A Preclinical Model of Sepsis-Induced Myopathy with Disuse in Mice

Summary

This murine model combines a septic insult with hindlimb muscle disuse to recapitulate the bedridden feature of the typical septic patient. The model represents a significant departure from previous models to study muscle dysfunction in sepsis and is a reproducible approach to addressing therapeutic strategies to treat this condition.

Abstract

Sepsis is a major cause of in-hospital deaths. Improvements in treatment result in a greater number of sepsis survivors. Approximately 75% of the survivors develop muscle weakness and atrophy, increasing the incidence of hospital readmissions and mortality. However, the available preclinical models of sepsis do not address skeletal muscle disuse, a key component for the development of sepsis-induced myopathy. Our objective in this protocol is to provide a step-by-step guideline for a mouse model that reproduces the clinical setting experienced by a bedridden septic patient. Male C57Bl/6 mice were used to develop this model. Mice underwent cecal ligation and puncture (CLP) to induce sepsis. Four days post-CLP, mice were subjected to hindlimb suspension (HLS) for seven days. Results were compared with sham-matched surgeries and/or animals with normal ambulation (NA). Muscles were dissected for in vitro muscle mechanics and morphological assessments. The model results in marked muscle atrophy and weakness, a similar phenotype observed in septic patients. The model represents a platform for testing potential therapeutic strategies for the mitigation of sepsis-induced myopathy.

Introduction

Sepsis is a life-threatening condition due to an overactive immune response that adversely affects multiple organ systems, resulting in a major burden to health systems worldwide¹. More recently, the in-hospital mortality linked to sepsis has decreased due to improved intensity care unit (ICU) management^1,2. However, approximately 75% of the patients surviving the initial septic insult develop skeletal muscle atrophy (e.g., reductions in cross-sectional area) and weakness (e.g., reductions in force production capacity)^3,4. This phenomenon has been characterized as sepsis-induced myopathy, highly linked to impaired physical activity and lack of independence to perform daily living tasks, leading to re-hospitalization and mortality within five years of the initial episode⁵.

Due to an aggressive and generalized infection, septic patients are exposed to prolonged periods of bed rest while recovering in the ICU. In this context, the skeletal muscle undergoes severe disuse, which likely exacerbates muscle atrophy and weakness^3,4. Currently, no treatment has effectively addressed sepsis-induced myopathy. The available preclinical models designed to address the myopathy have used cecal ligation and puncture (CLP)⁶, cecal slurry⁷, or injection of purified lipopolysaccharide (LPS), which is a component of the cellular wall in gram-negative bacteria⁸. Although these models succeed in delivering infection, they do not properly reproduce the muscle disuse observed in septic hosts beyond a natural reduction in physical activity observed in septic animals⁹.

The main objective of this study is to provide a detailed description of how to properly execute the model of sepsis-induced myopathy with disuse in mice. We demonstrate the feasibility of combining CLP as a model of sepsis with hindlimb suspension (HLS) as a model of disuse to study sepsis-induced myopathy in mice³. Furthermore, representative results of muscle mechanics and typical morphological changes in response to the model are also provided.

Protocol

The procedures have been reviewed and approved by the University of Florida IACUC (#202200000227). Male C57BL/6J mice, 17 weeks old, with a body mass ranging from 27 g to 34 g, were used for the present study. The experimental procedures and timeline outlined in this protocol are depicted in Figure 1. As indicated, the protocol spans a total of 11 days. Animals undergo survival surgery (CLP/Sham) on Day 0, followed by four days of fluid and analgesic support. On Day 4, animals begin HLS for a duration of 7 days. Terminal experiments are conducted on Day 11. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Cecal ligation and puncture (CLP)

