Metabolic enrichment of omega-3 polyunsaturated fatty acids does not reduce the onset of idiopathic knee osteoarthritis in mice.

OBJECTIVE: We evaluated the effect of a reduction in the systemic ratio of n-6:n-3 polyunsaturated fatty acids (PUFAs) on changes in inflammation, glucose metabolism, and the idiopathic development of knee osteoarthritis (OA) in mice. We hypothesized that a lower ratio of n-6:n-3 PUFAs would protect against OA markers in cartilage and synovium, but not bone. DESIGN: Male and female fat-1 transgenic mice (Fat-1), which convert dietary n-6 to n-3 PUFAs endogenously, and their wild-type (WT) littermates were fed an n-6 PUFA enriched diet for 9-14 months. The effect of gender and genotype on serum PUFAs, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and glucose tolerance was tested by 2-factor analysis of variance (ANOVA). Cortical and trabecular subchondral bone changes were documented by micro-focal computed tomography (CT), and knee OA was assessed by semi-quantitative histomorphometry grading. RESULTS: The n-6:n-3 ratio was reduced 12-fold and 7-fold in male and female Fat-1 mice, respectively, compared to WT littermates. IL-6 and TNF-α levels were reduced modestly in Fat-1 mice. However, these systemic changes did not reduce osteophyte development, synovial hyperplasia, or cartilage degeneration. Also the fat-1 transgene did not alter subchondral cortical or trabecular bone morphology or bone mineral density. CONCLUSIONS: Reducing the systemic n-6:n-3 ratio does not slow idiopathic changes in cartilage, synovium, or bone associated with early-stage knee OA in mice. The anti-inflammatory and anti-catabolic effects of n-3 PUFAs previously reported for cartilage may be more evident at later stages of disease or in post-traumatic and other inflammatory models of OA.

Long-term deficiency of circulating and hippocampal insulin-like growth factor I induces depressive behavior in adult mice: a potential model of geriatric depression.

Numerous studies support the hypothesis that deficiency of insulin-like growth factor I (IGF-1) in adults contributes to depression, but direct evidence is limited. Many psychological and pro-cognitive effects have been attributed to IGF-1, but appropriate animal models of adult-onset IGF-1 deficiency are lacking. In this study, we use a viral-mediated Cre-loxP system to knockout the Igf1 gene in either the liver, neurons of the CA1 region of the hippocampus, or both. Knockout of liver Igf1 reduced serum IGF-1 levels by 40% and hippocampal IGF-1 levels by 26%. Knockout of Igf1 in CA1 reduced hippocampal IGF-1 levels by 13%. The most severe reduction in hippocampal IGF-1 occurred in the group with knockouts in both liver and CA1 (36% reduction), and was associated with a 3.5-fold increase in immobility in the forced swim test. Reduction of either circulating or hippocampal IGF-1 levels did not alter anxiety measured in an open field and elevated plus maze, nor locomotion in the open field. Furthermore, local compensation for deficiencies in circulating IGF-1 did not occur in the hippocampus, nor were serum levels of IGF-1 upregulated in response to the moderate decline of hippocampal IGF-1 caused by the knockouts in CA1. We conclude that adult-onset IGF-1 deficiency alone is sufficient to induce a depressive phenotype in mice. Furthermore, our results suggest that individuals with low brain levels of IGF-1 are at increased risk for depression and these behavioral effects are not ameliorated by increased local IGF-1 production or transport. Our study supports the hypothesis that the natural IGF-1 decline in aging humans may contribute to geriatric depression.

Early-onset growth hormone deficiency results in diastolic dysfunction in adult-life and is prevented by growth hormone supplementation.

