IN VIVO ASSESSMENT OF FERROPTOSIS AND FERROPTOTIC STRESS IN MICE

Abstract

Ferroptosis is an iron-dependent, lipid peroxidation-driven regulated cell death that is triggered when cellular glutathione peroxidase 4 (GPX4)-mediated cellular defense is insufficient to prevent pathologic accumulation of toxic lipid peroxides. Ferroptosis is implicated in various human pathologies, including neurodegeneration, chemotherapy-resistant state of cancer cells, ischemia-reperfusion injury, and acute and chronic kidney diseases. Despite the fact that the ferroptotic process has been rigorously interrogated in multiple preclinical models and its importance is widely accepted, the lack of specific and readily available biomarkers to detect ferroptosis in vivo in mouse models makes it challenging to delineate its contribution to key pathologic events in vivo. Critical steps to practically evaluate ferroptosis include, but are not limited to, detecting increased cell death and pathologic accumulation of toxic lipid peroxides and testing augmentation of observed pathologic events by genetic inhibition of the glutathione-GPX4 axis or mitigation of pathologic process by ferroptosis inhibitors. Here, we describe methods to evaluate these key features of the ferroptotic process in mice in vivo. Specifically, we describe methods to detect toxic lipid peroxides (4-hydroxynonenal), cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] staining) as well as protocols to pharmacologically inhibit ferroptotic stress by liproxstatin-1. These protocols provide tools for understanding the ferroptotic process in mouse genetic or disease models.

BASIC PROTOCOL 1: HOW TO USE LIPROXSTATIN-1

BASIC PROTOCOL 2: HOW TO EVALUATE FERROPTOSIS IN MOUSE KIDNEYS

INTRODUCTION

Ferroptosis is an iron-dependent non-apoptotic form of regulated cell death that is triggered by pathologic accumulation of toxic lipid peroxides (Bayir et al., 2020; Conrad et al., 2021; Galluzzi et al., 2018; Ide et al., 2021; Jiang et al., 2021; Stockwell et al., 2017). Emerging evidence suggests that ferroptosis contributes to a wide spectrum of human diseases, such as neurodegeneration, cancers, ischemia-reperfusion (IR)-induced organ injury, stroke, traumatic brain injury, asthma, liver injury, acute and chronic kidney diseases (Alim et al., 2019; Conrad et al., 2021; Friedmann Angeli et al., 2014; Guan et al., 2021; Ide et al., 2021; Wenzel et al., 2017). Therefore, methods to evaluate the pathologic contribution of ferroptosis in preclinical disease models are crucial to understand this clinically relevant pathway and to translate experimental findings into the clinic.

Glutathione peroxidase 4 (GPX4) is a selenoenzyme that acts as the central regulator and the major defense enzyme against ferroptosis by detoxifying toxic lipid peroxides at the expense of reduced glutathione (Friedmann Angeli et al., 2014; W. S. Yang et al., 2014). However, multiple disease process significantly impairs and overwhelms this critical defense pathway, allowing the pathologic accumulation of lipid peroxides and subsequent cell membrane rupture (Jiang et al., 2021; Pedrera et al., 2021). The term “ferroptotic stress” is recently coined to describe cell states with the pathologic accumulation of lipid peroxides that triggers ferroptosis and other pathologic cellular events, such as maladaptive reprogramming of proximal tubular epithelial cells of the kidneys (Figure 1), (Alim et al., 2019; Brown et al., 2019; Ide et al., 2021). Importantly, emerging evidence suggests that cellular sensitivity of ferroptosis is regulated by multiple metabolic pathways and redox-regulating enzymes, identifying potential therapeutic targets to modulate ferroptosis sensitivity (Bayir et al., 2020; Jiang et al., 2021; Stockwell et al., 2017).

Figure 1. Molecular Regulatory Pathways of Ferroptosis.

Schematic illustration shows critical features of the ferroptotic process and the corresponding methods for their evaluation.

