Biomedical subjects

T Cowen

Publications and source records attributed to T Cowen.

At least 37 records · Page 2Linked to original sources

PubMed2000

Restricted diet rescues rat enteric motor neurones from age related cell death.

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.

Aging↗

PubMed1999

Differential effects of prepubertal chronic oestrogen treatment on the synthesis of noradrenaline in uterine myometrial and perivascular sympathetic nerves.

Previous studies have shown that chronic administration of oestrogen to prepubertal rats reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. The mechanisms underlying these changes are not known. In the present study we have analysed the effects of prepubertal chronic oestrogen treatment on the synthesis of noradrenaline in the rat uterine sympathetic nerves using biochemical and immunohistochemical approaches. Tyrosine hydroxylase activity was evaluated biochemically, by measuring the in vivo accumulation of dihydroxyphenylalanine (DOPA) in the presence of a DOPA-decarboxylase inhibitor. In addition, nerve fibres were visualised immunohistochemically using antibodies against tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DbetaH) and the general marker protein gene product 9.5 (PGP 9.5). After chronic oestrogen treatment, the total content of noradrenaline of the uterine horn was reduced, whereas the total content of DOPA was increased. In controls, TH-immunoreactive, DbetaH-immunoreactive and PGP 9.5-immunoreactive nerve fibres were distributed in both the circular and longitudinal myometrial layers and in the blood vessels of the intran-myometrial region. After chronic oestrogen treatment the only fibres recognised by the three antibodies were those associated with the blood vessels, but no myometrial-associated fibres could be recognised. These results suggest that noradrenaline synthesis is selectively reduced in myometrial-associated uterine sympathetic nerves, but is preserved in perivascular sympathetic nerves. The increased DOPA levels measured after chronic exposure to oestrogen was interpreted as the consequence of the substantial increase in size and number of blood vessels observed in the uterus of oestrogen-treated animals. A possible neurodegenerative effect of oestrogen on myometrial sympathetic fibres is discussed.

Animals↗

PubMed1998

Plasticity in adult and ageing sympathetic neurons.

The nature of neural plasticity and the factors that influence it vary throughout life. Adult neurons undergo extensive and continual adaptation in response to demands that are quite different from those of early development. We review the main influences on the survival, growth and neurotransmitter expression in adult and ageing sympathetic neurons, comparing these influences to those at work in early development. This "developmental" approach is proposed because, despite the contrasting needs of different phases of development, each phase has a profound influence on the mechanisms of plasticity available to its successors. Interactions between neurons and their targets, whether effector cells or other neurons, are vital to all of these aspects of neural plasticity. Sympathetic neurons require access to target-derived diffusible neurotrophic factors such as NGF, NT3 and GDNF, as well as to bound elements of the extracellular matrix such as laminin. These factors probably influence plasticity throughout life. In adult life, and even in old age, sympathetic neurons are relatively resistant to cell death. However, they continue to require target-derived diffusible and bound factors for their maintenance, growth and neurotransmitter expression. Failure to maintain appropriate neuronal function in old age, for example in the breakdown of homeostasis, may result partly from a disturbance of the dynamic, trophic relationship between neurons and their targets. However, there is no clear evidence that this is due to a failure of targets to synthesize neurotrophic factors. On the neural side of the equation, altered responsiveness of sympathetic neurons to neurotrophic factors suggests that expression of the trk and p75 neurotrophin receptors contributes to neuronal survival, maintenance and growth in adulthood and old age. Altered receptor expression may therefore underlie the selective vulnerability of some sympathetic neurons in old age. The role of neural connectivity and activity in the regulation of synthesis of target-derived factors, as well as in neurotransmitter dynamics, is reviewed.

Adult↗

PubMed1998

The effects of age on the overall population and on sub-populations of myenteric neurons in the rat small intestine.