OBJECTIVE: The primary goal of growth hormone (GH) replacement is to promote linear growth in children with growth hormone deficiency (GHD). GH and insulin-like growth factor-1 (IGF-1) are also known to have roles in cardiac development and as modulators of myocardial structure and function in the adult heart. However, little is known about cardiac diastolic function in young adults with childhood onset GH deficiency in which GH treatment was discontinued following puberty. The aim of the study was to evaluate the effects of long standing GHD and peri-pubertal or continuous GH replacement therapy on diastolic function in the adult dwarf rat. DESIGN: The dwarf rat, which possesses a mutation in a transcription factor necessary for development of the somatotroph, does not exhibit the normal peri-pubertal rise in GH around day 28 and was used to model childhood or early-onset GHD (EOGHD). In another group of male dwarfs, GH replacement therapy was initiated at 4 weeks of age when GH pulsatility normally begins. Ten weeks after initiation of injections, GH-treated dwarf rats were divided into 2 groups; continued treatment with GH for 12 weeks (GH-replete) or treatment with saline for 12 weeks. This latter group models GH supplementation during adolescence with GHD beginning in adulthood (adult-onset GHD; AOGHD). Saline-treated heterozygous (HZ) rats were used as age-matched controls. At 26 weeks of age, cardiac function was assessed using invasive or noninvasive (conventional and tissue Doppler) indices of myocardial contractility and lusitropy. RESULTS: Systolic function, as determined by echocardiography, was similar among groups. Compared with HZ rats and GH-replete dwarfs, the EOGHD group exhibited significant reductions in myocardial relaxation and increases in left ventricular filling pressure, indicative of moderate diastolic dysfunction. This was further associated with a decrease in the cardiac content of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), one of the important cardiac calcium regulatory proteins. Dwarfs supplemented with GH during the peri-adolescence stage, but not beyond (AOGHD), exhibited a subtle prolongation in the deceleration time to early filling. In contrast, continual GH replacement preserved diastolic function such that the cardiac phenotype of the GH-replete dwarfs resembled that of their age-matched HZ counterpart. DISCUSSION: Our data indicate that GHD during adolescence leads to overt diastolic dysfunction in early adulthood and this is prevented by continual GH replacement therapy. Since discontinuation of GH replacement following adolescence only mitigated the lusitropic deficits that were observed in untreated dwarfs, GH treatment into adulthood could be beneficial.

Early-onset GH deficiency results in spatial memory impairment in mid-life and is prevented by GH supplementation.

GH levels increase to high concentrations immediately before puberty then progressively decline with age. GH deficiency (GHD) originating in childhood is treated with GH supplementation to foster somatic development during adolescence. It is not clear if or how early GH replacement affects memory in adulthood, or whether it can prevent the cognitive deficits commonly observed in adults with childhood-onset GHD. Rats homozygous for the Dw-4 mutation (dwarf) do not exhibit the normal increase in GH at 4 weeks of age when GH levels normally rise and are used to model childhood or early-onset GHD (EOGHD). One group of these rats was injected with GH from 4 to 14 weeks of age to model GH supplementation during adolescence with GHD beginning in adulthood (adult-onset GHD; AOGHD). Another group received GH from 4 weeks throughout the lifespan to model normal lifespan GH (GH-replete). Age-matched, Dw-4 heterozygous rats (HZ) do not express the dwarf phenotype and were used as controls. At 8 and 18 months of age, spatial learning in the water maze was assessed. At 8 months of age all experimental groups were equally proficient. However, at 18 months of age, the EOGHD group had poor spatial learning compared to the AOGHD, GH-replete, and HZ groups. Our data indicate that GHD during adolescence has negative effects on learning and memory that emerge by middle-age unless prevented by GH supplementation.

Basal and hypercapnia-altered cerebrovascular perfusion predict mild cognitive impairment in aging rodents.

