Bleomycin

Experimental Mouse Model of Bleomycin-Induced Skin Fibrosis

Przemysław Błyszczuk,1 Anastasiia Kozlova,1 Zhongning Guo,1 Gabriela Kania,1 and Oliver Distler1,2
1Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland
2Corresponding author: [email protected]

Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin-induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger pro- gressive skin thickening, associated with excessive accumulation of collagen,
infiltration of immune cells, and formation of α-smooth muscle actin (α-SMA)- positive myofibroblasts. In this article, we provide a detailed protocol for the induction of skin fibrosis in experimental mice by bleomycin. Moreover, we describe procedures for processing and analyzing affected skin tissue, provide troubleshooting, highlight advantages and limitations of the presented model, and critically discuss representative results. ⃝C 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Intradermal bleomycin injections to induce skin fibrosis in mice.

Support Protocol: Mouse tissue collection for fibrosis evaluation and for other molecular assays
Basic Protocol 2: Evaluation of mouse skin thickness using Masson’s trichrome staining
Basic Protocol 3: Measurement of hydroxyproline content in skin tissue using a colorimetric assay
Basic Protocol 4: Evaluation of myofibroblasts in mouse skin by immunohis- tochemistry

Keywords: bleomycin . collagen . mouse model . skin fibrosis . systemic sclerosis

INTRODUCTION

Systemic sclerosis (SSc) is characterized by chronic fibrosis of the skin. In humans, the disease often affects multiple organs and then is characterized by high mortality (Jordan, Chung, & Distler, 2013). SSc is an autoimmune-mediated disease in which immune cells activate fibroblasts and other stromal cells for cytokine and extracellular matrix overproduction leading to vasculopathies and tissue fibrosis in the skin and in internal organs (Allanore et al., 2015; Jordan et al., 2013). The fibrotic phenotype especially in lungs, gastrointestinal tract, and the heart can lead to fatal organ dysfunctions.

Animal models represent an important preclinical platform to test molecular mecha- nisms and novel treatment strategies. There is, however, no single in vivo model that recapitulates the complexity of SSc. Skin fibrosis represents a main manifestation of SSc. There are several animal models of skin fibrosis mirroring the phenotype and pathogenesis of the disease in humans. Repetitive application with the cytotoxic agent bleomycin represents a commonly used mouse model of scleroderma. In this model, subcutaneous or intradermal injections of bleomycin trigger progressive thickening of the skin. A typical histopathological picture shows significant thickening of the dermis associated with excessive accumulation of type I collagen, fibroblasts, α-smooth muscle actin (α-SMA)-positive myofibroblasts, and various immune cells (Akhmetshina et al., 2008)—a scenario that is also observed in the skin during early stage SSc in humans (Allanore et al., 2015).

Bleomycin refers to a mixture of glycopeptides (referred to as bleomycin A2 and B2) derived from the bacterium Streptomyces verticillus. Bleomycin in the presence of iron and oxygen generates reactive oxygen species (ROS) and can damage DNA by causing single- and double-strand breaks (Chen, Ghorai, Kenney, & Stubbe, 2008). Because of its anti-proliferative properties, bleomycin is clinically used as a chemotherapeutic drug (Anthoney et al., 2004). In the body, bleomycin is inactivated by the enzyme bleomycin hydrolase. Low activity of bleomycin hydrolase in the skin and lungs (Chen, Chen, & He, 2012) sets the molecular basis for experimental models of bleomycin-induced fibrosis in these organs.

In this article, we describe a detailed protocol for the intradermal delivery of bleomycin to mice to generate an in vivo model of scleroderma (Basic Protocol 1). In addition, we describe how to harvest affected skin tissue (Support Protocol) and analyze skin thickness (Basic Protocol 2) as a primary endpoint for this model. Furthermore, we describe measurement of hydroxyproline content, a biochemical surrogate marker of tissue fibrosis (Basic Protocol 3) and an immunohistochemical method to detect myofibroblast-specific α-SMA (Basic Protocol 4) in the skin, which can be used as secondary endpoints.

