The etiology of radiation-induced cerebrovascular rare-faction remains unknown. In the present study, we examined the effect of whole-brain irradiation on endothelial cell (EC) proliferation/apoptosis and expression of various angiogenic factors in rat brain. F344×BN rats received either whole-brain irradiation (a single dose of 10 Gy γ rays) or sham irradiation and were maintained for 4, 8 and 24 h after irradiation. Double immunofluorescence staining was employed to visualize EC proliferation/apoptosis in brain. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), endothelial-specific receptor tyrosine kinase (Tie-2), and Ang-2 in brain were determined by real-time RT-PCR and immunofluores-cence staining. A significant reduction in CD31-immunore-active cells was detected in irradiated rat brains compared with sham-irradiated controls. Whole-brain irradiation significantly suppressed EC proliferation and increased EC apoptosis. In addition, a significant decrease in mRNA and protein expression of VEGF, Ang-1 and Tie-2 was observed in irradiated rat brains. In contrast, whole-brain irradiation significantly upregulated Ang-2 expression in rat brains. The present study provides novel evidence that whole-brain irradiation differentially affects mRNA and protein expression of VEGF, Ang-1, Tie-2 and Ang-2. These changes are closely associated with decreased EC proliferation and increased EC apoptosis in brain.

Emerging evidence has shown that oxidation of RNA including mRNA is elevated in several age-related diseases, although investigation of oxidized levels of individual RNA species has been limited. Recently we reported that an Aldehyde Reactive Probe (ARP) quantitatively reacts with oxidatively modified depurinated/depyrimidinated (abasic) RNA. Here we report a novel method to isolate oxidized RNA using ARP and streptavidin-beads. An oligo RNA containing abasic sites which were derivatized with ARP was pulled down by streptavidin-beads, whereas a control oligo RNA was not. In vitro oxidized RNA as well as total cellular RNA isolated from oxidatively stressed cells was also pulled down dependent on oxidation level, and concentrated in the pull-down fraction. Quantitative RT-PCR using RNA in the pull-down fraction demonstrated that several gene transcripts were uniquely increased in the fraction by oxidative stress. Thus, our method selectively concentrates oxidized RNA by pull-down and enables the assessment of oxidation levels of individual RNA species.

GH and its anabolic mediator, IGF1, are important not only in somatic growth but also in the regulation of brain function. Even though GH treatment has been used clinically to improve body composition and exercise capacity in adults, its influence on central nervous system function has only recently been recognized. This is also the case for children with childhood-onset GH deficiency (GHD) where GH has been used to stimulate bone growth and enhance final adult height. Circulating IGF1 is transported across the blood–brain barrier and IGF1 and its receptors are also synthesized in the brain by neurons and glial and endothelial cells. Nevertheless, the relationship between circulating IGF1 and brain IGF1 remains unclear. This study, using a GH-deficient dwarf rat model and peripheral GH replacement, investigated the effects of circulating IGF1 during adolescence on IGF1 levels in the brain. Our results demonstrated that hippocampal IGF1 protein concentrations during adolescence are highly regulated by circulating IGF1, which were reduced by GHD and restored by systematic GH replacement. Importantly, IGF1 levels in the cerebrospinal fluid were decreased by GHD but not restored by GH replacement. Furthermore, analysis of gene expression using microarrays and RT-PCR indicated that circulating IGF1 levels did not modify the transcription of Igf1 or its receptor in the hippocampus but did regulate genes that are involved in microvascular structure and function, brain development, and synaptic plasticity, which potentially support brain structures involved in cognitive function during this important developmental period.

The purpose of this study was to determine changes in klotho, endothelin (ET) receptors, and superoxide production in kidneys of aged rats and whether these changes are exacerbated in aged rats with cognitive impairment. Twenty aged rats (male, 27 months) were divided into an Old Impaired group (n = 9) and an Old Intact group (n = 11) according to a cognitive function test. A group of 12-month-old rats (n = 10) was used as a Young Intact group. Serum creatinine was increased significantly in the Old Impaired group, suggesting impaired renal function. Aged rats showed glomerulosclerosis and tubulointerstitialfibrosis. These pathological changes were markedly aggravated in the old cognitively impaired than in the old cognitively intact animals. Notably, aged rats demonstrated a significant decrease in klotho protein expression in renal cortex and medulla. Protein expression of IL-6, Nox2, ETa receptors and superoxide production were increased whereas mitochondrial SOD (MnSOD) and ETb receptors expression were decreased in kidneys of the aged rats. Interestingly, these changes were more pronounced in the old impaired than in the old intact rats. In conclusion, the aging-related kidney damage was exacerbated in aged rats with cognitive impairment. Klotho, ETB, and MnSOD were downregulated but ETa, IL-6, Nox2, and superoxide production were upregulated in the aging-related kidney damage. These changes were more pronounced in rats with cognitive impairment.

