Dietary DHA reduces downstream endocannabinoid and inflammatory gene expression and epididymal fat mass while improving aspects of glucose use in muscle in C57BL/6J mice.

OBJECTIVES: Endocannabinoid system (ECS) overactivation is associated with increased adiposity and likely contributes to type 2 diabetes risk. Elevated tissue cannabinoid receptor 1 (CB1) and circulating endocannabinoids (ECs) derived from the n-6 polyunsaturated acid (PUFA) arachidonic acid (AA) occur in obese and diabetic patients. Here we investigate whether the n-3 PUFA docosahexaenoic acid (DHA) in the diet can reduce ECS overactivation (that is, action of ligands, receptors and enzymes of EC synthesis and degradation) to influence glycemic control. This study targets the ECS tonal regulation of circulating glucose uptake by skeletal muscle as its primary end point. DESIGN: Male C57BL/6J mice were fed a semipurified diet containing DHA or the control lipid. Serum, skeletal muscle, epididymal fat pads and liver were collected after 62 and 118 days of feeding. Metabolites, genes and gene products associated with the ECS, glucose uptake and metabolism and inflammatory status were measured. RESULTS: Dietary DHA enrichment reduced epididymal fat pad mass and increased ECS-related genes, whereas it reduced downstream ECS activation markers, indicating that ECS activation was diminished. The mRNA of glucose-related genes and proteins elevated in mice fed the DHA diet with increases in DHA-derived and reductions in AA-derived EC and EC-like compounds. In addition, DHA feeding reduced plasma levels of various inflammatory cytokines, 5-lipoxygenase-dependent inflammatory mediators and the vasoconstrictive 20-HETE. CONCLUSIONS: This study provides evidence that DHA feeding altered ECS gene expression to reduce CB1 activation and reduce fat accretion. Furthermore, the DHA diet led to higher expression of genes associated with glucose use by muscle in mice, and reduced those associated with systemic inflammatory status.

Cardiovascular responses to phlebotomy and sitting in middle-aged and elderly subjects.

BACKGROUND: Responding appropriately to hypotensive challenges is an important determinant of health and functional independence in elderly individuals. Cardiovascular responses to phlebotomy and postural change were evaluated using a large database developed in a study designed to establish the safety of blood donation by older individuals. METHODS: The groups studied included 464 subjects aged 65 years and younger (range, 52 to 65 years) and 532 subjects more than 65 years old (range, 66 to 78 years old). Blood pressure and pulse rate measurements were followed by the withdrawal of 500 mL of blood. These measurements were repeated, first in the supine and then in the sitting position. RESULTS: Nearly all individuals studied remained hemodynamically stable after these two challenges. Age was not an independent predictor of blood pressure change after either phlebotomy or postural change. Large decreases in diastolic blood pressure were equally rare in both age groups. However, more older subjects (15.2%) exhibited a decline of 20 mm Hg or more in systolic blood pressure following phlebotomy, compared with the middle-aged group (6.9%). These age-related differences did not persist after controlling for the higher initial systolic blood pressures observed in the older subjects. Postphlebotomy postural change to the sitting position had little additional effect. CONCLUSIONS: These results indicate that the ability to respond to hypovolemia and postural change remains relatively intact in healthy elderly individuals. The higher prevalence of a significant drop in systolic blood pressure after phlebotomy, orthostasis, and possibly other homeostatic challenges in older subjects is probably due to the presence of higher basal blood pressure readings, including hypertension. In spite of these differences, blood donation is appropriate and should be encouraged in healthy elderly individuals in this age group.

Peripheral dopamine in pathophysiology of hypertension. Interaction with aging and lifestyle.

Dopamine, an ancestral catecholamine, is physiologically natriuretic and vasodilating, thus essentially protecting against hypertension. Its actions are overshadowed by the opposite effects of its main biological partner, norepinephrine, and this is accentuated with aging. Clinical observations combined with molecular biology approaches to catecholamine-synthesizing and catecholamine-metabolizing enzymes and receptors permit the identification of some inborn defects. Subtle changes in the dopamine-norepinephrine balance may account for the enhanced peripheral noradrenergic activity seen in the setting of decreased dopaminergic activity in advanced age. These changes may contribute to the diminished ability of the aged kidney to excrete a salt load, as well as to the finding that systolic blood pressure increases with age in populations with a high, but not in those with a low, intake of salt. The attainment of advanced age in Western societies with adverse lifestyle changes (mental rather than physical stress, excess salt intake, overeating, sedentarism) appears to facilitate the development of hypertension. The adaptation to all the preceding lifestyle changes necessitates an increased dopamine generation, which may initially compensate to maintain appropriate natriuresis and vasodilation since many patients with initial borderline essential hypertension express their sympathetic hyperfunction, in addition to increased norepinephrine release, by excessive dopamine release. However, the progression of hypertension is accompanied by a peripheral dopaminergic deficiency and diminished ability to excrete salt. This may represent an eventual inadequacy of a phylogenetically redundant system resulting in decreased natriuresis and vasodilation and may account for the responsiveness of established chronic hypertension to salt restriction, diuretics, and dopaminomimetic medication.

