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

doi:10.21203/rs.3.rs-4716297/v1

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

https://doi.org/10.21203/rs.3.rs-4716297/v1

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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

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.

Gross morphology, liver weight, and blood analysis

Figure 1A and 1B 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. 1B).

The average serum AST, ALT, and total iron levels were significantly higher in the iron overload group than in the control group (Fig. 1C 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. 2A). 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. 2B). 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. 3A). 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. 3A, 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. 3B). 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. 4A). 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. 3A, arrow), and hepatic stellate cells (Fig. 3B, 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. 4B). 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. 5A). 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. 5B). 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. 5D). The area and length of the mitochondria decreased in the iron-loaded group compared with the control group (Fig. 5C). 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. 6A). 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. 6B, 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. 6C 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. 6C). 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. 6D). 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. 6E). No significant differences in fenestration width and count were detected between the control and iron overload groups among 2-month-old mice.

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.

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 guidelines⁴⁸.

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.

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

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.

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

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Abstract

Figures

INTRODUCTION

RESULTS

Gross morphology, liver weight, and blood analysis

Infiltration of inflammatory cells increased significantly with age in the iron overload group

Hepatocytes exhibited decreased albumin expression and lost their intracellular organelles

Iron deposition was prominent in the old liver

Cell death was increased in the iron overload group, especially in old age

Collagen deposition around central vein increased in the iron-overloaded liver, particularly in old age

Fenestration of sinusoidal vessels disappeared in the aging iron-overloaded liver

DISCUSSION

METHODS

Animals

Tissue preparation

Immunostaining

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining

Hematoxylin and eosin (H&E) staining

Perl’s Prussian blue staining

Masson’s trichrome staining

Ultrastructure

Biochemical analysis

Statistical analyses

DATA AVAILABILITY

Abbreviations

Declarations

COMPETING INTERESTS:

ETHICS DECLARATIONS

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1