P van den Bosch de Aguilar

Fibronectin adsorption, conformation, and orientation on polystyrene substrates studied by radiolabeling, XPS, and ToF SIMS.

Protein adsorption is widely studied by a variety of techniques, but there still is little known about protein orientation and conformation after adsorption. This probably is due to the large number of parameters involved, such as the characteristics of the surface and the structure of the protein. In this study, the adsorption of fibronectin was investigated with three different techniques: radiolabeling, X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF SIMS) on polystyrene and oxidized polystyrene. The first two techniques have been widely used to study protein adsorption, allowing us to determine the amount of protein adsorbed on each surface. The ToF SIMS, however, is a technique just emerging for the study of protein adsorption. This study confirms its utility since ToF SIMS is found to be sensitive to the protein orientation and/or conformation at the surface. Indeed, the ToF SIMS peaks characteristic of the protein show differences in their reduced intensity between the two substrates. These differences, which are not detected by XPS, are attributed to different orientations and/or conformations of the protein.

Senile dementia of the Alzheimer type: Morphological and Immunocytochemical Studies.

Although recent progress in the isolation of neurofibrillary tangles (NFT) has been made (Ihara et al. 1983; Iqbal et al. 1984), little information is available on the biochemical nature of NFT, their precise relationship with neuron organelles and their etiopathogenesis. Difficulties in carrying out the biochemical analysis of NFT until now have been attributed to their unusual insolubility (Selkoe et al. 1982), although this property also seems a matter of debate (Iqbal et al. 1984). Ultrastructural studies have also clearly pointed out their unique morphological features (Terry 1963; Kidd 1963) Immunohistochemistry offers an interesting approach, since well-defined antibodies reacting with NFT in tissue sections (Anderton et al. 1982; Gambetti et al. 1983) have recently been described.

Topographical distribution in the adult rat brain of neurotrophic activities directed to central nervous system targets

Cortex, hippocampus, septum and striatum of day 18 rat embryos were grafted to several brain regions of young adult rats which had been lesioned in the chosen area 4 days earlier. Thirty days after transplantation, the grafts were fixed and morphometrically analysed under light microscope. The volumes, neuronal densities and total number of neurons of the transplants were compared. Each graft survived best when transplanted to its original region. Good survival was also achieved by heterotopic grafts between regions that are anatomically related. Striatal grafts showed reasonable survival only when transplanted to their original site. In a second series of experiences, the neurons from the same embryonic brain regions were cultured in a defined medium, to which was added tissue extracts from the lesioned regions of the adult brain. The neuronal survival was estimated. The in vitro results are closely related to those obtained in vivo. This experimental evidence agrees with the theory of the existence of a retrograde transport of NGF from the hippocampus to the septum, sustaining the survival of the latter. On the other hand, our results demonstrate the existence of other unidentified neurotrophic factors in the central nervous system which differ from one region to another.

Effects of aluminum exposure on behavioral parameters in the rat.

Adult rats were treated by intraperitoneal injection of aluminum gluconate for 3 months. Rats were submitted to the radial maze test to determine the influence of chronic aluminum intoxication on cognitive and noncognitive behavioral processes. Both learning abilities (working memory and reference memory) and rapidity (time spent to respond and to master a trial) were analyzed. Aluminum concentration was evaluated in the brain, serum, and liver to assess aluminum body burden. While hippocampus and neocortex showed a significant increase in aluminum concentration, aluminum treatment did never affect the animals performance during cue learning or when the insert cues were removed. The only behavioral difference observed was a decrease in rapidity: both the total time to finish a trial and the latency to make the first choice were lengthened in aluminum-intoxicated rats.

Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: relationship to cell adhesion.

