Special thanks to Claire Harrison and the Animal Care Facility at British Columbia Children&#39;s Hospital Research Institute (BCCHR) for their support in the animal work, as well as to Dr. Po-Yan Cheng for their guidance and input on animal monitoring and well-being.

<ol>
	<li>Liu, L., 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=Global,+regional,+and+national+causes+of+child+mortality+in+2000-13,+with+projections+to+inform+post-2015+priorities:+an+updated+systematic+analysis.">Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis.</a> The Lancet. 385 (9966), 430-440 (2015).</li><li>Wynn, J. L., 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=Increased+mortality+and+altered+immunity+in+neonatal+sepsis+produced+by+generalized+peritonitis.">Increased mortality and altered immunity in neonatal sepsis produced by generalized peritonitis.</a> Shock. 28 (6), 675-683 (2007).</li><li>Fink, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis.">Animal models of sepsis.</a> Virulence. 5 (1), 143-153 (2014).</li><li>Wynn, J. L., 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=Defective+innate+immunity+predisposes+murine+neonates+to+poor+sepsis+outcome+but+is+reversed+by+TLR+agonists.">Defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists.</a> Blood. 112 (5), 1750-1758 (2008).</li><li>Cuenca, A. G., 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=Critical+role+for+CXC+ligand+10/CXC+receptor+3+signaling+in+the+murine+neonatal+response+to+sepsis.">Critical role for CXC ligand 10/CXC receptor 3 signaling in the murine neonatal response to sepsis.</a> Infection and Immunity. 79 (7), 2746-2754 (2011).</li><li>Gentile, L. F., 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=Protective+immunity+and+defects+in+the+neonatal+and+elderly+immune+response+to+sepsis.">Protective immunity and defects in the neonatal and elderly immune response to sepsis.</a> Journal of Immunology. 192 (7), 3156-3165 (2014).</li><li>Cuenca, A. G., 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=Delayed+emergency+myelopoiesis+following+polymicrobial+sepsis+in+neonates.">Delayed emergency myelopoiesis following polymicrobial sepsis in neonates.</a> Innate Immunity. 21 (4), 386-391 (2015).</li><li>Gentile, L. F., 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=Improved+emergency+myelopoiesis+and+survival+in+neonatal+sepsis+by+caspase-1/11+ablation.">Improved emergency myelopoiesis and survival in neonatal sepsis by caspase-1/11 ablation.</a> Immunology. 145 (2), 300-311 (2015).</li><li>Wynn, J. L., 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+IL-17A+attenuates+neonatal+sepsis+mortality+induced+by+IL-18.">Targeting IL-17A attenuates neonatal sepsis mortality induced by IL-18.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (19), E2627-E2635 (2016).</li><li>Fallon, E. 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=Program+Cell+Death+Receptor-1-Mediated+Invariant+Natural+Killer+T-Cell+Control+of+Peritoneal+Macrophage+Modulates+Survival+in+Neonatal+Sepsis.">Program Cell Death Receptor-1-Mediated Invariant Natural Killer T-Cell Control of Peritoneal Macrophage Modulates Survival in Neonatal Sepsis.</a> Frontiers in Immunology. 8, 1469 (2017).</li><li>Young, W. 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=Improved+survival+after+induction+of+sepsis+by+cecal+slurry+in+PD-1+knockout+murine+neonates.">Improved survival after induction of sepsis by cecal slurry in PD-1 knockout murine neonates.</a> Surgery. 161 (5), 1387-1393 (2017).</li><li>Rincon, J. 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=Adjuvant+pretreatment+with+alum+protects+neonatal+mice+in+sepsis+through+myeloid+cell+activation.">Adjuvant pretreatment with alum protects neonatal mice in sepsis through myeloid cell activation.</a> Clinical &amp; Experimental Immunology. 191 (3), 268-278 (2018).</li><li>Starr, M. 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=A+new+cecal+slurry+preparation+protocol+with+improved+long-term+reproducibility+for+animal+models+of+sepsis.">A new cecal slurry preparation protocol with improved long-term reproducibility for animal models of sepsis.</a> PLoS ONE. 9 (12), e115705 (2014).</li><li>Hansen, L. 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=Deficiency+in+milk+fat+globule-epidermal+growth+factor-factor+8+exacerbates+organ+injury+and+mortality+in+neonatal+sepsis.">Deficiency in milk fat globule-epidermal growth factor-factor 8 exacerbates organ injury and mortality in neonatal sepsis.</a> Journal of Pediatric Surgery. 52 (9), 1520-1527 (2017).</li><li>Fujioka, K., 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=Induction+of+heme+oxygenase-1+attenuates+the+severity+of+sepsis+in+a+non-surgical+preterm+mouse+model.">Induction of heme oxygenase-1 attenuates the severity of sepsis in a non-surgical preterm mouse model.</a> SHOCK. 47 (2), 242-250 (2017).</li><li>Al Asmari, K. 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=Protective+effect+of+quinacrine+against+glycerol-induced+acute+kidney+injury+in+rats.">Protective effect of quinacrine against glycerol-induced acute kidney injury in rats.</a> BMC Nephrology. 18, PMC5273840 (2017).</li><li>Geng, 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=Differences+in+gene+expression+profiles+and+signaling+pathways+in+rhabdomyolysis-induced+acute+kidney+injury.">Differences in gene expression profiles and signaling pathways in rhabdomyolysis-induced acute kidney injury.