluation of exogenous agents applied by multiple injections.
In this laboratory, a silicone chamber model for peripheral nerve regeneration in adult rats has been developed and used to define basic principles of the regenerative events, such as the sequential stages being followed during 'spontaneous' regeneration in vivo and the role of neuronotrophic- and neurite-promoting factors as well as extracellular matrix molecules. Each of the defined stages seems amenable to experimental modulation. Previous attempts to enhance regeneration included increasing the volume of the nerve chambers along with the modification of fibrin matrix formation by prefilling with saline (PBS) or matrix precursors. We present here the results of a series of experiments on the effects of exogenous biochemical agents applied by multiple injections into these in vivo chambers. Out of a variety of agents screened, a mixture of laminin (L), testosterone (T), ganglioside GM 1 (G), and catalase (C) was shown to advance substantially the progress of regeneration in 16 day chambers, as compared to PBS-prefilled and PBS-injected controls. LTGC-treatment at day 0, 6, and 10 postimplantation caused an increasingly frequent occurrence of cellular elements in cross-sections obtained from the middle (S5) of the chambers (i.e. 5 mm from the proximal stump), which was 2-fold for vessels, 3-fold for Schwann cells, and 10-fold for axons. When only sections containing axons 3 mm from the proximal stump (S3) were compared in experimental and control groups, computerized area measurements also revealed an average 2-fold difference for the cross-sectional size of the whole regenerate, the endoneurium and the space occupied by blood vessels.
Animals↗
Human amnion membrane serves as a substratum for growing axons in vitro and in vivo.
The epithelial cell layer of human amnion membrane can be removed while the basement membrane and stromal surfaces remain morphologically intact. Such a preparation has been used as a substratum for the in vitro culture of dissociated neurons. Embryonic motor neurons from chick ciliary ganglion attached to both surfaces but grew extensive neurites only on the basement membrane. On cross sections of rolled amnion membranes, regenerating axons of cultured neurons were guided along pathways of basement membrane that were immunoreactive with an antibody to laminin. In addition, when rolled amnion membranes were implanted into a lesion cavity between the rat septum and hippocampus, cholinergic neurons extended axons through the longitudinally oriented implant into the hippocampus. Thus, this amnion preparation can serve as a bridge to promote axonal regeneration in vivo in damaged adult brain.
Amnion↗
Loss of GABAergic neurons in medial septum after fimbria-fornix transection.
Neurons in the medial septum of the rat brain undergo retrograde degeneration after transection of their projection to the hippocampal formation, the fimbria-fornix. This cell death has been characterized for both Nissl-stained neurons and acetylcholinesterase-stained neurons. The major cell type in the medial septum is GABAergic, and many of these GABAergic neurons project to the hippocampal formation. Because the fimbria-fornix transection causes more neuronal death than can be accounted for by the loss of cholinergic neurons, we have sought to determine if the GABAergic neurons undergo a cell death similar to that reported for the cholinergic neurons. We report here that GABAergic neurons are indeed lost after the transection but the time course is considerably slower than that for the cholinergic neurons.
Animals↗
Ionic responses and growth stimulation in rat astroglial cells: differential mechanisms of gangliosides and serum.
Rat astroglial cells respond to fetal calf serum (FCS) and gangliosides, including GM1, by undergoing proliferation. Here, we show that addition of FCS but not GM1 causes an increase in Na+, K+-pump activity, as measured by ouabain-sensitive 86Rb+ influx. The increase of Na+, K+-pump activity by FCS was due to increased Na+ influx (measured with 22Na+). This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+/H+ exchange. Amiloride also blocked the FCS-stimulated incorporation of [3H]thymidine into DNA. Two defined polypeptide growth factors, epidermal growth factor and fibroblast growth factor were also able to elicit an amiloride-sensitive Na+ influx and an ouabain-sensitive K+ uptake in these astroglial cells, in the presence of FCS or insulin. Thus, GM1 differs from serum and growth factors in the mechanisms by which these agents stimulate the proliferation of the astroglial cells used here.
