Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Summary

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

Abstract

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.^1-4

Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful⁵. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.

In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by ¹H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).⁶ The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.⁷

Hepatic metabolic profiling is measured directly from an intact liver biopsy using ¹H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism^7-10. This method can also be applied to any tissue biopsy^11,12.

Protocol

1. Colonization of germ-free animals and sample collection

1. Remove germ-free animals from isolators and house them in a conventional husbandry room in cages equipped with filter in front of the conventional animals which will serve as controls (Figure 1).
2. Mix half of the litter (3 days old) taken from the control conventional cage with the litter of the germ-free animals. Always keep 1/3 of the dirty conventional litter each time it is necessary to renew it in order to maintain a level of bacteria (keep it at least for 3 days).
3. Collect urine in a 1.5 mL microtube by handling the mouse over the tube and help micturition by gently ....

Discussion

In this protocol, we described a progressive colonization procedure in an open environment to further investigate the impact of gut microbiota on hepatic metabolism assessed by ¹H HR MAS NMR profiling of intact biopsy. Various methods of colonization have been described in the literature. The most common methods to colonize animals with a defined microbiota are oral gavage or contaminated drinking water^19,20. Fecal inoculation can also be used as previously described²¹. The colonization m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table of specific reagents and equipment:
2.5 mm microtube	New Era	NE-H5/2.5-V-Br
1.7 mm capillary tube	Sigma-Aldrich	NORS175001
Capillary adapter	New Era	NE-325-5/1.7
Extraction rod	New Era	NE-341-5
HR-MAS rotor BL4 with 50 μL spherical Teflon spacer kit	Bruker Corporation	HZ07213
Tool kit for 50 μL inserts	Bruker Corporation	B2950
Advance III 600 MHz NMR	Bruker Corporation
1H HR MAS NMR solid probe	Bruker Corporation
Deuterium oxide 99.9 %	Sigma-Aldrich	530867-1L
3-(trimethylsilyl)propionic acid-d4 (TSP)	Sigma-Aldrich	269913