</a> International Journal of Clinical and Experimental Pathology. 8 (11), 14087-14098 (2015).</li><li>Kim, J. 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=Macrophage+depletion+ameliorates+glycerol-induced+acute+kidney+injury+in+mice.">Macrophage depletion ameliorates glycerol-induced acute kidney injury in mice.</a> Nephron Experimental Nephrology. 128 (1-2), 21-29 (2014).</li><li>Nara, 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=Evaluations+of+lipid+peroxidation+and+inflammation+in+short-term+glycerol-induced+acute+kidney+injury+in+rats.">Evaluations of lipid peroxidation and inflammation in short-term glycerol-induced acute kidney injury in rats.</a> Clinical and Experimental Pharmacology and Physiology. 43 (11), 1080-1086 (2016).</li><li>Zager, R. A., Johnson, A. C. M., Lund, S., Hanson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure:+determinants+and+characteristics+of+the+injury-induced+hyperinflammatory+response.">Acute renal failure: determinants and characteristics of the injury-induced hyperinflammatory response.</a> American Journal of Physiology Renal Physiology. 291 (3), F546-F556 (2006).</li><li>Olfert, E. D., Godson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humane+Endpoints+for+Infectious+Disease+Animal+Models.">Humane Endpoints for Infectious Disease Animal Models.</a> Institute for Laboratory Animal Research Journal. 41 (2), 99-104 (2000).</li><li>Canadian Council on Animal Care. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching and testing. , (1998).</li><li>Nemzek, J. A., Xiao, H. Y., Minard, A. E., Bolgos, G. L., Remick, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humane+endpoints+in+shock+research.">Humane endpoints in shock research.</a> SHOCK. 21 (1), 17-25 (2004).</li><li>Morton, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+for+establishing+humane+endpoints.">A systematic approach for establishing humane endpoints.</a> Institute for Laboratory Animal Research Journal. 41 (2), 80-86 (2000).</li></ol>

Dr. James Wynn receives support from the National Institutes of Health (NIH)/National Institute of General Medical Sciences (R01GM128452) and the NIH/Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) (R01HD089939).

Postnatal neonatal mice have very limited mobility and fail to right themselves after being placed on their back, even when unchallenged. By DOL 7, the age of mice challenged in this model, a range of movement spanning from Rights-Mobile to FTR-Mobile was observed in unchallenged mice, with an important difference, namely that an unchallenged mouse at this age did not display FTR-Lethargic behavior. Only mice challenged with polymicrobial sepsis were observed to become FTR-Lethargic; therefore, this response can be a marker of disease severity. Being attentive to the cutoff of a 90&#176; angle from horizontal for hip movement allows for the consistent and accurate assignment of lethargic or mobile hip movement in mice. The time frame of 4 s to see if a mouse can right itself was selected because unchallenged mice were able to consistently right themselves within this time frame. Repeated measurement of the same mouse was avoided, while the time to right themselves and the measurement of hip mobility was limited to 4 s, to avoid excessively tiring the mouse, which could otherwise affect its ability to obtain food and warmth and could affect its prognosis to get better. Righting itself from both the left and the right side were observed, and the higher of the scores was used to determine if the mouse was at a humane endpoint, because some mice were found to display FTR-Nonmobile on one side yet have a higher mobility on the other side and be able to recover eventually.
The scoring system used to evaluate mouse health relied on the application of categorical cutoffs to what is a spectrum of movement and, therefore, could be prone to individual bias. Staff was trained together to ensure each person scored the mice the same; however, there will likely remain a level of subjectivity leading to variation. The consistency of scoring was evaluated by having seven researchers who had not previously performed the neonatal mouse monitoring learn the requirements outlined in this protocol and video and, then, independently assign behaviors and determine humane endpoint. A 97% accuracy was observed with scoring performed on 60 challenged mice, suggesting that individual bias does not play a substantial role in the behavioral assignments of this model. The presented behavioral monitoring protocol is based on observations of animals challenged on DOL 7, yet mice younger than 6 days in an unchallenged healthy state cannot consistently right themselves. Thus, the described humane endpoint criteria could not be applied directly to younger mice. If younger mice are used in this experimental model or if a different challenge model with different disease kinetics is applied, then suitable humane endpoint criteria must be developed and piloted to avoid the euthanasia of mice that would otherwise, eventually, recover. The scoring system displays a robust method of improving humane endpoint classification that, with testing and confirmation, could potentially be applied to other models.
