This work was supported by grants from Crohn&#8217;s and Colitis Foundation (D.C.M) and New York Crohn&#8217;s Foundation (D.C.M. and A.J.D.).&#160;The authors thank Ms. Carmen Ferrara for assistance with creating the video accompaniment to this article.

All procedures described here were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medicine.&#8221; To: &#8220;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
<ol>
	<li>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 the working channel (Figure 1C). 
	NOTE: Although the lens described here is 0&#176;, a 30&#176; lens can also be used for this purpose.</li>
	<li>Ensure that the working channel is in the open position and insert 3 Fr biopsy forceps through the working channel and advance it until the end of the sheath (while ensuring that it does not protrude out of the sheath) (Figure 1C). Attach the assembled endoscope to the light source and video imaging device per manufacturer&#8217;s instructions.</li>
	<li>&#160;Anesthetize the mouse with 5% isoflurane with oxygen in an induction chamber. Then move the mouse to an endoscopic staging platform containing a heating system (to prevent hypothermia) on its ventral side and maintain under anesthesia using a nose cone with 2% isoflurane with oxygen. Add vet ointment to the eyes to prevent dryness while under anesthesia. Ensure that the mouse is fully anesthetized by gently pinching the rear foot to test for a reflex. 
	NOTE: Any strain or either sex&#160;of mice can be used with this technique; however, it is preferable that mice are at least 8 weeks of age so they are large enough for the procedure.
	<ol>
		<li>Add vet ointment to the eyes to prevent dryness while under anesthesia.</li>
		<li>Ensure that the mouse is fully anesthetized by gently pinching the rear foot to test for a reflex.</li>
	</ol>
	</li>
	<li>Fill a 3 mL syringe with an attached rat gavage needle with room temperature phosphate-buffered saline (PBS). Insert needle approximately 1 cm into the mouse&#8217;s anus and gently infuse PBS to clear fecal material. Several fecal pellets should exit the mouse along with the PBS that was infused. 
	NOTE: Instillation of an excessive volume of PBS can result in foam formation in the lumen which could obscure viewing of the lumen.</li>
	<li>Insert the assembled endoscope 0.5 cm into the anus of the mouse. Advance the biopsy forceps into the lumen of the rectum until the full &#8216;jaws&#8217; (including hinge) of the forceps are beyond the end of the sheath (as observed on the video monitor) (Supplemental Video 1). Turn the forceps 90&#176; so that the jaws open in an east-west orientation (Supplemental Video 1).</li>
	<li>To biopsy, open the forceps and advance approximately 1 cm, close the forceps and in one smooth motion quickly pull back on the forceps while leaving them closed (Supplemental Video 1).
	<ol>
		<li>Avoid fully insufflating the colon when performing the biopsy. Therefore, leave the right side of the gas valve open during this step; although it should be noted that upon opening the forceps there is a risk of damaging the mucosa when the colon is in this position.</li>
	</ol>
	</li>
</ol>
2. Visualizing and measuring the wound bed
<ol>
	<li>Initiate video recording immediately after biopsy by pressing the foot pedal attached to the colonoscope recording device. Fully insufflate the colon by firmly pressing an index finger against the right side of the gas valve to completely cover the opening in order to force the air into the endoscope and thus into the colon. 
	NOTE: Although this protocol describes the use of an air pump in which the air flow is manually controlled, a peristaltic pump with a controlled air supply can also be used.</li>
	<li>Advance the forceps back out of the sheath, and into the rectal lumen while in the closed position (Supplemental Video 1). Place the forceps against the rectal wall immediately above the wound until the base of the jaws is aligned with the top edge of the viewing field (Figure 2 &#38; Supplemental Video 1). Continue fully insufflating the colon until a clear view of the wound can be observed. 
	NOTE: Care should be taken to extend the forceps far enough to reveal only the jaws of the forceps up to the base&#160; for every mouse being examined in order to ensure a consistent distance between the lens of the endoscope and the lesion (Figure 2, white arrow).</li>
	<li>If carrying out biopsies on multiple mice on the same day, remove the biopsied tissue of the previous mouse from the forceps (using the cleaning brush provided) and wipe down the lens sheath with 70% ethanol to clean it before inducing a wound in the next mouse. 
	NOTE: Although this protocol describes carrying out a single biopsy per mouse, multiple biopsies can be performed on a single mouse provided the tissue taken from the prior biopsy is removed from the forceps in order to ensure a full biopsy can be taken on subsequent biopsies.</li>
	<li>Place the mouse into a cage void of other mice and on top of a towel to keep the airway clear until it recovers from the anesthesia after completing the procedure. Monitor mouse to ensure that it recovers from the anesthesia as indicated by regaining activity. 
	NOTE: Two people are required to induce the wound and visualize the wound bed on day 0 (one operating the endoscope and the other operating the biopsy forceps) but only one individual is needed for visualizing wounds on subsequent days (as described below).</li>
	<li>Follow the same procedure for preparation of the mouse and colonoscopy for subsequent wound measurements (typically&#160;days 2, 4 and 6 following biopsy), as described above in sections 1.1-1.4, except for the induction of the wound.</li>
	<li>Locate the wound bed on the video monitor at these time points after inserting the endoscope, and advance the biopsy forceps (in the closed position) to immediately above the wound bed, insufflate the colon and initiate video recording, as described in sections 2.1 and 2.2 (Figure 2). 
	NOTE: It can be difficult to locate the wound bed more than 6 days following biopsy.</li>
