Protocol for assessing senescence-associated lung pathologies in mice

Summary Cellular senescence underlies tissue aging and aging-associated pathologies, as well as lung pathology. We and others have shown that elimination of senescent cells alleviates pulmonary diseases such as fibrosis and emphysema in animal models. We herein describe a protocol for assessing senescence-dependent lung phenotypes in mice. This protocol describes the use of ARF-DTR mice for semi-genetic elimination of lung senescent cells, followed by a pulmonary function test and the combination with pulmonary disease models to study lung pathologies. For complete details on the use and execution of this protocol, please refer to Hashimoto et al. (2016), Kawaguchi et al. (2021), and Mikawa et al. (2018).

SUMMARY Cellular senescence underlies tissue aging and aging-associated pathologies, as well as lung pathology. We and others have shown that elimination of senescent cells alleviates pulmonary diseases such as fibrosis and emphysema in animal models. We herein describe a protocol for assessing senescence-dependent lung phenotypes in mice. This protocol describes the use of ARF-DTR mice for semi-genetic elimination of lung senescent cells, followed by a pulmonary function test and the combination with pulmonary disease models to study lung pathologies. For complete details on the use and execution of this protocol, please refer to Hashimoto et al. (2016), Kawaguchi et al. (2021), and Mikawa et al. (2018).

BEFORE YOU BEGIN
The protocol below describes the specific steps for assessing senescence-dependent lung phenotypes in mice. In this protocol, we use a transgenic model, ARF-DTR mice, from which it is possible to eliminate p19 Arf -expressing cells using diphtheria toxin (DT)-mediated cell knockout system (Furukawa et al., 2006;Saito et al., 2001). p19 Arf plays an essential role in the induction of cellular senescence in rodent cells (Kamijo et al., 1997), and its expression increases during aging in the mouse tissues similarly to p16 INK4a (Krishnamurthy et al., 2004). ARF-DTR mice have transgene in which Arf exon 1b was replaced with genes encoding the diphtheria toxin receptor (DTR) fused to 2A peptide sequence and firefly luciferase (Figure 1). The luminescence signals observed in aged ARF-DTR mice are attributed to those in the lung, adipose, and testis tissues. We successfully eliminated p19 Arf -expressing cells from the lung tissue of ARF-DTR mice (> 6 months). While we used C57BL/6J background in all of the following analyses, we assume the protocols can also be adapted to Balb/c strain, as this strain has also been reported to show the similar aging-dependent changes in the lung structure and function as well as elastase-induced emphysema pathologies (Kawakami et al., 1984;Limjunyawong et al., 2015).
Institutional permission and oversight information for the animal study should be obtained. All animal experiments in this study were approved by the National Center for Geriatrics and Gerontology Animal Ethics Committee (approval numbers, 2-7, 3-2, 28-6, 29-24, 30-34 and 31-3).
Note: Male ARF-DTR mice and female wild-type mice are typically used for breeding so that ARF-DTR hemizygous offspring can efficiently be obtained.
2. Genotyping a. Cut the tails (5 mm) of pups and put in a 1.5 mL tube. b. Tail lysis i. Add 500 mL of tail lysis buffer and 2 mL Proteinase K.
iii. Vortex thoroughly, and check that the tail is completely lysed. c. Phenol/chloroform extraction of genomic DNA i. Add 500 mL of neutralized phenol/chloroform and vortex thoroughly.
iii. Transfer the upper phase to a new 1.5 mL tube. iv. Add 350 mL of isopropanol and invert until DNA precipitate forms. v. Centrifuge at 15,300 3 g for 5 min at room temperature and carefully remove and discard supernatant. vi. Add 0.5-1 mL of 70% ethanol and invert several times. vii. Centrifuge at 15,300 3 g for 5 min at room temperature and carefully remove and discard supernatant.  i. Perform multiplex PCR to amplify the transgene (DTR-Luc) and control allele using the primers described in the key resources table (KRT). PCR condition is described in materials and equipment.
Note: This genotyping PCR protocol is designed for the use of KOD Oneâ PCR Master Mix (TOYOBO) and a T100ä thermal cycler (Bio-Rad).
ii. Separate the PCR products by agarose gel electrophoresis. The amplicon sizes of control allele and transgene are 324 bp and 196 bp, respectively ( Figure 2). Aliquot stock solution into 600 mL/tube and store at À80 C for up to 1 year. Avoid repeated freeze-thawing.