Previous studies on ageing animal and human subjects have demonstrated a significant overall decline in neuronal numbers in the myenteric plexus of the enteric nervous system (ENS). Our study aimed to confirm this observation by counting myenteric neurons stained with the panneuronal markers PGP 9.5 and NADH-diaphorase. We also wished to examine the possibility that particular subpopulations of neurons are vulnerable. Therefore, we have immunostained and counted a number of nerve cell groups within the myenteric plexus of old and young Sprague Dawley rats using markers which reflect some of the neuronal phenotypes present, including ChAT and VIP. The number of neurons demonstrating NADH-diaphorase activity was significantly reduced (P < 0.05) by approximately 15 % in old rats. However, the number of neurons stained for PGP 9.5 immunohistochemistry was not reduced and demonstrated larger numbers of neurons than the NADH-diaphorase method. None of the other neuronal markers studied showed any significant reductions with age. In contrast to previous work, this study has gathered little evidence for extensive cell loss in the myenteric plexus of the aged rat, either in overall populations, or in any of the principal functional groups of neurons.

Aging↗

PubMed1998

In oculo transplants of myometrium from postpartum guinea pigs fail to support sympathetic reinnervation.

Sympathetic nerves to the enlarged fetus-containing region of the uterus undergo degenerative changes during late pregnancy and show slow regrowth after parturition. It is not known whether this unusual response of sympathetic nerves to smooth muscle hypertrophy is due to the sensitivity of short adrenergic neurons to hormonal changes, or whether the nerves respond to changes in the neurotrophic capacity of the target. We have investigated this question using in oculo transplantation. Small pieces of myometrium from the uterine horn of virgin guinea pigs, or from the region previously occupied by the placenta and fetus in postpartum guinea pigs, were transplanted into the anterior eye chamber. After 3 wk in oculo, the pattern of reinnervation of the transplants was assessed on whole mount stretch preparations stained for tyrosine hydroxylase. The histology of the transplants was examined in toluidine blue-stained semithin sections. Myometrial transplants from virgin donors and uterine artery transplants from both virgin and postpartum donors became organotypically reinnervated by sympathetic fibres from the host iris. In contrast, sympathetic nerves did not reinnervate myometrial transplants from postpartum donors, although they approached the transplants and became distributed in the surrounding connective tissue. All transplanted tissues showed a normal histological appearance. Both the myometrium and uterine artery from postpartum donors retained a hypertrophic appearance after 3 wk in oculo. We interpret these results to indicate that the degeneration of sympathetic nerves in late pregnancy, as well as their slow regrowth to the uterus after delivery, may be due to changes in uterine smooth muscle rather than a particular sensitivity of short adrenergic neurons to hormonal changes.

Animals↗

PubMed1997

Imaging and analysis of perivascular nerves in human mesenteric and coronary arteries: a comparison between epi-fluorescence and confocal microscopy.

Perivascular nerves supplying human arteries can be visualised after immunohistochemical staining for a variety of markers. The pattern and density of perivascular nerves vary with region, age and disease. Quantification of the nerve plexus, which may be performed by image analysis, is a prerequisite to assess differences in nerve density. The use of epi-fluorescence microscopy (EFM) presents difficulties in visualising the nerve plexus in certain tissues, which can affect the reliability with which specific staining can be localised and distinguished from non-specific staining. In this study, confocal scanning laser microscopy (CSLM) was used in parallel with EFM, in order to compare images from both techniques. In a comparison of identical areas of nerve plexuses of human mesenteric and coronary arteries stained for protein gene product (PGP) 9.5 and imaged using CSLM and EFM, higher values for area percent (area occupied by nerves), and intercept density (ID/mm, which reflects the number of nerve bundles detected) were found in CSLM images. Similar comparisons of unmatched epi-fluorescence and confocal images from a group of 45 mesenteric arteries revealed no significant difference for area percent, but significantly higher values for ID/mm in CSLM images. These findings illustrate that the better image quality in CSLM influences image analysis and can be very useful in studies of dynamic changes in nerve plexuses. We recommend CSLM for tissues that suffer from high background staining, such as human mesenteric and coronary arteries.

Adult↗

PubMed1997

Decreased neurofilament gene expression is an index of selective axonal hypotrophy in ageing.