With increasing age, a subset of otherwise healthy individuals undergoes impairments in learning and memory that have been termed mild cognitive impairment (MCI). The enhanced neuronal activity associated with learning and memory requires increased cerebral blood flow (CBF) to specific brain regions. However, the interactions between cerebral blood flow and MCI remain unclear. In this study, we address whether baseline or hypercapnia-induced (increased blood CO(2) levels) changes in CBF are modified with age, and whether these measures are predictive of cognitive status in rodents. Adult and aged rats were evaluated using a hippocampally-dependent task in a water maze. Aged rats were classified as memory-impaired or memory-intact based on performance comparisons with adult rats. Cerebral blood flow was assessed using flow-alternating inversion recovery (FAIR) magnetic resonance imaging (MRI), before and after breathing 10% CO(2). The transition period between CO(2) concentrations was examined with blood oxygen level dependent (BOLD) MRI. Separation of aged animals into memory-intact and impaired categories revealed increased basal perfusion in the dorsal hippocampus of memory-impaired versus memory-intact aged animals. Linear regression revealed that higher hippocampal perfusion was correlated with impaired memory in aged animals, and a logistic regression indicated that hippocampal perfusion predicted spatial memory ability. Several brain regions of aged rats demonstrated an attenuation of the perfusion increase normally observed in adult rats under hypercapnia. Memory-impaired animals were the primary contributor to this effect, as their perfusion response to hypercapnia was significantly reduced compared to adult animals. Aged, memory-intact animals were not significantly different from adults. BOLD MRI demonstrated a reduced response in aged animals to hypercapnia, with impaired animals being the primary contributor to the effect. A logistic regression model based on basal and hypercapnia perfusion correctly predicted cognitive status in 83.3% of animals tested. Our results indicate that age-related changes in vascular reactivity and perfusion are important contributing factors in memory impairment.

The ependymal route for insulin-like growth factor-1 gene therapy in the brain.

I.c.v. administration of the peptide insulin-like growth factor-1 (IGF-1) has been shown to be an effective neuroprotective strategy in the brain of different animal models, a major advantage being the achievement of high concentrations of IGF-1 in the brain without altering serum levels of the peptide. In order to exploit this therapeutic approach further, we used high performance recombinant adenoviral (RAd) vectors expressing their transgene under the control of the potent mouse cytomegalovirus immediate early (mCMV) promoter, to transduce brain ependymal cells with high efficiency and to achieve effective release of transgenic IGF-1 into the cerebrospinal fluid (CSF). We constructed RAd vectors expressing either a chimeric green fluorescent protein fused to HSV-1 thymidine kinase (TK/GFP)(fus), or the cDNA encoding rat IGF-1, both driven by the mCMV promoter. The vectors were injected into the lateral ventricles of young rats and chimeric GFP expression in brain sections was assessed by fluorescence microscopy. The ependymal cell marker vimentin was detected by immunofluorescence and nuclei were labeled with the DNA dye 4',6-diamidino-2-phenylindole. Blood and CSF samples were drawn at different times post-vector injection. In all cerebral ventricles, vimentin immunoreactive cells of the ependyma were predominantly transduced by RAd-(TK/GFP)(fus), showing nuclear and cytoplasmic expression of the transgene. For tanycytes (TK/GFP)(fus) expression was evident in their cytoplasmic processes as they penetrated deep into the hypothalamic parenchyma. I.c.v. injection of RAd-IGF-1 induced high levels of IGF-1 in the CSF but not in serum. We conclude that the ependymal route constitutes an effective approach for implementing experimental IGF-1 gene therapy in the brain.

Cognitive performance and age-related changes in the hippocampal proteome.

Declining cognitive performance is associated with increasing age, even in the absence of overt pathological processes. We and others have reported that declining cognitive performance is associated with age-related changes in brain glucose utilization, long-term potentiation and paired-pulse facilitation, protein expression, neurotransmitter levels, and trophic factors. However, it is unclear whether these changes are causes or symptoms of the underlying alterations in dendritic and synaptic morphology that occur with age. In this study, we examined the hippocampal proteome for age- and cognition-associated changes in behaviorally stratified young and old rats, using two-dimensional in-gel electrophoresis and MS/MS. Comparison of old cognitively intact with old cognitively impaired animals revealed additional changes that would not have been detected otherwise. Interestingly, not all age-related changes in protein expression were associated with cognitive decline, and distinct differences in protein expression were found when comparing old cognitively intact with old cognitively impaired rats. A large number of protein changes with age were related to the glycolysis/gluconeogenesis pathway. In total, the proteomic changes suggest that age-related alterations act synergistically with other perturbations to result in cognitive decline. This study also demonstrates the importance of examining behaviorally-defined animals in proteomic studies, as comparison of young to old animals regardless of behavioral performance would have failed to detect many cognitive impairment-specific protein expression changes evident when behavioral stratification data were used.

