Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse iBD models using near infrared (niR) detection. to address the limitation of visible light readouts-namely high background-IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. the increased sensitivity of niR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. in addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research. Ulcerative colitis (UC) and Crohn's disease (CD) are two main forms of both acute and chronic inflammatory bowel diseases (IBD) with complex disease etiology. While multiple anti-inflammatory treatment strategies are available to patients, there is active interest in the development of therapeutic strategies that improve epithelial repair and barrier function to reduce pro-inflammatory burdens 1-3. Several clinical studies suggest alterations in paracellular permeability and tight junction functions are key events in the pathogenesis of IBD 3-5. In addition to traditional readouts of disease severity such as body weight, colon length, and pathology score, screening and assessment of intestinal tight junction permeability is an increasingly important measurement in animal colitis models. Ratiometric measurements of sugar concentrations in urine provide gastrointestinal permeability data in the clinic, but data are highly variable and require significant urine collection at multiple timepoints 6,7. Other strategies utilize small molecule such as radiolabeled EDTA but require ionizing radiation and are more costly 8. Due to ease of handling, in vivo assessment of intestinal epithelial permeability in pre-clinical models largely relies on measuring serum fluorescein isothiocyanate (FITC)-labeled dextrans that are orally gavaged. However, 4 kDa FITC-dextran detection has limited sensitivity due to high blood autofluorescence and may prevent accurate differentiation between treatment groups in dose-response experiments, where absolute differences in fluorescence intensity may be low and/or near the limit of detection. Low molar absorptivity in addition to high

Although the dog is a common choice among nonrodent species in evaluation of compound safety for regulatory submission, information regarding the incidence of spontaneous or incidental microscopic changes in canine peripheral nerve is limited. A retrospective examination was performed of routine histologic preparations of sciatic nerve from eighty-one control dogs in toxicity studies ranging from ten days to three months in duration. Spontaneous background changes included digestion chambers, foci of vacuolation, nerve fibers circumscribed by proliferating Schwann cells (bands of Büngner), and small foci of myelin aggregation. The latter accounted for 91% of the microscopic changes and were noted in all sections examined. These changes were quantified, and the number per square millimeter of evaluable nerve tissue was determined for each slide. Densities of foci varied among the slides examined; no age- or sex-related trends were apparent. In addition, anatomic features of peripheral nerves including nodes of Ranvier, Schmidt-Lanterman incisures, Renaut bodies, and effects resulting from sectioning plane were noted. By demonstrating the range of effects observed within control animals, these observations provide a basis for recognition of possible compound-related effects in routine nerve preparations from dogs included in toxicity studies.

A painful, chronic condition, Rheumatoid Arthritis, is marked by bone erosion and soft tissue swelling at the joint. As treatments are investigated in pre-clinical models, characterizing disease progression is integral to assessing treatment efficacy. Here, in vivo and ex vivo micro-computed tomography (µCT) are used in parallel with traditional caliper score measurement to quantify physiological changes in the tarsal region in a murine, collagen-induced arthritis model. In vivo imaging methods, which are validated here through comparison to ex vivo and caliper methods, afford longitudinal analysis of both bone and soft tissue through a single image acquisition. This method removes the subjectivity of swelling quantification which is inherently associated with traditional caliper measurements. Histopathology offers an additional assessment of bone erosion and inflammation by providing a microscopic characterization of disease activity. In comparison to untreated animals, daily prednisolone (glucocorticoid) treatment is shown to restore bone volume, as reflected through in vivo and ex vivo µCT images, as well as histopathology. Prednisolone-associated reduction in inflammation is shown through in vivo µCT soft tissue volume measurements, paw caliper measurements, and histopathology. The findings reported here provide a comprehensive validation of in vivo µCT with a sensitivity that enables characterization of pre-clinical disease assessment in response to treatment in a murine, collagen-induced arthritis model.

Purpose To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR)-labeled targeted molecular imaging agents. Procedures. A human IgG1 with nanomolar binding affinity specific to the alternatively spliced D domain of tenascin C was generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix protein in the colon of mice given dextran sulfate sodium in the drinking water. The antibody reagent was labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate in vivo targeting specificity. Increasing doses of imaging agent were given to estimate the saturating dose. Results The NIR-labeled proteins successfully targeted colonic lesions in a murine model of colitis. Co-administration of a molar excess competing unlabeled dose reduced normalized uptake in diseased colon by > 70%. Near infrared ex vivo images of colon resected from diseased animals showed saturation at doses exceeding 1 nmol and was confirmed with additional quantitative ex vivo biodistribution. Cellular-level specificity and protein stability were assessed via microscopy. Conclusions Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.