Abbreviations: PUFA, polyunsaturated fatty acids; GPX4, glutathione/glutathione peroxidase 4: FSP1, ferroptosis suppressor protein 1, also known as AIFM2 (apoptosis-inducing factor mitochondria-associated 2); ACSL4, acyl-CoA synthetase long-chain family member 4; 4-HNE,4-hydroxynonenal; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

While many preclinical studies repeatedly identified the critical link between ferroptosis and multiple disease processes (Conrad et al., 2021), molecular characterization of ferroptosis in vivo is challenging. Unlike other regulated cell death pathways, there are no readily available biochemical markers of ferroptosis that can be applied universally across cell types and disease conditions (Bayir et al., 2020). For example, cleaved caspase 3 is a classical marker for detecting apoptosis, and phosphorylated mixed lineage kinase domain like pseudokinase (pMLKL) serves as a marker for necroptosis (Galluzzi et al., 2018). In fact, this lack of biomarkers may reflect the fact that ferroptosis is a functionally defined cell death process thus far (Bayir et al., 2020). The major functional hallmarks of ferroptosis are: (i) iron-dependency, (ii) lipid peroxidation-driven, and (iii) inhibitable by glutathione-peroxidase 4 (GPX4) activity (Jiang et al., 2021; Stockwell et al., 2017). Therefore, molecular characterization of ferroptosis in vivo requires the combined functional assessments of these pillars (Figure 1).

Critical steps to practically evaluate ferroptosis include, but are not limited to, detecting the evidence of increased cell death and pathologic accumulation of toxic lipid peroxides, then testing whether observed pathologic events are augmented by genetic inhibition of the glutathione-GPX4 axis or mitigated by pharmacological ferroptosis inhibitors (Figure 1). In addition, in some tissues and cell types, there are additional candidate biomarkers to evaluate ferroptosis and ferroptotic stress in mouse disease models that could supplement these functional evaluation steps (Chen et al., 2021; Feng et al., 2020; Stockwell et al., 2017). For example, acyl-CoA synthetase long-chain family member 4 (ACSL4) has been used widely as a biomarker to detect ferroptotic stress in the kidneys, one of the most vulnerable organs to ferroptotic stress and ferroptosis (Guan et al., 2021; Ide et al., 2021; Muller et al., 2017).

We have successfully used a combination of the following assays to detect the role of ferroptosis and ferroptotic stress in renal injury and repair process: (i) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining that detects both apoptosis and necrosis (Friedmann Angeli et al., 2014; Grasl-Kraupp et al., 1995; Ide et al., 2021); (ii) immunohistochemical detection of 4-hydroxynonenal (4-HNE) or malondialdehyde (MDA) for cellular lipid peroxidation state, (iii) immunostaining of ACSL4, (iv) genetic deletion of the Gpx4 gene, and (v) liproxstatin-1 to experimentally modulate ferroptotic stress level in vivo (Ide et al., 2021).

Here we describe detailed protocols to delineate ferroptosis and ferroptotic stress in mice in vivo. Basic protocol 1 details how to prepare and administer an in vivo active ferroptosis inhibitor, liproxstatin-1. Basic protocol 2 provides methods to localize cells undergoing ferroptotic stress in mice kidneys using immunostaining of 4-hydroxynonenal (4-HNE), a cytotoxic lipid peroxidation product.

BASIC PROTOCOL 1: HOW TO USE LIPROXSTATIN-1

Here we show how to prepare and administer liproxstatin-1 to evaluate the ferroptotic process in mouse disease models. The protocol was successfully applied to a mouse model of renal ischemia-reperfusion injury (IRI) and identified the role of ferroptotic stress in promoting the accumulation of proinflammatory proximal tubular cells after IRI and in triggering ferroptosis (Ide et al., 2021). The study identified a non-lethal role of ferroptotic stress in the kidney repair process using liproxstatin-1.

Materials

Liproxsatin-1 (Selleckchem, S7699)

Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, D2438, sterile filtered)

1X phosphate-buffered saline (PBS, Gibco, 10010-023)

Microcentrifuge tubes 1.5 ml

Vortex mixers

Pipettes (1000 μl and 20 μl)

26 G needle (BD, REF 305187)

1 ml syringe (BD, REF 309659)

Preparing liproxsatin-1 solution

• 1Prepare stock solution; The stock solution is prepared by dissolving liproxstatin-1 powder with DMSO at 50 μg/μl concentration using aseptic techniques under a cell culture hood. Note that the stock solution should be vortexed well and stored at −80 °C for long-term storage.

