Cooperative effects of gangliosides on trophic factor-induced neuronal cell recovery and synaptogenesis: studies in rodents and subhuman primates.
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Biomedical subjects
D Maysinger
Publications and source records attributed to D Maysinger.
Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its low-affinity receptor in cerebellum.
Deficits or excesses of thyroid hormones during critical periods of mammalian cerebellar development can lead to profound biochemical and morphological abnormalities in this system. The goal of this study was to investigate the effects of perinatal hypo- and hyperthyroidism on the ontogeny of nerve growth factor (NGF) and its low-affinity receptor (p75NGFR) in the rat cerebellum. The concentration of NGF and of p75NGFR immunoreactivity (IR) were measured, several days after birth, in cerebella of rats which had received propylthiouracil (PTU) or thyroxine. NGF concentration was markedly enhanced only on postnatal day 2 (P2) in hyperthyroid rats, whereas in hypothyroid (PTU-treated) rats NGF values were similar to age-matched controls. These observations suggest that thyroid hormone affects NGF synthesis during early periods of cerebellar development. In Purkinje cells of control animals, p75NGFR IR peaked at P10. In hypothyroid rats, the expression of p75NGFR was retarded, peaking at P15, whereas in hyperthyroid rats it was advanced, peaking at P8. The increased p75NGFR IR found in Purkinje cell bodies and the delayed disappearance of p75NGFR IR from the external granular layer of hypothyroid rats suggest different roles for thyroid hormone in the developing cerebellum. We conclude that p75NGFR and NGF are independently regulated by thyroid hormone during critical periods of cerebellar development. The effect of thyroid hormone deficiency on p75NGFR content in Purkinje cells may involve complex mechanisms such as impaired efficiency of axonal transport.
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Spreading depression induces c-fos-like immunoreactivity and NGF mRNA in the rat cerebral cortex.
Application of potassium chloride (KCl) to the brain surface elicits spreading depression which leads to a marked induction of the proto-oncogene c-fos in the treated cerebral cortex at the earliest time examined (90 min). High levels of c-fos immunoreactivity are observed up to 6 h after KCl treatment. The areas affected include the cingulate, entorhinal and frontoparietal cortex throughout the treated hemisphere. The c-fos expression preceded an increase in both NGFmRNA and NGF-like protein(s). A maximal increase in c-fos was detected within 3 h, whereas NGFmRNA peaked at 12 h and NGF-like protein(s) reached their maximum level 24 h after KCl application. The most prominent increase in NGFmRNA was measured in the entorhinal cortex (50-fold), but other cortical areas also showed a moderate increase of 2-3-fold. In conclusion, our results provide evidence that increases in c-fos and NGF expression are early adaptive responses following brain injury.
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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.
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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)
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Induction of c-FOS immunoreactivity in the hippocampus following potassium stimulation.
In microdialysis procedures high potassium ion concentrations are generally used to induce neurotransmitter release. However, the widespread effects, if any, of such a treatment have not been described. In order to establish a possible link between c-fos expression and stimulating conditions for neurotransmitter release in microdialysis procedures we administered KCl (100 mM) into the hippocampus. Proto-oncogene c-FOS-like immunoreactivity is upregulated in granule cells of the dentate gyrus, pyramidal cells of the hippocampus, cingulate, piriform and frontoparietal cortices at 2 h, but not 24 h after K+ administration. Neither implantation of the probes nor perfusion with artificial cerebrospinal fluid resulted in similar patterns of c-FOS immunoreactivity. In addition, we investigated whether the impairment of the cholinergic septohippocampal pathway would modify the K(+)-induced expression of the immediate early gene c-fos in the hippocampus. The expression of c-fos induced by KCl was not altered in the animals with fimbria-fornix lesion despite the marked decrease in acetylcholine release in the hippocampus. Glutamate concentrations measured in the same superfusates showed that a significantly greater glutamate release occurs in denervated hippocampi. Furthermore, abolishment of seizure-like activity (induced by KCl) in anesthetized animals did not alter expression of c-FOS immunoreactivity in the K(+)-stimulated hippocampi. The results from these studies confirm that most of the releasable acetylcholine of the hippocampus is linked to the fimbrial input and may suggest that c-FOS upregulation in this model does not respond to any cholinergic input from the medial septum via the fimbria-fornix.
