Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Summary

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Abstract

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Introduction

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to disease or surgery^1,2. After trauma, the gut undergoes a morphometric and functional adaptive response, characterized by crypt cell proliferation and increased nutrient absorption³. This step is critical in recovery, yet poorly understood. Experimental studies of the intestinal adaptive response have focused on the changes occurring after small bowel resection in mice, rats, and pigs, but understanding the molecular mechanism behind the adaptive response in other kinds of injuries (e.g.....

Protocol

All methods described were conducted in accordance with the guidelines of Danish legislation governing animal experimentation (1987). Studies were performed with the permission from the Danish Animal Experiments Inspectorate (2013-15-2934-00833) and the local ethical committee.

NOTE: Female C57BL/6J mice (~20−25 g) were obtained and housed eight per cage in standard 12 h light, 12 h dark cycle with free access to water and standard chow. Animals were left to acclimatize for one week before experiments began.

1. Induction of mucositis using 5-fluorouracil

1. Obtain 5-fluorouracil (5-FU) in a 50 mg/m....

Results

In the first experiment, we induced mucositis in mice at day 0 and sacrificed a group of mice each day for 5 consecutive days. When measuring the SI weight, we found that this parameter decreased from day 2 until day 4 suggesting a loss in the enterocyte mass. We also found that at day 5, the SI weight was not significantly different from day 0 (untreated mice) (Figure 1). The proliferation measured by the incorporation of BrdU was almost abolished at day 1 a.......

Discussion

Here, we demonstrate a widely accessible method to study SI injury and regeneration in a mouse model. A wide variety of preclinical animal models of intestinal injury exist, but it is vital we understand that each model is unique and that the endpoints must be appropriate to answer the research question. This model is excellent to study adaptive response to injury, but the endpoints should be modified when using the model as a pre-clinical model of mucositis. However, translation from animal models to patients is challen.......

Materials

Name	Company	Catalog Number	Comments
5-Fluorouracil	Hospira Nordic AB, Sweden	137853
Ketaminol®Vet	Merck, New Jersey, USA	511485
Rompun®Vet Xylazine	Rompunvet, Bayer, Leverkusen, Germany.	148999
10% nautral formalin buffer	Cell Path Ltd, Powys, United Kingdom	BAF-5000-08A
HistoClear	National Diagnostics, United Kingdom	HS-200
Pertex	HistoLab®, Sweden	840
BrdU	Sigma-Aldrich, Germany.	B5002
Tris/EDTA pH 9 buffer	Thermofisher scientific, Denmark	TA-125-PM4X
Peroxide Block	Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark	TL-060-QHDM
Rodent Block buffer	Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark	TL-060-QHDM
Monoclonal mouse anti-BrdU antibody	Thermofisher Scientific, Denmark.	MA1-81890
Lab Vision Antibody Diluent OP Quanto	Thermofisher Scientific, Denmark.	TA-125-ADQ
Horseradish peroxidase	Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark	TL-060-QHDM
DAB Quanto Substrate	DAB Substrate Kit, Thermofisher Scientific, Denmark	TA-125-QHDX
DAB Quanto Chromogen	DAB Substrate Kit, Thermofisher Scientific, Denmark	TA-125-QHDX
Zen Lite Software (Blue edition)	Carl Zeiss A/S		https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html
ImageJ Software	LOCI, University of Wisconsin		https://imagej.nih.gov/ij/