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 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)
<ol>
	<li>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.</li>
	<li>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 &#176;C, and the relative humidity (RH) is maintained between 30% and 60%. Ad libitum access to standard chow diet and water is ensured.</li>
	<li>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.</li>
	<li>Apply veterinary eye lubricant ointment to safeguard the animal&#39;s eyes from potential nosecone-induced damage or injury during surgery.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;mL&#160;for males and 0.5&#160;mL&#160;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.</li>
	<li>After the surgery, single house the animals in a clean cage on top of a heating mattress or a heating pad set at 35 &#176;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).</li>
	<li>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.</li>
</ol>
2. Hindlimb suspension (HLS)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 conditions12,13. 
	NOTE: Animal survival, discomfort, or distress will increase according to the severity of the infection.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>To enhance accuracy, measure xiphoid surface temperature and body weight twice per day11,15 and record along with the MMSS score sheet. 
	NOTE: Typical xiphoid surface temperature and body weight fluctuations are provided in Supplementary Figure 1.</li>
	<li>Consult the local IACUC for humane endpoints. 
	NOTE: To reproduce the results, the following criteria were used as endpoints: (1) Body weight loss &#62;40% from baseline. (2) Temperature &#60;30 &#176;C or reduction of &#62;5 &#176;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 &#8805;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.</li>
</ol>

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 authors have no conflicts of interest to disclose.

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

Research

JoVE Journal

Medicine

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.

371 Views.  University of Florida.  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.

Mouse Model for Studying Sepsis-Induced Myopathy with Hindlimb Suspension

Summary