RESUMEN
The objective of this article is to review the clinical presentation and neurobiology of degeneration of the enteric nervous system with emphasis on human data where available. Constipation, incontinence and evacuation disorders are frequently encountered in the ageing population. Healthy lower gastrointestinal function is essential for successful ageing as it is critical to maintaining independence and autonomy to pursue further activity. One clinical expression of enteric neurodegeneration is constipation. However, the aetiology may be multifactorial as disturbances of epithelial, muscle or neural function may all result from neurodegeneration. There is evidence of loss of excitatory (e.g. cholinergic) enteric neurons and interstitial cells of Cajal, whereas inhibitory (including nitrergic) neurons appear unaffected. Understanding neurodegeneration in the enteric nervous system is key to developing treatments to reverse it. Neurotrophins have been shown to accelerate colonic transit and relieve constipation in the medium term; they are also implicated in maintenance programmes in adult enteric neurons through a role in antioxidant defence. However, their effects in ageing colon require further study. There is evidence that 5-HT(2) and 5-HT(4) mechanisms are involved in development, maintenance and survival of enteric neurons. Further research is needed to understand and potentially reverse enteric neurodegeneration.
Asunto(s)
Envejecimiento/patología , Sistema Nervioso Entérico/patología , Intestinos/inervación , Intestinos/patología , Degeneración Nerviosa/patología , Anciano , Anciano de 80 o más Años , Envejecimiento/fisiología , Animales , Estreñimiento/patología , Estreñimiento/fisiopatología , Sistema Nervioso Entérico/fisiopatología , Femenino , Humanos , MasculinoRESUMEN
The objective of this article is to review the clinical presentation and neurobiology of degeneration of the enteric nervous system with emphasis on human data where available. Constipation, incontinence and evacuation disorders are frequently encountered in the ageing population. Healthy lower gastrointestinal function is essential for successful ageing as it is critical to maintaining independence and autonomy to pursue further activity. One clinical expression of enteric neurodegeneration is constipation. However, the aetiology may be multifactorial as disturbances of epithelial, muscle or neural function may all result from neurodegeneration. There is evidence of loss of excitatory (e.g. cholinergic) enteric neurons and interstitial cells of Cajal, whereas inhibitory (including nitrergic) neurons appear unaffected. Understanding neurodegeneration in the enteric nervous system is key to developing treatments to reverse it. Neurotrophins have been shown to accelerate colonic transit and relieve constipation in the medium term; they are also implicated in maintenance programmes in adult enteric neurons through a role in antioxidant defence. However, their effects in ageing colon require further study. There is evidence that 5-HT(2) and 5-HT(4) mechanisms are involved in development, maintenance and survival of enteric neurons. Further research is needed to understand and potentially reverse enteric neurodegeneration.
Asunto(s)
Envejecimiento/fisiología , Sistema Nervioso Entérico/fisiopatología , Enfermedades Neurodegenerativas/fisiopatología , Anciano/fisiología , Animales , Sistema Nervioso Entérico/patología , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/epidemiología , Enfermedades Neurodegenerativas/patología , Enfermedades Neuromusculares/tratamiento farmacológico , Enfermedades Neuromusculares/patología , Enfermedades Neuromusculares/fisiopatología , Neuronas/patología , Factores de Riesgo , Serotonina/fisiologíaRESUMEN
We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.