Neurotrophin receptor and tyrosine hydroxylase gene expression in aged sympathetic neurons.

Ribonuclease protection measurements revealed decreases of 26% in p75 neurotrophin receptor mRNA and 30% in trkA mRNA in superior cervical ganglia (SCG) of aged Long-Evans rats. These declines were not related to the presence of a spatial memory impairment, whose presence is known to strongly predict increased hypothalamic-pituitary-adrenal axis activity in these aged animals. A similar decrease with age was observed in p75, but not cyclophilin mRNA levels in SCG from F-344 inbred rats. In situ hybridization with paired sections from mature and aged F-344 rats revealed a 25% decline in the mean neuronal labeling index (LI) for p75 mRNA. In other paired sections, mean trkA LI decreased 16%, tyrosine hydroxylase (TH) LI increased 74% and cyclophilin LI did not change. Neuronal hypertrophy, p75 decreases and TH increases all occurred to a greatest extent in intermediate-sized neurons, resembling those innervating the pineal and cerebral vessels. In contrast to other SCG targets, this innervation is known to decline nearly 50% with aging. Retrograde tracer/in situ hybridization studies will be required to establish whether decreased p75 represents a marker for selective axonal regression and also to determine the significance of increased TH and neuronal hypertrophy.

NGF expression in the aged rat pineal gland does not correlate with loss of sympathetic axonal branches and varicosities.

The factors that determine the ability of some, but not all neurons, to sustain their axonal projections during aging remain largely unknown. Because sympathetic neurons remain responsive to nerve growth factor (NGF) in old age, it has been proposed that the selective decrease observed in the sympathetic innervation to some targets in aged rats may be the result of a deficit in target-derived NGF. In this study we utilized two different techniques to demonstrate decreased target innervation by sympathetic fibers in the aged rat pineal gland, which is an appropriate and relevant model for examining mechanisms of neuron-target interactions in aging. Tyrosine hydroxylase immunoreactive profiles were quantified in pineal glands of young and aged male Sprague-Dawley rats. The density of tyrosine hydroxylase-immunoreactive fibers was 30% lower in aged pineals, although the remaining fibers contained 20% more tyrosine hydroxylase-immunoreactivity. Othograde tracing of the pineal sympathetic innervation using biotinylated dextran revealed that average axon length, varicosity numbers, branch point numbers, and numbers of terminations were all decreased by approximately 50% in aged tissues, indicating possible functional deficits. These findings suggest that whole branches, along with their associated varicosities were lost in old age. A sensitive quantitative ribonuclease protection assay and a two-site ELISA assay were used to examine whether reduced NGF availability might correlate with sympathetic nerve atrophy. No significant differences were detected in either NGF mRNA or NGF protein levels when comparing young and aged pineal glands, suggesting that atrophy in aged sympathetic neurons is not causally related to reduced availability of NGF at the target. Our results indicate that mechanisms other than NGF expression need to be explored in order to explain the age-related axonal regression observed in this target.

Ovariectomy up-regulates neuronal neurofilament light chain mRNA expression with regional and temporal specificity.

