Effects of aging on the severity of liver injury in mice with iron overload

Effects of aging on the severity of liver injury in mice with iron overload | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of aging on the severity of liver injury in mice with iron overload So-Hyun Park, Soo-Jin Song, Jin-A Lee, Jung-A Shin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4716297/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract While iron is a vital component in the body, excessive iron leads to iron toxicity, which affects vital organs. In particular, the liver is considerably affected by iron toxicity because it stores the highest amount of iron in the body. Nonetheless, the relationship between iron overload and aging in the liver has not yet been clearly identified. This study aimed to observe the effects of aging on iron overload in the liver. Female C57BL/6J mice were randomly divided into vehicle control and iron overload groups ( n = 7–22 per group). The iron overload group was injected with Fe-dextran (0.5 g/kg) for 4 weeks. After the experimental period, liver and blood samples were obtained from 2-, 15-, and 22-month-old mice. Liver weight, iron deposition, structural changes, cell death, extracellular matrix deposition, and fenestration of sinusoidal vessels were analyzed and compared between the groups. Additionally, biochemical analyses (aspartate aminotransferase, alanine aminotransferase, and serum total iron levels) were performed. The iron overload group exhibited significant differences compared to the control group with age. In the elderly iron overload model, iron deposition, inflammatory cell infiltration, and cell death were significantly increased ( p < .0001). Moreover, deposition of the extracellular matrix and defenestration of sinusoidal fenestrae were observed among 22-month-old mice in the iron overload group. These results suggest that aging is a risk factor for iron-induced liver injury. Therefore, caution should be exercised when performing iron-related treatments in the elderly. Health sciences/Gastroenterology Health sciences/Risk factors iron overload aging liver Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Iron is an essential component of metabolism 1 – 3 that is required throughout the body, including enzymes, cell signaling, mitochondrial reaction, and immune function from the cellular level to the systemic level. 1 , 4 However, excessive iron causes iron overload toxicity 1 , 2 , 4 because it also generates oxidative stress, 2 , 5 , 6 affecting vital organs. 2 , 3 , 6 , 7 Consequently, iron is strictly regulated by numerous factors for homeostasis maintenance. 1 , 2 In particular, the liver plays a crucial role in iron homeostasis 3 , 5 but is greatly affected by iron toxicity because it stores most of the iron in the body. 2 , 3 , 5 Severe liver diseases associated with excessive iron, such as chronic liver disease, liver fibrosis, hepatocellular carcinoma, and even liver failure, have been reported. 3 , 7 Iron overload is a common complication that occurs during treatments for various diseases (e.g., dialysis for chronic kidney disease, chronic RBC transfusion for anemia, hematopoietic cell transplantation for hematologic diseases). 8 – 10 Due to iron overload, the liver may develop secondary hemochromatosis, hepatomegaly, and liver fibrosis as complications. 10 , 11 Some mouse studies have reported the results of iron overload in the liver. 12 – 22 Specifically, iron has been shown to cause liver damage. Other factors such as aging also influences the regulation of iron. 5 , 16 As age is an important risk factor in many diseases, an elderly person is more easily exposed to treatment-related iron, including iron supplementation therapy, dialysis, blood transfusion, and total parenteral nutrition. 2 , 23 – 26 Furthermore, aging itself can contribute to iron imbalance via physiologic changes. 27 Recent studies have reported the relationship between aging and iron overload in other organs including brain and retina. 28 , 29 Nonetheless, the relationship between aging and iron overload in the liver has not yet been clearly identified. The current study aimed to observe the effects of aging on iron overload in the liver. RESULTS Gross morphology, liver weight, and blood analysis Figure 1 A and 1 B show the gross anatomy of livers and the average liver weight, respectively. In general, the gross size and weight of livers increased with age. The livers of the iron overload group showed more hypertrophic changes and were pigmented with a dark color, as compared with those of the control group. In the iron overload group, the average liver weight in 15-month-old and 22-month-old mice is significantly increased, as compared with that in 2-month-old mice (Fig. 1 B). The average serum AST, ALT, and total iron levels were significantly higher in the iron overload group than in the control group (Fig. 1 C and D). No significant changes were observed between the iron overload group. The iron-overloaded liver showed hypertrophic hepatocytes filled with iron deposition as well as inflammatory cell infiltration throughout the liver In H&E staining, the normal hepatic structure was destroyed in the iron overload group, in contrast to the control group; furthermore, numerous clumps of large and round-shaped cells filled with iron deposition (arrow, brown color) were observed throughout the liver (Fig. 2 A). Inflammatory cells (arrowhead) infiltrated around the iron-deposited cells, and they were increased in 15-month-old and 22-month-old mice compared to 2-month-old mice in the iron overload group. Focal necrosis (asterisks) was observed in 15-month-old and 22-month-old mice in the iron overload group. Infiltration of inflammatory cells increased significantly with age in the iron overload group Immunostaining and cell counting for CD45-positive cells (red) indicated that inflammatory cells infiltrated more in the iron overload group than in the control group (Fig. 2 B). In the iron overload group, inflammatory cells were located around the iron-deposited hepatocytes and pericentral veins. With increased age, inflammatory cells infiltrated significantly. Inflammatory cell counts of 22-month-old and 15-month-old mice significantly increased in the iron overload group compared to the control group and within the iron overload group. No significant increase was noted between 15-month-old and 22-month-old mice in the iron overload group. Hepatocytes exhibited decreased albumin expression and lost their intracellular organelles Immunostaining for 22-month-old mice in the iron overload group showed that albumin, a marker of hepatocytes, was co-localized with clumps of cells filled with iron deposition, as observed by H&E staining (Fig. 3 A). Iba-1 (a marker of macrophages) and α-SMA (a marker of activated hepatic stellate cells) did not co-localize with the cells. Compared to light microscopy in the same area, immunostaining showed that hepatocytes with low iron accumulation expressed albumin, whereas hepatocytes with excessive iron accumulation had a significantly reduced intensity of albumin expression (Fig. 3 A, asterisks). A mass with macro-iron deposition was observed using light microscopy in the same cells. Macrophages, hepatic stellate cells, and α-SMA enclosed clumps of hepatocytes with excessive iron content. TEM revealed that the cytoplasm of hepatocytes was nearly fully filled with massive aggregates of iron (Fig. 3 B). Intracellular organelles (e.g., mitochondria, glycogen, and lipid droplets) hardly existed. The nucleus was pressed by iron accumulation and lost its normal shape. Iron aggregates were also found in other cells surrounding the hepatocytes. Hepatic microvilli, a feature of hepatocytes, were diminished. The iron aggregates had irregular shapes and a wide range of sizes. Iron deposition was prominent in the old liver Prussian blue staining enabled the visualization of the location and distribution of iron deposits (Fig. 4 A). In the control groups, little iron deposition was observed (blue color); however, the iron overload group exhibited intense iron deposition throughout the liver (deep blue color). The staining grade of iron deposition in the iron overload group had a mixed pattern and was either grade IV or V: 24 , 30 diffuse accumulation of increased iron in hepatocytes, Kupffer cells (Fig. 3 A, arrow), and hepatic stellate cells (Fig. 3 B, asterisks). The area of iron deposition increased with age ( p < .00001) and was more prominent in 22-month-old mice in the iron overload group than in 2-month-old and 15-month-old mice ( p < .00001 each). There was no significant difference between 2-month-old and 15-month-old mice in the iron overload group. Cell death was increased in the iron overload group, especially in old age TUNEL staining and TUNEL-positive cell counting indicated that the number of apoptotic cells substantially increased in 22-month-old mice in the iron overload group (Fig. 4 B). Dead cells were located at the site of iron deposition (asterisks). In contrast to the control group, the iron overload group showed a large amount of apoptotic cells (arrowhead). In particular, apoptotic cells were more numerous in 22-month-old mice than in 2-month-old and 15-month-old mice in the iron overload group. Immunostaining of GPX-4 showed that the signal intensity of GPX-4 decreased in the iron overload group, especially in 22-month-old mice (Fig. 5 A). In the iron overload group, ferroptotic cell death particularly occurred in the area of hepatocytes occupied by iron accumulation (white box and asterisk). In the control group, GPX-4 was expressed evenly throughout the liver, and there were no differences among the age groups. In contrast, the iron overload group showed two different areas: the area with the cluster of hepatocytes with massive iron showed decreased signal intensity of GPX-4 (asterisk), whereas the other area expressed GPX-4 as the control. The mean signal intensity of GPX-4 decreased in the iron overload group, as compared with the control group (Fig. 5 B). The 22-month-old mice in the iron overload group had significantly decreased GPX-4 intensity to compare to 2-month-old and 15-month-old mice in the iron overload group. TEM revealed ultrastructural changes in the mitochondria of hepatocytes. The control group had intact double membranes (inner and outer) and cristae, whereas the iron overload group had shrunken mitochondria with dense single membranes and vanishing cristae (Fig. 5 D). The area and length of the mitochondria decreased in the iron-loaded group compared with the control group (Fig. 5 C). No significant changes were observed between 22-month-old and 2-month-old mice in the iron overload group. Collagen deposition around central vein increased in the iron-overloaded liver, particularly in old age Masson’s trichrome staining showed that collagen deposition (blue) substantially increased near the central vein in 22-month-old mice in the iron overload group (Fig. 6 A). TEM revealed that the extracellular matrix (ECM), including collagen fibers, was deposited under the subendothelial space of the central veins and was prominent in 22-month-old mice in the iron overload group (Fig. 6 B, asterisks). In addition to the subendothelial space, the ECM of the iron overload group accumulated in the cells near the central vein. In the control group, the ECM increased with age, but intercellular deposition was hardly observed. Fenestration of sinusoidal vessels disappeared in the aging iron-overloaded liver TEM and SEM showed shrunken and disappearing fenestrae of sinusoidal vessels in the iron overload group (Fig. 6 C and D). Unlike the intact fenestrae (arrowhead) in the control group, closed or overlapping fenestrae (arrow) were often observed in 22-month-old mice in the iron overload group (Fig. 6 C). The control group had numerous fenestrae of the sinusoidal endothelial wall, whereas the iron overload group had fewer and smaller fenestrae with thicker vessel walls (Fig. 6 D). Furthermore, few fenestrae were present in 22-month-old mice in the iron overload group. The hepatic microvilli between the hepatocytes and sinusoidal vessels diminished with age in the iron overload group. Iron deposition and hypertrophic changes were also observed in endothelial cells in the iron overload group. The 22-month-old mice in the iron overload group exhibited significantly decreased width and count of sinusoidal fenestrations compared to the 22-month-old mice in the control group (Fig. 6 E). No significant differences in fenestration width and count were detected between the control and iron overload groups among 2-month-old mice. DISCUSSION The current study focused on how aging affects the liver during iron overload. Fe-dextran was administered intraperitoneally to three age groups—namely, young (2-month-old), old (15-month-old), and super-old (22-month-old) mice. The AST and ALT levels markedly increased in the iron overload group compared with the control group, which is similar to the results of other studies. 