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

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.

Representative 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 accomplished in our laboratory. The outcomes from the protocol are compared to matched animals undergoing CLP and/or sham surgeries, HLS and/or NA at 11 days after the initial surgery, except for the immune cell population, where experiments were performed at day 7 post-CLP. Data presented are mean± standard error.

A typical survival curve is presented in Figure 2. Young animals tolerate the protocol relatively well. The survival rate for a low-grade infection is around 80% to 85%. It is important to highlight that a more severe insult will result in higher mortality levels. To demonstrate the effectiveness of our CLP surgery in promoting infection, Figure 3 shows that CLP leads to an increase in the immune cell populations in animals' peritoneal lavage when compared to sham animals. Samples from peritoneal lavage were collected using sterile PBS at day 7 post-CLP and immediately analyzed using a Heska Element HT5 analyzer, which employs laser impedance and colorimetric methods for differential cell counts.

Body mass serves as an indicator of the severity of the model, and the combination of CLP and HLS typically leads to 11.8% ± 1.4 % of body mass loss³. Importantly, the model leads to a marked decrease in soleus mass when HLS is superimposed with CLP (Figure 4).

The model promotes a decrease in soleus cross-sectional area³ evidenced by a shift to the left in the cumulative fiber area, indicating that smaller fibers comprise a major part of the muscle section. For instance, when CLP and HLS are combined, 62% of the total fibers fall within the range of 600 µm² to 1600 µm², while sham animals undergoing HLS present 34% of the fibers in the same range. The average CSA in the Sham HLS group was 1359 µm² ± 41 µm², while CLP HLS presented an average CSA of 1233 µm² ± 67 µm². These findings are consistent with muscle atrophy. Representative images from soleus muscle morphology in response to the model are presented in Figure 5. To assess cross-sectional area (CSA), soleus muscles were frozen in the commercially available embedding medium (OCT), submerged in liquid nitrogen-cooled isopentane, and stored at -80 °C. Muscles mounted in OCT were sliced into 10 µm sections using a cryostat and stained with Hematoxylin and Eosin. Fiber CSA was measured by analyzing approximately 1000 fibers per animal and calculating the median area. Membrane boundaries of all visible fibers in each muscle section were traced using National Institutes of Health ImageJ software.

As a major hallmark of sepsis-induced myopathy, muscle weakness plays a critical role in the re-hospitalization of septic patients. The model demonstrates that disuse contributes to the development of myopathy, as seen in Figure 6. Muscle weakness in septic animals undergoing HLS can be observed in soleus force production ex vivo. While control animals (Sham NA) demonstrate a peak absolute force of 310 ± 14, mice subjected to CLP + NA exhibited a peak absolute force of 275.3 mN ± 41.1 mN. However, when HLS is added, the force decreases to 172.2 mN ± 19.11 mN, marking a notable ~40% decline in soleus contractile function. To access ex vivo forces, the soleus muscle was dissected. The muscle was placed in a hydrophobic Petri dish, bathed in Ringer's solution, and tied with nonabsorbable silk sutures. They were transferred to a tissue bath apparatus and stimulated at various frequencies (5 Hz, 15 Hz, 30 Hz, 50 Hz, 80 Hz, 120 Hz, 150 Hz, and 200 Hz).

Figure 1: Experimental design. Timeline representing the steps described in the protocol. Day 0: animal undergoes survival surgery (CLP or Sham); Day 4: animal undergoes HLS or remains in NA; Day 11: terminal experiments take place. CLP: cecal ligation and puncture. HLS: hindlimb suspension. NA: normal ambulation. Please click here to view a larger version of this figure.

Figure 2: Representative results of a typical survival curve of male C57Bl6, 17 weeks old, in response to the model. HLS does not increase mortality in this mild level of infection. CLP NA (n=5), CLP HLS (n=5) CLP: cecal ligation and puncture. HLS: hindlimb suspension. NA: normal ambulation. Please click here to view a larger version of this figure.