  For example, add 100 μl of DMSO to 5 mg liproxstatin-1.

  This solution shows a yellow discoloration, but this color change does not affect its pharmacological property in vivo and in vitro. We store this stock solution at −80 °C.

• 2Prepare working solution: The working solution is prepared by diluting the stock solution with 1X PBS to the final concentration of DMSO at 1 %.

  For example, we dissolve 5 μl stock solution in 500 μl of 1X PBS (total 505 μL). At first the color of the working solution is white and not transparent, but it soon becomes clear and transparent after rigorous vortexing.

Liproxsatin-1 injections to mice

• 3Administer the liproxstatin-1 working solution at 10 mg/kg body weight to mice intraperitoneally once daily. We use 26-gauge needles with 1 ml syringe for intraperitoneal injections.

  We use liproxstatin-1 under the clearance of Duke Occupational & Environmental Safety Office (OESO) and the approved IACUC protocol. Any specific hazard has not been identified based on the safety data sheet provided by the manufacturer. We follow the institutional standard operating procedure, such as using personal protective equipment (gowns and gloves) when injecting the drug into the mice. Additional drug-specific care is not required.

  We prepare the working solution just before going to the animal husbandry area and bring it at room temperature and light-shielded.

  We administer the same volume of vehicle solution (1 % DMSO in PBS) at the same schedule as the liproxstatin-1-treated group and use them as the control group.

  We commonly use C57BL6/J wild-type mice (RRID: IMSR_JAX:000664) or gene-modified mouse lines backcrossed to this background. Based on our experience and power calculation, we typically use 5 to 7 mice per group and did not observe any side effects up to 21 days of once-daily injections.

BASIC PROTOCOL 2: HOW TO EVALUATE FERROPTOSIS IN MOUSE KIDNEYS

As we discussed above, evaluating ferroptosis requires a combinatorial detection of cell death and the pathologic accumulation of lipid peroxides as a first key step. To detect cell death, we use TUNEL staining (Abcam: ab206386) following the manufacturer’s protocol with formalin-fixed paraffin-embedded (FFPE) tissue sections. It is important to note that TUNEL staining detects both apoptosis and necrosis (Grasl-Kraupp et al., 1995; M. Yang et al., 2014), (See Figure 2). Here, we show detailed steps to perform 4-hydroxynonenal (4-HNE) immunostaining to localize the cells with a high level of lipid peroxides using mouse kidney tissues.

Figure 2. 4-HNE immunostaining and TUNEL staining of ischemia-reperfusion injured kidneys.

(A) Immunostaining for 4-HNE using mouse monoclonal antibody. A representative image of 4-HNE is shown. We subjected the kidneys of NFE2–related factor 2 (Nrf2) knockout mice to unilateral ischemia-reperfusion injury (IRI, ischemic time for 45 min and reperfusion for 1 day). Abbreviations: CLK; contralateral uninjured kidney. Bouin-fixed tissue sections were stained with anti-4-HNE antibody (1:100) without antigen unmasking. Formalin-fixed tissue sections were stained with anti-4-HNE antibody (1:100) with antigen unmasking (pH6.0 citrate buffer, boiled for 10 min). The antigen unmasking procedure slightly improved the signal detection using FFPE sections (data not shown). Therefore, we highly recommend using Bouin-fixed sections for this antibody. (B) TUNEL staining of IR-injured kidneys. Note that TUNEL stains nuclei (a) and cytoplasm (b). We used kidney samples from C57BL/6J mice subjected to IRI surgery (ischemia time 45min, reperfusion 3 days). Scale bars: 50 μm.