PBS n/a 600 mL
Prepare the solution just before use. Injection volume: Body weight 3 10 mL/mouse (150 mg/kg)

Reagent Final concentration Amount
Diphtheria toxin 1 mg/mL 1 mg Aliquot stock solution into 10 mL/tube and store at À80 C for up to 1 year. Avoid freeze-thawing. This step describes how to monitor senescent cells by in vivo imaging.

Injectable DT solution
1. Prior to in vivo imaging, shave hair on the ventral side with an electric clipper ( Figure 3A). 2. Set up imaging settings at the IVIS spectrum imager system as follows; Exposure time: 3 min, Binning: Medium, F/Stop: 1, Emission Filter: Open, Field of View: B. 3. In vivo imaging a. Inject the luciferin (VivoGlo TM Luciferin, in vivo grade) intraperitoneally (i.p.) according to the manufacturer's instructions (https://www.promega.jp/products/luciferase-assays/reporter-assays/ vivoglo-luciferin-in-vivo-grade/?catNum=P1043#resources). b. Anesthetize mice with 2% isoflurane in air. c. Place the mice in the chamber and arrange them as necessary so that you can begin imaging ( Figures 3B and 3C). d. Perform the bioluminescence imaging 10 min post i.p. injection ( Figure 3D). Troubleshooting 1

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CRITICAL: Because dark colored hair impairs the luminescence detection, hair on the ventral side should be removed thoroughly before in vivo imaging.
Note: In ARF-DTR mice, luminescence reflecting senescent cells in the tissue is mainly obtained from lung, adipose tissue and testis.
4. To calculate light outputs, draw region of Interest (ROI) by Living image software ( Figure 3E).

Senescent cell elimination
Timing: $4 weeks This step describes how to eliminate senescent cells from lung tissue (Hashimoto et al., 2016). The experimental scheme for senescent cell elimination is described in Figure 4.
5. DT treatment a. Freshly prepare the DT solutions (5 mg/mL in PBS) and keep them on ice until use. b. Inject DT solution (50 mg/kg body weight) intraperitoneally twice with a 2-week interval.
CRITICAL: Do not use the refrozen DT solution.
6. Four weeks after DT injection, evaluate senescent cell elimination by in vivo imaging described in the steps 1-3. Troubleshooting 2 Note: In addition to in vivo imaging, we recommend assessing senescent cell elimination by gene expression analysis of senescent markers such as Ink4a, Arf and p21 by RT-qPCR.

Pulmonary function test
Timing: $1 h This step describes how to perform a pulmonary function test by the flexiVentâ system. 7. Euthanize mice by intraperitoneal injection of excess amount (100 mg/kg body weight) of pentobarbital sodium. 8. Cut and open the skin to expose the trachea ( Figure 5A). 9. Insert string under the trachea using small forceps (Figures 5B and 5C). 10. Make a small incision into the trachea ( Figure 5D). 11. Perform tracheal intubation using a 12 mm-long cannula ( Figure 5E). 12. Tie off the outside of the intubated site to prevent the cannula from falling out ( Figure 5F). 13. Connect the intubated cannula to a flexiVentâ system. Note: Diaphragm may be removed before connecting the flexiVentâ system to allow visual confirmation of the airflow into the lung tissue.
14. Ventilate the mouse at a respiratory rate of 150 breaths/min with a tidal volume of 10 mL/kg against a positive end expiratory pressure of 3 cmH 2 O. 15. Consecutively perform Deep inflation, Snapshot-150, Quickprime-3, and a pressure-volume loop with constant increasing pressure three times in each mouse. 16. Acquire parameters using a flexiVent software.
a. Calculate the dynamic compliance and resistance values using a single frequency forced oscillation technique. b. Calculate the static lung compliance value by fitting the Salazar-Knowles equation to the pressure volume loop. c. Obtain the tissue elastance and tissue damping values from respiratory system impedance data using a constant phase model.

