Optical Clearing Reveals TNBS-induced Morphological Changes of VGLUT2-positive Nerve Endings in the Colorectum

Sensitization of colorectal afferents and colorectal hypersensitivity have been observed in a mouse model of post-infectious irritable bowel syndrome via intracolonic treatment of 2,4,6-trinitrobenzenesulfonic acid (TNBS). In this study, we investigated the distribution and morphology of microscopic colorectal afferent endings before and after intracolonic treatment of TNBS. We genetically labeled predominantly extrinsic colorectal afferents using the vesicular glutamate transporter type 2 (VGLUT2) promoter. Then, we used an optical tissue clearing method of whole-mount colorectum to image labeled VGLUT2-nerve endings that are otherwise obscured in untreated samples. We used vector path tracing to quantify the density and degree of curliness of colorectal nerve endings in various sub-volumes of the colroectum captured via confocal imaging. VGLUT2-labeled nerve fibers were observed in all layers except the serosa and longitudinal muscle, although they were most abundantly present in the submucosa, myenteric plexus, and mucosa. Seven days after TNBS treatment, the density of VGLUT2-positive neural tissues were significantly reduced in these three layers. The TNBS-induced loss of VGLUT2 tissue density recovered in most cases by day 28 of TNBS injection. Interestingly, almost all VGLUT2-positive nerve endings in the submucosa displayed a much higher degree of curliness compared to those in other layers in both the control and treated colorectums. However, TNBS treatment significantly reduced the presence of severely curly nerve endings in the rectal region 28 days post-TNBS. The results from this morphological study along with previous findings of functional mechanotransduction studies point to the role of curly afferent endings in the submucosa in the encoding of noxious visceral stimuli i.e. in visceral nociception