STRATEGIC PLANNING
Prior Planning Experiments

Animal experimentation is regulated by law. Experimental procedures involving living animals should follow guidelines from the Directive 2010/63/EU of the European Par- liament on the protection of animals used for scientific purposes or the current National Institutes of Health (NIH) guidelines and must be approved by local authorities.

Technical Aspects

The mouse model of bleomycin-induced skin fibrosis takes typically 3 to 6 weeks. Bleomycin injections are performed every other day and require two operators. Experi- ments should be performed by pre-trained operators. To reduce variation, use of all mice for all experimental groups in one experiment is recommended.

Experimental Mice

Bleomycin-induced skin fibrosis should be performed using ?6-week-old C57BL/6 male mice. In principle, other inbred and outbred mice of both genders can be used in this model. However, genetics, age, and gender diversity as well as stress influence disease development and progression (Ruzehaji et al., 2015; Wei, Bhattacharyya, Tourtellotte, & Varga, 2011). Use of healthy, inbred, young (6 to 8 weeks old) mice of the same gender kept under proper housing conditions is recommended to reduce variations of obtained data. To minimize stress: (1) place mice into experimental cage or room at least 1 week prior to the experiment, (2) avoid combining adult (?5 weeks) males with unfamiliar
males due to increased aggression, and (3) remove additional or socially incompatible mice from experimental cages.

Bleomycin Dose Selection

Use of the optimal dose of bleomycin is essential to obtain the desired severity of skin lesions. Commercially available bleomycin contains variable ratios of bleomycin A2 and bleomycin B2 resulting in variable activity between lot numbers. It is highly recommended to perform pilot studies using three to five mice per group to determine the optimal dose for each individual lot of bleomycin and use the same batch throughout the whole experiment. Because strain, age, gender, and housing conditions can strongly affect susceptibility and severity of skin fibrosis, the optimal dose of bleomycin should be newly established when one or more of these parameters change.

Group Sizes

Use of sufficient number of animals per experimental group is needed to achieve con- clusive data. To define group sizes, statistical analysis of minimal sample sizes should be performed for primary endpoints. Use of correct input parameters (mean, standard deviation, and treatment effect size) is critical. Input parameters should be preferentially used from your own previous experiments or pilot studies using the same procedures rather than from literature data. We suggest using at least six mice per group to obtain statistically significant results on dermal thickness. In generally, use of less than three mice per experimental group is not recommended.

Experimental Endpoints

Identification of primary and secondary endpoints is useful for the statistical analysis of minimal sample sizes. Measurements of skin thickness represent a typical primary endpoint, while others, for example the number of immunopositive cells/mm2, hydrox- yproline content, number of α-SMA positive cells/mm2, are often considered as secondary endpoints. Other primary and secondary endpoints, such as prevalence of specific cell population in a defined organ or levels of specific biomolecules in plasma or serum can be defined depending on the aim of the study.

Group Allocation

Animals should be randomly allocated to experimental groups and distribution of relevant parameters such as age, body weight, gender, and skin phenotype (if applicable) should be controlled prior to performing experiments. In case of statistically significant differences for these parameters, mice should be re-allocated.

NOTE: All protocols using live animals must first be reviewed and approved by an Insti- tutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

INTRADERMAL BLEOMYCIN INJECTIONS TO INDUCE SKIN FIBROSIS IN MICE

In the mouse model of bleomycin-induced skin fibrosis, bleomycin is delivered every 2 days to the upper back of a mouse by intradermal injections. Continued injections of bleomycin result in inflammation and progressive skin fibrosis, which develops typically ~3 to 4 weeks after the first injection. This mouse model can be therefore used to design both preventive and therapeutic treatment approaches (Fig. 1). In the preventive approach, treatment begins at the same time as the initial bleomycin injection and skin fibrosis can be evaluated after 4 weeks. In the therapeutic treatment approach, mice receive continuous intradermal injections with bleomycin for 6 weeks, but treatment regimens begin 3 weeks after the first injection of bleomycin. The latter approach is more relevant to test anti-fibrotic drugs because of the established dermal fibrosis prior to treatment start, which better reflects the clinical scenario.