Asunto(s)
Envejecimiento/fisiología , Restricción Calórica , Plexo Mientérico/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/farmacología , Calbindina 2 , Calbindinas , Muerte Celular/efectos de los fármacos , Masculino , Plexo Mientérico/citología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/farmacología , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
OBJECTIVES: To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence. METHODS: Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured. RESULTS: Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered. CONCLUSIONS: Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Cartílago Articular/metabolismo , Catalasa/metabolismo , Condrocitos/metabolismo , Glutatión Peroxidasa/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismoRESUMEN
Many individuals experience gastrointestinal (GI) dysfunction more frequently as they age, and the segment of the human population that is growing the most rapidly is the 'oldest old', who are >/= 80 years old. There has recently been renewed interest in the age-related changes intrinsic to the gut, and these investigations may help physicians understand the 'normal' aged GI tract, as distinct from disordered bowel function that is the result of comorbid conditions and/or GI side effects of medications used to treat those conditions. In this concise review we summarize recent data that suggest age-related neurodegenerative changes in the enteric nervous system (ENS) are key to functional changes observed with advanced age. Morphological studies are reviewed that demonstrate clearly the loss of enteric neurones in both submucosal and myenteric plexuses in humans and in rodents. Recent studies that indicate selective preservation of nitrergic, but not cholinergic, neurones are reviewed, as are preliminary findings that intrinsic sensory neurones may be among the most 'age-labile' subpopulations of the ENS. Caloric restriction remains the only intervention known that prevents neurodegeneration of ageing in the ENS, and mechanisms involved in this phenomenon are discussed. The field of ageing research in enteric neurobiology is ripe for rapid progression from phenomenology of age-related losses of neurones and associated functional changes to discovery of therapeutic approaches that may help ameliorate deterioration of bowel function and thereby contribute significantly to improved quality of life in advanced age.
Asunto(s)
Anciano de 80 o más Años/fisiología , Envejecimiento , Sistema Nervioso Entérico/fisiología , Intestinos/inervación , Degeneración Nerviosa/fisiopatología , Anciano , Animales , Sistema Nervioso Entérico/patología , Humanos , Neuronas/patologíaRESUMEN
In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.
Asunto(s)
Estrógenos/farmacología , Factor de Crecimiento Nervioso/metabolismo , Plasticidad Neuronal , Neuronas Aferentes/fisiología , Receptor trkA/metabolismo , Útero/inervación , Adrenérgicos/farmacología , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Estrógenos/metabolismo , Femenino , Ganglios Espinales/anatomía & histología , Ganglios Espinales/citología , Guanetidina/farmacología , Inmunohistoquímica , Hibridación in Situ , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Simpatectomía Química , Útero/crecimiento & desarrollo , Útero/metabolismoRESUMEN
Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.
Asunto(s)
Fibras Adrenérgicas/metabolismo , Envejecimiento/fisiología , Neuronas/metabolismo , Receptor trkA/metabolismo , Receptor trkC/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Circulación Cerebrovascular , Hibridación in Situ , Iris/citología , Iris/inervación , Iris/metabolismo , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Ratas , Receptor de Factor de Crecimiento Nervioso , Receptor trkA/genética , Receptor trkC/genética , Receptores de Factor de Crecimiento Nervioso/genética , Transducción de Señal/fisiología , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/metabolismoRESUMEN
Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.
Asunto(s)
Estrógenos/farmacología , Miometrio/efectos de los fármacos , Sistema Nervioso Simpático/fisiología , Animales , Animales Recién Nacidos , Estrógenos/sangre , Femenino , Inmunohistoquímica , Iris/inervación , Microscopía Fluorescente , Miometrio/inervación , Miometrio/trasplante , Fibras Nerviosas/fisiología , Regeneración Nerviosa , Norepinefrina/metabolismo , Ovariectomía/métodos , Progesterona/sangre , Ratas , Ratas Wistar , Estadísticas no Paramétricas , Sistema Nervioso Simpático/efectos de los fármacos , Factores de Tiempo , Útero/efectos de los fármacos , Útero/inervaciónRESUMEN
Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.