12 – 14 , 21 , 22 Interestingly, the histological findings differed with age; however, AST and ALT levels did not significantly differ in the iron overload group. This implies that although the results of blood chemistry are similar in the young and old, the liver of the elderly is more susceptible to iron overload. Similar to previous studies, 15 , 16 , 18 , 20 clusters of iron-deposited hypertrophic lesions with necrotic changes were observed throughout the liver using H&E staining. Unlike previous iron overload studies that identified the Kupffer cells as the main iron deposition cells in iron overload, 3 , 12 , 15 , 19 , 23 , 31 , 32 our results indicated a mixed pattern, suggesting that iron was deposited in both the reticuloendothelial system and hepatocytes. 5 , 21 , 23 , 30 It is similar to the hepatocellular iron overload patterns described in other studies. 3 , 5 , 12 , 13 , 16 , 18 , 20 , 21 , 24 , 31 Because various factors, such as the amount of excessive iron, rate of iron deposition, and iron redistribution, determine iron overload, 15 , 23 , 33 our results seem to be attributable to differences in iron exposure period or dosage compared with recent studies. H&E staining and TEM revealed that iron-overloaded hepatocytes were filled with massive amounts of iron aggregates and had an abnormal structure, including the loss of intracellular organelles and hepatic microvilli. These iron aggregates can cause cellular damage via free radicals. 34 We found that inflammatory cells infiltrated the areas where iron accumulated, and the results for CD45-positive cells indicated that aging was associated with a higher propensity for inflammation during iron overload. Significantly more CD45-positive cells were present in the super-old iron-overloaded mice than in the old iron-overloaded mice. Recent studies reported that iron overload attracted inflammatory cells such as mononuclear phagocytes. This study suggests that aging aggravates inflammation in iron-overloaded livers. Prussian blue staining showed that old and super-old mice had a significant area of iron accumulation compared with young mice. Notably, the super-old mice had more extended iron-deposited areas. Given that the degree of iron deposition is closely associated with liver damage, 33 , 35 our findings suggest that aging may affect the severity of iron overload-induced liver damage. It is well known that iron is closely related to cell death via ROS. 36 , 37 As in previous studies, 14 , 16 , 20 – 22 TUNEL and GPX-4 results showed that cell death significantly increased throughout the liver in the iron overload groups compared to that in the control group in our study. Furthermore, we found that super-old mice were more susceptible to iron overload-related cell death than old and young mice. As previously reported, 22 , 37 – 39 it appears that ferroptosis occurs in hepatocytes under iron overload, based on the decrease in GPX-4 and ferroptosis-related mitochondrial damage. Considering that hepatocytes are primary cells in which excessive iron is deposited, 3 , 12 , 30 it seems that hepatocytes are liable for iron-dependent cell death. As liver cell death is thought to be one of the factors involved in liver disease, 36 , 40 our results imply that elderly people are more vulnerable to iron overload. Iron overload triggers liver fibrosis 3 , 13 , 23 , 24 . In Masson’s trichrome staining and TEM, the super-old iron overload group showed visible ECM deposition around the pericentral area with a score of I, 30 despite the same amount of iron administered as the young iron overload group. These findings suggest that aging plays a significant role in liver damage caused by iron overload. The fenestration of liver sinusoidal endothelial cells (LSECs) is regarded as a dynamic structure that can be easily affected by nearby environment. 41 – 43 Similar to the sinusoidal defenestration change caused by iron overload, 31 , 44 our results showed a loss of fenestration in the iron overload groups. In particular, almost no fenestration was observed in the super-old iron-overloaded mice. Similar to a previous study, which reported an increase in fenestration gaps (large fenestration > 300 nm) and no significant difference in diameter in old age, 42 we observed an increased mean fenestration diameter in super-old control mice, which was different from other studies reporting sinusoidal defenestration with aging. 41 , 43 , 45 It seems that the reason for this difference was due to measurement methods. We measured the average diameter, including gaps (large fenestration), whereas previous studies evaluated fenestration by porosity (area) or diameter, excluding gaps. Interestingly, the width and number of fenestrations were significantly decreased in the super-old iron overload and super-old control groups; however, there was no significant difference between the young groups. Our results imply that older patients are more affected by iron-mediated LSEC damage and are predicted to have more metabolic dysregulation due to sinusoidal defenestration. When iron overload occurs in the aging liver, massive amounts of iron accumulate in hepatocytes, and such iron deposition causes cellular damage and even liver cell death. Owing to hepatocyte death, the number of activated macrophages and hepatic stellate cells increase, attracting inflammatory cells, producing ECM, and thereby aggravating fibrotic changes in the liver. 3 , 19 , 23 , 36 Moreover, these activated macrophages and hepatic stellate cells are known to promote the defenestration of sinusoidal vessels. 46 As LSEC defenestration and ECM deposition are the initial features of liver fibrosis, 43 , 47 these can cause further liver injury. This study revealed that aging was a crucial factor for iron-induced liver damage. Specifically, iron deposition, inflammatory cell infiltration, cell death, ECM deposition, and defenestration of sinusoidal fenestrae were prominently observed in aging iron-overloaded livers. This finding implies that elderly patients should be carefully treated with iron-related therapies to minimize the risk of liver damage due to iron overload. Although we are the first to demonstrate a relationship between iron overloading in the liver and age, the underlying molecular mechanism has not been fully elucidated. Further investigations with various experimental conditions, including sex and iron concentrations, are required to reveal the mechanism. METHODS Animals All experiments were performed in compliance with the Institutional Animal Care and Use Committee of Ewha Woman’s University College of Medicine (Ethics approval number: EWHA MEDIACUC 21-003-8). This study was carried out in compliance with the ARRIVE guidelines 48 . In this study, female C57BL/6J mice purchased from ORIENT BIO Inc. (Seongnam, Korea) and the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea) were used to avoid the effects of sex, which is known to influence iron metabolism. 49 , 50 These mice were housed in cages in a specific pathogen-free, temperature- and light-controlled environment with free access to a normal diet and water. The mice were randomly divided into the control and iron overload groups. The iron overload group was intraperitoneally injected with Fe-dextran (0.5 g/kg) once a day for 4 weeks, whereas the control group was injected with normal saline at the same volume as the iron overload group. After 4 weeks of the injection period, the control and iron overload groups each had three different ages: 2-month-old, 15-month-old, and 22-month-old. Animals were fully anesthetized by respiratory anesthesia with a mixture of isoflurane and propylene glycol, and euthanized by transcardiac perfusion with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Mouse tissues were processed for immunohistochemical analysis and other histological examinations as described previously, 51 with minor modifications. Tissue preparation Cardiac blood sampling was performed before transcardiac perfusion. After euthanasia, the left and right medial lobes of the liver were excised. For fixation, the liver tissues were immersed in 4% paraformaldehyde in 0.1 M PBS at 4°C overnight. For immunohistochemistry, the tissues were processed (TP1020, Leica Microsystems, Germany), paraffin-embedded (HistoCore Arcadia, Leica Microsystems, Germany), and sectioned into 3 µm. For transmission electron microscopy (TEM), the liver tissues were sliced to approximately 1 mm × 1 mm × 1 mm. For scanning electron microscopy (SEM), the liver tissues were sliced into 200 µm. Immunostaining The livers were incubated with the following primary antibodies overnight at 4°C: anti-rabbit ionized calcium-binding adaptor molecule 1 (Iba-1; 1:3000, Wako, VA, USA) for macrophages, anti-mouse alpha-smooth muscle actin (α-SMA; 1:10000, Sigma, MO, USA) for hepatic stellate cells, anti-goat albumin (1:1000, Abcam, CA, USA) for hepatocytes, anti-rabbit CD45 (1:100, Abcam, CA, USA) for common leukocytes, and anti-rabbit glutathione peroxidase 4 (GPX-4; 1:100, Abcam, CA, USA) for ferroptosis. After washing twice in PBS for 10 min, the tissues were incubated with the following secondary antibodies for approximately 2 h at room temperature: goat anti-rabbit Cy5 (1:100, Jackson, PA, USA), goat anti-mouse Alexa 488 (1:200, Invitrogen, MO, USA), donkey anti-goat DyLight 405 (1:100, Jackson, PA, USA), goat anti-rabbit Cy3 (1:500, Jackson), and donkey anti-rabbit FITC (1:100, Jackson, PA, USA). Subsequently, the tissues were washed twice in PBS for 10 min (2 × 10 min) and mounted using the DAPI mounting medium (VECTASHIELD HardSet, Vector Laboratories, CA, USA). Digital images were obtained using a confocal microscope (LSM800, Carl Zeiss, Germany) with ZEN microscopy software (Carl Zeiss, Germany). Cell counting for CD45-positive cells and mean signal intensity measurement for GPX-4 were conducted using manual selection or semi-automatic tools in ImageJ software (NIH, Bethesda, MA, USA). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining To observe cell death, TUNEL staining was performed using the TUNEL kit (Merck Millipore, MA, USA) stored at -20°C. The livers were dehydrated as described above. After washing twice in PBS for 10 min, the cells were incubated in a solution containing 5X terminal deoxynucleotidyl transferase (TdT) equilibration buffer and distilled water at a ratio of 1:4 for 20 min at room temperature. The tissues were incubated in a TdT-labeling reaction mixture of TdT enzyme and fluorescein-FragEL at a ratio of 3:57 for 1 h at room temperature. After washing twice in PBS for 10 min, mounting was performed using the DAPI mounting medium. Digital TUNEL images were obtained using a confocal microscope (LSM800, Carl Zeiss, Germany) with ZEN microscopy software. TUNEL-positive cells were counted using Photoshop (Adobe Systems, CA, USA) adjustment functions and ImageJ software with semi-automatic selection tools. Hematoxylin and eosin (H&E) staining For H&E staining, the livers were immersed in xylene for 10 min twice and subsequently in 100% alcohol to 70% alcohol for 1 min each. Hematoxylin staining was performed for 7–10 min, followed by rinsing for approximately 5 min in warm running tap water. Eosin staining was carried out for 1–3 min, followed by rinsing several times in warm running tap water. After staining, the livers were immersed in 70% alcohol for 6–8 min, treated with 80% alcohol and 100% alcohol for 1 min each, and xylene twice for 3 min for dehydration. After mounting, the stained specimens were observed under a light microscope (BX50, Olympus, Japan). Perl’s Prussian blue staining Iron was visualized through Perl's Prussian blue staining. For Perl's Prussian blue staining, the tissues were hydrated as described above, and the livers were incubated in a mixture of 20% HCl and 10% potassium ferrocyanide (Daejung, Korea) at a ratio of 1:1 for 3 min at room temperature. After rinsing with running tap water, the sections were counterstained with Nuclear Fast Red (Vector Laboratories, CA, USA) for 3 min. The tissues were dehydrated as described above and mounted. Stained specimens were observed under a light microscope (BX50, Olympus, Japan). Iron deposition was quantified using semi-automatic tools in ImageJ software. Masson’s trichrome staining Connective tissue deposition was evaluated using Masson’s trichrome staining kit (Polysciences Inc., WA, USA). Hydration was performed in xylene twice for 5 min, followed by 100% alcohol twice for 3 min and 95% alcohol for 3 min. The tissues were incubated for 1 h in Bouin’s fixative solution preheated at 60°C in an oven and washed in warm running tap water for 5 min. The livers were immersed in a mixture of Weigert's iron hematoxylin A and B at a 1:1 ratio for 10 min and washed in warm running tap water for 5 min. Subsequently, the tissues were treated with Biebrich scarlet-acid fuchsin solution for approximately 3–5 min, followed by rinsing with distilled water. After immersion in a phosphotungstic/phosphomolybdic acid solution for 10 min, the tissues were directly immersed in an aniline blue solution for 40 min, followed by rinsing with distilled water; immersion in a 1% acetic acid solution for 1 min was then performed. Dehydration was carried out in 95% ethanol for 1 min, followed by 100% ethanol for 1 min and xylene for 2 min. Finally, the tissues were mounted, and the specimens were observed using a light microscope (BX50, Olympus, Japan). Ultrastructure For TEM, the liver tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After washing in 0.1 M phosphate buffer, they underwent a post-fix process with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4). They were dehydrated with ethanol and propylene oxide and embedded in epoxy resin. Ultrathin sections, approximately 60–70 nm in thickness, were cut by a diamond knife on an ultra-microtome (EMUC7, Leica Microsystems, Germany). The sections were contrasted with uranyl acetate, followed by lead citrate. The ultrastructure of the liver was observed by H-7650 TEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 80 kV. For SEM, the tissues were cut into smaller pieces and then prefixed with 2.5% glutaraldehyde at 4°C overnight. After washing in PBS, the tissues were incubated in 1% osmium tetroxide for 1 h. The samples were then washed and dehydrated in sequential concentrations with ethanol and critical point drying. After mounting onto stubs, sputter coating with gold (Quorum Technologies, England) was performed. The ultrastructure of the livers was observed using SEM (Sigma-300, Carl Zeiss, Germany). Biochemical analysis To observe changes in liver function, the serum levels of AST and ALT were analyzed in 2-, 15-, and 22-month-old mice using a chemistry autoanalyzer (cobas c702, Roche Diagnostics System, Switzerland). Serum total iron levels in 2-, 15-, and 22-month-old mice were assessed using the Iron/TIBC Reagent Kit (Pointe Scientific, MI, USA). Statistical analyses All statistical analyses were performed using GraphPad Prism 6 software (CA, USA) with a two-way analysis of variance and Tukey's test for multiple comparisons. Data were presented as mean ± standard deviation, with the statistical significance level set at a p -value of < .05. DATA AVAILABILITY The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Abbreviations ALT, alanine aminotransferase; AST, aspartate aminotransferase; ECM, extracellular matrix; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling Declarations COMPETING INTERESTS: The authors declare no competing interests. ETHICS DECLARATIONS This study was approved by the Institutional Animal Care and Use Committee of Ewha Woman’s University College of Medicine (EWHA MEDIACUC 21-003-8). All animal experiments were performed in compliance with the Institutional Animal Care and Use Committee of Ewha Woman’s University College of Medicine. Author Contribution J.S. conceived and designed the research. S.P. and S.S. performed most of experiments and contributed mouse models/samples. J.L. provided methodological and scientific assistance. S.P. and J.S. wrote the manuscript. Acknowledgement This study was supported by the National Research Foundation of Korea (grant number: NRF-2021R1C1C1008860). Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References NC, A. & PJ, S. Iron homeostasis. Annual review of physiology 69 (2007). https://doi.org/10.1146/annurev.physiol.69.031905.164337 Deugnier, Y., Brissot, P. & Loréal, O. Iron and the liver: update 2008. J Hepatol 48 Suppl 1, S113–123 (2008). https://doi.org/10.1016/j.jhep.2008.01.014 ER, A. & YM, S. Iron homeostasis in the liver. Comprehensive Physiology 3 (2013). https://doi.org/10.1002/cphy.c120016 CY, W. & JL, B. Liver iron sensing and body iron homeostasis. Blood 133 (2019). https://doi.org/10.1182/blood-2018-06-815894 S, M. et al. The Role of Iron and Iron Overload in Chronic Liver Disease. Medical science monitor: international medical journal of experimental and clinical research 22 (2016). https://doi.org/10.12659/msm.896494 J, L. et al. Ferroptosis: past, present and future. Cell death & disease 11 (2020). https://doi.org/10.1038/s41419-020-2298-2 Mancardi, D., Mezzanotte, M., Arrigo, E., Barinotti, A. & Roetto, A. Iron Overload, Oxidative Stress, and Ferroptosis in the Failing Heart and Liver. Antioxidants 10, 1864 (2021). Eschbach, J. W. & Adamson, J. W. Iron overload in renal failure patients: changes since the introduction of erythropoietin therapy. Kidney international. Supplement 69, S35-43 (1999). Majhail, N. S., Lazarus, H. M. & Burns, L. J. Iron overload in hematopoietic cell transplantation. Bone Marrow Transplantation 41, 997–1003 (2008). https://doi.org/Iron overload in hematopoietic cell transplantation10.1038/bmt.2008.99 Gattermann, N. The treatment of secondary hemochromatosis. Deutsches Ärzteblatt International 106, 499 (2009). Schafer, A. I. et al. Long-term Efficacy of Deferoxamine Iron Chelation Therapy in Adults With Acquired Transfusional Iron Overload. Archives of Internal Medicine 145, 1217–1221 (1985). https://doi.org/10.1001/archinte.1985.00360070091015 LG, V. & DR, P. Characterization of hepatic iron overload following dietary administration of dicyclopentadienyl iron (Ferrocene) to mice: cellular, biochemical, and molecular aspects. Experimental and molecular pathology 68 (2000). https://doi.org/10.1006/exmp.1999.2278 Arezzini, B., Lunghi, B., Lungarella, G. & Gardi, C. Iron overload enhances the development of experimental liver cirrhosis in mice. The International Journal of Biochemistry & Cell Biology 35, 486–495 (2003). https://doi.org/https://doi.org/10.1016/S1357-2725(02)00298-4 Gao, Y. et al. Mechanism of protective effects of Danshen against iron overload-induced injury in mice. Journal of Ethnopharmacology 145, 254–260 (2013). https://doi.org/https://doi.org/ 10.1016/j.jep.2012.10.060 Yatmark, P. et al. Iron distribution and histopathological characterization of the liver and heart of β-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone. Experimental and Toxicologic Pathology 66, 333–343 (2014). He, H. et al. Dual action of vitamin C in iron supplement therapeutics for iron deficiency anemia: prevention of liver damage induced by iron overload. Food & function 9, 5390–5401 (2018). Shu, W. et al. Iron Accumulates in Retinal Vascular Endothelial Cells But Has Minimal Retinal Penetration After IP Iron Dextran Injection in Mice. Investigative ophthalmology & visual science 60, 4378–4387 (2019). https://doi.org/10.1167/iovs.19-28250 Mandala, A. et al. PPARα agonist fenofibrate attenuates iron-induced liver injury in mice by modulating the Sirt3 and β-catenin signaling. American Journal of Physiology-Gastrointestinal and Liver Physiology 321, G262-G269 (2021). Y, K. et al. Iron-rich Kupffer cells exhibit phenotypic changes during the development of liver fibrosis in NASH. iScience 24 (2021). https://doi.org/10.1016/j.isci.2020.102032 Ma, W., Jia, L., Xiong, Q. & Du, H. Iron overload protects from obesity by ferroptosis. Foods 10, 1787 (2021). J, L. et al. The protective mechanism of resveratrol against hepatic injury induced by iron overload in mice. Toxicology and applied pharmacology 424 (2021). https://doi.org/10.1016/j.taap.2021.115596 Wu, A. et al. Fibroblast growth factor 21 attenuates iron overload-induced liver injury and fibrosis by inhibiting ferroptosis. Redox Biology 46, 102131 (2021). https://doi.org/https://doi.org/10.1016/j.redox.2021.102131 Deugnier, Y. & Turlin, B. Pathology of hepatic iron overload. World journal of gastroenterology 13, 4755–4760 (2007). https://doi.org/10.3748/wjg.v13.i35.4755 Akatsu, H. et al. Iron deposition in autopsied liver specimens from older patients receiving intravenous iron infusion. PloS one 15, e0237104 (2020). https://doi.org/10.1371/journal.pone.0237104 WJ, C., GP, K. & JP, G.-P. Role of Iron in Aging Related Diseases. Antioxidants (Basel, Switzerland) 11 (2022). https://doi.org/10.3390/antiox11050865 LV, B. et al. The impact of intravenous iron supplementation in elderly patients undergoing major surgery. BMC geriatrics 22 (2022). https://doi.org/10.1186/s12877-022-02983-y AA, W., A, J. & SJ, F.-T. Iron status in the elderly: A review of recent evidence. Mechanisms of ageing and development 175 (2018). https://doi.org/10.1016/j.mad.2018.07.003 Noh, B. et al. Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner. Aging Cell 22, e13977 (2023). https://doi.org/10.1111/acel.13977 Kumar, P. et al. Experimental oral iron administration: Histological investigations and expressions of iron handling proteins in rat retina with aging. Toxicology 392, 22–31 (2017). https://doi.org/https://doi.org/10.1016/j.tox.2017.10.005 Salomao, M. A. Pathology of Hepatic Iron Overload. Clin Liver Dis (Hoboken) 17, 232–237 (2021). https://doi.org/10.1002/cld.1051 Addo, L. et al. Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1852, 175–183 (2015). et al. Kupffer Cells and Blood Monocytes Orchestrate the Clearance of Iron–Carbohydrate Nanoparticles from Serum. International journal of molecular sciences 23, 2666 (2022). Hoffman, R., MD. Hematology: Basic Principles and Practice . Vol. 7th eddition 478–490 (Elsevier, 2018). Miyazaki, E. et al. Denatured H-ferritin subunit is a major constituent of haemosiderin in the liver of patients with iron overload. Gut 50, 413–419 (2002). https://doi.org/10.1136/gut.50.3.413 Gordeuk, V. R., Bacon, B. R. & Brittenham, G. M. Iron overload: causes and consequences. Annual review of nutrition 7, 485–508 (1987). H, Y. et al. Ferroptosis: Shedding Light on Mechanisms and Therapeutic Opportunities in Liver Diseases. Cells 11 (2022). https://doi.org/10.3390/cells11203301 BR, S. Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications. Cell 185 (2022). https://doi.org/10.1016/j.cell.2022.06.003 Chen, H.