Processes of peripheral nerve and neuromuscular repair.
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Animals↗
Biomedical subjects
G Toffano
Publications and source records attributed to G Toffano.
Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: I. Effects of cell density and of an adult mammalian striatal-derived neuronotrophic factor (SDNF).
The use of CNS cultures for detection and quantification of neuronotrophic activity in the CNS has been analyzed. In particular the development, i.e., neurotransmitter uptake characteristics, and survival of dopaminergic and GABAergic neurons in fetal mouse (E13)-dissociated mesencephalic cells cultured in serum-free, hormone-supplemented medium have been assessed as a function of culture time and cell density. At all times, more than 98% of the cells were classified as neurons on the basis of immunocytochemical criteria. Results indicate that the increase of cell density in vitro significantly enhances specific high-affinity dopamine uptake per dopaminergic cell and cell survival. This effect is not limited to the dopaminergic cells and suggests that the development of neurotransmitter-related traits and cell survival are influenced by cell density-derived trophic signals. The above-mentioned cultures and parameters have also been used to detect neuronotrophic activity in adult mammalian brain extracts or more purified preparations. In particular, bovine striatal extracts contain activity capable of increasing high-affinity neurotransmitter uptake parameters and cell survival of at least the dopaminergic and GABAergic neurons present in the culture system. The neuronotrophic activity from bovine striatum has been partially purified and is associated with a fraction whose main component is a basic protein of approximately 14 kDa.
Animals↗
Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: II. Modulatory effects of gangliosides.
This paper analyzes the effects of exogenously supplied GM1 on the development, i.e., specific neurotransmitter uptake capability and survival, of the dopaminergic neurons present in fetal mouse-dissociated mesencephalic cells. Exogenous GM1, but not asialo-GM1, sialic acid, or the oligosaccharide chain of GM1, enhances in a time- and concentration-dependent manner the specific 3H-dopamine uptake (increase of the apparent Vmax and decrease of the apparent Km value) and the long-term survival of the dopaminergic neurons. The GM1 effects on the behavior of the dopaminergic neurons require the presence of cell-derived neuronotrophic influences present within the culture system and are associated with an increase in the response of the cells to the trophic influences. GM1 effects are not limited to dopaminergic neurons, and depend on the stable association of the ganglioside molecule with the cells. It is suggested that GM1 is not a trophic agent per se, but rather potentiates neuronotrophic activities and/or exerts independent influences to which neurons respond only if appropriately supported.
Animals↗
Lysophosphatidylserine-dependent interaction between rat leukocytes and mast cells.
The production of lysophosphatidylserine has been studied in a population of rat peritoneal cells; 67% polymorphonuclear and 33% mononuclear leukocytes. Pulse-chase experiments with L-[U-14C]serine reveal a net lysophosphatidylserine production of 0.33 nmol/mg protein in 2 h of incubation. The source of lysophosphatidylserine is probably the phosphatidylserine of cells damaged during the incubation, since plasma membrane fragments obtained from the leukocytes yield higher lysophosphatidylserine production (1.9 nmol/mg protein in 1 h of incubation). Both leukocytes and plasma membranes show phosphatidylserine splitting activity when tested with vesicles of this phospholipid. In the presence of albumin a fraction of produced lysophosphatidylserine is recovered in the incubation medium. Under these conditions efficient incorporation of lysoderivative into surrounding leukocytes and conversion to phosphatidylserine requires cell activation by tetradecanoylphorbol acetate. In agreement with radiochemical data it is found that a suspension of leukocytes elicits histamine release when rat peritoneal mast cells and nerve growth factor are subsequently added. This typical, lysophosphatidylserine-dependent mast cell response is retained when leukocyte plasma membranes substitute the whole cells. These results suggest that leukocyte lysis at sites of tissue injury results in the production of a sufficient amount of lysophosphatidylserine to reach and activate surrounding mast cells.
Animals↗
Effect of lysophosphatidylserine on immunological histamine release.
The effect of lysophosphatidylserine on immunological histamine release has been studied in rat peritoneal mast cells actively sensitized with horse serum and in human basophils challenged with anti-IgE. In contrast to other lysophospholipids, lysophosphatidylserine enhances the immunological histamine release in rat mast cells. The effect shows the kinetics of a saturable process with an apparent Km for lysophosphatidylserine of 0.26 microM. A similar Km value (0.21 microM) is found when measuring the non-immunological histamine release activated by lysophosphatidylserine plus nerve growth factor. A comparison with phosphatidylserine shows that a half-maximal response to lysophosphatidylserine occurs at a concentration 4-times lower. In addition, the magnitude of the response is higher. At variance with rat mast cells, lysophosphatidylserine does not influence the histamine release elicited by immunological and non-immunological stimuli in human basophils. The histamine secretion in these cells is instead affected by a calcium ionophore or tetradecanoylphorbolacetate, a compound producing activation of protein kinase C.