BALF and lung tissue sampling
Timing: $1 h

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This step describes how to obtain bronchoalveolar lavage fluid (BALF) and lung tissue.
17. After pulmonary function test, connect a syringe containing 1 mL of 5 mM EDTA in PBS to the intubated cannula ( Figure 6). 18. Bronchoalveolar lavage fluid (BALF) sampling a. Gentle syringing for 3 times. b. Transfer the BALF to a 1.5 mL tube. c. Centrifuge at 5,800 3 g for 1 min at 4 C. d. Transfer the supernatant (BALF) to a new 1.5 mL tube, and store at -80 C until use. Troubleshooting 3 e. (if needed) Add 100 mL of CELLBANKERâ to precipitate (cell fraction), and store at -80 C until use.
Note: For longer storage of BALF cells, store the samples in liquid nitrogen.
19. Preparation for lung fixation a. After the BALF sampling, remove the syringe and connect a T-shaped stopcock to the intubated cannula ( Figure 7A). b. Connect a syringe filled with fixative solution (Mildformâ20N) to one side of the T-shaped stopcock ( Figure 7B). c. The other side is connected to a 1-m-long silicon tube ( Figure 7C) which is connected to a manometer and empty syringe (pressure-regulating syringe) through another T-shaped stopcock ( Figure 7D).
Note: In case of both RNA/protein extraction and tissue fixations are required from a single mouse, add the next step (step 20) before fixation.
20. Collecting the right lung lobes for RNA/protein extraction a. Clamp the right bronchus with forceps ( Figures 8A-8C). b. Excise the right lung lobes (4 lobes) for RNA extraction. c. Wash the right lung lobes twice with 10 mL of PBS. d. Mince the tissue into small pieces with sterile scissors in a 2 mL round-bottom tube ( Figures  9A and 9B). e. For RNA extraction, soak the tissue into RNAlaterâ and store at -30 C until use ( Figure 9C). f. For protein extraction, snap freezing by liquid nitrogen and store at -80 C until use. g. RNA/protein extraction using TRI REAGENTâ. Troubleshooting 4 21. Fix the lung tissue.

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This step describes how to assess senescence-dependent lung pathologies by combining with the pulmonary emphysema model using porcine pancreatic elastase (Mikawa et al., 2018). The experimental scheme for setting up pulmonary emphysema model is described in Figure 12.
Note: PPE administration should be done one week after first DT treatment.
24. One week after PPE administration, inject DT solution (50 mg/kg body weight) intraperitoneally (second DT treatment). 25. Two weeks after DT injection, perform the analyses described in the steps 7-22 (pulmonary function test, BALF isolation and tissue sampling).  Lung metastasis model

Timing: 2 weeks
This step describes how to assess senescence-dependent lung pathologies by combining with B16-F10 melanoma lung metastasis model (Kawaguchi et al., 2021). The experimental scheme for setting up lung metastasis model is described in Figure 13.
Note: Replace medium every 2-3 days, and a subcultivation ratio of 1:10 is recommended.
27. Prepare cell suspension for tail vein injection. a. Remove and discard culture media from flask. b. Briefly rinse the cell layer with PBS. c. Add 2 mL of trypsin-EDTA solution to the flask. d. Incubate the cells in a CO 2 incubator (37 C, 5% CO 2 ) until the cell layer is dispersed (usually within 2 min). e. Add 8 mL of complete growth medium and aspirate cells by gently pipetting. f. Transfer cells to a 50 mL tube and perform cell counting. g. Centrifuge at 400 3 g for 3 min at room temperature. h. Remove and discard the supernatant. i. Add 10 mL of PBS and wash cells by gently pipetting. j. Centrifuge at 400 3 g for 3 min at room temperature. k. Remove and discard the supernatant. l. Repeat steps i-k. m. Resuspend cells in PBS at a density of 2 3 10 6 cells/mL. 28. Inject 200 mL of B16-F10 cell suspension (4 3 10 5 cells /mouse) into the tail vein.
Note: B16-F10 injection should be done after DT treatment (twice with a 2-week interval).
29. DT treatment a. Freshly prepare the DT solutions (5 mg/mL in PBS) and keep them on ice until use. Note: When lung tissue is also required for other analysis (e.g., IHC), use the left lung lobe for metastasis site counting.