-J. et al. Response to iron overload in cultured hepatocytes. Scientific Reports 10, 21184 (2020). https://doi.org/10.1038/s41598-020-78026-6 X, C., PB, C., D, T. & R, K. Characteristics and Biomarkers of Ferroptosis. Frontiers in cell and developmental biology 9 (2021). https://doi.org/10.3389/fcell.2021.637162 Alison, M. R. & Sarraf, C. E. Liver cell death: patterns and mechanisms. Gut 35, 577–581 (1994). https://doi.org/10.1136/gut.35.5.577 Cogger, V. C., O'Reilly, J. N., Warren, A. & Le Couteur, D. G. A standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy. Journal of visualized experiments: JoVE , e52698 (2015). https://doi.org/10.3791/52698 Hunt, N. J. et al. Manipulating fenestrations in young and old liver sinusoidal endothelial cells. Am J Physiol Gastrointest Liver Physiol 316, G144-g154 (2019). https://doi.org/10.1152/ajpgi.00179.2018 K, S., LD, K., CF, H., P, M. & B, Z. The wHole Story About Fenestrations in LSEC. Frontiers in physiology 12 (2021). https://doi.org/10.3389/fphys.2021.735573 S, P., M, M., F, A., S, F. & E, T. Liver Sinusoidal Endothelial Cells at the Crossroad of Iron Overload and Liver Fibrosis. Antioxidants & redox signaling 35 (2021). https://doi.org/10.1089/ars.2020.8168 K, S. et al. Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study. Micron (Oxford, England : 1993) 150 (2021). https://doi.org/10.1016/j.micron.2021.103121 Bataller, R. & Brenner, D. A. Liver fibrosis. (2005). https://doi.org/10.1172/JCI24282 Ni, Y. et al. Pathological process of liver sinusoidal endothelial cells in liver diseases. World J Gastroenterol 23, 7666–7677 (2017). https://doi.org/10.3748/wjg.v23.i43.7666 Percie du Sert, N. et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. Journal of Cerebral Blood Flow & Metabolism 40, 1769–1777 (2020). Harrison-Findik, D. D. Gender-related variations in iron metabolism and liver diseases. World journal of hepatology 2 8, 302–310 (2010). Ikeda, Y. et al. Estrogen Regulates Hepcidin Expression via GPR30-BMP6-Dependent Signaling in Hepatocytes. PloS one 7 (2012). Park, H. et al. Increased Caveolin-2 Expression in Brain Endothelial Cells Promotes Age-Related Neuroinflammation. Mol Cells 45, 950–962 (2022). https://doi.org/10.14348/molcells.2022.0045 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4716297","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":332823223,"identity":"190d6700-3782-4c5b-9d0c-304284a36d2d","order_by":0,"name":"So-Hyun Park","email":"","orcid":"","institution":"Ewha Womans University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"So-Hyun","middleName":"","lastName":"Park","suffix":""},{"id":332823224,"identity":"187d0710-691d-4b3b-a093-b7b45ec99713","order_by":1,"name":"Soo-Jin Song","email":"","orcid":"","institution":"Ewha Womans University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Soo-Jin","middleName":"","lastName":"Song","suffix":""},{"id":332823225,"identity":"7f657986-e06b-4f04-aeaf-b26741ce1117","order_by":2,"name":"Jin-A Lee","email":"","orcid":"","institution":"Ewha Womans University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jin-A","middleName":"","lastName":"Lee","suffix":""},{"id":332823226,"identity":"d2a74728-461f-4cb5-9bb4-2143b9749c49","order_by":3,"name":"Jung-A Shin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYFCCA4wPgASYydhApBZmA1K1MLBJkKZFvvHws4qPbXfkGdgPP2CcuYcILQYHjpndnNn2zLCBJ82AccMzYrQwHDC7zbvtMNBNOQyMDw4Q47CG49+K/247bN/A/4ZILQwHzpgxM247nNggAbRlAzFaDA6cKZbs/Xc4uU3imcHBGUQ5bMbxjR9+nDls28+f/PBhD1EOk4CqYmOAxQ5BwN9AnLpRMApGwSgYwQAAtutADA1T/5cAAAAASUVORK5CYII=","orcid":"","institution":"Ewha Womans University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jung-A","middleName":"","lastName":"Shin","suffix":""}],"badges":[],"createdAt":"2024-07-10 07:25:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4716297/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4716297/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61806972,"identity":"c4477ef7-a293-45b2-8871-eb79adb70714","added_by":"auto","created_at":"2024-08-05 19:32:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3984837,"visible":true,"origin":"","legend":"\u003cp\u003eGross morphology and weight of the liver. (A) The iron overload group showed hepatomegaly and iron-pigmented dark-color changes. (B) Average liver weight of 15-month-old and 22-month-old mice significantly increased in comparison to 2-month-old mice in the iron overload group. (C and D) Serum total iron, alanine aminotransferase (ALT), and aspartate transaminase (AST) levels were significantly increased in the iron overload group compared with the control group. Scale bar = 1 cm in A. *\u003cem\u003ep\u003c/em\u003e\u0026lt;.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;.005, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4716297/v1/6e5e7cba0a85ec2b3bcd6e84.png"},{"id":61807332,"identity":"f963dfe9-ae7e-4a54-9cf7-44adb8b04928","added_by":"auto","created_at":"2024-08-05 19:40:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12683042,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin (H\u0026amp;E) staining and confocal microscopy labeled with CD45 (red) of the liver. (A) Compared with the control group, iron-deposited hypertropic hepatocytes (arrow) and lymphocyte infiltration (arrowhead) increased with age in the iron overload group throughout the liver. Focal necrosis (asterisks) was observed in 22-month-old mice in the iron overload group. (B) CD45 (red) showed that infiltration of inflammatory cells increased significantly with age in the iron overload group. Scale bar = 200 µm in A, 20 μm in B. *\u003cem\u003ep\u003c/em\u003e\u0026lt;.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;.005, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4716297/v1/a311c9df05c2753d9cecdc0e.png"},{"id":61806970,"identity":"fa0d3882-4853-4943-bb91-61d920a2d3dd","added_by":"auto","created_at":"2024-08-05 19:32:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9495712,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal microscopy, light microscopy, and transmission electron microscopy (TEM) of hepatocytes. (A) Labeled with albumin (blue), a-SMA (green), and Iba-1 (red). Albumin, a marker of hepatocytes, was co-localized with cells filled with iron deposition. Hepatocytes with excessive iron accumulation expressed significantly reduced intensity of albumin (asterisks). Iba-1 (a marker of macrophages) and α-SMA (a maker of activated hepatic stellate cells) did not co-localize with the cells. Iron was also deposited in the Kupffer cells (arrow). (B) TEM showed that hepatocytes lost their intracellular organelles. Iron was also deposited in the hepatic stellate cell (asterisks). Scale bar = 20 μm in A, 20 μm and 5 µm in B.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4716297/v1/069e62f84b396f943ffeeef7.png"},{"id":61806975,"identity":"5b3b3320-2e15-43c8-b3cb-ae1f7b4e5819","added_by":"auto","created_at":"2024-08-05 19:32:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13574196,"visible":true,"origin":"","legend":"\u003cp\u003ePrussian blue and TUNEL staining of serial sections. (A) In Prussian blue staining, deep blue color indicated massive iron deposition in the iron overload group. The area of iron deposition increased with age and was prominent in 22-month-old mice in the iron-overload group. (B) TUNEL staining showed that cell death (arrowhead) increased with age in the iron overload group and was located in the site where iron was deposited (asterisks). The TUNEL-positive cell count increased prominently in 22-month-old mice in the iron overload group. Scale bar = 200 μm in A, 20 μm in B. *\u003cem\u003ep\u003c/em\u003e\u0026lt;.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;.005, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4716297/v1/2ab7df97230b6bab5a1109e2.png"},{"id":61806974,"identity":"1b8d16bf-1939-4f72-b7b4-597a0dd22692","added_by":"auto","created_at":"2024-08-05 19:32:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14388154,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal microscopy labeled with GPX-4 (green) and transmission electron microscopy (TEM) for the mitochondria of hepatocytes. (A and B) GPX-4 showed that the signal intensity of GPX-4 decreased in the iron overload group compared with the control group, especially in 22-month-old mice. (C and D) TEM showed shrunken-sized mitochondria with a dense single membrane and vanished cristae in the iron overload group. Compared with the control group, the area and length of mitochondria decreased in the iron overload group. Scale bar = 20 μm in A, 5 μm in B. *\u003cem\u003ep\u003c/em\u003e\u0026lt;.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;.005, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4716297/v1/07396fbd0e4c891777e8eabd.png"},{"id":61806976,"identity":"6f6d211d-ae40-4665-a7bf-4b2ce2c5cda2","added_by":"auto","created_at":"2024-08-05 19:32:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16143698,"visible":true,"origin":"","legend":"\u003cp\u003eMasson’s trichrome, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) showing central vein and sinusoidal vessels of the liver. (A and B) Collagen deposition (blue in A, asterisks in B) around the central vein increased in the iron-overloaded liver, particularly in old age. (C) In contrast to the control group, fenestrations of sinusoidal vessels (arrowhead) disappeared in the iron overload group with age. In particular, closed and overlapped fenestrae (arrow) were observed in the 22-month-old iron overload group. (D) While there were numerous fenestrae in the control group, the iron overload group had fewer and smaller fenestrae with thickened vessel wall. (E) The 22-month-old iron overload group had significantly decreased width and count of sinusoidal fenestration compared with the control group. Scale bar = 200 µm in A, 10 µm in B and C, 1 µm in D. *\u003cem\u003ep\u003c/em\u003e\u0026lt;.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;.005, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4716297/v1/56d38548939862c73491b18d.png"},{"id":66550525,"identity":"b434ccb2-fb30-4c1d-81d8-8057ec10ac31","added_by":"auto","created_at":"2024-10-14 09:03:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":108984898,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4716297/v1/ce5f48de-d1ea-4365-b0fa-81ad04f42de9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of aging on the severity of liver injury in mice with iron overload","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIron is an essential component of metabolism\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e that is required throughout the body, including enzymes, cell signaling, mitochondrial reaction, and immune function from the cellular level to the systemic level.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e However, excessive iron causes iron overload toxicity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e because it also generates oxidative stress,\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e affecting vital organs.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Consequently, iron is strictly regulated by numerous factors for homeostasis maintenance.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e In particular, the liver plays a crucial role in iron homeostasis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e but is greatly affected by iron toxicity because it stores most of the iron in the body.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Severe liver diseases associated with excessive iron, such as chronic liver disease, liver fibrosis, hepatocellular carcinoma, and even liver failure, have been reported.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIron overload is a common complication that occurs during treatments for various diseases (e.g., dialysis for chronic kidney disease, chronic RBC transfusion for anemia, hematopoietic cell transplantation for hematologic diseases).\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Due to iron overload, the liver may develop secondary hemochromatosis, hepatomegaly, and liver fibrosis as complications.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Some mouse studies have reported the results of iron overload in the liver.\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Specifically, iron has been shown to cause liver damage.