Materials

Tissue cassettes (VWR, 18000-130)

Biopsy foam pads (VWR, 18000-262)

Bouin’s Fixative Solution, Picric Acid-Formalin-Acetic Acid Mixture (Ricca Chemical Company, 1120-1)

Xylene (VWR, MK866816)

Ethanol 200 PROOF (VWR, TX89125172DUK)

30% hydrogen peroxide (H₂O₂), (VWR, BDH7690–1)

Methanol (VWR, BDH 1135)

1x PBS (Fisher, BP399-20)

M.O.M.® (Mouse on Mouse) ImmPRESS® Polymer kit peroxidase (VECTOR, MP-2400)

Anti 4-HNE monoclonal antibody (Adipogen, Clone HNEJ-2, Ca. JAI-MHN-020P)

Vector® DAB (VECTOR, SK-4100)

Hematoxylin (VWR, 95057-844)

VectaMount® Permanent Mounting Medium (H-5000)

Microtome (Leica, HistoCore Biocut)

Adhesion microscope slides for immunohistochemistry (Matsunami, TOM-13)

Staining racks and vessels with lids (Sakura, Tissue-Tek® Manual Slide Staining Set, 4451)

60 °C incubator

Liquid Blocker –super PAP Pen (Electron Microscopy Sciences, 71312)

Parafilm (Bemis, PM-996)

Micro cover glasses (Matsunami, 2-171-16; 24 × 40 mm)

Transfer pipets (VWR, 16001-188)

Prepare mouse kidney samples

• 1Sacrifice mice according to the protocol approved by your local animal care committee and collect kidneys. We remove the capsule of the kidneys by peeing it off with forceps and cut them into pieces (typically 1/3 of the size of the kidneys) for better fixation. We cut kidneys by transverse section and always include hilum in all three portions so that we can observe both cortex and medulla.
• 2Fix decapsulated and sliced kidneys by immersion in Bouin’s solution in 5-ml tubes at 4 °C with rocking for 16 to 24 hours.

  CAUTION: Bouin’s solution is toxic and contains picric acid, which is explosive when it is dried out. Personal protective equipment (PPE) must be used when handling the solution. Bouin’s solution must be handled in a chemical fume hood.

  We observed better preservation of 4-HNE signal with tissues fixed with Bouin’s solution than 10 % neutral buffered formalin (NBF) as the manufacturer recommended (Figure 2A).

• 3Place fixed kidneys into a labeled tissue cassette with a biopsy foam and immerse the kidneys in 70 % ethanol solution for up to 7 days.

  70% ethanol solution decolorates the kidneys. We recommend changing the solution with a new 70% ethanol solution daily until the solution does not become yellow or tissues are processed to paraffin blocks (we typically change the 70% ethanol solution 5 to 7 times).

• 4Transfer the samples into a tissue processor (Sakura Tissue-Tek VIP-5 Vacuum Infiltration Processor) for dehydration, clearing, and infiltration for preparing paraffin-embedded tissue blocks.
  Tissue processing program information:

  1. 70% Alcohol, 20 min, mixed slowly
  2. 80% Alcohol, 25 min, mixed slowly
  3. 80% Alcohol, 25 min, mixed slowly
  4. 95% Alcohol, 30 min, mixed slowly
  5. 95% Alcohol, 30 min, mixed slowly
  6. 100% Alcohol, 45 min, mixed slowly
  7. 100% Alcohol, 45 min, mixed slowly
  8. 100% Xylene, 30 min, mixed slowly
  9. 100% Xylene, 30 min, mixed slowly
  10. 100% Xylene, 45 min, mixed slowly
  11. Paraffin, 45 min at 60 °C, mixed slowly
  12. Paraffin, 45 min at 60 °C, mixed slowly
  13. Paraffin, 45 min at 60 °C, mixed slowly
  14. Paraffin, 45 min at 60 °C, mixed slowly
      We store paraffin-embedded tissue blocks at 4 °C.

4-HNE immunostaining

Day 1

• 5Prepare FFPE sections at 5 μm thickness using microtome.
• 6Bake slides at 60 °C oven for about 30 min to 1 hour and proceed with deparaffinization.
• 7De-paraffinize and rehydrate; incubate slides in Xylene for 5 min twice and hydrate with graded ethanol solution (100 % ethanol for 5 min twice, 95 % ethanol for 4 min twice, and 70 % ethanol for 3 min). We use Sakura Manual Slide Staining Set for manual processing. Each backet needs approximately 250 mL of each solution to cover the slides. Then, rinse the slides using distilled water for 5 min.

  CAUTION: Xylene is toxic and highly flammable. Personal protective equipment (PPE) must be used when handling it. Use Xylene in a chemical fume hood.