Each preparation of cecal slurry or the use of a new mouse strain required the retitration of the cecal slurry dose to administer to achieve a similar lethal dose. Each preparation was standardized by the readout of interest, namely survival, rather than giving the same bacterial count. Each cecal slurry preparation&#39;s viable bacterial concentration varied slightly, potentially due to differences in the donor&#39;s commensal bacteria or due to variances in the weight left in the cell strainer of the cecal slurry stock postfiltration. During the titration of the cecal slurry, the first two litters were divided into two groups and each half of the litter were challenged with one of the two doses so that each of the doses would be tested in two litters. If the resulting survival rate did not match the required level, then the challenge dose was either increased or decreased by 5%-10% and the experiment repeated. Multiple litters were used to account for litter-to-litter differences that could cause resistance or increased susceptibility to sepsis across a litter. It was important to accurately titer the cecal slurry stock with each new preparation to ensure that the new titration of cecal slurry was comparable to previous cecal slurry preparations. Periods of excess noise and vibration, specifically during the compacting of asphalt and the construction of a nearby building and road, were observed to increase stress in the dams. This correlated with increased rates of cannibalization, and affected the mortality of the survival experiments, even affecting unchallenged mice, indicating that there can be extraneous impacts on neonatal survival that also need to be controlled for.
Prior methods for cecal slurry stock preparation included either the use of fresh cecal slurry or the preparation of frozen cecal slurry, using a variety of methods, including the storage in glycerol that would inevitably be transferred during the challenge. While the use of fresh cecal slurry provides the advantage of having a bacterial composition closest to original cecal contents, there is the risk of variance between individual donor mice due to the variation of commensal bacteria. While this was minimized by using cecal donors from the same vendor with minimal time between arrival and progression of the experiment, this could become a cost-prohibitive option for some laboratories and presented another timing logistics challenge in having age-matched mice available when commencing a cecal slurry experiment in neonatal mice that were 7 days old. An alternative method to using fresh cecal slurry was utilized, where multiple adult donors&#39; cecal contents were pooled, resuspended in D5W, frozen at -80 &#176;C without glycerol, and thawed one aliquot at a time for experiments. The utilization of adult donor cecal slurry to study neonatal sepsis could potentially transfer species of bacteria present in the cecal slurry that the neonatal mouse has not been exposed to, but it is a strategy that allows for the study of sepsis in neonatal mice and has been used to study neonatal mouse biology in the past13,14,15. Cecal slurry was diluted in D5W to provide nutrition to the bacteria, which allowed the establishment of an active infection once the bacteria were injected, and was done to mimic the availability of nutrients in the peritoneal cavity during necrotizing enterocolitis. Glycerol was not included as a stabilizing agent in freezing bacteria because of the potential negative side effects that could arise from glycerol injection alone. If glycerol had been included in the cecal slurry preparation, then the potential damage that glycerol alone could induce would need to be tested for by including a glycerol-only (lacking cecal slurry) injection in mice, which would have increased mouse usage. The bacteria viability of the cecal slurry stocks was tested after freezing the cecal slurry stock without glycerol and was found to be constant, with no change in bacteria concentration in separate aliquots of the same cecal slurry preparation stored at -80 &#176;C over a 6 month period. This suggests that the storage without glycerol is feasible in providing a consistent biological outcome. The use of a bulk-prepared frozen cecal slurry stock also allowed for the use of mice bred in-house, reducing cost and utilizing male mice that would otherwise be excess from breeding, therefore reducing mouse wastage.
The identification of failed challenges in mice was important to avoid adding extra noise to the system. After undergoing an intraperitoneal injection of cecal slurry, the mice were observed for the presence of a bulge underneath the skin, which indicated a failed injection that was actually subcutaneous. Mice were observed for leaks at the injection site, both immediately after needle removal and after allowing them to take a step after the injection, because mice would sometimes (rarely) leak only after moving the limb of the injection site by taking a step. The presence of a bulge or leak following the injection resulted in removing the mouse from the analysis. After all, either of these could result in a different outcome due to the incorrect amount of cecal slurry injected as a 5% difference in challenge dose has been observed to affect subsequent survival.
Cecal slurry challenge experiments often required varying target lethal doses with varying weight-adjusted doses. Due to this, injection volumes can range from as little as 20 &#181;L and up to 100 &#181;L. The proportionate experimental error associated with dead needle volume also changes along with the injection volume, increasing the difficulty to directly compare different doses. With the simple modification of standardizing the injection volume, this source of variance is removed from the experiment.
The neonatal mouse&#39;s behavioral monitoring system used in this protocol is the first of its kind. Researchers intent on conducting ethical research with newborn mice are often faced with the challenging lack of resources to assess the animal&#39;s well-being at this age. The presented intuitive and consistent monitoring system begins to address this knowledge gap. Importantly, this evidence-driven approach not only increases the quality of the experimental data obtained but, at the same time, also reduces the suffering of the experimental animals.