EXPECTED OUTCOMES
The below data ( Figure 14) represent expected results of lung phenotype by senescent cell elimination from ARF-DTR mice. Luciferase activity was detected in the lung area of the PBS-treated ARF-DTR mouse (left), which was hardly detectable in the DT-treated animal (right), suggesting that lung senescent cells expressing the transgene (luciferase and DT) were eliminated by DT.

LIMITATIONS
While the toxin receptor-mediated cell knockout system is a powerful tool to specifically eliminate senescent cells from tissues, there are some points that should be noted for the use of DT. First, continuous DT administration might produce anti-DT antibodies in the mouse body, which might neutralize the administered DT and prevent the elimination of senescent cells from tissues (Kimura et al., 2007). Thus, prolonged DT administration should be avoided. Second, in principle, DT should not work in mice without DTR, but frequent administration of DT shows toxicity probably due to the off-target effects. We thus recommend administrating DT with at least a 2-week interval. Elimination of senescent cells can be confirmed by in vivo imaging two days after DT injection. However, at least 2 weeks will be needed to assess the biological effect of senescent cell depletion in lung tissue.
In ARF-DTR mice, bioluminescence images can be mainly obtained in the lung and adipose tissue with aging. However, the expression level of ARF is increased in many other tissues during aging, suggesting that transgene expression in ARF-DTR mice does not fully mimic the expression pattern of the endogenous Arf. In male ARF-DTR mice, bioluminescence can be also obtained from testis regardless of aging and is decreased by DT treatment, indicating elimination of p19 Arf -expressing cells from testis. While no difference between male and female has been observed in the above lung experiments, results should be carefully interpreted when ARF-DTR mice are utilized for other types of analysis.
While the presence of senescent cells in lung tissue can be confirmed as early as 6 months old by in vivo imaging, respiratory function has not yet declined at that time. Therefore, to evaluate agedependent changes in lung function, it is recommended to use ARF-DTR mice older than 12 months Figure 14. Elimination of p19 Arf -expressing cells from lung tissue Twelve-month-old ARF-DTR mice were intraperitoneally injected with PBS (left) or DT (right). Luciferase activity was monitored by in vivo imaging 2 days after the injection. Scale bar; 10 mm. of age. Disease models (e.g., emphysema model) will be needed to see the effects of senescent cell elimination in younger animals.

TROUBLESHOOTING Problem 1
No or very weak bioluminescence detected during in vivo imaging (step 3).

Potential solution
This could be mostly caused by incomplete substrate injection. Repeat the luciferin injection to solve the problem.
Additionally, dark colored hair highly affects optical imaging by blocking and absorbing the photon. It is essential to shave the hair thoroughly around the area to observe when using the black mice. Alternatively, the use of white hair background is recommended.

Problem 2
Failure in senescent cells elimination (steps 5 and 6)

Potential solution
In most cases the possible reason is losing bioactivity of DT. The freshly prepared DT solution should be used at all times. Do not use the refrozen DT solution, because DT is very sensitive to freezethawing.

Problem 3
Blood contamination in the BALF (step 18)

Potential solution
Carefully cut and open the skin to expose the trachea (step 8). If blood vessels are damaged and bleeding occurs, flush with PBS before tracheostomy (step 9).

Problem 4
Low RNA yield and quality (step 20)

Potential solution
Low yield and quality may be caused by excess amounts of tissue. To improve the yield and quality, reduce the amount of starting material. Homogenize tissue samples in 1 mL of TRI reagent per 50-100 mg of tissue. More than 1 mg per mg of lung tissue with OD260/OD280 >1.9 is expected.

Problem 5
Unclear lung structure from the tissue section (step 22).

Potential solution
In most cases the possible reason is insufficient lung inflation. This could be caused by a leakage of fixative solution from the clamped site of the right bronchus. To avoid the failure, we recommend tying off the clamped site of the right bronchus with string.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Masataka Sugimoto (msugimot@ncgg.go.jp).

Materials availability
This study did not generate new unique reagents. ARF-DTR mouse is available upon request to read contact.

Data and code availability
This study did not generate/analyze [datasets/code].