Problem:
Abdominal pain frequently arises from aberrant neural regulation of the gastrointestinal (GI) tract, yet what we know about the anatomical arrangement and physiological mechanisms governing visceral nociceptive afferent axons is still very little. This incomplete understanding hinders the development of effective treatments that tackle pain in the digestive tract, especially chronic abdominal pain.
While vagal afferent innervation of the stomach along with its mechanical receptors in rats and mice have been extensively documented, our knowledge of the precise spatial distribution of nociceptive afferent innervation and spinal afferents in the stomach remains limited. This limitation arises due to the lack of advanced techniques that enable precise labeling, visualization, tracing, and digital characterization of different types of spinal afferent terminal structures, as well as IHC labeling of nociceptive axons in the whole stomach flat-mount.
A more comprehensive understanding of the structural organization of nociceptive afferents within the stomach is needed, if we are to effectively modulate physiological responses and harness bioelectronic therapies for the treatment of various conditions associated with gastric pain.
Solution:
Comprehensive labeling of CGRP axons in the flat-mount of whole stomach muscular layers at single cell/axon/varicosity scale.
Figure 1: Summary of the main findings of this study. CGRP-IR axons form an extensive terminal network in both ventral and dorsal mouse stomachs. The innervation aims at different targets, including the vasculature, myenteric plexus, and longitudinal/circular muscles.
Digital reconstruction of CGRP-IR axon innervation of a mouse stomach on a 3D organ scaffold.
Figure 2: Digital reconstruction of CGRP-IR axon innervation of a mouse stomach on a 3D organ scaffold. The digitization highlights the vasculature surfacing the muscular wall as well as the dense innervation of these axons on different gastric targets. Each axon is represented by a different color. Click here for an animated demonstration of the scaffold.
Digital characterization of single spinal afferent axons and terminals; anterograde tracing and Neurolucida reconstruction.
Figure 3: A. Digital reconstruction of the spinal afferent axons in the ventral stomach of a representative rat. Each individual axon is represented by a different color. B. Neurolucida tracing of a single spinal afferent axon innervating the fundic circular muscle. B1, B2. Brightfield photomicrographs of selected regions in B. C, D. Spinal afferent axons also innervate individual neurons within the myenteric ganglia (C) and blood vessels (D). Abbreviation: LES = lower esophageal sphincter.
By employing anterograde tracer injection into the left DRG, IHC labeling and utilizing a combination of imaging methods - including confocal and Zeiss Imager M2 microscopy, Neurolucida 360 tracing, and the integration of axon tracing data into a 3D stomach scaffold - we thoroughly examined CGRP-IR axons and their terminals in the entire muscular layers of the mouse stomach.
Remarkably, this enabled the first instance in which we have presented a topographical distribution map of nociceptive CGRP-IR axon innervation (Fig 1 & Fig 2), and labeled spinal afferent axons and their terminal structures (Fig 3), within the complete muscular layers of the stomach in such fine detail at a cellular/axonal/varicosity scale.
The impact:
This work establishes an anatomical groundwork for future functional mapping of nociceptive axons, anatomical remodeling in different diseases, and the facilitation of treatments for abdominal pain such as bioelectronics. Therefore, the findings of our two recent studies provide an anatomical characterization of neural circuitry in the GI tract that underlies peripheral nociceptive and GI disorders which gives insight to critical pain pathways.
Additionally, this research provides a strong groundwork for upcoming quantitative assessments of CGRP-IR axon innervation and spinal afferents at various locations within the stomach in both normal and pathological conditions. The combination of density analysis and the scaffold common coordinate framework will prove invaluable for comparing the structural organization of nociceptive pathways among diverse populations, including gender comparisons, mucosa vs submucosa layers and remodeling under pathological conditions.
From a clinical perspective, neural manipulation begins with the need to create a difference in the treatment of pain and tension, as well as the need to enhance proper functioning of the central, peripheral, and autonomic nervous system. And thus, a comprehensive neural map of different circuitries that process specific kinds of information like this serves as a guide to neurologists, neurosurgeons, and therapists, whose interventions are more target-specific and, when combined with other modalities, provide a more long-lasting treatment effect.
