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Biomedical subjects

P Aebischer

Publications and source records attributed to P Aebischer.

At least 91 records · Page 5Linked to original sources
PubMed1995

Laminin oligopeptide derivatized agarose gels allow three-dimensional neurite extension in vitro.

The phenotypic expression of various neural cells is influenced by extracellular matrix (ECM) molecules. This study aims to develop a three-dimensional gel tailored to support neurite extension from neural cells. Laminin-derived (LN) oligopeptides CDP-GYIGSR, a 19-mer IKVAV containing sequence, GRGDSP, a cocktail of the three aforementioned LN peptides (PEPMIX), and a control peptide sequence GGGGG were covalently linked to an agarose hydrogel backbone using the bi-functional coupling agent 1'1, carbonyldiimidazole. Embryonic day 9 chick DRGs and PC12 cells were suspended in three dimensions in underivatized and derivatized agarose gels and neurite extension was analyzed. Agarose gels derivatized with CDPGYIGSR and PEPMIX enhanced neurite outgrowth from DRGs while GRGDSP and IKVAV derivatized gels inhibited neurite extension when compared to underivatized agarose gels. The IKVAV derivatized gels significantly enhanced neurite outgrowth from PC12 cells in comparison to underivatized and other LN peptide derivatized gels. Agarose hydrogels carrying covalently immobilized LN oligopeptides thus evoke specific responses from cells which contain receptors to the peptides used. Agarose hydrogels derivatized with neurite promoting peptide sequences may find applications in various models of in vivo regeneration of nervous tissue.

Animals↗
PubMed1995

Polymer encapsulated cell lines genetically engineered to release ciliary neurotrophic factor can slow down progressive motor neuronopathy in the mouse.

Ciliary neurotrophic factor (CNTF) has recently generated great interest due to its potential as a therapeutic agent for the treatment of human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Because the systemic half-life of CNTF is only in the order of a few minutes, continuous delivery of this trophic factor could be attractive or even necessary in the therapy of these diseases. One promising technique involves the polymer encapsulation of cells which have been genetically modified to secrete neurotrophic factors. The polymer capsules can be implanted into animals and effect the slow release of the protein for several months. The encapsulation technique immuno-isolates the foreign cells from host immune cells and at the same time prevents tumour formation by the transplanted cells. In this study, we have used progressive motoneuronopathy (pmn) mice to determine the extent to which encapsulated cell lines secreting CNTF could alter the course of the disease. pmn/pmn homozygotes present severe loss of myelinated motor fibres and a significant reduction of facial motoneuron cell bodies. The mice develop weakness of the hindlimbs and die during the sixth week after birth. We found that CNTF delayed the disease progression by increasing the survival time by 40% and by improving motor function as assessed by three behavioural tests. Moreover, histological counts of the phrenic nerve myelinated axons and facial nucleus motoneurons indicated a significant reduction of motoneuron loss. These results suggest that polymer-encapsulated cells releasing neurotrophic factors may provide a potential delivery system for treating neurodegenerative diseases such as ALS.

Animals↗
PubMed1995

Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo.

Fas/APO-1 (CD95) is a cell surface receptor which mediates apoptosis when ligated by specific antibodies or by its recently cloned natural ligand, FasL. We have studied the cytotoxic potential of FasL in vivo using Fas/APO-1-expressing Yac-1 cells as targets. Supernatant harvested from Neuro-2a cells transfected with the murine FasL cDNA contains FasL and transduces a potent apoptotic signal to Yac-1 cells in vitro. Specificity of FasL-mediated cytotoxicity was confirmed by competition assays using soluble Fas or anti-Fas/APO-1 F(ab')2 fragments which specifically interfere with FasL-Fas/APO-1 interactions. Intraperitoneal injection of FasL-containing supernatant efficiently killed Yac-1 target cells which had been implanted in capsules into the peritoneal cavity of mice. Analysis of the target cells revealed DNA fragmentation and nuclear changes typical of apoptosis. As previously shown, intraperitoneal injection of anti-Fas/APO-1 antibodies caused liver failure (Ogasawara, J., Watanabe, F.R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S., Nature 1993. 364:806) and was observed at doses which did not reduce Yac-1 cell viability. In contrast, FasL did not induce histopathology in the liver when applied intraperitoneally at doses cytotoxic for Yac-1 cells. However, intravenous administration of FasL induced lethal liver hemorrhages and hepatocyte apoptosis. Thus, locally applied FasL kills tumor cells very efficiently without systemic toxicity and may therefore represent a candidate for local tumor treatment.

