Macrophages have multiple roles in the heart including immune surveillance and extracellular matrix remodeling. Aging increases both collagen deposition and macrophage number in the heart; however, rodent models used to study cardiac macrophages have age-related comorbidities such as atherosclerosis and hypertension. The Fischer 344 rat does not develop these conditions with aging; therefore, the purpose of this study was to evaluate macrophage number and polarization in the hearts of aged (24-month) and young (6-month) Fischer 344 rats. Paraffin-embedded hearts were assessed for collagen deposition and immunolabeled for CD68, CD163, CD206, and galectin-3. Compared with young rats, significantly greater collagen deposition was observed in the old rats. There were no significant differences in CD68+ or CD163+ cells between age groups, but both CD206+ and galectin-3+ cells were more numerous in the aged animals. Double-immunofluorescence studies demonstrated that galectin-3 colocalized with both CD68 and CD163, suggesting that galectin-3 is found in cardiac macrophages. Further colocalization studies demonstrated similar proportions of CD68+/CD163-, CD68+/CD163+, and CD68-/CD163+ cells between age groups, suggesting that aging does not affect macrophage polarization. As CD206+ and galectin-3+ cells promote fibrosis, these results warrant future studies that delineate the specific roles of these cells in the aged heart.

Abstract Ferroptosis is a form of cell death characterized by a pro‐oxidative cellular milieu and iron‐dependent lipid peroxidation. Ferroptosis has been implicated in various forms of liver injury, in keeping with the major role of the liver in iron metabolism. Limited research has addressed potential differences in ferroptosis mediators with age and sex, especially in an in vivo model. The goal of this investigation was to evaluate hepatic labile iron and mediators of ferroptosis with ageing in both sexes. Because female animals generally display greater antioxidant defences than males, we hypothesized that females would display a phenotype resistant to ferroptosis. Here, we determined iron contents, protein expression of ferroptosis mediators and measures of oxidative injury in liver samples from 12‐ and 24‐month‐old male and female Fischer 344 rats. In comparison to males, the livers of female rats at both ages contained more non‐haem iron, which was associated with greater ferritin heavy chain expression and attenuated expression of transferrin receptor‐1. In female rats, the 24‐month‐old group had higher contents of thiobarbituric acid reactive substances compared with their 12‐month‐old counterparts, yet similar contents of labile iron. These results suggest a disconnect between labile iron contents and oxidative injury with age. Female animals also displayed greater expression of acyl‐CoA synthetase long‐chain family member 4 (ACSL4), a modulator of ferroptosis, and greater abundance of high molecular weight 4‐hydroxnonenal‐modified proteins. These results demonstrate clear differences in iron and ferroptosis mediators between sexes and suggest that female rats of this strain might be more susceptible to ferroptosis.

Objective: Although health and wellness behaviors are associated with positive body image, research is limited regarding the relationship between sleep and positive body image. We propose that negative affective states may link sleep and body image. Specifically, we examined whether better sleep may relate to positive body image through reductions in negative affective experiences. Participants: Participants were 269 undergraduate women. Methods: Cross-sectional surveys were administered. Results: We found correlations in the expected directions between sleep, positive body image variables (i.e., body appreciation, appearance evaluation, and appearance orientation), and negative affective states (i.e., depression, anxiety, and stress). There were group differences in negative affective states and body image based on adequate sleep. Data supported indirect effects of sleep through depression on appearance evaluation, and through depression and stress on body appreciation, respectively. Conclusions: Our findings indicate sleep warrants further research attention as a wellness behavior related to more positive body image.

