Loss of visceral pain following colorectal distension in an endothelin-3 deficient mouse model of Hirschsprung's disease

Abstract

Endothelin peptides and their endogenous receptors play a major role in nociception in a variety of different organs. They also play an essential role in the development of the enteric nervous system. Mice with deletions of the endothelin-3 gene (lethal spotted mice, ls/ls) develop congenital aganglionosis. However, little is known about how nociception might be affected in the aganglionic rectum of mice deficient in endothelin-3. In this study we investigated changes in spinal afferent innervation and visceral pain transmission from the aganglionic rectum in ls/ls mice. Electromyogram recordings from anaesthetized ls/ls mice revealed a deficit in visceromotor responses arising from the aganglionic colorectum in response to noxious colorectal distension. Loss of visceromotor responses (VMRs) in ls/ls mice was selective, as no reduction in VMRs was detected after stimulation of the bladder or somatic organs. Calcitonin gene related peptide (CGRP) immunoreactivity, retrograde neuronal tracing and extracellular afferent recordings from the aganglionic rectum revealed decreased colorectal spinal innervation, combined with a reduction in mechanosensitivity of rectal afferents. The sensory defect in ls/ls mice is primarily associated with changes in low threshold wide dynamic range rectal afferents. In conclusion, disruption of endothelin 3 gene expression not only affects development and function of the enteric nervous system, but also specific classes of spinal rectal mechanoreceptors, which are required for visceral nociception from the colorectum.

Figure 1. Loss of visceromotor responses to rectal distension in ls/ls mice

A, electromyographic recording of visceromotor responses to incremental rectal distension in a control C57BL/6 mouse (up to 120 mmHg). B, rectal distension up to 120 mmHg did not evoke a measurable visceromotor response in ls/ls mouse, although somatic stimuli (tail pinch, hindlimb pinch, paw pinch and whisker pinch) were effective. C, quantification of visceromotor responses to rectal distension in control and ls/ls mice. D, distension pressures over 200 mmHg evoked small visceromotor responses in some, but not all ls/ls mice.

Figure 2. ls/ls mice show normal visceromotor responses to bladder distension

A, visceromotor responses in control mouse to graded bladder distensions (20–100 mmHg). B, similar responses were seen in ls/ls mice. C, averaged visceromotor responses did not differ significantly in controls and ls/ls mice. D and E, similar CGRP immunoreactivity from spinal afferents innervating the control and ls/ls mouse bladder detrusor muscle. F, no statistical difference was found in total density of CGRP immunoreactivity between the bladders of control and ls/ls mice when measured as a percentage of field of view.

Figure 3. Changes in visceromotor response evoked by focal electrical stimulation of the exposed colorectum in ls/ls mice

A, electromyographic responses evoked by calibrated tail pinch and hindlimb pinch did not differ between C57BL/6 and ls/ls mice. B, averaged responses to tail or hindlimb pinch for C57BL/6 and ls/ls mice showed no significant differences. C, focal electrical stimulation (1 Hz, 0.4 ms, 60 V, 10 s) of the surface of the exposed control rectum elicited brief visceromotor responses with latencies of ∼50 ms. D, VMRs elicited from the aganglionic colorectum were of reduced intensity. E, the average number per second of multiunit action potentials were significantly reduced in ls/ls mice.

Figure 4. DiI retrograde tracing and CGRP immunoreactivity reveals differences in spinal innervation from the aganglionic rectum of ls/ls mice

A, retrograde neuronal tracing from the rectum, 2–4 mm from the anal sphincter revealed DiI labelled sensory nerve cell bodies in dorsal root ganglia, with the peak distribution at L5 and L6 in both C57BL/6 and ls/ls mice. There were significantly less DRG labelled neurons in ls/ls mice. B and C, CGRP immunoreactivity in control mouse colorectum (B) and in aganglionic ls/ls colorectum (C). Dense immunoreactivity is present in the myenteric plexus of control mice, but it is significantly reduced in the aganglionic colorectum of ls/ls mice. D, CGRP immunoreactivity was significantly reduced in ls/ls mouse rectum, when measured as a percentage of field of view.

