3-D neurohistology of transparent tongue in health and injury with optical clearing

Abstract

Tongue receives extensive innervation to perform taste, sensory, and motor functions. Details of the tongue neuroanatomy and its plasticity in response to injury offer insights to investigate tongue neurophysiology and pathophysiology. However, due to the dispersed nature of the neural network, standard histology cannot provide a global view of the innervation. We prepared transparent mouse tongue by optical clearing to reveal the spatial features of the tongue innervation and its remodeling in injury. Immunostaining of neuronal markers, including PGP9.5 (pan-neuronal marker), calcitonin gene-related peptide (sensory nerves), tyrosine hydroxylase (sympathetic nerves), and vesicular acetylcholine transporter (cholinergic parasympathetic nerves and neuromuscular junctions), was combined with vessel painting and nuclear staining to label the tissue network and architecture. The tongue specimens were immersed in the optical-clearing solution to facilitate photon penetration for 3-dimensiontal (3-D) confocal microscopy. Taking advantage of the transparent tissue, we simultaneously revealed the tongue microstructure and innervation with subcellular-level resolution. 3-D projection of the papillary neurovascular complex and taste bud innervation was used to demonstrate the spatial features of tongue mucosa and the panoramic imaging approach. In the tongue injury induced by 4-nitroquinoline 1-oxide administration in the drinking water, we observed neural tissue remodeling in response to the changes of mucosal and muscular structures. Neural networks and the neuromuscular junctions were both found rearranged at the peri-lesional region, suggesting the nerve-lesion interactions in response to injury. Overall, this new tongue histological approach provides a useful tool for 3-D imaging of neural tissues to better characterize their roles with the mucosal and muscular components in health and disease.

Keywords: neural network; neurohistology; neuromuscular junction; optical clearing; papilla; skeletal muscle; tongue innervation; tongue lesion.

Figure 1

Optical clearing increases light transmission of mouse tongue specimens. (A,B) Change of tongue optical property after the clearing process. The entire tongue (panel A) and cross-section (panel B; thickness: 200 μm) were immersed in saline and optical-clearing solution prior to taking the images. Solutions were removed in panel (A) to avoid light reflection. In panel (B), the specimen was held by two coverslips and a spacer filled with immersion solution. (C,D) Transmitted light micrographs of the tongue specimens in saline and optical-clearing solution. (E) Increase in light transmission across the optically cleared tongue cross-section over a spectrum of wavelengths. Colored lines indicate the wavelengths of laser lines used in confocal microscopy. Results are presented as mean ± standard deviation (n = 8). (F,G) Confocal micrograph of the optically cleared tongue and the standard H&E image. The confocal micrograph consists of fluorescence signals derived from nuclear staining (propidium iodine) and tissues' autofluorescence (excited by the 488-nm laser). Both the confocal and H&E images show the tongue mucosal and muscular structures.

Figure 2

Deep-tissue microscopy of the tongue microstructure, vasculature, and innervation with high definition. (A) Extended imaging depth in tongue microscopy with optical clearing. Upper panels show the signals of blood vessels in the optically cleared tongue cross-section, while in the opaque specimen (lower panels) the signals drastically declined as the focal plane progressed into the tissue. The blue and red in the images are the range indicators of signal intensity, showing the locations with no signals and saturated signals, respectively. (B) In-depth projection of the vascular signals derived from the optically cleared tongue. 360° presentation of the image stack is shown in Video S3. (C) Neurovascular complex in the core of filiform papilla. Video S4 shows a 360° projection of the image stack. (D) Microstructure and innervation of fungiform papilla. Left: in-depth projection of a fungiform papilla in between the filiform papillae. Right: projection of the fungiform papilla and its innervation. Asterisk indicates the taste bud at the top of the papilla. A 2-D micrograph is placed at the background to indicate the location of the epithelium. A fly-through presentation of the image stack is shown in Video S5. (E,F) Zoom-in examination of taste buds in transparent tongue epithelium. Arrows in panel (E) and squashed circles in panel (F) indicate taste pores, which connected the exterior and interior domains of the epithelium. Pan-neuronal marker PGP9.5 and sensory nerve marker CGRP were used to reveal the taste bud innervation. Green: nuclei. Red: capillaries. White: PGP9.5-labeled nerves. Yellow: CGRP-labeled sensory nerves. A 360° projection of the sensory nerve network is shown in Video S6

