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.

In vivo expression of the intermediate filament peripherin in rat motoneurons: modulation by inhibitory and stimulatory signals.

Peripherin is a type III intermediate filament which, in contrast to the neurofilaments, is strongly up-regulated after nerve injury. Although peripherin expression is stimulated in vitro by neurotrophins and cytokines, little is known about its in vivo regulation. In this report, we show that the in vivo down-regulation of peripherin expression to normal levels during regeneration closely correlates with target reconnection in rat facial motoneurons. Prevention of reconnection, by transection and suture, results in the persistence of strong peripherin expression for prolonged periods of up to 11months. This contrasts with the modulation of the p75 low-affinity neurotrophin receptor, whose expression returns to normal even in the absence of reconnection. We further demonstrate that blockade of the axonal transport in non-injured motoneurons increases the expression of peripherin. Blockade of the axonal transport simultaneously to, or after injury of, facial motoneurons does not abolish the axotomy-induced peripherin up-regulation. These data demonstrate that the in vivo expression of peripherin is normally restrained by a distal retrogradely transported inhibitory signal. Thus, peripherin up-regulation results primarily from a lack of supply in this factor. Our results show that stimulatory factors released at the injury site are not required for the initial up-regulation and maintenance of high peripherin expression. However, they appear to enhance this increase during the acute post-lesion phase. Peripherin expression is thus finely tuned by both glial cell-derived stimulatory and distal inhibitory signals that reflect neuron-target interactions.

PEG embedding for immunocytochemistry: application to the analysis of immunoreactivity loss during histological processing.

We have developed a protocol for the production and longterm storage of polyethylene glycol (PEG) sections for immunocytochemistry. Sections obtained by this protocol allow immunolabeling for many different antigens, such as intermediate filaments, macrophage markers, or neurotransmitter enzymes. Standard histological staining can also be performed on these sections. This fixation-embedding system may therefore be of interest for histopathology of rare specimens, as well as for experimental research. Multiple labeling can be performed either on the same section or on consecutive thin sections, thus allowing a more thorough analysis of precious experimental material. We compare the advantages of PEG vs cryostat or vibratome sections. This protocol has been used to study the inactivation of antigenicity by paraffin embedding. We have identified the infiltration by paraffin as the antigenicity inactivating step, not dehydration or high temperature as generally thought.

Axonal regeneration after peripheral nerve grafting and fibrin-fibronectin-containing matrix implantation on the injured septohippocampal pathway of the adult rat: a light and electron microscopic study.

The damaged septohippocampal pathway was utilized to study the axonal regeneration of injured neurons. Semipermeable tubes, 2-mm long, were placed in the axis of the transected septohippocampal pathway of adult rats. In a first series of experiments, empty tubes were implanted. Even six weeks after the operation, no regenerated axons were observed in the conduit. In a second series of experiments, in order to validate our approach, segments of pre-degenerated sciatic nerves were introduced into the tubes. Under these experimental conditions, acetylcholinesterase (AChE)-containing regenerated axonal processes were detected in the grafted sciatic nerves. Glial fibrillary acidic protein (GFAP)-immunodetection showed that astroglial cells and astrocyte processes were able to progress on and into the peripheral grafts. At the electron microscopic level, axons were observed in close contact with Schwann cells which myelinated some of them. In some other cases, unmyelinated axons were also present at the surface of reactive astroglial cells filled by numerous intermediate filaments. These central glial cells had migrated among the sciatic nerve collagen fibers. No axon was detected without glial cell contact. In a third series of experiments, we implanted semipermeable tubes previously filled with a fibrin-fibronectin-containing matrix provided by peripheral regeneration chambers. One week after the implantation of the tubes containing this peripheral substrate, different cell types were observed migrating into the conduit and replacing the fibrin-fibronectin-containing matrix. Among these cells astrocytes were present as revealed by GFAP-immunocytochemistry and electron microscopic examinations. During the following weeks, axons were detected in contact with the reactive astroglial cells. AChE-histochemistry showed that axons were able to cross the two millimeter distance separating the septal part and the hippocampal part of the lesion site. GABA (γ-aminobutyric acid)-ergic fibers were also detected in the regenerated structure. These experiments show that cellular or acellular substrates provided by the PNS can promote the regeneration of CNS GABAergic and cholinergic neurons. Our observations suggest that astrocytes can take an important part, after their migration or after extending processes, in the axonal regeneration in the adult CNS of the rat, possibly in furnishing a cellular terrain for the progression of growth cones over a distance of two millimeters and in maintaining regenerated axons at least until the sixth week after the operation.

