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Biomedical subjects

D Maysinger

Publications and source records attributed to D Maysinger.

At least 19 recordsLinked to original sources
PubMed1995

Developmental deficiency of the cholinergic system in congenitally hyperammonemic spf mice: effect of acetyl-L-carnitine.

The sparse-fur (spf) mutant mouse has an X-linked deficiency of hepatic ornithine transcarbamylase (OTC) and develops hyperammonemia in the postnatal period similar to that seen in human patients. We studied the effect of congenital hyperammonemia on the development of cerebral cholinergic parameters such as choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and high-affinity choline uptake (HACU) in spf mice. The serum ammonia levels of spf mutant mice were significantly elevated after weaning compared with control animals. ChAT activity levels started decreasing in mutant spf mice from the age of 30 days (i.e., immediately after weaning); it reached significantly lower levels in the adult animals. HACU was consistently lower (P < .01) in spf/Y mice compared with controls up to the adult stage. However, there were no marked changes in the activity of AChE between control and hyperammonemic spf mice. The levels of beta-NGF, which is essential for cholinergic differentiation and function, were significantly lower in different brain regions of adult mutant mice compared with normal controls. A treatment of spf/spf breeding females with acetyl-L-carnitine, at a dose of 1.5 mM in drinking water, starting from day 1 of conception, resulted in a significant restoration of ChAT activity levels in some brain regions of the spf/Y offspring. The beta-NGF levels were also significantly elevated after supplementation with ALCAR in mutant mice compared with untreated mutant mice. These data are suggestive of a neurotrophic property of ALCAR during cholinergic deficiency caused by congenital hyperammonemia.

Acetylcarnitine↗
PubMed1994

In vitro effects of brain derived neurotrophic factor released from microspheres.

The principal objective of these studies was to microencapsulate brain derived neurotrophic factor (BDNF) and to test its biological effects in vitro. Two types of microspheres with BDNF were prepared using biodegradable polymers: chitosan and poly-(L-lactic acid):poly-(L-glycolic acid). Depending on the procedure, sizes ranged from 1 micron to 1 mm as determined by an image analysis system. The biological assays confirmed that the released BDNF remains biologically active in vitro. Microencapsulation of BDNF and other trophic factors may represent a viable alternative to currently employed delivery systems in vivo and holds potential for eventual therapy of neurodegenerative disorders, in particular for peripheral neuropathies and CNS disorders where supplementation of neurotrophins retards or even prevents neural degeneration.

Animals↗
PubMed1994

Neocortical infarction in subhuman primates leads to restricted morphological damage of the cholinergic neurons in the nucleus basalis of Meynert.

The aim of the present study was to investigate the long-term effect of cortical infarction on the subhuman primate (Cercopithecus aethiops) basal forebrain. The lesion, carried out by cauterizing the pial blood vessels supplying the left fronto-parieto-temporal neocortex, induced retrograde degenerative processes within the ipsilateral nucleus basalis of Meynert. The morphometrical analysis revealed that significant shrinkage of cholinergic neurons and loss of neuritic processes were localized within the intermediate regions of the nucleus basalis. The average cross-sectional areas of choline acetyltransferase-immunoreactive neurons in the intermedio-ventral (Ch4iv) and intermedio-dorsal (Ch4id) nucleus basalis were decreased to 62.5 +/- 9.5 and 58.0 +/- 8.6%, respectively, of the sham-operated values. Although an apparent loss of Nissl-stained magnocellular neurons in Ch4iv and Ch4id was found by applying a quantitative analysis based on a perikaryal-size criterion, data obtained by the quantification of immunostained material failed to reveal any significant decrease of cholinergic cell density. Results are discussed in view of future application of this ischemic model to study processes of retrograde degeneration following cortical target removal and to assess potential neurotrophic and neuroprotective properties of pharmacologic agents.

Animals↗
PubMed1994

Encapsulated genetically engineered fibroblasts: release of nerve growth factor and effects in vivo on recovery of cholinergic markers after devascularizing cortical lesions.

