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.
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.
Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to disease or surgery1,2. 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., chemical or bacterial) is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Experimentally, different approaches have been used to describe the complex molecular and cellular index of small intestinal pathology, including histopathological scoring and measuring the outcome of injury. Despite this, what is absent from the literature is a detailed description of how to perform the procedures that are needed to obtain reproducible data. When identifying factors involved in adaptation, such as gut hormones, an easy, low cost, and reproducible animal model is warranted and here we suggest using a model of chemotherapy-induced intestinal mucositis (CIM).
One of the simplest and very informative endpoints of both injury and adaptation is to measure the mass of the small intestine (SI). We know that a hallmark of mucositis is apoptosis of enterocytes, time-dependent villus atrophy and reduced mitosis. Therefore, examining intestinal morphology is highly relevant in preclinical models4,5. In humans, a decline in plasma citrulline, a marker of functioning enterocytes, correlates with toxicity scores and inflammatory markers6 in addition to the absorptive capacity7, suggesting this amino acid is an excellent biomarker of mucositis. Citrulline can be measured in both mice and rats, and has shown excellent correlations with villus length8, crypt survival9, and radiation-induced mucositis10.
A major advantage of measuring plasma citrulline is the ability to collect repeated measurements from one animal. However, multiple blood sampling in mice is restricted to a total blood volume of 6 µL/g/week and requires general anaesthesia. This unfortunately also limits the use of citrulline measurements in mice. Furthermore, the measurement of citrulline requires high-performance liquid chromatography11,12, which is costly and time-consuming. Recently, we showed that citrulline levels in mice correlate significantly with SI weight (p < 0.001) (unpublished data), making citrulline a direct measurement reflecting enterocyte mass. A limitation to the measurement of SI weight is the necessity for the mice to be sacrificed and thus no repeated measurements within the same mouse are possible. Still the method provides the possibility to perform a variety of other tissue analyses directed to the research question, and these facts can conceivably make up for the additional use of animals. We, therefore, suggest using SI weight as an easy, low-cost, and fast biomarker of injury and adaptation in mice. To ensure reproducibility and acceptable analytic variation, the intestines should be carefully removed from the animal, flushed with saline, emptied and dried before weighing. In this article, we show exactly how this procedure is performed.
Another hallmark of mucositis is the loss of the proliferating cells in the crypts and a compensatory hyperproliferation during the regenerative period3. The cellular marker Ki67 has been frequently used to determine fast proliferative cells by means of immunohistochemistry13. Even though Ki67 is a simple marker of proliferation, it has a tendency for imprecision as Ki67 is present during all active phases of the cell cycle (G1, S, G2, and M)14. Specific labelling is essential to detect replicating cells, which is why we suggest in situ incorporation of 5-bromo-2'-deoxyuridine (BrdU), a synthetic analogue of thymidine, as it is largely restricted to replicating cells in the S-phase15. BrdU is injected in the animals 150 minutes before sacrificing and cells can be subsequently detected with immunohistochemistry using BrdU specific antibodies. In this method article, we show exactly how to measure the area of BrdU immunopositive cells within a crypt using a free image software.
Morphologic and functional changes are often studied in 5-FU induced mucositis models, where the intestinal adaptation is assessed by villus height and crypt depth. During this study, we found that during the acute phase of mucositis, which is equal to the injury phase, proliferation measured by BrdU incorporation is not correlated with crypt depth. In contrast to this, crypt depth is significantly correlated with proliferation seen in the repair phase of mucositis, 3 to 5 days after induction. This suggests that the acute phase of mucositis is not measurable by crypt depth alone. We suggest that when using proliferation as an endpoint in the acute phase of mucositis mice, BrdU incorporation should preferably be used but when quantitating hyperproliferation in the later stage during the regenerative phase, crypt depth is a reasonable alternative to BrdU incorporation. The goal of this study was to describe this model in a way that it can be used by all researchers, both in the field of oncology but especially researchers not familiar with intestinal injury models.
The described model can be used to phenotype transgenic models according to the adaptive response using body weight, SI weight and crypt depth as endpoints. As an example, we show here how we used the model of 5-fluorouracil (5-FU) induced mucositis in a cellular knock out model with insufficient L-cell secretion16. Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are intestinal hormones co-secreted from the enteroendocrine L-cells in response to food intake17,18. GLP-2 is recognized as an important factor for intestinal healing, the regulation of mucosal apoptosis and the improvement of the barrier function of the SI19,20,21,22. Based on the literature, we hypothesized that endogenous hormones are essential for compensatory hyperproliferation occurring in the adaptive response after injury.
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
2. Tissue collection
3. Small intestine histology
4. Measurement of crypt depth and/or villus height
5. BrdU quantification (proliferation) by immunohistochemistry
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 and day 2, but at day 4 and day 5 there were approximately four-fold and five-fold increases in proliferation, respectively (Figure 2). This hyperproliferation was also illustrated when measuring the crypt depth (Figure 3). What is not illustrated by measuring crypt depth is the loss in proliferating cells at day 1 and day 2, where crypt depth was reduced by approximately 13% but was not significantly different from the healthy mice. We could show that, in the regenerative phase of mucositis there was a strong correlation between BrdU incorporation and crypt depth, but this did not count for the acute phase indicating that crypt depth as an endpoint might not be suitable in the acute phase of mucositis (Figure 4).
