Chapter 16

Chapter 16 Experimental Model of HindLimb Suspension-Induced Skeletal Muscle Atrophy in Rodents Gabriel Nasri Marzuca-Nassr, Kaio Fernando Vitzel, Gilson Masahiro Murata, Jose´ Luis Ma´rquez, and Rui Curi Abstract Due to the difficulty of performing research protocols that reproduce human skeletal muscle disuse conditions, an experimental animal model of “hindlimb suspension” (or hindlimb unloading) was developed. This method was created in the 1970s and utilizes rats and mice to mimic space flight and bed rest in humans. It provides an alternative to investigate mechanisms associated with skeletal muscle mass loss and interventions designed to attenuate atrophy induced by hindlimb unloading. The mentioned protocol also allows investigating quality of bones and changes in several physiological parameters such as blood pressure, heart rate, plasma or tissue lipid composition, and glycemia. Key words Skeletal muscle atrophy, Hindlimb unloading, Bed rest, Muscle mass loss, Muscle disuse

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Introduction Skeletal muscle atrophy is characterized in clinical practice by loss of muscle mass and decreased strength, leading to impairment in physical performance [1–3]. The atrophic process is sustained by a decrease in protein synthesis and/or an increase in protein degradation pathways, producing a protein turnover imbalance that favors muscle mass loss [4–6]. Several conditions can lead to skeletal muscle atrophy, including sepsis, cancer, disuse, immobilization, and aging. Skeletal muscle mass loss reduces physical capacity and impairs glycemic and energy metabolism control efficiency. These factors aggravate the aforementioned conditions and increase the occurrence of comorbidities and mortality [7, 8]. Mechanical unloading causes muscle atrophy in common situations (e.g., bed rest) and specific events (e.g., space flight). Due to the difficulty of mimicking such conditions in humans, for research purposes, animal experimental models were developed [9]. The one that mimics space flight and bed rest is “hindlimb suspension” (also

Paul C. Guest (ed.), Pre-Clinical Models: Techniques and Protocols, Methods in Molecular Biology, vol. 1916, https://doi.org/10.1007/978-1-4939-8994-2_16, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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referred to as hindlimb unloading). This model was first described in the 1970s and has been widely used in both female and male rodents of different ages [10]. Knockout and transgenic mice (such as MAFbx and MuRF1 knockout mice [11] and FAT-1 transgenic mice [12]) have also been submitted to hindlimb suspension. Several interventions (e.g., dietary supplementation, electrical stimulation, physical exercise, and reloading) were also tested in this experimental model as an attempt to prevent or attenuate disuseinduced skeletal muscle atrophy and to investigate the mechanisms associated with skeletal muscle mass loss [12–14]. Hindlimb suspension is also used to examine bone mass quality loss and changes in blood pressure, heart rate, plasma or tissue lipid composition, and glycemia under skeletal atrophy [15, 16]. Under disuse, skeletal muscles of the hind limbs composed mainly of oxidative/type I/slow muscle fibers suffer greater atrophy as compared with glycolytic/type II/fast muscle fibers. We [12, 14] and others [17, 18] have reported muscle mass loss in the following order: soleus > gastrocnemius > plantaris > tibialis anterior > extensor digitorum longus (EDL). We also found a decrease in water and food intake, fat depot weight, cross-sectional areas of fibers and muscles, force production and protein synthesis markers and an increase in protein degradation markers in skeletal muscles from rats submitted to hindlimb suspension [12–14]. There are other experimental models to study skeletal muscle atrophy in animals such as denervation, immobilization, obesity, and diabetes [9, 19, 20]. Immobilization and denervation lead to a greater percentage of muscle weight loss after 14 days as compared with hindlimb suspension [11]. However, to mimic prolonged hospitalization situations, hindlimb suspension is recognized as the most appropriate animal model [9]. Denervation deprives muscles of nerve signaling, which does not occur during unloading caused by bed rest. Immobilization can cause swelling or ulceration of the limb without it being readily observable by the experimenter [21]. In the diabetes model, there are systemic alterations that interfere with results and give rise to a different etiology of the muscle atrophic process [19]. Hindlimb suspension leads to a decrease in the muscle mass by mechanical unloading while still allowing hind limb movement in opposition to the other experimental models cited above. The aim of this chapter is to describe the animal model of hindlimb suspension-induced skeletal muscle atrophy.

Skeletal Muscle Atrophy Model

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Materials Animals

2.2 HindLimb Suspension (Fig. 1)

1. Rats or mice (see Note 1). 1. Plastic box (acrylic; approx. 30 cm 28 cm 38 cm). 2. Metal line. 3. Metal wire (diameter ~2.2 mm). 4. Screws. 5. Metal stops. 6. Pulley with hook. 7. Small metal chain. 8. Gravity feed water bottle. 9. Metal grid. 10. Strong adhesive tape (gray). 11. Metal binder clips/clamps. 12. Cutter knife. 13. Pliers. 14. Scissors. 15. Laboratory gas burner. 16. Protection gloves.