Figure 3

In-depth microscopy with tile scanning reveals nerves remodeling in response to tongue injury. (A–C) Stitching of transmitted light and confocal micrographs across the tongue section. Panel (A) shows a protrusive lesion at the left side of the section (inset: gross view of the diseased tongue). Pan-neuronal marker PGP9.5 (panel B) was used to reveal the tongue innervation. Overlay of panels (A,B) is shown in Figure S1. Arrows in panels (B,C) indicate the remodeled skeletal muscle fibers—revealed by the staining background and confirmed in the transmitted light micrograph—entering the lesion domain (circles in panels C,D). Asterisks in panel (B) indicate the landmark fungiform papillae and their innervation (positive control of PGP9.5 staining). (D) Zoom-in examination of the condensed PGP9.5⁺ nerve fibers in the lesion domain (the cyan box in panel B).

Figure 4

Perivascular sympathetic innervation of mouse tongue in injury. (A,B) Gross view of the 4-nitroquinoline 1-oxide-treated tongue. Squashed circles indicate an early-stage lesion. In the gross view, perivascular sympathetic innervation of tongue arterioles was prominent in both the diseased and normal domains (arrows in panel B). Cyan: sympathetic marker TH. (C,D) Zoom-in examination of sympathetic nerves encircling the tongue arterioles. The morphology indicates the sympathetic control of local blood flow. (E,F) Zoom-in examination of perivascular sympathetic innervation of capillaries in tongue mucosa (two examples). Particularly, in panel (F), the in-depth projection shows the sympathetic nerves ascending from the skeletal muscle domain to innervate the fungiform papilla in the mucosa. (G,H) TH-labeled sympathetic nerves encircling the arteriole underneath the diseased epithelium. Arrows indicate the perivascular sympathetic innervation.

Figure 5

Cholinergic innervation of mouse tongue and its remodeling in injury. (A,B) Gross view of the VAChT-labeled cholinergic nerves in the tongue. Panel (A) reveals three features: (1) the association of the VAChT⁺ parasympathetic varicosities and axons with the von Ebner's gland (green asterisk) and its secretory ducts (arrows indicate the ducts extending toward the mucosal surface), (2) the association of the parasympathetic varicosities with large blood vessels (yellow asterisks), and (3) the aggregation of VAChT⁺ neuromuscular junctions in the skeletal muscle domain (squashed circle). The same aggregation and alignment of tongue neuromuscular junctions can be seen in panel (B), in which the transmitted light micrograph shows the muscle fibers. Some VAChT⁺ neuromuscular junctions were found scattered under the submucosa, indicating the extension of the muscle fibers to this area. (C) Zoom-in examination of cholinergic nerves in the tongue muscle domain. The paired staining and projection reveal the perivascular parasympathetic nerves with VAChT⁺ varicosities following the capillaries (asterisks, upper panel) and the cholinergic neuromuscular junctions (arrows, lower panel). (D,E) Imaging of the VAChT-labeled cholinergic nerves underneath the diseased epithelium. Panel (D): transmitted light micrograph of a tongue lesion; inset: illustration of the protrusive epithelial remodeling. Panel (E): abundant VAChT⁺ parasympathetic nerve fibers and neuromuscular junctions underneath the tongue lesion. Arrows indicate the remodeled alignment of the VAChT⁺ neuromuscular junctions. Geometrically, the protrusive lesion creates a “core” (illustrated in the inset and marked as the circles in the images) with condensed nerve density, similar to the intra-lesional region shown in panels 3B,D.