Sepsis is a leading cause of human newborn infectious deaths1. Because newborn sepsis is poorly understood, little progress has been made in both the identification of at-risk newborns early during the disease and the development of efficacious treatments or prophylaxes. This necessitates the use of animal models of sepsis to better understand the process and test possible interventions. Furthermore, adult rodents respond differently to sepsis, with statistically significant differences in the number of bacteria to administer to obtain the same lethal dose (LD) and differences in the resulting host response as compared to newborns2. Thus, neonatal sepsis has to be studied in neonates. Several adult sepsis models have been used in sepsis research. These include an intravenous challenge with specific organisms implicated in adult human sepsis or cecal ligation and puncture (CLP). CLP is an endogenous challenge model where the cecum is surgically isolated, ligated, and punctured to allow leakage of intestinal contents into the peritoneum, eventually leading to the systemic dissemination of microbes and their products3. However, the surgical procedure required to establish CLP is lethal to newborn animals; therefore, an alternate method is necessary to mimic the polymicrobial challenge of CLP to induce neonatal sepsis. The cecal slurry model for neonatal polymicrobial sepsis was developed to address this need, whereby the cecal contents of animals are harvested, suspended in sterile dextrose 5% in water (D5W), and intraperitoneally injected into newborn mice2. This has, since, become an increasingly popular model to study sepsis in both newborn and adult animals and has substantially advanced mechanistic insights in the disease&#39;s process4,5,6,7,8,9,10,11,12,13,14,15.
Given the increasing use of this model and desire of researchers to directly compare results across publications, there is a need for the technical aspects to be well described and standardized across studies. Standardization applies to three aspects of the model, namely, i) the preparation of the cecal slurry stock, ii) the preparation of the challenge aliquots for injection into the experimental animals, and iii) the definition of the humane endpoint whereby animals are deemed nonsurvivors in challenge experiments. Specifically, methods to prepare the cecal slurry stock are often referenced to the original article introducing the model2. A brief summary of that model is that cecal contents from adult mice were harvested, suspended in sterile D5W to a concentration of 80 mg/mL, and used within 2 h to inject the experimental animals. This original model used mice of the same age, from the same vendor location, which were housed in their respective research facilities for less than 2 weeks prior to harvesting cecal contents. The use of in-house bred mice, although reducing the cost from regular vendor delivery and allowing for the use of excess mice of a broader range of sex and age, also substantially increased donor-to-donor variability. This motivated the development of an alternative technique, whereby cecal contents from multiple mice were pooled together to prepare a large stock, which was then aliquoted and stored at -80 &#176;C13. This alternate method was adapted by multiple groups14,15. However, that adaptation resulted in some technical variations, both in the storage media used (10% or 15% glycerol, or D5W alone) and in the strategy of filtration to remove particulate (multistage filtration through a 860 &#181;m and, then, a 190 &#181;m filter, or individual filtrations through 100 &#181;m or 70 &#181;m filters)13,14,15. The injection of glycerol alone could potentially cause harm, given that 25%-50% glycerol injections have been used as a rodent model of renal injury16,17,18,19,20. To avoid unintended side effects of glycerol, the cecal slurry stock preparation for mice in this study is frozen in D5W without glycerol, and tests of bacterial viability from storage at -80 &#176;C are performed. The filtration strategy used in this study is one pass through a 70 &#181;m filter, which has not been directly compared to the other filtration strategies listed.
Lethal weight-adjusted doses of injected cecal slurry may vary from facility to facility and should be titered out to the desired lethality for individual groups. With different challenge doses, the accompanying challenge volumes change by necessity. However, this methodological detail has not been reported before. Furthermore, strategies for standard procedures, such as intraperitoneal injection, are rarely elaborated on within the literature, but individual techniques may affect whether newborn mice leak when injected and impact their final outcomes.