In the second study, we examined mucostis in our transgenic mouse model with insufficient L cell secretion. Mice with deficient GLP-1 and GLP-2 showed a severe loss of body weight (BW) and a decrease in SI weight in the recovery phase, which was highly significant compared to the wild type (WT) 5-FU mice (p < 0.01) (Figure 5A,B). Furthermore, the transgenic mice failed to show compensatory hyperproliferation; crypts were significantly shorter than in both WT mice and healthy controls. Contrary to this the WT mice showed an increase in crypt depth as a sign of hyperproliferation (Figure 5C).
Figure 1: Small intestinal weight. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and the intestinal weight was measured as described. Results are shown as mean ± standard error of the mean (SEM). n = 13. This figure has been modified from Hytting-Andreasen et al.16. Please click here to view a larger version of this figure.
Figure 2: BrdU immunopositive cells per crypt for the duodenum, jejunum, and ileum of the SI. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and BrdU incorporation was quantified by immunohistochemistry as described. Results are shown as mean ± SEM. n = 13. *p < 0.05, ***p < 0.001 compared to day 0 (analysis of variance [ANOVA] followed by Dunnett multiple comparison test). ***p < 0.001 compared to day 0 (ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.16. Please click here to view a larger version of this figure.
Figure 3: Measurement of crypt depth. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and crypt depth was measured as described. Results are shown as mean ± SEM. n = 13. *p < 0.05, ***p < 0.001 compared to day 0 (ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.16. Please click here to view a larger version of this figure.
Figure 4: Correlation of crypt depth and BrdU. Crypt depth (in duodenum, jejunum, and ileum) is correlated to BrdU incorporation at each day of sacrifice using two-tailed Pearson correlation test. Please click here to view a larger version of this figure.
Figure 5: GLP-1 and GLP-2 deficient mice fail to regenerate after acute mucositis. (A) Change in BW (%), (B) SI weight (g), and (C) crypt depth in ileum (µm). Results are shown as mean ± SEM. Tg = transgenic mice; WT = wild type mice; 5-FU = 5-fluorouracil. n = 4−8. *p < 0.05, **p < 0.01, ***p < 0.001 compared to healthy control (WT saline), a = p < 0.05, aa = p < 0.01, aaa = p < 0.001 compared to WT 5-FU (two-way ANOVA followed by Bonferroni multiple comparison test [BW] or ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.16. Please click here to view a larger version of this figure.
Supplementary Figure 1: Small intestinal tissue before the induction of mucositis, during the acute phase and the recovery phase of mucositis. (A) Haemotoxylin and eosin (H&E) staining and (D) BrdU staining in untreated mice. The black dotted line in panel A exemplifies a well-orientated crypt, whereas the dotted green line demonstrates a well-orientated villus. (B) H&E staining and (E) BrdU staining during the acute phase of mucositis. (C) H&E staining and (F) BrdU staining during the recovery phase after the induction of mucositis. Scale bar = 100 μm. Please click here to download this file.
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 challenging23. Our suggested endpoints of SI weight and proliferation should be limited to the study of adaptive response only. The study of endogenous factors often requires the use of transgenic mice, and even if small bowel resection is possible in mice1, this model could be an alternative to avoid post-operative mortality. When applying this model to transgenic mice, it is important to watch the mice carefully and monitor their weight every day. During this study, some of the mice experienced a weight loss of up to 30%, which is quite substantial. To avoid high mortality in sensitive phenotypes, we suggest performing pilot studies in transgenic mice, since dose adjusting might be necessary.
A critical step within the described method is the removal and weighing of the SI. It is important that the removal and handling be performed in the same manner and by the same researcher each time to avoid large inter-assay variations.
The consistency of crypt and villus selection is important to avoid variance and bias when measuring crypt depth and villus height. When embedding the tissue in paraffin, the intestines are positioned in an upright position to make transverse cuts, thus increasing the possibility for intact villi and crypts. After cutting of the tissue, a well-orientated crypt and/or villus is selected. Selection is based on the full visualization of the whole crypt and villus in the same plane and the presence of clear borders of cells within the crypt and villus. A limitation to this method is the somewhat subjective approach when selecting a well-orientated crypt since the selection of a well-orientated crypt and/or villi is made after cutting the tissue. A previous study24 has presented an alternative method to overcome this limitation, where they use microdissection. In this method, villi and crypts are selected while observing under a microscope, prior to the tissue being cut, thus making it possible to ensure that an intact crypt and villi are being dissected from the tissue.
In contrast to previous methods used to quantity BrdU positive cells25,26, this protocol describes the area of BrdU positive cells per crypt, which provides a fast way to quantitate proliferative cells within each crypt. This technique, however, may be somewhat restrictive since it requires a more profound knowledge of the software suggested for the measurement of this technique. A future application of this protocol could be to create a more automatic generated method to quantify and measure the BrdU positive cells.
The authors have nothing to disclose.
This work was supported by an unrestricted grant from the Novo Nordisk Center for Basic Metabolic Research and the Lundbeck Foundation.
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/ |