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R M Lindsay

Publications and source records attributed to R M Lindsay.

At least 19 recordsLinked to original sources
PubMed1997

The effect of delta-gluconolactone, an oxidised analogue of glucose, on the nonenzymatic glycation of human and rat haemoglobin.

Nonenzymatic glycation of proteins and oxidative stress are considered independent factors important in the development of the complications of diabetes but may be interrelated by the process of autoxidative glycation. This pathway involves monosaccharide autoxidation to a reactive ketoaldehyde analogue and subsequent reaction with protein to form a ketoimine adduct. This study demonstrates that delta-gluconolactone (delta-GL), an oxidised analogue of glucose, is a potent glycating agent in vitro of haemoglobin present in blood samples from insulin-dependent diabetic and non-diabetic human subjects and from spontaneously diabetic, insulin-dependent BB/Edinburgh (BB/E) rats. The percentage glycated haemoglobin after incubation (37 degrees C, 5 h) with delta-GL (25 mmol/l) was significantly (P < 0.002) higher than that observed using an equimolar concentration of glucose. Intravenous administration of delta-GL (1 g/kg) to non-diabetic BB/E rats also significantly increased glycation of haemoglobin (6.0 +/- 0.1% vs 4.9 +/- 0.1%, P < 0.01) whereas intravenous injection of an identical dose of glucose had no significant effect (5.1 +/- 0.1% vs 5.0 +/- 0.2%). These results support the hypothesis that nonenzymatic glycation of proteins involves attachment by both native and oxidised monosaccharides. Further investigation of the interactions between diabetes-associated increases in oxidative stress and glycation on the development and progression of the vascular complications of diabetes is necessary.

Animals↗
PubMed1997

Endogenous NGF and CNTF levels in human peripheral nerve injury.

Nerve growth factor (NGF) is trophic to sensory and sympathetic fibres, and ciliary neurotrophic factor (CNTF) to motoneurones, in animal models of peripheral nerve injury: NGF excess produces hyperalgesia. In this first study of injured human nerves and sensory ganglia, we quantified and localized endogenous NGF and CNTF in 59 neonate and adult patients with brachial plexus and peripheral nerve injury. NGF levels were generally depleted in injured nerves, but relatively preserved acutely in nerve segments distal to injury. NGF immunostaining was observed in Schwann cells in distal nerve segments with pockets of high levels in some neuromas. CNTF levels and immunostaining in Schwann cells were markedly decreased distally within days of injury. We propose that early local administration of NGF and CNTF-like agents may help prevent degenerative changes in injured nerves, while at later stages local anti-NGF treatment (e.g. of some neuromas) may ameliorate chronic pain.

Adolescent↗
PubMed1997

Hepatocyte growth factor promotes motor neuron survival and synergizes with ciliary neurotrophic factor.

Hepatocyte growth factor (HGF) has been shown to function as a potent mitogen for a variety of cells, transducing its signal through the c-met tyrosine kinase receptor. Ciliary neurotrophic factor (CNTF) is a cytokine that has been shown to promote survival of motor neurons. We show here that c-met mRNA is present in the embryonic rat spinal cord. Peak expression of c-met (at E14) coincides with the period of naturally occurring cell death in motor neurons, suggesting a possible role of HGF in the regulation of this process. Utilizing a neuron-enriched culture system, we established that HGF, like CNTF, stimulates choline acetyltransferase (CAT) activity in motor neurons. When co-administered to motor neuron cultures, saturating concentrations of HGF and CNTF produced a synergistic increase in CAT levels. We show that this synergy reflects enhanced motor neuron survival. Exposure of motor neuron cultures to the cytostatic agent vincristine markedly decreased CAT levels; co-treatment with HGF and CNTF (but not either factor alone) restored CAT activity to control levels. Our findings indicate that HGF is a survival factor for motor neurons, that it acts synergistically with CNTF, and that HGF and CNTF can together be neuroprotective in the face of vincristine toxicity.

Animals↗
PubMed1997

Neurotrophin-3 administration attenuates deficits of pyridoxine-induced large-fiber sensory neuropathy.

