The study of inflammatory bowel disease models in laboratory animals (namely mice) is largely based on pathology and histological scores of random transverse or longitudinal thin intestinal sections. Here we illustrate in detail the approach and the protocols we have implemented using SM to profile, image and assay functionally focal pathologies (target lesions analysis) which has allowed us to demonstrate that target functional characterization of gastrointestinal (GI) health (using MPO activity as a biochemical example) is complementary and superior to understand disease mechanisms in 3D compared to traditional blind analysis of histological specimens. SM-imaging and stereoscopic vision, like in the 3D-movie industry is advantageous in that the tissues can be examined in real time (fresh soon after collection) without special staining. Our innate ability to understand the world in three dimensions has allowed us, through SM, to sample 3D-focal/structural pathologies for functional characterization and so phenotype the various inflammatory patterns in the intestinal tract of not only mice, but other animals.
The detailed protocol was validated and tested to minimize test variability and allow for a more comparable platform for inflammatory testing relying on SM and MPO as very rapid and cost effective technique. In short, this protocol explains how the intestinal mucosa is examined using SM, how small tissues are dissected in real time without the aid of staining from freshly dissected animals, how the sample is stored or immediately homogenized and mixed with a lysis buffer, and the activity measured using an chemically simple enzyme substrate and optic density. If reagents are prepared, up to 24 sample sets can be harvested, homogenized and tested in 4-5 h. Importantly, virtually any other tissue can be tested to be used as a within animal controlled test. Here we use mice as test model. Examples of MPO activity variability due to temperature of incubation (Figure 5)
and the relevance of integrated MPO and SM to elucidate spatial diversity within the same mouse are here illustrated (Figure 6) ). Although we have described in great detail a protocol to quantify MPO precisely, and to control for potential gut microbiota confounding effects due to cage-to-cage variability, the most important aspect of 3D-target phenotyping relies on the observer’s ability to discern a given 3D-SM pathological lesion from among 10 possible abnormal 3D-SM categories and normal SM-mucosal anatomy as recently described. Therefore, it becomes critical that the observer recognizes the principles, rationale and limitations to the use of SM for intestinal 3D-target phenotyping.
Principles and rationale for SM vision in GI research
From a medical perspective, SM allows the visualization of microscopic surfaces magnified and dilated in all dimensions giving the observer an accurate sense of its real 3D topographical appearance (12,13,14). Such perception allows observers to assess and precisely manipulate most specimens without the aid of specialized devices. Although micromanipulators are available and needed to handle SM-magnified structures as small as single oocytes, our study focused on examining intestinal tissues at the villous level (villous width: 125-150 µm), and therefore was conducted harvesting tissues (~10 mg) by hand with the aid of scalpels or >26G needles as cutting/handling instruments to emphasize its potential for rapid implementation (6). In the early 1800s, stereoscopic images were famous as they recreated depth maps in people’s minds as optical photographic illusions. Stereoscopic vision was recreated when paired-pictures taken simultaneously with two-lens cameras (with lens separated by a distance equal to the distance between the human eyes) were seen simultaneously within a certain distance from the eyes. Currently, 3D-movie industries rely on this innate virtue of the brain to reconstruct structures in human minds. Stereomicroscopes were later invented by gradually improving separate magnifying optical pathways intended as telescopic extension of our eyes.
With acquired learning from real scenarios, our brain is highly accurate at predicting distances that separate objects if presented as SM images. A priori knowledge acquired as humans learn from the environment is what allows deciphering if an image is a real object or a miniaturized replica. The information needed by the brain to make predictions is multifactorial. They include color, contrast, shades, scale (familiar size), occlusion (how much an object is occluded by a more forefront object), perspective (the difference between images acquired from each eye), parallax (the change of interrelationships between objects when the observer slightly shifts its position side to side), focus (the time it requires for our eye lenses to adjust and focus on a distant with respect to a close object), and convergence (the degree of strabismus or eye angulation toward the nose required to focus on close objects). Because SM benefits from previous learning, the purpose of the present manuscript is to illustrate in detail necessary aspects to make the protocols and derived knowledge more uniform.
Strengths and limitations
With respect to the strengths, limitations, and the future development of SM-based technologies compared to histological procedures, SM analysis of intestinal tissues has advantages that cannot be captured by histological approaches as mentioned (i.e., the latter examines only very thin cross-sections in 2D). Because SM is a new approach, there are not automated analyses available yet; however, automation is expected to expand the phenotyping possibilities with SM. Currently, we are testing existing video analysis algorithms to stream the scoring of increasing number of SM videos. For such purposes, there is a growing body of literature in histological pathology to serve as foundation image analysis strategy for SM automation. The choice of manual versus automated analysis when available in the future will ultimately depend on the user based on the volume of samples to process and the labor-computational time available for investment. In our laboratory, SM video imaging of fixed GI specimens delays the submission of samples for histopathology in average 12-48 h when experimental groups have 24 mice (although, each mouse SM video takes 2-5 minutes to be completed, and 1-10 minutes to be analyzed). The delay however is logistically irrelevant as it always increases our certainty that the phenotype is exactly characterized; certainty that we always lacked relying only on histology. Complemented, SM and histology have been tremendously powerful to characterize the structural phenotype of intestinal diseases in all our tested mouse models. Aside from the delay in sample submission for histology, no other limitations are evident. SM is highly intuitive, inexpensive, and easily implemented for image capturing and analysis by no specialists making it suitable to be used as a real time readout when mice are used experimentally in fields outside of gastroenterology.
With respect to MPO activity variability, the present protocol seeks to quantify the total amount of oxidative activity in the tissue homogenate as a surrogate of inflammation or oxidative stress as a marker of inflammation and inflammatory cell infiltration while controlling for flora and 3D-architecture, something that has not been addressed in the past. A more specific antibody capture activity assay (4) is currently available that could be combined with the SM-dissection of our 3D-microscopic lesions samples to determine the concentrations of both extracellular and intracellular MPO activity using anti-MPO specific antibodies. MPO-antibody capture activity assays have increased sensitivity, but they will not improve the variability due to the local spatial variability we have recently discovered exist in the intestinal tract of mice using SM (6, 15). To control for reagent-related variability, we have developed the protocol here described to allow the rapid testing of several samples from a particular experiment in the same run, which can be standardized by using a control MPO reagent (e.g., Sigma M6908) to create a standard curve to make testing quantitative, use anti-MPO binding antibodies to increase test sensitivity, and re-scale the estimated values using a range between 1 and 2 as recently described(1) to make relative comparisons possible.
In conclusion, stereomicroscopic 3-D target biochemical (MPO) phenotyping is a cost-effective rapid strategy that can be used as a complementary research and diagnostic methodology in therapeutic and pathologic studies of intestinal inflammation.