Biomedical subjects

S Ambrosio

Publications and source records attributed to S Ambrosio.

49 records · Page 3Linked to original sources

PubMed1992

HPLC analysis of hexosamine phosphates in biological samples.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.

Animals↗

PubMed1992

Neuronal changes in the nigrostriatal pathway of 1-methyl-4-phenylpyridine-treated mice.

Thirty young-adult mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 30 mg/kg/day for 2 days and sacrificed 24 hours later in order to determine striatal catecholamines and to study morphological changes in the nigrostriatal pathway. Immunohistological techniques were also used with polyclonal antibodies for glial fibrillary acidic protein (GFAP) and tyrosine hydroxylase (TH), and monoclonal antibodies for two subunits of neurofilaments. Silver impregnation demonstrated conspicuous neuronal changes affecting cellular processes from substantia nigra in all treated mice. Terminal and axonal degeneration were also observed in striata. These changes were associated with a moderate to marked gliosis. The TH immunoreactivity was normal in cell bodies of substantia nigra but was decreased in striata from MPTP-treated mice. These data indicate that in mice the deterioration of dendritic and axonal neuropil may constitute a significant causal factor of MPTP neurotoxicity.

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

PubMed1991

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat brain.

The concentration of fructose 2,6-bisphosphate in the brain remained stable during starvation and early stages of ischaemia, but decreased in diabetes or after lengthened ischaemia. 6-Phosphofructo-1-kinase activity was also decreased in diabetic and ischaemic animals, whereas 6-phosphofructo-2-kinase was not modified. The concentration of the bisphosphorylated metabolite seems to be remarkably constant under a wide variety of experimental conditions, suggesting that it plays an essential role in the basal activation of 6-phosphofructo-1-kinase. Purified 6-phosphofructo-2-kinase also showed fructose-2,6-bisphosphatase activity with an activity ratio similar to that of the purified heart isoenzyme. The brain enzyme also has a net charge similar to that of the heart isoenzyme. Its activity is not modified by sn-glycerol 3-phosphate, and it is more sensitive to citrate than the liver or muscle isoenzyme. Moreover, the enzyme from brain, similarly to that from heart and muscle, is not modified by the cyclic AMP-dependent protein kinase or protein kinase C. A near-full-length cDNA probe from liver hybridized with RNA from brain and heart. In both cases, a major band of 6.8 kb of RNA and a minor one of 4 kb of RNA were detected. All these properties support the hypothesis that brain contains a different isoenzymic form from that of liver and muscle, and it is probably related to the heart isoform.

Animals↗

PubMed1991

Fructose 2,6-bisphosphate in hypoglycemic rat brain.

Fructose 2,6-bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6-bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6-bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6-bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6-phosphofructo-2-kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6-phosphate). These results suggest that fructose 2,6-bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6-phosphofructo-1-kinase and glycolysis.

Animals↗

PubMed1991

Extracellular levels of adenosine and its metabolites in the striatum of awake rats: inhibition of uptake and metabolism.

The level of purines in the striatum of awake, freely moving rats was studied using microdialysis. The calculated extracellular concentration of adenosine and its metabolites inosine and hypoxanthine was very high immediately after implantation of the dialysis probe but decreased within 24 h to a level which remained stable for two days. Using in vitro calibration to determine the relative recovery of the dialysis probes we estimated resting levels in the striatal extracellular space to be 40, 110 and 580 nM, respectively. Inhibition of adenosine deaminase by deoxycoformycin produced a significant 1.4-fold increase in extracellular adenosine levels and a fall in inosine and hypoxanthine. A combination of three uptake blockers (dipyridamole, lidoflazine and nitrobenzylthioinosine), caused a 4.5-fold increase in extracellular adenosine levels without any change in inosine or hypoxanthine levels. After uptake inhibition deoxycoformycin did not have any significant effect. The present results show that the microdialysis technique can be used to determine levels of purines in the extracellular fluid of defined brain regions in awake animals. The high levels recorded during the first several hours after implantation may be artefactually high and reflect trauma. The results also show that adenosine levels can be altered in vivo by inhibitors of adenosine transport and adenosine deaminase. The present results indicate that the physiological adenosine level in striatal extracellular space is in the range 40-460 nM.

Adenosine↗

PubMed1989

Acute effects of 1-methyl-1,4-phenylpyridinium ion (MPP+) on purine metabolism in rat striatum studied in vivo using the microdialysis technique.

In order to obtain further insight into the interactions between the purinergic and dopaminergic pathways in the striatum, we studied both metabolisms simultaneously, using a microdialysis technique in 1-methyl-1,4-phenylpyridinium ion (MPP+) unilaterally-denervated conscious rats. In these rats the contralateral side was used as control. The perfusates were collected every 20-25 min using 4 mm dialysis probes, implanted in each striatum, and assayed for dopamine and purine metabolites. After MPP+ administration, all adenosine metabolites - with the exception of uric acid - and dopamine levels were significantly increased in the extracellular medium. However, the time-course change in dopamine level did not correlate with the adenosine and inosine time-courses, suggesting a different mechanism of liberation in response to MPP+ administration.

1-Methyl-4-phenylpyridinium↗

PubMed1989

Dopamine D1 and D2 receptors visualized in MPTP treated C57 mice by in vitro autoradiography: lack of evidence of receptor modifications in parkinsonian mice.