Acetylcholine↗
Primate nucleus basalis of Meynert p75NGFR-containing cholinergic neurons are protected from retrograde degeneration by the ganglioside GM1.
The effects of unilateral devascularizing lesions of the neocortex in primates (Cercopithecus aethiops) on the immunoreactivity of choline acetyltransferase and the low-affinity nerve growth factor receptor (p75NGFR) were investigated in cell bodies of the nucleus basalis of Meynert. Choline acetyltransferase enzymatic activity was measured in the dissected ipsi- and contralateral nucleus basalis of Meynert as well as in the remaining cortex adjacent to the lesion. Cortically lesioned animals displayed a shrinkage of p75NGFR-immunoreactive cholinergic cell bodies in only the intermediate portion of the nucleus basalis of Meynert as well as a depletion of choline acetyltransferase activity in this cellular complex. In contrast, cortically lesioned monkeys treated with monosialoganglioside did not reveal a significant loss of choline acetyltransferase activity or shrinkage of nucleus basalis of Meynert cholinergic neurons, but rather a modest hypertrophy. These results are discussed in relation to a possible use of putative trophic agents in the repair of the damaged central nervous system.
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Long-term protective effects of human recombinant nerve growth factor and monosialoganglioside GM1 treatment on primate nucleus basalis cholinergic neurons after neocortical infarction.
Neocortical infarction induces biochemical and morphological retrograde degenerative changes in cholinergic neurons of the rat nucleus basalis magnocellularis [Sofroniew et al. (1983) Brain Res. 289, 370-374]. In the present study, this lesion model has been reproduced in the non-human primate (Cercopithecus aethiops) to investigate whether degenerative changes affecting the cortex surrounding the lesioned area and the ipsilateral basal forebrain are prevented by the early administration of recombinant human nerve growth factor alone or in combination with the monosialoganglioside GM1. Six months after surgery and treatment, the monkeys were processed either for biochemistry (choline acetyltransferase assay) or immunocytochemistry. In lesioned vehicle-treated animals, choline acetyltransferase activity significantly decreased by 28% in the cortex surrounding the injured area and by 31% in the ipsilateral nucleus basalis of Meynert when compared with values of sham-operated monkeys. These biochemical changes were fully prevented with the administration of nerve growth factor alone or in combination with the monosialoganglioside GM1. The morphometrical analysis revealed a significant shrinkage of cholinergic neurons (61 +/- 1.4% of sham-operated cell size) and loss of neuritic processes (59 +/- 10% of sham-operated values) within the intermediate nucleus basalis region of lesioned vehicle-treated animals. Although a protection of the cholinergic cell bodies within the nucleus basalis was found with both treatments, a significant recovery of the neuritic processes (84 +/- 7.2% of sham-operated values) was assessed only in the double-treated monkeys. These results indicate that the early administration of nerve growth factor alone or in combination with the monosialoganglioside GM1 induces a long-term protective effect on the nucleus basalis cholinergic neurons in cortical injured non-human primates.
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Effects of coencapsulated NGF and GM1 in rats with cortical lesions.
Nerve growth factor (NGF) and monosialoganglioside GM1 were microencapsulated in biodegradable co-polymer poly(L-lactide):co-glycolide and administered locally to rats with unilateral cortical devascularizing lesions. Microcapsules were placed directly onto the lesioned cortical surface and rats were sacrificed 30 days post-operatively. Biochemical and quantitative immunocytochemical analyses revealed effective protection from degenerative changes of nucleus basalis magnocellularis (NBM) cholinergic neurones on the lesioned side as well as a significant increase in choline acetyltransferase activity in the frontoparietal cortex surrounding the lesion. Results of this study strongly suggest that the topical application of microcapsules of a biodegradable polymer containing a mixture of two neuroprotective factors is a viable alternative to the use of osmotic minipumps for delivery of these agents into the CNS. Indeed, this approach might find clinical applications.
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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↗
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↗
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.
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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.
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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↗
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.
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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↗
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↗
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.
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