Summary

Оценка изменения Печеночная Метаболический Во Прогрессивная Колонизация зародыш без мыши по 1 Н-ЯМР-спектроскопии

Published: December 15, 2011
doi:

Summary

Прогрессивная процедура колонизации описывается для дальнейшей оценки его влияния на метаболизм печеночных хоста. Колонизация контролируется, не invasively путем оценки экскреции микробных метаболитов совместного с помощью ЯМР основе метаболических профилей в то время как печеночный метаболизм оценивается Высокое разрешение магического угла Спиннинг (HR MAS) ЯМР профилирования интактных биопсии.

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 metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Protocol

1. Колонизация неинфицированных животных и отбора образцов Удалить неинфицированных животных из изоляторов и их размещение в обычной комнате хозяйства в клетках, оборудованных фильтром перед обычных животных, которые будут служить в качестве контроля (рис. 1). Смешайте поло…

Discussion

В этом протоколе, мы описали прогрессивные процедуры колонизации в открытую среду для дальнейшего изучения воздействия микрофлоры кишечника на печеночный метаболизм оценивается 1 H HR MAS ЯМР профилирования интактных биопсии. Различные методы колонизации были описаны в литератур…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Все спектров ЯМР используется в качестве иллюстративных примеров взяты из ранее опубликованных исследований 7, которая выполнена при финансовой поддержке компании Nestle.

Materials

Table of specific reagents and equipment:

Name of the reagent Company Catalogue number Comments
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 HZ07213
Tool kit for 50 μL inserts Bruker B2950
Advance III 600 MHz NMR Bruker
1H HR MAS NMR solid probe Bruker
Deuterium oxide 99.9 % Sigma-Aldrich 530867-1L
3-(trimethylsilyl)propionic
acid-d4 (TSP)
Sigma-Aldrich 269913

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Cite This Article
Heath, P., Claus, S. P. Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy. J. Vis. Exp. (58), e3642, doi:10.3791/3642 (2011).

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