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Analysis of intestinal adaptation gene expression by cDNA expression arrays

JPEN: Journal of Parenteral and Enteral Nutrition, Nov/Dec 2000 by Erwin, Christopher R, Falcone, Richard A Jr, Stern, Lawrence E, Kemp, Christopher J, Warner, Brad W

ABSTRACT. Background: As a tool for determining gene expression on a genomic scale, cDNA microarrays are a promising new technology that can be applied to the study of complex physiologic processes. The objective of this study was to characterize the expression of individual genes and patterns of gene expression that might provide insight into the mechanism of intestinal adaptation after massive small bowel resection. Methods: Male ICR mice underwent a 50% proximal small bowel resection (SBR) or sham operation. After 3 days, the remnant ileum was harvested, weighed, and RNA extracted. Changes in gene expression were detected utilizing Clontech Atlas mouse cDNA expression arrays. Some of these changes were confirmed by reverse

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transcriptase-polymerase chain reactions (RT-PCR) and Northern blots. Results: Analysis of these cDNA arrays revealed changes in the expression of multiple genes, including those involved in cell cycle regulation, apoptosis, DNA synthesis, and transcriptional regulation. The patterns of expression were consistent with the increased cell proliferation and apoptosis observed during intestinal adaptation. A large number of genes not previously associated with intestinal adaptation were identified. Conclusions: This technology may facilitate the elucidation of the intricate cellular mechanisms underlying intestinal adaptation. (journal of Parenteral and Enteral Nutrition 24:311-316, 2000)

The loss of a significant length of small bowel is a major clinical problem that occurs in a wide spectrum of medical and surgical conditions. After massive small bowel resection (SBR), an important compensatory response to the acute loss of absorptive surface area occurs and is termed adaptation. This physiologic process ultimately results in an increase in small bowel length and diameter. The mechanisms and mediators of intestinal adaptation are not well understood but many factors including luminal nutrients, pancreaticobiliary secretions, and various hormones and growth factors probably play a role.1Understanding the pathophysiology of intestinal adaptation is critical in the development of therapeutic strategies aimed at augmenting this important response.

The molecular biologic investigation of the mechanisms governing intestinal adaptation previously has been performed on an individual gene basis. The small number of genes that have been and can be considered limits this approach.2,3 In addition, genes whose functions are not intuitively related to adaptation may not be accorded appropriate consideration. A global view of the cellular events that transpire during intestinal adaptation would be more useful.

With the advent of cDNA microarray technology, the study of gene expression on a genomic scale is now possible.4-6 Because of hardware and clone set require ments, large-scale cDNA microarray technology continues to be limited to only a few laboratories. A more accessible alternative is the cDNA filter array. Although filter arrays usually have a smaller clone set and are less sensitive than microchip arrays, they include only known genes, grouped by function, simplifying the interpretation of results. In addition, they are commercially available, relatively affordable, and require no specialized equipment.

In this study, mouse cDNA filter arrays were used to characterize the changes in gene expression that occur during intestinal adaptation. The observed patterns of gene expression may serve as the basis for future experiments that further define the regulatory mechanisms involved in this important physiologic process.

MATERIALS AND METHODS

The Children's Hospital Research Foundation Institutional Animal Care and Use Committee approved a protocol for this study.

Operative Procedure

ICR male mice at least 60 days of age and weighing 25 to 30 g were randomly assigned to receive either a 50% proximal SBR with reanastomosis or sham operation (transection of the bowel with reanastomosis only) as our laboratory has described previously in detail.7 Both sham and SBR reanastomosis are approximately 15 cm from the ileocecal junction.

Collection of Samples

Three days after the operative procedure, mice were killed with an intramuscular injection of ketamine: xylazire:acepromazine (4:1:1) followed by cervical dislocation. The abdominal cavity was opened and a 4-cm segment of ileum removed at a point 2-cm distal to the anastomosis. The isolated ileum was gently rolled with cotton swabs and flushed with cold (4 deg. C) normal saline to remove the luminal contents, blotted dry, weighed, quick frozen in liquid nitrogen, and stored at - 70 deg C for subsequent RNA extraction.

RIVA Preparation

The tissue (n = 6 each for sham and SBR) was homogenized and total RNA isolated using TRIzol reagent (GIBCO BRL, Gaithersburg, MD) with two additional phenol/chloroform extractions followed by DNase I treatment. Poly(A+) RNA (mRNA) was isolated from pooled total RNA with oligo(dT) cellulose columns (GIBCO BRL). For both total RNA and poly(A+) RNA, concentrations were determined spectrophotometrically at A^sub 260^ and all samples checked on agarose/formaldehyde gels.

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Analysis of intestinal adaptation gene expression by cDNA expression arrays

JPEN: Journal of Parenteral and Enteral Nutrition, Nov/Dec 2000 by Erwin, Christopher R, Falcone, Richard A Jr, Stern, Lawrence E, Kemp, Christopher J, Warner, Brad W

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