Axonal atrophy may reflect earlier and more reversible events in neurodegeneration and ageing than somatic atrophy. Innervation density by sympathetic fibres from the rat superior cervical ganglion (SCG) to the middle cerebral artery (MCA) decreases dramatically in old age, while that to the iris is largely unchanged. In situ hybridization was used in conjunction with retrograde tracers to examine the role of the neuronal cytoskeleton in this selective axonal vulnerability. Using a riboprobe complementary to neurofilament light chain (NF-L) mRNA, there was a 22-25% decrease in the mean grain density in aged neurones when all neurones were examined. A small subset of these neurones was shown to project to the MCA and another to the iris. In young SCG, both subpopulations expressed intermediate grain densities for NF-L mRNA. In MCA-projecting neurones, there was a 40% decline in grain density with ageing (p < 0.05), with no change in iris-projecting neurones. Our results demonstrate that major decreases in NF-L expression may represent cellular markers of selective axonal hypotrophy by aged neurones.

Aging↗

PubMed1997

Responses of mature and aged sympathetic neurons to laminin and NGF: an in vitro study.

Whilst the potent effects of NGF and laminin on developing neurons are well documented, relatively little is known about the effects of, or altered availability of or altered responsiveness to, these substances on the growth of adult neurons. We have therefore examined this question using explant cultures of sympathetic neurons from the superior cervical ganglion (SCG) of mature and aged rats. Explants were grown on substrata containing different doses of laminin, either with or without added NGF in culture medium containing FCS. Individually, laminin and NGF had relatively small effects on neurite outgrowth and length, which tended to be reduced in old neurons. In contrast, laminin in the presence of exogenous NGF exerted a powerful effect on nerve growth which was substantially greater than the sum of the effects of the individual factors. This synergy was evident in all experimental groups and was greatest in old explants at high doses of laminin, where growth was comparable to that of mature neurons. The dose-response curve of old neurons to laminin in the presence of added NGF indicated reduced responsiveness. These results suggest that variations in the availability of laminin and/or exogenous NGF, together with altered patterns of neuronal responsiveness, may contribute to impaired neuronal plasticity in old age.

Aging↗

PubMed1996

Extracellular matrix molecules influence innervation density in rat cerebral blood vessels.

We have investigated the contribution of the acellular component (i.e., the extracellular matrix, or ECM) in determining the innervation density of peripheral target tissue, and the possible involvement of ECM molecules in age-related axonal atrophy. We addressed this issue using cerebral blood vessels where both the overall density and the density of sympathetic innervation decrease by 50% in aged rats. In order to study the capacity of the ECM to influence pattern and density of innervation, cerebral blood vessels from young (6 weeks) and old (24 months) rats were repeatedly freeze-thawed to kill the cellular component of the vessel wall before transplantation in oculo into young host rats. After 4 weeks, the nature and degree of reinnervation of transplants by host nerves was studied by quantitative immunohistochemistry using PGP9.5 (a general neuronal marker) or CGRP (calcitonin gene-related peptide, a sensory marker), or catecholamine histochemistry using the glyoxylic acid technique to visualise noradrenergic sympathetic nerves. The density of reinnervation was measured by computerized image analysis. The PGP-immunoreactive reinnervation on old transplanted cerebral blood vessels was significantly less dense compared to young ones. However, nerve density on both young and old transplants was lower than on control, ex vivo blood vessels. CGRP-positive nerves on both young and old transplants were sparse, but similar to control tissues, whereas catecholamine-containing nerves were very sparse on all transplants, and significantly less than on control blood vessels. These results suggest that alterations occur in the aged ECM, that make it less receptive to innervation. However, they also indicate that, along with the ECM, factors produced by the cellular components of the blood vessel wall are essential, at least for the maintenance of the sympathetic innervation.

Aging↗

PubMed1996

Transplanted sweat glands from mature and aged donors determine cholinergic phenotype and altered density of host sympathetic nerves.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

Adrenergic Fibers↗

PubMed1996

Levels of NGF protein do not correlate with changes in innervation of the rat iris in old age.