Aging promotes oxidative stress in vascular endothelial and smooth muscle cells, which contribute to the development of cardiovascular diseases. NF-E2–related factor 2 (Nrf2) is a transcription factor, which is activated by reactive oxygen species in the vasculature of young animals, leading to adaptive upregulation of numerous reactive oxygen species detoxifying and antioxidant genes. The present study was designed to elucidate age-associated changes in the homeostatic role of Nrf2-driven free radical detoxification mechanisms in the vasculature of nonhuman primates. We found that carotid arteries of aged rhesus macaques (Macaca mulatta, age: ≥20 years) exhibit significant oxidative stress (as indicated by the increased 8-iso-PGF2α and 4-HNE content and decreased glutathione and ascorbate levels) as compared with vessels of young macaques (age: ∼10 years) that is associated with activation of the redox-sensitive proinflammatory transcription factor, nuclear factor-kappaB. However, age-related oxidative stress does not activate Nrf2 and does not induce Nrf2 target genes (NQO1, GCLC, and HMOX1). In cultured vascular smooth muscle cells (VSMCs) derived from young M mulatta, treatment with H2O2 and high glucose significantly increases transcriptional activity of Nrf2 and upregulates the expression of Nrf2 target genes. In contrast, in cultured vascular smooth muscle cells cells derived from aged macaques, H2O2– and high glucose–induced Nrf2 activity and Nrf2-driven gene expression are blunted. High glucose–induced H2O2 production was significantly increased in aged vascular smooth muscle cells compared with that in vascular smooth muscle cells from young M mulatta. Taken together, aging is associated with Nrf2 dysfunction in M mulatta arteries, which likely exacerbates age-related cellular oxidative stress, promoting nuclear factor-kappaB activation and vascular inflammation in aging.

Age-related cognitive decline occurs without frank neurodegeneration and is the most common cause of memory impairment in aging individuals. With increasing longevity, cognitive deficits, especially in hippocampus-dependent memory processes, are increasing in prevalence. Nevertheless, the neurobiological basis of age-related cognitive decline remains unknown. While concerted efforts have led to the identification of neurobiological changes with aging, few age-related alterations have been definitively correlated to behavioral measures of cognitive decline. In this work, adult (12 Months) and aged (28 months) rats were categorized by Morris water maze performance as Adult cognitively Intact, Aged cognitively Intact or Aged cognitively Impaired, and protein expression was examined in hippocampal synaptosome preparations. Previously described differences in synaptic expression of neurotransmission-associated proteins (Dnm1, Hpca, Stx1, Syn1, Syn2, Syp, SNAP25, VAMP2 and 14-3-3 eta, gamma, and zeta) were confirmed between Adult and Aged rats, with no further dysregulation associated with cognitive impairment. Proteins related to synaptic structural stability (MAP2, drebrin, Nogo-A) and activity-dependent signaling (PSD-95, 14-3-3θ, CaMKIIα) were up- and down-regulated, respectively, with cognitive impairment but were not altered with increasing age. Localization of MAP2, PSD-95, and CaMKIIα demonstrated protein expression alterations throughout the hippocampus. The altered expression of activity- and structural stability-associated proteins suggests that impaired synaptic plasticity is a distinct phenomenon that occurs with age-related cognitive decline, and demonstrates that cognitive decline is not simply an exacerbation of the aging phenotype.

Mitochondrial dysfunction and oxidative stress are thought to play important roles in mammalian aging. Resveratrol is a plant-derived polyphenol that exerts diverse anti-aging activities, mimicking some of the molecular and functional effects of dietary restriction. This review focuses on the molecular mechanisms underlying the mitochondrial protective effects of resveratrol, which could be exploited for the prevention or amelioration of age-related diseases in the elderly.