Figure 5. Characteristics of functional classes of distension-sensitive rectal afferents in wild-type and ls/ls mice

A, wild-type mouse: unit 1 is a muscular-mucosal afferent (sensitive to stroking and probing with light von Frey hairs); unit 2 is a serosal afferent (only activated by 200–500 mg von Frey hair probing. B, superimposed action potentials (×7) of units 1 and 2 confirm single unit recordings. C, recording from a different nerve reveals a muscular afferent (activated by small distensions) and a serosal afferent, with a typically higher distension threshold. D, superimposed action potentials confirm single unit recordings and show characteristic shapes of action potentials. E, plot of action potential amplitude and half-duration for each class (muscular, muscular-mucosal and serosal) show consistent differences in profiles. Typically, serosal afferents had a characteristic smaller amplitude and longer duration than muscular-mucosal afferents. F, comparison of total stretch-evoked firing from all functional classes of afferents (multi-unit recording from rectal nerves) for control C57BL/6 and ls/ls mice. Total stretch sensitivity of afferents was significantly reduced in ls/ls mice compared with controls.

Figure 7. Properties of high threshold serosal rectal afferents in C57BL/6 and ls/ls mice

A, the stretch sensitivity of high threshold rectal afferents did not differ between control and ls/ls mice (note the low rate of firing by comparison with Fig. 6E and G), although the sensitivity of serosal afferents to von Frey hair probing was significantly reduced in ls/ls mice at high intensity stimuli (B). C, numbers of serosal afferents in each rectal nerve did not differ significantly between ls/ls and control mice. D, comparison of thresholds to circumferential distension between 3 classes of afferents in both wild-type and ls/ls mice show that serosal afferents had significantly higher thresholds than either muscular or muscular-mucosal afferents. E and F, the relative proportions of each class of afferents recorded from rectal nerves in control and ls/ls mice differed, reflecting the reduced numbers of low threshold wide-dynamic range fibres. G shows relative contribution of low threshold (LT) and high threshold (HT) afferents on the y-axis in the total mean firing frequency (in Hz) in response to stretch. High threshold afferents contribute little to the total rectal nerve firing even at maximum stretch. H and I show that responses to stretch of low threshold capsaicin-sensitive and capsaicin-resistant rectal afferents were both significantly reduced in ls/ls mice.

Figure 6. Differences in firing of low threshold wide dynamic range distension-sensitive rectal afferents in control and ls/ls mice

A, two typical muscular-mucosal afferents from a control mouse, responded to both von Frey hair stroking and stretch. B, both units responded to capsaicin (10 μm) (superimposed action potentials shown in inset). C, a muscular-mucosal unit in an ls/ls mouse showed normal responses to mucosal stroking but diminished response to distension by a 10 g load, compare to A. D, the total count of low threshold mechanoreceptors (a mixture of muscular and muscular mucosal afferents) in recorded rectal nerve trunks showed a significant reduction in ls/ls mice compared to control animals. E and F, the stretch and probing sensitivity of muscular-mucosal afferents was significantly reduced compared to controls. G and H, muscular afferents of ls/ls mice showed a similar reduction in stretch and probe-induced firing compared to controls. Note that stretch-induced responses in both muscular and muscular-mucosal afferents did not saturate across the entire range of loads applied. For this reason, these two classes were both considered to belong to low threshold wide dynamic range mechanoreceptors.

Figure 8. Compliance of the aganglionic ls/ls colorectum did not differ from that of the control C57BL/6 bowel

A, typical example of force generated during ramp distension by imposed changes in length (100 μm s⁻¹, 4 mm) with averaged traces shown in B. C, typical example of lengthening of control colorectum evoked by a 40 g load (distension by imposed load) with averaged data in D not showing any difference between control and ls/ls mice. Changes in compliance are unlikely to explain different stimulus–response functions of mechanoreceptors between control and mutant mice.