Immunohistochemical characterization of interstitial cells and their spatial 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, yet their function in the esophagus remains unknown. The mouse esophagus has been described as primarily skeletal muscle; however, ICC have been identified in this region. This study characterizes the distribution of skeletal and smooth muscle cells (SMCs) and their spatial relationship to ICC, PDGFRα⁺ cells, and intramuscular motor neurons in the mouse esophagus. SMCs occupied approximately 30% of the distal esophagus, but their density declined in more proximal regions. Similarly, ANO1⁺ intramuscular ICC (ICC-IM) were distributed along the esophagus, with density decreasing proximally. While ICC-IM were closely associated with SMCs, they were also present in regions of skeletal muscle. Intramuscular, submucosal, and myenteric PDGFRα⁺ cells were densely distributed throughout the esophagus, yet only intramuscular PDGFRα⁺ cells in the lower esophageal sphincter (LES) and distal esophagus expressed SK3. ICC-IM and PDGFRα⁺ cells were closely associated with intramuscular nNOS⁺, VIP⁺, VAChT⁺, and TH⁺ neurons and GFAP⁺ cells resembling intramuscular enteric glia. These findings suggest that ICC-IM and PDGFRα⁺ cells may have roles in regulating esophageal motility due to their close proximity to each other and to skeletal muscle and SMCs, although further functional studies are needed to explore their role in this region. The mixed muscular composition and presence of interstitial cells in the mouse distal esophagus is anatomically similar to the transitional zone found in the human esophagus, and therefore, motility studies in the mouse may be translatable to humans.

Keywords: Glia; Interstitial cells of Cajal; Motility; Neurons; PDGFRα+ cells.

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

a Anatomy 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. b Removal 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)).

Fig. 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. b The 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.

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

Double immunohistochemical (IHC) labeling of 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)

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

a Double 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 Double 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) double IHC 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).

Fig. 5. Nitrergic and peptidergic innervation of the mouse esophagus

Double IHC labeling on cryosections of WT mouse esophagus with a pan neuronal antibody, Anti-Hu (red) and antibodies against nNOS or VIP (green), show the presence of nitrergic and peptidergic neurons in the submucosal region, within the circular and longitudinal muscle layers and on the myenteric plane between the circular and longitudinal muscles. A sparse myenteric plexus is visible in the distal a,d, middle, b,e and proximal c,f regions of the esophagus as identified with the anti-Hu antibody (red). Antibodies against nNOS and VIP were used to determine the extent of nitrergic and peptidergic innervation respectively. Panels ai-ci show the presence of nNOS⁺ nerve fibers (green) in the myenteric plexus throughout the entire length of the esophagus. nNOS⁺ nerve fibers are also present in the submucosal and intramuscular regions. Panels di-fi show VIP⁺ nerve fibers (green) in the submucosa and within the muscle layers. VIP⁺ nerve fibers were only evident in the myenteric plexus in the distal esophagus di. Smaller panels indicated by i are magnified images of the panels to their left. (CM= Circular muscle layer, LM= Longitudinal muscle layer, *= myenteric plexus, # = submucosa)

Fig. 6. Cholinergic and adrenergic innervation of the mouse esophagus

Double IHC labeling on cryosections of WT mouse esophagus with an anti-Hu antibody (red) and antibodies against vesicular acetylcholine transporter (VAChT) a-c or tyrosine hydroxylase (TH) d-f nerve fibers (green), show the presence of cholinergic and adrenergic neurons in the submucosal region, within the circular and longitudinal muscle layers and on the myenteric plane. Antibodies against VAChT and TH were used to determine the extent of cholinergic and adrenergic innervation respectively. Panels ai-ci show the presence of cholinergic nerve fibers (green) in the submucosa and within the muscle layers. Cholinergic nerves are also evident in the myenteric plexus (Panels ai, ci), however the innervation was sparse. Panels di-fi show abundant submucosal and intramuscular TH⁺ neurons (green). TH⁺ neurons were abundant in the myenteric plexus within the distal, middle and proximal esophagus. Smaller panels indicated by i are magnified images of the panels to their left. (CM= Circular muscle layer, LM= Longitudinal muscle layer, *= myenteric plexus, # = submucosa)

Fig. 7. Intramuscular nitrergic and peptidergic 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 a-b or an antibody against vasoactive intestinal peptide (VIP) c-d (green) demonstrates the presence of nitrergic neurons and peptidergic neurons within smooth muscle and around skeletal muscle regions throughout the LES and esophagus a-d. a Double IHC labeling using nNOS (green) and Kit (red) revealed that ICC-IM are closely associated with intramuscular 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). c Double IHC labeling with VIP (green) and Kit (red) antibodies revealed that ICC-IM are closely associated with VIP⁺ nerve fibers in the LES clasp (i), distal (ii), middle (iii) and proximal esophagus (iv). d shows similar associations between VIP⁺ nerve fibers (green) and PDGFRα-IM (red) in the LES clasp (i) and throughout the esophagus (ii-iv).

Fig. 8. Intramuscular cholinergic and adrenergic 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) a,b or tyrosine hydroxylase (TH, green) c,d showing cholinergic and adrenergic nerve fibers in the LES clasp (i) and throughout the esophagus (ii-iv). a Co-labeling of the same preparations with a Kit antibody (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 with VAChT (green) and PDGFRα (red) antibodies revealed that PDGFRα-IM also formed close contacts with intramuscular cholinergic neurons in the LES clasp (i), and throughout the esophagus (ii-iv). c Co-labeling with Kit antibody (red) demonstrated close associations between intramuscular TH⁺ nerve fibers and ICC-IM in the mouse LES clasp (i), distal (ii), middle (iii) and proximal esophagus (iv). d Double IHC labeling with TH (green) and PDGFRα (red) antibodies revealed that PDGFRα-IM also made contact with adrenergic neurons in the LES clasp (i), and throughout the esophagus (ii-iv).

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

a Double IHC 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 Double IHC 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).