Orientation of cell adhesion and growth on patterned heterogeneous polystyrene surface.

Studies of neurite outgrowth or cell migration, two important processes in neuronal networks formation, are facilitated by cell culture models capable of orientating cellular growth and of designing a well-defined cellular pattern. Heterogeneous polystyrene surfaces composed of oxygen plasma-treated stripes (PSox) with a low hydrophobicity separated by non-treated areas (PS) have these properties. In this study, to guide cell growth, we developed a cell culture model using these supports and we identified the molecular factors involved in cellular orientation. When the heterogeneous supports were not coated, proteins from a serum culture medium were required for cells to line up on PSox. On the other hand, cell orientation on coated surfaces was clearly influenced by competitive adsorption of adhesive proteins such as fibronectin or collagen and anti-adhesive molecules as pluronic F68 or albumin. Attachment factors were adsorbed on PSox stripes while adsorption of anti-adhesive molecules on the most hydrophobic PS areas prevented cell adhesion or growth. Thus, we describe the preparation of a cell culture substrate that succeeded in orientating cell growth and that led to a line of cells on adhesive PSox stripes ranging from 2 to 100 microns width.

A new in vivo model to study the influence of the microenvironment in the regeneration of the central nervous system.

In order to study the 'in vivo' regenerative capacity of the central nervous system, a semipermeable tube was placed in the axis of the lesioned nigrostriatal pathway of adult rats. In spite of a correct positioning of the tube, no growing central nervous processes were observed within the tube after 3 to 6 weeks when it was left empty. However, when the lumen of the tube was previously filled with a pre-degenerated sciatic nerve, unmyelinated and myelinated fibers were observed growing in the peripheral graft. Since the content of the tube can be modified, it appears that this model can be used to test the capability of cellular or acellular microenvironments to promote the 'in vivo' regeneration of the mammalian central nervous system fibers.

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.

Axonal growth and glial migration from co-cultured hippocampal and septal slices into fibrin-fibronectin-containing matrix of peripheral regeneration chambers: a light and electron microscope study.

In order to investigate whether a fibrin-fibronectin-containing matrix of a peripheral regeneration chamber could promote the growth of central nervous system neurons, hippocampal and septal slices were co-cultured in the presence of this acellular substrate. In introducing the peripheral matrix into a 2-mm-long tube between hippocampal and septal slices, a spatio-temporal sequence of cell migration and axonal growth was described by light and electron microscopy. Axons were able to elongate directly into the flocculent material constituting the matrix and a possible neurite-promoting activity was implicated in this process as axonal growth was not detected in direct contact with rat plasma coagulated with calcium, or chicken plasma coagulated with thrombin, used as control matrices. However, in the 3 different substrates tested, astrocytes were able to migrate and dilated astroglial processes containing intermediate filaments were detected. Axonal processes were observed growing on the glial cell surface. GFAP-positive phagocytic cells, that could be of the same origin as astrocytes, were involved in matrix removing. Neuronal growth and glial migration arose from hippocampal and septum slices and acetylcholinesterase-containing fibers were seen in the bridging structure suggesting that cholinergic axons were able to progress to the hippocampal slice. This technique appeared to provide a model in which axonal growth and cell migration can be studied 'in vitro' in a 3-dimensional environment.

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.

A new model for quantification of microvascular regeneration after a lesion of the rat cerebral cortex.

The purpose of this study is to validate a method for the quantification of the neovascularization in the vicinity of a lesion made in the cerebral rat cortex. A cavity, made by aspiration in the occipital cortex of young rats, induces around the lesion a parenchymal and vascular reaction. The parenchymal reaction is characterized by cellular necrosis and gliosis. The vascularization is more dense around the cavity than in normal cortex. Morphometric analysis indicates, 8 days after the lesion, a 130% increase of the total length of the vessels in comparison to the contralateral normal cortex.

Neuron migration within the radial glial fiber system of the developing murine cerebrum: an electron microscopic autoradiographic analysis.