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Taping (Fig. 1)

1. Containment (e.g., animal restraining tube) or anaesthesia (e.g., ketamine and xylazine, isoflurane, sodium pentobarbital). 2. Metal wire (diameter ~2.2 mm) for rats or paperclip (diameter ~0.726 mm) for mice.

Fig. 1 Photos of materials used in the present experimental protocol, taken during development of the articles published by Marzuca-Nassr et al. [12–14]

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3. Waterproof tape (Leukoplast®, 1.25 cm 5 m or 2.5 cm 5 m). 4. Protractor ruler. 5. Permanent marker.

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Methods

3.1 Building HindLimb Suspension Cage (Figs. 2 and 3)

1. Design a cage of type and dimensions to comply with local ethics guidelines for care and use of laboratory animals (see Notes 1 and 2). 2. Heat one end of the metal line and traverse it through the upper sidewalls of the box, along the length of the cage (Fig. 3a). 3. Insert a metal stop, pulley with hook and another metal stop (in that order) in the metal line during the process (Figs. 3b–h) (see Note 3).

Fig. 2 Schematic drawing of the special cage (a ventilated lid or metal wire should be considered at the top of the cage)


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Fig. 3 Photos of special cage building taken during development of the articles by Marzuca-Nassr et al. [12–14]

4. Adjust the stops on both ends of the metal line at a distance of 5 cm from the side of the cage (Figs. 2 and 3e) (see Note 4). 5. With the cutter knife, make two rectangle-shaped holes (5 cm 2 cm) in a lower sidewall, the same size as the metal binder clips (Fig. 2). 6. Insert the clips with food pellets from the outside of the box (Fig. 3l) (see Note 4). 7. Using wire and pliers, keep the clip in a semi-open position so that the food pellets can be placed. 8. Between the two rectangles, make a hole in which the metal nozzle of the water bottle can be inserted (Figs. 2 and 3j–l). 9. Use a wire support to keep the water bottle in an upright inverted position (Figs. 3k and m). 10. If the rat is large (over 200 g), use two inverted boxes held together by large metal binder clips (Figs. 3n and o). 11. For smaller rats or mice, one box is suitable (Figs. 3m and p).

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Fig. 4 Photos of tape technique taken during development of the articles by Marzuca-Nassr et al. [12–14]

12. In both cases, small holes must be made on top of the cage to allow ventilation and/or to place the metal wire (Fig. 2). 13. In both cases, a metal grid will be placed next to the floor of the box (Fig. 2) (see Note 5). 3.2 Taping Technique for HindLimb Suspension (Fig. 4)

1. An assistant should contain the animal while the researcher executes the taping technique (see Note 6). 2. Cut three strips of waterproof tape (~1 cm wide and 3 cm long) and place these around the tail starting from the base, leaving a 0.5 cm gap between them (Fig. 4b) (see Note 7). 3. Pliers can be used to shape a wire piece (~2 mm diameter and ~5 cm long) according to Fig. 4c. 4. Place the wire over the three strips and fix it with three new strips (~1 cm wide and ~4 cm long) overlapping the previous ones. 5. The “loop” end of the wire should be directed towards the tip of the tail (Figs. 4d–f) (see Notes 8–10). 6. Use a small metal chain to connect the “loop” end of the wire to the pulley (Fig. 4c). 7. Use a protractor to measure 30 of suspension between the body of the animal and the floor of the cage (see Note 11). 8. Monitor the animals twice a day throughout the experiment (see Notes 12–14). 9. Water and food should be changed every 2 days or when necessary (see Note 15).


3.3 Animal Maintenance During HindLimb Suspension

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1. The animals should be kept under controlled environmental conditions (22 1 C, with light-dark periods of 12 h) with food (standard diet for rodents) and water available ad libitum. 2. The animals have to be kept in individual cages for at least 3 days before starting the hindlimb suspension protocol (see Note 15). 3. The animals have to be randomly distributed into two groups: (1) control and (2) hindlimb suspension. 4. The animals of the control group are kept in an individual cage, similar to the ones used by the hindlimb suspension group (see Note 16). 5. The food pellets must be provided the same way as for the hindlimb suspension group (attached to binder clips in the floor of the cage) (Figs. 3l or p) (see Notes 16 and 17).

