Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Summary

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

Abstract

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Introduction

The gastrointestinal (GI) tract is a complex organ system given its multiple functions, host cell types (e.g. epithelial, immune, stromal, etc.) as well as trillions of microbes. In light of this complexity, diseases of the GI tract often involve the interplay of all of these factors. For example, inflammatory bowel diseases (IBD) are associated with cycles of inflammation and remission in the GI tract, involving the activation of inflammatory cells, dysbiosis, and epithelial repair^1,2,3,4,5,

Protocol

All procedures described here were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medicine.” To: “All procedures described here were approved by the Institutional Animal Care and Use Committees of Weill Cornell Medicine and Stony Brook University.

1. Colonoscopy and wound induction

1. Pre-assemble the components of the endoscope by first inserting the 1.9 mm rigid bore endoscope into the sheath (Figure 1A-B). Attach the air pump (to provide colonic insufflation) using the tubing provided, to the gas valve on the left side of the sheath next to ....

Results

The small items (lens, sheath, biopsy forceps) needed to carry out biopsies are shown in Figure 1 along with indicators of proper assembly of these components. Figure 2 shows representative images of acceptable views of the wound bed in order to accurately quantify the size of the wound bed and closure rate of the wound. An example of an ex vivo view of the wound bed is shown in Figure 3A inclusive of indicators of the perimeter of .......

Discussion

Ensuring consistent and accurate biopsies as well as measurements of wound size are of paramount importance when attempting to effectively evaluate the rate of wound closure in this model. Therefore, several measures should be taken to be confident that the procedures are being correctly carried out. First, the depth of the biopsy must not be too shallow or deep. If too shallow, there will not be a sufficient window to evaluate wound closure. Figure 2 demonstrates an optimal biopsy depth and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biopsy forceps, 3 Fr	Karl Storz	61071ZJ
Coloview Tower system	Karl Storz	contact company
Examination sheath, 9 Fr, Kit	Karl Storz	61029DK
Hopkins telescope, 0', 1.9 mm x 10 cm	Karl Storz	64301AA
isofluorane	Covetrus	2905
methylene blue	Sigma-Aldrich	M9140
micro iris scissors	Integra	18-1619
NIH ImageJ	NIH	N/A	software available for free download from: https://imagej.nih.gov/ij/
Pawfly MA-60 aquarium pump	Amazon	N/A
scalpal with #10 blade	Hill-Rom	372610