Female Sprague Dawley rats were obtained from Taconic farms (Germantown, NY) at 20 days of age. At 21 days of age, rats were given an ip injection of 50 mg 1-methyl-1-nitrosourea/kg body weight (Ash Stevens, Detroit, MI) [26
]. Rats were group-housed in an environmentally controlled room maintained at 22°C and 50% relative humidity with a 12 h light–dark cycle. They were fed a modification of the AIN-93G diet formulation [27
] and deionized water ad libitum. The study was terminated 9 weeks following carcinogen injection. The work followed ethical guidelines approved by the Colorado State University Animal Care and Use Committee.
2.1 Whole Mount Preparation
At necropsy, rats were skinned, and a midline incision was made in the lower abdominal wall to expose the internal organs. A second incision was made at the pubic symphysis, and the pelvic bones were spread apart to reveal organs at the base of the abdomen. Reproductive organs were removed in toto and the gastrointestinal tract exposed. The colon was cut at the anus, carefully lifted out of the abdomen and excised above the ileo-cecal valve to visually maintain the anatomical orientation of the tissue. Colon length was measured in millimeters from the anus to the cecum, and residual colonic mesentery was removed using Adson forceps (George Tiemann & Co, Hauppauge, NY, Cat. No. 105-235-1). The cecum, anus, and rectum were removed, and the colon was trisected starting at the distal end into three equal lengthwise segments that were operationally defined as representing descending, transverse, and ascending regions, respectively. The colon was briefly immersed in phosphate-buffered saline (PBS) pH 7.4, and hardened fecal pellets were removed using a gentle milking action of the colonic tissue between the index finger and thumb. A longitudinal incision running the entire length of the colon was made using a small pair of scissors (George Tiemann & Co, Cat. No. 105-411), which had been modified in house using a bench grinder to blunt and smooth both tips in order to prevent perforation of the colon and minimize disruption of the fragile colonic mucosa while making the incision. Large deposits of soft fecal material were removed using Adson forceps, and the tissue was washed in PBS to remove residual fecal material from the colonic mucosa.
The colon was laid serosa side down onto a Kimwipe® to wick away excess moisture. This process was repeated a few times by gently lifting the colon and placing it on a dry section of the Kimwipe® until the serosal surface became tacky, i.e., increased resistance noted when separating the colon from the Kimwipe®. Whole mounts of colonic tissue segments were prepared by laying the serosal surface down onto 75 × 25 mm 3-aminopropyltriethoxysilane-treated microscope slides (Statlab Medical Products, McKinney, TX, Cat. No. 418). Adson forceps were used to carefully stretch the four corners of the tissue toward the edges of the slide. Once tacked down at the corners, Adson forceps were used to stretch the areas lying in between by carefully grasping a small portion of underlying serosa at the edge of the colon and slightly lifting the tissue off the glass while pulling toward the edge of the slide, being careful not to damage the delicate mucosa nor introduce air bubbles between the tissue and glass. This process was quickly repeated along all four sides until the tissue was stretched completely flat across the surface of the slide. Whole mounts were exposed to air for a period of 4 min prior to immersion in fixative to prevent tissue detachment from the glass slide. Whole mounts were fixed in 10% neutral buffered formalin for 24 h, rinsed in tap water for 15 min, and stored in 70% ethanol.
2.2 Methylene Blue Staining and Image Acquisition
Colon whole mounts were removed from 70% ethanol, rinsed in deionized water, and stained in 0.05% methylene blue (Sigma-Aldrich, St. Louis, MO., Cat No. M9140) for 3 min. Whole mount slides were rinsed in running deionized water until all excess methlyene blue stain had been removed. Digital images of stained colon whole mounts were captured using a customized image acquisition system (North Central Instruments, Inc., Plymouth, MN) consisting of a 3.0 megapixel CMOS digital camera (Clemex Technologies, Inc. Longueuil, Canada) mounted on a Z16 APO monocular zoom lens 16:1 with a magnification range of ×0.57–9.2 (Leica Microsystems, Inc., Bannockburn, IL). The camera and lens were mounted on a Z motor (Leica Microsystems, Inc.) attached to a transmitted light base with a 100 × 100 mm motorized stage (Clemex Technologies, Inc.). An X–Y control box and joystick (Clemex Technologies, Inc.) in conjunction with a Pentium 4 desktop PC (Dell, Round Rock, TX) and Captiva v4.0 software (Clemex Technologies, Inc.) were used for image acquisition (Figure ).