Animals↗
Developmental and age-related changes in D1-dopamine receptors and dopamine content in the rat striatum.
The relationship between the postnatal development of dopaminergic (DAergic) nerve endings and the maturation of D1 DA receptors in the rat striatum was analyzed by measuring the content of DA and dihydroxyphenylacetic acid (DOPAC), two biochemical markers of DAergic nerve terminal proliferation, and the ontogenetic changes in [3H]SCH 23390 binding sites. DA-stimulated adenylate cyclase (AC) activity was also measured in order to characterize the coupling of [3H]SCH 23390 binding sites to the responses mediated by the activation of D1 DA receptors. Striatal levels of DA and DOPAC, as well as the density and affinity of [3H]SCH 23390 binding sites and DA-stimulated AC activity were also measured in senescent rats. The striatal content of DA increased slowly after birth, reaching adult levels by postnatal day 60 and remaining constant through adulthood and senescence (up to 20 months of age). The density of [3H]SCH 23390 binding sites increased 14-fold from birth to postnatal day 35, when a peak value was reached, whereas a significant decrease was observed in the striatum of aged rats. In contrast, the affinity of D1 DA receptors for [3H]SCH 23390 remained unchanged from birth through senescence. The stimulation of cyclic AMP formation induced by 100 microM DA increased 4-fold from birth to postnatal day 14, when the maximal responsiveness to DA was observed and then returned to adult levels. No significant alterations were observed in the Km values during development, whereas the stimulatory effect of 100 microM DA on AC activity was significantly decreased in senescent rats.(ABSTRACT TRUNCATED AT 250 WORDS)
3,4-Dihydroxyphenylacetic Acid↗
Differential effect of ganglioside GM1 on rat brain phosphoproteins: potentiation and inhibition of protein phosphorylation regulated by calcium/calmodulin and calcium/phospholipid-dependent protein kinases.
The monosialoganglioside GM1 displays complex effects on protein phosphorylation of rat cerebral cortex membrane preparations. The exogenous ganglioside at a concentration of 350 microM in absence of calcium only stimulated the phosphorylation of a protein of MW = 64,000. In presence of 1 mM calcium a twofold effect is observed irrespective of the phosphoprotein considered. In particular there is an enhancement of 32P incorporation in four major phosphoproteins of MW = 160,000, 140,000, 64,000 and 50,000 in presence of GM1 compared with that observed with calcium alone. The maximal stimulating effect is achieved with a ganglioside concentration of 35 microM. This effect is inhibited by the addition of 100 microM trifluoperazine (TFP), a phenothiazine known to inhibit calmodulin and protein kinase-C activities. These four proteins represent the major substrates for the calcium/calmodulin-dependent protein kinase with the MW = 64,000 and 50,000 proteins co-migrating with the autophosphorylated subunits of this enzyme. In addition, the ganglioside inhibited the phosphorylation of three proteins with MW = 86,000, 20,000 and 14,000. The electrophoretic properties of these phosphoproteins are similar to the autophosphorylated form of protein kinase-C and to the rat myelin basic proteins, respectively. The effect of the ganglioside on their phosphorylation is not influenced by TFP. Finally, a protein with an apparent molecular weight of 46,000 shows also an increased phosphorylation in presence of GM1. The reported results indicate that exogenous GM1 can have profound effects on different kinases such as the calcium/calmodulin dependent protein kinase, the protein kinase-C and also some unknown calcium-independent protein kinases.
Animals↗
Age-dependent changes in the mechanisms controlling prolactin secretion and phosphatidylinositol turnover in male rats: effect of phosphatidylserine.
The present study was undertaken in order to better characterize the functional state of anterior pituitary gland in young and old rats by using prolactin secretion and incorporation of radioactive phosphate into phosphatidylinositol (PI) as markers. The in vitro incorporation of radiolabeled phosphate into anterior pituitary PI was significantly (p less than 0.01) greater in young (3-5 months) than in aged (24-25 months) male Sprague-Dawley rats. No significant difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Also, the extent of prolactin secretion from isolated pituitary was significantly greater in young than in aged rats, while the prolactin pituitary content was significantly higher in aged animals. In vitro dopamine (DA) decreased the incorporation of 32P into PI, both in young and old pituitary glands, and inhibited prolactin secretion into the incubation medium. Brain cortex-phosphatidylserine (BC-PS), a pharmacologically active purified phospholipid, capable of stimulating the dopaminergic system in the hypothalamus and of decreasing prolactin secretion both in humans and rats in vitro and in vivo, inhibited the incorporation of labeled phosphate into PI of pituitary glands from either young or old rats, but did not alter the prolactin secretion from the glands incubated in vitro. Baseline prolactin plasma levels did not differ significantly between young and old rats either when blood was collected from the trunk after decapitation or underwent sampling from chronically cannulated rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
Reparative processes in aged brain.
