This work was supported by NIH R21 AG072011 to OL.

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 ...

Mouse Model for Studying Sepsis-Induced Myopathy with Hindlimb Suspension

<ol>
	<li>Prescott, H. C., Angus, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+recovery+from+sepsis.">Enhancing recovery from sepsis.</a> JAMA. 319 (1), 62-75 (2018).</li><li>Stortz, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+clinical+outcomes+and+the+immunocatabolic+phenotype+of+chronic+critical+illness+after+sepsis+in+surgical+intensive+care+unit+patients.">Benchmarking clinical outcomes and the immunocatabolic phenotype of chronic critical illness after sepsis in surgical intensive care unit patients.</a> J Trauma Acute Care Surg. 84 (2), 342-349 (2018).</li><li>Laitano, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+hindlimb+disuse+on+sepsis-induced+myopathy+in+mice.">The impact of hindlimb disuse on sepsis-induced myopathy in mice.</a> Physiological Rep. 9 (14), e14979 (2021).</li><li>Callahan, L. A., Supinski, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis-induced+myopathy.">Sepsis-induced myopathy.</a> Crit Care Med. 37, S354-S367 (2009).</li><li>Cuthbertson, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+and+quality+of+life+in+the+five+years+after+severe+sepsis.">Mortality and quality of life in the five years after severe sepsis.</a> Crit Care. 17 (2), R70 (2013).</li><li>Schmitt, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+contribute+to+long-term+tissue+repletion+following+surgical+sepsis.">Muscle stem cells contribute to long-term tissue repletion following surgical sepsis.</a> J Cachexia Sarcopenia Muscle. 14 (3), 1424-1440 (2023).</li><li>Owen, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+muscle+weakness+and+mitochondrial+dysfunction+in+the+absence+of+sustained+atrophy+in+a+preclinical+sepsis+model.">Chronic muscle weakness and mitochondrial dysfunction in the absence of sustained atrophy in a preclinical sepsis model.</a> eLife. 8, e49920 (2019).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+implicated+in+sepsis-induced+muscle+weakness+and+ameliorated+with+metformin.">Cellular senescence implicated in sepsis-induced muscle weakness and ameliorated with metformin.</a> Shock. 59 (4), 646 (2023).</li><li>Granger, J. I., Ratti, P. L., Datta, S. C., Raymond, R. M., Opp, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis-induced+morbidity+in+mice:+effects+on+body+temperature,+body+weight,+cage+activity,+social+behavior+and+cytokines+in+brain.">Sepsis-induced morbidity in mice: effects on body temperature, body weight, cage activity, social behavior and cytokines in brain.</a> Psychoneuroendocrinology. 38 (7), 1047-1057 (2013).</li><li>Ruiz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+modeling+in+mice:+ligation+length+is+a+major+severity+factor+in+cecal+ligation+and+puncture.">Sepsis modeling in mice: ligation length is a major severity factor in cecal ligation and puncture.</a> Intensive Care Med Exp. 4 (1), 22 (2016).</li><li>Laitano, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xiphoid+surface+temperature+predicts+mortality+in+a+murine+model+of+septic+shock.">Xiphoid surface temperature predicts mortality in a murine model of septic shock.</a> Shock (Augusta, Ga). 50 (2), 226-232 (2018).</li><li>Park, S., Brisson, B. K., Liu, M., Spinazzola, J. M., Barton, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+IGF-I+excels+in+promoting+functional+muscle+recovery+from+disuse+atrophy+compared+with+pro-IGF-IA.">Mature IGF-I excels in promoting functional muscle recovery from disuse atrophy compared with pro-IGF-IA.</a> J App Physiol. 116 (7), 797-806 (2013).</li><li>Spradlin, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+muscle+Igf1+exacerbates+disuse+atrophy+weakness+in+mice.">Deletion of muscle Igf1 exacerbates disuse atrophy weakness in mice.</a> J App Physiol. 131 (3), 881-894 (2021).</li><li>Shrum, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+scoring+system+to+evaluate+sepsis+severity+in+an+animal+model.">A robust scoring system to evaluate sepsis severity in an animal model.</a> BMC Res Notes. 7 (1), 233 (2014).</li><li>Mai, S. H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature+and+mouse+scoring+systems+as+surrogate+markers+of+death+in+cecal+ligation+and+puncture+sepsis.">Body temperature and mouse scoring systems as surrogate markers of death in cecal ligation and puncture sepsis.</a> Intensive Care Med Exp. 6 (1), 20 (2018).</li><li>Vankrunkelsven, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+muscle+weakness+in+a+mouse+model+of+critical+illness:+does+fibroblast+growth+factor+21+play+a+role.">Development of muscle weakness in a mouse model of critical illness: does fibroblast growth factor 21 play a role.</a> Skelet Muscle. 13 (1), 12 (2023).</li><li>Supinski, G., Stofan, D., Nethery, D., Szweda, L., DiMarco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apocynin+improves+diaphragmatic+function+after+endotoxin+administration.">Apocynin improves diaphragmatic function after endotoxin administration.</a> J Appl Physiol. 87 (2), 776-782 (1999).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+leads+to+impaired+mitochondrial+calcium+uptake+and+skeletal+muscle+weakness+by+reducing+the+micu1:mcu+protein+ratio.">Sepsis leads to impaired mitochondrial calcium uptake and skeletal muscle weakness by reducing the micu1:mcu protein ratio.</a> Shock (Augusta, Ga). 60 (5), 698-706 (2023).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+NAT10+protects+against+sepsis-induced+skeletal+muscle+atrophy+by+inhibiting+ROS/NLRP3.">Targeting NAT10 protects against sepsis-induced skeletal muscle atrophy by inhibiting ROS/NLRP3.</a> Life Sci. 330, 121948 (2023).</li><li>Mankowski, R. T., Laitano, O., Clanton, T. L., Brakenridge, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+treatment+strategies+of+acute+myopathy+and+muscle+wasting+after+sepsis.">Pathophysiology and treatment strategies of acute myopathy and muscle wasting after sepsis.</a> J Clinical Med. 10 (9), 1874 (2021).</li><li>Oliveira, J. R. S., Mohamed, J. S., Myers, M., Brooks, M. J., Alway, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hindlimb+suspension+and+reloading+on+gastrocnemius+and+soleus+muscle+mass+and+function+in+geriatric+mice.">Effects of hindlimb suspension and reloading on gastrocnemius and soleus muscle mass and function in geriatric mice.</a> Exp Gerontol. 115, 19-31 (2019).</li><li>Ashare, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-1+improves+survival+in+sepsis+via+enhanced+hepatic+bacterial+clearance.">Insulin-like growth factor-1 improves survival in sepsis via enhanced hepatic bacterial clearance.</a> Am J Respir Crit Care Med. 178 (2), 149-157 (2008).</li><li>Starr, M. E., Saito, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+increase+in+food+spilling+by+laboratory+mice+may+lead+to+significant+overestimation+of+actual+food+consumption:+implications+for+studies+on+dietary+restriction,+metabolism,+and+dose+calculations.">Age-related increase in food spilling by laboratory mice may lead to significant overestimation of actual food consumption: implications for studies on dietary restriction, metabolism, and dose calculations.</a> J Gerontol A Biol Sci Med Sci. 67 (10), 1043-1048 (2012).</li><li>Crowell, K. T., Soybel, D. I., Lang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restorative+mechanisms+regulating+protein+balance+in+skeletal+muscle+during+recovery+from+sepsis.">Restorative mechanisms regulating protein balance in skeletal muscle during recovery from sepsis.</a> Shock. 47 (4), 463-473 (2017).</li></ol>