We assess whether reactive oxygen species production and resistance to oxidative stress might be causally involved in the exceptional longevity exhibited by the ocean quahog Arctica islandica. We tested this hypothesis by comparing reactive oxygen species production, resistance to oxidative stress, antioxidant defenses, and protein damage elimination processes in long-lived A islandica with the shorter-lived hard clam, Mercenaria mercenaria. We compared baseline biochemical profiles, age-related changes, and responses to exposure to the oxidative stressor tert-butyl hydroperoxide (TBHP). Our data support the premise that extreme longevity in A islandica is associated with an attenuated cellular reactive oxygen species production. The observation of reduced protein carbonyl concentration in A islandica gill tissue compared with M mercenaria suggests that reduced reactive oxygen species production in long-living bivalves is associated with lower levels of accumulated macromolecular damage, suggesting cellular redox homeostasis may determine life span. Resistance to aging at the organismal level is often reflected in resistance to oxidative stressors at the cellular level. Following TBHP exposure, we observed not only an association between longevity and resistance to oxidative stress–induced mortality but also marked resistance to oxidative stress–induced cell death in the longer-living bivalves. Contrary to some expectations from the oxidative stress hypothesis, we observed that A islandica exhibited neither greater antioxidant capacities nor specific activities than in M mercenaria nor a more pronounced homeostatic antioxidant response following TBHP exposure. The study also failed to provide support for the exceptional longevity of A islandica being associated with enhanced protein recycling. Our findings demonstrate an association between longevity and resistance to oxidative stress–induced cell death in A islandica, consistent with the oxidative stress hypothesis of aging and provide justification for detailed evaluation of pathways involving repair of free radical–mediated macromolecular damage and regulation of apoptosis in the world's longest-living non-colonial animal.

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.

The discovery that in invertebrates, disruption of the insulin/insulin-like growth factor (IGF)-1 pathway extends life span and increases resistance to oxidative injury led to the hypothesis that IGF-1 signaling may play a role in regulating cellular reactive oxygen species production, oxidative stress resistance, and consequentially, organismal life span in mammals. However, previous studies testing this hypothesis in rodent models of IGF-1 deficiency yielded controversial results. The Lewis dwarf rat is a useful model of human growth hormone (GH)/IGF-1 deficiency as it mimics many of the pathophysiological alterations present in human GH/IGF-1–deficient patients as well as elderly individuals. Peripubertal treatment of Lewis dwarf rats with GH results in a significant extension of life span. The present study was designed to test the role of the GH/IGF-1 axis in regulating cellular oxidative stress and oxidative stress resistance, utilizing primary fibroblasts derived from control rats, Lewis dwarf rats and GH-replete dwarf rats. Measurements of cellular dihydroethidium and C-H2DCFDA fluorescence showed that cellular O2·− and peroxide production were similar in each group. Fibroblasts from control and Lewis dwarf rats exhibited similar antioxidant capacities and comparable sensitivity to H2O2, rotenone, high glucose, tunicamycin, thapsigargin, paraquat, and mitomycin, which cause apoptosis through increasing oxidative stress, mitochondrial damage, ATP depletion, and/or by damaging DNA, lipids and proteins. Fibroblasts from GH-replete rats exhibited significantly increased antioxidant capacities and superior resistance to H2O2, rotenone and bacterial lipopolysaccharide–induced cell death compared with cells from Lewis dwarf rats, whereas their sensitivity to the other stressors investigated was not statistically different. Thus, low circulating IGF-1 levels present in vivo in Lewis dwarf rats do not elicit long-lasting alterations in cellular reactive oxygen species generation and oxidative stress resistance, whereas life span–extending peripubertal GH treatment resulted in increased antioxidant capacity and increased resistance to cellular injury caused by some, but not all, oxidative stressors.