Animals↗
PubMed1995

Hydrogel-based three-dimensional matrix for neural cells.

The ability to organize cells in three dimensions (3D) is an important component of tissue engineering. This study sought to develop an extracellular matrix (ECM) equivalent with a physicochemical structure capable of supporting neurite extension from primary neural cells in 3D. Rat embryonic day 14 striatal cells and chick embryonic day 9 dorsal root ganglia extended neurites in 3D in agarose hydrogels in a gel concentration-dependent manner. Primary neural cells did not extend neurites above a threshold agarose gel concentration of 1.25% wt/vol. Gel characterization by hydraulic permeability studies revealed that the average pore radius of a 1.25% agarose gel was 150 mm. Hydraulic permeability studies for calculating average gel pore radius and gel morphology studies by environmental and scanning electron micrography showed that the average agarose gel por size decreased exponentially as the gel concentration increased. It is hypothesized that the average gel porosity plays an important role in determining the ability of agarose gels to support neurite extension. Lamination of alternating nonpermissive, permissive, and nonpermissive gel layers facilitated the creation of 3D neural tracts in vitro. This ability of agarose hydrogels to organize, support, and direct neurite extension from neural cells may be useful for applications such as 3D neural cell culture and nerve regeneration. Agarose hydrogel substrates also offer the possibility of manipulating cells in 3D, and may be used as 3D templates for tissue engineering efforts in vitro and in vivo.

Animals↗
PubMed1995

Bovine serum albumin conformation on methyl and amine functionalized surfaces compared by scanning force microscopy.

We investigated the adsorption of albumin on chemically modified gold surfaces by scanning force microscopy operating both in contact and noncontact mode. The surface modification was performed with thiol-based self-assembling molecules carrying amine or methyl groups. The albumin on the aminoethanethiol-coated gold formed a uniform layer and single molecules could be distinguished. On the dodecanethiol-coated surface the protein adsorbed in aggregates or single isolated molecules depending on the incubation time. The width of the albumin molecule on both surface was similar, but the height was much lower on the amine than on the methyl surface. This was interpreted as a difference in the conformation of albumin depending on the substrate, and could explain the promotion of cell adhesion on amine-treated polymers coated with albumin.

Adsorption↗
PubMed1995

Synthesis and characterization of fluoropolymeric substrata with immobilized minimal peptide sequences for cell adhesion studies. I.

In this work, poly(tetrafluoroethylene-co-hexafluoropropylene) (also known as fluorinated ethylene propylene; FEP) was functionalized at the surface using a radio frequency glow discharge plasma. This particular surface modification produced controlled densities of hydroxyl functionality on the FEP surface. These surface hydroxyl groups provided sites for the covalent attachment of minimal peptide sequences, that are specific for neuronal attachment. FSCA, ATR-FTIR, ToF-SIMS, and fluorescence spectroscopy were used to evaluate peptide reaction efficiencies and to verify that intact peptide sequences were covalently attached to the FEP surfaces. These modified substrata were then used to study the cell attachment and response to covalently bound minimal peptide sequences. Cell attachment and differentiation results using NG108-15 and PC12 neuronal cell lines are presented in the adjoining paper by Ranieri et al.

Amino Acid Sequence↗
PubMed1995

Neuronal cell attachment to fluorinated ethylene propylene films with covalently immobilized laminin oligopeptides YIGSR and IKVAV. II.

