Differential behavioral syndrome evoked in the rats after multiple doses of SSRI fluoxetine with selective MAO inhibitors rasagiline or selegiline.

This study investigated whether rasagiline and selegiline (MAO-B inhibitors) induce serotonin syndrome in fluoxetine-treated rats. Rats received rasagiline (0.1, 0.5, 2.0 mg/kg), or selegiline (0.8, 4.0, 16.0 mg/kg) (doses reflecting the clinical ratio of 1:8 base) in drinking water for 28 days. During the last 21 days, they received injections of fluoxetine 10 mg/kg (controls received water only, then saline injections; a fluoxetine only group received water only then fluoxetine). Serotonin syndrome was assessed using neurological severity score (NSS), food intake and weight gain. Mean NSS significantly increased, and weight and food consumption significantly decreased in rats receiving fluoxetine alone compared with controls. Selegiline 16 mg/kg but not rasagiline (regardless of dose) exacerbated these effects. We concluded that selegiline's amphetamine-like metabolites may increase synaptic cathecholamines and possibly serotonin, aggravating fluoxetine's effect. Rasagiline is devoid of this effect and may therefore be safer for use with serotonergic drugs in parkinsonian patients.

Rasagiline is neuroprotective in an experimental model of brain ischemia in the rat.

The neuroprotective effects of intravenous rasagiline were investigated in a rat model of stroke. Middle cerebral artery (MCA) occlusion was performed in male rats and the short- (neurological severity score [NSS], infarct size), intermediate- (cognition) and long-term (necrotic area) effects were assessed. A bolus (3 mg/kg) of rasagiline followed by a 3-h infusion (3 mg/kg/h), initiated immediately after MCA occlusion, reduced infarct size by 48.6% and NSS by 32.7% relative to saline treatment. Cognitive function, tested in a water maze 2-3 weeks after occlusion, also significantly improved compared with saline-treated controls. Necrotic brain area was 35-50% smaller with rasagiline than with saline following a single bolus dose. The single bolus rasagiline dose was as effective as a rasagiline bolus followed by rasagiline infusion in short-term outcomes. The neuroprotective effect of rasagiline was fully reproducible when administered at 2 h following occlusion but not after 4 h.

Neuroprotective effect of rasagiline in a rodent model of Parkinson's disease.

Sprague-Dawley rats received a unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum and were treated daily for 6 weeks with increasing doses of monoamine oxidase type B inhibitor rasagiline [R(+)-N-propargyl-1-aminoindane] or saline (controls). Both doses of rasagiline markedly increased the survival of dopaminergic neurons in the lesioned substantia nigra, compared to controls (+97% and +119%, respectively). Treatment with the lower dose of rasagiline also abolished the motor stereotypies associated with nigrostriatal lesion. Our study supports the neuroprotective potential of chronic rasagiline administration in an experimental model of Parkinson's disease (PD).

Rasagiline, a monoamine oxidase-B inhibitor, protects NGF-differentiated PC12 cells against oxygen-glucose deprivation.

In our in vitro model, rasagiline a selective irreversible monoamine oxidase-B (MAO-B) inhibitor, protected nerve growth factor (NGF)-differentiated PC12 cells from cell death under oxygen and glucose deprivation (OGD). The severity of the OGD insult, as expressed by cell death, was time-dependent. Exposure of the cells to OGD for 3 hr followed by 18 hr of reoxygenation caused about 30-40% cell death. Under these conditions, the neuroprotective effect of rasagiline was dose-dependent: rasagiline reducing OGD-induced cell death by 68% and 80% at 100 nM and 1 microM, respectively. The neuroprotective effect of rasagiline was also observed when added after the OGD insult (55% reduction in cell death). Under rasagiline treatment, there was a lesser decrease in ATP content in cultures exposed to OGD compared with that in untreated cultures. OGD followed by reoxygenation resulted in a several fold increase in PGE(2) release into the extracellular medium. Rasagiline (100 nM-1 microM) markedly inhibited OGD-induced PGE(2) release. Clorgyline, a monoamine oxidase-A (MAO-A) inhibitor, did not protect NGF-differentiated PC12 cells against OGD-induced cell death. As NGF-differentiated PC12 cells contain exclusively MAO type A, these data suggest that the neuroprotective effect of rasagiline under OGD conditions is independent of MAO inhibition.

Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers and Mash-1-dependence.