Asunto(s)
Estrógenos/farmacología , Sistema Nervioso Simpático/crecimiento & desarrollo , Útero/inervación , Animales , Estrógenos/metabolismo , Femenino , Factor de Crecimiento Nervioso/análisis , Factor de Crecimiento Nervioso/biosíntesis , Norepinefrina/análisis , Norepinefrina/biosíntesis , Ratas , Ratas Wistar , Sistema Nervioso Simpático/anatomía & histología , Sistema Nervioso Simpático/efectos de los fármacos , Útero/anatomía & histología , Útero/efectos de los fármacosRESUMEN
Data are presented in support of the idea of antagonistic pleiotropy that features which are adaptive during early life may become maladaptive during the ageing process, when selective pressure is reduced. A model of selective vulnerability to age-related neurodegeneration involving neighbouring subpopulations of vulnerable and protected sympathetic neurons is presented. The two groups of neurons are morphologically and physiologically distinct, indicating advantageous adaptation to particular functions. Neurotrophin signalling is investigated in these different groups of neurons, revealing significant differences between them: neurotrophic factor expression in their target tissues is markedly different, same as their neurotrophin uptake characteristics. Preliminary evidence is presented that the mechanism linking neurotrophin signalling and age-related neurodegeneration may involve the capacity of neurons to buffer free radical generation, hence reducing the effects of attrition by free radical damage.
Asunto(s)
Envejecimiento/metabolismo , Supervivencia Celular/fisiología , Mamíferos/metabolismo , Músculo Liso/inervación , Degeneración Nerviosa/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Ganglio Cervical Superior/metabolismo , Animales , Radicales Libres/metabolismo , Humanos , Mamíferos/anatomía & histología , Modelos Neurológicos , Degeneración Nerviosa/fisiopatología , Neuronas/citología , Ganglio Cervical Superior/citologíaRESUMEN
We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red-nerve growth factor (DTR-NGF) conjugate. DTR-NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR-NGF labelling in SCG neurons was shown to be dose-dependent with an EC(50) of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR-NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85-93%) reduction in numbers of DTR-NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.
Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Sistema Nervioso Simpático/metabolismo , Animales , Anticuerpos Bloqueadores , Transporte Axonal/fisiología , Dextranos , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Iris/inervación , Iris/metabolismo , Masculino , Microscopía Confocal , Ratas , Ratas Sprague-Dawley , Sistema Nervioso Simpático/citología , XantenosRESUMEN
By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.
Asunto(s)
Proteínas de Neoplasias , Factores de Crecimiento Nervioso/metabolismo , Neuronas/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Ganglio Cervical Superior/citología , Clorometilcetonas de Aminoácidos/farmacología , Androstadienos/farmacología , Animales , Apoptosis/fisiología , Inhibidores de Caspasas , Supervivencia Celular , Células Cultivadas , Cromonas/farmacología , Medio de Cultivo Libre de Suero , Inhibidores Enzimáticos/farmacología , Microinyecciones , Microscopía Fluorescente , Morfolinas/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Oligodesoxirribonucleótidos Antisentido/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , WortmaninaRESUMEN
Adult growth hormone deficient patients are known to exhibit reduced sweating and their ability to thermoregulate is diminished. Treatment of these patients with recombinant human growth hormone (r-hGH) is claimed to reverse these abnormalities. We have investigated this claim, as well as the mechanism underlying these altered sweating responses in GH-deficient patients as part of a placebo-controlled study on the effects of 6-12 months r-hGH therapy. Skin biopsies were obtained from these subjects and changes in morphology and innervation parameters for the eccrine sweat glands were examined. These included histochemistry for acetylcholinesterase (AChE) and immunohistochemistry for the neuropeptide vasoactive intestinal polypeptide (VIP) and for PGP9.5, a general neuronal marker. Sweat gland acinar size and periacinar innervation were measured by computerised image analysis. The patients underwent pilocarpine iontophoresis sweat rate tests and their serum insulin-like growth factor 1 (IGF-1) levels were assessed. Since active acromegaly involves excess GH secretion and hyperhidrosis, skin biopsies and sweat tests were also carried out on a group of these patients, as well as on control subjects. We have demonstrated a sweating defect in adult GH-deficiency which is accompanied by a reduction in AChE and VIP levels in the nerve supply to sweat glands. Following r-hGH therapy, an increase in AChE and VIP staining is seen in the sudomotor nerves accompanied by restoration of sweat rates and serum IGF-1 levels. Hence, normalization of sweat gland function includes recovery of sudomotor synapse constituents. A trophic effect of GH on sweat gland epithelium and/or on the associated nerves is proposed, supported by the observation that in acromegaly the size of sweat gland acini and the density of innervation to the sweat glands was greater than in controls.
