Abstract
Interest in the communication between the gastrointestinal tract and central nervous system, known as the gut–brain axis, has prompted the development of quantitative analytical platforms to analyze microbe- and host-derived signals. This protocol enables investigations into connections between microbial colonization and intestinal and brain neurotransmitters and contains strategies for the comprehensive evaluation of metabolites in in vitro (organoids) and in vivo mouse model systems. Here we present an optimized workflow that includes procedures for preparing these gut–brain axis model systems: (stage 1) growth of microbes in defined media; (stage 2) microinjection of intestinal organoids; and (stage 3) generation of animal models including germ-free (no microbes), specific-pathogen-free (complete gut microbiota) and specific-pathogen-free re-conventionalized (germ-free mice associated with a complete gut microbiota from a specific-pathogen-free mouse), and Bifidobacterium dentium and Bacteroides ovatus mono-associated mice (germ-free mice colonized with a single gut microbe). We describe targeted liquid chromatography–tandem mass spectrometry-based metabolomics methods for analyzing microbially derived short-chain fatty acids and neurotransmitters from these samples. Unlike other protocols that commonly examine only stool samples, this protocol includes bacterial cultures, organoid cultures and in vivo samples, in addition to monitoring the metabolite content of stool samples. The incorporation of three experimental models (microbes, organoids and animals) enhances the impact of this protocol. The protocol requires 3 weeks of murine colonization with microbes and ~1–2 weeks for liquid chromatography–tandem mass spectrometry-based instrumental and quantitative analysis, and sample post-processing and normalization.
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Acknowledgements
This study was supported by an NIH K01 K12319501 grant and Global Probiotic Council 2019-19319 (M.A.E.). This work was also supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant P30-DK-56338 to Texas Medical Center Digestive Disease Center, Gastrointestinal Experimental Model Systems), NIH U01CA170930 grant (J.V.) and unrestricted research support from BioGaia AB (Stockholm, Sweden) (J.K.S. and J.V.). The Texas Children’s Hospital Department of Pathology and Immunology provides salary support to Texas Children’s Microbiome Center-Metabolomics and Proteomics Mass Spectrometry Laboratory staff, and purchased all of the reagents, the consumables and durable supplies, and the LC–MS/MS equipment described. The authors thank K. Prince (Pathology at Texas Children’s Hospital) for her assistance with the graphics contained in this manuscript.
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Concept and design (T.D.H., S.J.H., M.A.E. and A.M.H.); intellectual contribution (T.D.H., S.J.H., M.A.E., N.O., J.K.S. and A.M.H.); data acquisition (T.D.H., S.J.H., M.A.E., F.I., W.R., B.L., N.O. and J.K.S.); data analysis and interpretation (T.D.H., S.J.H., M.A.E., M.B., F.I., W.R., B.L., K.M.H., N.O., J.K.S. and A.M.H.); drafting manuscript (T.D.H., S.J.H. and M.A.E.), editing manuscript (T.D.H., S.J.H., M.A.E., M.B., F.I., W.R., B.L., K.M.H., N.O., J.K.S., J.V. and A.M.H.); funding (M.A.E., J.V. and A.M.H.).
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J.V. and J.K.S. receive unrestricted research support from BioGaia AB, a Swedish Probiotic Company. J.K.S. is a consultant for Pastuer21, Inc. J.V. also serves on the scientific advisory boards of Biomica, an Israeli informatics enterprise, Seed, a United States-based probiotics/prebiotics company, and Plexus Worldwide, a United States-based nutrition company. T.D.H. sits on the editorial advisory board and serves as an associate editor with the STAR Protocols journal (Cell Press). All remaining authors have no financial relationships to disclose.
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Engevik, M. A. et al. Cell Mol. Gastroenterol. Hepatol. 11, 221–248 (2020): https://doi.org/10.1016/j.jcmgh.2020.08.002
Horvath, T. D. et al. iScience 25, 104158 (2022): https://doi.org/10.1016/j.isci.2022.104158
Luck, B. et al. Biomolecules 11, 1091 (2021): https://doi.org/10.3390/biom11081091
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Horvath, T.D., Haidacher, S.J., Engevik, M.A. et al. Interrogation of the mammalian gut–brain axis using LC–MS/MS-based targeted metabolomics with in vitro bacterial and organoid cultures and in vivo gnotobiotic mouse models. Nat Protoc 18, 490–529 (2023). https://doi.org/10.1038/s41596-022-00767-7
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DOI: https://doi.org/10.1038/s41596-022-00767-7
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