	<li>Advance the forceps into the lumen on these subsequent days to the same distance as on day 0 to ensure a consistent distance between the lens of the endoscope and the lesion across days. Ensuring a consistent distance between the lens and lesion will enhance the accuracy of measurements over time. 
	NOTE: One individual can carry out measurements on these days (endoscope is operated with the right hand and the biopsy forceps are advanced with the left hand).</li>
	<li>Once all measurements are completed, open the video recordings of the serial colonoscopies in video editing software that enables the creation of still shots from video.</li>
	<li>Advance the video to a frame showing a point in time when the wound bed can be easily visualized, the closed forceps are above the wound bed and against the rectal wall and the wall is taut. Take a snapshot of this frame and code the file name to ensure that measurements of the wound beds are carried out in a blinded manner.</li>
	<li>Open the images with coded file names in NIH ImageJ to quantify the size of the wound bed. Under the Analyze tab, select Set Measurements and check the Area box.
	<ol>
		<li>Select the Freehand selections tool from the main menu bar and draw a perimeter around the wound (see Figure 2). Under the Analyze tab select Measure and the value of that measurement will automatically populate in the Results window.</li>
	</ol>
	</li>
	<li>Export the results into a spreadsheet after completing measurements for all images, by selecting Save As under the File tab within the Results window and changing the extension to become a spreadsheet file.</li>
	<li>Calculate the size of the wound on days subsequent to the induction of the wound (i.e. days 2, 4 and 6) relative to the size on day 0 (immediately after wounding) in a spreadsheet. Accomplish this by dividing the size of the wound on each day by the size of the wound on day 0 and converting that value to a percentage. 
	NOTE: Under normal conditions using this method of colonoscopic viewing, the greatest closure of the wound is observed after the first 2 days (~75% reduction in size) with more gradual closure on days 4 and 6 (~80% and ~95% reduction in size, respectively).</li>
</ol>
3. Collection of the wound bed for molecular analysis
<ol>
	<li>Euthanize mouse using CO2 asphyxiation followed by cervical dislocation (or equivalent technique) on the selected day following biopsy.</li>
	<li>Harvest the distal region of the colon by opening the skin and abdominal muscle to expose the body cavity. Place closed scissors under the colon and gently lift to release it from the underlying mesentery and then cut the colon at its midpoint and at the anus to remove it from the mouse.</li>
	<li>Flush fecal content using a rat gavage needle attached to a 20 mL syringe filled with ice-cold 1x PBS then lay down the colon onto filter paper.</li>
	<li>Cut open the colon longitudinally on filter paper ensuring that the mesenteric side is face down against the filter paper. Apply 0.2% methylene blue to the mucosa using a squeeze pipet while tissue is still on the filter paper and then drain off excess methylene blue. View the colon under a dissecting microscope and locate the wound bed (Figure 3A).</li>
	<li>Using 4 inch micro iris scissors, cut around the edge of the wound bed (Figure 3A, dashed circle) being careful not to cut into the muscle layer (unless the muscle is desired) and transfer the dissected wound bed to a tube using fine point tweezers for snap-freezing or desired storage method. 
	NOTE: The amount of tissue collected in this manner is enough for extracting RNA for RNA-seq or equivalent analysis.</li>
</ol>
4. Preparation of tissue for histological analysis
<ol>
	<li>Euthanize the mouse and harvest the colon as described in step 3.1-3.2.</li>
	<li>Cut open the colon longitudinally on filter paper ensuring that the mesenteric side is face down against the filter paper. Gently apply 4% paraformaldehyde (or fixative of choice) using a squeeze pipet and cover the tissue with parafilm. Leave flat in a sealed container for 4-6 hours.</li>
	<li>Transfer tissues to 70% ethanol for storage. When ready to process the tissues, remove parafilm and apply 0.2% methylene blue to the mucosa using a squeeze pipet while tissue is still on filter paper. Then drain off excess methylene blue.</li>
	<li>View the colon under a dissecting microscope and locate the wound bed (Figure 3A). Using a scalpel with a #10 blade, cut directly through the center of the wound bed and continue cutting through the remainder of the colon in a straight line, such that the colon has been cut in half, length-wise (Figure 3A, black line).</li>
	<li>Process the colon and then paraffin embed (either one or both sides that remain after cutting) such that the side that was cut by the scalpel (the side that was the center of wound bed before bisection) is face down in the paraffin. Proceed to cutting sections and staining for desired stain(s) or maker(s).</li>
	<li>If cryosections are required for a given staining, harvest the colon as described in step 3.1 and open longitudinally on filter paper, ensuring that the mesenteric side is face down against the filter paper.</li>
	<li>Bisect the wound bed within the freshly harvested colon as described in step 4.5 and embed the colon (either one or both sides that remain after cutting) such that the side that was cut by the scalpel is face down in a base mold half-filled with tissue freezing medium.</li>
	<li>Secure the tissue in place with fine tweezers and place the base mold onto a metal plate on top of dry ice to harden the freezing medium. Once the bottom portion of the medium is frozen (and the tissue is held in place), release the tissue and fill the remaining volume of the base mold with freezing medium and place back onto dry ice.
	<ol>
		<li>After the entire volume of freezing medium is frozen, transfer to a -80 &#176;C freezer until sectioning.</li>
	</ol>
	</li>
	<li>For paraffin or frozen sections, cut and stain additional sections by H&#38;E to ensure that the wound bed has been captured on the section before proceeding to study-specific staining. Figure 3B shows an example of a section in which the wound bed can be clearly observed.</li>
</ol>