Animal welfare, including a definition of humane endpoint, is a central aspect of this model and in any model of infection and inflammation in rodents21. In 1998, the Canadian Council on Animal Care (CCAC) published extensive guidelines for humane endpoint selection, defining the humane endpoint as &#34;any actual or potential pain, distress, or discomfort should be minimized or alleviated by choosing the earliest endpoint that is compatible with the scientific objectives of the research&#34;22. Others also caution that humane endpoints must be established based on scientific justification rather than on a subjective interpretation of the animal&#39;s state alone21. While there is a wealth of resources for clinical, behavioral, and body-condition sign-based criteria for humane endpoint, even in the context of infection and inflammation specifically21,23,24, none of these, including the CCAC guidelines for humane endpoint22,&#160;mention newborn mice. Thus, objectively and scientifically justified humane endpoints are much more difficult to establish for newborn animals, given both their limited behavioral capabilities and the lack of evidence from criteria like weight loss, which is commonly used for adult mice. Currently, the criteria for the humane endpoint used for 5- to 12-day-old neonatal mice in the cecal slurry literature all reference back to the original manuscript that introduced the model2. In this original paper, the definition of humane endpoint for newborn animals was based on two criteria; namely, the location of a mouse outside of the nest (scattering) and the lack of milk spots had been seen to result in death within hours. A complicating matter in assigning a humane endpoint is that milk spots become difficult to see in mouse strains with dark fur, such as the commonly employed C57BL/6J strain, after the first week of life, while sick animals are monitored until the 14th day of life (DOL). Further, dead animals can be found postchallenge when applying these criteria (own observation; unpublished); thus, a more rigorous definition of humane endpoint is necessary to alleviate suffering to experimental animals and avoid mortality in situations where the outcome could be accurately discerned earlier.
All three methodological aspects of the cecal slurry model are presented in a standard operating procedure detailing the preparation of cecal slurry stock, a method for injecting experimental animals that keeps the injection volume constant between doses and reduces the risk of leaks, and a definition of humane endpoint for 7- to 12-day-old mice based on a system of behavioral modeling. Behavioral information of mouse health scores from over 240 experimental animals was collected and grouped by final survival outcome, demonstrating an evidence-driven definition of humane endpoint. The suffering of experimental animals is reduced by identifying moribund neonatal mice at the earliest possible time point, while biologically significant survival outcomes can be inferred by observing key variables. The visual representation of both cecal slurry preparation and neonatal mouse behaviors will serve as an excellent resource to any group studying sepsis or newborn challenge model animals.

<table border="0" cellpadding="0" cellspacing="0" style="width:609px;" width="610">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">0.1 - 20 &#956;L pipette tips</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">732-0799</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">1.8 mL Microcentrifuge tube</td>
			<td style="width:95px;">Costar</td>
			<td style="width:123px;">3621</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">100 - 1000 &#956;L pipette tips</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">732-0801</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:222px;">1 - 200 &#956;L pipette tips</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">732-0800</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">15 mL Centrifuge tube</td>
			<td style="width:95px;">FroggaBio</td>
			<td style="width:123px;">TB15-25</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:84px;width:222px;">23G1 needles</td>
			<td style="width:95px;">Becton Dickinson</td>
			<td style="width:123px;">305145</td>
			<td style="width:171px;">only the needle, not the syringe, used for pinning mouse to styrofoam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">28G 0.5 mL Insulin syringe</td>
			<td style="width:95px;">BD</td>
			<td style="width:123px;">329461</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">2 mL Cryogenic vial</td>
			<td style="width:95px;">Corning</td>
			<td style="width:123px;">430488</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">50 mL Centrifuge tube</td>
			<td style="width:95px;">Fisher scientific</td>
			<td style="width:123px;">14-432-22</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">5 mL pipette</td>
			<td>Costar</td>
			<td style="width:123px;">4487</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">6 - 10 week old C57BL/6J adult mice</td>
			<td style="width:95px;">Jackson Laboratories</td>
			<td style="width:123px;">664</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">7 + day old C57BL/6J neonatal mice</td>
			<td style="width:95px;">Bred in house</td>
			<td style="width:123px;">n.a</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">70 &#956;m Cell strainer</td>
			<td style="width:95px;">Falcon</td>
			<td style="width:123px;">352350</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Defibrinated Sheep&#39;s Blood</td>
			<td style="width:95px;">Dalynn</td>
			<td style="width:123px;">HS30-500</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Dextrose 5% Water (D5W)</td>
			<td style="width:95px;">Baxter</td>
			<td style="width:123px;">JB0080</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Dissecting forceps</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">&#160;82027-386</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Dissecting Scissors, Sharp Tip</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">&#160;82027-592</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">Dissecting Scissors, Sharp/Blunt Tip</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">82027-594</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">Ethanol (HistoPrep 95% Denatured Ethyl Alcohol)</td>
			<td style="width:95px;">Fisherbrand</td>
			<td style="width:123px;">HC11001GL</td>
			<td style="width:171px;">diluted to 70% with double distilled water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">Ethanol-proof marker; Lab marker</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">52877-310</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">EZ Anesthesia Vaporizer</td>
			<td style="width:95px;">EZ Anesthesia</td>
			<td style="width:123px;">EZ-155</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:222px;">Germinator 500, Dry sterilize surgicial instrument (Hot bead sterilizer)</td>
			<td style="width:95px;">Braintree Scientific</td>
			<td style="width:123px;">GER 5287-120V</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">Isoflurane</td>
			<td style="width:95px;">Fresenius Kabi</td>
			<td style="width:123px;">CP0406V2</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Micro Spatula</td>
			<td style="width:95px;">Chemglass</td>
			<td style="width:123px;">CG-1983-12</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Pipette-Aid</td>