Chronic treatment of adult rats for 2-3 weeks with high doses of pyridoxine (vitamin B6) produced a profound proprioceptive loss, similar to that found in humans overdosed with this vitamin or treated with the chemotherapeutic agent cisplatin. Pyridoxine toxicity was manifest as deficits in simple and precise locomotion and sensory nerve function and as degeneration of large-diameter/large-fiber spinal sensory neurons. As assessed quantitatively in a beam-walking task and by EMG recording of H waves evoked by peripheral nerve stimulation, coadministration of the neurotrophic factor neurotrophin-3 (NT-3; 5-20 mg . kg-1 . d-1, s.c.) during chronic pyridoxine treatment largely attenuated the behavioral and electrophysiological sequelae associated with pyridoxine toxicity. Furthermore, NT-3 administration prevented degeneration of sensory fibers in the dorsal column of the spinal cord. These data are consistent with the evidence that NT-3 is a target-derived neurotrophic factor for muscle sensory afferents and suggest that pharmacological doses of NT-3 may be beneficial in the treatment of large-fiber sensory neuropathies.

Animals↗
PubMed1997

Effects of BDNF infusion on the regulation of TrkB protein and message in adult rat brain.

Exposure of embryonic CNS neurons to BDNF in vitro causes down-regulation of TrkB protein and mRNA, and an attenuation of functional responses to acute neurotrophin stimulation. In order to investigate ligand-mediated regulation of TrkB in vivo, we infused BDNF into the midbrain, near the periaquaductal grey-dorsal raphe (PAG-DR), or into the olfactory bulb of adult rats. Midbrain infusion of BDNF produced analgesia that was sustained for the duration of BDNF delivery. Analysis of TrkB receptor levels revealed that at the point when the maximal analgesic effect of BDNF was obtained, there was a concommitant 75% decrease in full-length TrkB protein at the infusion site. After discontinuation of infusion, levels of TrkB recovered toward base line. Interestingly, TrkB protein levels were not accompanied by decreased trkB mRNA levels. To determine if BDNF infusion decreased TrkB protein levels in other brain areas and whether trkB mRNA might be down-regulated in the cell bodies of neurons projecting to the infusion site, we infused BDNF into the olfactory bulb. Following a 12-day infusion of BDNF, TrkB protein levels decreased within the bulb to a similar extent as in the PAG-DR. This decrease in receptor protein, however, was not accompanied by decreased trkB mRNA levels in the olfactory cortex, which is afferent to the bulb. Taken together, our data suggest that decreases in TrkB receptor protein at the site of BDNF infusions in the adult brain represent receptor turnover, but this is not associated with altered expression of trkB mRNA or attenuation of the pharmacological effects of BDNF.

Age Factors↗
PubMed1997

Antidepressant-like effect of brain-derived neurotrophic factor (BDNF).

Previous studies have shown that infusion of brain-derived neurotrophic factor (BDNF) into the midbrain, near the PAG and dorsal/median raphe nuclei, produced analgesia and increased activity in monoaminergic systems. Alterations in monoaminergic activity have also been implicated in the pathogenesis and treatment of depression. The present studies examined the ability of centrally administered BDNF to produce antidepressant-like activity in two animal models of depression, learned helplessness following exposure to inescapable shock and the forced swim test. In the learned helplessness paradigm, vehicle-infused rats pre-exposed to inescapable shock (veh/shock) showed severe impairments in escape behavior during subsequent conditioned avoidance trials, including a 47% decrease in the number of escapes and a 5 fold increase in escape latency, as compared to vehicle-infused rats which received no pre-shock treatment (veh/no shock). Midbrain BDNF infusion (12-24 micrograms/day) reversed these deficits, and in fact, BDNF-infused rats pre-exposed to inescapable shock (BDNF/shock) showed escape latencies similar to veh/no shock and BDNF/no shock rats. In the forced swim test, BDNF infusion decreased the immobility time by 70% as compared to vehicle-infused controls. Non-specific increases in activity could not account for these effects since general locomotor activity of BDNF- and vehicle-infused animals was not different. These findings demonstrate an antidepressant-like property of BDNF in two animal models of depression, which may be mediated by increased activity in monoaminergic systems.

