Preclinical Evaluation of a Microwave-Based Accessory Device for Colonoscopy in an In Vivo Porcine Model with Colorectal Polyps.

BACKGROUND AND AIMS: Colonoscopy is currently the most effective way of detecting colorectal cancer and removing polyps, but it has some drawbacks and can miss up to 22% of polyps. Microwave imaging has the potential to provide a 360° view of the colon and addresses some of the limitations of conventional colonoscopy. This study evaluates the feasibility of a microwave-based colonoscopy in an in vivo porcine model. METHODS: A prototype device with microwave antennas attached to a conventional endoscope was tested on four healthy pigs and three gene-targeted pigs with mutations in the adenomatous polyposis coli gene. The first four animals were used to evaluate safety and maneuverability and compatibility with endoscopic tools. The ability to detect polyps was tested in a series of three gene-targeted pigs. RESULTS: the microwave-based device did not affect endoscopic vision or cause any adverse events such as deep mural injuries. The microwave system was stable during the procedures, and the detection algorithm showed a maximum detection signal for adenomas compared with healthy mucosa. CONCLUSIONS: Microwave-based colonoscopy is feasible and safe in a preclinical model, and it has the potential to improve polyp detection. Further investigations are required to assess the device's efficacy in humans.

Genome Editing in Pigs.

The generation of genetically engineered (GE) pigs for disease modeling and xenotransplantation has been massively facilitated by the discovery of the CRISPR/Cas9 system. For livestock, genome editing is a powerful tool when used in combination with either somatic cell nuclear transfer (SCNT) or microinjection (MI) into fertilized oocytes. To generate either knockout or knock-in animals using SCNT, genome editing is carried out in vitro. This has the advantage that fully characterized cells are being employed to generate cloned pigs, predetermining their genetic makeups. However, this technique is labor-intensive and, hence, SCNT is better suited for more challenging projects such as the generation of multi-knockout- and knock-in pigs. Alternatively, CRISPR/Cas9 is introduced directly into fertilized zygotes via microinjection to produce knockout pigs more rapidly. Finally, the embryos are each transferred into recipient sows to deliver GE piglets.Both techniques, SCNT and MI, are technically challenging and therefore require skilled expertise, especially when applied for porcine embryos. Here, we present a detailed laboratory protocol for the generation of knockout and knock-in porcine somatic donor cells for SCNT and knockout pigs via microinjection. We describe the state-of-the-art method for isolation, cultivation, and manipulation of porcine somatic cells, which can then be used for SCNT. Moreover, we describe the isolation and maturation of porcine oocytes, their manipulation by microinjection, and the embryo transfer into surrogate sows.

TNF ΔARE Pigs: A Translational Crohn's Disease Model.

BACKGROUND AND AIMS: Crohn's disease [CD] is a major subtype of inflammatory bowel diseases [IBD] with increasing incidence and prevalence. Results of studies using available small and large animal models are often poorly translatable to patients, and few CD models show small intestinal pathology. Due to its similarities to humans, the pig has emerged as a highly suitable translational disease model, particularly for testing novel nutritional and technological interventions. Our goal was to develop a physiologically relevant porcine CD model to facilitate translation of findings and interventions towards the clinic. METHODS: We generated pigs bearing a 93-bp deletion of the adenosine-uracil-rich element [ARE] and a constitutive-decay element within the 3' untranslated region of the TNF gene. Comparative analysis of physiological, molecular, histological and microbial characteristics was performed between wild-type, TNFΔARE/+ and TNFΔARE/ΔARE animals. Alterations in the microbiome were compared to the TNFΔARE mouse model and IBD patients. RESULTS: TNF ΔARE pigs recapitulate major characteristics of human CD, including ulcerative transmural ileocolitis, increased abundance of proinflammatory cytokines, immune cell infiltration and dysbiotic microbial communities. 16S rRNA gene amplicon sequencing revealed enrichment in members belonging to Megasphaera, Campylobacter, Desulfovibrio, Alistipes and Lachnoclostridum in faecal or mucosa-associated bacteria compared to wild-type littermates. Principal components analysis clustering with a subset of TNFΔARE/+ mice and human IBD patients suggests microbial similarity based on disease severity. CONCLUSIONS: We demonstrate that the TNFΔARE pig resembles a CD-like ileocolitis pathophenotype recapitulating human disease. The ability to conduct long-term studies and test novel surgical procedures and dietary interventions in a physiologically relevant model will benefit future translational IBD research studies.