Aging results in accumulation of M1 and M2 hepatic macrophages and a differential response to gadolinium chloride.

Macrophages have vital roles in innate immunity by modulating the inflammatory response via their ability to alter their phenotype from pro-inflammatory (M1) to anti-inflammatory (M2). Aging increases activation of the innate immune system, and macrophage numbers increase in the aged liver. Since macrophages also produce free radical molecules, they are a potential source of age-related oxidative injury in the liver. This study evaluated macrophage phenotype in the aged liver and whether the increase in the number of macrophages with aging is associated with enhanced hepatic oxidative stress. Hepatic macrophage phenotype and oxidative stress were evaluated 2 days after a single intraperitoneal injection of saline or gadolinium chloride (GdCl3, 10 mg/kg) in young (6 months) and aged (24 months) Fischer 344 rats. GdCl3 has been shown to decrease the expression of macrophage-specific markers and impair macrophage phagocytosis in the liver. Saline-treated aged rats demonstrated greater numbers of both M1 (HO-1+/iNOS+) and M2 (HO-1+/CD163+) macrophages, without evidence of a phenotypic shift. GdCl3 did not alter levels of dihydroethidium fluorescence or malondialdehyde, suggesting that macrophages are not a major contributor to steady-state levels of oxidative stress. However, GdCl3 decreased M1 and M2 macrophage markers in both age groups, an effect that was attenuated in aged rats. In old animals, GdCl3 decreased iNOS expression to a greater extent than HO-1 or CD163. These results suggest a novel effect of aging on macrophage biology and that GdCl3 shifts hepatic macrophage polarization to the M2 phenotype in aged animals.

Basic recovery aids: what's the evidence?

This review summarizes the evidence base for using compression, massage, caloric replacement, cold, and heat as exercise recovery aids in sport.

Altered expression of iron regulatory proteins with aging is associated with transient hepatic iron accumulation after environmental heat stress.

An increasing body of evidence suggests that dysregulation of iron metabolism contributes to age-related pathologies. We have previously observed increased hepatic iron with aging, and that environmental heat stress stimulates a further increase in iron and oxidative liver injury in old rats. The purpose of this study was to determine a mechanism for the increase in hepatic iron in old rats after heat stress. Young (6 mo) and old (24 mo) Fischer 344 rats were exposed to two heating bouts separated by 24 h. Livers were harvested after the second heat stress, and protein levels of the iron import protein, transferrin receptor-1 (TFR1), and the iron export protein, ferroportin (Fpn) were determined by immunoblot. In the nonheated condition, old rats had lower TFR1 expression, and higher Fpn expression. After heat stress, TFR1 declined in the old rats, and iron chelation studies demonstrated that this decline was dependent on a hyperthermia-induced increase in iron. TFR1 did not change in the young rats after heat stress. Since TFR1 is inversely regulated by iron, our results suggest that the increase in intracellular iron with aging and heat stress lower TFR1 expression. Fpn expression increased in both age groups after heat stress, but this response was delayed in old rats. This delay in the induction of an iron exporter suggests a mechanism for the increase in hepatic iron and oxidative injury after heat stress in aged organisms.

A cluster of exertional rhabdomyolysis affecting a Division I Football team.