1. After obtaining the animals from the commercial source, let them acclimate in the animal facility for at least 1 week before conducting CLP (or sham) surgeries. This will help minimize the stress associated with transportation.
2. Group-house the mice, adhering to local IACUC guidelines.
   NOTE: As a general direction, the animals are housed in a maximum of 5 mice per cage until the day of surgery. Standard cages, measuring 7.25 inches in width, 11.75 inches in length, and 5 inches in height, are utilized and furnished with corncob bedding. A 12h : 12h light-dark cycle is maintained, with lights on at 7 AM and off at 7 PM. The housing temperature is kept at 20-22 °C, and the relative humidity (RH) is maintained between 30% and 60%. Ad libitum access to standard chow diet and water is ensured.
3. To perform CLP, anesthetize the animal with isoflurane (2.5%, 500 mL/min) in an induction chamber. Confirm anesthesia by pinching the paw with tweezers. Once in deep anesthesia, as confirmed by the absence of reflex withdrawal from paw pinching, transfer the animal to continued anesthesia using a nose cone (2.5%, 100-125 mL/min).
   NOTE: Aseptic techniques should be employed throughout the whole procedure.
4. Apply veterinary eye lubricant ointment to safeguard the animal's eyes from potential nosecone-induced damage or injury during surgery.
5. To clean the surgical site, use commercially available hair remover. Remove the fur from the lower abdomen only, avoiding overexposure of the skin.
   NOTE: Alternatively, animal hair clippers can be used, but care must be taken to avoid skin damage.
6. Once the surgical site is exposed, cleanse the area with three applications of povidone-iodine (or an equivalent germicidal scrub), followed by a rinse with 70% alcohol in between each application.
7. Administer a single dose of 3.25 mg/kg of sustained-release buprenorphine or equivalent, according to the analgesic treatment approved by your local IACUC.
8. Transfer the mouse to the surgical area. Isolate the surgical site using an adhesive drape. Under deep anesthesia, make a ventral midline incision (~2 cm) in the skin using a scalpel blade.
   1. Use scissors to separate the skin from the muscle layer. Using the scalpel blade, make a smaller incision (~1 cm) in the muscle layer. Once the bowels are visualized, using blunt forceps, locate the cecum and exteriorize it.
9. Once exteriorized, ligate the cecum using a sterile 5-0 polyglactin absorbable suture. Consider the area of the ligated cecum, defined as the distance from the distal end of the cecum to the ligation point, as it will contribute to the severity of the infection. To reproduce the results presented here, tie the cecum 1 cm from its distal point.
   NOTE: Ligating a larger cecum area will result in increased severity¹⁰.
10. Using a 27 G needle, perforate the cecum through and through, allowing the fecal content to leak. With caution, gently squeeze the cecum to externalize the fecal content. To perform sham surgery, follow the same steps exposing the animal cecum. However, do not ligate nor puncture the cecum.
    NOTE: The needle gauge directly affects the severity of the infection. To produce a low-grade infection, 26 G to 28 G needles are recommended. Please note that using thicker needle gauges will result in an increased mortality rate, and animals may not tolerate the subsequent hindlimb suspension phase of the protocol.
11. Relocate the cecum into the abdominal cavity. Close the muscle layer with a sterile 5-0 absorbable suture. Close the skin with a 5-0 nylon, nonabsorbable suture. After the skin suture is complete, provide sterile saline (1 mL for males and 0.5 mL for females) via subcutaneous injection in the back of the animal.
    NOTE: For closing the muscle layer, a continuous suture technique is recommended, whereas for the skin layer, an interrupted suture technique is recommended. Consult and conform with your local IACUC guidelines for suturing in survival surgery.
12. After the surgery, single house the animals in a clean cage on top of a heating mattress or a heating pad set at 35 °C. Observe the mouse every 15 min throughout the initial hour following anesthesia recovery, after which it can be returned to the housing facility.
    NOTE: Provide a minimum amount of chow on the cage floor to allow animals ad libitum food without affecting the surgical site. After returning to the facility, animals are checked twice a day following the septic animal assessment (step 3).
13. Provide sterile saline and analgesic support over the following four days to allow the surgical incision to heal.
    NOTE: Monitoring xiphoid surface temperature and body weight daily helps to keep accurate records of the sepsis severity¹¹.

2. Hindlimb suspension (HLS)

1. To perform the HLS, investigators must follow the local IACUC ethics guidelines. This includes ensuring the usage of appropriate cage dimensions and flooring, which are crucial aspects for accommodating animal locomotion, eating, and drinking habits in HLS conditions.
   NOTE: 4 days of recovery after CLP is recommended for wound healing.
2. Following 4 days of recovery from CLP or sham surgery, anesthetize the animal under light isoflurane flow (2.5%, 100-125 mL/min). Attach the mouse tail to a short metal chain using foam tape. Place the metal chain parallel to the tail while the foam tape firmly embraces the tail and chain together.
3. To ensure the suspension of the hindlimbs, attach the metal chain to a hook connected to a crossbar along the center of the cage. Additionally, affix a second small bar that can move along the crossbar to allow greater movement ability for the animal.
   NOTE: Animals must be able to move via their forelimbs by utilizing the metal grid on the cage floor.
4. Adjust the height of the suspended limbs to prevent the contact of the paws with the chow pellets. Monitor the animals and clean the shaved area around the sutured skin by hand with a water-soaked cotton swab at least twice daily during the suspension period.
   NOTE: Cleaning is crucial to avoid infection at the surgery site, especially from urine scalding due to the raised body position.
5. To reproduce the results, ensure that the animals undergo 7 days of hindlimb suspension. The duration was determined based on previous time course studies showing the minimum time required for hindlimb suspension to elicit meaningful effects on skeletal muscles in non-septic conditions^12,13.
   NOTE: Animal survival, discomfort, or distress will increase according to the severity of the infection.