Amiloride↗
Association of laminin with heparan and chondroitin sulfate-bearing proteoglycans in neurite-promoting factor complexes from rat schwannoma cells.
The present studies were undertaken to confirm the presence and identity of a putative proteoglycan associated with laminin in neurite-promoting factor complexes isolated from rat schwannoma cell conditioned medium. Sucrose density gradient centrifugation of the complex resolved two laminin-associated Na2[35S]O4-labeled peaks which were termed Pools A and B. Both pools had nearly all their [35S] cpms associated with glycosaminoglycan, contained heparan sulfate-proteoglycan core protein antigen and displayed a similarly high neurite promoting potency relative to their laminin contents. However, Pool A contained about twice as many [35S] cpms and twice as much proteoglycan core protein per laminin than Pool B. Seventy percent of Pool A cpms was associated with heparan sulfate and 30% with chondroitin sulfate whereas the inverse was true for Pool B. Treatment with heparitinase and/or chondroitinase ABC caused laminin in either pool to elute at lower salt concentrations from DEAE cellulose. In SDS-PAGE the [35S] cpms of both pools ran with the same mobility as laminin but could be separated from laminin under reducing conditions. The Pool A cpms remained at 900 KD and the Pool B cpms spread over the 200-900 KD range. By rotary shadowing electron microscopy, Pool B fractions contained primarily cross-shaped laminin images, often associated with proteoglycan-like images. Pool A fractions contained i) dense, aggregated images including intact laminin from which emanated proteoglycan-like strands, ii) circular images bearing globular domains and less commonly, iii) distorted cross-shaped laminin-like images. These studies support the existence of at least two forms of laminin-proteoglycan complexes which differ in biochemical, immunochemical and ultrastructural characteristics.
Animals↗
A small-gauge cannula device for continuous infusion of exogenous agents into the brain.
A method is described for the construction of an intraventricular or intraparenchymal cannula device, which when connected to an Alzet osmotic pump, can be used for the continuous infusion of experimental solutions into the brain. A 33-gauge, stainless-steel cannula is encased within a dental acrylic stabilization platform prior to stereotaxic implantation, and after implantation, the platform is glued to the animal's skull using cyanoacrylate adhesive. This procedure provides for the long-term stability (at least 4 weeks) of the small-gauge cannula without the need for additional stabilization skull screws, thus minimizing damage to surrounding tissues by the cannula and postsurgical trauma to the animal. Using the stock model 2002 Alzet pump to infuse artificial cerebral spinal fluid at a flow rate equal to 0.5 microliter/h, an inflammatory tissue reaction around the cannula tip was consistently found after 2 weeks of continuous intraparenchymal infusion. However, the inflammatory reaction could be significantly reduced or eliminated by decreasing the flow rate to approximately 0.25 microliter/h, using a modified Alzet pump. Alternatively, the stock 0.5 microliter/h pump could be used without causing parenchymal damage if the cannula tip was implanted into the lateral ventricle.
Animals↗
An examination of ciliary neuronotrophic factors from avian and rodent tissue extracts using a blot and culture technique.
We have previously reported a technique for determining the apparent molecular weight (Mr) of ciliary neuronotrophic factors (CNTFs) in crude extracts. This method involves SDS-polyacrylamide gel electrophoresis of the extract. Western blotting and culture of purified ciliary ganglion neurons on the paper containing the blotted lane. Neurons will survive only if in direct contact with the trophic factor band and the surviving neurons, when stained with a vital dye, will outline the CNTF band thereby indicating the Mr of the active polypeptide. Here we have modified this 'blot and culture' technique by including Mr standard proteins in the same electrophoretic lane with the samples, identifying the proteins by staining the nitrocellulose blot with Amido black, marking the standard bands with pinholes and destaining the blot prior to seeding neurons onto it. The active CNTF polypeptides can then be identified by their ability to support the 24-h survival of cultured ciliary neurons. This modified technique was used to determine the Mr of CNTF activities in several chick and rat tissue extracts of selected developmental ages and to ascertain if the two forms of CNTF are exclusive to chick and rat, embryonic and adult, or eye and nerve tissues. We report that the above modifications permitted a more accurate method for Mr determination than the previous method, only two apparent forms of CNTF were recognized, the Mr found for each form is 25 kDa and 28 kDa, both forms can be present in chick and rat tissues and from embryonic and adult sources and the 28 kDa form is predominant in rat while the 25-kDa form is predominant in chicken tissues.