Endogenous neurotrophic factors have been proposed to play an important role in degenerative diseases and aging. In aged rats, neurotrophic activity induced by partial lesion of nigro-striatal pathways seems to be lower compared to young rats when assayed in vitro on cultured mesencephalic dissociated neuronal cells. In parallel experiments, we could also observe an age-dependent delay in spontaneous recovery of striatal tyrosine hydroxylase activity after lesion. Both experimental evidences suggest a low-potential neuroplasticity in aged rats. Nevertheless, the aging brain seems to be responsive to pharmacological manipulation. Long-term phosphatidylserine treatment has been shown to maintain the integrity of neuronal structures altered by the aging process. Putative mechanisms of action underlying these effects are presented.
Aging↗
Activity of phosphatidylserine on memory retrieval and on exploration in mice.
Recent human and animal studies have reported about neurochemical and behavioral changes occurring during aging. Bovine brain phosphatidylserine (BC-PS) administered in vivo was reported to produce changes in the metabolic status of the brain and in behavioral performances. Within the Albino-Swiss strain of mice two subtypes have been shown to exist with different learning and memory retention abilities. These animals also have different reactions to various psychoactive drugs. The experiments presented here deal with the hypothesis that BC-PS may show different effects according to the starting emotional status of the animals. The technique of intracerebral injection was employed to ensure active concentrations of BC-PS in the brain. The results reported here show that BC-PS clearly improves performances in the subtype of mice with poor exploratory activity.
Administration, Oral↗
The effect of GM1 ganglioside on coerulospinal, noradrenergic, adult neurons and on fetal monoaminergic neurons transplanted into the transected spinal cord of the adult rat.
GM1 ganglioside, thyroxine and hydrocortisone were tested for their ability to improve the survival and growth of fetal locus coeruleus noradrenergic neurons in the transected, adult spinal cord. GM1 alone was also tested for its effect on fetal mesencephalic dopaminergic neurons implanted into a small dorsolateral cavity at the L2 region of the cord previously transected at the T9-T10 region. None of the substances tested had any measurable effect on either of the fetal implants. However, in the GM1- and thyroxine-treated animals the somatic dendrites of the axotomized, noradrenergic, coerulospinal neurons appeared more robust, and more intensely fluorescent, compared to their appropriate controls. GM1 also caused a pronounced sprouting of the axotomized monoaminergic (catecholaminergic and serotonergic) fibres in the rostral region of the cord adjacent to the transection site. All of the mesencephalic dopaminergic implants survived in both the GM1-treated animals and their saline-injected controls. However, their development was apparently not influenced by GM1. The results indicate that GM1 and thyroxine can enhance those aspects of the reactive mechanisms of mature, axotomized, noradrenergic coerulospinal neurons that promote their regeneration. As such, GM1 could become a useful tool in current attempts to foster the regeneration of damaged monoaminergic neurons in the mammalian CNS.
Adrenergic Fibers↗
Intracerebral quinolinic acid injection in the rat: effects on dopaminergic neurons.
Intrastriatal infusion of the endogenous excitotoxin quinolinic acid (QUIN) leads to the degeneration of neuronal cell bodies around the injection site. Dopaminergic afferents not only survive the toxic insult but react by increasing their activity in the acute and subacute phases following the injection of QUIN. Measurements of the tissue concentrations of acidic dopamine metabolites, and determinations of L-DOPA accumulation after DOPA-decarboxylase inhibition, indicate an increased dopamine turnover within 90 min after the administration of 50 micrograms QUIN. At the later timepoints examined (6 h, 4 and 11 days after QUIN), dopaminergic parameters are increased in the injected striatum only while no changes can be detected in the homolateral substantia nigra. Local norepinephrine levels are elevated 4 and 11 days after an intrastriatal QUIN injection but remain unchanged at distant sites or earlier postinjection periods. The acute increase in nigrostriatal activity may be mediated by an excessively stimulated, yet functional striatonigral feedback loop whereas subsequent changes represent local reactions of dopaminergic nerve terminals secondary to neuronal degeneration in the striatum. In accordance with this interpretation, no monoaminergic changes can be observed in the hypothalamus 4 days following the local injection of 50 micrograms QUIN, a dose which does not cause neuronal necrosis in this brain area. These data are concordant with, and are discussed in the context of, a possible involvement of QUIN in the pathogenesis of Huntington's disease.