OBJECTIVE: To identify risk factors for exertional rhabdomyolysis (ER) among collegiate football athletes. We hypothesized that a back squat workout triggered ER in some players, and that the risk of ER was altered by players' characteristics or other exposures. DESIGN: Case report and case-control study. SETTING: National Collegiate Athletic Association Division I Football Program and an academic medical center. PARTICIPANTS: National Collegiate Athletic Association Division I football players. INDEPENDENT VARIABLES: Characteristics, performance during the implicated workout, and exposures of players. MAIN OUTCOME MEASURES: Exertional rhabdomyolysis was the primary outcome; the hypotheses were formulated before data were collected. RESULTS: Initial serum creatine kinase and creatinine values ranged from 96,987 to 331,044 U/L and from 1.0 to 3.4 mg/dL, respectively. The risk of ER increased as the time and number of sets needed to complete 100 back squats increased [odds ratio (OR), 1.11; 95% confidence interval (CI), 1.03-1.19; P = 0.0051 and OR, 1.33; 95% CI, 1.09-1.63; P = 0.0056, respectively]. Affected players were significantly more likely than unaffected players to report that they went to muscle failure (P = 0.006), did not think they could complete the workout (P = 0.02), and performed extra squats (P = 0.02) during the back squat assignment. For athletes playing skilled or semiskilled positions, the risk of ER increased as the percent body weight lifted increased [OR (corresponding to a 10% increase), 1.77; 95% CI, 1.06-2.94; P = 0.0292]. Drinking protein shakes after the implicated workout was associated with a decreased risk (OR, 0.70; 95% CI, 0.51-0.96; P = 0.0284); the odds decreased about 30% per shake. CONCLUSIONS: Percent body weight lifted, the number of sets, and time needed to complete 100 back squats were significantly associated with increased risk of ER. Affected athletes were more likely to report going to muscle failure, thinking they could complete the workout, and performing extra squats during the back squat assignment. Consuming protein shakes after the implicated workout was associated with a decreased risk. Clinicians, athletes, and athletic program staff must know risk factors for ER and early symptoms of ER.

Altered accumulation of hepatic mitochondrial antioxidant proteins with age and environmental heat stress.

Aging impairs overall physiological function, particularly the response to environmental stressors. Repeated heat stress elevates reactive oxygen species and macromolecular damage in the livers of aged animals, likely due to mitochondrial dysfunction. The goal of this investigation was to determine potential mechanisms for mitochondrial dysfunction after heat stress by evaluating key redox-sensitive and antioxidant proteins (Sirt-3, MnSOD, Trx-2, and Ref-1). We hypothesized that heat stress would result in greater mitochondrial abundance of these proteins, but that aging would attenuate this response. For this purpose, young (6 mo) and old (24 mo) Fisher 344 rats were exposed to heat stress on two consecutive days. During each heating trial, colonic temperature was elevated to 41°C during the first 60 min, and then clamped at this temperature for 30 min. Nonheated animals served as controls. At 2 and 24 h after the second heat stress, hepatic mitochondria were isolated from each animal, and then immunoblotted for Sirt-3, acetylated lysine residues (Ac-K), MnSOD, Trx-2, and Ref-1. Aging increased Sirt-3 and lowered Ac-K. In response to heat stress, Sirt-3, Ac-K, MnSOD, and Ref-1 increased in mitochondrial fractions in both young and old animals. At 2 h after the second heat stress, mitochondrial Trx-2 declined in old, but not in young animals. Our results suggest that some components of the response to heat stress are preserved with aging. However, the decline in Trx-2 represents a potential mechanism for age-related mitochondrial damage and dysfunction after heat stress.NEW & NOTEWORTHY Our results suggest heat stress-induced mitochondrial translocation of Sirt-3, MnSOD, and Ref-1 in young and old animals. Aged rats experienced a decline in Trx-2 after heat stress, suggesting a potential mechanism for age-related mitochondrial dysfunction.

Renal Iron Accumulation and Oxidative Injury With Aging: Effects of Treatment With an Iron Chelator.

Dysregulation of iron metabolism in the kidney may contribute to age-related increases in renal oxidative stress and dysfunction. This study assessed the effects of short-term iron chelation on markers of iron status, oxidative stress, inflammation, and autophagy in the kidneys of old rats. Old Fischer 344 rats (24 months) were treated with deferoxamine (DFO; 200 mg/kg, twice daily for 4.5 days); saline-treated young (6 months) and old rats served as controls. Renal nonheme iron was significantly higher in the old rats, with iron localized in the renal cortex. Ferritin levels were elevated in the kidneys of old rats, while expression of several antioxidant enzymes and mitochondrial proteins were reduced and protein carbonyls increased compared to young rats. DFO treatment significantly reduced ferritin levels, and increased transferrin receptor-1 protein, but did not affect nonheme iron content or protein carbonyls, nor did it reverse age-related changes in antioxidant enzymes and mitochondrial proteins. Although short-term DFO treatment did not mitigate the age-related increase in iron content and oxidative damage, this work demonstrates that old rats respond appropriately to DFO, suggesting that optimization of iron chelation regimens could be useful in improving renal homeostasis with aging.