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Acromegalia/fisiopatología , Trastornos del Crecimiento/tratamiento farmacológico , Trastornos del Crecimiento/fisiopatología , Hormona del Crecimiento/administración & dosificación , Hormona de Crecimiento Humana/deficiencia , Sudoración/fisiología , Acetilcolinesterasa/análisis , Acromegalia/patología , Adulto , Antígenos de Diferenciación/análisis , Biopsia , Femenino , Trastornos del Crecimiento/patología , Humanos , Técnicas In Vitro , Factor I del Crecimiento Similar a la Insulina/metabolismo , Iontoforesis , Masculino , Persona de Mediana Edad , Agonistas Muscarínicos/farmacología , Pilocarpina/farmacología , Glándulas Sudoríparas/inervación , Glándulas Sudoríparas/patología , Glándulas Sudoríparas/fisiología , Sistema Nervioso Simpático/química , Sistema Nervioso Simpático/enzimología , Sistema Nervioso Simpático/fisiopatología , Tiroxina/sangre , Triyodotironina/sangre , Tirosina 3-Monooxigenasa/análisis , Ubiquitina Tiolesterasa , Péptido Intestinal Vasoactivo/análisisRESUMEN
The survival and growth of embryonic and postnatal sympathetic neurons is dependent on both NGF and NT3. While it has been established that adult sensory neurons survive independently of neurotrophins, the case is less clear for adult sympathetic neurons, where the studies of survival responses to neurotrophins have relied upon using long-term cultures of embryonic neurons. We have previously established a method to culture purified young (7 day) and adult (12 week) sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) in order to examine their survival and growth responses to neurotrophins. We now show that by 12 weeks after birth virtually all neurons (90%) survive for 24 h in the absence of neurotrophins. Neuron survival is unaffected by treatment with anti-NGF antibodies (anti-NGF) or with the tyrosine kinase inhibitor, K252a, confirming the lack of dependence on extrinsic neurotrophins. Duration of neuron survival in culture increases significantly between E19 and day 7 and week 12 posnatally, and is similarly unaffected by the presence of anti-NGF or K252a. Saturating concentrations of NGF and NT3 are equipotent in promoting neurite extension and branching. However, we find that NGF is more potent than NT3 in promoting neurite growth, irrespective of postnatal age. The growth-promoting effects of NGF and NT3 are almost entirely blocked by K252a, demonstrating that these effects are mediated via activation of Trk receptors, which therefore appear to remain crucial to plasticity of adult neurons. Our results indicate that maturing neurons acquire protection against cell death, induced in the absence of neurotrophin, while retaining their growth responsiveness to these factors.
Asunto(s)
Animales Recién Nacidos/fisiología , Proteínas de Drosophila , Factor de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/embriología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Embrión de Mamíferos/fisiología , Desarrollo Embrionario y Fetal , Proteínas de Insectos/fisiología , Masculino , Factor de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neurotrofina 3/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factores de TiempoRESUMEN
In vitro studies of dissociated neurons have provided crucial data regarding the regulation of plasticity in embryonic and perinatal neurons from both central and peripheral nervous systems. There have been few attempts to apply these methods to adult or aged neurons and the methods that have been reported have not been able to dissect the possible confounding contributions of non-neuronal cells and serum. Furthermore, quantitative assays of cultured neurons, particularly of their growth, have rarely been described. We report here the development of a novel method for the dissociation, purification and culture of sympathetic neurons from the adult and aged rat SCG under serum free conditions and in defined media. The technique results in a relatively high yield of viable, growing neurons. We describe methods for assaying the total yield of neurons, the proportion of surviving neurons and the proportion of neurons initiating neurite outgrowth after plating. A novel semiautomated assay of neurite outgrowth is outlined using image analysis of composite images of immunofluorescence-stained single neurons.