Animals↗
Reversal of scopolamine-induced amnesia by phosphatidylserine in rats.
Scopolamine (2 mg/kg IP) and propranolol (55 mg/kg IP), given before a single learning trial, reduce retention of a passive avoidance response in rats. Phosphatidylserine, 30-60 mg/kg IP, antagonizes the amnesic effect of scopolamine but not that of propranolol. The retention of the passive avoidance response is not affected by phosphatidylserine given alone. The results indicate that this phospholipid selectively counteracts the action of scopolamine on passive avoidance acquisition, probably via a cholinergic mechanism.
Amnesia↗
Phospholipids in inflammatory synovial effusions.
The concentration of phospholipids and proteins was determined in 23 inflammatory synovial fluids obtained from human knee joints. The synovial fluid to plasma phospholipid ratio (0.48 and 0.37 at high and low inflammatory state) was lower than the value found for the total protein content (0.68 and 0.53, respectively) indicating that phospholipids were more discriminated than proteins in their transfer from plasma to the synovial space. Constant amounts of phosphatidylinositol were found in all synovial fluids, whereas trace amounts of lysophosphatidylethanolamine and phosphatidylserine were more frequent in the active inflammatory state. A decrease in the relative amounts of phosphatidylcholine and phosphatidylinositol with respect to plasma suggested the possibility of phospholipid hydrolysis in the synovial compartment. In agreement, determinations of phospholipase activity disclosed the presence of a phospholipase A2 in the fluid phase of synovial effusions. Phospholipid derivatives formed in the synovial space may thus contribute to the amplification of the inflammatory response.
Arthritis↗
Characterization of macrophages elicited by intraperitoneal injection of hyaluronate.
Hyaluronate of 120,000 molecular weight has been injected in the peritoneal cavity of mice to study its effect on migration of inflammatory cells in vivo. After one day a dose-dependent granulocyte migration is observed. Three days later the number of granulocytes is greatly reduced and macrophages form about half of the total cell population. Hyaluronate-elicited macrophages show a decreased 5'-nucleotidase and an increased acid phosphatase activity as compared to resident macrophages. The production of superoxide anion in response to the phorbol ester tetradecanoyl-phorbolacetate, and the phagocytic activity are also enhanced. Macrophages elicited by hyaluronate secrete growth factor(s) for non-lymphoid mesenchymal cells. It is concluded that hyaluronate in vivo stimulates the migration of inflammatory cells, thus causing the recruitment of a population of stimulating macrophages. These effects may explain previous reports on the acceleration of wound healing by hyaluronate.
5'-Nucleotidase↗
Inhibition of protein kinase activity by apomorphine.
The effect of apomorphine (1-20 microM) on protein kinase activity was studied in extracts from rat peritoneal mast cells and brain tissue. Apomorphine inhibited the cyclic AMP-dependent and the calcium-plus phosphatidylserine-dependent protein kinase activity with an IC50 between 1 and 6 microM, depending on the tissue and on the protein kinase involved. This effect might explain previous results on the apomorphine-induced inhibition of histamine release in rat peritoneal mast cells.
Animals↗
Phorbol 12-myristate 13-acetate induces prolactin secretion from rat anterior pituitary gland by the activation of protein kinase-C.
The purpose of this study was to characterize the action of phorbol 12-myristate 13-acetate (PMA), a tumor-promoting agent, on rat anterior pituitary gland, focusing the attention on prolactin secretion. PMA elicited a significant increase in prolactin secretion without affecting phosphatidylinositol turnover, considered as an early post-receptor event controlling PRL secretion. However incubation of anterior pituitary glands with PMA caused a loss of protein kinase-C activity in cytoplasm concomitant with an increased enzyme activity in the membrane. The action of PMA on prolactin secretion seems to be mainly dependent from the redistribution and activation of protein kinase-C. In fact, the phorbol ester did not affect pituitary cAMP and cGMP metabolism either in basal conditions or after theophylline.
Animals↗
Central nervous system repair and nerve cell cultures.
Is anatomical and functional repair following injury or disease in the adult brain and spinal cord possible, and to what extent? Current evidence concerning neuronal plasticity of mature neurons suggests that opportunities may exist provided the variables are understood. Highly informative are the cellular and molecular processes regulated, in the mature CNS, by growth-promoting molecules directed to neurons. In this field neuronal cell culture methodology has proven to be indispensable and its application may in the future contribute significantly to the development of novel therapeutical strategies.
Animals↗