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.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
02 2023
02 2023
Historique:
received:
23
11
2020
accepted:
26
07
2022
pubmed:
10
11
2022
medline:
14
2
2023
entrez:
9
11
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 36352124
doi: 10.1038/s41596-022-00767-7
pii: 10.1038/s41596-022-00767-7
doi:
Types de publication
Journal Article
Review
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
490-529Subventions
Organisme : NCI NIH HHS
ID : U01 CA170930
Pays : United States
Informations de copyright
© 2022. Springer Nature Limited.
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