Figure 1 Schematic presentation of experimental designs for the preventive treatment (A) and therapeutic treatment (B) models of bleomycin-induced skin fibrosis. i.d., intradermally.

Materials

Experimental mice: 6- to 8-week-old C57BL/6 females (The Jackson Laboratory) Isoflurane vaporizer (anesthesia system for rodents; Funnel-Fill, VetEquip) 1 unit/ml bleomycin solution (see recipe) Sterile 0.9% sodium chloride (NaCl) solution Electric hair clipper for small animals Non-toxic permanent marker pen, black Hypodermic 27-gauge needle 1-ml syringe NOTE: 1 unit = 1 unit USP = 1000 IU First bleomycin injection (one operator).
1. Anesthetize mouse.This procedure should be performed for one mouse at a time. (For general mouse anesthesia guidelines see Current Protocols article: Adams & Pacharinsak, 2015.)
2. Shave upper back of a mouse (~2 cm × 2 cm) using an electric hair clipper.Shaving has to be repeated regularly every 7 to 10 days or as needed.
3. Draw a 1-cm × 1-cm square on the shaved skin using permanent marker pen (see Fig. 2A).
Drawing should be repeated as soon as it fades.
4. Inject 100 µl bleomycin solution (1 unit/ml) intradermally at position 1 shown in Figure 2B. Inject control mice with 100 µl 0.9% NaCl.
For intradermal injection, invade the skin with a needle horizontally (10° to 15° angle) and press plunger of the syringe once the bevel has completely penetrated (but has not gone through) the skin. Injection should be directed to the center of the square.
5. Place mouse back in its respective cage and wait until it becomes conscious and fully active.
Do not keep unconscious mice with active ones in one cage.
Second bleomycin injections (two operators)
6. For Operator 1: Restrain mouse by holding the base of the tail and neck.
7. For Operator 2: Quickly inject 100 µl bleomycin solution (1 unit/ml) or 0.9% NaCl (for control mice) intradermally in the appropriate position; injections should be performed cyclically into positions 1 to 5 as shown in Figure 2B.
For positions 1 to 4, injections should be directed to the center of the square.
8. Repeat steps 6 to 7 every 2 days for 4 or 6 weeks.
Four-week treatment with bleomycin is used for a preventive approach and 6-weeks treat- ment for therapeutic approaches (see Fig. 1).

Figure 2 Injection and harvesting strategies in the mouse model of bleomycin-induced skin fibrosis. Panel (A) shows mice during treatment with saline or bleomycin. Panel (B) shows a scheme for bleomycin (or saline) injections. Injections are performed every other day starting from position 1 to position 5 and are further continued cyclically. Arrows indicate direction of injections. Panel (C) indicates the strategy for harvesting of skin tissue samples for histology (two samples) and skin biopsies (six samples) for other assays, such as hydroxyproline measurements and various molecular downstream applications.

MOUSE TISSUE COLLECTION FOR FIBROSIS EVALUATION AND OTHER MOLECULAR ASSAYS

To evaluate skin fibrosis, histological and immunohistochemical analyses, as well as measurements of hydroxyproline content, tissue collection is routinely performed. Skin tissue biopsies can be collected also for multiple molecular downstream applications including RNA and protein analyses. In general, mice should be sacrificed and tissue collected 24 hr after the last bleomycin injection.
Additional Materials (also see Basic Protocol 1) Euthanasia system for rodents (see Current Protocols article: Donovan & Brown, 2006).

1. Euthanize mouse.
Use one of the approved methods for mouse euthanasia, such as inhalation with carbon dioxide or overdose with anesthetics. Follow the most recent guidelines for the respective methods (see also Current Protocols article: Donovan & Brown, 2006).
2. Place mouse in a prone position. Stretch and fix all four limbs.
If necessary, shave the skin.
3. Using a scalpel, cut out the marked square (marked in Basic Protocol 1; 1 cm ×
1 cm) of the skin and place in a Petri dish.
Keep the epidermis to the top. Do not dissect dermis from the subcutaneous tissue.
4. Take up to six skin biopsies using a 3-mm disposable biopsy punch from positions S1 to S6 as shown in Figure 2C and transfer them into the collection tubes.
For the hydroxyproline assay, snap-freeze biopsies (in collection tubes) in liquid nitrogen and store at −80°C. For other applications follow the respective protocol.
5. Using a scalpel, cut the middle section of the harvested skin, divide into two equal pieces as shown in Figure 2C, and fix in 4% buffered formalin at room temperature. After overnight fixation, transfer tissue into 50% v/v ethanol and embed in paraffin.