Material surfaces that can mediate cellular interactions by the coupling of specific cell membrane receptors may allow for the design of a biomaterial that can control cell attachment, differentiation, and tissue organization. Cell adhesion proteins have been shown to contain minimum oligopeptide sequences that are recognized by cell surface receptors and can be covalently immobilized on material surfaces. In this study, cell attachment to fluorinated ethylene propylene (FEP) films functionalized with the laminin-derived oligopeptides, YIGSR and a 19-mer IKVAV-containing sequence, was assessed using NG108-15 neuroblastoma and PC12 cells. A radiofrequency glow discharge (RFGD) process that replaces the FEP surface fluorine atoms with reactive hydroxyl functionalities was used to activate the film surfaces. The oligopeptides were then covalently coupled to the surface by their C-terminus using a standard nucleophilic substitution reaction. The covalent attachment of the oligopeptides to the FEP surface was verified using electron spectroscopy for chemical analysis (ESCA). Receptor-mediated NG108-15 cell attachment on the YIGSR-modified films was determined using competitive binding assays. Average cell attachment on the oligopeptide immobilized films in medium containing soluble CDPGYIGSR was reduced by approximately a factor of 2, compared to cell attachment in serum-free medium alone. No significant decrease in cell attachment was noted in medium containing the mock oligopeptide sequence CDPGYIGSK. FEP films immobilized with the 19-mer IKVAV sequence demonstrated a higher percentage of receptor mediated cell attachment on the film surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in adult rat thoracic spinal cord.

We previously demonstrated that Schwann cells (SCs) in semipermeable guidance channels promote axonal regeneration in adult rat spinal cord transected at the mid-thoracic level. Propriospinal but not supraspinal axons grew into these channels. Here, we tested the ability of exogenous brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) to promote axonal regeneration in this novel model. The two neurotrophins were delivered simultaneously into the channel by an Alzet minipump at a rate of 12 micrograms/day for each neurotrophin for 14 of 30 days tested; phosphate-buffered saline, the vehicle solution, was used as a control. Significantly more myelinated nerve fibers were present in SC/neurotrophin grafts than in SC/vehicle grafts (1523 +/- 292 vs 882 +/- 287). In the graft, at least 5 mm from the rostral cord-graft interface, some nerve fibers were immunoreactive for serotonin, a neurotransmitter specific to raphe-derived axons in rat spinal cord. Fast blue retrograde tracing from SC/neurotrophin grafts revealed labeled neurons in 10 nuclei of the brain stem, 67% of these being in the lateral and spinal vestibular nuclei. The mean number of labeled brain stem neurons in the SC/neurotrophin group (92; n = 3) contrasted with the mean in the SC/vehicle group (6; n = 4). Our results clearly demonstrate that BDNF and NT-3 infusion enhanced propriospinal axonal regeneration and, more significantly, promoted axonal regeneration of specific distant populations of brain stem neurons into grafts at the mid-thoracic level in adult rat spinal cord.

Animals↗
PubMed1995

Treatment of amyotrophic lateral sclerosis using a gene therapy approach.

Neurotrophic factors which act on motor neurons may provide an efficient treatment for amyotrophic lateral sclerosis. In order to overcome the difficulty of administering proteins into the central nervous system, it is possible to use a gene therapy approach, i.e. the polymer encapsulation of cells which have been genetically engineered to release one of these neurotrophic factors. The polymer-encapsulated cells can be implanted subcutaneously or even intrathecally and permit continuous, slow release of proteins. There is no rejection of the cells due to their isolation by a semi-permeable membrane, no risk of tumor formation and the polymer device can be removed in the event of a problem.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Bcl-2 overexpression prevents motoneuron cell body loss but not axonal degeneration in a mouse model of a neurodegenerative disease.