The present analysis provides direct evidence in the mouse that in the course of course of neocortical histogenesis, contact between migrating neurons and the surfaces of radial glial fibers is both invariant and relatively selective. The analysis characterizes in detail the migratory behavior of the individual migrating cell with respect to the overall radial glial fiber system as this system varies systematically in its structure with ascent through the strata of the cerebral wall. A quantitative study of the relationships between the radial glial fibers confidently identified by their glycogen content and the migrating neurons marked autoradiographically by injection of [3H]thymidine was also performed at the ultrastructural level on tangential sections at different pallial levels in E16 and E17 embryos. The overall set of observations lend support to the hypothesis that radial glial fibers act specifically as guides to neuronal migration and illustrate the nature of the cell-to-cell interaction which serves this cellular process critical to neocortical histogenesis.

Ageing of the spinal ganglion neurons in the rat: a radioautographic study following injection of [3H]lysine.

After administration of [3H]lysine to 3- and 24-month-old rats, radioautography demonstrates a significantly less important uptake in the A-type spinal ganglion neurons and in the old animals. This is in agreement with the existence of at least two functional categories of neurons in the spinal ganglion and suggests that protein synthesis is diminished in the old animals. The very fact that incorporation varies between animals of the same age sustains the hypothesis according to which amplitude of ageing is essentially an individual physiological-dependent process.

Spontaneous perikaryal neurofilament phosphorylation in the septofimbrial nucleus of the rat.

Phosphorylation of the 200 kDa neurofilament peptide NF-H usually only occurs in axons. We describe the spontaneous presence of phosphorylated NF-H in a population of small spindle-shaped neurons of the rat septofimbrial nucleus. A similar phenomenon has been observed in axotomized neurons and in human neurodegenerative diseases. Our observations, as well as previous studies by other authors, indicate that perikaryal neurofilament phosphorylation is not necessarily linked to pathological conditions.

Effects of dibutyryl cyclic adenosine monophosphate, ACTH(4-10) and gangliosides on nerve fiber regeneration after rat sciatic nerve tubulization.

Tubulization of sectioned rat sciatic nerves was used to evaluate the effect of dibutyryl cyclic adenosine monophosphate (db-cAMP), a fragment of the adrenocorticotrophic hormone (ACTH(4-10] and gangliosides on the regrowth of injured nerve processes. Four weeks post-operative, the number of myelinated fibers was counted at different levels of the lesioned sciatic nerves and the endoneurium surface of the bridging structure in the tube was measured. Animals treated with gangliosides and db-AMPc showed statistically significant differences compared to vehicle treated rats. The gangliosides have shown a beneficial effect but db-cAMP diminished the number of myelinated fibers. In this case, an implication of Schwann cell mitosis and migration is discussed. In spite of better results obtained in ACTH(4-10)-treated animals compared to controls, the differences did not attain a statistically significant level. These experiments confirm the beneficial effect of gangliosides on peripheral nerve regeneration and reveal a negative effect of db-cAMP in the regeneration chamber model.

Heparin treatment increases 9-kb MAP2 mRNA levels in neuronal cell cultures.

When neuronal adhesion is reduced by heparin, cultured neurons detach from the substratum and form clusters connected by neuritic fascicles. 24 h after treatment, Northern blots analysis revealed an increase in the expression of the 9-kb MAP2 mRNA in the heparin-treated neuronal cultures. MAP2 being associated with the cross-bridges between microtubules, this increased expression could be an attempt of the cells to rigidify their cytoskeleton to compensate for the reduced attachment. However, the MAP2 protein content was not increased after a 24-h heparin treatment. We discuss possible explanations for this observation and their implications on MAP2 metabolism.

The abilities of the rat brain to sustain the survival of implanted or cultured embryonic dorsal root ganglion neurons depend on the selected region.

The purpose of the present study was to examine the regional distribution of trophic activity in the lesioned adult rat brain on implanted peripheral neurons as target cells. Embryonic dorsal root ganglia (DRG) were implanted into selected regions of previously lesioned adult rat brains. The survival of these neurons was quantified after thirty days. In another experiment embryonic DRG neurons were cultured for 24 h together with tissue extracts of the same lesioned brain regions and the neuronal survival was estimated. We report here that all the tested regions, i.e. the occipital, parietal and frontal cortex, the hippocampus and the striatum, exert a trophic effect on embryonic DRG neurons both 'in vivo' and 'in vitro'. This effect is twice as high in the hippocampus as in the striatum, the other cortical regions showing intermediate values. The in vitro results are qualitatively the same as the in vivo results.