There have been several reports describing elevation of oxidized RNA in aging or age-related diseases, however RNA oxidation has been assessed solely based on 8-hydroxy-guanosine levels. In this study, Aldehyde Reactive Probe (ARP), which was originally developed to detect DNA abasic sites was used to assess RNA oxidation. We found that ARP reacted with depurinated tRNAPhe or chemically synthesized RNA containing abasic sites quantitatively to as little as 10 fmoles, indicating that abasic RNA is recognized by ARP. RNA oxidized by Fenton-type reactions, γ-irradiation, or peroxynitrite increased ARP reactivity dose-dependently, indicating that ARP is capable of monitoring oxidized RNA mediated by reactive oxygen species or reactive nitrogen species. Furthermore, oxidative stress increased levels of ARP reactive RNA in cultured cells. These results indicate the versatility of the assay method for biologically relevant oxidation of RNA. Thus, we have developed a sensitive assay for analysis of oxidized RNA.

Whole brain radiation therapy (WBRT) is commonly used for treatment of primary and metastatic brain tumors; however, cognitive impairment occurs in 40–50% of brain tumor survivors. The etiology of the cognitive impairment following WBRT remains elusive. We recently reported that radiation-induced cerebrovascular rarefaction within hippocampal subregions could be completely reversed by systemic hypoxia. However, the effects of this intervention on learning and memory have not been reported. In this study, we assessed the time-course for WBRT-induced impairments in contextual and spatial learning and the capacity of systemic hypoxia to reverse WBRT-induced deficits in spatial memory. A clinical fractionated series of 4.5Gy WBRT was administered to mice twice weekly for 4 weeks, and after various periods of recovery, behavioral analyses were performed. To study the effects of systemic hypoxia, mice were subjected to 11% (hypoxia) or 21% oxygen (normoxia) for 28 days, initiated 1 month after the completion of WBRT. Our results indicate that WBRT induces a transient deficit in contextual learning, disruption of working memory, and progressive impairment of spatial learning. Additionally, systemic hypoxia completely reversed WBRT-induced impairments in learning and these behavioral effects as well as increased vessel density persisted for at least 2 months following hypoxia treatment. Our results provide critical support for the hypothesis that cerebrovascular rarefaction is a key component of cognitive impairment post-WBRT and indicate that processes of learning and memory, once thought to be permanently impaired after WBRT, can be restored.

In humans, growth hormone deficiency (GHD) and low circulating levels of insulin-like growth factor 1 (IGF-1) significantly increase the risk for cerebrovascular disease. Genetic growth hormone (GH)/IGF-1 deficiency in Lewis dwarf rats significantly increases the incidence of late-life strokes, similar to the effects of GHD in elderly humans. Peripubertal treatment of Lewis dwarf rats with GH delays the occurrence of late-life stroke, which results in a significant extension of life span. The present study was designed to characterize the vascular effects of life span-extending peripubertal GH replacement in Lewis dwarf rats. Here, we report, based on measurements of dihydroethidium fluorescence, tissue isoprostane, GSH, and ascorbate content, that peripubertal GH/IGF-1 deficiency in Lewis dwarf rats increases vascular oxidative stress, which is prevented by GH replacement. Peripubertal GHD did not alter superoxide dismutase or catalase activities in the aorta nor the expression of Cu-Zn-SOD, Mn-SOD, and catalase in the cerebral arteries of dwarf rats. In contrast, cerebrovascular expression of glutathione peroxidase 1 was significantly decreased in dwarf vessels, and this effect was reversed by GH treatment. Peripubertal GHD significantly decreases expression of the Nrf2 target genes NQO1 and GCLC in the cerebral arteries, whereas it does not affect expression and activity of endothelial nitric oxide synthase and vascular expression of IGF-1, IGF-binding proteins, and inflammatory markers (tumor necrosis factor alpha, interluekin-6, interluekin-1β, inducible nitric oxide synthase, intercellular adhesion molecule 1, and monocyte chemotactic protein-1). In conclusion, peripubertal GH/IGF-1 deficiency confers pro-oxidative cellular effects, which likely promote an adverse functional and structural phenotype in the vasculature, and results in accelerated vascular impairments later in life.

Background

Age-related cognitive dysfunction, including impairment of hippocampus-dependent spatial learning and memory, affects approximately half of the aged population. Induction of a variety of neuroinflammatory measures has been reported with brain aging but the relationship between neuroinflammation and cognitive decline with non-neurodegenerative, normative aging remains largely unexplored. This study sought to comprehensively investigate expression of the MHC II immune response pathway and glial activation in the hippocampus in the context of both aging and age-related cognitive decline.