Bcl-2 and its analogs protect different classes of neurons from apoptosis in several experimental situations. These proteins may therefore provide a means for treatment of neurodegenerative diseases. We examined the effects of Bcl-2 overexpression in a genetic mouse model with motor neuron disease (progressive motor neuronopathy/pmn). Pmn/pmn mice lose motoneurons and myelinated axons, and die at 6 weeks of age. When these mice were crossed with transgenic mice that overexpress human Bcl-2, there was a rescue of the facial motoneurons with a concomitant restoration of their normal soma size and expression of choline acetyltransferase. However, Bcl-2 overexpression did not prevent degeneration of myelinated axons in the facial and phrenic motor nerves and it did not increase the life span of the animals. Since Bcl-2 acts strictly on neuronal cell body survival without compensating for nerve degeneration in pmn/pmn/bcl-2 mice, this proto-oncogene would not in itself be sufficient for treatment of neurodegenerative diseases where axonal impairment is a major component.

Animals↗
PubMed1994

Glial cell line-derived neurotrophic factor (GDNF), a new neurotrophic factor for motoneurones.

Glial cell line-derived neurotrophic factor (GDNF) has been postulated to be a specific dopaminergic neurotrophic factor since it selectively enhances the survival of dopaminergic neurones in vitro. We report here that GDNF can also act as a neurotrophic factor for motoneurones. GDNF released by GDNF-transfected BHK cells increases the activity of choline acetyltransferase (ChAT) in cultures from embryonic rat ventral mesencephalon containing cholinergic neurones from cranial motor nuclei and in cultured spinal motoneurones. Furthermore, local application of polymer-encapsulated BHK cells releasing GDNF to transected facial nerve in newborn rats diminishes the death of motoneurones normally occurring after axotomy in the neonatal period. The present results indicate that GDNF may have a therapeutic potential in human motoneurone diseases such as amyotrophic lateral sclerosis.

Animals↗
PubMed1994

The astrocyte inhibition of peripheral nerve regeneration is reversed by Schwann cells.

Schwann cell transplantation into the lesioned or demyelinated central nervous system (CNS) is being extensively explored as an approach to favorably influencing repair in the CNS. Under a variety of circumstances, however, the CNS glial microenvironment appears to offer an unfavorable terrain for the promotion of neurite elongation and for Schwann cell differentiation. Due to the heterogeneity of the cellular contents at injury sites, the specific role of each cell type present in limiting Schwann cell function is unclear. The damaged peripheral nervous system, a system capable of substantial regeneration (and free of the potentially negative influence of oligodendrocytes), represents a valuable model in which to specifically evaluate the influence of astrocytes on Schwann cell function. In the present study, purified cortical astrocyte populations were seeded into semipermeable guidance channels alone or in combination with adult Schwann cell populations to determine their effects on regeneration across an 8-mm gap in the transected sciatic nerve of the adult rat. Channels were prepared with (or without) a defined cellular content, implanted in inbred Lewis rats and evaluated after 3 weeks. Channels seeded with astrocytes alone impeded regeneration, regardless of the maturity of the astrocytes (7-8 days vs 28 days in culture) and their seeding density (40 vs 80 x 10(6) cells/ml). On the other hand, Schwann cells derived from adult sciatic nerve seeded at similar densities enhanced the regenerative process. Regenerative capacity was diminished when astrocytes were combined with Schwann cells; the rate of regeneration increased as the number of Schwann cells in the astrocyte/Schwann cell mixture increased. Immunostaining of the nerve stumps related to astrocyte-seeded channels and of the regenerated tissue in the astrocyte-Schwann cell-seeded channels indicated that astrocytes had migrated into the proximal nerve stump; only a few astrocytes remained within the regenerated cable. The present experiments show that although astrocytes alone inhibit nerve regeneration, Schwann cells are able to partially overcome this inhibition if they are provided in sufficient numbers. We believe these observations will be valuable in considering clinical strategies to use autologous Schwann cell transplantation to influence CNS regeneration.

Acrylonitrile↗
PubMed1994

Functional recovery in hemiparkinsonian primates transplanted with polymer-encapsulated PC12 cells.

