Name: a controlled mouse model for neonatal polymicrobial sepsis
Uploaded: 2019-01-27T14:00:00.000+00:00
Duration: 14 min 54 s
Description: Watch this Scientific Journal Video about A Controlled Mouse Model for Neonatal Polymicrobial Sepsis at JoVE.com

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.

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	<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><tbody><tr><td>0.1 - 20 &amp;mu;L pipette tips</td><td>VWR</td><td>732-0799</td><td></td></tr><tr><td>1.8 mL Microcentrifuge tube</td><td>Costar</td><td>3621</td><td></td></tr><tr><td>100 - 1000 &amp;mu;L pipette tips</td><td>VWR</td><td>732-0801</td><td></td></tr><tr><td>1 - 200 &amp;mu;L pipette tips</td><td>VWR</td><td>732-0800</td><td></td></tr><tr><td>15 mL Centrifuge tube</td><td>FroggaBio</td><td>TB15-25</td><td></td></tr><tr><td>23G1 needles</td><td>Becton Dickinson</td><td>305145</td><td>only the needle, not the syringe, used for pinning mouse to styrofoam</td></tr><tr><td>28G 0.5 mL Insulin syringe</td><td>BD</td><td>329461</td><td></td></tr><tr><td>2 mL Cryogenic vial</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>50 mL Centrifuge tube</td><td>Fisher scientific</td><td>14-432-22</td><td></td></tr><tr><td>5 mL pipette</td><td>Costar</td><td>4487</td><td></td></tr><tr><td>6 - 10 week old C57BL/6J adult mice</td><td>Jackson Laboratories</td><td>664</td><td></td></tr><tr><td>7 + day old C57BL/6J neonatal mice</td><td>Bred in house</td><td>n.a</td><td></td></tr><tr><td>70 &amp;mu;m Cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Defibrinated Sheep&#039;s Blood</td><td>Dalynn</td><td>HS30-500</td><td></td></tr><tr><td>Dextrose 5% Water (D5W)</td><td>Baxter</td><td>JB0080</td><td></td></tr><tr><td>Dissecting forceps</td><td>VWR</td><td>&amp;nbsp;82027-386</td><td></td></tr><tr><td>Dissecting Scissors, Sharp Tip</td><td>VWR</td><td>&amp;nbsp;82027-592</td><td></td></tr><tr><td>Dissecting Scissors, Sharp/Blunt Tip</td><td>VWR</td><td>82027-594</td><td></td></tr><tr><td>Ethanol (HistoPrep 95% Denatured Ethyl Alcohol)</td><td>Fisherbrand</td><td>HC11001GL</td><td>diluted to 70% with double distilled water</td></tr><tr><td>Ethanol-proof marker; Lab marker</td><td>VWR</td><td>52877-310</td><td></td></tr><tr><td>EZ Anesthesia Vaporizer</td><td>EZ Anesthesia</td><td>EZ-155</td><td></td></tr><tr><td>Germinator 500, Dry sterilize surgicial instrument (Hot bead sterilizer)</td><td>Braintree Scientific</td><td>GER 5287-120V</td><td></td></tr><tr><td>Isoflurane</td><td>Fresenius Kabi</td><td>CP0406V2</td><td></td></tr><tr><td>Micro Spatula</td><td>Chemglass</td><td>CG-1983-12</td><td></td></tr><tr><td>Pipette-Aid</td><td>Drummond</td><td>4-000-100</td><td></td></tr><tr><td>Rainin Classic Pipette PR-1000</td><td>Rainin</td><td>17008653</td><td></td></tr><tr><td>Rainin Classic Pipette PR-20</td><td>Rainin</td><td>17008650</td><td></td></tr><tr><td>Rainin Classic Pipette PR-200</td><td>Rainin</td><td>17008652</td><td></td></tr><tr><td>Scale</td><td>Sartorius</td><td>BL 150 S</td><td></td></tr><tr><td>Specimen forceps</td><td>VWR</td><td>82027-440 / 82027-442</td><td></td></tr><tr><td>Square 1000 mL Storage Bottle</td><td>Corning</td><td>431433</td><td></td></tr><tr><td>Styrofoam board</td><td>Any</td><td>n.a</td><td></td></tr><tr><td>Sure-Seal Mouse/Rat euthanasia chamber</td><td>Euthanex</td><td>EZ-178</td><td></td></tr><tr><td>Tryptic Soy Agar</td><td>Sigma-Aldrich</td><td>22091-2.5KG</td><td></td></tr><tr><td>VX-200 Lab Vortex Mixer</td><td>Labnet International</td><td>S0200</td><td></td></tr><tr><td>weigh paper</td><td>Fisherbrand</td><td>09-898-12B</td><td></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 ...

a-controlled-mouse-model-for-neonatal-polymicrobial-sepsis

Research

JoVE Journal

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

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

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 ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

Medicine

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

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 Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

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

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

A Neonatal Mouse Spinal Cord Compression Injury Model

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Probiotic Studies in Neonatal Mice Using Gavage

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development