Figure 3: Representative results of immune cell population in peritoneal lavage of septic and sham animals performed 7 days after CLP. The key immune cell population is high in CLP animals, demonstrating that the mild to moderate CLP approach is effective in inducing sepsis. Data is presented as mean ± standard error. WBC: white blood cells. NEU: neutrophils. LYM: lymphocytes. MONO: monocytes. EOS: eosinophils. BAS: basophils. CLP: cecal ligation and puncture (Unpaired t-test was used). Please click here to view a larger version of this figure.

Figure 4: Changes in soleus mass. CLP HLS induces higher waste in soleus muscles. CLP NA (n = 7), CLP HLS (n = 4). Data is presented as mean ± standard error. Representative results of Soleus mass in response to the model. CLP: cecal ligation and puncture. HLS: hindlimb suspension. An unpaired t-test was used. Please click here to view a larger version of this figure.

Figure 5: Representative results of muscle cross-sectional area in response to the model. (A) CLP HLS induces muscle atrophy, as evidenced by the left shift in the fiber area. Data is presented as mean ± standard error. Cumulative soleus fiber area of sham and septic animals in response to the model. Sham HLS (n = 3), CLP HLS (n = 4). (B) Hematoxylin and eosin staining of soleus muscle from sham and septic animals. CLP: cecal ligation and puncture. HLS: hindlimb suspension. Scale bars = 250 µm. Please click here to view a larger version of this figure.

Figure 6: Representative results of soleus muscle force-frequency curve in response to the model. (A) Absolute force-frequency curve. CLP HLS leads to major impairment in soleus absolute and peak forces. Sham NA n = 3, CLP NA n = 3, CLP HLS n = 3. (B) Peak absolute force in response to model. Sham NA (n = 3), CLP NA (n = 3), CLP HLS (n = 3). Data are presented as mean ± standard error. CLP: cecal ligation and puncture. HLS: hindlimb suspension. Please click here to view a larger version of this figure.

Supplementary Figure 1: Typical xiphoid surface temperature and body weight fluctuations. Please click here to download this File.

Supplementary File 1: Modified Murine Sepsis Score (MMSS). Please click here to download this File.

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 its pathophysiology^7,8,16,17. Although this protocol primarily details findings from the soleus muscle, it is entirely feasible to extend ex vivo assessments to other muscular types, for example, the extensor digitorum longus (EDL), and to conduct in situ evaluations across a diverse array of muscle groups.

Patients experiencing sepsis-induced myopathy remain bedridden for long periods of time, recovering from the septic insult¹. In general, 75% of the survivors develop myopathy and often are readmitted to the ICU because of falls or muscle-weakness related problems^1,3. Here, this significant gap is addressed by offering an alternative tool to study sepsis-induced myopathy.

This model can be used to study the mechanisms contributing to sepsis-induced myopathy, as well as new strategies to mitigate the symptoms. Potential mechanisms underlying sepsis-induced myopathy include myeloid cell infiltration³, dysfunctional mitochondria¹⁸, overproduction of reactive oxygen species¹⁹, and impaired muscle regenerative capacity⁶. Future investigations must address these pathways incorporating the disuse as an active component of myopathy, given that disuse exacerbates muscle atrophy and weakness in septic patients^3,20. Additionally, the model allows the researchers to investigate key aspects of myopathy further, such as when the onset of muscle dysfunction occurs. Finally, potential therapeutic targets can be assessed throughout the time course of the model to identify strategies that may prevent, attenuate, and/or fully reverse symptoms.