Estrogens can influence the survival, plasticity and function of many adult neurons. Many of these effects, such as neurite outgrowth and increased dendritic spine density, are mediated by changes in neuronal cytoskeletal architecture. Since neurofilament proteins play a key role in the maintenance and remodeling of the neuronal cytoskeleton, we postulated that changes in neurofilament light chain mRNA may parallel some of the alterations in neuronal architecture which follow bilateral ovariectomy. We measured neurofilament light chain mRNA levels using a ribonuclease protection assay at two time-points after ovariectomy in mature female rats. One week after ovariectomy, neurofilament light chain mRNA levels (corrected for glucose-6-phosphate dehydrogenase mRNA) did not differ from sham-operated animals in the five brain regions examined (hypothalamus, striatum, hippocampus, frontal cortex and occipital cortex). Four months after ovariectomy, neurofilament light chain mRNA levels were similarly unchanged in the hypothalamus and striatum. In contrast, statistically significant increases in neurofilament light chain mRNA expression were observed in the three regions receiving basal forebrain projections (hippocampus, frontal cortex and occipital cortex). In situ hybridization demonstrated increases in neurofilament light chain mRNA expression involving subpopulations of smaller medial septal neurons. There also appeared to be an increased number of larger septal neurons following long-term ovariectomy. We propose that atrophic changes involving basal forebrain projection fibers are followed by compensatory axonal growth by other 'intact' basal forebrain neurons. Increased neurofilament light chain mRNA expression and somatic hypertrophy in medial septal neurons may both be reflective of the need to sustain an axonal network which is larger and more complex. In contrast, increased neurofilament light chain mRNA expression observed in basal forebrain targets following long-term ovariectomy may be reflective of compensatory changes taking place in local neurons.

Recovery of function following unilateral denervation, but not unilateral decentralization, of the pineal gland as indicated by measurements of pineal melatonin content and urinary melatonin metabolites.

The rat pineal gland is an attractive system for studies on the capacity of neural systems to recover following partial injury, allowing both for the creation of precise subtotal lesions and for the measurement of recovery of function at the cellular level. The pineal gland receives overlapping sympathetic innervation from the right and left internal carotid nerves from neurons whose cell bodies are located in the two superior cervical ganglia. This innervation regulates several aspects of pineal metabolism in a circadian fashion, with the most dramatic being a marked increase in the night-time activity of N-acetyltransferase, a key enzyme regulating the rate of melatonin synthesis. We have previously shown that a highly divergent pattern takes place in the night-time activity of this enzyme following two different unilateral lesions of the sympathetic innervation to the gland. Thus, following a unilateral lesion of the internal carotid nerve (unilateral denervation), there is an initial decline in N-acetyltransferase activity; however, normal activity is again seen during the second and subsequent nights. In contrast, a unilateral lesion of the cervical sympathetic trunk, the nerve that innervates the superior cervical ganglion (unilateral decentralization), results in "permanent" impairment of N-acetyltransferase activity. In the present study, we report that the functional capacity of the entire pathway for melatonin synthesis is similarly affected following these lesions, as reflected by the levels of melatonin and of its precursor N-acetylserotonin in the pineal gland, as well as the levels of the main melatonin metabolite 6-hydroxy-melatonin in the urine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Functional recovery and collateral neuronal sprouting examined in young and aged rats following a partial neural lesion.

The rat pineal gland was chosen as a model system to study how aging affects the capacity of surviving neurons to compensate for partial destruction of a neural pathway. The pineal gland receives bilateral overlapping sympathetic innervation from the two internal carotid nerves, whose activity regulates several aspects of pineal metabolism in a circadian fashion. The most dramatic of these is the marked nighttime increase in the activity of N-acetyltransferase, the rate-limiting enzyme in melatonin synthesis. These features allow for the pineal gland to be used as a model system for studies on neuronal plasticity, since it is possible to create specific partial neural lesions and to evaluate functional recovery subsequently at the cellular level. We examined the activity of N-acetyltransferase and the content of melatonin in the pineal gland as indices of pineal function at various time points after unilateral surgical denervation (lesion of one of the two internal carotid nerves) in 4-month-(young) and 25-month-old (aged) rats. At both ages, the nighttime levels of the two parameters were significantly lower 8 h after this lesion than in sham-operated animals of the same age, indicating impaired function. When examined at later time points (i.e., 1.5 and 10 days after this lesion), both young and aged animals exhibited full recovery in these two parameters. Measurement of specific neuronal uptake of [3H]norepinephrine was utilized as an index of the number of sympathetic varicosities innervating the pineal gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Interventional stroke therapies in the elderly: are we helping?

BACKGROUND AND PURPOSE: It is unclear whether endovascular therapies for the treatment of AIS are being offered or are safe in older adults. The use and safety of endovascular interventions in patients older than 75 years of age were assessed. MATERIALS AND METHODS: A retrospective review of patients with AIS 75 years or older (n = 37/1064) was compared with a younger cohort (n = 70/1190) by using an established data base. Admission and discharge NIHSS scores, rates of endovascular treatment, SICH, in-hospital mortality, and the mBI were assessed. RESULTS: Rates of endovascular treatments were significantly lower in older patients (5.9% in the younger-than-75-year versus 3.5% in the older-than-75-year cohort, P = .007). Stroke severity as measured by the NIHSS score was equivalent in the 2 age groups. The mBI at 12 months was worse in the older patients (mild or no disability in 52% of the younger-than-75-year and 22% in the 75-year-or-older cohort, P = .006). Older patients had higher rates of SICH (9% in younger-than-75-year versus 24% in the 75-year-or-older group, P = .04) and in-hospital mortality (26% in younger-than-75-year versus 46% in the 75-year-or-older group, P = .05). CONCLUSIONS: Patients older than 75 years of age were less likely to receive endovascular treatments. Older patients had higher rates of SICH, disability, and mortality. Prospective randomized trials are needed to determine the criteria for selecting patients most likely to benefit from acute endovascular therapies.