The goal of the current study was to examine associations between hormonal contraceptive use and indicators of well-being including body image, eating behavior, sleep and energy level. Drawing on a health protection framework, we expected that individuals who use hormonal contraceptives would be more attuned to health and report more positive health attitudes and behaviors on these dimensions. Undergraduate college women (N = 270; M = 19.39 years, SD = 2.43, range 18-39 years) from diverse racial/ethnic and sexual orientation groups completed a survey online. Measures included hormonal contraception use, body image, weight control behavior, breakfast consumption, sleep behavior, and daytime energy level. Nearly 1/3 (30.9%) of the sample reported current hormonal contraceptive use, with most users reporting use of birth control pills (74.7%). Women who used hormonal contraceptives reported significantly higher appearance orientation and body surveillance, lower average energy, more frequent night awakenings, and more naps. Longer duration of hormonal contraceptive use was significantly related to higher body surveillance, and engaging in more unhealthy weight control behavior. Hormonal contraceptive use is not related to indicators of greater well-being. Rather, hormonal contraceptive use is related to greater attention to appearance, lower daytime energy, and some indicators of poorer sleep quality. Clinicians who prescribe hormonal contraceptives should attend to body image, sleep and energy concerns among users.

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.

Liver macrophages serve important roles in iron homeostasis through phagocytosis of effete erythrocytes and the export of iron into the circulation. Conversely, intracellular iron can alter macrophage phenotype. Aging increases hepatic macrophage number and nonparenchymal iron, yet it is unknown whether age-related iron accumulation alters macrophage number or phenotype. To evaluate macrophages in a physiological model of iron loading that mimicked biological aging, young (6 mo) Fischer 344 rats were given one injection of iron dextran (15 mg/kg), and macrophage number and phenotype were evaluated via immunohistochemistry. A separate group of old (24 mo) rats was treated with 200 mg/kg deferoxamine every 12 h for 4 days. Iron administration to young rats resulted in iron concentrations that matched the values and pattern of tissue iron deposition observed in aged animals; however, iron did not alter macrophage number or phenotype. Aging resulted in significantly greater numbers of M1 (CD68+) and M2 (CD163+) macrophages in the liver, but neither macrophage number nor phenotype were affected by deferoxamine. Double-staining experiments demonstrated that both M1 (iNOS+) and M2 (CD163+) macrophages contained hemosiderin, suggesting that macrophages of both phenotypes stored iron. These results also suggest that age-related conditions other than iron excess are responsible for the accumulation of hepatic macrophages with aging.

The liver plays a pivotal role in the regulation of iron metabolism through its ability to sense and respond to iron stores by release of the hormone hepcidin. Under physiologic conditions, regulation of hepcidin expression in response to iron status maintains iron homeostasis. In response to tissue injury, hepcidin expression can be modulated by other factors, such as inflammation and oxidative stress. The resulting dysregulation of hepcidin is proposed to account for alterations in iron homeostasis that are sometimes observed in patients with liver disease. This review describes the effects of experimental forms of liver injury on iron metabolism and hepcidin expression. In general, models of acute liver injury demonstrate increases in hepcidin mRNA and hypoferremia, consistent with hepcidin's role as an acute-phase reactant. Conversely, diverse models of chronic liver injury are associated with decreased hepcidin mRNA but with variable effects on iron status. Elucidating the reasons for the disparate impact of different chronic injuries on iron metabolism is an important research priority, as is a deeper understanding of the interplay among various stimuli, both positive and negative, on hepcidin regulation. Future studies should provide a clearer picture of how dysregulation of hepcidin expression and altered iron homeostasis impact the progression of liver diseases and whether they are a cause or consequence of these pathologies.

Aging is associated with chronic, low-grade inflammation that adversely affects physiological function. The liver regulates systemic inflammation; it is a source of cytokine production and also scavenges bacteria from the portal circulation to prevent infection of other organs. The cells with primary roles in these functions, hepatic macrophages, become more numerous in the liver with “normal” aging (i.e., in the absence of disease). Here, we demonstrate evidence and potential mechanisms for this phenomenon, which include augmented tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) expression in the liver. Also, we discuss how an age-related impairment in autophagy within macrophages leads to a pro-oxidative state and ensuing production of proinflammatory cytokines, particularly interleukin 6 (IL-6). Given that the liver is a rich source of macrophages, we posit that it represents a major source of the elevated systemic IL-6 observed with aging, which is associated with physiological dysfunction. Testing a causal role for liver macrophage production of IL-6 during aging remains a challenge, yet interventions that have targeted macrophages and/or IL-6 have demonstrated promise in treating age-related diseases. These studies have demonstrated an age-related, deleterious reprogramming of macrophage function, which worsens pathology. Therefore, hepatic macrophage accrual is indeed a cause for concern, and therapies that attenuate the aged phenotype of macrophages will likely prove useful in promoting healthy aging.