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Notes 1. Ensure that all approvals are in place with the appropriate institutional authorities prior to beginning experiments. In this study, animal handling was conducted according to the Guiding Principles for the Care and Use of Laboratory Animals and approved by the ethics committee of the Universidad Cato´lica del Maule, Chile and Universidade de Saõ Paulo, Brazil. 2. The animal has to be able to move around the box, so the pulley needs to allow a 360 rotation. The height of the cage depends on the weight and size of the animals providing enough room for an appropriate set up of the experimental model. 3. This will prevent the pulley from reaching the sidewalls. 4. If using an acrylic cage that cannot be cut, attach the metal binder clips with food on the cage floor, using strong adhesive tape. Place the clip in the corner of the cage but be sure that the stops will not prevent the animal from reaching the food with its front legs and face. It does not allow the animals to climb over the food and support their hind limbs. It also prevents them from urinating or defecating over the pellets. 5. A metal wire ~1.5 cm high has to be added to the floor to allow the animal to grip with its forelimbs to move and for urine and feces to pass to the sector under the metal wire. 6. An animal restraining tube or anesthesia may be used if there is no assistance or to reduce animal stress. The same procedure has to be used in control animals (sham).

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7. In mice, the strips are 0.5 cm wide and 1.5 cm long leaving a 0.25 cm gap between them. 8. For security, two strips can be placed over the one described in line with the tail. 9. In mice, the wire can be replaced by a paperclip. 10. A permanent marker to write on the tape and identify the animal can be used. In addition, the edges of the taping in the tail to monitor if the taping moves throughout the days of experimentation can be marked (Fig. 4f). 11. Forelimb muscle is used as control [10]. 12. If any sign of pain or stress occurs, the animal should be removed from the experiment. 13. To weigh the animals during the hindlimb suspension period, remove the metal chain gently from the pulley for the animal not applying any load on the hind limbs. Then, in an adapted container (with a horizontal metal line and a pulley on the upper part, similar to the cage) on the scale, place the animal in the hindlimb suspension position. At the end of the experiment, remove all the taping and weigh the animal. This value needs to be reduced from the body weight acquired during the hindlimb suspension period. 14. Animals can receive different treatments during the experiment. After the hindlimb suspension period, the taping can be removed to continue with a reloading phase. 15. Food and water consumption has to be monitored after isolating animals in individual cages. Withdrawal from a social environment may incur in stress [15], affecting food and water intake [13], with direct influence on body composition and muscle mass. The first 24 h of hindlimb suspension are critical for animal adaptation to cage. After the first 3 days, the body weight has to be evaluated and compared with the initial values. Animals that do not manage to adapt themselves to the new conditions (e.g., body weight lower than the baseline; rat-tail clinical feature; hair, eyes, and facial appearances of the animals with any indication of pain or discomfort) should be removed from the study. 16. Control animals should not be housed in groups (4–5 animals) and in regular commercial cages. This condition is not comparable to hindlimb suspension cage environment. 17. This affects physical activity levels and feeding of the animal, having effects on body composition that are not associated with hindlimb suspension.


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Acknowledgments The authors are grateful to Jose´ Roberto Mendonça for the excellent technical assistance. This research was supported by Becas Chile (CONICYT), Universidad de La Frontera, FAPESP, CNPq, CAPES, and Dean’s Office for Post-graduate Studies and Research of the Cruzeiro do Sul University. References 1. Rudrappa SS, Wilkinson DJ, Greenhaff PL, Smith K, Idris I, Atherton PJ (2016) Human skeletal muscle disuse atrophy: effects on muscle protein synthesis, breakdown, and insulin resistance-A qualitative review. Front Physiol 7:361. https://doi.org/10.3389/fphys.2016. 00361 2. Dirks ML, Backx EM, Wall BT, Verdijk LB, van Loon LJ (2016) May bed rest cause greater muscle loss than limb immobilization? Acta Physiol (Oxf) 218:10–12 3. Backx EMP, Horstman AMH, Marzuca-Nassr GN, van Kranenburg J, Smeets JS, Fuchs CJ et al (2018) Leucine supplementation does not attenuate skeletal muscle loss during leg immobilization in healthy, Young Men Nutrients 10 (5). pii: E635. doi: https://doi.org/10.3390/ nu10050635 4. Jackman RW, Kandarian SC (2004) The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 287(4):C834–C843 5. Sandri M (2008) Signaling in muscle atrophy and hypertrophy. Physiology (Bethesda) 23:160–170 6. Bonaldo P, Sandri M (2013) Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech 6(1):25–39 7. Atherton PJ, Greenhaff PL, Phillips SM, Bodine SC, Adams CM, Lang CH (2016) Control of skeletal muscle atrophy in response to disuse: clinical/pre-clinical contentions and fallacies of evidence. Am J Physiol Endocrinol Metab 311(3):E594–E604 8. Dirks ML, Wall BT, van de Valk B, Holloway TM, Holloway GP, Chabowski A et al (2016) One week of bed rest leads to substantial muscle atrophy and induces whole-body insulin resistance in the absence of skeletal muscle lipid accumulation. Diabetes 65 (10):2862–2875 9. Powers SK, Kavazis AN, McClung JM (2007) Oxidative stress and disuse muscle atrophy. J Appl Physiol (1985) 102(6):2389–2397 10. Morey-Holton ER, Globus RK (2002) Hindlimb unloading rodent model: technical

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