EVALUATION OF MOUSE SKIN THICKNESS USING MASSON’S TRICHROME STAINING

Skin thickness often represents the primary endpoint in the mouse model of bleomycin- induced skin fibrosis. Increased thickness of the dermis can be visualized by a number of different histological staining methods. Masson’s trichrome staining specifically labels the major dermis component collagen (green or blue) and therefore it should be considered as a primary option.

Materials

Paraffin-embedded skin tissue (see Support Protocol) Xylene.Microscope (100× magnification) equipped with a camera Computer with software for picture analysis (e.g., AvioVision 4.8).
1. Perform Masson’s trichrome staining on a section of skin tissue; perform all steps at room temperature.
In the described method of Masson’s trichrome staining, nuclei are brown-black; cytoplasm and muscle are red; collagen is green; muscles are light red; and erythrocytes are bright red.
2. Using a microtome, prepare 4-µm thick sections of paraffin-embedded skin tissue on a glass microscope slide. Cut skin tissue with the whole dermis and place sections on the slide in a horizontal orientation.
3. Deparaffinize paraffin-embedded skin tissue on the slide:
a. Incubate slide in xylene 5 min. Repeat once using fresh xylene.
b. Incubate slide in 100% ethanol 3 min. Repeat twice using fresh 100% ethanol.
c. Incubate slide in 95% ethanol 3 min. Repeat once using fresh 95% ethanol.
d. Incubate slide in 80% ethanol 3 min.
e. Incubate slide 10 s with 1% acetic acid.
4. Stain skin tissue section on the slide:
a. Incubate slide in Weigert’s iron hematoxylin solution 3 min.
b. Rinse slide under running tap water 10 min.
c. Stain with Ponceau acid fuchsin solution with azofloxin 5 min.
d. Rinse slide twice with fresh solution of 1% acetic acid.
e. Incubate slide in 3% phosphomolybdic acid in 2% Orange G for 5 min.
f. Rinse slide twice with fresh solution of 1% acetic acid.
g. Incubate slide in 1% light green in 0.2% acetic acid 30 s.
h. Rinse slide twice with fresh solution of 1% acetic acid.
5. Dehydrate stained skin tissue section and mount slide:
a. Wash slide in 80% ethanol 10 s.
b. Wash slide in 95% ethanol 10 s.
c. Wash slide in 100% ethanol 3 min. Repeat twice using fresh 100% ethanol.
d. Incubate slide in xylene 5 min. Repeat once using fresh xylene.
e. Place one drop of Pertex mounting medium and place coverslip on the slide.
Take pictures and evaluate skin thickness
6. Take three random, non-overlapping pictures for each skin biopsy at 100× magni- fication; the skin tissue should cover >75% of picture area with epidermis oriented horizontally.
7. Using the appropriate software (for example AvioVision 4.8), measure thickness of the green labeled dermis thickness in three sections of each picture by measuring the distance from the epidermis to the subcutaneous tissue. Perform measurements perpendicularly to the epidermis as shown in the Figure 3A.
There are six pictures from each mouse (i.e., eighteen measurements/mouse in total).
8. Average all measurements of dermal thickness obtained from the skin of each mouse. Perform measurements by two independent analyzers and calculate the average.

Figure 3 Skin thickness in bleomycin-induced skin fibrosis. Eight-week-old male C57BL/6 mice were intradermally injected with bleomycin or saline every other day for 4 weeks. Murine skin paraffin sections were stained according to the Masson’s trichrome staining protocol. Green stain- ing indicates collagen. Panel (A) shows representative pictures of skin obtained from saline and bleomycin treated mice with indication of skin thickness by red bars. Magnification 100×. Panel (B) Quantification of skin thickness of saline and bleomycin treated mice (n = 6 for each group). Data are represented as means with standard deviation. p value was calculated with Student’s t test.
There are at least two skin biopsies from each mouse used for this analysis (i.e., six pictures/mouse in total). Pictures should be blinded to the examiner for measuring dermal thickness.