Aging results in increased autophagy of mitochondria and protein nitration in rat hepatocytes following heat stress.

The natural breakdown of cells, tissues, and organ systems is a significant consequence of aging and is at least partially caused by a decreased ability to tolerate environmental stressors. Based on quantitative ultrastructural analysis using transmission electron microscopy and computer imaging, we show significant differences in hepatocyte morphology between young and old rats during a 48-hr recovery period following a 2-day heat stress protocol. Mitochondrial injury was greater overall in old compared with young rats. Autophagy was observed in both young and old rats, with autophagy greater overall in old compared with young hepatocytes. Lipid peroxidation and protein nitration were evaluated by localization and quantification of 4-hydroxy-2-nonenal (4-HNE)-modified protein adducts and 3-nitrotyrosine (3-NT) levels, respectively. Levels of 3-NT but not 4-HNE-protein adducts were significantly elevated in hepatocytes of old rats in comparison with young at 90 min after heat stress, suggesting a major role for reactive nitrogen species in the pathology observed at this time point. These results show a differential response of hepatocyte mitochondria to heat stress with aging, as well as greater levels of both autophagic and nitrative damage in old vs young hepatocytes. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

1962-2007: a cell stress odyssey.

The induction of a cellular stress response was first observed in 1962 in a set of serendipitous experiments in Drosophila melanogasterlarvae, which led to the discovery of a family of intracellular polypeptides known as heat shock proteins (HSPs). These highly conserved proteins are present in both prokaryotic and eukaryotic species, suggesting that they play important roles in fundamental cellular processes. Moreover, these proteins are induced in response to a range of stimuli, implicating HSPs as important modifying factors in an organism's response to a variety of physiological conditions. HSPs were initially regarded as intracellular molecules mediating cytoprotective, regulatory and chaperoning functions. However, the past two decades have seen an explosion of information related to the cell stress response, with a primary focus on molecular chaperones, which are a class of multifunctional intracellular proteins that assist in folding and assembly of other proteins. Stress proteins have also been identified on cell surfaces and in extracellular fluids, and are now viewed as potential immunomodulators, pro-inflammatory signalling molecules, and anti-inflammatory proteins in disease states. This chapter serves as an overview of the rapidly expanding world of cell stress proteins and aims to provide the reader with a foundation for more detailed presentations in subsequent sections of this book.

Chronic antioxidant enzyme mimetic treatment differentially modulates hyperthermia-induced liver HSP70 expression with aging.

One postulated mechanism for the reduction in stress tolerance with aging is a decline in the regulation of stress-responsive genes, such as inducible heat shock protein 72 (HSP70). Increased levels of oxidative stress are also associated with aging, but it is unclear what impact a prooxidant environment might have on HSP70 gene expression. This study utilized a superoxide dismutase/catalase mimetic (Eukarion-189) to evaluate the impact of a change in redox environment on age-related HSP70 responses to a physiologically relevant heat challenge. Results demonstrate that liver HSP70 mRNA and protein levels are reduced in old compared with young rats at selected time points over a 48-h recovery period following a heat-stress protocol. While chronic systemic administration of Eukarion-189 suppressed hyperthermia-induced liver HSP70 mRNA expression in both age groups, HSP70 protein accumulation was blunted in old rats but not in their young counterparts. These data suggest that a decline in HSP70 mRNA levels may be responsible for the reduction in HSP70 protein observed in old animals after heat stress. Furthermore, improvements in redox status were associated with reduced HSP70 mRNA levels in both young and old rats, but differential effects were manifested on protein expression, suggesting that HSP70 induction is differentially regulated with aging. These findings highlight the integrated mechanisms of stress protein regulation in eukaryotic organisms responding to environmental stress, which likely involve interactions between a wide range of cellular signals.