Enteric and sympathetic neurons have previously been proposed to be lineally related. We present independent lines of evidence that suggest that enteric neurons arise from at least two lineages, only one of which expresses markers in common with sympathoadrenal cells. In the rat, sympathoadrenal markers are expressed, in the same order as in sympathetic neurons, by a subset of enteric neuronal precursors, which also transiently express tyrosine hydroxylase. If this precursor pool is eliminated in vitro by complement-mediated lysis, enteric neurons continue to develop; however, none of these are serotonergic. In the mouse, the Mash-1-/- mutation, which eliminates sympathetic neurons, also prevents the development of enteric serotonergic neurons. Other enteric neuronal populations, however, including those that contain calcitonin gene related peptide are present. Enteric tyrosine hydroxylase-containing cells co-express Mash-1 and are eliminated by the Mash-1-/- mutation, consistent with the idea that in the mouse, as in the rat, these precursors generate serotonergic neurons. Serotonergic neurons are generated early in development, while calcitonin gene related peptide-containing enteric neurons are generated much later. These data suggest that enteric neurons are derived from at least two progenitor lineages. One transiently expresses sympathoadrenal markers, is Mash-1-dependent, and generates early-born enteric neurons, some of which are serotonergic. The other is Mash-1-independent, does not express sympathoadrenal markers, and generates late-born enteric neurons, some of which contain calcitonin gene related peptide.

Vimentin immunoreactive glial cells in the fish optic nerve: implications for regeneration.

The poor regenerative ability of neurons of the central nervous system in mammals, as compared with their counterpart in fish or amphibians, is thought to stem from differences in their immediate nonneuronal environment and its response to axonal injury. We describe one aspect of the environmental response to axonal injury in a spontaneously regenerating system--the fish optic nerve. The aspect under investigation was the reaction of glial cells at the injury site. This was examined by the use of antibodies that specifically recognize vimentin in fish glial cells. In the present study, affinity-purified vimentin antibodies were raised against a nonconserved N-terminal 14-amino acid peptide, which was predicted from the nucleotide sequence of vimentin. These antibodies were found to react specifically with glial cells in vitro. Moreover, the antivimentin antibodies stained both the optic nerve and the optic tract, but with different patterns. Specificity of the antibodies was verified by protein immunoblotting, tissue distribution, and labeling patterns. After injury, vimentin immunoreactivity initially disappeared from the site of the lesion due to cell death. Early signs of glial cell migration toward the injury site were evident a few days later. It is suggested that the reappearance of vimentin-positive glial cells at the site of injury is associated with axonal elongation across it, and that they contribute to the regenerative ability of the fish optic nerve.

Axonal regeneration is associated with glial migration: comparison between the injured optic nerves of fish and rats.

The central nervous systems of mammals and fish differ significantly in their ability to regenerate. Central nervous system axons in the fish readily regenerate after injury, while in mammals they begin to elongate but their growth is aborted at the site of injury, an area previously shown to contain no glial cells. In the present study we compared the ability of glial cells to migrate and thus to repopulate the injured area in fish and rats, and used light and electron microscopy in an attempt to correlate such migration with the ability of axons to traverse this area. One week after the optic nerve was crushed, both axonal and glial responses to injury were similar in fish and rat. In both species glial cells were absent in the injured area (indicated by the disappearance of glial fibrillary acidic protein and vimentin immunoreactive cells from the site of injury in rat and fish, respectively), while at the same time axonal growth, indicated by expression of the growth-associated protein GAP-43, was restricted to the proximal part of the nerve. In fish, 2 weeks after the crush, GAP-43 staining (i.e., growing axons) was seen at the site of injury, in association with migrating vimentin-positive glial cells. One week later the site of injury in the fish optic nerve was repopulated by vimentin-positive glial cells, and GAP-43-positive axons had already traversed the site of injury and reached the distal part of the nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Disappearance of astrocytes and invasion of macrophages following crush injury of adult rodent optic nerves: implications for regeneration.

Injury to the mammalian central nervous system results in loss of function because of its inability to regenerate. It has been postulated that some axons in the mammalian central nervous system have the ability to regenerate but fail to do so because of the inhospitable nature of surrounding glial cells. For example, mature oligodendrocytes were shown to inhibit axonal growth, and astrocytes were shown to form scar tissue that is nonsupportive for growth. In the present study we report an additional phenomenon which might explain the failure of axons to elongate across the site of the injury, namely, the absence of astrocytes from the crush site between the glial scar and the distal stump. Astrocytes began to disappear from the injury site as early as 2 days after the injury. After 1 week the site was necrotic and contained very few glial cells and numerous macrophages. Disappearance of glial cells was demonstrated in both rabbit and rat optic nerves by light microscopy, using antibodies directed against glial fibrillary acidic protein, and by transmission electron microscopy. Results are discussed with reference to possible implications of the long-lasting absence of astrocytes from the injury site, especially in view of the differences between the present findings in rodents and our recent observations in fish.

L1 immunoreactivity in the developing fish visual system.

Previous studies have suggested that L1, the cell adhesion molecule, is present in the regenerating fish optic nerve. The present study was undertaken in order to determine whether L1 is expressed by fish neurons and specifically by non-myelinated axons, using fish retinal explants in vitro and the developing fish visual system in vivo. In vitro, the nonmyelinated axons emerging from retinal explants showed L1 immunoreactivity and in vivo, L1 immunoreactive sites were found to be associated with areas rich in non-myelinated axons. At embryonic stage 23, as the eye developed and optic nerve axons began to elongate towards the tectum, L1-like immunoreactivity was seen both in optic nerve and in plexiform layers of the retina. At this stage and until hatching, the cellular layers within the retina showed little or no staining relative to the layers containing axons or dendrites. After hatching, L1 immunoreactivity was also observed in the ganglion cell layer, but upon maturation both the retina and the optic nerve lost most of their L1 immunoreactivity. We therefore suggest that non-myelinated axons of the fish visual system express L1 during development, which is lost after myelination and presumably reappears during regeneration.