Agrin becomes concentrated at neuroeffector junctions in developing rodent urinary bladder.

The formation of somatic neuromuscular junctions in skeletal muscle is regulated by an extracellular matrix protein called agrin. Here, we have examined the expression and localization of agrin during development of the rodent urinary bladder, as a first step to examining its possible role at autonomic neuroeffector junctions in smooth muscle. We have found that agrin is expressed on the surface of developing smooth muscle cells and in the basement membrane underlying the urothelium. More importantly, agrin is progressively concentrated at parasympathetic varicosities during postnatal development and is present at virtually all junctions in mature muscle. Reverse transcription/polymerase chain reaction analysis has shown that pelvic ganglion neurons that innervate the bladder express LN/z8 agrin, whereas bladder smooth muscle expresses LN/z- agrin. Together, these results demonstrate that nerve and/or muscle agrin becomes localized at cholinergic parasympathetic varicosities in smooth muscle, where it could play a role in the maturation of the neuroeffector junction.

Estrogen replacement raises rat CRP without evidence of complement activation.

Given current controversies regarding anti- and pro-inflammatory effects of estrogen, there is a need to explore relationships between gonadal hormones and inflammation using appropriate animal models. It has been proposed that rats are not appropriate for such research since, contrary to the effect of estrogen in humans, earlier animal studies had reported that estrogen downregulates serum C-reactive protein (rCRP) levels in the rat. With these considerations in mind, we re-examined the effects of estrogen withdrawal and replacement on CRP expression and complement activation in the rat. F-344 rats underwent bilateral ovariectomy or sham surgery at 9-10 months of age. Four months later, ovariectomized rats were treated with traditional high-dose 17beta-estradiol (Hi-E2) capsules, lower-dose (Lo-E2) 17beta-estradiol capsules, or placebo capsules for 7 days prior to sacrifice. Levels of plasma rat C-reactive protein (rCRP) were significantly lower in ovariectomized vs. sham-operated animals (415.5 +/- 10.6 vs. 626.6 +/- 23.0 mg/L, p < 0.001). Estrogen replacement significantly raised rCRP levels in ovariectomized animals (690.0 +/- 28.0 mg/L in Lo-E2 and 735.5 +/- 35.8 mg/L in Hi-E2, respectively, p < 0.001). Plasma rCRP levels correlated significantly with both hepatic rCRP (r = 0.79, p < 0.001) and serum estradiol (r = 0.70, p < 0.001) levels. However, no significant differences were observed in indices of complement activation (C4b/c) or CRP-complement complex generation (rCRP-C3 complex). In the mature female rat, ovariectomy reduces and estrogen replacement raises rCRP. Effects of estrogen on plasma rCRP induction are mediated, at least in part, through hepatic mechanisms and do not appear to require or be associated with complement activation.

Alterations in target innervation and collateral sprouting in the aging sympathetic nervous system.

The ability to sustain appropriate target innervation and to undergo collateral sprouting following losses of related neural inputs may favor the maintenance of normal function in adult and aged organisms. Young (4 month old) rats underwent a unilateral sympathetic denervation of the pineal gland and 1 day later exhibited an approximately 50% decrease in the area fraction represented by tyrosine hydroxylase (TH)-immunoreactive profiles in this target tissue. Ten days after this lesion, the density of TH immunoreactivity increased to over 80% of control values. In aged (25 month old) animals, endogenous fluorescence produced by the presence of lipofuscin was subtracted from the captured image, revealing a more than 50% decrease in innervation density to this target tissue with aging. The density of TH-immunoreactive profiles decreased by approximately one-half in aged animals lesioned 1 day earlier. However, 10 days after a unilateral denervation it was still approximately one-half of that obtained in control aged rats, providing morphologic support for a failure in collateral sprouting with aging in this system.

Transynaptic regulation of low-affinity p75 nerve growth factor receptor mRNA precedes and accompanies lesion-induced collateral neuronal sprouting.