MEASUREMENT OF HYDROXYPROLINE CONTENT IN SKIN TISSUE USING A COLORIMETRIC ASSAY

Skin fibrosis is characterized by the excessive accumulation of collagen in the der- mis. Hydroxyproline is largely restricted to collagen; therefore collagen content can be indirectly assessed by measuring hydroxyproline content in the skin biopsy. Hydrox- yproline is formed by the post-translational hydroxylation of the amino acid proline and can be detected by biochemical reactions using a simple and sensitive colorimetric assay.

Materials

Prepare skin biopsy samples

1. Incubate 3-mm fresh frozen skin biopsy in 500 µl 6 M HCl at 120°C for 3 hr.
Tissue should dissolve completely.
2. Adjust pH to 5 to 7 using 6 M NaOH and 6 M HCl. Check pH with pH indicator strips.
3. Transfer 125 µl of this lysate into a new container and add 125 µl distilled water.
Samples should be run in duplicate.
The remaining lysate can be stored at −80°C.
Prepare hydroxyproline standards
4. Using 100 µg/ml hydroxyproline stock and distilled water, prepare 250 µl of the following hydroxyproline standards: 40, 30, 20, 15, 10, 5, and 1 µg/ml. Use distilled water as a blank.
Standards and blank should be run in duplicate or triplicate.
Measure hydroxyproline
5. Add 125 µl of 0.06 M chloramine T solution to 250 µl sample, standard, or blank prepared in the previous step. Vortex and incubate 20 min at room temperature.
Contains toxic methoxyethanol: Wear nitrile gloves. Prepare chloramine T solution fresh.
6. Add 125 µl of 3.15 M perchloric acid, vortex, and incubate 5 min at room temperature.
Perchloric acid is toxic: Wear nitrile gloves.
7. Add 125 µl of 0.2 g/ml p-dimethylaminobenzaldehyde, vortex, and incubate 20 min at 60°C. Cool to room temperature.
Contains toxic methoxyethanol: Wear nitrile gloves. Prepare p-dimethylaminobenzaldehyde solution fresh.
Samples should become red or orange for standards or skin biopsies, and yellow for blank.
8. Vortex samples and transfer 200 µl into flat bottom 96-well plates and measure absorbance at 557 nm using a standard microplate spectrophotometer.
9. Create a standard curve using values obtained from blank and hydroxyproline stan- dards and determine hydroxyproline concentration in biopsy samples.
Original skin biopsy lysates are diluted 1:1 (see step 3), therefore obtained results have to be multiplied by 2.

EVALUATION OF MYOFIBROBLASTS IN MOUSE SKIN BY IMMUNOHISTOCHEMISTRY

Fibroblast-to-myofibroblast transition is commonly observed in fibrotic processes. In- creased numbers of dermal myofibroblasts are a characteristic feature of fibrotic skin. Myofibroblasts are characterized by expression of α-SMA, which upregulates their con-
tractile activity and represents a reliable marker of these cells. α-SMA-positive myofibroblasts can be detected using immunohistochemical analysis. Quantification of α-SMA-positive myofibroblasts in the dermis can be therefore used to characterize active fibrosis in the skin.