Animals↗
PubMed1997

Effects of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 on the laminar distribution of transganglionically fransported choleragenoid in the spinal cord dorsal horn following transection of the sciatic nerve in the adult rat.

Spinal cord projections from transected sciatic nerves treated with different neurotrophins were investigated in the adult rat following injections of choleragenoid into the proximal stump of the injured nerve. Transganglionically transported choleragenoid labelled primary afferent fibres in all spinal cord dorsal horn laminae except the outer part of lamina II (II(o)), which is almost devoid of labelling. Transection of the sciatic nerve, however, resulted in intense transganglionic choleragenoid labelling in lamina II(o) and in lamina I. In this study, the sciatic nerve was transected bilaterally and 4erve growth factor (6 or 24 microg), brain-derived neurotrophic factor (20 microg), neurotrophin-3 (27 microg) or cytochrome C (8 microg; control substance) was applied unilaterally during postoperative survival times of eight, 16 and 32 days. The animals received bilateral injections of choleragenoid into the injured nerve two days before they were killed. The effect of the axotomy and neurotrophin treatment was evaluated by analysing the extent of choleragenoid and substance P immunoreactivity in the somatotopically appropriate spinal cord dorsal horn regions. At eight days' postoperative survival, laminae I and II(o) on the transected, non-treated side showed much more intense choleragenoid-like immunoreactivity compared to the contralateral transected, nerve growth factor-treated (6 and 24 microg) side. A similar situation was also found in cases treated with the higher dose (24 microg) at 16 days but to a lesser degree when the lower (6 microg) dose was used. After 32 days' survival, there was no detectable side difference in the choleragenoid labelling pattern. At 16 days' survival, the mean area of choleragenoid-positive ganglion cell body profiles in the L5 dorsal root ganglion of the transected, non-treated side was significantly smaller than the mean area of the transected, nerve growth factor-treated (24 microg) neurons. An axotomy-induced depletion of substance P-like immunoreactivity was seen from eight days' survival and onwards, whereas on the nerve growth factor-treated side a clearcut substance P depletion was not observed until 32 days. Brain-derived neurotrophic factor, neurotrophin-3 and cytochrome C had no detectable effects on the distribution of choleragenoid labelling or substance P-like immunoreactivity in the dorsal horn following sciatic nerve transection. In conclusion, peripheral nerve injury-induced expansion of primary afferent choleragenoid labelling in the spinal cord dorsal horn is counteracted by treating the axotomized nerve with nerve growth factor.

Animals↗
PubMed1997

In vivo and in vitro evidence of altered nitric oxide metabolism in the spontaneously diabetic, insulin-dependent BB/Edinburgh rat.