\u003c/p\u003e \u003cp\u003eOther factors such as aging also influences the regulation of iron.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e As age is an important risk factor in many diseases, an elderly person is more easily exposed to treatment-related iron, including iron supplementation therapy, dialysis, blood transfusion, and total parenteral nutrition.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Furthermore, aging itself can contribute to iron imbalance via physiologic changes.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Recent studies have reported the relationship between aging and iron overload in other organs including brain and retina.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Nonetheless, the relationship between aging and iron overload in the liver has not yet been clearly identified. The current study aimed to observe the effects of aging on iron overload in the liver.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGross morphology, liver weight, and blood analysis\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB show the gross anatomy of livers and the average liver weight, respectively. In general, the gross size and weight of livers increased with age. The livers of the iron overload group showed more hypertrophic changes and were pigmented with a dark color, as compared with those of the control group. In the iron overload group, the average liver weight in 15-month-old and 22-month-old mice is significantly increased, as compared with that in 2-month-old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe average serum AST, ALT, and total iron levels were significantly higher in the iron overload group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). No significant changes were observed between the iron overload group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe iron-overloaded liver showed hypertrophic hepatocytes filled with iron deposition as well as inflammatory cell infiltration throughout the liver\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn H\u0026amp;E staining, the normal hepatic structure was destroyed in the iron overload group, in contrast to the control group; furthermore, numerous clumps of large and round-shaped cells filled with iron deposition (arrow, brown color) were observed throughout the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Inflammatory cells (arrowhead) infiltrated around the iron-deposited cells, and they were increased in 15-month-old and 22-month-old mice compared to 2-month-old mice in the iron overload group. Focal necrosis (asterisks) was observed in 15-month-old and 22-month-old mice in the iron overload group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInfiltration of inflammatory cells increased significantly with age in the iron overload group\u003c/h2\u003e \u003cp\u003eImmunostaining and cell counting for CD45-positive cells (red) indicated that inflammatory cells infiltrated more in the iron overload group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In the iron overload group, inflammatory cells were located around the iron-deposited hepatocytes and pericentral veins. With increased age, inflammatory cells infiltrated significantly. Inflammatory cell counts of 22-month-old and 15-month-old mice significantly increased in the iron overload group compared to the control group and within the iron overload group. No significant increase was noted between 15-month-old and 22-month-old mice in the iron overload group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHepatocytes exhibited decreased albumin expression and lost their intracellular organelles\u003c/h2\u003e \u003cp\u003eImmunostaining for 22-month-old mice in the iron overload group showed that albumin, a marker of hepatocytes, was co-localized with clumps of cells filled with iron deposition, as observed by H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Iba-1 (a marker of macrophages) and α-SMA (a marker of activated hepatic stellate cells) did not co-localize with the cells. Compared to light microscopy in the same area, immunostaining showed that hepatocytes with low iron accumulation expressed albumin, whereas hepatocytes with excessive iron accumulation had a significantly reduced intensity of albumin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, asterisks). A mass with macro-iron deposition was observed using light microscopy in the same cells. Macrophages, hepatic stellate cells, and α-SMA enclosed clumps of hepatocytes with excessive iron content.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTEM revealed that the cytoplasm of hepatocytes was nearly fully filled with massive aggregates of iron (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Intracellular organelles (e.g., mitochondria, glycogen, and lipid droplets) hardly existed. The nucleus was pressed by iron accumulation and lost its normal shape. Iron aggregates were also found in other cells surrounding the hepatocytes. Hepatic microvilli, a feature of hepatocytes, were diminished. The iron aggregates had irregular shapes and a wide range of sizes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIron deposition was prominent in the old liver\u003c/h2\u003e \u003cp\u003ePrussian blue staining enabled the visualization of the location and distribution of iron deposits (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In the control groups, little iron deposition was observed (blue color); however, the iron overload group exhibited intense iron deposition throughout the liver (deep blue color). The staining grade of iron deposition in the iron overload group had a mixed pattern and was either grade IV or V:\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e diffuse accumulation of increased iron in hepatocytes, Kupffer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, arrow), and hepatic stellate cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, asterisks). The area of iron deposition increased with age (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.00001) and was more prominent in 22-month-old mice in the iron overload group than in 2-month-old and 15-month-old mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.00001 each). There was no significant difference between 2-month-old and 15-month-old mice in the iron overload group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell death was increased in the iron overload group, especially in old age\u003c/h2\u003e \u003cp\u003eTUNEL staining and TUNEL-positive cell counting indicated that the number of apoptotic cells substantially increased in 22-month-old mice in the iron overload group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Dead cells were located at the site of iron deposition (asterisks). In contrast to the control group, the iron overload group showed a large amount of apoptotic cells (arrowhead). In particular, apoptotic cells were more numerous in 22-month-old mice than in 2-month-old and 15-month-old mice in the iron overload group.\u003c/p\u003e \u003cp\u003eImmunostaining of GPX-4 showed that the signal intensity of GPX-4 decreased in the iron overload group, especially in 22-month-old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the iron overload group, ferroptotic cell death particularly occurred in the area of hepatocytes occupied by iron accumulation (white box and asterisk). In the control group, GPX-4 was expressed evenly throughout the liver, and there were no differences among the age groups. In contrast, the iron overload group showed two different areas: the area with the cluster of hepatocytes with massive iron showed decreased signal intensity of GPX-4 (asterisk), whereas the other area expressed GPX-4 as the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mean signal intensity of GPX-4 decreased in the iron overload group, as compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The 22-month-old mice in the iron overload group had significantly decreased GPX-4 intensity to compare to 2-month-old and 15-month-old mice in the iron overload group.\u003c/p\u003e \u003cp\u003eTEM revealed ultrastructural changes in the mitochondria of hepatocytes. The control group had intact double membranes (inner and outer) and cristae, whereas the iron overload group had shrunken mitochondria with dense single membranes and vanishing cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The area and length of the mitochondria decreased in the iron-loaded group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). No significant changes were observed between 22-month-old and 2-month-old mice in the iron overload group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCollagen deposition around central vein increased in the iron-overloaded liver, particularly in old age\u003c/h2\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining showed that collagen deposition (blue) substantially increased near the central vein in 22-month-old mice in the iron overload group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). TEM revealed that the extracellular matrix (ECM), including collagen fibers, was deposited under the subendothelial space of the central veins and was prominent in 22-month-old mice in the iron overload group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, asterisks). In addition to the subendothelial space, the ECM of the iron overload group accumulated in the cells near the central vein. In the control group, the ECM increased with age, but intercellular deposition was hardly observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFenestration of sinusoidal vessels disappeared in the aging iron-overloaded liver\u003c/h3\u003e\n\u003cp\u003eTEM and SEM showed shrunken and disappearing fenestrae of sinusoidal vessels in the iron overload group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D). Unlike the intact fenestrae (arrowhead) in the control group, closed or overlapping fenestrae (arrow) were often observed in 22-month-old mice in the iron overload group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The control group had numerous fenestrae of the sinusoidal endothelial wall, whereas the iron overload group had fewer and smaller fenestrae with thicker vessel walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, few fenestrae were present in 22-month-old mice in the iron overload group. The hepatic microvilli between the hepatocytes and sinusoidal vessels diminished with age in the iron overload group. Iron deposition and hypertrophic changes were also observed in endothelial cells in the iron overload group.\u003c/p\u003e \u003cp\u003eThe 22-month-old mice in the iron overload group exhibited significantly decreased width and count of sinusoidal fenestrations compared to the 22-month-old mice in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). No significant differences in fenestration width and count were detected between the control and iron overload groups among 2-month-old mice.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe current study focused on how aging affects the liver during iron overload. Fe-dextran was administered intraperitoneally to three age groups\u0026mdash;namely, young (2-month-old), old (15-month-old), and super-old (22-month-old) mice.\u003c/p\u003e \u003cp\u003eThe AST and ALT levels markedly increased in the iron overload group compared with the control group, which is similar to the results of other studies.\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Interestingly, the histological findings differed with age; however, AST and ALT levels did not significantly differ in the iron overload group. This implies that although the results of blood chemistry are similar in the young and old, the liver of the elderly is more susceptible to iron overload.