Expression of integrins by murine MSC80 Schwann cell line: relationship to cell adhesion and migration.

Schwann cells (Sc) are one of the most important factors promoting regeneration of both the peripheral and the central nervous system. They provide a permissive environment for neurite outgrowth and the making of this environment requires interactions between Sc and extracellular matrix proteins that are mediated via integrin receptors. This study characterized, by immunoprecipitation, the integrins expressed by the mouse MSC80 Sc line. Our results showed that MSC80 Sc expressed alpha1beta1, alpha5beta1 and alpha6beta1 integrins as well as the alpha v-subunit associated with an unidentified 80-90 kDa beta-subunit. Adhesion and migration assays revealed a hierarchy of protein influences that are dependent upon the type of cellular behaviour. Integrin expression correlated with MSC80 Sc line adhesion and migration on extracellular matrix proteins. The MSC80 Sc line expressed a pattern of integrins which allowed adherence on vitronectin and collagen IV, and faster migration on merosin and laminin. As the integrin pattern and the behaviour of MSC80 on ECM were similar to primary Sc, MSC80 are a potential abundant source of Sc for further in vitro and in vivo experiments.

Regeneration of lesioned cholinergic septal neurons of the adult rat can be promoted by peripheral nerve grafts and a fibrin-fibronectin-containing matrix of peripheral regeneration chambers.

Axonal regeneration of septal cholinergic neurons was examined after lesion of the septohippocampal pathway of the adult rat and implantation of tubes containing peripheral cellular or acellular substrates. After empty tube implantation, no regenerated structures were observed in the conduit. However, after implanting tubes filled with sections of predegenerated sciatic nerves or a fibrin-fibronectin-containing matrix provided by peripheral regeneration chambers, numerous regenerated axons were detected 6 weeks after the operation. At the electron microscopic level, regenerated axons were observed in the grafted sciatic nerves in contact with Schwann cells but also in contact with astrocytes which were able to migrate and send processes into the graft. After fibrin-fibronectin-containing-matrix implantation, the regenerated structure between septum and hippocampus was composed mainly of fibroblasts, astrocytes, and regenerated axons associated to these central glial cells.

Ultrastructural changes in brain parenchyma during normal aging and in animal models of aging.

During aging, the brain parenchyma of animals and humans share many similarities, both in the gray and the white matter. Unfortunately, until now, neither aged animals nor animal models reproduce the two hallmarks of aging of the human brain: senile plaques and tangles. Therefore, observations performed on animals are limited to some aspects of the involutive process which affects brain parenchyma during aging and their appropriateness to the human situation. One striking aspect concerns the occurrence of vacuolated necrotic cells whose number increases with advancing age. These cells can constitute markers of the brain involutive process and they characterize, both in animal and human, the more vulnerable areas of the brain affected by the neuronal rarefaction. Experimental animal models can be used to study the various conditions which sustain the cell survival and to determine, at the cellular level, the factors leading the brain parenchyma to an irreversible state of degradation.

Suloctidil increases the rat brain cortex microvascular regeneration after a lesion.

A "cavity" lesion made by aspiration in the rat occipital cortex induces a parenchymal and a vascular reaction in its vicinity. The first was mainly characterized by cellular necrosis and gliosis, the second by an increase of the vascular network. In vehicle treated rats, a 50% significant increase of the vascular network was observed around the cavity 4 days after the lesion, in comparison to the uninjured contralateral cortex. The effects of a vasoactive substance, suloctidil, on the vascular reaction was studied in the brain cortex. A single oral dose of suloctidil (30 mg/kg; 2 hours before the sacrifice) gave the same effect as the vehicle group. After 8 days of suloctidil oral administration (30 mg/kg; twice daily: 4 days before lesion and 4 days after) a significant increase (123%) of the vascular network was observed around the cavity. The hypothetical ways by which a chronic treatment of suloctidil induces this increase of the neovascularization observed after cortical lesion are discussed.