The mechanisms responsible for dysregulation of iron metabolism in response to ethanol ingestion are poorly understood. Relatively brief ethanol exposures in rodents are associated with reduced hepatic hepcidin expression without increases in hepatic iron content. This study evaluated the effects of long-term ethanol treatment on hepatic iron metabolism in two mouse strains. Ethanol was administered in the drinking water to C57BL/6 and BALB/c mice for up to 11 months. Hepatic histology and iron concentrations (HIC) were assessed, along with expression of relevant genes and proteins by real-time RT-PCR and western blot, respectively. The livers of ethanol-consuming mice of both strains showed mild steatosis without inflammation or fibrosis. Stainable hepatocyte iron was modestly increased in both strains ingesting ethanol, although hepatic iron concentrations were significantly higher only in C57BL/6 mice. Long-term ethanol did not affect hepcidin mRNA (Hamp1 or Hamp2) in either strain, nor was the expression of several oxidative stress-responsive genes (glutamate cysteine ligase, gamma-glutamyl transpeptidase, heme oxygenase-1 and growth differentiation factor 15) altered in response to ethanol, suggesting that oxidative stress and suppression of hepcidin expression in short-term ethanol feeding models may be transient phenomena that resolve as mice adapt to ethanol exposure. This murine model of chronic ethanol ingestion demonstrates modest increases in hepatic iron without changes in hepcidin expression, markers of oxidative stress or significant histologic liver injury. Further investigations are needed to characterize the mechanisms of dysregulated iron metabolism resulting from chronic ethanol ingestion.

Aging is accompanied by a pro‐inflammatory state that adversely affects physiological function in many organs. Macrophages, innate immune cells of the myeloid lineage, exist on a phenotypic spectrum ranging from pro‐inflammatory (M1) to anti‐inflammatory (M2), and play a major role in tissue homeostasis throughout life. Previously, we have shown that aging increases the number of macrophages of both phenotypes (M1 and M2) in the liver; however, in other organs, there are conflicting findings as to the effects of aging on macrophage number and polarization. Therefore, the purpose of this study was to determine the effects of aging on macrophage abundance and phenotype in the kidneys and spleen. We hypothesized that aging would increase the number of M1 macrophages, and decrease the number of M2 macrophages in each organ. To investigate this, we utilized immunohistochemistry to detect the M1 marker, inducible nitric oxide synthase (iNOS), and the M2 marker, cluster of differentiation 206 (CD206), in kidney and spleen sections from young (6 months) and old (24 months) male Fischer 344 rats. The number of cells were counted in 6–7 visual fields, the area of the field was determined, and the results are expressed as the number of cells per square millimeter. In the kidney, we detected diffuse iNOS staining in tubular cells, however there was no visible staining in cells with morphologic characteristics of macrophages in either age group. Cells positive for CD206 were detected most frequently in the medulla, with no significant differences in number between young (3.3 cells/mm2) and old (4.1 cells/mm2) animals. In the spleen, macrophages resided in the marginal zone, and in the red pulp. We observed a trend (p=0.07) for fewer splenic iNOS+ macrophages in young animals, compared to the old (0.015 cells/mm2 vs. 0.072 cells/mm2). However, there was no difference in splenic CD206+ macrophages between young and old groups (59 vs 57 cells/mm2). Unlike the liver, we have observed similar numbers of M2 macrophages between young and old animals in the kidneys and spleen, which reflects the heterogeneous nature of aging. Our results have also identified a unique M2 macrophage population in the kidney, which resides in the renal medulla.Support or Funding InformationFaculty Development Grant, Penn State Abington. Erickson Discovery Grant, Penn State University.