NAD(P)H oxidase-induced oxidative stress in sympathetic ganglia of apolipoprotein E deficient mice.

Superoxide anion (O2*-) is increased throughout the arterial wall in atherosclerosis. The oxidative stress contributes to lesion formation and vascular dysfunction. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived O2*- is increased in nodose sensory ganglia and sympathetic ganglia of apolipoprotein E deficient (apoE-/-) mice, an established animal model of atherosclerosis. O2*- measured ex vivo by L-012-enhanced chemiluminescence was increased by 79+/-17% in whole sympathetic ganglia from apoE-/- mice (n=5) compared with sympathetic ganglia from control mice (n=5) (P<0.05). In contrast, O2*- was not elevated in nodose ganglia from apoE-/- mice. Dihydroethidium staining confirmed the selective increase in O2*- in sympathetic ganglia of apoE-/- mice, and revealed the contribution of both neurons and non-neuronal cells to the O2*- generation. We investigated the enzymatic source of increased O2*- in sympathetic ganglia of apoE-/- mice. The mRNA expression of gp91phox, p22phox, p67phox, and p47phox subunits of NAD(P)H oxidase measured by real time RT-PCR was increased approximately 3-4 fold in sympathetic ganglia of apoE-/- mice (n=5) compared with control ganglia (n=5). NADPH oxidase activity measured by lucigenin chemiluminescence was increased by 68+/-12% in homogenates of sympathetic ganglia from apoE-/- mice (n=7) compared with control ganglia (n=7) (P<0.05). The results identify sympathetic ganglia as a novel site of oxidative stress in atherosclerosis, and suggest that upregulation of NAD(P)H oxidase is the source of increased O2*- generation. We speculate that oxidative stress in sympathetic ganglia may contribute to impaired baroreflex control of sympathetic nerve activity.

Heat-induced liver injury in old rats is associated with exaggerated oxidative stress and altered transcription factor activation.

A decline in stress tolerance is a hallmark of aging. For instance, older organisms showed extensive hepatic damage, along with increased morbidity and mortality, after environmental heating. We hypothesized that hyperthermic challenge would produce exaggerated oxidative stress in old animals, leading to increased hepatic injury. After a heat-stress protocol, time-course changes in reactive oxygen species (ROS) levels, oxidative damage markers, glutathione (GSH)/glutathione disulfide (GSSG) ratios, and activation of stress-response transcription factors (AP-1 and NF-kappaB) were measured in young and old rats. A small, transient increase in hepatic oxidative damage, with minimal injury, was observed in young rats. However, old rats showed widespread hepatic injury that was manifested over a 24 h period after heating. This pathology was preceded by elevated steady-state levels of ROS, along with large increases in lipid peroxidation products, prolonged hepatic DNA oxidation damage, aberrant GSH/GSSG profiles, and altered activation patterns for AP-1. These data indicate that young animals have an effective oxidation-reduction buffering system in the liver that provides protection from oxidative damage to intracellular macromolecules under stress conditions. In sharp contrast, an environmental challenge in older animals produces exaggerated oxidative stress and alterations in signal transduction pathways, which can contribute to cellular dysfunction and age-related reductions in stress tolerance.

Redox modulation of the liver with chronic antioxidant enzyme mimetic treatment prevents age-related oxidative damage associated with environmental stress.