Genetically engineered rat fibroblasts producing nerve growth factor (NGF) were encapsulated in alginate-polylysine-alginate gels with the objective to produce viable "minifactories" continuously producing and secreting NGF into the rat brain. Microencapsulated fibroblasts (NGF secretors and NGF non-secretors) were placed onto the surface of the lesioned rat cortex (unilateral devascularizing lesion) and animals were sacrificed 30 days after surgery. Fibroblasts NGF-non secreters normally produce tumors after implantation, therefore, they were irradiated prior to encapsulation. Three other experimental groups were studied in parallel: non-lesioned (controls), lesioned rats receiving "empty" alginate spheres and lesioned animals without treatment and microspheres. Biochemical analysis of microdissected brain tissues of lesioned animals treated with encapsulated NGF-secretor fibroblasts showed a significant increase in choline acetyltransferase (ChAT) activity in cortices adjacent to the lesion but not far from it (entorhinal cortex). This may indicate a gradient of concentration of the released NGF and/or differential responsivity of lesioned vs non-lesioned target tissue. ChAT enzymatic activity in the microdissected nucleus basalis magnocellularis (NBM) was significantly decreased (P < 0.05) in all lesioned animals treated with spheres without fibroblasts and those with fibroblasts not secreting NGF. Morphometric analysis of ChAT-IR and low affinity NGF-receptor IR cholinergic neurons in the middle portion of the NBM shows a prevention of neuronal shrinkage and extensive neuropil in animals treated with microencapsulated NGF-secretor fibroblasts. The results of this study demonstrate that NGF from encapsulated genetically engineered fibroblasts can be secreted for at least long enough to prevent degenerative changes of cholinergic neurons in the NBM.

Alginates↗
PubMed1994

Chemiluminometric determination of choline-related substances in pharmaceutical preparations by dot-blot.

A simple and reliable method of assaying succinylcholine chloride, oxtriphylline (choline theophyllinate) and acetycholine chloride in pharmaceutical preparations, based on conversion to choline, is provided by combination of chemiluminometry of choline and dot-blot technique. Recoveries of 102 and 97% for choline chloride in Quelicin injection and Choledyl 200 tablets matrices, respectively, and of 99% for acetylcholine chloride in Miochol solution matrix could be achieved using simple choline chloride standards in phosphate buffer pH 8.6. Accordingly, matrix-matched standards were found redundant. Favourable results obtained in preparations-matched media including limits of detection of 37-39 pmol microliters-1 of choline chloride, together with accuracies of 0-2% and RSDs ranging from 4 to 7% are the further evidence of the suitability of the method.

Acetylcholine↗
PubMed1993

Microencapsulation of genetically engineered fibroblasts secreting nerve growth factor.

We demonstrated that genetically modified fibroblasts can be encapsulated into biocompatible, biodegradable spheres retaining their viability and capacity to continuously secrete nerve growth factor (NGF) for at least two months. Genetically engineered rat fibroblasts producing NGF were encapsulated in an alginate-polylysine gel with the ultimate objective of improving transplantation methodologies. Cultures were suspended in a sodium alginate solution and the suspension was extruded drop-wise into a solution of calcium chloride. Morphological properties of the spheres were assessed by light and electron microscopy. The spheres had a homogenous external membrane, without fibroblasts, protruding from the surface of the capsular membrane. The NGF determinations in culture media showed that encapsulated fibroblasts continued to synthesize NGF for at least 60 days. We also confirmed that secreted NGF was biologically active, by assessing the induction of choline acetyltransferase (ChAT) activity in dissociated embryonic rat septal cultures. These results encourage further studies using in vivo models to determine the value of applying microencapsulated genetically modified cells secreting trophic factors as a therapeutic strategy for central nervous system (CNS) injuries.

Animals↗
PubMed1993

Effects of acidic fibroblast growth factor on cholinergic neurons of nucleus basalis magnocellularis and in a spatial memory task following cortical devascularization.

