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.
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.
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 repair1,2,3,4,5,6,7. Having appropriate model systems to study IBD and other inflammatory conditions of the GI tract is critical for elucidating the pathogenesis of disease. Several models exist to study IBD pathogenesis including genetically engineered mice and the use of chemicals such as dextran sodium sulfate (DSS) in rodents8,9,10. Limitations of these models include an inability to precisely control the induction of inflammation as well as difficulties in evaluating wound healing. Alternative methods to mimic aspects of IBD pathogenesis could prove useful for developing therapies.
Colonoscopic-guided pinch biopsies in mice offer a useful model system to study the pathogenesis of the inflammatory response, wound healing, as well as host-microbe interactions in the colon. This approach was first used as an experimental tool in 2009, which demonstrated its utility for studying the acute inflammatory response and wound healing in the gut11. Subsequent studies utilized this technique to evaluate the roles of different signaling pathways as well as the gut microbiota, in colonic wound healing11,12,13,14,15,16,17,18. More recently, our group used this model to investigate the importance of sphingosine-1-phosphate signaling and bacteria in the acute response to colonic injury19. Although useful, carrying out colonoscopic-guided pinch biopsies in mice and evaluating subsequent tissue changes can be technically challenging. For instance, perforation of the bowel can occur upon induction of injury and ensuring consistent measurements of the wound bed through serial colonoscopies can be difficult. Additionally, orienting the colonic tissue properly to visualize the wound bed for histological or immunohistochemical analyses can be challenging. Although some information does exist regarding these methods18,20, a precise step-wise description of these techniques along will visual aids promises to enhance the reliability and broader utility of this model. Here, we present a detailed method to carry out colonoscopic-guided pinch biopsies in mice, track wound closure over time and prepare the tissue to enable histologic and molecular analyses of the wound bed. Creating a standard method to carry out these techniques can expand the use of this model to study previously uninvestigated mediators that are potentially important for GI inflammation and wound repair.
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
2. Visualizing and measuring the wound bed
3. Collection of the wound bed for molecular analysis
4. Preparation of tissue for histological analysis
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 the wound bed (indicating the region to excise for molecular analysis) and where to cut the tissue in order to enable visualization of the wound bed upon sectioning. Figure 3B shows a representative image of an H&E-stained section in which the wound bed can be clearly observed. Supplemental Video 1 provides a view of the biopsy procedure from inside the mouse’s colon.
Figure 1: Items needed to carry out biopsies. (A) Image of lens (a), sheath (b) and biopsy forceps (c). (B) Insertion of the lens into the sheath. (C) Insertion of forceps through the working channel of the sheath (solid arrow). Dashed arrow indicates correct location for attachment of air pump. Please click here to view a larger version of this figure.
Figure 2: Colonoscopic images of wound bed following biopsy. Still images were created from video recordings of colonoscopies immediately after biopsy (Day 0) and 2, 4, and 6 days later. Blue lines indicate the edges of the wound beds at each time point. The arrow indicates the correct length of extension of the forceps into the lumen to ensure proper distance from the lens to the rectal wall, in order to ensure consistent measurements of the wound bed over time. Please click here to view a larger version of this figure.
Figure 3: Ex vivo and sectioned images of wound bed. (A) A colon was harvested from a mouse 2 days following biopsy, stained with 0.2% methylene blue and imaged under a dissecting microscope. The dashed circle indicates the edges of the wound bed. The black line indicates the proper location to bisect the wound bed/colon prior to embedding for sectioning. (B) Representative image of an H&E-stained section of a wound bed. Asterisks indicate wound bed and arrows indicate intact crypts adjacent to wound bed indicating the borders of the injured region. Please click here to view a larger version of this figure.
Supplemental Video 1: Biopsy procedure and wound bed imaging. Please click here to download this video.
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 size on day 0. Note the clear distinction between the mucosa around the wound bed and the tissue that remains beneath the wound bed (Figure 2). If the biopsy is too deep, perforation can occur thus resulting in an unevaluable mouse. Upon insufflation after biopsy on day 0, if the mouse’s abdomen becomes severely distended, perforation has occurred and the mouse cannot be used for evaluation and should be euthanized. It is helpful if the same individual carries out the biopsies for a given study to further ensure consistency. Second, it is critical to carry out the biopsy in the rectum and not in a more proximal region. Given that the rectum is thicker than more proximal regions of the colon, there is a markedly reduced chance of perforation. A mark should be made on the outside of the sheath 0.5 cm from the end and the endoscope should only be inserted up until that point to ensure that the biopsy forceps will not extend beyond the rectum. Third, having the right amount of insufflation both during the biopsy and for visualizing the wound bed is essential. During biopsy, if the colon is too distended the colonic wall will be too taut and there will not be a sufficient amount of mucosa to biopsy. Therefore, it is suggested that the individual operating the endoscope not press their index finger against the open end of the gas valve, in order to allow for minimal air flow into the colon. However, the opposite approach should be used when visualizing wound beds for the purposes of measuring wound size. Once the wound is located, fully insufflate the colon by pressing the index finger firmly against the open end of the gas valve and hold it there until a desired view is obtained. The colonic wall should be as taut as possible for this purpose. Of note, full insufflation of the colon is important for also ensuring consistent lateral views of the wound bed. Lastly, it is critical to ensure that a consistent distance is maintained between the lens and the wound to enable accurate measurements over multiple colonoscopies in the same mice. A closer distance will artificially make the wound bed appear larger than it is and a farther distance will make it appear smaller. Therefore, using the biopsy forceps as a guide to maintaining distance is very helpful.
In addition to the major considerations to take into account when using this model, there are more minor points to be aware of that can also impact the ability to perform this procedure effectively. For example, it should be made certain that the colonic lumen is clear when performing colonoscopies. Although fecal material is cleared by flushing with PBS, additional material can descend into the rectum after inserting the endoscope. Moreover, when visualizing the wound bed immediately after biopsy, blood can obscure the field. Therefore, it is sometimes necessary to remove the endoscope from the rectum, flush the colon with PBS to clear the luminal blood and reinsert the endoscope to visualize the wound bed. In certain cases, blood and other luminal content can become adhered to the lens, obscuring the field. In these cases, the endoscope should be removed from the mouse and the lens should be wiped using the brush before continuing imaging.
Prior to the advent of the colonoscopic-guided pinch biopsy model, researchers had limited model systems to investigate colonic wound healing. One approach was to evaluate the recovery from exposure to chemical inducers of colonic injury such as DSS8. However, this approach does not allow for precise control of the extent of injury being induced nor the location of the injury throughout the colon. Moreover, precise measurements of mucosal healing in real-time can be challenging with these chemical models. Although useful, the biopsy model also has limitations. For instance, operators are limited to carrying out wounding in the distal colon. This limitation prevents studies of small intestinal ulceration, an important clinical issue. Additionally, although this technique recapitulates some aspects of IBD pathogenesis, it cannot be considered a true model of this disease. Of technical consideration, it can be challenging to induce consistent wound sizes across mice, generate evaluable wounds, or locate wound beds at the later stages of the wound healing process. When taking these points into consideration, it is advisable to begin studies with additional mice to account for the loss of unevaluable samples.
The authors have nothing to disclose.
This work was supported by grants from Crohn’s and Colitis Foundation (D.C.M) and New York Crohn’s Foundation (D.C.M. and A.J.D.). The authors thank Ms. Carmen Ferrara for assistance with creating the video accompaniment to this article.
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 |