Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site.

Monoclonal antibodies were made against a truncated form of human laminin isolated from placenta. 12 antibodies were isolated and characterized. All antibodies stained basement membranes in placenta and immunoprecipitated laminin from media of cultured choriocarcinoma cells. Three antibodies, 3E5, 4C7, and 4E10, partially blocked the neurite-promoting activity of laminin. Addition of a second antibody, goat anti-mouse IgG, caused more complete blocking of the activity. Two of the blocking antibodies, 4C7 and 4E10, reacted with epitopes within the globular domain at the end of the long arm of laminin, and the third one, 3E5, reacted at the end of the rod-like portion of the long arm adjacent to the globular domain, as shown by electron microscopy after rotary shadowing. Five nonblocking antibodies used in the same test reacted with epitopes in other domains of the molecule. Blocking antibodies 3E5 and 4E10 could be used in immunoblotting and both antibodies reacted with the same polypeptides in pepsin fragments of human laminin, the predominant polypeptides being approximately 400 kD. When a crude extract of human amnion was used as a source of intact laminin, the 4E10 antibody detected a single polypeptide of approximately 400 kD. A nonblocking antibody, 2E8, which reacted at the center of the laminin cross, reacted predominantly with a 200-kD polypeptide in human laminin fragments and exclusively with a 200-kD polypeptide in amnion extract and in rat laminin. Our results with human laminin match the results by Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468, in which the neurite-promoting activity of mouse laminin resides at the end of the long arm, which is also the site for heparin binding. However, since the active fragments of human laminin did not bind to heparin, the neurite-promoting site should be different from the heparin-binding site. Our results further suggest that the neurite-promoting site may be contained in or close to the 400-kD component of laminin.

Regulation of alpha 2 beta 1-mediated fibroblast migration on type I collagen by shifts in the concentrations of extracellular Mg2+ and Ca2+.

Extracellular Ca2+ can reverse the Mg(2+)-dependent, alpha 2 beta 1-mediated adhesion of WI38 human fibroblasts to type I collagen substrates. Affinity chromatography data also demonstrate that Ca2+ can specifically elute the fibroblast alpha 2 beta 1 integrin bound to type I collagen-Sepharose in Mg2+. In modified Boyden chamber migration assays, Mg2+ alone supports the alpha 2 beta 1-mediated migration of fibroblasts on type I collagen substrates, while Ca2+ does not. However, a twofold enhancement in migration was observed when combinations of the two cations were used, with optimal migration observed when the Mg2+/Ca2+ ratio was higher than one. Inhibitory mAbs directed against various integrin subunits demonstrate that these observed cation effects appear to be mediated primarily by alpha 2 beta 1. These data, together with reports that under certain physiological conditions significant fluctuations in the concentrations of extracellular Ca2+ and Mg2+ can take place in vivo, suggest that the ratio between these two cations is involved in the up- and downregulation of integrin function, and thus, may influence cell migratory behavior.

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.

Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands.

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

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.

RGDN peptide interaction with endothelial alpha5beta1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles.

The ability of an integrin-binding Arg-Gly-Asp-Asn (RGDN)- containing peptide to influence vascular tone by interacting with the alpha5beta1 integrin was studied using rat skeletal muscle arterioles. After blockade of beta3 integrin function, isolated arterioles with spontaneous tone showed concentration-dependent vasoconstrictions to topical application of GRGDNP, a peptide that shows a greater ability to interact with alpha5beta1 than with alphavbeta3. The constriction to GRGDNP (2.1 mM) was inhibited by blocking alpha5 integrin function, and was intensified by blocking beta3 integrin function. In contrast, GRGDSP, a peptide that interacts better with alphavbeta3, was unable to induce sustained constrictions. Removal of the endothelium abolished the vasoconstriction in response to GRGDNP, suggesting that the response was due to release of an endothelium-dependent factor. Indeed, blockade of ETA endothelin receptors with BQ-610 (1 microM), similar to removal of the endothelium and alpha5 integrin blockade, inhibited the vasoconstriction. These data indicate that interaction of RGD peptides, and in particular the RGDN sequence with endothelial cell alpha5beta1, causes endothelin-mediated arteriolar vasoconstriction. These results indicate that integrins are novel signaling receptors within the vascular wall that affect vasomotor tone, and may play an important role in vascular control.

A latent Mr 94,000 gelatin-degrading metalloprotease induced during differentiation of HL-60 promyelocytic leukemia cells: a member of the collagenase family of enzymes.