			<td>Drummond</td>
			<td style="width:123px;">4-000-100</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Rainin Classic Pipette PR-1000</td>
			<td style="width:95px;">Rainin</td>
			<td style="width:123px;">17008653</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Rainin Classic Pipette PR-20</td>
			<td style="width:95px;">Rainin</td>
			<td style="width:123px;">17008650</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Rainin Classic Pipette PR-200</td>
			<td style="width:95px;">Rainin</td>
			<td style="width:123px;">17008652</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Scale</td>
			<td style="width:95px;">Sartorius</td>
			<td style="width:123px;">BL 150 S</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">Specimen forceps</td>
			<td style="width:95px;">VWR</td>
			<td style="width:123px;">82027-440 / 82027-442</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Square 1000 mL Storage Bottle</td>
			<td style="width:95px;">Corning</td>
			<td style="width:123px;">431433</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Styrofoam board</td>
			<td style="width:95px;">Any</td>
			<td style="width:123px;">n.a</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">Sure-Seal Mouse/Rat euthanasia chamber</td>
			<td style="width:95px;">Euthanex</td>
			<td style="width:123px;">EZ-178</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:222px;">Tryptic Soy Agar</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:123px;">22091-2.5KG</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:222px;">VX-200 Lab Vortex Mixer</td>
			<td style="width:95px;">Labnet International</td>
			<td style="width:123px;">S0200</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">weigh paper</td>
			<td style="width:95px;">Fisherbrand</td>
			<td style="width:123px;">09-898-12B</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
</table>

a controlled mouse model for neonatal polymicrobial sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

Cecal slurry viability stored at -80 &#176;C can be tested over time by serially diluting and plating aliquots of cecal slurry stock on 5% sheep&#39;s blood tryptic soy agar followed by 24 h of aerobic incubation at 37 &#176;C. Subsequent counting of culturable colony-forming unit (CFU) content of a cecal slurry preparation was found not to change over a 6 month period, and the viability was not affected by prolonged storage at -80 &#176;C (Figure 2). Each donor mouse resulted, on average, in enough cecal slurry to challenge three to four litters (data not shown).
Mice challenged at DOL 7 with cecal slurry to induce polymicrobial sepsis began to reach the humane endpoint within 12 h of the challenge, and polymicrobial sepsis was mostly resolved by 48 h postchallenge, as observed in a Kaplan-Meier survival curve combined from data from over 200 challenged mice (Figure 1A). The lethality was dependent on the challenge dose administered, with a 5% change in challenge dose resulting in a roughly 15% difference in survival rate (Figure 1B). The mouse body weight was measured at each monitoring visit. Weight loss was seen in all challenged animals, being nondiscriminatory between mice that ended up surviving and those that did not during the initial 24 h postchallenge (Figure 1C). After 24 h, most surviving animals began to regain their weight, while all nonsurvivors continued to lose weight and moved to their humane endpoint. However, a small proportion of surviving animals that had retained their righting reflex also continued to lose weight or failed to gain weight, until the end of the experiment, even losing as much as 20% of their initial body weight within 40 h of the challenge. As there was an overlap of weight loss between mice that ended up surviving and those that did not, the change in weight or a threshold of weight loss could not be used as a criterion for humane endpoint while still maintaining the goal of accurately dividing survivors from nonsurvivors.
The behavior of mice was monitored as outlined in the protocol and in Table 2. Snapshots of the health categories are displayed (Figure 3A-C). These photos show the different health categories of mice who failed to right themselves after being placed on their back and outline the difference between FTR-Mobile and FTR-Lethargic, which is an important distinction. Unchallenged healthy mice of this age do not display FTR-Lethargic activity; therefore, this health category is a marker of disease and a response to challenge. Sick mice displayed FTR-Lethargic symptoms (Figure 3B) and could regress toward FTR-Nonmobile (Figure 3C), where the upper leg remains parallel with the bottom leg, with little to zero hip rocking movement, which is one of the criteria for humane endpoint. The mice might also recover, gaining increased hip movement and becoming FTR-Mobile (Figure 3A). The righting reflex and mobility scores were determined for both the left and right side of each mouse, and the highest score was utilized to determine whether the mouse had reached a humane endpoint. Behavioral information was collected from over 240 animals challenged with a lethal dose 60 (LD60) of cecal slurry, and 144 humane endpoints were observed (Figure 3D-F and Table 1). This evidence-driven approach was used to define and refine the humane endpoint across four disease stages, categorized by the experimenters based on both behavioral differences between survivors and nonsurvivors and by the fraction of humane endpoints reached during each time frame. During early experiments, FTR-Nonmobile mice that had no hip movement were consistently found dead within 4-6 h of this behavior being observed. In the collection of the presented information, an FTR-Nonmobile health score was used as criterion for a humane endpoint. From 12-21 h postchallenge, while FTR-Nonmobile mice were euthanized, both surviving and nonsurviving animals displayed very similar behavioral patterns and could not be distinguished in any other way (Figure 3D). From 21-48 h postchallenge, the majority of surviving mice regained their righting reflex, while fewer than 1% of the FTR behaviors observed were in animals that would go on to survive the experiment (Figure 3E). Thus, mice that failed to right themselves from both sides became an additional criterion for humane endpoint during this time. Between 12 and 20 h postchallenge, 12.5% of the total number of humane endpoints were observed, versus 80.5% between 20 and 48 h, and 7% after 48 h (Table 1). A distinguishing feature between mice that ended up surviving and that eventually worsened to a humane endpoint was the loss of the righting reflex, independent of hip mobility (Figure 3F). Indeed, between 20 and 48 h after the challenge, a total of 121 mice had failed to right themselves from both sides, with 116 of these mice eventually progressing to a humane endpoint (which represents a 96% accuracy in identifying mice that would not recover). Beyond 48 h after the challenge, 11 mice were observed to fail to right themselves from both sides, and 10 of these progressed to a humane endpoint (a 91% accuracy). Beyond 20 h after the challenge, the number of mice that lost the righting reflex for both sides predicts the final outcome with an accuracy of more than 90%; therefore, this has been added to the humane endpoint criteria, to identify nonrecovering mice earlier and reduce mouse suffering (Table 1).