1. Altered vasoreactivity may contribute significantly to the pathogenesis of diabetic vascular complications. This study investigated the effect of (a) insulin-related diabetes, and (b) chronic in vivo administration of N(omega)-nitro-L-arginine ester (L-NAME), a nitric oxide (NO) synthase inhibitor, on mean arterial pressure and in vitro vascular reactivity to noradrenaline in mesenteric arterial bed preparations from spontaneously diabetic, insulin-dependent and treated BB rats, the best animal model of insulin-dependent mellitus (IDDM) currently available. Four groups of animals from the Edinburgh colony (BB/E) of spontaneous diabetic BB rats were studied: age-matched (mean +/- s.e. mean = 156 +/- 2d) non-diabetic (glycated haemoglobin = 3.8 +/- 0.1%) and insulin-treated diabetic (glycated haemoglobin = 6.2 +/- 0.5%; duration of diabetes = 56 +/- 4 d) groups were either L-NAME treated (oral dose = 27 +/- 1 mg kg-1 d-1; duration of treatment from 30 until 153 days of age) or untreated. Although our diabetic BB/E rats do not achieve overall normoglycaemia, individual adjustment of the daily insulin dose administered to every diabetic rat achieves better glycaemic control than previous groups studying altered vascular reactivity and endothelial dysfunction in this animal model of diabetes. 2. Mean arterial pressure (measured directly via indwelling carotid arterial cannulae) was not significantly different between non-diabetic (116 +/- 3 mmHg; n = 10) and diabetic (122 +/- 2 mmHg; n = 12) BB/E rats. L-NAME treatment significantly (P < 0.001) increased mean arterial pressure in both groups (165 +/- 6 mmHg; n = 9 and 142 +/- 4 mmHg; n = 6 respectively) but the degree of hypertension observed in L-NAME-treated diabetic rats was significantly (P < 0.01) attenuated compared to non-diabetic rats treated with L-NAME. 3. Mesenteric arterial bed preparations were cannulated under anesthesia, excised and intralumenally perfused ex vivo with noradrenaline (0.2-20 microM). Basal perfusion pressures were not significantly different in mesentery preparations from non-diabetic (27.0 +/- 2.6 mmHg) and diabetic (27.1 +/- 3.2 mmHg) BB/E rats. There was no significant difference in maximal response above basal perfusion pressure (MAX) or pEC50, defined as the negative log of the agonist concentration required to give 50% of the maximal response above basal perfusion pressure, to noradrenaline in untreated non-diabetic (166 +/- 7 mmHg and 5.74 +/- 0.05 respectively) and diabetic (170 +/- 11 mmHg and 5.59 +/- 0.05) BB/E rats. 4. In vivo treatment of non-diabetic and diabetic BB/E rats with L-NAME had no significant effect on basal perfusion pressure (25.9 +/- 4.3 mmHg and 28.5 +/- 3.9 mmHg respectively). L-NAME treatment in vivo increased (P < 0.001) MAX to noradrenaline of non-diabetic rats (224 +/- 8 mmHg) but did not affect the value for diabetic rats (178 +/- 14 mmHg). L-NAME treatment did not alter after the pEC50 values in either group (5.71 +/- 0.05 and 5.65 +/- 0.05). 5. Consistent with previous studies using vascular preparations from spontaneously diabetic BB rats, mesentery preparations from diabetic BB/E rats (n = 12) exhibited a significantly reduced vasodilator response to acetylcholine (F value = 4.4, P < 0.05) across the concentration range studied compared to non-diabetic BB/E rats (n = 12) although there was no significant difference in maximal relaxation (diabetic 53.1 +/- 4.3% vs non-diabetic 55.7 +/- 5.5%) or pEC50, (diabetic 6.92 +/- 0.25 vs non-diabetic 7.49 +/- 0.22). There was no significant (F value = 0.8, P > 0.1) difference in the response to GTN between preparations from non-diabetic and diabetic rats (maximal relaxation: 49.6 +/- 3.7% vs 48.5 +/- 4.3%; pEC50: 7.84 +/- 0.12 vs 7.89 +/- 0.22 respectively). 6. In conclusion, vascular responsiveness to noradrenaline is not impaired in spontaneously diabetic BB/E rats with significantly better glycaemic control than those used in previous studies. However, following chronic L-NAME treatment, diabetic BB/E rats exhibit attenuated hypertension and an absence of enhanced vascular responsiveness to noradrenaline in vitro compared to similarly treated non-diabetic rats. These results, together with the significantly impaired endothelium-dependent vasodilatation and unchanged endothelium-independent vasodilatation in vitro of preparations from diabetic BB/E rats, are consistent with the hypothesis that functional changes in the synthesis and metabolism of NO (rather than altered vascular responsiveness to NO) occur in diabetes. Our results indicate that good glycaemic control alone is insufficient to prevent these abnormalities in NO availability and further studies to characterize the origin of these changes are necessary.

Animals↗
PubMed1996

Ciliary neurotrophic factor induces down-regulation of its receptor and desensitization of signal transduction pathways in vivo: non-equivalence with pharmacological activity.