Isolation and sequence analysis of two intermediate filament cDNA clones from fish optic nerve.

The high post-traumatic regenerative ability of fish central nervous system has been partially attributed to the hospitable nature of the surrounding non-neuronal cells and their appropriate response to injury. Uncovering the correlation between fish non-neuronal cell structure and behavior might yield a better understanding of what makes them supportive to axonal growth. Towards this goal, structural proteins expressed by fish non-neuronal cells need to be characterized. In the present study we isolated cDNA clones encoding fish intermediate filaments which are prominent structural proteins in astrocytes. Among the isolated clones, one was identified as fish vimentin and another was found identical to the cloned fish keratin 8. Results are discussed with respect to the use of these cDNAs for further understanding of fish non-neuronal cell plasticity.

Glial fibrillary acidic protein in the fish optic nerve.

The intermediate filament glial fibrillary acidic protein (GFAP) is the predominant cytoskeletal protein of mature glial cells in the mammalian nervous system. The nervous systems of lower vertebrates, such as fish, have been examined for the presence of GFAP and several investigators have shown that goldfish (Carassius auratus) brain contains GFAP-positive astrocytes. The same studies have demonstrated that, in contrast to the brain, the optic nerve of goldfish did not show any GFAP immunoreactivity, suggesting that this intermediate filament protein is not expressed in fish optic nerve astrocytes. The present study shows, however, that the monoclonal antibodies to porcine GFAP react with the optic nerve of carp (Cyprinus carpio), another member of the goldfish family. These antibodies to porcine GFAP cross react with rat brain and carp optic nerve, yielding a band of approximately 52 kDa in both species. Northern blot analysis using mouse GFAP DNA probe revealed that carp optic nerve RNA contains two transcripts of 2.3 and 2.1 kb, which hybridize with the mouse GFAP probe. Injury to the carp optic nerve was followed by a decrease of GFAP immunoreactivity from neural tissue and a strong expression around blood vessels and connective tissues. On the basis of these observations and within the limitation of the techniques it is reasonable to conclude that the carp optic nerve expresses GFAP immunoreactivity and that the pattern of expression of this intermediate filament protein is altered after injury. Such an alteration might be relevant to the process of regeneration.

Immunological evidence that the neural adhesion molecule L1 is expressed in fish brain and optic nerve: possible association with optic nerve regeneration.

In the mammalian peripheral nervous system (PNS), expression of the neural adhesion molecule L1 on Schwann cells and neurons has been correlated with axonal growth during development and regeneration. The present study was undertaken to examine whether a similar correlation exists between a lesion-induced increase of L1 expression and regenerative capacity in the central nervous system (CNS). The fish optic nerve was used as a model for a successfully regenerating region of the CNS. Immunochemical and immunohistological experiments carried out with immunoaffinity purified polyclonal antibodies, generated against L1 from mouse brain, showed that carp optic nerve and brain, but not liver, contained L1 immunoreactivity. Western blot analysis of brain tissue yielded one distinct band at 200 kDa, while a double band at 200 kDa and two low-molecular weight bands at 120 and 100 kDa, possibly degradation products, were seen in the optic nerve. Immunohistological examination of normal optic nerves revealed L1 immunoreactivity, predominantly associated with connective tissue boundaries of nerve fascicles and with blood vessels, as well as inside axonal fascicles. L1 immunoreactivity was increased by 25%, 8 days after crushing of the optic nerve, as determined by radioimmunoassay on a nerve segment distal to the site of injury and compared with untreated control nerves. Increased levels of L1 were also seen by immunohistology and found to be predominantly associated, as in the normal nerve, with connective tissue boundaries and blood vessels. These observations suggest that a lesion-induced increase in L1 expression in the fish optic nerve is associated with axonal regrowth in the CNS.

Biochemical and biomechanical properties of avian callus after local administration of dihydroxylated vitamin D metabolites.

In vitamin D-fed chicks 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were implanted into experimentally-produced fractures of the mid-tibia. The mechanical and biochemical properties of the tibia were evaluated for two weeks, including torsion tests, measurement of alkaline phosphatase activity, 45Ca incorporation, and Ca2+ content. Both dihydroxylated metabolites of vitamin D3 had a direct effect on endochondral bone formation. 24,25(OH)2D3 strengthened the callus, and raised alkaline phosphate activity in the first seven days after fracture. 1,25(OH)2D3 decreased the strength of the callus concomitant with a reduction in 45Ca incorporation. It is suggested that local application of 24,25(OH)2D3 into fractures may accelerate healing and prevent non-union.

Responses of rachitic cartilage cells to metabolites of vitamin D3.

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.