The treatment of HL-60 promyelocytic leukemia cells with phorbol esters (12-O-tetradecanoylphorbol-13-acetate) results in the appearance of cell substrate adhesion and the release of a Mr 94,000 gelatin-degrading metalloprotease. The appearance of the metalloprotease in the culture medium directly correlates with the timing and extent of cell substrate adhesion over a 24-h period. Anti-Mr 94,000 metalloprotease blocking antibodies were unable to interfere with the HL-60 cell substrate adhesion induced by 12-O-tetradecanoylphorbol-13-acetate, although they were able to specifically remove the Mr 94,000 gelatin-degrading activity from either HL-60 or U-937 cell-conditioned medium. A purified metalloprotease preparation was found to be predominantly latent and activated by organomercurials, acid treatment (pH 2 to 3.6), or 8 M urea. The activating effect of the latter two denaturing treatments suggests that conformational changes may be the common activating mechanism. The different treatments also caused the appearance of lower molecular weight gelatin-degrading bands (in gelatin zymogram gels) in a manner consistent with the autocatalytic cleavage that occurs with other collagnase proenzymes during activation. Edman degradation of a cyanogen bromide fragment from the Mr 94,000 metalloprotease provided the amino acid sequence [PR(C)GVPD] which is present in type I collagenase, stromelysin, and transin proenzyme sequences and partially conserved (V----N substitution) in the type IV collagenase proenzyme. This sequence has been reported to be important in the maintenance of the latent state of the transin proenzyme (R. Sanchez-Lopez et al., J. Biol. Chem., 263: 1892-11899, 1988) and is a sequence unique to collagenase proenzymes. The N-terminal sequence of the Mr 94,000 metalloprotease (AP-QDQST) is unique and distinct from other collagenases. Thus, the Mr 94,000 metalloprotease from HL-60 cells appears to be a distinct and new member of the collagenase family of proteases.

Molecular Signaling Pathways Controlling Vascular Tube Morphogenesis and Pericyte-Induced Tube Maturation in 3D Extracellular Matrices.

During capillary network formation, ECs establish interconnecting tubes with defined lumens that reside within vascular guidance tunnels (physical spaces generated during EC tubulogenesis). Pericytes are recruited to EC tubes within these tunnels and capillary basement membrane deposition occurs to facilitate tube maturation. Here, we discuss molecular mechanisms controlling EC tubulogenesis demonstrating the involvement of integrins, MT1-MMP, extracellular matrix, Cdc42, Rac1, Rac2, k-Ras, Rap1b, and key downstream effectors including Pak2, Pak4, IQGAP1, MRCKß, and Rasip1. These molecules activate kinase cascades controlling EC tube formation, in conjunction with growth factor receptor signaling, which involve PKCɛ, Src family, Raf, Mek, and Erk kinases. These molecules and signaling cascades stimulate EC lumen and tube formation by: regulating MT-MMP-dependent lumen expansion and vascular guidance tunnel formation; generation of intracellular vacuoles/vesicles to create EC apical membranes; and establishing cytoskeletal polarity with acetylated tubulin distributed subapically (and F-actin basally) to facilitate vacuole trafficking/fusion in a polarized, perinuclear region. Using defined serum-free models, we have demonstrated that human EC tubulogenesis and EC-pericyte tube coassembly requires five exogenously applied growth factors which are SCF, IL-3, SDF-1α, FGF-2, and insulin (Factors). Also, we have demonstrated that EC-derived PDGF-BB and HB-EGF are necessary for pericytes to proliferate, recruit to tubes, and induce basement membrane assembly. Finally, we have shown that VEGF fails to directly stimulate EC tubulogenesis. In contrast, it acts as an upstream EC primer of downstream "Factor"-induced tubulogenic and EC-pericyte tube coassembly by upregulating c-Kit, IL-3Rα, and CXCR4 as well as PDGF-BB and HB-EGF expression.

The alpha4beta1 integrin can mediate leukocyte adhesion to casein and denatured protein substrates.

An understanding of the binding specificity of leukocyte integrins is important to determine the range of ligands that interact with these receptors during inflammatory processes. In this study we show that the alpha4beta1 integrin can interact with casein and denatured albumin and promote leukocyte adhesion through these interactions. This was demonstrated with the use of blocking antibodies directed to alpha4beta1 and peptide adhesion competitors containing the alpha4beta1 binding tripeptide, Leu-Asp-Val (LDV). Consistent with this data, the adhesion is completely divalent cation-dependent and is stimulated by known activators of leukocyte integrin function, namely phorbol ester and the beta1 integrin activating antibody, 8A2. It is interesting to note that neither bovine alpha-casein or human albumin contain an LDV site (present in the CS-1 site of alternatively spliced fibronectin) or an IDS site (present in VCAM-1) yet they promote adhesion through this integrin. Furthermore, alpha4beta1 directly binds to Sepharose columns containing casein, casein fragments, or denatured albumin but does not bind columns containing native albumin. These data suggest that the binding specificity for the alpha4beta1 integrin is considerably broader than previously realized. This work has implications for how subsets of leukocytes may interact with damaged proteins during tissue injury and inflammation.

