A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Abstract

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.

BCG vaccination–induced emergency granulopoiesis provides rapid protection from neonatal sepsis

“…Instead, the number of neutrophils recruited to the lung correlated with reduced bacterial burden, as well as accelerated weight recovery postchallenge (fig. S7J and K), both established markers of improved outcome in this model (29).…”

“…Neonatal mice were used to allow for a controlled and standardized model to study relevant pathways, with survival and bacterial burden standing as measurements of disease severity. Mouse survival assays had sample size calculated as described (29). Survival assays required a minimum of 17 mice per group to detect a 40% increase in survival in mice challenged with a dose lethal for 65% of recipients (LD 65 ).…”

Robust health-score based survival prediction for a neonatal mouse model of polymicrobial sepsis

“…Polymicrobial sepsis was induced using a modified version of the model first described by Wynn et al . in 2007 and recently outlined on video in the Journal of Visualized Experiments [19,20]. Briefly, cecal material was extruded from adult male mouse ceca (aged 6–10 weeks), diluted in dextrose 5% water (D5W) to 160 mg slurry / mL D5W, filtered through a 70 μm sterile strainer, pooled, aliquoted, and frozen at -80 o C. Aliquots were thawed at room temperature, diluted further in D5W to the desired weight-adjusted dose, and kept on ice prior to intraperitoneal (IP) injection.…”

“…Nesting material was rubbed in hands to transfer that litter’s smell to the gloves, and then each mouse was individually scruffed and placed on their back as visualized in Brook et al . 2019 [20]. Health scores arose naturally out of a need to define a humane endpoint for extremely ill neonatal pups.…”

“…Righting reflex measurements were further standardized by adding a four-second cutoff, determined by the observation that more time was unnecessary, and less time risked inappropriately assigning ‘fail to right’ to a healthy pup. Previous work has shown minimal variance in score assignments between trained individuals[20]. For simplicity of recording and interpretation, these observations were transformed into ranked scores from 0–5, as presented in Table 1.…”