Methods

Three independent cohorts of adult (12-13 months) and aged (26-28 months) F344xBN rats were behaviorally characterized by Morris water maze testing. Expression of MHC II pathway-associated genes identified by transcriptomic analysis as upregulated with advanced aging was quantified by qPCR in synaptosomal fractions derived from whole hippocampus and in hippocampal subregion dissections (CA1, CA3, and DG). Activation of astrocytes and microglia was assessed by GFAP and Iba1 protein expression, and by immunohistochemical visualization of GFAP and both CD74 (Ox6) and Iba1.

Results

We report a marked age-related induction of neuroinflammatory signaling transcripts (i.e., MHC II components, toll-like receptors, complement, and downstream signaling factors) throughout the hippocampus in all aged rats regardless of cognitive status. Astrocyte and microglial activation was evident in CA1, CA3 and DG of intact and impaired aged rat groups, in the absence of differences in total numbers of GFAP+ astrocytes or Iba1+ microglia. Both mild and moderate microglial activation was significantly increased in all three hippocampal subregions in aged cognitively intact and cognitively impaired rats compared to adults. Neither induction of MHCII pathway gene expression nor glial activation correlated to cognitive performance.

Conclusions

These data demonstrate a novel, coordinated age-related induction of the MHC II immune response pathway and glial activation in the hippocampus, indicating an allostatic shift toward a para-inflammatory phenotype with advancing age. Our findings demonstrate that age-related induction of these aspects of hippocampal neuroinflammation, while a potential contributing factor, is not sufficient by itself to elicit impairment of spatial learning and memory in models of normative aging. Future efforts are needed to understand how neuroinflammation may act synergistically with cognitive-decline specific alterations to cause cognitive impairment.

This review focuses on molecular, cellular, and functional changes that occur in the vasculature during aging; explores the links between mitochondrial oxidative stress, inflammation, and development of vascular disease in the elderly patients; and provides a landscape of molecular mechanisms involved in cellular oxidative stress resistance, which could be targeted for the prevention or amelioration of unsuccessful vascular aging. Practical interventions for prevention of age-associated vascular dysfunction and disease in old age are considered here based on emerging knowledge of the effects of anti-inflammatory treatments, regular exercise, dietary interventions, and caloric restriction mimetics.

Decreased cognitive performance reduces independence and quality of life for aging individuals. Healthy brain aging does not involve significant neuronal loss, but little is known about the effects of aging at synaptic terminals. Age-related cognitive decline likely reflects the manifestation of dysregulated synaptic function and ineffective neurotransmission. In this study, hippocampal synaptosomes were enriched from Young-adult (3 months), Adult (12 months), and Aged (26 months) Fischer 344 × Brown Norway rats, and quantitative alterations in the synaptoproteome were examined by 2-DIGE and MS/MS. Bioinformatic analysis of differentially expressed proteins identified a significant effect of aging on a network of neurotransmission-regulating proteins. Specifically, altered expression of DNM1, HPCA, PSD95, SNAP25, STX1, SYN1, SYN2, SYP, and VAMP2 was confirmed by immunoblotting. 14-3-3 isoforms identified in the proteomic analysis were also confirmed due to their implication in the regulation of the synaptic vesicle cycle and neurotransmission modulation. The findings of this study demonstrate a coordinated downregulation of neurotransmission-regulating proteins that suggests an age-based deterioration of hippocampal neurotransmission occurring between adulthood and advanced age. Altered synaptic protein expression may decrease stimulus-induced neurotransmission and vesicle replenishment during prolonged or intense stimulation, which are necessary for learning and the formation and perseverance of memory.

The present study was designed to examine the effect of aging on radiation-induced expression of pro-inflammatory mediators in rat brain. Male F344×BN rats (4, 16, and 24 months of age) received either whole brain irradiation with a single dose of 10Gy γ-rays or sham-irradiation, and were maintained for 4, 8, and 24 h post-irradiation. The mRNA expression levels of various pro-inflammatory mediators such as cytokines, adhesion molecules, chemokine, and matrix metalloproteinase were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The acute inflammatory responses to irradiation, including overexpression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9) were markedly attenuated in the hippocampus of middle-aged and old rats compared with young groups. Specifically, a significant age-dependent decrease in TNF-α expression was detected 8 and 24 h after irradiation and a similar age-related attenuation was observed in IL-1β, ICAM-1, and VCAM-1 expression 4 and 8 h post-irradiation. MCP-1 expression was reduced 4 h post-irradiation and MMP-9 expression at 8 h post-irradiation. These results provide evidence for the first time that radiation-induced pro-inflammatory responses in the brain are suppressed in aged animals.

