Immunohistochemical characterization of interstitial cells and their relationship to motor neurons within the mouse esophagus

Abstract

Interstitial cells of Cajal (ICC) and PDGFRα⁺ cells regulate smooth muscle motility in the gastrointestinal (GI) tract. However, their role(s) in esophageal motility are still unclear. The mouse esophagus has traditionally been described as almost entirely skeletal muscle in nature though ICC have been identified along its entire length. The current study evaluated the distribution of skeletal and smooth muscle within the esophagus using a mouse selectively expressing eGFP in smooth muscle cells (SMCs). The relationship of SMCs to ICC and PDGFRα⁺ cells was also examined. SMCs declined in density in the oral direction however SMCs represented ~ 25% of the area in the distal esophagus suggesting a likeness to the transition zone observed in humans. ANO1⁺ intramuscular ICC (ICC-IM) were distributed along the length of the esophagus though like SMCs, declined proximally. ICC-IM were closely associated with SMCs but were also found in regions devoid of SMCs. Intramuscular and submucosal PDGFRα⁺ cells were densely distributed throughout the esophagus though only intramuscular PDGFRα⁺ cells within the LES and distal esophagus highly expressed SK3. ICC-IM and PDGFRα⁺ cells were closely associated with nNOS⁺, VIP⁺, VAChT⁺ and TH⁺ neurons throughout the LES and distal esophagus. GFAP⁺ cells resembling intramuscular enteric glia were observed within the muscle and were closely associated with ICC-IM and PDGFRα⁺ cells, occupying a similar location to c. These data suggest that the mouse esophagus is more similar to the human than thought previously and thus set the foundation for future functional and molecular studies using transgenic mice.

Keywords: Interstitial cells of Cajal; PDGFRα+ cells; glia; motility; neurons.

Figure 1. Gross Anatomy of the mouse esophagus and gastroesophageal junction

aAnatomy of the mouse esophagus in situ, demonstrates the cervical, thoracic and abdominal portions of the intact esophageal body, image shows the esophagus traversing the diaphragm to join the stomach at the gastroesophageal region. bRemoval of the esophagus and stomach from the mouse for dissection. c the esophagus is pinned out as a flat sheet with LES intact, the boundaries of proximal, middle and distal esophagus are indicated and the LES clasp is identified with a white arrow. d Trichrome staining of sagittal sections of the intact mouse esophagus and stomach show longitudinal muscle layer (LM), and circular muscle layer (CM) (pink) of the esophagus arranged perpendicular to one another, the LES clasp muscle is identified by the green dashed line at the gastroesophageal junction (adapted from (Drumm et al., 2022)).

Figure 2. Contractile activities differ in the distal esophagus and LES

Isometric tension recordings were carried out on strips of distal esophagus and LES circular muscle (TTX present throughout). Example traces of contractile activity are shown. The baseline is indicated by a dotted line. a The distal esophagus was stretched by 1 g (st) and after an equilibration period generated rhythmic phasic contractions that persisted in the presence of TTX. bThe LES was stretched to 0.25 g (st) and after a short equilibration period generated a sustained contraction or ‘tone’ and no phasic activity. This activity was also insensitive to TTX.

Figure 3. SMCs, ICC, and PDGFRα⁺ cells are present throughout the mouse LES and esophagus, and form close contacts with one another

Immunohistochemistry on cryosections of SmMHC/^eGFP+ mouse esophagus shows presence of SMCs between skeletal muscle fibers in the proximal a, middle b and distal esophagus c as well as in a distinct band located beneath the mucosa (muscularis mucosae). Panels d-f show the presence of Kit⁺ ICC-IM within SMC bundles and in close proximity to skeletal muscle fibers in the proximal d, middle e and distal esophagus f, the density of ICC-IM was greatest in the LES clasp and distal esophagus f, but this declined in density proximally d-e. Panels g-i show two populations of PDGFRα⁺ cells, one located within the muscle (intramuscular) and another that forms a network in the submucosal region. These cells were located within SMC bundles and alongside skeletal muscle fibers in the proximal g, middle h and distal esophagus i. The density of both intramuscular and submucosal PDGFRα⁺ cells remained constant throughout the esophagus. Smaller panels indicated by i are higher magnification images of the panels to their left. (* = submucosa, # = muscularis mucosae)