  Please note that antigen unmasking (pH6.0) is not necessarily and not effective in detecting 4-HNE using this protocol.

• 8Block endogenous peroxidase activity. Incubate slides in 0.3 % H₂O₂ in methanol for 20 min at room temperature.
• 9Wash slides in PBS solution for 2 min twice.
• 10Incubate slides in working solution of prepared M.O.M Mouse IgG Blocking Reagent for 1 hour.

  Preparation of M.O.M. Mouse IgG Blocking Reagent working solution: add 2 drops of M.O.M.

  Blocking Reagent stock solution to 2.5 ml of PBS.

• 11Wash in PBS for 2 min twice.
• 12Incubate slides in M.O.M Normal Horse Serum 2.5 % for 5 min.
• 13Incubate slides in the diluted primary antibody solution overnight at 4 °C.

  Although MOM kit instruction recommends the incubation time for 30 min, we typically extend incubation time to overnight at 4 °C to enhance the signal detection. Appropriate use of negative control is critical to exclude potential false-positive results, such as a combination of slides without the primary antibody incubation and slides with normal kidneys with primary antibody.

  The manufacturer’s recommended concentration of primary antibody is 10 – 20 μg/ml (stock concentration 100 μg/ml). However, we use this antibody at 1 – 2 μg/ml (1:50 – 1:100 of stock solution diluted by the blocking buffer provided by the kit). We recommend titrating the concentration to determine the optimal concentration with a low background signal.

  Covering sections with a piece of parafilm helps the sections secure from drying.

Day 2

• 14Wash slides in PBS for 2 min twice.
• 15Incubate slides in M.O.M. ImmPRESS Reagent for 10 min.

  We usually incubate the sections for a longer time (ex. for 30 min).

• 16Wash slides in PBS for 2 min twice.
• 17
  Prepare DAB (3,3-Diaminobenzidine) solution; add the following reagents to 5.0 ml of distilled water

  • Add 2 drops of Vector DAB Reagent 1
  • Add 4 drops of Vector DAB Reagent 2
  • Add 2 drops of Vector DAB Reagent 3
• 18Incubate slides in DAB solutions for 5–10 min at room temperature.

  We recommend optimizing the incubation time by testing different incubation durations.

• 19Wash slides in tap water for 5 min.
• 20Counterstain the slides with hematoxylin (1x solution) at room temperature for 15 to 30 sec.

  Rinse slides with running water for about 1 to 2 min to remove residual hematoxylin.

  We recommend evaluating the staining level under microscope before going to the next step to ensure the optimal counter staining. You can repeat the STEP20 to obtain the desired staining level.

• 21Dehydrate the slides by incubating them with 95% ethanol for 30 sec, 100% ethanol for 30sec twice, and then twice in 100% Xylene.
• 22Place a coverslip over the tissues with a permanent mounting medium.

  We apply one drop of mounting medium onto the slide by a transfer pipet. Slowly tip a coverslip on to the slide from the long-edge of the slide to another side to pushing any bubbles away.

  If bubbles are on the sections, you can incubate the slides in 100% Xylene to remove the coverslip and repeat the STEP 22.

COMMENTARY

Background information

While multiple potent ferroptosis inhibitors are available (Stockwell et al., 2017), ferrostatin-1 and liproxstatin-1 are the two commonly used ferroptosis inhibitors (Dixon et al., 2012; Friedmann Angeli et al., 2014). These reagents act as radical trapping antioxidants, effectively reducing membrane lipid peroxidation and inhibiting the ferroptotic death process. Ferrostatin-1 was first identified as a potent ferroptosis inhibitor through a high-throughput chemical screening that inhibits erastin-induced ferroptosis in human fibrosarcoma HT-1080 cells (Dixon et al., 2012). Although ferrostatin-1 is highly effective in cell culture models, it may not be suitable for in vivo mouse disease models due to its low metabolic stability originating from its ester moiety (Hofmans et al., 2016). Improved ferrostatins with better stability and ADME (absorption, distribution, metabolism, and excretion) profiles have been developed for in vivo use (Devisscher et al., 2018; Hofmans et al., 2016; Linkermann et al., 2014). Liproxstatin-1 is another potent ferroptosis inhibitor identified through high-throughput screening using tamoxifen-inducible Gpx4 knockout mouse embryonic fibroblasts (Friedmann Angeli et al., 2014). Liproxstatin-1 potently scavenges lipid peroxides and effectively prevents ferroptosis both in cell culture and in vivo mouse disease models. Assessment of ADME parameters supports effectiveness with in vivo administration in mouse disease models. The reported half-life (t1/2) is 4.6 hours after 1.0 mg/kg intravenous injection, and its bioavailability is 52% (Friedmann Angeli et al., 2014). Indeed, we and others successfully employed liproxstatin-1 in the genetic model of ferroptosis in mice (Friedmann Angeli et al., 2014; Ide et al., 2021). Improved ferrostatins or liproxstatin-1 are valuable tools to evaluate the ferroptotic process in mouse disease models.