One of the difficulties in the management of sepsis-induced myopathy lies in the lack of effective skeletal muscle recovery strategies^1,20. A reloading phase, where the animal can be released from the HLS, would offer a valuable model of patients after ICU discharge that is yet to be explored. Although it was demonstrated that after HLS, the hindlimb muscles regain normal size and strength in healthy animals²¹, in the context of sepsis, skeletal muscle regenerative capacity is compromised^3,6, leading to ineffective regeneration in response to rehabilitation²⁰. For instance, sepsis is associated with low levels of insulin-like growth factor-1 (IGF-1)²², which is required for proper muscle regeneration after reloading from HLS¹³. The incorporation of a reloading phase is straightforward (i.e., it involves letting go of animals from the suspension apparatus and observing the recovery from injury) and will allow researchers to explore potential mechanisms of muscle regeneration following a septic insult. Furthermore, rehabilitation strategies can be added to the model, improving the potential for new treatment discoveries.

The severity of sepsis is a critical aspect of the protocol, as many researchers use thicker needle gauges or a wider ligation area to induce a more severe level of sepsis. In the approach herein described, we delivered a lower-grade infection mimicking a mild to moderate level of sepsis. While a more severe infection may lead to greater atrophy and weakness, the mortality rate also increases exponentially. Delivering a near-lethal level of infection to the host does not translate to the patient who survives the infection and is affected by the myopathy. Finally, antibiotic support can be used to improve the mortality in severe levels of sepsis. However, it may represent a mechanistic interference with basic science approaches and require the addition of further control groups.

Despite being the first protocol to combine septic and disuse stressors, the model is not without limitations. Even though the model is designed to study hindlimbs, the forelimbs are somewhat overloaded as this represents the only means of locomotion in the cage. Furthermore, the tilted body position modifies the hemodynamic responses, which can limit research examining the systemic effects of the model. Additionally, monitoring food and water intake over HLS and/or CLP phases may add valuable information about the course of the muscle dysfunction seen in this model. However, measuring food intake in mice is challenging, mainly when pelleted or ground chow is used. Normally, changes in chow weight are reported to quantify food intake. However, there are several issues to be accounted for regarding food intake²³. If pelleted or ground chow is used, it's crucial to consider the crumbs, which can make up a substantial part of the weight difference before and after feeding. Mice often pick up smaller pellets or fragments and transport them to their nests within the cage. These tiny crumbs can blend with bedding, making them challenging to notice. Using a cage with a wire mesh bottom allows for the collection of crumbs underneath. While ground chow can deter food hoarding in nests, it might lead to more spillage. Wet mash, created by mixing water with ground chow, reduces spillage but alters the diet's taste, potentially affecting intake due to changes in palatability. A previous study in mice demonstrated that food intake did not differ between CLP (with 25 G needle) and Sham on day 5 after surgery²⁴. Nevertheless, if this pattern persists with the introduction of hindlimb suspension, it remains to be determined. Another limitation of this model is the absence of antibiotics and intravenous fluid resuscitation treatment to emulate the treatment received by the patient in the ICU. While adding these treatments could indeed make the model more reflective of clinical scenarios, they also introduce additional variables that need to be controlled. The current model already encompasses a significant number of variables, and each new element added increases the complexity of the experiment, the number of mice required, and the complexity of statistical treatment. Introducing antibiotics, for example, while clinically relevant, could affect various aspects of the sepsis response and recovery, complicating the interpretation of the specific effects of skeletal muscle recovery post-sepsis.

Despite these limitations, the model offers a significant departure from previous models used to address sepsis-induced myopathy. For instance, casting, denervation, and pharmacological-induced limb paralysis can deliver disuse to the hindlimbs. However, they do not mimic the patient setup and lack the ability to offer a post-treatment phase, such as a reloading phase.

In conclusion, the protocol presents a novel preclinical model for studying sepsis-induced myopathy, incorporating a crucial factor often overlooked in previous studies: the role of disuse in worsening myopathy. By replicating a patient's bedridden state by introducing unloading of the hindlimbs, this model mirrors key aspects of the condition inducing muscle atrophy and weakness. This innovation provides a platform to explore mechanisms underlying myopathy, incorporating disuse as an active element for studying muscle dysfunction and potential therapeutic targets in sepsis.

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