Integrin expression in human melanoma cells with differing invasive and metastatic properties.

During the process of tumor cell invasion and metastasis, tumor cells are known to interact with extracellular matrix proteins, endothelial cells, platelets and other organ-specific structures. Integrins are cell surface molecules which mediate cell-matrix and cell-cell interactions and are likely to be important for tumor cell survival and dissemination. The purpose of this study was to characterize the integrin and proteolytic enzyme repertoire from low (A375P), medium (A375M) and high metastatic (A375SM) human melanoma cell lines. These cell lines are also invasive through human amniotic membranes in vitro and their invasiveness parallels the reported metastatic phenotype. The types and levels of expression of the various integrin receptors were analysed by quantitative immunoprecipitation using a panel of monoclonal antibodies directed to known integrin subunits. In addition, cDNA probes to the integrin subunits were used in quantitative northern blot analysis. These data show that the integrin alpha v beta 3 increases 50- to 100-fold as these cells progress to a more metastatic phenotype. alpha 4 beta 1 levels also appeared to increase several fold, while other beta 1 integrins did not differ in their expression levels. The increased alpha v beta 3 expression in the more metastatic cells resulted in an increased adhesion to vitronectin and fibrinogen substrates in cell attachment assays. However, alpha v- and beta 3-specific antibodies did not inhibit A375 cell invasion through the amnion. Each cell line was found to release similar quantities of a 72-kDa gelatinase/type IV collagenase and tissue type plasminogen activator. These results suggest that during the progression of these tumor cells from a low to high metastatic phenotype, marked changes in integrin expression occurred which may facilitate interactions with platelets, endothelial cells and specific extracellular matrix proteins to promote metastasis.

Multifocal verruciform xanthoma of the upper aerodigestive tract in a child with a systemic lipid storage disease.

We report the clinical, light-microscopic, and ultrastructural features of a case of multifocal verruciform xanthoma in the upper aerodigestive tract of a child with a systemic lipid disorder. Lipid storage cells were found in liver, bone marrow, and as a component of verruciform xanthomas. To our knowledge this represents the first case of verruciform xanthoma reported in (a) a child, (b) as a multifocal lesion in the upper aerodigestive tract, (c) associated with a systemic lipid disorder, and (d) with ultrastructural evidence of lipid accumulation within endothelial cells. Although this patient presented with lesions involving the tongue and larnyx, subsequently lesions were found in the bone marrow and liver. Two months later more lesions were discovered on the epiglottis, posteior tongue, right glottis, and in grossly normal peritonsillar mucosa. Six months later a new oral lesion developed. Based upon these observations, we speculate that the pathogenesis of verruciform xanthoma involves accumulation of excess lipid in subepithelial sites which is scavenged by macrophages. Lipid-laden macrophages release epithelial growth factors that lead to epithelial hyperplasia. Depending on the degree of epithelial hyperplasia, the gross appearance of verruciform xanthomas may be flat, sessile, papillary, or verrucous.

"Minified" Goldmann applanating prism for tonometry in monkeys and humans.

The tip diameter of the standard Goldmann applanating prism was reduced from 7.0 mm to 4.0 or 4.5 mm, but the endpoint of intraocular pressure measurement-applanation of a circle of cornea having a diameter of 3.06 mm-was not changed. The "minified" tonometers exhibited standard mechanical calibration characteristics in three standard configurations. The 4.5-mm tonometer was calibrated for cynomolgus monkey eyes in vivo by open and closed stopcock manometry. By open stopcock manometry, between pressures of 4 and 70 mm Hg, IOP(tonometer) = 1.01 IOP(manometer)-0.72, with very little scatter or curvature. During closed stopcock manometry, between pressures of 5 and 55 mm Hg, IOP(tonometer)=1.07 IOP(manometer)-1.32, again with very little scatter or curvature. The tonometer elevated manometric IOP by an average of about 7.5%; the percentage pressure elevation decreased slightly as pressure increased. The 4.0-mm tonometer was calibrated against a standard tonometer in living human eyes. Over the pressure range from 0 to 75 mm Hg, IOP(minitonometer)=0.98 IOP(standard tonometer)+ 0.82, with very little scatter or curvature. Similar comparison of two standard tonometers showed a virtually identical relationship and scatter. We conclude that minified Goldmann tonometers provide accurate and reprodible measurements of IOP in cynomolgus monkey and human eyes.