Recently, patterned polystyrene surfaces containing hydrophobic (PS) and more hydrophilic (PSox) areas have been shown to be capable of directing cellular growth, which is mainly due to the competitive adsorption of adhesive and antiadhesive molecules. In this article, the competitive adsorption between a pluronic surfactant and fibronectin was studied on homogeneous PS or PSox substrates conditioned with mixtures containing increasing concentrations of one of the two molecules. Radiolabeling and X-ray photoelectron spectroscopy techniques showed that fibronectin adsorption increased on both surfaces if the fibronectin concentrations increased in the conditioning mixture. In contrast, fibronectin adsorption decreased on PSox and did not occur on PS surfaces when pluronic concentrations increased in the coating mixture. A comparison of these data with pheochromocytoma and Schwann cells cultured on patterned surfaces showed that the direction of cell growth on PSox areas depended first on the relative concentrations of the two components in the mixtures, and second, on their ratio; the best concentration ratio probably depends on the cells ability to recondition its support.

Reduced axonal transport of the G4 molecular form of acetylcholinesterase in the rat sciatic nerve during aging.

Abstract: Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40–60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.

Rat sciatic nerve regeneration within an acrylic semipermeable tube and comparison with a silicone impermeable material.

Sectioned rat sciatic nerves were placed in tubes to study the regrowth of injured nerve processes. First, we characterized quantitatively the regeneration of myelinated fibers at different levels of an acrylic semipermeable tube, from two to 27 weeks postoperatively. From the fifth week, myelinated fiber counts at mid-tube level were equal to the value of an intact nerve, but at the distal part the number of fibers exceeded both mid-tube level and unsectioned nerve values. At the proximal part of the tube an important fiber disorganization was observed. Second, we have compared acrylic semipermeable and totally impermeable silicone tubes at four and 27 weeks postoperatively. In terms of the number of myelinated fibers and the surface of the endoneurium at the mid-tube level, the most effective tube was the impermeable one. This study points out the importance of the tube wall permeability in nerve regeneration.

A new plasma-based method to promote cell adhesion on micrometric tracks on polystyrene substrates

A new procedure has been developed in order to obtain heterogeneous polymer surfaces for the promotion of cell adhesion. For this purpose, a microelectronic photosensitive resin was spin coated on polystyrene (PS) substrates. The resin was then submitted to UV light irradiation through a mask and partially developed. The sample was further submitted to a plasma oxygen discharge prior to dissolution of the remaining resin. The characterization by time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), and dynamic contact angle (DCA) allowed us to conclude that hydrophilic paths were created on the more hydrophobic PS substrate together with the complete removal of the resin. In order to optimize cell adhesion contrast, the modified surfaces were then conditioned with a solution containing both a surfactant (pluronic F68) and a protein. Two different proteins were tested (collagen I and fibronectin). PC12 cell cultures on those conditioned surfaces showed that cell adhesion occurs only on the hydrophilic tracks. ToF SIMS spectra and images recorded on those substrates revealed the presence of the proteins only in the hydrophilic tracks. In the same time, the surfactant is suspected to adsorb mainly on the hydrophobic areas of the samples.

Perikaryal neurofilament phosphorylation in axotomized and 6-OH-dopamine-lesioned CNS neurons.

The axon reaction in the central nervous system was studied using a monoclonal antibody to phosphorylated neurofilaments. Axotomy was performed by cutting the nigrostriatal pathway. We were able to show that phosphorylated epitopes of neurofilaments, that are usually restricted to axons, could be detected in the perikarya and dendrites of axotomized neurons as early as 3 days postlesion. These neurons remained labelled up to 17 days after axotomy and in some cases even up to 6 weeks. The cytoplasmic changes appearing in the lesioned neurons 8 days after axotomy seem to indicate that these neurons will probably degenerate or survive only in an atrophied, non-functional state as they are unable to regenerate their sectioned axon. Neurochemical lesions, using the neurotoxin 6-OH-dopamine, were performed to establish whether this reaction of perikaryal neurofilament phosphorylation may be a non-specific phenomenon accompanying neuronal degeneration or injury. Although cell loss was important, no labelled neurons could be observed following 6-OH-dopamine treatment. These results indicate that the induction of perikaryal neurofilament phosphorylation is a response to selective types of neuronal injury and concerns selective neuronal populations.

Etude ultrastructurale des neurones ganglionnaires spinaux au cours du vieiUissement chez le rat

The chronology of the alterations impairing the cytology of the spinal ganglion neurons was analysed in 3- to 32-month-old male rats. A type neurons are impaired earlier and more severely than B neuro