Growth hormone treatment attenuates age-related changes in hippocampal short-term plasticity and spatial learning.

Downregulation of the growth hormone/insulin-like growth factor-1 (IGF-1)axis is one of the most robust biomarkers of mammalian aging. Reports have suggested that age-related changes in secretion of growth hormone and IGF-1 contribute to the development of some peripheral characteristics of the aged phenotype including decreased bone density and lean body mass. Recent work has focused on the identification of a role for age-related reductions in growth hormone and IGF-1 in the development of cognitive impairments associated with aging. In the current study, we report that aged (30 month-old) Brown Norway x Fisher rats demonstrate impairments in spatial learning compared with adult (10 month-old) animals, and that 4-month treatment with growth hormone (300 microg twice daily) attenuates age-related learning impairments. After 6 months of treatment, we employed an extracellular paired-pulse protocol to investigate age-related changes in hippocampal short-term plasticity, and found that aged rats exhibit significantly increased paired-pulse ratios (PPRs) at an interpulse interval of 50 ms compared with adult rats. Long-term growth hormone administration restored PPRs in aged animals to values comparable to those observed in adult controls. Since the age-related changes observed in PPR may result from decreases in hippocampal inhibitory tone mediated by GABA(A) receptors, we assessed GABA(A) receptor subunit expression by immunoblot analysis. Data revealed significant age-related decreases in GABA(A) receptor alpha-1 subunit expression which were attenuated by growth hormone treatment. However, hippocampal levels of the gamma2 subunit, glutamic acid decarboxylase (GAD)(65), and GAD(67) protein concentrations were not significantly affected by age or growth hormone treatment. In conclusion, we suggest that age-related decreases in growth hormone and IGF-1 contribute to cognitive decline, in part, via alterations in hippocampal short-term plasticity. Changes in plasticity may reflect a shift in the balance of hippocampal inhibitory and excitatory function.

Effects of age and insulin-like growth factor-1 on neuron and synapse numbers in area CA3 of hippocampus.

Age-related effects associated with the hippocampus include declines in numbers of neurons and synapses in the dentate gyrus and area CA1, and decreased cognitive ability as assessed with the Morris water maze. The present study quantified both neuron and synapse number in the same tissue block of area CA3 of the hippocampus. No investigations of both density of neurons and synapses together in area CA3 of hippocampus have been performed previously, despite its importance as the terminal field of dentate gyrus mossy fibers, the second synapse in the trisynaptic circuit in the hippocampus. Numerical density of neurons and synapses were assessed in 4-, 18-, and 29-month-old rats receiving infusions of saline into the lateral ventricle and in 29-month-old rats receiving infusions of insulin-like growth factor-1 (IGF-1). Numerical density of neurons of the stratum pyramidale of CA3 of hippocampus remained constant across the life span as did the numerical density of synapses in stratum lucidum of area CA3. Despite the reported role of IGF-1 in synaptogenesis and improvements in behavior with age, ventricular infusion of this growth factor did not affect the numerical density of neurons or synapses in 29-month-old rats when compared to saline-infused old rats. Further, reported effects of IGF-1 on adult neurogenesis in the dentate gyrus are not reflected in an IGF-1-related increase in synapse density in this region.

Intracerebroventricular infusion of insulin-like growth factor-I ameliorates the age-related decline in hippocampal neurogenesis.