a Workstation used to acquire high-resolution composite images of stained whole mount preparations. b Colonic tissue whole mount on a glass microscope slide stained with methylene blue, bar = 1 mm.
Methylene blue-stained colon whole mounts were placed on a 6-mm thick sheet of white acrylic plastic (Gagne, Inc. Johnson City, NY) mounted on top of the motorized stage to act as a diffuser. Specimens were trans-illuminated using a 20 V/150 W halogen lamp light source (Volpi, Auburn, NY) with a daylight filter mounted at the rear of the base. A series of seamless tiled Z stack images (×1.6 objective) were captured automatically using the motorized stage in conjunction with the Captiva 4.0 software. The software automatically merged the tiled Z stack images together into a single uniformly focused composite image based on a best contrast algorithm (Figure ). Resulting images were saved as TIF files. High-resolution image acquisition enabled the user to easily identify ACF containing one or more crypts (Figure ).
a–h Examples of ACF in methylene blue-stained colon whole mount. ACF are easily distinguished from surrounding normal colonic mucosa, bars = 100 μm.
2.3 Digital Extraction of ACF
Composite digital images were opened in Photoshop® v9.0.1 (Adobe Systems, Inc., San Jose, CA) and saved as PSD files (Photoshop® native format with support for layers), one file for each of the three whole mount slide preparations per animal representing ascending, transverse, and descending colon. ACF were individually circumscribed on a separate transparent layer using the Photoshop® pencil tool with a contrasting bright color in conjunction with an Intuos 3 stylus and digital tablet (Wacom Technology Corp., Vancouver, WA). ACF were digitally excised from the image by selecting the transparent area outside the circumscribed borders using the magic wand tool and then applying the inverse command to select only the colored borders of the circumscribed ACF. The background layer was activated and that layer, via copy command, was used to create a new transparent layer containing only the excised ACF. The image was duplicated and flattened to remove all layers except the excised ACF on a white background. The resulting image was saved as a separate TIF file for image analysis.
2.4 HID-AB Staining
Methylene blue-stained colon whole mounts were stained with HID-AB as described by Caderni et al. [28
] to look for evidence of mucin depletion in ACF. Images of HID-AB-stained colon whole mounts were acquired as outlined above and added to PSD files as a separate layer and aligned with the original methylene blue-stained images. Once aligned, the same layer used to circumscribe methylene blue-stained ACF was used to extract HID-AB-stained ACF as a separate layer using the technique described above, and the resulting image was saved as a separate TIF file for image analysis. The use of separate aligned layers permitted each image to be toggled on or off at will enabling qualitative assessment of the same ACF in both methylene blue and HID-AB stained whole mounts.
2.5 Image Analysis
Separate macros were developed in Image-Pro Plus® v188.8.131.52 (Media Cybernetics Inc., Bethesda, MD) to streamline analysis of ACF from both methylene blue and HID-AB-stained image files. In both macros, individual ACF were automatically selected for analysis from imported images. The methylene blue macro presented the user with multiple renderings of each ACF, which included the methylene blue-stained ACF image, a surface plot image depicting ACF topography with crypt detail, a pseudo-colored surface plot image showing changes in both topography and density and a high-contrast grayscale image with software identified crypt areas marked in red (Figure ). The user was presented with options to toggle marked crypt areas on/off and split or join marked areas prior to analysis based on visual characteristics present in the other renderings. A number of morphometric parameters were measured, which included area, density, integrated optical density, maximum diameter, and roundness [(perimeter2)/(4*π *area)] of each ACF. The same parameters were applied to crypts lying within each ACF, and the total numbers of crypts per ACF were also measured.