The ability of acidic fibroblast growth factor to elicit a trophic response in the nervous system of the rat was tested in vitro and in vivo. Treatment of cultured septal cells with acidic fibroblast growth factor resulted in an elongation of glial processes as assessed by immunostaining for glial fibrillary acidic protein. Increased choline acetyltransferase was also observed. The responses to acidic fibroblast growth factor in vivo were studied in rats trained in a spatial memory task, using the Morris water maze. Randomly selected animals were subjected to unilateral cortical devascularization. This lesion results in partial unilateral infarction of the neocortex, and in retrograde degeneration of the nucleus basalis magnocellularis. Animals were tested post-lesion for memory retention and were then killed for morphological studies. Intracerebroventricular administration of acidic fibroblast growth factor (0.6 microgram/h for seven days starting at surgery) prevented the lesion-induced impairment in this test, and reduced the nucleus basalis magnocellularis cholinergic degeneration, as assessed by morphometric choline acetyltransferase-like immunoreactivity and radioenzymatic assay for choline acetyltransferase activity. The preservation of the phenotype of injured cholinergic neurons of the nucleus basalis magnocellularis by acidic fibroblast growth factor was indicated by the maintenance of the cross-sectional area of cell bodies and mean length of neuritic processes one month after surgery. The effect of acidic fibroblast growth factor in non-cholinergic cells remains to be investigated. It is suggested that acidic fibroblast growth factor may alleviate the lesion-induced deficit in the memory retention task by preventing disruption of functional connections between nucleus basalis magnocellularis and intact cortical areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Microencapsulated nerve growth factor: effects on the forebrain neurons following devascularizing cortical lesions.

In this study, we report the effects of nerve growth factor (NGF) delivered into the CNS via a novel delivery system for prolonged, controlled release. The effectiveness of NGF incorporated in the biodegradable microspheres was investigated in the rat model for central cholinergic degeneration. Mature male rats were unilaterally lesioned by disruption of the pia arachnoid vessels and vehicle (alginate microspheres without NGF) and microencapsulated NGF was placed at the site of the lesion. Choline acetyltransferase (ChAT) activity was measured in the nucleus basalis magnocellularis (NBM) and cortex in the (a) non-lesioned control animals; (b) lesioned animals treated with 'empty' microspheres and (c) lesioned animals treated with microspheres containing NGF, 30 days following surgery. Similarly lesioned animals received NGF via permanently installed cannulae in order to compare the novel route of administration with the more conventional one. Immunocytochemical results showed an absence of the cholinergic cell body shrinkage in the NBM otherwise observed in lesioned animals. Furthermore, an increase in intensity of ChAT immunostaining in NGF-treated, lesioned animals was evident. The present results stress the experimental therapeutic possibilities of novel delivery systems for administration of trophic factors in the CNS.

Alginates↗
PubMed1992

Effects of nerve growth factor on cortical and striatal acetylcholine and dopamine release in rats with cortical devascularizing lesions.

The effects of intraventricular nerve growth factor (NGF) or saline treatments on extracellular acetylcholine (ACh), dopamine (DA) and adenosine (Ade) levels in the cortex and striatum of rats with unilateral devascularizing cortical lesions were studied in vivo with microdialysis. The devascularizing cortical lesion produced a decrease in extracellular ACh levels in both cortex and striatum as compared to those in normal rats, while the NGF treatment produced a significant increase in ACh levels in both regions. NGF could even increase cortical ACh levels in normal rats. The cortical lesion produced a decrease in extracellular DA in the cortex, while the NGF treatment appeared to reverse this effect. No significant changes in DA were observed in the striatum. The present study gives evidence that a unilateral cortical devascularizing lesion leads to changes in extracellular ACh and DA levels in cortex and striatum and that these changes could be reversed with intraventricular NGF treatment.

Acetylcholine↗
PubMed1992

Trophic factor effects on cholinergic innervation in the cerebral cortex of the adult rat brain.