Despite the widespread use of polypeptide growth factors as pharmacological agents, little is known about the extent to which these molecules regulate their cognate cell surface receptors and signal transduction pathways in vivo. We have addressed this issue with respect to the neurotrophic molecule ciliary neurotrophic factor (CNTF). Administration of CNTF in vivo resulted in modest decreases in levels of CNTFRalpha mRNA and protein in skeletal muscle. CNTF causes the rapid tyrosine phosphorylation of LIFRbeta and gp130 and the induction of the immediate-early gene, tis11; injection of CNTF 3-7 h after an initial exposure failed to re-stimulate these immediate-early responses, suggesting a biochemical desensitization to CNTF not accounted for by decreased receptor protein. To determine whether the desensitization of immediate-early responses caused by CNTF resulted in a functional desensitization, we compared the efficacy of multiple daily injections versus a single daily dose of CNTF in preventing the denervation-induced atrophy of skeletal muscle. Surprisingly, injections of CNTF every 6 h, which falls within the putative refractory period for biochemical responses, resulted in efficacy equal to or greater than injections once daily. These results suggest that although much of the CNTF signal transduction machinery is down-regulated with frequent CNTF dosing, biological signals continue to be recognized and interpreted by the cell.

Animals↗
PubMed1996

Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease.

Huntington disease is a dominantly inherited, untreatable neurological disorder featuring a progressive loss of striatal output neurons that results in dyskinesia, cognitive decline, and, ultimately, death. Neurotrophic factors have recently been shown to be protective in several animal models of neurodegenerative disease, raising the possibility that such substances might also sustain the survival of compromised striatal output neurons. We determined whether intracerebral administration of brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, or ciliary neurotrophic factor could protect striatal output neurons in a rodent model of Huntington disease. Whereas treatment with brain-derived neurotrophic factor, nerve growth factor, or neurotrophin-3 provided no protection of striatal output neurons from death induced by intrastriatal injection of quinolinic acid, an N-methyl-D-aspartate glutamate receptor agonist, treatment with ciliary neurotrophic factor afforded marked protection against this neurodegenerative insult.

Animals↗
PubMed1996

Role of neurotrophins and trk receptors in the development and maintenance of sensory neurons: an overview.

The neurotrophins are a family of polypeptide neuronal growth factors related to the prototypical neurotrophic factor, nerve growth factor (NGF). In mammals this gene family encompasses NGF, brain-derived neurotrophic factor (BDNF) and neurotrophins-3 and -4/5, (NT-3, NT-4/5). The neurotrophins initiate signal transduction in responsive cells by ligand induced dimerization and activation of one of the Trk family of receptor tyrosine kinases; NGF being specific for TrkA, BDNF and NT-4/5 for TrkB, and TrkC the preferred receptor for NT-3. In accord with differential patterns of distribution of Trk receptors in peripheral ganglia, the neurotrophins show both distinct and overlapping specificity towards subpopulations of sensory neurons of both neural crest and neural placode origin. In vitro and in vivo studies, and transgenic mice baring targeted null mutations of the neutrophin genes have established that BDNF, NT-3 and NT-4/5, like NGF, play critical roles as classical target-derived survival factors for subclasses of developing sensory neurons. However, much broader effects of neurotrophins on sensory neurons are now evident, including paracrine and autocrine actions on neuroblast proliferation, phenotypic differentiation, and survival and regeneration in the adult. This article provides an overview of the discovery and properties of the neurotrophin family, their receptors and their actions and specificity for both distinct and overlapping subpopulations of spinal and cranial sensory neurons.

Animals↗
PubMed1996

Non target-derived roles of the neurotrophins.

The hypothesis that target-derived neurotrophic factors are essential for the survival, differentiation and maintenance of sensory, sympathetic and motor neurons has been well supported by analysis of mice bearing null mutations in the neurotrophins and their receptors. However, the localization of brain-derived neurotrophic factor (BDNF) in a population of dorsal root ganglia (DRG) sensory neurons (Ernfors et al. 1990b; Ernfors & Persson 1991; Schecterson & Bothwell 1992) suggested the additional possibility that BDNF could act in a paracrine or autocrine manner to mediate neuronal survival. We tested this hypothesis in cultured adult DRG neurons, which survive as single cells in microwells in the absence of added trophic factors (Lindsay 1988). About 35% of these neurons were specifically killed by BDNF antisense oligonucleotide administration in a dose-dependent manner, with no effect of sense oligonucleotides. Antisense administration was accompanied by an 80% decrease in BDNF protein levels over the first 24 h of treatment (Acheson et al. 1995). The BDNF autocrine loop that we propose to be present in sensory neurons may be representative of a broader phenomenon in the nervous system as a whole, where the balance of neurotrophic support may shift during development from target-derived to paracrine or autocrine modes. Perhaps as a consequence of this developmental shift, the survival of both peripheral nervous system (PNS) and central nervous system (CNS) neurons in the adult is less affected by axotomy or target removal when compared to their response during development.