Animals↗
Catalase protection of neuronal survival in vitro is not directed to the accumulation of peroxides in the culture medium.
Walicke et al. (1986, J. Neurosci. 6, 1114-1121) have shown that catalase can replace the pyruvate requirement for survival of CNS neurons cultured in vitro. Since presently the only known function of catalase is the enzymatic degradation of hydrogen peroxide to water and oxygen, the simplest interpretation of the ability of catalase to support neuronal survival would be that catalase removes from the culture medium hydrogen peroxide. To test this hypothesis 8-day embryonic chick forebrain cells were cultured for 24 hr in a modified Eagle's Basal Medium with the serum-free supplement N1 (HEBM/N1) in the presence or absence of Phenol Red, 20 micrograms/ml catalase, 1 mM pyruvate, and/or 25 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) on a polyornithine-laminin substratum. The various media were then assayed for peroxide content using the potassium iodide method described by Wang and Nixon (1978, In Vitro 14, 714-722). The present data reveal that (1) HEBM/N1 normally contains approximately 50 microM peroxides, little of which is hydrogen peroxide, (2) the organic peroxide levels accumulating in this medium are not reduced by either catalase or pyruvate, and (3) medium modifications can reduce to no longer detectable levels the peroxides accumulating in the medium, but catalase or pyruvate is still required for neuronal survival. We conclude that catalase must exert its survival-promoting action at levels other than peroxides accumulating in the culture medium.
Animals↗
Exogenous administration of neuronotrophic factors in vivo protects central nervous system neurons against axotomy induced degeneration.
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Acetylcholinesterase↗
Inhibition of growth of Entamoeba histolytica by allicin, the active principle of garlic extract (Allium sativum).
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Disulfides↗
Differential effects of nerve growth factor and ciliary neuronotrophic factor on catecholamine storage and catecholamine synthesizing enzymes of cultured rat chromaffin cells.
The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8-day-old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4-day culture period in a dose-dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.
Adrenal Medulla↗
Dissociation of the stellate morphology from intracellular cyclic AMP levels in cultured rat brain astroglial cells: effects of ganglioside GM1 and lysophosphatidylserine.
Secondary microcultures of newborn rat cerebrum astroglial (AG) cells, maintained in a serum-free, chemically defined medium, were treated with various agents known to elevate intracellular cyclic AMP (cAMP) levels. Earlier studies had shown these drugs to induce a process-bearing (stellate) morphology in the AG cells, a response that was antagonized by the presence of gangliosides. One millimolar dibutyryl cyclic AMP (dBcAMP), 10 microM forskolin, 12 nM cholera toxin, and 30 microM isoproterenol all raised intracellular cAMP levels, from basal values of 3 pmol/10(6) cells to 30-30,000 pmol/10(6) cells, depending on the agent tested. dBcAMP caused the greatest elevation, and forskolin the least. The timing and/or the level of the AMP response did not precisely correlate with those of the stellation response. Values of ED50 with the four agents, as determined for the cAMP response, were always higher than stellation ED50 values in all treatments, and ED50 did not correlate with the maximal levels of cyclic AMP induced by the four agents. The capacity of ganglioside GM1 to block the stellation response to the four agents was not accompanied by a similar capacity to block the cAMP responses. Lysophosphatidylserine (lysoPS) had the capacity to induce AG cell stellation as well, without altering the basal level of cAMP. Both lysoPS and gangliosides, therefore, may act directly on the cellular machinery underlying the stellation response without involving changes in intracellular AMP.
Animals↗