A reduction in stress tolerance is a hallmark of the aging process, and the lowered functional capacity observed in aged organisms is associated with an increased rate of oxidative stress and a greater susceptibility of aged tissues to oxidative injury. In this report, we show that chronic systemic administration of a superoxide dismutase (SOD)/catalase mimetic (EUK-189), delivered over a 1 month period via osmotic pump, prevents heat stress-induced liver injury by dramatically decreasing oxidative damage in aged animals. Widespread liver injury was present in old but not young vehicle-treated rats in response to a 2 day heating protocol. However, SOD/catalase mimetic treatment markedly decreased the hyperthermia-induced liver injury associated in old animals. The reversal of damage with EUK-189 was associated with an improvement in intracellular redox status and a striking reduction in hepatocellular lipid peroxidation. EUK-189 treatment also blocked the activation of activator protein-1 (AP-1), which is a redox-sensitive early response transcription factor involved in the regulation of cellular stress responses. These results demonstrate that oxidative stress plays a unique role in age-related hyperthermic injury and suggest that therapeutic strategies aimed at improving redox potential, such as chronic SOD/catalase mimetic treatment, can prevent the oxidative-mediated damage associated with environmental stress.

Thiol supplementation in aged animals alters antioxidant enzyme activity after heat stress.

Declines in oxidative and thermal stress tolerance are well documented in aging systems. It is thought that these alterations are due in part to reductions in antioxidant defenses. Although intracellular thiols are major redox buffers, their role in maintaining redox homeostasis is not completely understood, particularly during aging, where the reliance on antioxidant enzymes and proteins may be altered. To determine whether thiol supplementation improved the antioxidant enzyme profile of aged animals after heat stress, young and old Fischer 344 rats were treated with N-acetylcysteine (NAC; 4 mmol/kg ip) 2 h before heat stress. Liver tissue was collected before and 0, 30, and 60 min after heat stress. Aging was associated with a significant decline in tissue cysteine and glutathione (GSH) levels. There was also an age-related decrease in copper-zinc superoxide dismutase activity. Heat stress did not alter liver GSH, glutathione disulfide, or antioxidant enzyme activity. With NAC treatment, old animals took up more cysteine than young animals as reflected in an increase in liver GSH and a corresponding decrease in glutamate cysteine ligase activity. Catalase activity increased after NAC treatment in both age groups. Copper-zinc superoxide dismutase activity did not change with heat stress or drug treatment, whereas manganese superoxide dismutase activity was increased in old animals only. These data indicate that GSH synthesis is substrate limited in old animals. Furthermore, aged animals were characterized by large fluctuations in antioxidant enzyme balance after NAC treatment, suggesting a lack of fine control over these enzymes that may leave aged animals susceptible to subsequent stress.

Inhibition of oral cancer cell growth by adenovirusMnSOD plus BCNU treatment.

We hypothesized that inhibitors of peroxide removal, such as BCNU, an indirect inhibitor of glutathione peroxidase (GPx), and 3-amino-1,2,4-triazole (AT), a direct inhibitor of catalase (CAT), should cause toxicity to cancer cells after manganese superoxide dismutase (MnSOD) overexpression due to elevated peroxide levels. In vitro, hamster cheek pouch carcinoma cells (HCPC-1) and human oral squamous carcinoma cells (SCC-25) were infected with various combinations of adenovirus containing MnSOD cDNA (AdMnSOD). Cells were then treated with or without BCNU and assayed for viability using Annexin/PI staining and flow cytometry. In AdMnSOD plus BCNU-treated SCC-25 and HCPC-1 cells, a 30-60% decrease in cell viability was observed compared to BCNU alone. In vivo, HCPC-1 and SCC-25 xenografts were allowed to grow to approximately 70 mm(3) and 10(9) plaque forming units (pfu) of AdMnSOD were injected directly into the tumors. Two days later, 15 or 30 mg/kg BCNU was injected intratumorally. Tumor growth was greatly inhibited (4- to 20-fold) by this combined treatment, as well as increasing animal survival. Tumor volume could be decreased further by giving multiple doses of AdMnSOD or inhibiting catalase activity with AT. These results suggest that, by using these combination therapies, a significant decrease in tumor mass can be achieved.

Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance.

Cells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). The precise functions of HSPs are unknown, but there is considerable evidence that these stress proteins are essential for survival at both normal and elevated temperatures. HSPs also appear to play a critical role in the development of thermotolerance and protection from cellular damage associated with stresses such as ischemia, cytokines, and energy depletion. These observations suggest that HSPs play an important role in both normal cellular homeostasis and the stress response. This mini-review examines recent evidence and hypotheses suggesting that the HSPs may be important modifying factors in cellular responses to a variety of physiologically relevant conditions such as hyperthermia, exercise, oxidative stress, metabolic challenge, and aging.

Selected contribution: Differential expression of stress-related genes with aging and hyperthermia.

Aging is associated with a reduced capacity to cope with physiological stress. To study the molecular mechanisms associated with the decline in stress tolerance that accompanies aging, differences in gene expression between young and old Fischer 344 rats under euthermic control conditions or in response to hyperthermic challenge were evaluated using a cDNA array containing 207 stress-related genes. In the nonstressed control condition, aging resulted in selective upregulation of stress protein genes and transcripts involved in cell growth, death, and signaling, along with a downregulation of genes involved in antioxidant defenses and drug metabolism. Heat stress resulted in a broad induction of genes in the antioxidant and drug metabolism categories and transcripts involved in DNA, RNA, and protein synthesis for both age groups. Old animals had a robust upregulation of genes involved in cell growth, death, and signaling after heat challenge, along with a blunted expression of stress-response genes. In contrast, young animals had a strong induction of stress-response genes after hyperthermic challenge. Changes in expression of selected genes were confirmed by RT-PCR analysis. These findings suggest that aging results in altered gene expression in response to heat stress that is indicative of decreased stress protein transcription and increased expression of oxidative stress-related genes. Thus our findings support the postulate that transcriptional changes in response to a physiological challenge such as hyperthermia contribute to the loss of stress tolerance in older organisms.

The nitric oxide synthase inhibitor N(G)-nitro-L-arginine decreases defibrillation-induced free radical generation.

OBJECTIVES: To demonstrate that nitric oxide (NO) contributes to free radical generation after epicardial shocks and to determine the effect of a nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NNA), on free radical generation. BACKGROUND: Free radicals are generated by direct current shocks for defibrillation. NO reacts with the superoxide (O(2).(-)) radical to form peroxynitrite (O=NOO(-)), which is toxic and initiates additional free radical generation. The contribution of NO to free radical generation after defibrillation is not fully defined. METHODS AND RESULTS: Fourteen open chest dogs were studied. In the initial eight dogs, 40 J damped sinusoidal monophasic epicardial shocks was administered. Using electron paramagnetic resonance, we monitored the coronary sinus concentration of ascorbate free radical (Ascz.(-)), a measure of free radical generation (total oxidative flux). Epicardial shocks were repeated after L-NNA, 5 mg/kg IV. In six additional dogs, immunohistochemical staining was done to identify nitrotyrosine, a marker of reactive nitrogen species-mediated injury, in post-shock myocardial tissue. Three of these dogs received L-NNA pre-shock. After the initial 40 J shock, Ascz.(-) rose 39+/-2.5% from baseline. After L-NNA infusion, a similar 40 J shock caused Ascz.(-) to increase only 2+/-3% from baseline (P<0.05, post-L-NNA shock versus initial shock). Nitrotyrosine staining was more prominent in control animals than dogs receiving L-NNA, suggesting prevention of O=NOO(-) formation. CONCLUSIONS: NO contributes to free radical generation and nitrosative injury after epicardial shocks; NOS inhibitors decrease radical generation by inhibiting the production of O=NOO(-).

The nitric oxide synthase inhibitor N(G)-nitro-L-arginine decreases defibrillation-induced free radical generation.