The bilateral sympathetic innervation of the rat pineal gland from the two superior cervical ganglia (SCG) is a useful model system to investigate the mechanisms by which intact neurons compensate for neuronal losses. Cutting of the internal carotid nerve (ICN) on one side has been shown to result in the removal of approximately one-half of the innervation to the pineal gland within 2 days. This denervation is followed by the development of collateral neuronal sprouting from the contralateral "intact" SCG, most of which takes place during the next 2 days. Using a solution hybridization protection assay, levels of low-affinity NGF receptor p75NGFR mRNA (pg/microgram total RNA) were found to be increased 25%, with no change in cyclophilin mRNA, in the SCG contralateral to the lesion performed 1 or 3 days earlier. In situ hybridization with a 35S riboprobe complementary to p75NGFR mRNA demonstrated a large increase in this mRNA in some cells of this intact SCG at both 1 and 3 days after a contralateral ICN cut lesion. The clustering of these cells toward the rostral portion of the SCG suggests that they may overlap with the population of sympathetic neurons which provides innervation to bilaterally innervated structures such as the pineal gland. The nature of the signals involved in the regulation of NGF receptor mRNA levels and their role in initiating and maintaining collateral sprouting remain to be fully established. Nevertheless, the time course of the changes in mRNA levels suggests that regulation of the low-affinity NGF receptor gene may be involved in the sequence of events associated with the collateral sprouting response by intact sympathetic nerve cells following partial denervation.

Decline in response to nicotine in aged rat striatum: correlation with a decrease in a subpopulation of nicotinic receptors.

Specific and reproducible changes involving the cholinergic and dopaminergic systems have been described in both the aging rodent and the human nervous system. Nevertheless, relatively little information is available on changes in nicotinic cholinergic receptors occurring in normal aging, and there have been few attempts to correlate alterations in receptor densities with changes in nicotinic actions. We have utilized the nicotine-mediated stimulation of endogenous dopamine efflux in a striatal slice preparation as a functional index of responsiveness to nicotine in aging. Following incubation with nicotine, this efflux was significantly lower in 25-month-old (aged) as opposed to 4-month-old (young) rats. In contrast, the release of striatal dopamine following a high-potassium stimulus was similar at both ages. Binding studies in young and aged animals did not reveal any significant change with age in the total number of striatal nicotinic receptors recognized by either [3H]nicotine or the neuronal nicotinic antagonist 125I-neuronal bungarotoxin. However, there was a nearly 80% decline in the subpopulation of striatal nicotinic receptors jointly recognized by both nicotine and neuronal bungarotoxin, but not by alpha-bungarotoxin. Quantitative autoradiography demonstrated declines with age in this receptor subtype in several brain regions examined. Decrements in this specific subpopulation of nicotinic receptors or in the nerve cells expressing these receptors may contribute to the functional declines that take place in the aging motor and visual systems.

Experience of an incontinence clinic for older women: no apparent age limit for potential physical and psychological benefits.

Urinary incontinence (UI) is a common but undertreated condition in older women. Although a variety of noninvasive interventions is available, older women may be hesitant to seek care for UI because of misconceptions about normal aging and treatment futility. We sought to evaluate the effectiveness of a UI clinic specifically tailored to the needs of older women to promote a sense of empowerment and to enhance satisfaction with treatment and outcome. We describe a case series of 52 women between the ages of 65 and 98 who were evaluated at the Geriatric Incontinence Clinic at the McGill University Health Centre over a 1-year period. A standardized telephone questionnaire was administered by a nurse consultant 6 months after each subject's final visit to assess patient satisfaction and current incontinence status. Forty-five women (86%) were available for telephone follow-up and completed the questionnaire. Mean age was 80 years, with urge incontinence in 45%, mixed incontinence (stress and urge) in 33%, impaired bladder emptying with urge symptoms in 10%, and other diagnoses in 12%. Overall, a mean reduction of 1.4 incontinent episodes per day was reported. At follow-up, 30% of the subjects reported being cured of their incontinence, 30% had improved, 20% were the same, and 20% were worse. Over 85% of all women reported satisfaction with their new incontinence status. Women of all ages, independent of the type of UI, type of treatment, and cognitive status, were able to achieve reductions in incontinence symptoms. All patients who had worsened were noncompliant with treatment recommendations at follow-up. Older women can derive significant benefit from a UI assessment. Neither advanced age nor category of incontinence precludes improvements or enhanced satisfaction with treatment. Efforts to improve targeting and compliance may improve outcomes.