Critical Parameters and Troubleshooting

The following troubleshooting guide includes common problems around liproxstatin-1 preparation and 4-HNE immunostaining (See Table 1 and 2).

Table 1.

Troubleshooting guide for Liproxstatin-1 treatment (Protocol 1)

Problem	Possible Cause	Solution
Insoluble, unclear solution	Insufficient mixing	Vortex it well again
	Wrong concentration	Calculate the concentration again

Table 2.

Troubleshooting guide for 4-HNE immunostaining (Protocol 2)

Problem	Possible Cause	Solution
Poor signal	Primary antibody concentration is too low	Use a higher concentration of primary antibody
	Insufficient antibody access to target antigen	Try antigen retrieval using antigen unmasking solutions
	HRP-DAB reaction is too weak.	Extend incubation time in DAB solution
High background	Endogenous peroxidase activity	Extend 0.3% H2O2 incubation time (>20min)
	HRP-DAB reaction is too strong.	Shorten incubation time in DAB solution

Abbreviations: HRP, horse-radish peroxidase.

Understanding Results

Liproxsatin-1 administration to mice

We previously used the liproxstatin-1 treatment protocol (protocol 1) to inhibit ferroptosis and ferroptotic stress-induced accumulation of inflammatory proximal tubular cells in a mouse renal IRI model (Ide et al., 2021). We used a combination of TUNEL staining (cell death), 4-HNE and malondialdehyde (lipid peroxidation), SOX9/VCAM1 staining (inflammatory proximal tubular cells) as readouts of the effect. Daily administration of liproxstatin-1 starting from 1-hour prior to IRI ameliorated these pathologic changes and overall damage of Gpx4-deficient kidneys that underwent IRI (Ide et al., 2021).

4-HNE immunostaining

Bouin’s solution is recommended for fixing tissues to use the anti-4-HNE mouse monoclonal antibody. As shown in Figure 2, IR injured-kidneys fixed in Bouin’s solution show a much stronger 4-HNE signal compared to 10% NBF-fixed kidneys (overnight fixation). Specificity of the antibody signal should be also tested using uninjured tissues (e.g., sham-treated group, contralateral kidneys of unilateral renal IRI model) and the tissue sections without incubating the primary antibody.

As discussed above, increased 4-HNE signal alone does not support the presence of ferroptosis. Thus, a combinatorial assessment is crucial. We recommend detecting cell death using TUNEL staining in addition to evaluating lipid peroxidation status, 4-HNE staining or other methods to evaluate lipid peroxidation for detecting ferroptosis.

Note; we use the TUNEL assay kit from Abcam (ab206386) and follow instructions provided by the manufacturer. To avoid misinterpretation due to false-positive and negative results, it is important to prepare positive control (tissue sections incubated with DNase1) and negative control slides (ex. normal kidney sections). Also, please note that the TUNEL signal is detected both in nuclei and cytoplasm. Cytoplasmic TUNEL signal is likely from karyorrhexis during necrotic cell death (Figure 2B), (M. Yang et al., 2014).

Time Considerations

Protocol 1; Preparing liproxstatin-1 solution takes about an hour.

Protocol 2; It takes a few days to prepare Bouin’s solution-fixed paraffin-embedded mouse kidney samples. Additional 2 days are required to perform 4-HNE immunostaining.