Iron is implicated in the pathogenesis of a number of human liver diseases. Hereditary hemochromatosis is the classical example of a liver disease caused by iron, but iron is commonly believed to contribute to the progression of other forms of chronic liver disease such as hepatitis C infection and nonalcoholic fatty liver disease. In this review, we present data from cell culture experiments, animal models, and clinical studies that address the hepatotoxicity of iron. These data demonstrate that iron overload is only weakly fibrogenic in animal models and rarely causes serious liver damage in humans, calling into question the concept that iron overload is an important cause of hepatotoxicity. In situations where iron is pathogenic, iron-induced liver damage may be potentiated by coexisting inflammation, with the resulting hepatocyte necrosis an important factor driving the fibrogenic response. Based on the foregoing evidence that iron is less hepatotoxic than is generally assumed, claims that assign a causal role to iron in liver injury in either animal models or human liver disease should be carefully evaluated.

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.

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.

Background Aging is characterized by increases in inflammation and oxidative stress, conditions that are exacerbated by environmental factors such as diet. In this study, we investigated the effects of a trans -fatty acid (TFA) diet on the liver in adult (25 wk) and old (60 wk) senescence-accelerated mice (SAMP8 strain) of both sexes. Our goal was to assess the effects of the diet on protein markers of inflammation and oxidative stress in the liver. Methods Male and female mice were placed on life-long diets containing similar amounts of total fat (17%), with differing amounts of TFA: 2% (moderate TFA group) or 0.2% of total energy from TFA (control diet group). At the indicated ages, livers were harvested and evaluated for markers of inflammation and oxidative stress, as well as for enzymes of fat metabolism via immunoblotting. Relative densities of protein bands were determined and compared via a three-factor ANOVA. Results Compared to males, females demonstrated significantly lower inflammatory protein expression (ICAM-1, MCP-1, COX-2), along with lower expression of the DNA damage marker, Gadd153, and the oxidative stress marker, HO-1. Female mice demonstrated higher expression of antioxidant enzymes (SOD-1, SOD-2, and Ref-1) and lipogenic enzymes (FASN, ACLY) compared to male mice. While HO-1 was elevated in the female mice fed the TFA diet compared to controls, the diet did not affect other markers of oxidative stress or inflammation. However, the diet was associated with significant increases in FASN and ACLY in adult (25 wk) male mice. Conclusions Our results suggest sexually dimorphic protein expression in the liver, with female mice demonstrating lower inflammation and increased oxidative stress defenses. Additionally, considering that FASN and ACLY contribute to hepatic lipogenesis, our results suggest a potential mechanism for the dyslipidemia in adult male mice that is associated with TFA diets.

Although iron‐catalysed oxidative damage is presumed to be a major mechanism of injury leading to cirrhosis and hepatocellular carcinoma in hemochromatosis, these events have been difficult to recapitulate in an animal model. In this study, we evaluated regulators of hepatocarcinogenesis in a rodent model of chronic iron overload. Sprague–Dawley rats were iron loaded with iron dextran over 6 months. Livers were harvested and analysed for markers of oxidative stress, as well as the following proteins: p53, murine double minute 2, the Shc proteins p66, p52, p46; β‐catenin, CHOP, C/EBPα and Yes‐associated protein. In this model, iron loading is associated with hepatocyte proliferation, and indices of oxidative damage are mildly increased in tandem with augmented antioxidant defenses. Alterations potentially favouring carcinogenesis included a modest but significant decrease in p53 levels and increases in p52, p46 and β‐catenin levels compared with control livers. Countering these factors, the iron‐loaded livers demonstrated a significant decrease in CHOP, which has recently been implicated in the development of hepatocellular carcinoma, as well as a reciprocal increase in C/EBPα and decrease in Yes‐associated protein. Our results suggest that chronic iron overload elicits both tumour suppressive as well as tumour‐promoting mechanisms in rodent liver. Copyright © 2015 John Wiley & Sons, Ltd.

