Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Summary

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Abstract

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Introduction

Animal models have greatly enriched our understanding of numerous intestinal pathologies. The laboratory mouse (Mus musculus) has emerged as a prime animal model in biomedical research due to its abundant genetic and genomic information and is readily available in transgenic and knockout strains. In addition to enhancing understanding disease pathogenesis, animal models are also importantly used for testing drug candidates as well as preclinical diagnostic or therapeutic interventions. However, despite the variety of mouse models mimicking human disease, many diagnostic and interventional options that are routinely used in patient care are not available for m....

Protocol

All animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) according to German Animal Protection Law.

1. Materials and Experimental Setup

1. Animal care
   1. Use female or male mice of any strain weighing 20 to 25 g and house them according to local animal care legislation.
   2. Feed mice with special chow for rodents and apply alfalfa-free chow at least three days prior to fluorescence examinations to minimize endoluminal auto-fluorescence.
   3. Provide autoclaved drinking water ad libitum.
2. Induction of acute DSS-induced colitis

Results

In vivo monitoring of intestinal wound healing
During routine endoscopy, mucosal wounds were induced mechanically by miniature biopsy forceps with a diameter of 3 French (equals 1 mm; Figure 1A). Subsequently, wound healing was monitored by daily endoscopic examinations and quantified by measurement of the residual wound area using image editing software, e.g., ImageJ (Figure 1B). The individual wound closure over time is expressed .......

Discussion

Epithelial wound healing is an ongoing process. Continuous physiological exfoliation of surface cells within the gastrointestinal mucosa occurs requiring frequent regeneration of epithelial cells ¹⁶. Consequently, impaired wound healing has an immense impact on several diseases including gastrointestinal ulcers and ¹⁷ anastomotic leakage ¹⁸. Evaluation of molecular background as well as potential drug candidates to stimulate epithelial healing may only be incompletely performed in cell cu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Alfalfa-free diet	Harlan Laboritories, Madison, USA	2014
Azoxymethane (AOM)	Sigma-Aldrich, Deisenhofen, Germany	A5486
Bepanthen eye ointment	Bayer, Leverkusen, Germany	80469764
Dextran sulphate sodium (DSS)	TdB Consulatancy, Uppsala, Sweden	DB001
Eosin	Sigma-Aldrich, Deisenhofen, Germany	E 4382
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich, Deisenhofen, Germany	E 9884
Falcon Tube 50 ml	BD Biosciences, Erembodegem, Belgium	352070
Florene 100 V/V	Abbott, Wiesbaden, Germany	B506
Haematoxylin	Sigma-Aldrich, Deisenhofen, Germany	HHS32-1L
Isopentane (2-Methylbutane)	Sigma-Aldrich, Deisenhofen, Germany	M32631-1L
Methylene blue	Merck, Darmstadt, Germany	1159430025
O.C.T. Tissue Tek compound	Sakura, Zoeterwonde, Netherlands	4583
Omnican F - canula	Braun, Melsungen, Germany	9161502
Phosphate buffered saline, PBS	Lonza, Verviers, Belgium	4629
Sodium Chloride 0.9%	Braun, Melsungen, Germany	5/12211095/0411
Standard diet	Altromin, Lage, Germany	1320
Tissue-Tek Cryomold	Sakura, Leiden, Netherlands	4566
Vitro – Clud	R. Langenbrinck, Teningen, Germany	04-0002
Equipment
AIDA Control	Karl Storz - Endoskope, Tuttlingen, Germany	20 096020
Bandpass filter	Semrock, Rochester, USA	HC 716/40
Bandpass filter	Semrock, Rochester, USA	HC 809/81
Biopsy Forceps, 3 Fr., 28 cm	Karl Storz - Endoskope, Tuttlingen, Germany	61071ZJ
Dell Monitor	Dell, Frankfurt am Main, Germany	U2412Mb
Examination Sheath, 9 Fr.	Karl Storz - Endoskope, Tuttlingen, Germany	61029D
Examination Sheath, 9 Fr.	Karl Storz - Endoskope, Tuttlingen, Germany	61029C
Fiber Optic Light Cable, 3.5 mm	Karl Storz - Endoskope, Tuttlingen, Germany	69495NL
Fluorescein Blue Filter System	Karl Storz - Endoskope, Tuttlingen, Germany	20100032
Fluorescein Barrier Filter	Karl Storz - Endoskope, Tuttlingen, Germany	20100033
Foot switch	Karl Storz - Endoskope, Tuttlingen, Germany	20010430
HOPKINS Telescope, 1.9 mm, Length 10 cm	Karl Storz - Endoskope, Tuttlingen, Germany	1830231
SCB D-light P	Karl Storz - Endoskope, Tuttlingen, Germany	20 133720
SCB tricam SL II	Karl Storz - Endoskope, Tuttlingen, Germany	20 2230 20
Tubing set instruments VETPUMP II	Karl Storz - Endoskope, Tuttlingen, Germany	69811
Tricam PDD PAL	Karl Storz - Endoskope, Tuttlingen, Germany	20221037
UniVet Porta	Groppler Medizintechnik, Deggendorf, Germany	BKGM 0451
Vetpump 2	Karl Storz - Endoskope, Tuttlingen, Germany	69321620