Cross-species neural grafting of cell lines immunoisolated by a permselective polymer membrane represents a promising source of neuroactive molecules for the treatment of neurodegenerative diseases. Utilization of a cell line of xenogeneic origin is advantageous since the transplanted cells will be rejected by the host immune system in case of breakage of the immunoisolating envelope. Polymer-encapsulated PC12 cells, a dopaminergic cell line derived from a rat pheochromocytoma, were transplanted in five Macaca fascicularis monkeys which had been previously rendered hemiparkinsonian by a unilateral carotid injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Well-preserved, tyrosine hydroxylase positive encapsulated PC12 cells were observed in the lesioned striatum for up to 5 months after implantation. Four out of five monkeys which received polymer-encapsulated PC12 cells showed significant behavioral improvement, whereas three monkeys implanted with either encapsulated bovine chromaffin cells or empty polymer envelopes showed no amelioration.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1994

Seizure suppression in kindling epilepsy by intracerebral implants of GABA- but not by noradrenaline-releasing polymer matrices.

Gamma-aminobutyric acid (GABA)-releasing polymer matrices were implanted bilaterally, immediately dorsal to the substantia nigra, in rats previously kindled in the amygdala. Two days after implantation, rats with GABA-releasing matrices exhibited only focal limbic seizures in response to electrical stimulation, whereas animals with control matrices devoid of GABA had generalized convulsions. GABA release from the polymer matrices was high during the first days after implantation, as demonstrated both in vitro and, using microdialysis, in vivo. The anticonvulsant effect was no longer observed at 7 and 14 days at which time GABA release was found to be low. In a parallel experiment, polymer matrices containing noradrenaline (NA) were implanted bilaterally into the hippocampus of rats with extensive forebrain NA depletion induced by an intraventricular 6-hydroxydopamine injection. No effect on the development of hippocampal kindling was observed, despite extracellular NA levels exceeding those of rats with intrahippocampal locus coeruleus grafts that have previously been shown to retard kindling rate. The results indicate that GABA-releasing implants located in the substantia nigra region can suppress seizure generalization in epilepsy, even in the absence of synapse formation and integration with the host brain. In contrast, the failure of NA-releasing polymer matrices to retard the development of seizures in NA-depleted rats suggests that such an effect can only be exerted by grafts acting through a well-regulated, synaptic release of NA.

Animals↗
PubMed1994

Chitosan as a matrix for mammalian cell encapsulation.

Cells encapsulated in a semi-permeable polymer membrane display enhanced viability and proliferation when sequestered along with a supportive matrix. In the present study, anionic sodium alginate and cationic chitosan were used as supportive matrices to study their influence on the growth of both anchorage dependent and independent cell types. A phaeochromocytoma-derived cell line, PC12, and two fibroblast cell lines, R208F and R208N.8, were encapsulated in sodium alginate microcapsules, crosslinked chitosan microcapsules and 60:40 acrylonitrile-vinylchloride copolymer (PAN/PVC) macrocapsules containing precipitated chitosan as an internal matrix. Microcapsules were maintained for 2 wk in vitro and macrocapsules were maintained for 4 wk. Both the PC12 and the R208F cells proliferated in the alginate microcapsules. Catecholamine release was detected from PC12 cell-loaded microcapsules. In contrast, the R208N.8 cells were not supported by the alginate and the only viable R208N.8 cells seen in this system protruded from the alginate matrix into the surrounding cationic poly-l-lysine lamina. All three cell lines grew poorly in crosslinked chitosan microcapsules yet they exhibited excellent viability when sequestered in PAN/PVC macrocapsules containing an internal matrix of precipitated chitosan. Significant catecholamine levels were detected from macroencapsulated PC12 cells while macroencapsulated R208N.8 cells released nerve growth factor (NGF) as demonstrated by a qualitative bioassay. This study shows that a cationic hydrogel, precipitated chitosan, supports attachment and spreading of fibroblasts when used as a matrix in the lumen of a PAN/PVC macrocapsule.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylic Resins↗
PubMed1994

Spatial control of neuronal cell attachment and differentiation on covalently patterned laminin oligopeptide substrates.