\u003c/p\u003e \u003cp\u003eSimilar to previous studies,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e clusters of iron-deposited hypertrophic lesions with necrotic changes were observed throughout the liver using H\u0026amp;E staining. Unlike previous iron overload studies that identified the Kupffer cells as the main iron deposition cells in iron overload,\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e our results indicated a mixed pattern, suggesting that iron was deposited in both the reticuloendothelial system and hepatocytes.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e It is similar to the hepatocellular iron overload patterns described in other studies.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Because various factors, such as the amount of excessive iron, rate of iron deposition, and iron redistribution, determine iron overload,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e our results seem to be attributable to differences in iron exposure period or dosage compared with recent studies.\u003c/p\u003e \u003cp\u003eH\u0026amp;E staining and TEM revealed that iron-overloaded hepatocytes were filled with massive amounts of iron aggregates and had an abnormal structure, including the loss of intracellular organelles and hepatic microvilli. These iron aggregates can cause cellular damage via free radicals.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e We found that inflammatory cells infiltrated the areas where iron accumulated, and the results for CD45-positive cells indicated that aging was associated with a higher propensity for inflammation during iron overload. Significantly more CD45-positive cells were present in the super-old iron-overloaded mice than in the old iron-overloaded mice. Recent studies reported that iron overload attracted inflammatory cells such as mononuclear phagocytes. This study suggests that aging aggravates inflammation in iron-overloaded livers.\u003c/p\u003e \u003cp\u003ePrussian blue staining showed that old and super-old mice had a significant area of iron accumulation compared with young mice. Notably, the super-old mice had more extended iron-deposited areas. Given that the degree of iron deposition is closely associated with liver damage,\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e our findings suggest that aging may affect the severity of iron overload-induced liver damage.\u003c/p\u003e \u003cp\u003eIt is well known that iron is closely related to cell death via ROS.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e As in previous studies,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e TUNEL and GPX-4 results showed that cell death significantly increased throughout the liver in the iron overload groups compared to that in the control group in our study. Furthermore, we found that super-old mice were more susceptible to iron overload-related cell death than old and young mice. As previously reported,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e it appears that ferroptosis occurs in hepatocytes under iron overload, based on the decrease in GPX-4 and ferroptosis-related mitochondrial damage. Considering that hepatocytes are primary cells in which excessive iron is deposited,\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e it seems that hepatocytes are liable for iron-dependent cell death. As liver cell death is thought to be one of the factors involved in liver disease,\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e our results imply that elderly people are more vulnerable to iron overload.\u003c/p\u003e \u003cp\u003eIron overload triggers liver fibrosis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In Masson\u0026rsquo;s trichrome staining and TEM, the super-old iron overload group showed visible ECM deposition around the pericentral area with a score of I,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e despite the same amount of iron administered as the young iron overload group. These findings suggest that aging plays a significant role in liver damage caused by iron overload.\u003c/p\u003e \u003cp\u003eThe fenestration of liver sinusoidal endothelial cells (LSECs) is regarded as a dynamic structure that can be easily affected by nearby environment.\u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Similar to the sinusoidal defenestration change caused by iron overload,\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e our results showed a loss of fenestration in the iron overload groups. In particular, almost no fenestration was observed in the super-old iron-overloaded mice. Similar to a previous study, which reported an increase in fenestration gaps (large fenestration\u0026thinsp;\u0026gt;\u0026thinsp;300 nm) and no significant difference in diameter in old age,\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e we observed an increased mean fenestration diameter in super-old control mice, which was different from other studies reporting sinusoidal defenestration with aging.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e It seems that the reason for this difference was due to measurement methods. We measured the average diameter, including gaps (large fenestration), whereas previous studies evaluated fenestration by porosity (area) or diameter, excluding gaps. Interestingly, the width and number of fenestrations were significantly decreased in the super-old iron overload and super-old control groups; however, there was no significant difference between the young groups. Our results imply that older patients are more affected by iron-mediated LSEC damage and are predicted to have more metabolic dysregulation due to sinusoidal defenestration.\u003c/p\u003e \u003cp\u003eWhen iron overload occurs in the aging liver, massive amounts of iron accumulate in hepatocytes, and such iron deposition causes cellular damage and even liver cell death. Owing to hepatocyte death, the number of activated macrophages and hepatic stellate cells increase, attracting inflammatory cells, producing ECM, and thereby aggravating fibrotic changes in the liver.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Moreover, these activated macrophages and hepatic stellate cells are known to promote the defenestration of sinusoidal vessels.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e As LSEC defenestration and ECM deposition are the initial features of liver fibrosis,\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e these can cause further liver injury.\u003c/p\u003e \u003cp\u003eThis study revealed that aging was a crucial factor for iron-induced liver damage. Specifically, iron deposition, inflammatory cell infiltration, cell death, ECM deposition, and defenestration of sinusoidal fenestrae were prominently observed in aging iron-overloaded livers. This finding implies that elderly patients should be carefully treated with iron-related therapies to minimize the risk of liver damage due to iron overload. Although we are the first to demonstrate a relationship between iron overloading in the liver and age, the underlying molecular mechanism has not been fully elucidated. Further investigations with various experimental conditions, including sex and iron concentrations, are required to reveal the mechanism.\u003c/p\u003e "},{"header":"METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experiments were performed in compliance with the Institutional Animal Care and Use Committee of Ewha Woman\u0026rsquo;s University College of Medicine (Ethics approval number: EWHA MEDIACUC 21-003-8). This study was carried out in compliance with the ARRIVE guidelines\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, female C57BL/6J mice purchased from ORIENT BIO Inc. (Seongnam, Korea) and the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea) were used to avoid the effects of sex, which is known to influence iron metabolism.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e These mice were housed in cages in a specific pathogen-free, temperature- and light-controlled environment with free access to a normal diet and water. The mice were randomly divided into the control and iron overload groups. The iron overload group was intraperitoneally injected with Fe-dextran (0.5 g/kg) once a day for 4 weeks, whereas the control group was injected with normal saline at the same volume as the iron overload group. After 4 weeks of the injection period, the control and iron overload groups each had three different ages: 2-month-old, 15-month-old, and 22-month-old. Animals were fully anesthetized by respiratory anesthesia with a mixture of isoflurane and propylene glycol, and euthanized by transcardiac perfusion with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Mouse tissues were processed for immunohistochemical analysis and other histological examinations as described previously,\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e with minor modifications.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTissue preparation\u003c/h2\u003e \u003cp\u003eCardiac blood sampling was performed before transcardiac perfusion. After euthanasia, the left and right medial lobes of the liver were excised. For fixation, the liver tissues were immersed in 4% paraformaldehyde in 0.1 M PBS at 4\u0026deg;C overnight. For immunohistochemistry, the tissues were processed (TP1020, Leica Microsystems, Germany), paraffin-embedded (HistoCore Arcadia, Leica Microsystems, Germany), and sectioned into 3 \u0026micro;m. For transmission electron microscopy (TEM), the liver tissues were sliced to approximately 1 mm \u0026times; 1 mm \u0026times; 1 mm. For scanning electron microscopy (SEM), the liver tissues were sliced into 200 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining\u003c/h2\u003e \u003cp\u003eThe livers were incubated with the following primary antibodies overnight at 4\u0026deg;C: anti-rabbit ionized calcium-binding adaptor molecule 1 (Iba-1; 1:3000, Wako, VA, USA) for macrophages, anti-mouse alpha-smooth muscle actin (α-SMA; 1:10000, Sigma, MO, USA) for hepatic stellate cells, anti-goat albumin (1:1000, Abcam, CA, USA) for hepatocytes, anti-rabbit CD45 (1:100, Abcam, CA, USA) for common leukocytes, and anti-rabbit glutathione peroxidase 4 (GPX-4; 1:100, Abcam, CA, USA) for ferroptosis. After washing twice in PBS for 10 min, the tissues were incubated with the following secondary antibodies for approximately 2 h at room temperature: goat anti-rabbit Cy5 (1:100, Jackson, PA, USA), goat anti-mouse Alexa 488 (1:200, Invitrogen, MO, USA), donkey anti-goat DyLight 405 (1:100, Jackson, PA, USA), goat anti-rabbit Cy3 (1:500, Jackson), and donkey anti-rabbit FITC (1:100, Jackson, PA, USA). Subsequently, the tissues were washed twice in PBS for 10 min (2 \u0026times; 10 min) and mounted using the DAPI mounting medium (VECTASHIELD HardSet, Vector Laboratories, CA, USA).\u003c/p\u003e \u003cp\u003eDigital images were obtained using a confocal microscope (LSM800, Carl Zeiss, Germany) with ZEN microscopy software (Carl Zeiss, Germany). Cell counting for CD45-positive cells and mean signal intensity measurement for GPX-4 were conducted using manual selection or semi-automatic tools in ImageJ software (NIH, Bethesda, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTerminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining\u003c/h2\u003e \u003cp\u003eTo observe cell death, TUNEL staining was performed using the TUNEL kit (Merck Millipore, MA, USA) stored at -20\u0026deg;C. The livers were dehydrated as described above. After washing twice in PBS for 10 min, the cells were incubated in a solution containing 5X terminal deoxynucleotidyl transferase (TdT) equilibration buffer and distilled water at a ratio of 1:4 for 20 min at room temperature. The tissues were incubated in a TdT-labeling reaction mixture of TdT enzyme and fluorescein-FragEL at a ratio of 3:57 for 1 h at room temperature. After washing twice in PBS for 10 min, mounting was performed using the DAPI mounting medium.\u003c/p\u003e \u003cp\u003eDigital TUNEL images were obtained using a confocal microscope (LSM800, Carl Zeiss, Germany) with ZEN microscopy software. TUNEL-positive cells were counted using Photoshop (Adobe Systems, CA, USA) adjustment functions and ImageJ software with semi-automatic selection tools.