Animals↗
PubMed1996

BDNF increases monoaminergic activity in rat brain following intracerebroventricular or intraparenchymal administration.

We have previously demonstrated alterations in serotonin metabolism within descending pathways following infusion of brain-derived neurotrophic factor (BDNF) into the midbrain, near the periaqueductal gray and dorsal and median raphe nuclei. The aim of the present study was to extend these studies to include a comprehensive regional examination of monoamine (serotonin, dopamine and norepinephrine) and metabolite levels in discrete areas of the intact, adult rat forebrain following direct intraparenchymal midbrain BDNF infusion. We have compared neurochemical changes following midbrain infusion of BDNF to those obtained following intracerebroventricular (i.c.v.) infusion. Significant increases in levels of 5-HIAA and/or the 5-HIAA/5-HT ratio were found in all areas examined including the hippocampus, cortex, striatum, n. accumbens, substantia nigra and hypothalamus following both midbrain and i.c.v. infusion. Changes in dopaminergic activity were also observed, but displayed more regional specificity, i.e. changes were found primarily within the striatum and cortex. The two infusion sites produced similar patterns of neurochemical effects although the magnitude of the changes did vary in some areas. These results suggest that BDNF increased synthesis and/or turnover of serotonin, and to a lesser extent dopamine, in the mature rat forebrain. Furthermore, these data point to possible functional roles for BDNF in neuropsychiatric and neurodegenerative conditions which involve a dysregulation of these monoamine systems.

Animals↗
PubMed1996

A continuous striatal infusion of 6-hydroxydopamine produces a terminal axotomy and delayed behavioral effects.

Rat models of Parkinson's disease typically employ a rapid nigral injection of 6-hydroxydopamine (6-OHDA) to produce a near-complete loss of nigrostriatal dopamine neurons, and thus, model end stage disease. The present report describes the use of a continuous, low dose infusion of 6-OHDA into the striatum which produces a terminal axotomy of nigrostriatal dopamine neurons and protracted behavioral response. A solution of 6-OHDA in 0.4% ascorbate, delivered at 37 degrees C from osmotic minipumps, was stable for 8 days as determined by its retained toxicity to a dopaminergic neuroblastoma cell line. The continuous infusion of 0.2 mu g 6-OHDA per h did not affect the striatal uptake of [3H]%GABA, [3H]choline, or [3H]glutamate but reduced [3H]dopamine uptake by 55% within 1.5 days after the start of the infusion. The striatal infusion of 6-OHDA produced a dose-dependent reduction of striatal dopamine and DOPAC levels but did not alter HVA, 5-HT, or 5-HIAA. An increase in amphetamine-induced ipsiversive rotations occurred within 1.5 days after the acute striatal injection of 20 mu g or 30 mu g of 6-OHDA but required 4 days to develop with the continuous 6-OHDA infusion. The topography of the lesion mapped by [3H]mazindol binding showed that, beginning by 1.5 days, a diffuse depletion of terminals encompassed much of the striatum in the 30 mu g acute injection group, whereas in the continuously infused rats, the lesion was apparent only by 4 days and was restricted to a smaller and more completely lesioned area. Unlike acutely lesioned animals, continuously infused rats revealed no obvious loss of dopamine neurons in the pars compacta by 5 weeks after 6-OHDA. The continuous striatal infusion of 6-OHDA can produce a topographically limited terminal axotomy of dopamine neurons and a protracted behavioral impairment.

Animals↗
PubMed1996

Astrocytes infected with replication-defective adenovirus containing a secreted form of CNTF or NT3 show enhanced support of neuronal populations in vitro.