Insulin-like growth factor-I (IGF-I), a functionally important neurotrophic factor, impacts tissues throughout the body including the central nervous system. In addition to the significant proportion of IGF-I that is synthesized in the liver and released into the plasma, IGF-I is expressed locally in tissues. The present study investigated the relationship between plasma and local brain levels of IGF-I in two well-characterized models of decreased IGF-I. The first is an adult-onset growth hormone deficiency (AOGHD) model, and the second is a caloric restriction (CR) model. In the first cohort of animals from both models, the hippocampus was removed from the brain immediately following decapitation, and in the second cohort, the animals were perfused transcardially with phosphate buffered saline to remove cerebral blood prior to harvesting the hippocampus. Our results demonstrated that although the plasma IGF-I levels were decreased in the CR and AOGHD rats compared to controls, the hippocampal IGF-I levels did not differ among the groups. These data suggest that local brain IGF-I levels are regulated in a different manner than plasma IGF-I levels.

This review focuses on mitochondrial abnormalities that occur in the vasculature during aging and explores the link between mitochondrial oxidative stress, chronic low-grade vascular inflammation, increased rate of endothelial apoptosis, and development of vascular diseases in the elderly. Therapeutic strategies targeting the mitochondria for prevention of age-associated vascular dysfunction and disease in old age are considered here based on emerging knowledge of the vasoprotective effects of caloric restriction, caloric restriction mimetics, the GH/IGF-1 axis, and mitochondria-targeted antioxidants.

The distinctions between senescence and disease are blurred in the literature of evolutionary biology, biodemography, biogerontology and medicine. Theories of senescence that have emerged over the past several decades are based on the concepts that organisms are a byproduct of imperfect structural designs built with imperfect materials and maintained by imperfect processes. Senescence is a complex mixture of processes rather than a monolithic process. Senescence and disease have overlapping biological consequences. Senescence gives rise to disease, but disease does not give rise to senescence. Current data indicate that treatment of disease can delay the age of death but there are no convincing data that these interventions alter senescence. An understanding of these basic tenets suggests that there are biological limits to duration of life and the life expectancy of populations and reveal biological domains where the development of interventions and/or treatments may modulate senescence.

Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin-like growth factor 1 (IGF-1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF-1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF-1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF-1 significantly improved axon number, diameter, and density. IGF-1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF-1 treatment.

Purpose

Pro-inflammatory environments in the brain have been implicated in the onset and progression of neurological disorders. In the present study, we investigate the hypothesis that brain irradiation induces regionally specific alterations in cytokine gene and protein expression.

Materials and methods

Four month old F344 × BN rats received either whole brain irradiation with a single dose of 10 Gy γ-rays or sham-irradiation, and were maintained for 4, 8, and 24 h following irradiation. The mRNA and protein expression levels of pro-inflammatory mediators were analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. To elucidate the molecular mechanisms of irradiation-induced brain inflammation, effects of irradiation on the DNA-binding activity of pro-inflammatory transcription factors were also examined.

Results

A significant and marked up-regulation of mRNA and protein expression of pro-inflammatory mediators, including tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1), was observed in hippocampal and cortical regions isolated from irradiated brain. Cytokine expression was regionally specific since TNF-α levels were significantly elevated in cortex compared to hippocampus (57% greater) and IL-1β levels were elevated in hippocampus compared to cortical samples (126% greater). Increases in cytokine levels also were observed after irradiation of mouse BV-2 microglial cells. A series of electrophoretic mobility shift assays (EMSA) demonstrated that irradiation significantly increased activation of activator protein-1 (AP-1), nuclear factor-κB (NF-κB), and cAMP response element-binding protein (CREB).

Conclusion

The present study demonstrated that whole brain irradiation induces regionally specific pro-inflammatory environments through activation of AP-1, NF-κB, and CREB and overexpression of TNF-α, IL-1β, and MCP-1 in rat brain and may contribute to unique pathways for the radiation-induced impairments in tissue function.