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eFor H\u0026amp;E staining, the livers were immersed in xylene for 10 min twice and subsequently in 100% alcohol to 70% alcohol for 1 min each. Hematoxylin staining was performed for 7\u0026ndash;10 min, followed by rinsing for approximately 5 min in warm running tap water. Eosin staining was carried out for 1\u0026ndash;3 min, followed by rinsing several times in warm running tap water. After staining, the livers were immersed in 70% alcohol for 6\u0026ndash;8 min, treated with 80% alcohol and 100% alcohol for 1 min each, and xylene twice for 3 min for dehydration. After mounting, the stained specimens were observed under a light microscope (BX50, Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePerl\u0026rsquo;s Prussian blue staining\u003c/h2\u003e \u003cp\u003eIron was visualized through Perl's Prussian blue staining. For Perl's Prussian blue staining, the tissues were hydrated as described above, and the livers were incubated in a mixture of 20% HCl and 10% potassium ferrocyanide (Daejung, Korea) at a ratio of 1:1 for 3 min at room temperature. After rinsing with running tap water, the sections were counterstained with Nuclear Fast Red (Vector Laboratories, CA, USA) for 3 min. The tissues were dehydrated as described above and mounted. Stained specimens were observed under a light microscope (BX50, Olympus, Japan). Iron deposition was quantified using semi-automatic tools in ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMasson\u0026rsquo;s trichrome staining\u003c/h2\u003e \u003cp\u003eConnective tissue deposition was evaluated using Masson\u0026rsquo;s trichrome staining kit (Polysciences Inc., WA, USA). Hydration was performed in xylene twice for 5 min, followed by 100% alcohol twice for 3 min and 95% alcohol for 3 min. The tissues were incubated for 1 h in Bouin\u0026rsquo;s fixative solution preheated at 60\u0026deg;C in an oven and washed in warm running tap water for 5 min. The livers were immersed in a mixture of Weigert's iron hematoxylin A and B at a 1:1 ratio for 10 min and washed in warm running tap water for 5 min. Subsequently, the tissues were treated with Biebrich scarlet-acid fuchsin solution for approximately 3\u0026ndash;5 min, followed by rinsing with distilled water. After immersion in a phosphotungstic/phosphomolybdic acid solution for 10 min, the tissues were directly immersed in an aniline blue solution for 40 min, followed by rinsing with distilled water; immersion in a 1% acetic acid solution for 1 min was then performed. Dehydration was carried out in 95% ethanol for 1 min, followed by 100% ethanol for 1 min and xylene for 2 min. Finally, the tissues were mounted, and the specimens were observed using a light microscope (BX50, Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eUltrastructure\u003c/h2\u003e \u003cp\u003eFor TEM, the liver tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After washing in 0.1 M phosphate buffer, they underwent a post-fix process with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4). They were dehydrated with ethanol and propylene oxide and embedded in epoxy resin. Ultrathin sections, approximately 60\u0026ndash;70 nm in thickness, were cut by a diamond knife on an ultra-microtome (EMUC7, Leica Microsystems, Germany). The sections were contrasted with uranyl acetate, followed by lead citrate. The ultrastructure of the liver was observed by H-7650 TEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 80 kV.\u003c/p\u003e \u003cp\u003eFor SEM, the tissues were cut into smaller pieces and then prefixed with 2.5% glutaraldehyde at 4\u0026deg;C overnight. After washing in PBS, the tissues were incubated in 1% osmium tetroxide for 1 h. The samples were then washed and dehydrated in sequential concentrations with ethanol and critical point drying. After mounting onto stubs, sputter coating with gold (Quorum Technologies, England) was performed. The ultrastructure of the livers was observed using SEM (Sigma-300, Carl Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical analysis\u003c/h2\u003e \u003cp\u003eTo observe changes in liver function, the serum levels of AST and ALT were analyzed in 2-, 15-, and 22-month-old mice using a chemistry autoanalyzer (cobas c702, Roche Diagnostics System, Switzerland). Serum total iron levels in 2-, 15-, and 22-month-old mice were assessed using the Iron/TIBC Reagent Kit (Pointe Scientific, MI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 6 software (CA, USA) with a two-way analysis of variance and Tukey's test for multiple comparisons. Data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, with the statistical significance level set at a \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALT, alanine aminotransferase; AST, aspartate aminotransferase; ECM, extracellular matrix; H\u0026amp;E, hematoxylin and eosin; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eETHICS DECLARATIONS\u003c/h2\u003e \u003cp\u003e This study was approved by the Institutional Animal Care and Use Committee of Ewha Woman\u0026rsquo;s University College of Medicine (EWHA MEDIACUC 21-003-8). All animal experiments were performed in compliance with the Institutional Animal Care and Use Committee of Ewha Woman\u0026rsquo;s University College of Medicine.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.S. conceived and designed the research. S.P. and S.S. performed most of experiments and contributed mouse models/samples. J.L. provided methodological and scientific assistance. S.P. and J.S. wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was supported by the National Research Foundation of Korea (grant number: NRF-2021R1C1C1008860).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNC, A. \u0026amp; PJ, S. Iron homeostasis. Annual review of physiology 69 (2007). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.physiol.69.031905.164337\u003c/span\u003e\u003cspan address=\"10.1146/annurev.physiol.69.031905.164337\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeugnier, Y., Brissot, P. \u0026amp; Lor\u0026eacute;al, O. Iron and the liver: update 2008. J Hepatol 48 Suppl 1, S113\u0026ndash;123 (2008). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhep.2008.01.014\u003c/span\u003e\u003cspan address=\"10.1016/j.jhep.2008.01.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eER, A. \u0026amp; YM, S. Iron homeostasis in the liver. Comprehensive Physiology 3 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cphy.c120016\u003c/span\u003e\u003cspan address=\"10.1002/cphy.c120016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCY, W. \u0026amp; JL, B. Liver iron sensing and body iron homeostasis. Blood 133 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood-2018-06-815894\u003c/span\u003e\u003cspan address=\"10.1182/blood-2018-06-815894\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS, M. \u003cem\u003eet al.\u003c/em\u003e The Role of Iron and Iron Overload in Chronic Liver Disease. Medical science monitor: international medical journal of experimental and clinical research 22 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12659/msm.896494\u003c/span\u003e\u003cspan address=\"10.12659/msm.896494\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ, L. \u003cem\u003eet al.\u003c/em\u003e Ferroptosis: past, present and future. Cell death \u0026amp; disease 11 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41419-020-2298-2\u003c/span\u003e\u003cspan address=\"10.1038/s41419-020-2298-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMancardi, D., Mezzanotte, M., Arrigo, E., Barinotti, A. \u0026amp; Roetto, A. Iron Overload, Oxidative Stress, and Ferroptosis in the Failing Heart and Liver. Antioxidants 10, 1864 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEschbach, J. W. \u0026amp; Adamson, J. W. Iron overload in renal failure patients: changes since the introduction of erythropoietin therapy. Kidney international. Supplement 69, S35-43 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajhail, N. S., Lazarus, H. M. \u0026amp; Burns, L. J. Iron overload in hematopoietic cell transplantation. \u003cem\u003eBone Marrow Transplantation\u003c/em\u003e 41, 997\u0026ndash;1003 (2008). https://doi.org/Iron overload in hematopoietic cell transplantation10.1038/bmt.2008.99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGattermann, N. The treatment of secondary hemochromatosis. Deutsches \u0026Auml;rzteblatt International 106, 499 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchafer, A. I. \u003cem\u003eet al.\u003c/em\u003e Long-term Efficacy of Deferoxamine Iron Chelation Therapy in Adults With Acquired Transfusional Iron Overload. Archives of Internal Medicine 145, 1217\u0026ndash;1221 (1985). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1001/archinte.1985.00360070091015\u003c/span\u003e\u003cspan address=\"10.1001/archinte.1985.00360070091015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLG, V. \u0026amp; DR, P. Characterization of hepatic iron overload following dietary administration of dicyclopentadienyl iron (Ferrocene) to mice: cellular, biochemical, and molecular aspects. Experimental and molecular pathology 68 (2000). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/exmp.1999.2278\u003c/span\u003e\u003cspan address=\"10.1006/exmp.1999.2278\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArezzini, B., Lunghi, B., Lungarella, G. \u0026amp; Gardi, C. Iron overload enhances the development of experimental liver cirrhosis in mice. The International Journal of Biochemistry \u0026amp; Cell Biology 35, 486\u0026ndash;495 (2003). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/S1357-2725(02)00298-4\u003c/span\u003e\u003cspan address=\"10.1016/S1357-2725(02)00298-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, Y. \u003cem\u003eet al.\u003c/em\u003e Mechanism of protective effects of Danshen against iron overload-induced injury in mice. Journal of Ethnopharmacology 145, 254\u0026ndash;260 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/\u003c/span\u003e\u003cspan address=\"https://doi.org/https://doi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jep.2012.10.060\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2012.10.060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYatmark, P. \u003cem\u003eet al.\u003c/em\u003e Iron distribution and histopathological characterization of the liver and heart of β-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone. Experimental and Toxicologic Pathology 66, 333\u0026ndash;343 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, H. \u003cem\u003eet al.\u003c/em\u003e Dual action of vitamin C in iron supplement therapeutics for iron deficiency anemia: prevention of liver damage induced by iron overload. Food \u0026amp; function 9, 5390\u0026ndash;5401 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShu, W. \u003cem\u003eet al.\u003c/em\u003e Iron Accumulates in Retinal Vascular Endothelial Cells But Has Minimal Retinal Penetration After IP Iron Dextran Injection in Mice. Investigative ophthalmology \u0026amp; visual science 60, 4378\u0026ndash;4387 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1167/iovs.19-28250\u003c/span\u003e\u003cspan address=\"10.1167/iovs.19-28250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMandala, A. \u003cem\u003eet al.\u003c/em\u003e PPARα agonist fenofibrate attenuates iron-induced liver injury in mice by modulating the Sirt3 and β-catenin signaling. American Journal of Physiology-Gastrointestinal and Liver Physiology 321, G262-G269 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY, K. \u003cem\u003eet al.\u003c/em\u003e Iron-rich Kupffer cells exhibit phenotypic changes during the development of liver fibrosis in NASH. \u003cem\u003eiScience\u003c/em\u003e 24 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.isci.2020.102032\u003c/span\u003e\u003cspan address=\"10.1016/j.isci.2020.102032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa, W., Jia, L., Xiong, Q. \u0026amp; Du, H. Iron overload protects from obesity by ferroptosis. Foods 10, 1787 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ, L. \u003cem\u003eet al.