The spatial control of neuronal cell attachment and differentiation via specific receptor mediated interactions, may provide an effective means for the in vitro reconstruction of neuronal cell architecture. In this study, receptor-specific oligopeptide sequences derived from the extracellular matrix (ECM) molecule laminin, a potent neural cell attachment and differentiation promoter were covalently bound on fluorinated ethylene propylene (FEP) films. The degree of receptor-specific cell attachment and the ability to spatially control neurite outgrowth by covalently patterning the oligopeptide sequences on the FEP film surface were assessed. FEP films were first chemically activated with a Radio Frequency Glow Discharge (RFGD) process that covalently replaces the surface fluorine atoms with reactive hydroxyl groups. Oligopeptides containing the YIGSR sequence from the B1 chain of laminin and the water soluble oligopeptide containing the IKVAV sequence (CSRARKQAASIKVAVSADR) from the A chain were covalently bound to the hydroxylated FEP films. Electron Spectroscopy for Chemical Analysis (ESCA) verified the covalent attachment of the oligopeptides to the material surface. The degree of receptor mediated NG108-15 cell attachment on immobilized CDPGYIGSR films was determined using competitive binding media. A 78% reduction in cell attachment was observed on films containing CDPGYIGSR in the cell plating medium. Only a 23% reduction in cell attachment was noted on films plated in medium containing a mock CDPGYIGSK sequence. FEP films immobilized with the IKVAV oligopeptide sequence were shown to mediate PC12 cell attachment and a competitive binding medium also significantly attenuated cell attachment on the immobilized films. The spatial patterning of these oligopeptide sequences to the FEP surface was shown to localize cell attachment and neurite extension on the patterned pathways. The surrounding unmodified FEP surface was inhibitory in serum containing medium and prevented cellular interactions outside the oligopeptide modifications. The spatial immobilization of laminin oligopeptides on FEP films provides a means to organize the attachment and differentiation of neuronal cells in a receptor-specific manner.

Animals↗
PubMed1994

The functional characteristics of Schwann cells cultured from human peripheral nerve after transplantation into a gap within the rat sciatic nerve.

The use of human Schwann cells (SCs) in transplantation to promote regeneration in central and peripheral neural tissues must be preceded by efforts to define the factors that regulate their functional expression. Adult-derived human SCs can be isolated and purified in culture, but the culture conditions that allow their full differentiation have not yet been defined. We tested the functional capacity of these cells to enhance axonal regeneration and myelinate regenerating axons in vivo by transplanting them into the damaged PNS of an immune-deficient rat. SCs were purified from human peripheral nerve obtained from organ donors. Semi-permeable guidance channels were filled with a 30% Matrigel containing solution with or without human SCs suspended at a density of 80 x 10(6) cells/ml. Channels were implanted within an 8 mm gap of the transfected sciatic nerve of nude female rats for a period of 4 weeks. Survival of the transplanted human SCs was established by dissociating nerve explants taken from the regenerated cable (after first placing them in culture for 5 d) and staining individual cells for a primate-specific NGF receptor (PNGFr) and S 100. Only one-half of the S 100-positive cells stained for the PNGFr, which indicated that the regenerated cable contained an approximately equal number of human and rat (host) SCs. The presence of some human myelin segments was confirmed by immune staining with an HNK-1 antibody that specifically labels human but not rat myelin. The majority of the myelin segments in the regenerated cable, however, were produced by the rat SCs. The number of myelinated axons and the cross-sectional area of the cable were significantly greater in channels seeded with human SCs when compared to channels containing the diluted Matrigel solution alone. We conclude that purified cultured human SCs can survive and substantially enhance axonal regeneration when transplanted into the injured PNS of an immune-deficient rat. Some of the transplanted human SCs are capable of myelinating regenerating rat axons but are less successful than the host SCs.

Animals↗