The dentate gyrus of the hippocampus is one of few regions in the adult mammalian brain characterized by ongoing neurogenesis. Significantly, recent studies indicate that the rate of neurogenesis in the hippocampus declines with age, perhaps contributing to age-related cognitive changes. Although a variety of factors may influence the addition of new neurons in the adult dentate gyrus, the mechanisms responsible for the age-related reduction remain to be established. Insulin-like growth factor-I (IGF-I) is one promising candidate to regulate neurogenesis in the adult and aging brain since it influences neuronal production during development and since, like the rate of neurogenesis, it decreases with age. In the current study, we used bromodeoxyuridine labeling and multilabel immunofluorescence to assess age-related changes in neuronal production in the dentate gyrus of adult Brown Norway x Fischer 344 rats. In addition, we investigated the relationship between changes in neurogenesis and the age-dependent reduction in IGF-I by evaluating the effect of i.c.v. infusion of IGF-I on neurogenesis in the senescent dentate gyrus. The analyses revealed an age-dependent reduction in the number of newly generated cells in the adult dentate subgranular proliferative zone and, in addition, a 60% reduction in the differentiation of newborn cells into neurons. Restoration of IGF-I levels in senescent rats significantly restored neurogenesis through an approximately three-fold increase in neuronal production. The results of this study suggest that IGF-I may be an important regulator of neurogenesis in the adult and aging hippocampus and that an age-related decline in IGF-I-dependent neurogenesis could contribute to age-related cognitive changes.

Growth hormone increases regional coronary blood flow and capillary density in aged rats.

In this study, we examined the effects of age and growth hormone replacement on both coronary blood flow and capillary density. Blood flow was measured by using [(14)C]-iodoantipyrine in three groups of anesthetized Brown Norway x Fischer 344 rats: young vehicle-treated animals (6 months; n = 13), old vehicle treated animals (30 months; n = 9), and old animals treated with bovine growth hormone (200 microg/kg) twice a day for 30 days (30 months; n = 7). Capillary density was measured by color segmentation analysis of sections stained for platelet endothelial cell adhesion molecule-1. In all regions examined, coronary blood flow decreased with age, and growth hormone administration resulted in an increase in flow compared to vehicle-treated animals. Capillary density decreased with age in the apex and the left ventricular middle segment. In response to growth hormone administration, capillary density increased significantly in the apex but not in other regions of the heart. Our results demonstrate that growth hormone enhances regional myocardial blood flow in the aged heart and suggest that part of this effect could be due to an increase in capillary density.

Assessment of physical function in aging rodents: a strategy for improving preclinical testing.

Geriatric medicine has recently focused on the biological mechanisms contributing to disability in the activities of daily living (ADLs), with special emphasis given to the study of sarcopenia, an age-related decline in muscle mass. Explaining the etiology of sarcopenia may provide useful information for the development of targeted interventions, especially those that are pharmacological in nature. However, exploratory studies aimed at evaluating the long-term effects of a particular intervention are costly and time consuming in a clinical setting. Therefore there is a need for preclinical testing of the efficacy of pharmacotherapies. This review provides (1) example of factors that contribute to the incidence of disability; (2) the conceptualization of a widely accepted human model of disability applied to an animal model; and (3) information on the potential advantages that may be realized from such translational research.

Growth hormone reverses age-related cardiac myofilament dysfunction in rats.

We tested the hypotheses that aging is associated with a reduction in overall cardiac contractility and myofilament force generation that could be reversed with growth hormone (GH) replacement. Three groups of male Brown-Norway rats were studied: young (Y(SAL): 8 mo old, n = 13), old (O(SAL): 28 mo old, n = 13), and old GH-treated (O(GH): 28 mo old, n = 12; 300 microg bovine GH, twice a day for 30 days). The left ventricular (LV) pressure-volume relation was derived in isolated hearts, after which isolated trabecular muscles from these hearts were permeabilized and maximal myofilament force generation (Fmax) was measured. LV developed pressures at a LV volume of 0.3 ml were significantly depressed with age: 84 +/- 6 vs. 71 +/- 6 mmHg (Y(SAL) vs. O(SAL), respectively, P = 0.001) and not restored by GH (69 +/- 4 mmHg). Fmax was reduced in the aged hearts: 47.5 +/- 3.12 vs. 35.9 +/- 3.03 mN/mm2 (Y(SAL) vs. O(SAL), respectively, P = 0.014) but was restored with GH replacement to 46.7 +/- 3.12 mN/mm2 (O(SAL) vs. O(GH), P = 0.021). Our results suggest that cellular myofilament contractility is reduced with aging and restored with GH replacement.