\u003c/em\u003e The protective mechanism of resveratrol against hepatic injury induced by iron overload in mice. Toxicology and applied pharmacology 424 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.taap.2021.115596\u003c/span\u003e\u003cspan address=\"10.1016/j.taap.2021.115596\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A. \u003cem\u003eet al.\u003c/em\u003e Fibroblast growth factor 21 attenuates iron overload-induced liver injury and fibrosis by inhibiting ferroptosis. Redox Biology 46, 102131 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.redox.2021.102131\u003c/span\u003e\u003cspan address=\"10.1016/j.redox.2021.102131\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeugnier, Y. \u0026amp; Turlin, B. Pathology of hepatic iron overload. World journal of gastroenterology 13, 4755\u0026ndash;4760 (2007). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3748/wjg.v13.i35.4755\u003c/span\u003e\u003cspan address=\"10.3748/wjg.v13.i35.4755\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkatsu, H. \u003cem\u003eet al.\u003c/em\u003e Iron deposition in autopsied liver specimens from older patients receiving intravenous iron infusion. PloS one 15, e0237104 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0237104\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0237104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWJ, C., GP, K. \u0026amp; JP, G.-P. Role of Iron in Aging Related Diseases. Antioxidants (Basel, Switzerland) 11 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox11050865\u003c/span\u003e\u003cspan address=\"10.3390/antiox11050865\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLV, B. \u003cem\u003eet al.\u003c/em\u003e The impact of intravenous iron supplementation in elderly patients undergoing major surgery. BMC geriatrics 22 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12877-022-02983-y\u003c/span\u003e\u003cspan address=\"10.1186/s12877-022-02983-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAA, W., A, J. \u0026amp; SJ, F.-T. Iron status in the elderly: A review of recent evidence. \u003cem\u003eMechanisms of ageing and development\u003c/em\u003e 175 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mad.2018.07.003\u003c/span\u003e\u003cspan address=\"10.1016/j.mad.2018.07.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoh, B. \u003cem\u003eet al.\u003c/em\u003e Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner. Aging Cell 22, e13977 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/acel.13977\u003c/span\u003e\u003cspan address=\"10.1111/acel.13977\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, P. \u003cem\u003eet al.\u003c/em\u003e Experimental oral iron administration: Histological investigations and expressions of iron handling proteins in rat retina with aging. Toxicology 392, 22\u0026ndash;31 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.tox.2017.10.005\u003c/span\u003e\u003cspan address=\"10.1016/j.tox.2017.10.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalomao, M. A. Pathology of Hepatic Iron Overload. Clin Liver Dis (Hoboken) 17, 232\u0026ndash;237 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cld.1051\u003c/span\u003e\u003cspan address=\"10.1002/cld.1051\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAddo, L. \u003cem\u003eet al.\u003c/em\u003e Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1852, 175\u0026ndash;183 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eet al.\u003c/em\u003e Kupffer Cells and Blood Monocytes Orchestrate the Clearance of Iron\u0026ndash;Carbohydrate Nanoparticles from Serum. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e 23, 2666 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffman, R., MD. \u003cem\u003eHematology: Basic Principles and Practice\u003c/em\u003e. Vol. 7th eddition 478\u0026ndash;490 (Elsevier, 2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyazaki, E. \u003cem\u003eet al.\u003c/em\u003e Denatured H-ferritin subunit is a major constituent of haemosiderin in the liver of patients with iron overload. Gut 50, 413\u0026ndash;419 (2002). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/gut.50.3.413\u003c/span\u003e\u003cspan address=\"10.1136/gut.50.3.413\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGordeuk, V. R., Bacon, B. R. \u0026amp; Brittenham, G. M. Iron overload: causes and consequences. Annual review of nutrition 7, 485\u0026ndash;508 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH, Y. \u003cem\u003eet al.\u003c/em\u003e Ferroptosis: Shedding Light on Mechanisms and Therapeutic Opportunities in Liver Diseases. Cells 11 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells11203301\u003c/span\u003e\u003cspan address=\"10.3390/cells11203301\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBR, S. Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications. Cell 185 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2022.06.003\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2022.06.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, H.-J. \u003cem\u003eet al.\u003c/em\u003e Response to iron overload in cultured hepatocytes. Scientific Reports 10, 21184 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-78026-6\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-78026-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX, C., PB, C., D, T. \u0026amp; R, K. Characteristics and Biomarkers of Ferroptosis. Frontiers in cell and developmental biology 9 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fcell.2021.637162\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2021.637162\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlison, M. R. \u0026amp; Sarraf, C. E. Liver cell death: patterns and mechanisms. Gut 35, 577\u0026ndash;581 (1994). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/gut.35.5.577\u003c/span\u003e\u003cspan address=\"10.1136/gut.35.5.577\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCogger, V. C., O'Reilly, J. N., Warren, A. \u0026amp; Le Couteur, D. G. A standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy. \u003cem\u003eJournal of visualized experiments: JoVE\u003c/em\u003e, e52698 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3791/52698\u003c/span\u003e\u003cspan address=\"10.3791/52698\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunt, N. J. \u003cem\u003eet al.\u003c/em\u003e Manipulating fenestrations in young and old liver sinusoidal endothelial cells. Am J Physiol Gastrointest Liver Physiol 316, G144-g154 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/ajpgi.00179.2018\u003c/span\u003e\u003cspan address=\"10.1152/ajpgi.00179.2018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK, S., LD, K., CF, H., P, M. \u0026amp; B, Z. The wHole Story About Fenestrations in LSEC. Frontiers in physiology 12 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphys.2021.735573\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2021.735573\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS, P., M, M., F, A., S, F. \u0026amp; E, T. Liver Sinusoidal Endothelial Cells at the Crossroad of Iron Overload and Liver Fibrosis. Antioxidants \u0026amp; redox signaling 35 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/ars.2020.8168\u003c/span\u003e\u003cspan address=\"10.1089/ars.2020.8168\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK, S. \u003cem\u003eet al.\u003c/em\u003e Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study. \u003cem\u003eMicron (Oxford, England\u003c/em\u003e: 1993) 150 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micron.2021.103121\u003c/span\u003e\u003cspan address=\"10.1016/j.micron.2021.103121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBataller, R. \u0026amp; Brenner, D. A. Liver fibrosis. (2005). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1172/JCI24282\u003c/span\u003e\u003cspan address=\"10.1172/JCI24282\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi, Y. \u003cem\u003eet al.\u003c/em\u003e Pathological process of liver sinusoidal endothelial cells in liver diseases. World J Gastroenterol 23, 7666\u0026ndash;7677 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3748/wjg.v23.i43.7666\u003c/span\u003e\u003cspan address=\"10.3748/wjg.v23.i43.7666\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePercie du Sert, N. \u003cem\u003eet al.\u003c/em\u003e The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. Journal of Cerebral Blood Flow \u0026amp; Metabolism 40, 1769\u0026ndash;1777 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrison-Findik, D. D. Gender-related variations in iron metabolism and liver diseases. World journal of hepatology 2 8, 302\u0026ndash;310 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkeda, Y. \u003cem\u003eet al.\u003c/em\u003e Estrogen Regulates Hepcidin Expression via GPR30-BMP6-Dependent Signaling in Hepatocytes. PloS one 7 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, H. \u003cem\u003eet al.\u003c/em\u003e Increased Caveolin-2 Expression in Brain Endothelial Cells Promotes Age-Related Neuroinflammation. Mol Cells 45, 950\u0026ndash;962 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14348/molcells.2022.0045\u003c/span\u003e\u003cspan address=\"10.14348/molcells.2022.0045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"iron overload, aging, liver","lastPublishedDoi":"10.21203/rs.3.rs-4716297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4716297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile iron is a vital component in the body, excessive iron leads to iron toxicity, which affects vital organs. In particular, the liver is considerably affected by iron toxicity because it stores the highest amount of iron in the body. Nonetheless, the relationship between iron overload and aging in the liver has not yet been clearly identified. This study aimed to observe the effects of aging on iron overload in the liver.\u003c/p\u003e \u003cp\u003eFemale C57BL/6J mice were randomly divided into vehicle control and iron overload groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7\u0026ndash;22 per group). The iron overload group was injected with Fe-dextran (0.5 g/kg) for 4 weeks. After the experimental period, liver and blood samples were obtained from 2-, 15-, and 22-month-old mice. Liver weight, iron deposition, structural changes, cell death, extracellular matrix deposition, and fenestration of sinusoidal vessels were analyzed and compared between the groups. Additionally, biochemical analyses (aspartate aminotransferase, alanine aminotransferase, and serum total iron levels) were performed.\u003c/p\u003e \u003cp\u003eThe iron overload group exhibited significant differences compared to the control group with age. In the elderly iron overload model, iron deposition, inflammatory cell infiltration, and cell death were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.0001). Moreover, deposition of the extracellular matrix and defenestration of sinusoidal fenestrae were observed among 22-month-old mice in the iron overload group.\u003c/p\u003e \u003cp\u003eThese results suggest that aging is a risk factor for iron-induced liver injury. Therefore, caution should be exercised when performing iron-related treatments in the elderly.\u003c/p\u003e","manuscriptTitle":"Effects of aging on the severity of liver injury in mice with iron overload","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 19:32:24","doi":"10.21203/rs.3.rs-4716297/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"06e06a03-1d4b-4821-9b5d-7a8ad0ba5bd5","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35239282,"name":"Health sciences/Gastroenterology"},{"id":35239283,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2024-10-14T08:53:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-05 19:32:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4716297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4716297","identity":"rs-4716297","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}