Journal of Visualized Experiments www.jove.com Copyright © 2019 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License January 2019 | 143 | e59074 | Page 1 of 9 Video Article Probiotic Studies in Neonatal Mice Using Gavage Freddy Francis 1 , Natallia Varankovich 2 , Byron Brook 1 , Nelly Amenyogbe 1 , Rym Ben-Othman 2 , Bing Cai 2 , Danny Harbeson 1 , Aaron C. Liu 1 , Ben Dai 2 , Shelly McErlane 4 , Kris Andrews 4 , Tobias R. Kollmann* 1,2 , Pinaki Panigrahi* 3 1 Department of Experimental Medicine, University of British Columbia 2 Department of Pediatrics, University of British Columbia 3 Department of Epidemiology and Pediatrics, University of Nebraska Medical Centre 4 Animal Care Services, University of British Columbia * These authors contributed equally Correspondence to: Freddy Francis at fredfrancis@me.com URL: https://www.jove.com/video/59074 DOI: doi:10.3791/59074 Keywords: Immunology and Infection, Issue 143, Probiotics, Prebiotics, Gavage, Neonate, Microbiome, Prophylaxis Date Published: 1/27/2019 Citation: Francis, F., Varankovich, N., Brook, B., Amenyogbe, N., Ben-Othman, R., Cai, B., Harbeson, D., Liu, A.C., Dai, B., McErlane, S., Andrews, K., Kollmann, T.R., Panigrahi, P. Probiotic Studies in Neonatal Mice Using Gavage. J. Vis. Exp. (143), e59074, doi:10.3791/59074 (2019). Abstract 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. Video Link The video component of this article can be found at https://www.jove.com/video/59074/ Introduction In infants, early probiotic exposure has been associated with immunomodulatory effects leading to reduced incidence of diseases like necrotizing enterocolitis, atopic dermatitis and sepsis 1,2,3,4,5 . However, the mechanism behind this immunomodulatory response is challenging to explore given the limitation to sampling in newborn human trials (i.e., sequential blood draws and biopsies). Neonatal mouse models can help study the mechanism of action involved in neonatal immune regulation associated with probiotic use and changes in the intestinal microbiota. Unfortunately, most mouse models for probiotics have largely focused on adult mice; however, the impact of probiotics is likely to be highest early in life, suggesting models specific for this age group will be useful 3,6 . In addition, neonatal mouse models are better suited to study diseases and interventions intended for application in early life of human infants as they are expected to more closely mimic a developing immune and microbial system 7,8,9,10 . The aim was to study the extent and patterns of probiotic colonization of neonatal mice with a focus on the mechanistic interaction between the host and its microbiome. Suitable descriptions of newborn models were not found in the literature, and thus a need for the development of robust and standardized method was addressed. Established methods of oral administration of various compounds to newborn mice include maternal transfer of desired compounds through milk by treating the water source for pregnant dams 11 or using feeding needles to facilitate administration of desired compounds into the oropharynx 12 . These methods are useful for experiments that do not have precise dosage requirements and where the treatment is readily ingested by the recipient mouse. Probiotics are often administered in conjunction with a prebiotic such as galactooligosaccharide and fructooligosaccharide (FOS) that serve as a source of nutrition for probiotic bacteria; these additive compounds make the solution viscous and challenging to administer via the above-mentioned methodologies. Devising a method to administer precise amounts of probiotics and prebiotics to newborn mice starting as early as the first day of life (DOL) was necessary. In the process of developing the gavage technique, the possibility of colonization-spread (as observed in other probiotic studies between the treatment and the control arms 13,14,15,16 ) was tested and the relative abundance of colonized Lactobacillus plantarum (LP) in the intestines of pups with different gavage schedules was assessed. The probiotic