Purpose

The prevalence of bladder dysfunctions increases with age. In humans it is difficult to separate changes related to exogenous factors from those directly related to the aging process. Some confounding variables can be avoided by studying age related changes in an animal model. We evaluated the impact of age on bladder function in vivo and in vitro, and characterized the corresponding morphological changes.

Materials and Methods

Young (4 to 6 months old) and old (older than 28 to 30 months) male Fischer/Brown Norway rats were used in the study. Cystometric studies were done in conscious, freely moving rats. After cystometry tissue strips from the bladder body were used in in vitro studies of muscarinic receptor activation and electrical field stimulation, and histological examination.

Results

Old rats had higher bladder weight than young rats but the bladder-to-body weight ratio did not change. We noted significant age related differences in 8 of 10 cystometric parameters. Old rats had increased bladder capacity, post-void residual volume, micturition volume and frequency, baseline and intermicturition pressure, and spontaneous activity but decreased micturition pressure. Bladder strip responses to carbachol and electrical field stimulation were significantly lower in old than in young rats. Histological examination revealed urothelial thinning, lower muscle mass and higher collagen content in the bladders of old vs young rats.

Conclusions

Physiological aging alters bladder function in male rats even when external factors remain constant. Thus, in old rats bladder capacity, post-void residual urine and spontaneous activity are higher, and responses to muscarinic receptor stimulation and electrical field stimulation are lower than in young rats. Such changes correspond to findings in aging human bladders, supporting the view that the Fischer/Brown Norway rat is a useful model in which to study age related bladder function changes.

Purpose

Vision loss is one of the most common complications of aging, even in individuals with no diagnosed ocular disease. Increasing age induces structural alterations and functional impairments in retinal neurons and microvasculature linked to the activation of proinflammatory signaling pathways. Commonalities between the effects of aging and those observed with diabetes, including visual impairment, vascular dysfunction, and increased inflammatory response, have led to the hypothesis that diabetes-associated pathologies reflect an “advanced aging” phenotype. The goal of this study was to investigate the effects of aging on retinal mRNA expression of neurovascular and inflammatory transcripts previously demonstrated to be regulated with diabetes.

Methods

The relative expression of 36 genes of interest previously identified as consistently regulated with diabetes was assessed in retinas of Young (3 month), Adult (12 month), and Aged (26 month) Fischer 344 x Brown Norway (F1) hybrid rats using quantitative PCR. Serum samples obtained at sacrifice were assayed to determine serum glucose levels.

Results

Eleven inflammation- and microvascular-related genes previously demonstrated to be upregulated in young diabetic rats (complement component 1 s subcomponent [C1s], chitinase 3-like 1 [Chi3L1], endothelin 2 [Edn2], guanylate nucleotide binding protein 2 [Gbp2], glial fibrillary acidic protein [Gfap], intracellular adhesion molecule 1 [Icam1], janus kinase 3 [Jak3], lipopolysaccharide-induced TNF factor [Litaf], complement 1-inhibitor [Serping1], signal transducer and activator of transcription 3 [Stat3], tumor necrosis factor receptor subfamily member 12a [Tnfrsf12a]) demonstrated progressively increasing retinal expression in aged normoglycemic rats. Additionally, two neuronal function–related genes (glutamate receptor ionotropic NMDA 2A [Grin2a] and polycomb group ring finger 1 [Pcgf1]) and one inflammation-related gene (pigment epithelium-derived growth factor [Pedf]) displayed patterns of expression dissimilar to that previously demonstrated with diabetes.

Conclusions

The commonalities in retinal age-related and diabetes-induced molecular alterations provide support for the hypothesis that diabetes and aging engage some common para-inflammatory processes. However, these results also demonstrate that while the retinal genomic response to diabetes and aging share commonalities, they are not superimposable phenotypes. The observed changes in retinal gene expression provide further evidence of retinal alterations in neurovascular and inflammatory processes across the adult rat lifespan; this is indicative of para-inflammation that may contribute to the functional impairments that occur with advanced age. The data also suggest the potential for an additive effect of aging and diabetes in the development of diabetic complications.

Intracerebroventricular 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 the chimeric protein (TK/GFP)fus (green fluorescent protein fused to HSV1 thymidine kinase) 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 DAPI. 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. Intracerebroventricular 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.