Predicting length of stay for psychiatric diagnosis-related groups using neural networks.

OBJECTIVE: To test the effect of diagnosis on training an artificial neural network (ANN) to predict length of stay (LOS) for psychiatric patients involuntarily admitted to a state hospital. DESIGN: A series of ANNs were trained representing schizophrenia, affective disorders, and diagnosis-related group (DRG) 430. In addition to diagnosis, variables used in training included demographics, severity of illness, and others identified to be significant in predicting LOS. RESULTS: Depending on diagnosis, ANN-predictions compared with actual LOS indicated accuracy rates ranging from 35% to 70%. The validity of ANN predictions was determined by comparing LOS estimates with the treatment team's predictions at 72 hours following admission, with the ANN predicting as well as or better than did the treatment team in all cases. CONCLUSIONS: One problem in traditional approaches to predicting LOS is the inability of a derived predictive model to maintain accuracy in other independently derived samples. The ANN reported here was capable of maintaining the same predictive efficiency in an independently derived cross-validation sample. The results of ANNs in a cross-validation sample are discussed and the application of this tool in augmenting clinical decision is presented.

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.

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.

Human amnion membrane as a substratum for cultured peripheral and central nervous system neurons.

We report here on the use of human amnion membrane as a substratum for the culture of neuronal cells. Pieces of amnion membrane were bound to nitrocellulose paper as a supporting material, seeded with neurons, and cultured for 1-4 days. Neurons and neurites were visualized after fixation by immunoperoxidase staining using an anti-neurofilament monoclonal antibody. Neurons from embryonic chick ciliary and dorsal root ganglia and fetal rat hippocampus were cultured on either the basement membrane or stromal surface of the amnion membrane. Neurons adhered to both surfaces but extended neurites only on the basement membrane surface. Neurons survived and continued to grow neurites in serum-free medium for at least 4 days. When cultured for 4 days on the basement membrane surface in the presence of 10% fetal calf serum, the ciliary ganglion neurons survived but neurite growth was markedly inhibited, while dorsal root ganglion neurons survived, hypertrophied and grew an extensive network of neurites. Other experiments addressed the question whether the basement membrane surface had an ability to guide growing neurites. Amnion membranes were folded, frozen, cross-sectioned using a cryostat, and placed on the nitrocellulose to give irregular patterns of basement membrane juxtaposed to the collagenous stroma. Ciliary ganglion neurons after 4 days in culture had initiated and extended neurites in patterns which corresponded and were limited to those areas visualized by indirect immunofluorescence staining using anti-laminin antibodies. Thus, in vivo assembled human amnion basement membranes appear to contain signals that both promote and guide neuritic growth from previously axotomized embryonic peripheral and central nervous system neurons. The amnion membrane represents a novel tool for the culture of neuronal cells in vitro and potentially could be used as a neurite-promoting bridging material in vivo for regeneration studies.

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.

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.

Rat amnion membrane matrix as a substratum for regenerating axons from peripheral and central neurons: effects in a silicone chamber model.

An extracellular matrix preparation, the human amnion membrane matrix (hAMM) can serve as a neurite-promoting substratum for cultured peripheral and central neurons, and also as a support for axonal growth in experimentally injured adult brain in vivo. In the present study, we tested similar materials as bridges in a silicone chamber model for the regeneration of sciatic nerve in the adult rat. Since hAMM elicited an inflammatory response, we developed a rat amnion membrane matrix (rAMM), which proved to be an excellent neurite-promoting substratum for cultured ganglionic and spinal cord neurons. The rAMM was coiled and inserted in the 10 mm gap between the two nerve stumps from the silicone chambers. At 16 days after implantation, temporal progress of regeneration was grossly similar as in saline-prefilled control chambers. However, rAMM-prefilled chambers displayed significantly higher number of vessels and a markedly different geometry of the regenerate: an endoneurium, surrounded by a perineurial-like cell layer, was formed outside the largely preserved central portion of the rAMM coil. After longer regeneration times (28 days), a rAMM core was no longer detected, but some rAMM-like materials remained interspersed in the endoneurium. The overall organization of the regenerate and the number of myelinated axons at this time were similar to those of control chambers, although the endoneurial cross-sectional area was larger in the rAMM chambers. One specimen, however, displayed the very patterns for which the experiments were designed, namely an array of numerous, myelinated axons tracing the spiraling spaces between consecutive lamellae of the rAMM coil.