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-0 Ethicon Coated Vicryl</td>
			<td>Ethicon</td>
			<td>D5792</td>
			<td>Absorbable suture used for closure of muscle layer and for ligation of the cecum.</td>
		</tr>
		<tr>
			<td>4-0 Ethilon Black 18&#34;&#160;</td>
			<td>Ethicon</td>
			<td>662G</td>
			<td>Non absorbable suture for closure of the skin layer.</td>
		</tr>
		<tr>
			<td>BD&#160; PrecisionGlide Needle 26-28 G</td>
			<td>BD</td>
			<td>305136 for 27g needle</td>
			<td>Needle for puncturing the cecum.</td>
		</tr>
		<tr>
			<td>C57BL/6J mice&#160;</td>
			<td>Jackson Laboratory&#160;</td>
			<td>strain #000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>Puritan</td>
			<td>S-18991</td>
			<td>Swabs for topical application of iodine.</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>(Leica CM1950)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dynarex Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td>Topical Antiseptic Liquid for Skin and Mucosa</td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof (100%)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>To make 70% ethanol for cleaning skin.</td>
		</tr>
		<tr>
			<td>Hindlimb Suspension Cages</td>
			<td>Custom Made</td>
			<td>N/A</td>
			<td>These custom made cages will be highlighted in the video recordings of the MS.</td>
		</tr>
		<tr>
			<td>Optixcare Eye Lube</td>
			<td>Optixcare</td>
			<td></td>
			<td>Eye lube for protection during survival surgery.</td>
		</tr>
		<tr>
			<td>Scalpel blades #11</td>
			<td>Fine Science</td>
			<td></td>
			<td>Blade used to make incisions on skin and muscle.</td>
		</tr>
		<tr>
			<td>Skin-Trac</td>
			<td>Zimmer</td>
			<td>736579</td>
			<td>Foam tape for fixing the tail to the suspension apparatus.</td>
		</tr>
		<tr>
			<td>SomnoSuite Low-Flow Digital Vaporizer</td>
			<td>Kent Scientific Corporation</td>
			<td>SS-01</td>
			<td>Vaporizer for Isoflurane Anesthesia</td>
		</tr>
		<tr>
			<td>Tissue bath apparatus&#160;</td>
			<td>Aurora Scientific</td>
			<td>Model 800A, Dual Mode Muscle Lever 300C</td>
			<td></td>
		</tr>
	</tbody>
</table>

a preclinical model of sepsis-induced myopathy with disuse in mice

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.

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.

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...

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.

Sepsis is a major cause of in-hospital deaths. Improvements in treatment result in a greater number of sepsis survivors. Approximately 75% of the ...

a-preclinical-model-of-sepsis-induced-myopathy-with-disuse-in-mice