The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on D1 and D2 dopamine receptors was assayed using in vitro quantitative autoradiography. D1 receptor subtype was labeled using 1 nM of 3H-SCH 23390 and D2 receptor subtype was labeled using 0.4 nM of 3H-spiroperidol. The results are compared to the effect of MPTP on the striatal levels of dopamine and its metabolites, in BL C57 mice. While 2 and 5 doses of MPTP 30 mg/kg/day intraperitoneally reduced the content of striatal dopamine and its metabolites, no modifications were detected in D2 receptor subtype in any cerebral area studied. However, D1 receptors were reduced in the substantia nigra 24 hr after the last of 2 doses, but not later. We suggest a compensatory mechanism of the surviving dopaminergic neurones as well as the participation of spare receptors. This would explain the lack of receptor modification after the lesion obtained as seen by the striatal reduction of dopamine and metabolites content, after MPTP administration.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1989

Acute peripheral catecholaminergic changes in rat after MPTP and MPP+ treatment.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its main metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the peripheral catecholaminergic system of the rat were investigated. MPTP and MPP+ injections (20 mg/kg i.p.) caused a marked acute depletion of heart noradrenaline, up to 75% twelve hours after the administration, and a decrease of adrenal gland adrenaline. The time-course of the effect of MPTP and MPP+ is reported, together with a decrease in the tyrosine hydroxylase activity after MPTP treatment, more evident in the adrenal glands. Pargyline (50 mg/kg i.p.) is not able to prevent such a neurotoxic peripheral effect.

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

PubMed1988

Acute effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on catecholamines in heart, adrenal gland, retina and caudate nucleus of the cat.

The short-term (24 h) effects of 10 mg/kg of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on cat catecholamine levels of heart, adrenal gland, retina, and caudate nucleus were studied. Significant differences in catecholamines, including adrenomedullar adrenaline, heart noradrenaline, and retinal dopamine, were observed. No differences were found in caudate nucleus dopamine. In this organ, the levels of dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were significantly lower after MPTP treatment. Biochemical changes caused by MPTP with respect to its effects on the peripheral catecholaminergic organs are discussed.

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

PubMed1987

Peripheral effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its main metabolite 1-methyl-4-phenylpyridinium ion (MPP+) in the rat.

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its main metabolite 1-methyl-4-phenyl-pyridinium ion (MPP+) on catecholamine concentrations was tested in some peripheral organs of the rat. Acute injection of either compound caused a marked depletion of heart noradrenaline (NA). MPP+ had more effect than the parent compound. MPTP had no effect on adrenal catecholamines while its metabolite was effective, but only after several hours. Repeated MPTP and MPP+ injections (20 mg/kg X 5) resulted in marked depletion of heart NA and adrenal gland adrenaline. Clear recovery was observed in both organs 10 days after treatment.

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

PubMed1987

Different toxicity of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) on the nigrostriatal and mesolimbic pathways.

The influence of MPTP (30 mg/kg X 2) on dopamine content and tyrosine hydroxylase activity in striatal, limbic and cortical areas of C-57/B16 mice was studied. General dopamine depletion was observed 24 h after the treatment, but 30 days after administration recovery was complete in all but the striatum. At 24 h, tyrosine hydroxylase activity was decreased only in striatum. It is proposed that MPTP acts differently in striatum and in other dopaminergic areas.

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

PubMedDate not supplied

Plasticity of the nigrostriatal system in MPTP-treated mice. A biochemical and morphological correlation.

In order to compare the recovery capacity of the nigrostriatal system between adult and old mice, MPTP hydrochloride was administered to 48 BL/C57 male mice, which were sacrificed 24 h or 10 d after the second dose. The animals were divided into four groups, based on age (adult or old) and moment of sacrifice (24 h or 10 d). The detailed morphology of the neurons and the cellular processes of the substantia nigra pars compacta and the striatum were studied using the Golgi method. Immunostaining with a polyclonal glial fibrillary acidic protein antiserum using the peroxidase-antiperoxidase technique was performed to study the glial response. Striatal catecholamines were determined to correlate the biochemical data with the morphological changes. Significant neuronal changes of cellular processes were observed in substantia nigra pars compacta from all MPTP-treated mice, consisting of swelling and distortion of cellular bodies, discontinuous thickness, and nodulations of dendrites with baded aspect. Axons showing focal swelling and nodulations were also found in the neuropil of silver impregnated striata. Marked gliosis with reactive astrocytes in substantia nigra and striatum from all the old treated mice was found. Recovery was only observed in adult mice sacrificed 10 d after withdrawal. At this time, all the old MPTP-treated mice showed marked neuronal changes and a persistent marked gliosis. As expected, 24 h after the MPTP treatment, a marked depletion of dopamine and its metabolites was found in all the animals; at 10 d, the depletion was partially reversed in the adult group. These data correlate well with the observed morphological changes. Our results suggest that, in mice, deterioration of dendritic and axonal neuropil constitutes a significant causal factor of the MPTP neurotoxicity. These features are related to the age of the animals and the integrity of the plasticity phenomena, which appear to be altered in old mice.

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

PubMedDate not supplied

Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor.

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.

3T3 Cells↗