Insulin-like growth factor-1 selectively increases glucose utilization in brains of aged animals.

Previous studies indicate that insulin-like growth factor-1 is an important neurotrophic agent and that decreases in brain concentrations of IGF-1 and the type 1 IGF receptor have an important role in the age-related decline in memory, neuronal function and possibly dendritic architecture. In this study, we assessed the effects of age and IGF-1 replacement on local cerebral glucose utilization (LCGU). Three groups of male Brown-Norway rats (7, 18 and 28 months of age) were implanted with Alzet minipumps and either saline or IGF-1 (50ng/0.5 microliter/hour) was infused into the lateral ventricle for 28 days. On day 28, LCGU was measured by infusion of 2-[(14)C]deoxyglucose during the dark phase of the light/dark cycle. Results indicate that glucose utilization significantly decreased with age throughout the brain including the anterior cingulate, sensorimotor and retrosplenial cortex, CA1, CA3 and dentate gyrus of hippocampus and several regions of the hypothalamus. Administration of IGF-1 to aged animals increased rates of LCGU in the anterior cingulate of the cortex (14.2%), CA1 region of the hippocampus (11.0%) and the arcuate nucleus of the hypothalamus (12.0%). Our results indicate that although glucose utilization decreases with age throughout the brain, the effects of IGF-1 infusion are manifest only in specific brain regions. Since IGF-1 has been shown to reverse the age-related decrease in memory, these results suggest that despite the wide distribution of the type 1 IGF receptor the actions of IGF-1 on glucose utilization are highly localized. Additionally, the close association between glucose utilization and excitatory amino acid activity suggests that IGF-1 may act on specific neural pathways to increase glutamate activity in brain regions associated with learning and memory.

Reduction in the chondrocyte response to insulin-like growth factor 1 in aging and osteoarthritis: studies in a non-human primate model of naturally occurring disease.

OBJECTIVE: Although the development of osteoarthritis (OA) is closely associated with aging, the mechanism for this association is not clear. This study was designed to determine the effects of aging and OA on the chondrocyte response to stimulation with insulin-like growth factor 1 (IGF-1) in a non-human primate model of naturally occurring OA. METHODS: Chondrocytes were isolated from cartilage removed separately from the medial and lateral femoral condyles and tibial plateaus of cynomolgus monkeys at the time of necropsy. Each joint site was scored histologically on a scale of 0-7 for OA pathologic changes. Isolated chondrocytes were cultured in alginate in serum-free medium and stimulated with IGF-1 or des(1-3) IGF-1, which has a much lower affinity for IGF binding proteins (IGFBP) than IGF-1. Response was measured as the ability to stimulate sulfate and proline incorporation. RESULTS: Cartilage samples from 34 monkeys ranging in age from 6.7 years to 27 years and with histologic scores ranging from 0 to 7 were analyzed. A significant decline in the response to IGF-1 was noted with both increasing age and increasing OA score. Controlling for the OA score, the estimated effect of age on IGF-1 response, measured by total sulfate incorporation, was a decline of 3.81% per year (P = 0.0001), or a 75% decline over 20 years as a monkey ages from young to older adult. Controlling for age, the effect of OA score was significant only for proline incorporation in the alginate matrix (estimated slope coefficient +/-standard error -15.9 +/- 7.2; P = 0.03), suggesting a negative effect of OA on retention of 3H-proline-labeled proteins in the matrix. There was a significantly reduced response to des(1-3) IGF-1 with increasing age, but no effect of OA score on response to des(1-3) IGF-1. There was no effect of age on cell viability. CONCLUSION: These results demonstrate a significant age-related decline in the chondrocyte response to IGF-1. The finding that increasing OA score was associated with a reduced response to intact IGF-1 but not des(1-3) IGF-1 suggests a role of increased production of inhibitory IGFBP in OA. Since the cells from older animals had a reduced response to both forms of IGF-1, the mechanism of the reduced response with age cannot be attributed to changes in IGFBP. Age-related changes in IGF receptors or, more likely, age-related alterations in intracellular signal transduction may also be involved.