We have examined the hypothesis that alterations in target levels of NGF protein lead to selective changes in peripheral nerve fibres in maturity and old age. NGF levels were assayed using 2-site ELISA in the iris of young and old rats. NGF levels in the iris showed no significant change in old age and did not correlate with age-related changes in the density of NGF-sensitive irideal sympathetic and sensory nerve fibres. Following sympathectomy, NGF levels increased substantially in old but not in young irises, possibly because the lower density of aged sensory nerves resulted in reduced capacity for NGF uptake. Content and uptake of NGF in sympathetic (SCG) neurones was shown to be reduced in old age, while NGF content of the sensory (trigeminal) ganglion increased in old age. Thus NGF levels in neurones, as well as targets, fail to correlate with peripheral nerve fibre growth or atrophy.

Aging↗

PubMed1996

Decreased neurofilament gene expression is an index of selective axonal hypotrophy in ageing.

Aging↗

PubMed1996

Transplanted sweat glands from mature and aged donors determine cholinergic phenotype and altered density of host sympathetic nerves.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH)). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

Adrenergic Fibers↗

PubMed1996

Target-specific differences in the dendritic morphology and neuropeptide content of neurons in the rat SCG during development and aging.

Our purpose in this work was to investigate the role of target tissues in the regulation of dendritic morphology from sympathetic neurons during development and aging. Neurons were retrogradely labeled from three targets, the iris, the submandibular gland (SMG), and the middle cerebral artery (MCA). They were then fixed and intracellularly injected to demonstrate their dendritic arborizations. Dendritic geometry varied quantitatively in sympathetic neurons innervating different target tissues at all stages of development. Neurons innervating the iris had the largest cell bodies and most extensive dendritic arborizations, whereas the vasomotor neurons were the smallest. The number of primary dendrites, however, did not vary significantly between the different neuronal populations. The growth of dendritic arborizations during postnatal development and their atrophy in old age were not concordant in the different neuron populations we studied. Neurons innervating the MCA and the iris ceased dendritic growth early in postnatal development, whereas the dendritic complexity of neurons supplying the SMG increased well into adulthood. By contrast, dendritic atrophy was seen in aged MCA- and SMG-projecting neurons but not in those innervating the iris, suggesting, with other evidence, correlated and distinct patterns of growth and atrophy in axons and dendrites of mature sympathetic neurons projecting to different targets. Swollen dendrites and protuberances on cell soma were a prominent feature of aged neurons. In addition to the target-specific variation in neuronal morphology, we observed diversity in neurotransmitter phenotype. For example, neuropeptide Y was expressed in iridial but not SMG-projecting neurons. These results show a range of age- and target-specific differences in the dendritic morphology and neuropeptide content of sympathetic neurons that may be a result of differing trophic interactions with their target tissues.

Aging↗

PubMed1996

Can the neurotrophic hypothesis explain degeneration and loss of plasticity in mature and ageing autonomic nerves?

The causes of age-related degeneration in the peripheral nervous system remain unclear. The search for clues has focused on developmental mechanisms and particularly on the neurotrophic hypothesis and its principal player, nerve growth factor, reduced levels of which are thought to cause degeneration of some autonomic and central neurons in old age. Nerve growth factor may well be important in the mature and ageing nervous system, but recent experiments on sympathetic nerves in ageing rats suggest that lack of NGF is not the only limiting factor in neuronal growth and survival. Other candidates include laminin, which is bound in the extracellular matrix and may act in synergy with NGF to regulate neuronal maintenance and growth in maturity. Reduced, region-specific patterns of availability of one or both of these substances may underlie age-related degeneration in autonomic nerves. Different combinations of these factors may influence particular aspects of neuronal plasticity, such as collateral sprouting and regeneration. In addition to extrinsic factors, it appears increasingly likely that altered neuronal responsiveness to neurotrophic factors in old age contributes to structural and functional deficits in autonomic nerves.

Aging↗

PubMed1996

Influence of age and anti-nerve growth factor treatment on the sympathetic and sensory innervation of the rat iris.