Materials

Paraffin-embedded skin tissue (see Support Protocol) Xylene 100%, 95%, 80%, 70%, and 50% ethanol
Antibody diluent: Ready-to-use antibody diluent for immunohistochemistry with background-reducing components (Dako)
Blocking solution: 10% goat serum diluted in antibody diluent
Anti-α-SMA antibody solution: Monoclonal IgG2a mouse anti-α-SMA antibody, clone 1A4, diluted 1:750 in in antibody diluent
Secondary antibody solution: Alkaline phosphatase-conjugated polyclonal goat anti-mouse IgG diluted 1:50 in TBS
Alkaline phosphatase substrate: VECTOR Red Alkaline Phosphatase (Red AP) Substrate Kit (Vector Laboratories)
Meyer’s hematoxylin (Avantor) Pertex mounting medium (Histolab) Tris-buffered saline (TBS)
Tris-buffered saline with 0.05% Tween 20 (TBST)
Microtome
Glass microscope slides Coverslips
Hydrophobic barrier pen Humidity chamber
Microscope (400× magnification) equipped with a camera Computer with software for picture analysis
Perform α-SMA immunohistochemistry staining on a section of skin tissue
NOTE: In the described method, nuclei are blue and α-SMA-positive staining is red.
1. Using a microtome, prepare a 4-µm thick section of paraffin-embedding skin tissue on a glass microscope slide.
2. Deparaffinize and rehydrate paraffin-embedding skin tissue on the slide:
a. Incubate slide in xylene 10 min. Repeat twice using fresh xylene.
b. Incubate slide in 100% ethanol 6 min. Repeat using fresh 100% ethanol.
c. Incubate slide in 95% ethanol 3 min. Repeat using fresh 95% ethanol.
d. Incubate slide in 80% ethanol 3 min.
e. Incubate slide in 70% ethanol 3 min.
f. Incubate slide in 50% ethanol 3 min.
g. Incubate slide in TBS 5 min. Proceed to the next step immediately.
Stain skin tissue section on the slide
NOTE: Deparaffinized and rehydrated tissue is fragile. Handle with care and prevent tissue drying.
3. Using a hydrophobic barrier pen, draw a circle around skin tissue.
Adding staining solutions inside the circle minimizes the volume of antibodies used.
4. Incubate specimen in 100 µl blocking solution at room temperature, 1 hr.
During this step, keep slides in humidity chamber to prevent evaporation of blocking solution.
5. Remove blocking solution.
6. Incubate specimen in 100 µl of anti-α-SMA antibody solution at room temperature, 1 hr.
During this step, keep slides in humidity chamber to prevent evaporation of antibody solution.
7. Remove antibody solution.
8. Wash slide in TBST 5 min. Repeat using fresh TBST.
9. Incubate specimen in 100 µl secondary antibody solution at room temperature, 30 min.
During this step, keep slides in humidity chamber to prevent evaporation of antibody solution.
10. Wash slide in TBS 5 min. Repeat using fresh TBS.
11. Incubate specimen in 100 µl alkaline phosphatase substrate at room temperature for
~10 min.
Observe signal development in real time to determine incubation time for optimal signal- to-background ratio.
12. Wash slide in distilled water 5 min.
13. Incubate specimen in Meyer’s hematoxylin at room temperature for 45 s.
14. Rinse slide under running tap water 5 min. Dehydrate stained skin tissue section and mount the slide.
15. Wash slide in 80% ethanol 10 s.
16. Wash slide in 95% ethanol 10 s.
17. Incubate slide in 100% ethanol 3 min. Repeat twice using fresh 100% ethanol.
18. Incubate slide in xylene 5 min. Repeat using fresh xylene.
19. Place one drop of Pertex mounting medium and place coverslip on the slide.
Take pictures to quantify α-SMA-positive myofibroblasts in the dermis
20. Take six random, non-overlapping pictures for each skin piece at 400× magnification.
Take pictures of the dermis (above subcutaneous tissue) only.
21. Count manually the number of α-SMA-positive myofibroblasts as shown in Figure 4.
Vascular smooth muscle cells are also positive for α-SMA. In contrast to dermal myofi- broblasts, vascular smooth muscle cells form (small) outer walls of arteries. Therefore, α-SMA-positive smooth muscle cells must be excluded from the quantification.
22. Average the quantifications of α-SMA-positive myofibroblasts obtained from skin of one mouse (twelve pictures/mouse). Perform measurements by two independent analyzers and calculate the average.

Figure 4 Alpha smooth muscle actin (α-SMA)-positive myofibroblasts in the skin. Representative immunohistochemistry of α-SMA-positive cells in skin sections of 8-week-old male C57BL/6 mice intradermally injected with bleomycin or saline every other day for 4 weeks. α-SMA-positive cells are stained in red. Magnification 400×.There are two skin biopsies obtained from each mouse, i.e., twelve pictures/mouse in total). Sections should be analyzed in a blinded manner.Do not use standard deviations or standard errors for the averaged value obtained from one mouse.