Aging is associated with reduced tolerance to physiological stressors such as hyperthermia. In animal models, heat stress is associated with increased oxidative DNA damage in the livers of old rats. In this study, we evaluated the expression of redox factor‐1 (Ref‐1), a DNA repair enzyme, and thioredoxin‐1 (Trx‐1), an antioxidant protein. We hypothesized that these proteins would be induced by heat stress in young animals, and that aging would attenuate this response. Young (6 mo) and old (24 mo) male Fischer 344 rats were exposed to a two‐heat stress protocol, and livers were harvested at several time points after the second heat stress. Ref‐1 and Trx‐1 were evaluated by immunoblot and immunohistochemistry. In young rats, Ref‐1 was induced immediately (0 h) after heat stress, and returned to control levels at 2h. We observed no heat‐induced elevation of Ref‐1 in old rats; however, Ref‐1 was increased ~2‐fold in old, compared to young rats at all time points. At 2h after heat stress, Trx‐1 was increased in old rats, but there was no change in young rats. In tissue sections, Trx‐1 was localized to Kupffer cells in both age groups. Also, we observed frequent ductular reactions in the old rats that were positive for both Ref‐1 and Trx‐1. The impairment in the induction of Ref‐1 suggests a mechanism for the increased oxidative DNA damage observed in old rats after heat stress. Furthermore, the observation of ductular reactions positive for both Ref‐1 and Trx‐1 demonstrates a proliferative cellular niche that develops with aging.

Aging is associated with reduced tolerance to physiological stressors such as hyperthermia. In animal models, heat stress is associated with increased oxidative damage in the livers of old rats. In this study, we evaluated the expression of redox factor-1 (Ref-1), a DNA repair enzyme, and thioredoxin-1 (Trx-1), an antioxidant protein. We hypothesized that these proteins would be induced by heat stress in young animals, and that aging would attenuate this response. Young (6 mo) and old (24 mo) male Fischer 344 rats were exposed to a two-heat stress protocol, and livers were harvested at several time points after the second heat stress. Ref-1 and Trx-1 were evaluated by immunoblot and immunohistochemistry. In young rats, Ref-1 was induced by ~50% immediately (0 h) after heat stress, and returned to control levels at 2 h. We observed no change in Ref-1 after hyperthermia in old rats; however, aging was associated with a 2-fold increase in Ref-1 expression. At 2 h after heat stress, Trx-1 was increased in old rats, but there was no change in young rats. In tissue sections, we observed frequent ductular reactions in the old rats that were positive for both Ref-1 and Trx-1. The impairment in the induction of Ref-1 suggests a mechanism for the increased oxidative injury observed in old rats after heat stress. Furthermore, the observation of ductular reactions positive for both Ref-1 and Trx-1 demonstrates a proliferative cellular niche that develops with aging.

Aging results in exacerbated mitochondrial damage and cellular dysfunction after exposure to physiological challenges such as heat stress. Additionally, induction of mitochondrial stress‐defense proteins is blunted after heat stress with aging. The sirtuins are deacetylase enzymes implicated in stress defense and longevity. Sirtuin‐3 (SIRT3), which has been shown to reduce oxidative injury, is a mitochondrial enzyme that deacetylates mitochondrial proteins. Since little is known about the response of hepatic SIRT3 to physiological stress, the goal of the present study was to characterize protein levels of SIRT3 in young (6 mo) and old (24 mo) Fischer 344 rats after heat stress. Animals were exposed to a two‐heat stress protocol, and liver mitochondria were isolated at 2 and 24 h after the second heat stress. Nonheated animals served as controls. SIRT3 protein expression and global protein acetylation were determined by immunoblot analysis. Aging was associated with an increase in SIRT3 protein, and a trend for a decrease in protein acetylation. In each of the two age groups, heat stress increased both SIRT3 and global protein acetylation, which suggests that the increase in protein acetylation overwhelms the deacetylase activity of SIRT3. The increase in SIRT3 suggests that it is a stress‐responsive protein and warrants further inquiry into the acetylation status of selected mitochondrial proteins.Grant Funding Source: Supported by NIH R01 AG‐12350