We have investigated alterations in the nerve supply to the iris of aged rats and the role of endogenous nerve growth factor in these changes. The overall density of nerve fibres, and the density of calcitonin gene-related peptide containing sensory nerves, were decreased by over 20% on the aged iris, as measured by computerized image analysis on immunostained preparations, while the density of sympathetic innervation was maintained. Whilst the majority of nerves supplying the iris (sympathetic, sensory and parasympathetic) are known to respond to exogenous nerve growth factor during development and in adulthood, the role of endogenous, target-derived nerve growth factor in nerve maintenance in maturity and old age awaits confirmation. Our results showed that localized treatment with anti-nerve growth factor of iridial nerve terminals did not affect sympathetic or sensory neurons in young rats, but caused a dramatic reduction of sympathetic nerve density on irides of old rats. The effect of anti-nerve growth factor treatment on the sensory innervation of old irides was less obvious. We conclude that aged sympathetic nerves are more susceptible to nerve growth factor deprivation than young ones, or than young or aged sensory neurons, perhaps as a result of reduced responsiveness to nerve growth factor with age. Since sympathetic innervation is maintained, whilst sensory innervation is decreased in the aged iris, age-related changes in innervation are unlikely to be due to altered availability of endogenous nerve growth factor.

Aging↗

PubMed1996

Perivascular nerves of the human basal cerebral arteries: I. Topographical distribution.

In the present study the topographical distribution of the intrinsic nerve plexuses of the basal cerebral arteries in humans was quantified and the relation between vessel diameter and nerve density was investigated. Whole-mount preparations of various segments of the basal cerebral arteries from middle-aged patients were stained for protein gene product (PGP) 9.5. The deep nerve plexuses, located at the adventitial-medial border, were quantified by image analysis. Confocal scanning laser microscopy was used to study nerve plexuses throughout the adventitia. Transverse cryostat sections were stained for PGP 9.5, tyrosine hydroxylase and neurofilament, and quantified. The results showed a three-layered configuration of the adventitial nerves. Measurements on whole-mounts demonstrated that nerve densities were highest in the posterior communicating artery (PCom), and next highest in the proximal parts of the posterior cerebral artery (PCA) and anterior choroidal artery. There appeared to be no clear relation between nerve density and vessel diameter. The measurements on sections confirmed the high nerve densities in the PCom and PCA. Tyrosine hydroxylase- and neurofilament-immunoreactivities appeared to demonstrate separate subpopulations of the overall nerve plexuses, representing sympathetic and, possibly, sensory fibers, respectively. Densities of both subgroups generally followed those of PGP 9.5-immunoreactive nerves. Transmission electron microscopy suggested motor function of the deep nerve plexuses. The results indicate a stronger neuronal influence on this part of the cerebral circulation than hitherto reported. It is concluded that human basal cerebral arteries display a topographical distribution of deep perivascular nerves, and that nerve density is determined by locality rather than by vascular diameter.

Adult↗

PubMed1996

Perivascular nerves of the human basal cerebral arteries: II. Changes in aging and Alzheimer's disease.

In the present study the intrinsic nerve plexuses of the basal cerebral arteries, derived from aged non-Alzheimer's and aged Alzheimer's disease patients were quantified and compared. A previous study described and quantified nerve density on similar arteries from healthy middle-aged patients. Whole-mount preparations of various segments of the basal cerebral arteries were stained for protein gene product 9.5. The deep nerve plexuses, located at the adventitial-medial border, were quantified by image analysis. Transverse cryostat sections were stained for various markers and quantified. Measurements on whole mounts demonstrated that nerve densities were highest in the posterior communicating artery and in the postcommunicating part of the posterior cerebral artery (PCA) for both aged and Alzheimer's groups. Statistical comparison showed a tendency toward decreased nerve density with aging, which was significant for the internal carotid artery, precommunicating part of the PCA, and the anterior choroidal artery in both non-Alzheimer's and Alzheimer's aged groups. In addition, in Alzheimer's patients nerve density was significantly lower in the precommunicating part of the anterior cerebral artery compared with the healthy aged group. Measurements on sections confirmed the tendency to decreased innervation with aging. It is concluded that densities of deep perivascular nerves of human basal cerebral arteries are subject to localized changes caused by aging and Alzheimer's disease.

Aged↗