The frequency that mice need monitoring changes over time, due to different rates of death postchallenge, and is outlined in Table 1. A mouse was considered to be at its humane endpoint at any point if it had failed to right itself and displayed nonmobile hip movement on both sides, or if the mouse was found scattered from the nest, was unable to right itself, and had lethargic hip movement. Mice with either of these conditions were not expected to be able to rejoin the litter and have been observed to be FTR-Nonmobile within 4-6 h. Starting 20 h after the challenge, a new humane endpoint was added because the presented information shows that the vast majority of mice that FTR from both sides ends up succumbing to disease.
The videos, tables, and resources presented in this manuscript are an effective teaching resource for the correct behavioral assignment of challenged mice. Seven experimenters were asked to watch the training video and read both the protocol and the tables before assigning behaviors to 60 challenged animals. The identification of humane endpoint assignment was accurate both for distinguishing FTR-Nonmobile mice from mice that displayed the other behaviors (Figure 4A) and FTR mice from mice that were able to right themselves within the allowable time frame (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58574/58574fig1.jpg" /> 
Figure 1: Kaplan-Meier survival curve, cecal slurry dose titration, and weight change following the cecal slurry challenge. (A)&#160;Survival outcome of neonatal C57BL/6J mice challenged with an intraperitoneal cecal slurry injection at DOL 7. The data for this figure were combined from independent experiments using multiple challenge doses, ranging from 0.7 to 1.3 mg of cecal slurry per gram body weight was administered to these mice. (B) Neonatal mice challenged with 0.80 to 0.95 mg of cecal slurry per gram body weight from one cecal slurry preparation display a dose-dependent relationship between the amount of cecal slurry given and the percentage of survival.(C) The percentage of change in weight compared to the challenge weight, with the dotted line denoting a 20% loss of weight from the time of the challenge. <a href="https://www.jove.com/files/ftp_upload/58574/58574fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58574/58574fig2.jpg" /> 
Figure 2: CFU concentration in cecal slurry stock stored at -80 &#176;C does not change over a 6 month period. The effect of the cecal slurry age on CFU concentration was tested using linear regression. Each point represents one aliquot of the same cecal slurry preparation, serially diluted and plated over a 6 month period. <a href="https://www.jove.com/files/ftp_upload/58574/58574fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58574/58574fig3.jpg" /> 
Figure 3: Hip mobility categories of mice that fail to right themselves and of animal behaviors at various times postchallenge. Mice that have been challenged with sepsis, when placed on their back, will display signs of morbidity that can be measured by the degree of hip movement. (A)&#160;A fail to right (FTR)-Mobile mouse shows hip rocking movement of their upper leg exceeding 90&#176; angle from horizontal. (B)&#160;An FTR-Lethargic mouse shows hip rocking movement but does not exceed 90&#176; angle from horizontal at any point during the 4 s of monitoring. (C)&#160;Some FTR-Nonmobile mice will extend their leg, bending at the knee, but will show very little (less than 10&#176; angles) to zero hip rocking movement, and the legs will remain parallel to each other. (D) Animal behaviors 12-21 h postchallenge show that only FTR-Nonmobile behaviors separate survivors from nonsurvivors. (E)&#160;From 21 to 48 h postchallenge, only 4 out of the 592 observed FTR behaviors (0.67%) belong to survivors, allowing the righting reflex to predict the final outcome and be used as a new criterium for humane endpoint. (F)&#160;Beyond 48 h postinfection, 6 out of 131 mice (4.55%) that had a righting reflex went on to become part of the FTR group and were sacrificed by the end of the experiment, justifying sustained monitoring throughout the course of recovery. <a href="https://www.jove.com/files/ftp_upload/58574/58574fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58574/58574fig4.jpg" /> 