Age-related synaptic changes in sensorimotor cortex of the Brown Norway X fischer 344 rat.

Investigations of age-related changes in synapse density have yielded contradictory conclusions. The goal of the present study was to determine whether there is a significant decline in the number of cortical synapses in old age. Therefore, brains from 10-, 15-, and 32-month-old Brown Norway X Fischer 344 rats were prepared for electron microscopy and synapses were counted in a stereotaxically-identified region of sensorimotor cortex. Within this cortical area, synapses were counted in layers 2 and 4 and the data have been presented both as number of synapses per volume of neuropil and as the ratio of synapses per neuron. Results indicated that there was a decline in synapse density between 15 and 32 months in layer 2, but not in layer 4. This decline was significant not only for total synapses, but also for subcategories of synapses when classified by presynaptic features or postsynaptic element. Specifically, there was a significant decline in presumptive inhibitory synaptic terminals, i.e., those containing nonround synaptic vesicles, as well as a significant decline in synapses that contact dendritic spines.

Chronic [D-Ala2]-growth hormone-releasing hormone administration attenuates age-related deficits in spatial memory.

The age-related decline in growth hormone is one of the most robust endocrine markers of biological aging and has been hypothesized to contribute to the physiological deficits observed in aged animals. However, there have been few studies of the impact of this hormonal decline on brain aging. In this study, the effect of long-term subcutaneous administration of [D-Ala2]-growth hormone-releasing hormone (GHRH) on one measure of brain function, memory, was investigated. Animals were injected daily with 2.3 microg of [D-Ala2]-GHRH or saline from 9 to 30 months of age, and the spatial learning and reference memory of animals were assessed by using the Morris water maze and compared with those of 6-month-old animals. Results indicated that spatial memory decreased with age and that chronic [D-Ala2]-GHRH prevented this age-related decrement (24% improvement in the annulus-40 time and 23% improvement in the number of platform crossings compared with saline treated, age-matched controls; p < .05 each). No changes were noted in sensorimotor performance. [D-Ala2]-GHRH attenuated the age-related decline in plasma concentrations of insulinlike growth factor-1 (IGF-1) (p <.05). These data suggest that growth hormone and IGF-1 have important effects on brain function, that the decline in growth hormone and IGF-1 with age contributes to impairments in reference memory, and that these changes can be reversed by the chronic administration of GHRH.

Age and insulin-like growth factor-1 modulate N-methyl-D-aspartate receptor subtype expression in rats.

N-Methyl-D-aspartate (NMDA) receptors have been reported to have an important role in synaptic plasticity and neurodegeneration. Two major subtypes of these receptors, NMDAR1 and NMDAR2, are present in brain and heterogeneity of these receptors have been reported to define specific functional responses. In this study, the effects of age and chronic insulin-like growth factor-1 (IGF-1) administration on NMDA receptor density and subtype expression were investigated in frontal cortex, CA1, CA2/3 and the dentate gyrus of the hippocampus of young (10 months), middle-aged (21 months) and old (30 months) male Fisher 344xBrown Norway (F1) rats. No age-related changes in (125)I-MK-801 binding or NMDAR1 protein expression were observed in hippocampus or frontal cortex. However, analysis of NMDAR2A and NMDAR2B protein expression in hippocampus indicated a significant decrease between 21 and 30 months of age and administration of IGF-1 increased these receptor subtypes. In cortex, NMDAR2A and NMDAR2B protein expression were not influenced by age or IGF-1 treatment, although NMDAR2C protein expression decreased with age and this decline was not ameliorated by IGF-1 administration. These data demonstrate that NMDA receptor subtypes are altered with age in a regional and subtype specific manner. We conclude that both age and IGF-1 regulate the expression of NMDA receptor subtypes and suggest that age-related changes in NMDA receptor heterogeneity may result in functional changes in the receptor that have relevance for aging.