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The authors have nothing to disclose.

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...

The gastrointestinal (GI) tract is a complex organ system&#160;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.

<table><tbody><tr><td>Biopsy forceps, 3 Fr</td><td>Karl Storz</td><td>61071ZJ</td><td></td></tr><tr><td>Coloview Tower system</td><td>Karl Storz</td><td>contact company</td><td></td></tr><tr><td>Examination sheath, 9 Fr, Kit</td><td>Karl Storz</td><td>61029DK</td><td></td></tr><tr><td>Hopkins telescope, 0&#039;, 1.9 mm x 10 cm</td><td>Karl Storz</td><td>64301AA</td><td></td></tr><tr><td>isofluorane</td><td>Covetrus</td><td>2905</td><td></td></tr><tr><td>methylene blue</td><td>Sigma-Aldrich</td><td>M9140</td><td></td></tr><tr><td>micro iris scissors</td><td>Integra</td><td>18-1619</td><td></td></tr><tr><td>NIH ImageJ</td><td>NIH</td><td>N/A</td><td>software available for free download from: https://imagej.nih.gov/ij/</td></tr><tr><td>Pawfly MA-60 aquarium pump</td><td>Amazon</td><td>N/A</td><td></td></tr><tr><td>scalpal with #10 blade</td><td>Hill-Rom</td><td>372610</td><td></td></tr></tbody></table>

performing colonoscopic-guided pinch biopsies in mice and evaluating subsequent tissue changes

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 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 ...

Watch this Scientific Journal Video about Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes at JoVE.com

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

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 ...

performing-colonoscopic-guided-pinch-biopsies-mice-evaluating

Research

JoVE Journal

Immunology and Infection

4.4K Views.  Stony Brook University, Stony Brook, NY. Deze methode biedt een ideale aanpak voor het bestuderen van colonische wondgenezing en de moleculaire en histologische veranderingen die optreden na letsel. Deze methode kan ook licht werpen op IBD pathogenese. Het belangrijkste voordeel van deze techniek is om de locatie van letsel en de timing van het wondgenezingsproces nauwkeurig te beheersen.Personen die deze methode voor het eerst uitvoeren, kunnen moeite hebben om de colonoscoop in de dikke darm van de muis in te brengen en con...

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

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Cancer Research

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Medicine

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

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.

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

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.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

Colonoscopic Guided Pinch Biopsy in a Mouse Model: A Technique to Visualize and Extract Tissue Samples from Mouse Colon

Source: Montrose, D. C., et al. <a href="https://www.jove.com/v/60949">Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes.</a> J. Vis. Exp. (2021)
This video demonstrates the protocol for performing colonoscope-guided pinch biopsy in the mouse. This technique helps visually analyze the colon lumen and collect tissue samples for detailed studies.

Source: Montrose, D. C., et al. Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes. J. Vis. Exp. (2021)
This ...

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

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