Figure 4: Instructional resources result in accurate behavioral classification by independent experimenters. Experimenters trained by watching video accompanying this protocol categorized videos of 60 neonatal mice into different health groups. (A) The ability to distinguish a humane endpoint was determined and an average of 97% of behaviors was accurately categorized as FTR-Nonmobile or not, while only 1% of FTR-Nonmobile mice were misidentified. Two percent of the mice were falsely identified as FTR-Nonmobile. (B) The identification of the second humane endpoint criterium of correctly distinguishing between FTR mice or those having the ability to right themselves within 4 s of being placed on their back was assigned correctly in 97% of the scorings, while only 0.96% of the mice were incorrectly assigned as righting themselves and 2% of mice were incorrectly assigned as FTR. <a href="https://www.jove.com/files/ftp_upload/58574/58574fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disease Stage</td>
			<td>A: High morbidity, no mortality</td>
			<td>B:&#160; High morbidity, low mortality</td>
			<td>C: High morbidity, high mortality</td>
			<td>D: Low morbidity, low mortality</td>
		</tr>
		<tr>
			<td>Hours post challenge</td>
			<td>0&#8722;12</td>
			<td>12&#8722;20</td>
			<td>20&#8722;48</td>
			<td>&#62;48</td>
		</tr>
		<tr>
			<td>Monitoring frequency</td>
			<td>2 h post challenge</td>
			<td>every 4&#8722;6 h</td>
			<td>Every 4&#8722;6 h, 8 h, unattended overnight</td>
			<td>1&#8722;2 times daily, more if needed</td>
		</tr>
		<tr>
			<td>Proportion of total humane endpoints observed&#160;</td>
			<td>0/144</td>
			<td>18/144</td>
			<td>116/144</td>
			<td>10/144</td>
		</tr>
		<tr>
			<td>Percentage of humane endpoints observed</td>
			<td>0%</td>
			<td>12.5%</td>
			<td>80.5%</td>
			<td>7%</td>
		</tr>
		<tr>
			<td rowspan="3">Humane endpoint criteria</td>
			<td colspan="2">1. FTR&#8722;Nonmobile on both sides</td>
			<td colspan="2">1. FTR&#8722;Nonmobile on both sides</td>
		</tr>
		<tr>
			<td colspan="2">2. Scattered from nest and is FTR&#8722;Lethargic</td>
			<td colspan="2">2. Scattered from nest and is FTR&#8722;Lethargic</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">3. FTR on both left or right side (with any mobility score)</td>
		</tr>
	</tbody>
</table>
Table 1: Frequency of monitoring and humane endpoint criteria in the different stages of disease. Monitoring frequency, humane endpoints observed, the percentage of humane endpoints, and humane endpoint criteria during different stages of disease.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Righting Reflex</td>
			<td>Mobility</td>
			<td>Time limit to right after being placed on back</td>
			<td>Time limit to measure amount of movement (mobile / lethargic / nonmobile)</td>
			<td>Mobility scoring Criteria</td>
		</tr>
		<tr>
			<td rowspan="3">Rights</td>
			<td>Mobile</td>
			<td rowspan="6">4 s&#160;</td>
			<td rowspan="3">An additional 8 s</td>
			<td>The mouse takes multiple steps in a row, maintaining forward momentum, and explores its environment. Pup will not fall over.</td>
		</tr>
		<tr>
			<td>Lethargic</td>
			<td>The mouse can take a step but will stop and pause before taking another. Pup may fall over.</td>
		</tr>
		<tr>
			<td>Nonmobile</td>
			<td>The mouse does not take any steps after righting itself. Pup may fall over.</td>
		</tr>
		<tr>
			<td rowspan="3">Fail to right</td>
			<td>Mobile hips</td>
			<td rowspan="3">The same 4 s used to measure righting reflex</td>
			<td>Has energetic hip movement with the upper leg rotating beyond 90&#176; from horizontal at least once within 4 s.</td>
		</tr>
		<tr>
			<td>Lethargic hips</td>
			<td>Hip movement up to but not beyond 90&#176; from horizontal.</td>
		</tr>
		<tr>
			<td>Nonmobile hips</td>
			<td>Limbs may move by extending and retracting but the hips will not rotate. Pup looks very sickly.&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: Monitoring table and criteria in determining the health score of mice. The provided criteria were used to define health category groups to mice, and to reduce individual variance in assigning health scores.

Watch this Scientific Journal Video about A Controlled Mouse Model for Neonatal Polymicrobial Sepsis at JoVE.com

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

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Immunology and Infection

10.8K Views.  University of British Columbia. This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice. 

Video: A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...