Background Information

In addition to bleomycin-induced skin fi- brosis, there are a number of other available animal models of SSc. For example, trans- genic mice, such as TSK-1, TSK-2, Fra-2- tg, and many others spontaneously develop an SSc-like phenotype in the skin and in in- ternal organs (Beyer, Schett, Distler, & Dis- tler, 2010). Chronic graft-versus-host disease (GVHD) represents another important model of SSc. In the GVHD model, the SSc phe- notype is induced by bone marrow trans- plantation between specific mouse strains (Jaffee & Claman, 1983). In comparison to other SSc models, bleomycin-induced skin fi- brosis is mostly limited to a fibrotic pheno- type in the skin only but increased doses of bleomycin injected subcutaneously can trig- ger fibrosis also in other organs including lungs (Yoshizaki et al., 2008). Furthermore, a mouse model of bleomycin-induced skin fi- brosis is applicable in many mouse strains and is particularly useful to test novel anti- inflammatory and anti-fibrotic medications. A well-defined course of disease progression fol- lowing bleomycin treatment allows the testing of therapeutic drugs not only in the preventive but also in the therapeutic approach, i.e., once skin fibrosis is pre-established (Akhmetshina, Venalis, et al., 2009).

The first study describing bleomycin- induced skin thickening in mice was re- ported in 1999 (Yamamoto et al., 1999). In the original paper, the authors injected BALB/c mice subcutaneously with 100 µl of 1 mg/ml bleomycin dose every other day over a period of 4 weeks. Using this pro- tocol, skin fibrosis was successfully induced in several other inbred mouse strains in- cluding C57BL/6, C3H/He, A/J, and DBA/2 (Yamamoto, Kuroda, & Nishioka, 2000). The overall bleomycin treatment scheme with some modifications has been reproduced in many laboratories and is still widely used today. Commonly, C57BL/6, BALB/c, or C3H/He mice are treated with bleomycin ev- ery other day or 5 days a week for 3 to 4 weeks by subcutaneous or intradermal in- jections (Akhmetshina, Dees, et al., 2009; Avouac et al., 2011, 2013; Distler et al., 2007; Kitaba et al., 2012; Ohashi & Yamamoto, 2015; Singh, Kasam, Sontake, Wynn, & Madala, 2017). Importantly, different knock- out or transgenic mice can be also used for bleomycin-induced skin thickening.

Critical Parameters

Successful induction of the fibrotic phe- notype in the skin by repetitive bleomycin injection is dependent on two key factors:
(1) quality and dosage of bleomycin and (2) health status of experimental mice. A proper dosage of injected bleomycin plays a critical role in disease induction. Due to lot- to-lot variations in bleomycin activity, it is highly recommended to follow a unit/ml rather than a mg/ml regimen of injected bleomycin. However, despite the stated specific bleomycin activity in units (USP or IU), there are still lot- to-lot and manufacturer-to-manufacturer dif- ferences in disease induction efficacy. It is therefore recommended that different lots not be used in the same set of experiments. Fur- thermore, repeated freezing and thawing of bleomycin and/or inappropriate storage might result in its reduced activity.

