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.

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...

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

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.

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 ...

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.

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JoVE Journal

Medicine

495 Views.  University of Florida. 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 myo...

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

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Preclinical Controlled Cortical Impact Model for Traumatic Hemorrhage Contusion and Neuroinflammation

Cerebral contusion is a severe medical problem affecting millions of people worldwide each year. There is an urgent need to understand the pathophysiological mechanism and to develop effective therapeutic strategy for this devastating neurological disorder. Intraparenchymal hemorrhage and post-traumatic inflammatory response induced by initial physical impact can aggravate microglia/macrophage activation and neuroinflammation which subsequently worsen brain pathology. We provide here a controlled cortical impact (CCI) protocol that can reproduce experimental cortical contusion in mice by using a pneumatic impactor system to deliver mechanical force with controllable magnitude and velocity onto the dural surface. This preclinical model allows researchers to induce moderately severe focal cerebral contusion in mice and to investigate a wide range of post-traumatic pathological progressions including hemorrhage contusion, microglia/macrophage activation, iron toxicity, axonal injury, as well as short-term and long-term neurobehavioral deficits. The present protocol can be useful for exploring the long-term effects of and potential interventions for cerebral contusion.

Intraparenchymal hemorrhage and neuroinflammation accompanied by cerebral contusion can trigger severe secondary brain injury. This protocol details a mouse controlled cortical impact (CCI) model allowing researchers to study hemorrhage contusion and post-traumatic immune responses and explore potential therapeutics.

Cerebral contusion is a severe medical problem affecting millions of people worldwide each year. There is an urgent need to understand the ...

A Preclinical Model of Exertional Heat Stroke in Mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...