OBJECTIVES: to demonstrate that nitric oxide (NO) contributes to free radical generation after epicardial shocks and to determinethe effect of a nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NNA), on free radical generation. BACKGROUND: Free radicals are generated by direct current shocks for defibrillation. NO reacts with the superoxide (O2*-) radical to for peroxynitrite (O = NOO-), which is toxic and initiates additional free radical generation. The contribution of NO to free radical generation after defibrillation is not fully defined. METHODS AND RESULTS: Fourteen open chest dogs were studied. In the initial eight dogs, 40 J damped sinusoidal monophasic epicardial shocks was administered. Using electron paramagnetic resonance, we monitored the coronary sinus concentration of ascorbate free radical (Asc*-), a measure of free radical generation (total oxidative flux). Epicardial shocks were repeated after L-NNA, 5 mg/kg IV. In six additional dogs, immunohistochemical staining was done to identify nitrotyrosine, a marker of reactive nitrogen species-mediated injury, in post-shock myocardial tissue. Three of these dogs received L-NNA pre-shock. After the initial 40 J shock, Asc*- rose 39 +/- 2.5% from baseline. After L-NNA infusion, a similar 40 J shock caused Asc*- to increase only 2 +/- 3% form baseline (P < 0.05, post-L-NNA shock versus initial shock). Nitrotyrosine staining was more prominent in control animals than dogs receiving L-NNA, suggesting prevention of O = NOO- formation. CONCLUSION: NO contributes to free radical generation and nitrosative injury after epicardial shocks; NOS inhibitors decrease radical generation by inhibiting the production of O = NOO-.

Heat stress stimulates hepcidin mRNA expression and C/EBPα protein expression in aged rodent liver.

Elevations in hepatic iron content occur with aging and physiological stressors, which may promote oxidative injury to the liver. Since dysregulation of the iron regulatory hormone, hepcidin, can cause iron accumulation, our goal was to characterize the regulation of hepcidin in young (6 mo) and old (24 mo) Fischer 344 rats exposed to environmental heat stress. Liver and blood samples were taken in the control condition and after heating. Hepcidin expression did not differ between young and old rats in the control condition, despite higher levels of hepatic iron and IL-6 mRNA in the latter. Following heat stress, pSTAT3 increased in both groups, but C/EBPα and hepcidin mRNA increased only in old rats. Despite this, serum iron decreased in both age groups 2 h after heat stress, suggesting hepcidin-independent hypoferremia in the young rats. The differential regulation of hepcidin between young and old rats after hyperthermia may be due to the enhanced expression of C/EBPα protein in old rats. These data support the concept of "inflammaging" and suggest that repeated exposures to stressors may contribute to the development of anemia in older individuals.

Investigating autophagy: quantitative morphometric analysis using electron microscopy.

Autophagy is a compensatory pathway involving isolation and subsequent degradation of cytosolic material and organelles in eukaryotic cells.(1) The autophagic process can provide a "housekeeping" function by removing damaged proteins and organelles in a selective or nonselective fashion in order to exert a protective effect following stress.(2) Remarkably, after being discovered to be much more of a targeted process than a random one, the role of autophagy became implicated in many normal cellular and disease processes.(3) Several methodologies are routinely employed to monitor the entire autophagic process.(4) Microtubule-associated protein light chain 3, a mammalian homolog of yeast Atg8, has been widely used as a specific marker to monitor autophagy in numerous cell types.(5) While monitoring autophagic flux is extremely important, it is also beneficial to perform a detailed analysis by electron microscopy (EM) to evaluate changes in various autophagic structures, quantify the areas involved, and determine if any particular organelle(s) or area of the cell cytoplasm is being targeted for degradation.(6) The following article describes methods to localize and quantify subcellular areas of autophagy using transmission EM. Also discussed are methods for subcellular localization of specific proteins by employing immunogold EM; this method becomes particularly useful in detecting early changes in cellular homeostasis that may occur before later signs of cellular insult can be observed morphologically.