Figure 4. ICC-IM and PDGFRα⁺-IM cells are closely associated throughout the esophagus and LES

a Dual IHC labeling of whole-mount mouse esophagus and LES with antibodies against Kit (red) and the Ca²⁺ activated Cl” channel, ANO1 (green) revealed that ICC-IM highly express ANO1 in the mouse LES (i), distal (ii), middle (iii) and proximal esophagus (iv). b Dual IHC labeling of whole-mount mouse esophagus and LES with antibodies against PDGFRα (red) and the small conductance Ca²⁺ activated K⁺ channel, SK3 (green) revealed that PDGFRα⁺-IM in the LES clasp highly express SK3 channels (i). Occasionally PDGFRα⁺ cells in the distal (ii), middle (iii) and proximal esophagus (iv) did express SK3 however this was less common than in the LES clasp, instead SK3 expression was noted in vascular like structures (indicated by white asterisks *). c ANO1 (green) and PDGFRα (red) dual labeling shows a close association between ANO1⁺ ICC-IM and PDGFRα-IM in the mouse LES (i), distal esophagus (ii), middle esophagus (iii) and proximal esophagus (iv).

Figure 5. Nitrergic nerve fibers are closely associated with ICC-IM and PDGFRα-IM

IHC labeling of whole-mount LES and esophagus with a neuronal nitric oxide synthase (nNOS) antibody (green) demonstrates the presence of nitrergic neurons within smooth muscle and around skeletal muscle regions throughout the LES and esophagus a-b. a Dual IHC labeling using nNOS (green) and Kit (red) revealed that ICC-IM are closely associated with nitrergic nerve fibers in the LES clasp (i), distal (ii), middle (iii) and proximal esophagus (iv). b shows similar intimate associations between nNOS⁺ nerve fibers (green) and PDGFRα-IM (red) in the LES clasp (i) and throughout the esophagus (ii-iv).

Figure 6. VIPergic nerve fibers are closely associated with ICC-IM and PDGFRα-IM

IHC labeling in the mouse whole-mount LES and esophagus with an antibody for vasoactive intestinal peptide (VIP, green) demonstrates the presence of VIP⁺ nerve fibers within smooth muscle and around skeletal muscle bundles a-b. a Dual IHC labeling for VIP (green) and Kit (red) revealed that ICC-IM are closely associated with VIP⁺ nerve fibers in the LES clasp (i), distal (ii), middle (iii) and proximal esophagus (iv). b shows similar associations between VIP⁺ nerve fibers (green) and PDGFRα-IM (red) in the LES clasp (i) and throughout the esophagus (ii-iv).

Figure 7. Cholinergic nerve fibers are closely associated with ICC-IM and PDGFRα-IM

IHC labeling in the mouse whole-mount LES and esophagus using an antibody against vesicular acetylcholine transporter (VAChT, green). Panels a-b show VAChT⁺ nerve fibers in the LES clasp (i) and throughout the esophagus (ii-iv). a Co-labeling of the same preparations with Kit (red) demonstrated very close associations between cholinergic nerve fibers (green) and ICC-IM (red) in the mouse LES clasp (i), distal (ii), middle (iii) and proximal esophagus (iv). b Co-labeling of VAChT (green) and PDGFRα (red) revealed that PDGFRα-IM also formed close contacts with cholinergic neurons in the LES clasp (i), and throughout the esophagus (ii-iv).

Figure 8. Adrenergic nerve fibers are closely associated with ICC-IM and PDGFRα-IM

Tyrosine hydroxylase (TH) antibody labeling was used to identify adrenergic nerve fibers in whole-mount mouse LES and esophagus. Panels a-b show the presence of TH⁺ nerve fibers (green) in the LES clasp (i) and throughout the esophagus (ii-iv). a Co-labeling with Kit (red) demonstrated close associations between TH⁺ nerve fibers and ICC-IM in the mouse LES clasp (i), distal (ii), middle (iii) and proximal esophagus (iv). b Co-labeling of TH (green) and PDGFRα (red) revealed that PDGFRα-IM also made contact with adrenergic neurons in the LES clasp (i), and throughout the esophagus (ii-iv).

Figure 9. GFAP+ cells form close contacts with ICC-IM and PDGFRα-IM

a Co-labeling of whole-mount mouse esophagus and LES with antibodies against Kit (red) and glial fibrillary acidic protein (GFAP, green) revealed that ICC-IM make contact with GFAP⁺ cells in the LES (i) and esophagus (ii-iv). b Co-labeling of whole-mount mouse LES and esophagus tissues antibodies against PDGFRα (red) and GFAP (green) revealed that PDGFRα-IM also formed close contacts with GFAP⁺ cells in the LES clasp (i), and esophagus (ii-iv). GFAP⁺ cells were found within muscle bundles and varied in morphology, from spindle-shaped cells with distinct cell bodies and long projections (ai-aiv, bi, biii) to clusters of branched cells that appeared to form basket-like-structures (bii, biv).