3. Septic animal assessment

NOTE: Assessing the clinical condition of the animal is a key aspect of keeping track of severity post CLP/sham surgeries. Also, as required by IACUC, humane endpoints must be established for animal welfare. To address these concerns and provide standards for daily animal care, directions for performing animal assessment using the Modified Murine Sepsis Score (MMSS) were used¹⁴.

1. Use the MMSS (Supplementary File 1) to assess the animal. Note that for each category, a score of 0 represents a healthy animal. Score the animal twice a day from 0 to 3 according to the severity of the infection.
2. To enhance accuracy, measure xiphoid surface temperature and body weight twice per day^11,15 and record along with the MMSS score sheet.
   NOTE: Typical xiphoid surface temperature and body weight fluctuations are provided in Supplementary Figure 1.
3. Consult the local IACUC for humane endpoints.
   NOTE: To reproduce the results, the following criteria were used as endpoints: (1) Body weight loss >40% from baseline. (2) Temperature <30 °C or reduction of >5 °C from previous value. (3) A score of 3 in the following: Response to stimulus, level of consciousness, or respiration quality. (4) Total daily MSSS ≥17. The assessment described here is designed to be performed post-surgery and in animals undergoing normal ambulation. It is recommended to refrain from handling animals undergoing HLS to prevent contact between their hindlimbs and surfaces. After the final assessment, euthanize the animal as per local animal ethics committee recommendations.

Results

For the representative data shown in the results, male C57BL/6J mice, 17 weeks old, with a body mass ranging from 27 to 34 g, were used. The entire protocol takes eleven days to complete and consists of the surgery intervention (CLP or sham), saline and analgesic support (days 0 to 4), and the HLS disuse (days 4 to 11). Terminal experiments can be performed at any point over the suspension phase. To better understand the impact of the model on skeletal muscle function, the results are compiled from several experiments ac...

Discussion

The current protocol provides technical guidelines for the implementation of a new preclinical model of sepsis-induced myopathy. All materials and important steps are described in detail for the reproduction of the model. This approach can reproduce the skeletal muscle dysfunction observed in septic patients, highlighting the role of disuse as a crucial component in worsening myopathy. Thus far, the majority of the preclinical studies addressing sepsis-induced myopathy did not include the disuse as an active component of...

Materials

Name	Company	Catalog Number	Comments
4-0 Ethicon Coated Vicryl	Ethicon	D5792	Absorbable suture used for closure of muscle layer and for ligation of the cecum.
4-0 Ethilon Black 18"	Ethicon	662G	Non absorbable suture for closure of the skin layer.
BD  PrecisionGlide Needle 26-28 G	BD	305136 for 27g needle	Needle for puncturing the cecum.
C57BL/6J mice	Jackson Laboratory	strain #000664
Cotton Tipped Applicators	Puritan	S-18991	Swabs for topical application of iodine.
Cryostat	(Leica CM1950)
Dynarex Povidone Iodine Prep Solution	Dynarex	1415	Topical Antiseptic Liquid for Skin and Mucosa
Ethanol 200 Proof (100%)	Fisher Scientific		To make 70% ethanol for cleaning skin.
Hindlimb Suspension Cages	Custom Made	N/A	These custom made cages will be highlighted in the video recordings of the MS.
Optixcare Eye Lube	Optixcare		Eye lube for protection during survival surgery.
Scalpel blades #11	Fine Science		Blade used to make incisions on skin and muscle.
Skin-Trac	Zimmer	736579	Foam tape for fixing the tail to the suspension apparatus.
SomnoSuite Low-Flow Digital Vaporizer	Kent Scientific Corporation	SS-01	Vaporizer for Isoflurane Anesthesia
Tissue bath apparatus	Aurora Scientific	Model 800A, Dual Mode Muscle Lever 300C