combining cecal ligation and puncture (clp) with hindlimb suspension (hls) to study sepsis-induced myopathy in mice

Mouse Model for Studying Sepsis-Induced Myopathy with Hindlimb Suspension

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) management1,2. 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 weakness3,4. 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.

Author Spotlight: Advanced Integrated Model for Sepsis-Induced Myopathy and Single-Cell Metabolic Analysis

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

Our research focuses on understanding the mechanisms behind sepsis induced myopathy. Current models overlook the disuse component, which is crucial for bad reading septic patients. We aim to integrate this aspect for a more accurate disease model.We have significantly advanced the field by combining cecal ligation and puncture model with high limb suspension. This integrated approach allow us to study the systematic effect of sepsis and the impact of muscle disuse on sepsis induced myopathy, providing a more realistic model for understanding the disease progression and potential interventions. Our protocol combines cecal ligation and puncture with high limb suspension to replicate the clinical scenario of human sepsis more accurately in mice.By simulating muscle atrophy and weakness seen in septic patients, our approach provides a comprehensive model for studying the interplay between sepsis and muscle function, allowing for more targeted investigations into therapeutic interventions.

Combining Cecal Ligation and Puncture (CLP) with Hindlimb Suspension (HLS) to Study Sepsis-Induced Myopathy in Mice

To begin, transfer the prepared anesthetized mouse to the surgical area and isolate the surgical site with an adhesive drape. Under deep anesthesia, use a scalpel blade to make a ventral midline incision in the skin. Next, use scissors to separate the skin from the muscle layer.With the help of a scalpel blade, make a smaller incision in the muscle layer. Once the bowels are visualized, use blunt forceps to locate the cecum and exteriorize it. Then ligate the cecum with a sterile 5/0 polyglactin absorbable suture.Using a 27-gauge needle, perforate the secum through and through allowing the fecal content to leak. Gently squeeze the secum to externalize the fecal content with caution. Afterward, relocate the secum into the abdominal cavity and close the muscle layer with a sterile 5/0 absorbable suture.Then use a 5/0 nylon non-absorbable suture to close the skin. After that, provide sterile saline via subcutaneous injection in the back of the animal. After four days of recovery from CLP, or sham surgery, use foam tape to attach the anesthetized mouse tail to a short metal chain.Then attach the metal chain to a hook connected to a crossbar along the center of the cage. Affix a second small bar that can move along the crossbar to allow the animal greater movement ability. Then adjust the height of the suspended limbs to prevent the paws from contacting the chow pellets.Ensure that the animals can move via their forelimbs using the metal grid on the cage floor. CLP animals demonstrated increased immune cell populations in their peritoneal lavage compared to sham animals. CLP and HLS lead to approximately 11.8%of body mass loss.A marked decrease in soleus mass was observed when HLS was superimposed with CLP, indicating that smaller fibers comprise a major part of the muscle section. Compared to the control animals, CLP-HLS caused significant impairment in both the absolute and peak forces of the soleus.

combining-cecal-ligation-puncture-clp-with-hindlimb-suspension-hls-to

Research

JoVE Journal

Medicine

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.

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.