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Técnicas Citológicas , Neurociencias/métodos , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/fisiología , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular , Medio de Cultivo Libre de Suero/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Factor de Crecimiento Nervioso/administración & dosificación , Factor de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/fisiología , Neurotensina/administración & dosificación , Neurotensina/farmacología , Ratas , Ratas Sprague-Dawley , Células de Schwann/citología , Células de Schwann/fisiologíaRESUMEN
The light microscopical and ultrastructural morphology of the innervation of the major cerebral arteries and pial vessels is described, including the origins of the different groups of nerve fibres and their characteristic neurotransmitter phenotype. Species and region specific variations are described and novel data regarding the parasympathetic innervation of cerebral vessels are presented. The dynamic nature, or plasticity, of cerebrovascular innervation is emphasized in describing changes affecting particular subpopulations of neurons during normal ageing and in Alzheimer's disease. The molecular controls on plasticity are discussed with particular reference to target-associated factors such as the neurotrophins and their neuronal receptors, as well as extracellular matrix related factors such as laminin. Hypotheses are presented regarding the principal extrinsic and intrinsic influences on plasticity of the cerebrovascular innervation.
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Envejecimiento/patología , Enfermedad de Alzheimer/patología , Arterias Cerebrales/inervación , Plasticidad Neuronal , Telencéfalo/irrigación sanguínea , Seno Cavernoso/inervación , Humanos , Degeneración Nerviosa , Vías Nerviosas , Neurotransmisores/fisiología , Células Receptoras Sensoriales/fisiologíaRESUMEN
Mutant Caenorhabditis elegans in which the age-1 and daf-2 genes (involved in insulin-receptor-like signalling) are expressed at low levels exhibit extended lifespan. Wolkow and colleagues recently showed that restricted re-expression of age-1 and daf-2 genes in neurons of these mutants rescues wild-type lifespan as effectively as ubiquitous re-expression. Low levels of insulin-like signalling in neurons might control longevity by enhancing protection against free radical damage. However, in mammalian cells (including neurons) reduced insulin-like signalling is generally thought to be deleterious to antioxidant defence and to neuron survival. Here we discuss the new work and several hypotheses to explain this apparent contradiction.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Longevidad/genética , Fosfatidilinositol 3-Quinasas , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas del Helminto/genética , Insulina/metabolismo , Receptor de Insulina/genética , Transducción de SeñalRESUMEN
Altered neuronal responses to trophic factors may play a role in neuronal maintenance in adulthood and may also be involved in neuronal atrophy in old age. We have investigated this issue in the rat iris, studying responsiveness of sympathetic and sensory iridial nerves to a range of NGF concentrations in mature and aged rats. We show here that growth responses of sensory nerves to NGF, as measured by quantitative immunohistochemistry and image analysis, were unchanged in old rats. In contrast, there was a small but significant reduction in responsiveness of aged sympathetic neurons. The shapes of the dose-response curves for sensory and sympathetic neurons were different, with a larger response over a narrower range of concentrations in sensory neurons. Lower levels of p75 immunoreactivity were observed in iridial nerves from old compared to young rats. NGF treatment had no effect on receptor staining in young rats but restored 'young' levels of p75 staining in old rats. Our results do not support the hypothesis of a primary role for NGF in maintenance or atrophy of nerves in ageing.