Neurotrophic factors have been shown to ameliorate neuronal death in several in vitro and in vivo models of neurodegenerative disease. However, delivery of polypeptide growth factors to compromised neurons in the CNS is problematic as the blood-brain barrier prevents systemic delivery, and chronic in-dwelling cannulae are required for intraparenchymal delivery. To circumvent these problems and specifically target neurotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) vectors that contain a secretable form of ciliary neurotrophic factor (sCNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCNTF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose-dependent expression of sCNTF or NT-3 mRNA was detected 7 days after infection. The levels of mRNA expressed in transgenic astrocytes was dependent on virus titer and increased with increasing virus concentration. sCNTF or NT-3 protein was also detected in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad secreted neurotrophic factors at a rate of approximately 120 pg/10(6) cells/h and 350 pg/10(6) cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose ganglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolayers pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with Lac-Z/Ad. This study demonstrates that, using replication-defective adenovirus, primary astrocytes can be efficiently engineered to secrete bioactive sCNTF or NT-3, resulting in enhanced survival of responsive peripheral and central neuronal populations.

Adenoviridae↗
PubMed1996

CNTF induces raphe neuronal precursors to switch from a serotonergic to a cholinergic phenotype in vitro.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that mediates survival and differentiation of neurons as well as many other cell types. In this study, CNTF and leukemia inhibitory factor (LIF) reduced the apparent number of primary serotonergic neurons in E14 raphe culture by 90% as determined by immunocytochemistry for serotonin (5HT). The reduction in 5HT cell number was not due to neuronal loss as removal of CNTF after 4 days in culture resulted in a partial restitution of the serotonergic phenotype. In the RN46A serotonergic cell line which is induced to become serotonergic by brain-derived neurotrophic factor (BDNF), the addition of CNTF suppressed tryptophan hydroxylase and 5HT synthesis and increased choline acetyl transferase (ChAT) expression by 6-fold and ChAT activity by 20- to 30-fold over 12 days. As with the primary neurons, removal and replacement of CNTF with BDNF after 4 days resulted in a partial restitution of 5HT expression. Moreover, other members of the CNTF-cytokine family that use gp130 and/or LIF receptor beta as their signal transducing receptors-LIF, oncostatin M, interleukin 6, and interleukin 11-had similar effects on increasing ChAT activity and reducing 5HT expression in RN46A cells. Analysis of 5HT levels showed no significant difference in the amount of serotonin between wild-type and CNTFR alpha knockout mice at birth, suggesting that the potential to switch phenotype mediated through CNTFR alpha is a latent property of neuroepithelial precursors in the raphe nucleus.

Acetylcholinesterase↗
PubMed1996

Prenatal glucocorticoid exposure leads to offspring hyperglycaemia in the rat: studies with the 11 beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone.

Recent human epidemiological studies have linked low birth weight with a substantially increased risk of non-insulin-dependent diabetes mellitus in later life. These data suggest that the intrauterine environment plays a crucial role in determining later glucose homeostasis, but the mechanism is unknown. We have proposed that exposure of the fetus to excess maternal glucocorticoids may underpin the epidemiological findings. Normally placental 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD-2) protects the fetus from the normally higher maternal levels of glucocorticoids by inactivating corticosterone and cortisol to inert 11-keto products. Here we show that administration of carbenoxolone, an inhibitor of placental 11 beta-HSD 2, to pregnant rats, leads to a significant reduction in average birth weight (20% fall). At 6 months of age, the male offspring of carbenoxolone-treated pregnancies had similar weights to controls, but showed significantly higher fasting plasma glucose (6.0 +/- 0.3 vs 4.8 +/- 0.2 mmol/l; p < 0.01) and exhibited significantly greater plasma glucose (10% higher) and insulin (38% higher) responses to an oral glucose load. These effects of carbenoxolone require intact maternal adrenal glands suggesting that inhibition of feto-placental 11 beta-HSD 2 is key. These data support the notion that deficiency of placental 11 beta-HSD, by exposing the fetus to excess maternal glucocorticoids, reduces growth and predisposes to hyperglycaemia in later life.

11-beta-Hydroxysteroid Dehydrogenases↗