The cholinergic pathway ascending from the nucleus basalis magnocellularis (NBM) to the cortex has been implicated in several important higher brain functions such as learning and memory. Following infarction of the frontoparietal cortical area in the rat, a retrograde atrophy of cholinergic cell bodies and fiber networks occurs in the basalocortical cholinergic system. We have observed that neuronal atrophy in the NBM induced by this lesion can be prevented by intracerebroventricular administration of exogenous nerve growth factor (NGF) or the monosialoganglioside GM1. In addition, these agents can upregulate levels of cortical choline acetyltransferase (ChAT) activity in the remaining cortex adjacent to the lesion site. Furthermore, an enhancement in cortical high-affinity 3H-choline uptake and a sustained in vivo release of cortical acetylcholine (ACh) after K+ stimulation are also observed after the application of neurotrophic agents. Moreover, these biochemical changes in the cortex are accompanied by an anatomical remodeling of cortical ChAT-immunoreactive fibers and their synaptic boutons.

Animals↗
PubMed1992

Inhibition of high affinity choline uptake in the rat brain by neurotoxins: effect of monosialoganglioside GM1.

Mustard derivatives of ethyl-choline and hemicholinium-3 have been suggested as possible specific cholinergic neurotoxins. In this study a structural analog of hemicholinium-3, a,a'-bis[di(2-chloroethyl)amino]-4,4'-2-biacetophenone (toxin 7), was added to synaptosomes prepared from the cortex, striatum or hippocampus of rat brain. Synaptosomal high affinity choline uptake (HACU) was significantly decreased in a dose-dependent manner by addition of toxin 7, while synaptosomal uptake of GABA or dopamine was not changed. Incubation of cortical synaptosomes with the monosialoganglioside GM1 prevented the decrease in HACU seen following administration of toxin 7. This preventative effect of GM1 was greater if GM1 was added prior to or concomitant with toxin 7, than if GM1 was added following toxin 7. Two newly synthesized hemicholinium-3 analogs, 4-[3'-di(2-chloroethyl)aminopropionyl]biphenyl (toxin 5) and 4-[3'-di(2-bromoethyl)aminopropionyl]biphenyl (toxin 6) caused a large decrease in HACU when added to cortical synaptosomes, this decrease was significantly greater than that seen with the same dose of toxin 7 or ethyl-choline aziridinium (AF64A). Ultrastructural changes in the synaptosomal membrane following incubation with toxin 7 or toxin 7 with GM1 were examined by electron microscopy. Development of a compound which is both a potent neurotoxin, and is specific for cholinergic neurons will allow new insights into the normal function of the cholinergic system in the CNS and provide animal models of disease states in which cholinergic degeneration is an important element.

Animals↗
PubMed1992

Grafting of genetically modified cells: effects of acetylcholine release in vivo.

In this study, microdialysis was used to investigate functional recovery of central cholinergic neurons in the forebrain of rats with cortical devascularizing lesions. Mature male rats were unilaterally lesioned by disruption of the pia arachnoid vessels and genetically modified fibroblasts secreting nerve growth factor (NGF) were placed at the site of the lesion. One month following surgery, microdialysis probes were installed in the remaining cortex and were perfused with artificial cerebrospinal fluid (csf) containing neostigmine (5 nM) and/or KCl (100 mM). The basal (non-stimulated) release of acetylcholine (ACh) in the cortex was similar in all experimental groups, whereas KCl stimulated release of ACh was significantly augmented (P < 0.05) in the ipsilateral remaining cortex in lesioned animals that have been implanted with fibroblasts secreting NGF. These results suggest that NGF secreted by genetically engineered fibroblasts modulates neuroplasticity in the adult mammalian CNS and may favour recovery of cortical function following injury.

Acetylcholine↗
PubMed1992

Recovery of nucleus basalis cholinergic neurons by grafting NGF secretor fibroblasts.

The effects of nerve growth factor (NGF) secreted from genetically modified fibroblasts was studied in vitro, using dissociated septal cells and in vivo, in rats bearing unilateral cortical devascularizing lesions. Transfected fibroblasts expressing nerve growth factor (NGF) were co-cultured with rat embryonic cholinergic cells of the septal region. This in vitro system showed that NGF secretor cells produce biologically active NGF, as determined by increasing choline acetyltransferase (ChAT) activity in septal culture after seven days. The potential therapeutic value of applying grafts of transfected fibroblasts expressing NGF in the model of retrograde atrophy of cholinergic neurons of the nucleus basalis magnocellularis (NBM) was assessed following partial devascularizing lesions of the cerebral cortex. We observed an increase in ChAT activity in the remaining cortex and a partial protection of the ipsilateral NBM, as determined by morphometric and biochemical studies.