The effects of growth hormone and IGF-1 deficiency on cerebrovascular and brain ageing.

Research studies clearly indicate that age-related changes in cellular and tissue function are linked to decreases in the anabolic hormones, growth hormone and insulin-like growth factor (IGF)-1. Although there has been extensive research on the effects of these hormones on bone and muscle mass, their effect on cerebrovascular and brain ageing has received little attention. We have also observed that in response to moderate calorie restriction (a treatment that increases mean and maximal lifespan by 30-40%), age-related decreases in growth hormone secretion are ameliorated (despite a decline in plasma levels of IGF-1) suggesting that some of the effects of calorie restriction are mediated by modifying the regulation of the growth hormone/IGF-1 axis. Recently, we have observed that microvascular density on the surface of the brain decreases with age and that these vascular changes are ameliorated by moderate calorie restriction. Analysis of cerebral blood flow paralleled the changes in vasculature in both groups. Administration of growth hormone for 28 d was also found to increase microvascular density in aged animals and further analysis indicated that the cerebral vasculature is an important paracrine source of IGF-1 for the brain. In subsequent studies, administration of GHRH (to increase endogenous release of growth hormone) or direct administration of IGF-I was shown to reverse the age-related decline in spatial working and reference memory. Similarly, antagonism of IGF-1 action in the brains of young animals impaired both learning and reference memory. Investigation of the mechanisms of action of IGF-1 suggested that this hormone regulates age-related alterations in NMDA receptor subtypes (e.g. NMDAR2A and R2B). The beneficial role of growth hormone and IGF-1 in ameliorating vascular and brain ageing are counterbalanced by their well-recognised roles in age-related pathogenesis. Although research in this area is still evolving, our results suggest that decreases in growth hormone and IGF-1 with age have both beneficial and deleterious effects. Furthermore, part of the actions of moderate calorie restriction on tissue function and lifespan may be mediated through alterations in the growth hormone/IGF-1 axis.

Effects of moderate caloric restriction on cortical microvascular density and local cerebral blood flow in aged rats.

The present study was designed to assess the impact of moderate caloric restriction (60% of ad libitum fed animals) on cerebral vascular density and local cerebral blood flow. Vascular density was assessed in male Brown-Norway rats from 7-35 months of age using a cranial window technique. Arteriolar density, arteriole-arteriole anastomoses, and venular density decreased with age and these effects were attenuated by moderate caloric restriction. Analysis of local cerebral blood using [14C]iodoantipyrine indicated that basal blood flow decreased with age in CA1, CA3 and dentate gyrus of hippocampus; similar trends were evident in cingulate, retrosplenal, and motor cortex. Basal blood flow was increased in all brain regions of moderate caloric restricted old animals (compared to old ad libitum fed animals) and no differences were observed between ad libitum fed young and caloric restricted older animals. In response to a CO2 challenge to maximally dilate vessels, blood flow increased in young and old ad libitum fed animals, but a similar increase was not observed in caloric restricted old animals. We conclude that a decrease in cerebral vasculature is an important contributing factor in the reduction in blood flow with age. Nevertheless, vessels from young and old animals have the capacity to dilate in response to a CO2 challenge and, after CO2, no differences are observed between the two age-groups. These results are consistent with the hypothesis that aged animals fail to adequately regulate local cerebral blood flow in response to physiological stimuli. Moderate caloric restriction increases microvascular density and cerebral blood flow in aged animals but tissues exhibit little or no increase in blood flow in response to CO2 challenge. The cause of this deficient response may indicate that vessels are maximally dilated in aged calorically restricted animals or that they fail to exhibit normal regulatory control.