Figure 3 a Methylene blue-stained ACF enlarged to show detail. b Surface plot enhanced image of a showing ACF topography with crypt detail. c Surface plot image of a with pseudo color applied to show changes in both topography and density. d High-contrast image (more ...)
The HID-AB macro used three different segmentation thresholds based on hue, saturation, and intensity to isolate areas within each ACF and place them into three separate classes based on color: HID (dark brown), AB (blue), and unstained (absence of brown or blue color). Class areas were measured and expressed as a percent of the total area for each ACF. Unstained areas, representing ≥85% of the total area of each ACF, were operationally defined as MDF. Morphometric data from both macros were exported via DDE to an Excel spreadsheet.
2.6 Whole Mount Tissue Processing, Paraffin-Embedding, and Microtomy
Colon whole mounts on glass slides were placed in Tissue Tek® plastic slide racks (VWR, West Chester, PA, Cat. No. 25608-868) and processed in an automatic tissue processor using an abbreviated processing schedule and infiltrated with molten paraffin. Whole mounts were bisected down the long axis, and each half was trisected yielding six pieces of tissue per slide; tissues were embedded as separate blocks, mucosa side down. Five-micron serial sections were cut from each block, mounted onto 3-aminopropyltriethoxysilane-treated glass microscope slides, and stained with hematoxylin and eosin (H&E) according to normal laboratory protocol. Images of H&E sections were acquired as mentioned above and added to the PSD file as a separate layer. This layer was aligned with previously captured methylene blue and HID-AB layers, thus allowing qualitative assessment of ACF across all three staining techniques.
2.7 β-catenin Immunohistochemistry
Sections were cut at 5 μm and mounted on 3-aminopropyltriethoxysilane-treated slides and heat-immobilized in a 60°C oven for 20 min. Sections were deparaffinized in three changes of xylene, hydrated in a series of graded ethanols, rinsed in deionized water followed by three rinses in Tris-buffered saline (TBS) [50 mM Tris–HCl, 150 mM NaCl, pH 7.6 with 0.05% Tween 20 (Dako, Carpinteria, CA, Cat. No. S1968 and S1966)]. Subsequent steps were carried out at room temperature using an Autostainer (Dako, Carpenteria, CA). Anti-β-catenin (BD Biosciences, San Jose, CA, Cat. No. 610153) 1:50 was applied and incubated for 1 h followed by two rinses in TBS. FITC donkey anti-mouse Fab'2 (Jackson ImmunoResearch Laboratories, West Grove, PA, Cat. No. 715-096-151) 1:100 in 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Cat. No. 017-000-121) was applied and incubated for 30 min followed by two rinses in TBS. 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, Cat. No. D1306) 300 nM was applied and incubated for 10 min followed by two rinses in TBS. Slides were rinsed in two changes of deionized water for 1 min and allowed to air dry under a fume hood in the dark.
Images were acquired using a Zeiss Axiocam HRm camera (Carl Zeiss, Thornwood, NY) coupled to a Zeiss Axioskope II microscope (Carl Zeiss). Multi-channel acquisition within the Axiovison v4.1 software (Carl Zeiss) was used to obtain separate images at a magnification ×400 for both FITC (filter excitation and emission of 480 and 535 nm, respectively) and DAPI (filter excitation and emission of 350 nm and 460 nm, respectively) stained areas, which were rendered as a composite image containing both fluorophores.
2.8 Statistical Analyses
Descriptive statistics, ANOVA, and multivariate analysis of variance were performed using SYSTAT v12.02 (SYSTAT Software, Inc., Chicago, IL).