Animals↗
PubMed1990

Effects of treatment with microencapsulated monosialoganglioside GM1 on cortical and striatal acetylcholine release in rats with cortical devascularizing lesions.

The present study shows a novel administration form of the monoganglioside GM1, which following microencapsulation in human serum albumin was topically applied on cortical regions damaged by devascularization in rats. The effects of microencapsulated GM1 on extracellular levels of acetylcholine, choline and dopamine in the cortex and in the striatum were analyzed using in vivo microdialysis. Cholinergic neurons in the nucleus basalis magnocellularis were studied immunohistochemically using monoclonal antibodies raised against choline acetyltransferase (ChAT). It was found that cortical devascularizing lesions produced a decrease in extracellular levels of cortical acetylcholine and choline, and retrograde morphological changes in cholinergic neurons in the nucleus basalis magnocellularis. GM1 promoted (1) recovery of the retrograde morphological changes produced by the decortication in the nucleus basalis magnocellularis and (2) a parallel increase in cortical acetylcholine release. No changes were observed in the striatum, nor on cortical or striatal dopamine levels simultaneously measured in the same perfusates.

Acetylcholine↗
PubMed1990

Acetylcholine release in vivo: effects of chronic treatment with monosialoganglioside GM1.

Rats with unilateral cortical devascularizing lesions were treated with the monosialoganglioside GM1 in two different ways. One group of animals received GM1 (5 mg/kg/day, for 7 days), through a permanent cannula implanted intracerebroventricularly (i.c.v.) and connected to an osmotic minipump. The other group was treated with microencapsulated GM1 placed directly onto the surface of the lesioned cortex. The effect of GM1, administered into the lateral ventricle and supracortically, on the release of ACh in vivo was studied, using a microdialysis system combined with sensitive high performance liquid chromatography (HPLC). The release of acetylcholine and choline was studied in the cortex and striatum of the rat under nonstimulated (basal) and KCl (100 mM)-stimulating conditions. The non-stimulated release of acetylcholine was only measurable in the presence of neostigmine and was found to be about 30 microM in the cortex and approximately 10 times greater in the striatum. A large concentration of KCl led to a remarkable increase of acetylcholine in the control (C) and vehicle-treated lesioned groups (V i.c.v., V cap: 11-13 fold), but was greater in the GM1-treated groups (GM1 i.c.v., GM1 cap: 20-25 fold). In contrast, KCl-stimulated release of ACh in striata from GM1-treated lesioned groups was significantly less (5-10 fold), compared to the unlesioned controls (C: 16 fold) and lesioned vehicle-treated rats (V i.c.v. and V cap: 16-18 fold). The release of choline was not increased significantly by large concentrations of KCl present in the perfusion medium. In all experimental groups, and in both structures of the brain, ratios between stimulated and non-stimulated release of choline was between 0.7-1.2.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1990

Three-dimensional reconstruction and quantitative evaluation of devascularizing cortical lesions in the rat.

The use of a reconstruction method suitable for three-dimensional (3-D) representations of the cortex for the quantitative evaluation of unilateral devascularizing cortical lesions is reported, demonstrating that 3-D reconstructions can be used for quantitative evaluation of the extent of cortical lesions, in addition to simply visualizing shapes. The procedure allowed the definition of precise quantitative parameters characterizing the volumes and surface areas of the cortical regions involved. It was shown that digitized serial sections from atlases can be utilized for multiple correlation studies by superimposing morphological and biochemical parameters. More specifically, the enzymatic activity of choline acetyltransferase (ChAT) in the nucleus basalis magnocellularis (NBM), and morphological parameters (number and cross-sectional areas) of the ChAT-positive immunoreactive cholinergic neurons, were related to the numerical values for the volume and surface area of the cortical lesions. The applicability of this method goes beyond the scope illustrated in this study. For example, various morphological anomalies in the brain could be quantified; changes in various cortical and subcortical structures could be followed during development; and the 3-D size, shape and position of a graft could be related to the number of viable foetal cells.

Animals↗