Use of healthy, young (<8-week-old), age- and sex-matched mice of the same genetic background as well as proper housing and social conditions are decisive for obtaining reproducible results. It is recommended that recent guidelines on mouse housing be fol- lowed (Current Protocols article: Burkholder et al., 2012). Moreover, monitoring of exper- imental mice prior to and during experiments should not be neglected because aggressive in- dividuals not only cause stress but can also physically injure the skin of other animals. Both of these incidents can influence the qual- ity of the obtained data. Removing aggressive mice from the experimental cage is recom- mended. As mouse age, sex, genetics, hous- ing conditions, and the specific lot-dependent bleomycin activity represent variable param- eters, performing a pilot study in order to optimize the protocol for each laboratory is recommended. Troubleshooting Statistical analyses In general, mouse experiments are per- formed using relatively small group sizes and therefore are often underpowered and associ- ated with the risk of false-negative results (type II error). The overall effect in the mouse model of bleomycin-induced skin fibrosis should be analyzed using primarily skin thickness data or another pre-defined primary endpoint. Analy- ses of secondary endpoints are helpful to better characterize the observed skin phenotype. Use of proper statistical analysis is crucial for appropriate interpretation of the results. As a rule, normally distributed data should be analyzed using one-tailed Student’s t-test (for two groups) or one-way analysis of variance (ANOVA; for more than two groups), whereas non-normally distributed data should be ana- lyzed using the Mann-Whitney U test (for two groups) or the Kruskal-Wallis test by ranks (for more than two groups). Accordingly, the data should be analyzed first for normal dis- tribution. It is expected that primary and sec- ondary endpoint data for the reference group are normally distributed. Non-normal distri- bution of skin thickness in the mouse model of bleomycin-induced skin fibrosis is observed when disease is not induced in some animals or in the case when group sizes are too small. When analyzing more than two groups, post- hoc tests for multiple comparisons have to be performed. Following ANOVA, it is recom- mended that Fishers Least Significant Differ- ence test be used, due to the small group sizes. Figure 5 Hydroxyproline content in bleomycin-induced skin fibrosis. 8-week-old male C57BL/6 mice were intradermally in- jected with bleomycin or saline every other day for 4 weeks. Collagen content was assessed by hydroxyproline assay from one skin biopsy per mouse of saline and bleomycin treated mice (n = 6 for each group). Data are repre- sented as means with standard deviation. p value was calculated with Student’s t test. Understanding Results Intradermal injections of bleomycin into the upper back of a mouse induce skin thick- ening and fibrotic changes. Herein, we pro- vide experimental results obtained from 8- week-old male C57BL/6 mice intradermally injected with bleomycin or saline every other day for 4 weeks. All experimental procedures followed protocols described in this article. Presented data show typical results for ex- perimental setup with low group sizes (n = 6). Experimental mice were analyzed for skin thickening as a primary endpoint. Representa- tive Masson’s trichrome staining pictures are shown in Figure 3A. As shown in Figure 3B, the overall skin thickness of the bleomycin-treated group is significantly increased in comparison to the control group. A feature of note is some vari- ability in the skin thickness of mice treated with bleomycin. In fact, one mouse treated with bleomycin showed no increase in skin thickness. In addition to skin thickness data, measurements of hydroxyproline content from one skin biopsy per mouse reflecting a sec- ondary endpoint are presented in Figure 5. Data show significantly higher hydroxypro- line levels in skin biopsies obtained from bleomycin-treated mice. In this case, there was high variability observed in the control group. In order to reduce such variability, analysis of more skin biopsies from one mouse is rec- ommended. The fact that this model can be associated with certain variations should be considered when planning new experiments. Time Considerations As indicated above, mouse treatment varies between 3 and 6 weeks depending on the type of experiment and is defined during planning. Consider that analysis of primary and sec- ondary endpoints in the bleomycin-induced skin fibrosis model is also time consuming. Preparation of histological and immunohis- tochemical skin tissue sections requires 2 to 3 working days. In a routine manner, mea- surements of the skin thickness or counting of immunopositive cells are completed in 15 to 20 min per slide (six pictures per slide have to be analyzed). Hydroxyproline measurements can be assessed during 1 to 2 working days. Together, the analysis of typical primary and secondary endpoints for studies involving 20 to 30 mice can be completed within 8 to 10 working days, including statistical analysis. Acknowledgments O.D. received funds from the Swiss Na- tional Science Foundation, Skintegrity, Novar- tis, Herzog-Egli Foundation, Helmut Horten Foundation, and Forschungskredit UZH. Literature Cited Adams, S. & Pacharinsak, C. (2015). Mouse anesthesia and analgesia. Current Protocols in Mouse Biology, 5, 51-63. doi: 10.1002/ 9780470942390.mo140179. Akhmetshina, A., Dees, C., Busch, N., Beer, J., Sarter, K., Zwerina, J., . . . Distler, J. H. (2009). 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