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Fibras Adrenérgicas/efectos de los fármacos , Envejecimiento/fisiología , Iris/inervación , Factor de Crecimiento Nervioso/farmacología , Neuronas Aferentes/efectos de los fármacos , Fibras Adrenérgicas/química , Fibras Adrenérgicas/fisiología , Animales , Anticuerpos , Péptido Relacionado con Gen de Calcitonina/análisis , Péptido Relacionado con Gen de Calcitonina/inmunología , Masculino , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas Aferentes/química , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/análisis , Receptor de Factor de Crecimiento Nervioso/inmunología , Receptor trkA/análisis , Receptor trkA/inmunología , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/inmunologíaRESUMEN
Retrograde tracing and immunohistochemistry was used in rats to investigate whether the ganglia in the cavernous sinus contribute to cerebrovascular innervation. The cavernous sinus ganglia in rat include the cavernous part of the pterygopalatine ganglion (PGC) and small cavernous ganglia (CG). The tracers, fluorogold and fast blue, were applied to the middle cerebral artery in eight rats. After 1 to 4 days, the cavernous sinuses were dissected out and studied as whole mount preparations and sections. A moderate number of labeled neurons were visible in the ipsilateral PGC and CG. Furthermore, fibers in the cavernous nerve plexus and abducens nerve were labeled, suggesting that the pathway from the cavernous sinus ganglia to the cerebral arteries runs through the cavernous plexus and then retrogradely along the abducens nerve to the internal carotid artery. Selected sections were immunohistochemically stained for the cholinergic marker, vesicular acetylcholine transporter (VAChT). Most cells in the PGC and CG were VAChT-immunoreactive, some of which also contained tracer. It is concluded that in rat, the cavernous sinus ganglia, consisting of the PGC and small CG, contribute to parasympathetic cerebrovascular innervation and that the cavernous nerve plexus and abducens nerve are involved in the pathway from these ganglia to the cerebral arteries.
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Seno Cavernoso/inervación , Arterias Cerebrales/inervación , Ganglios/fisiología , Proteínas de Transporte de Membrana , Sistema Nervioso Parasimpático , Proteínas de Transporte Vesicular , Acetilcolinesterasa/análisis , Animales , Transporte Axonal , Proteínas Portadoras/análisis , Histocitoquímica , Inmunohistoquímica , Masculino , Vías Nerviosas/anatomía & histología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
BACKGROUND: Alone among autonomic neurones, enteric neurones are known to be vulnerable to age related cell death; over 50% may be lost in aging rodents. A previous study demonstrated unexpectedly that neurones of the myenteric plexus from rats fed a restricted diet appeared not to suffer from extensive cell death in contrast with previous studies of ad libitum fed animals. AIMS: To compare myenteric neurone numbers in the ileum of young and aging male Sprague-Dawley rats fed either ad libitum or a restricted diet. METHODS: Neurones were counted in whole mount preparations of rat ileum stained immunohistochemically for the pan-neuronal marker PGP9.5, for choline acetyltransferase, or for nitric oxide synthase, or with NADH or NADPH histochemistry. RESULTS: Neurone numbers in the rat myenteric plexus were substantially affected by the dietary regimen: ad libitum feeding (50-60 g per day of standard rat chow) resulted in the death of about 50% of myenteric neurones in 24 month Sprague-Dawley rats, while numbers were unchanged when the daily dietary intake was halved between the ages of six and 24 months. Animals fed a double restricted diet (15 g per day) showed no cell loss at 30 months, as well as the predicted increase in longevity. Neurone loss was largely complete by 16 months in ad libitum fed animals. Numbers of cholinergic (possibly motor) neurones, as demonstrated by choline acetyltransferase immunohistochemistry, were substantially reduced in ad libitum fed aging rats but not in animals fed a restricted diet. Loss of cholinergic neurones after ad libitum feeding was confirmed by reduced numbers of neurones of a size range matching that of cholinergic neurones. CONCLUSIONS: Ad libitum feeding of adult rats has adverse effects on the survival of myenteric neurones, neurone loss commencing before 16 months of age. Cholinergic neurones appear to be particularly vulnerable to the effects of diet. Restricting dietary intake from six months of age prevents neurone loss almost entirely up to 30 months of age in these rats.