Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal hypersensitivity

Abstract

Afferent input contributes significantly to the pain and colorectal hypersensitivity that characterize irritable bowel syndrome. In the present study, we investigated the contributions of mechanically sensitive and mechanically insensitive afferents (MIAs; or silent afferents) to colorectal hypersensitivity. The visceromotor response to colorectal distension (CRD; 15-60 mmHg) was recorded in mice before and for weeks after intracolonic treatment with zymosan or saline. After CRD tests, the distal colorectum with the pelvic nerve attached was removed for single-fiber electrophysiological recordings. Colorectal afferent endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. Intracolonic zymosan produced persistent colorectal hypersensitivity (>24 days) associated with brief colorectal inflammation. Pelvic nerve muscular-mucosal but not muscular mechanosensitive afferents recorded from mice with colorectal hypersensitivity exhibited persistent sensitization. In addition, the proportion of MIAs (relative to control) was significantly reduced from 27% to 13%, whereas the proportion of serosal afferents was significantly increased from 34% to 53%, suggesting that MIAs acquired mechanosensitivity. PGP9.5 immunostaining revealed no significant loss of colorectal nerve fiber density, suggesting that the reduction in MIAs is not due to peripheral fiber loss after intracolonic zymosan. These results indicate that colorectal MIAs and sensitized muscular-mucosal afferents that respond to stretch contribute significantly to the afferent input that sustains hypersensitivity to CRD, suggesting that targeted management of colorectal afferent input could significantly reduce patients' complaints of pain and hypersensitivity.

Fig. 1.

Visceromotor responses (VMRs) to colorectal distension (CRD). VMRs were normalized to the baseline (day 0) response at 60 mmHg. A and B: responses to CRD (15–60 mmHg) recorded over time from 11 saline (control)-treated mice (A) and 11 zymosan (Zym)-treated mice (B). VMRs were unchanged after saline treatment (F_5,50 = 0.32) but significantly increased at all times except day 3 after Zym treatment (F_5,40 = 4.37, P = 0.003).

Fig. 2.

F4/80-immunoreactive macrophages (amorphous cells in red) were mainly detected in the lamina propria of the mucosa in control tissue. Tissue sections were counterstained with 4′,6-diamidino-2-phenylindole (nuclei in blue). Four and twenty-four hours after the third daily intracolonic treatment with Zym, an accumulation of macrophages at the bottom of the crypts and in the submucosa in the distal colorectum was evident (boxed area), which resolved by day 6. The isotype control antibody, rat κ monoclonal IgG_2b (1:1,000, Abcam, Cambridge, MA), showed no staining, indicating that the immunostaining with F4/80 antibody was not due to nonspecific binding of immunoglobulins to Fc receptors expressed on immune cells.

Fig. 3.

Protein gene product (PGP)9.5 immunostaining of the distal colorectum. A–C: PGP9.5-immunoreactive nerve fibers in A were quantified as areas (proportions) with respect to the colorectal mucosal and submucosal area (B), which were normalized by control mean PGP9.5 proportions (C). Intracolonic treatment with Zym did not affect the density of PGP9.5 immunoreactivity.

Fig. 4.

Classes of pelvic nerve afferent colorectal endings. Afferent endings were identified by electrical stimulation (e-stim; arrows indicate stimulus artifacts) and classified based on responses to mechanical stimuli. Mechanically insensitive afferents (MIAs) do not respond to mechanical stimuli. All four classes of mechanosensitive endings responded to probing (0.4–1.4 g). Muscular/mucosal (mus/mucos) endings also responded to mucosal stroking (10 mg) and circumferential stretch (0–170 mN). Muscular (mus) endings also responded to stretch, and mucosal (mucos) endings also responded to stroking. Serosal endings did not respond to either stroking or stretch.

Fig. 5.

Proportions of colorectal pelvic nerve afferents after the intracolonic instillation of saline or Zym. A and B: proportions of afferent recorded from mice 24 days after saline (A) or Zym (B) treatment and tested for colorectal hypersensitivity (data shown in Fig. 1, A and B, respectively).

Fig. 6.

Responses of serosal afferents to graded probing (0.4, 1, and 1.4 g) in mice treated with saline or Zym. A and B: Zym did not affect the response magnitude (A) but did produce reduced proportions of fibers activated by 0.4-g probing (B). *P < 0.05.

Fig. 7.

Effect of Zym on stretch-sensitive afferents. Stretch-sensitive muscular and muscular-mucosal afferents were tested with ramped circumferential stretch (0–170 mN at 5 mN/s), corresponding to intraluminal pressures of 0–45 mmHg (7). Action potentials were evenly binned into three bins and plotted as responses to stretch. A and B: responses of muscular/mucosal afferents to stretch were significantly increased after Zym treatment (A; F_1,63 = 5.415, P < 0.03), whereas responses of muscular afferents to stretch were unaffected (B; F_1,96 = 0.107). *P < 0.05.