Inhibition of CXCR4 in Spinal Cord and DRG with AMD3100 Attenuates Colon-Bladder Cross-Organ Sensitization

Abstract

Background: Cross-sensitization of pelvic organs is one theory for why symptoms of gut sickness and interstitial cystitis/bladder pain syndrome overlap. Experimental colitis has been shown to trigger bladder hyperactivity and hyperalgesia in rats. The chemokine receptor CXCR4 plays a key role in bladder function and central sensitization. We aim to study the role of CXCR4 and its inhibitor AMD3100 in colon-bladder cross-organ sensitization.

Methods: The colitis model was established by rectal infusion of trinitrobenzene sulfonic acid. Western blot and immunofluorescence were used to assess the expression and distribution of CXCR4. Intrathecal injection of AMD3100 (a CXCR4 inhibitor) and PD98059 (an ERK inhibitor) were used to inhibit CXCR4 and downstream extracellular signal-regulated kinase (ERK) in the spinal cord and dorsal root ganglion (DRG). Intravesical perfusion of resiniferatoxin was performed to measure the pain behavior counts of rats, and continuous cystometry was performed to evaluate bladder voiding function.

Results: Compared to the control group, CXCR4 was expressed more in bladder mucosa and colon mucosa, L6-S1 dorsal root ganglion (DRG), and the corresponding segment of the spinal dorsal horn (SDH) in rats with colitis. Moreover, intrathecal injection of the AMD3100 suppressed bladder overactivity, bladder hyperalgesia, and mastocytosis symptoms caused by colitis. Furthermore, AMD3100 effectively inhibited ERK activation in the spinal cord induced by experimental colitis. Finally, treatment with PD98059 alleviated bladder overactivity and hyperalgesia caused by colitis.

Conclusion: Increased CXCR4 in the DRG and SDH contributes to colon inflammation-induced bladder overactivity and hyperalgesia partly via the phosphorylation of spinal ERK. Treatment targeting the CXCR4/ERK pathway might provide a potential new approach for the comorbidity between the digestive system and the urinary system.

Keywords: ERK; colitis; pain; sensory afferents; urgency; visceral hypersensitivity.

Figure 1

Changes in histology and MPO activity in the colon and bladder during experimental colitis. (A) Colon and bladder tissues are stained with HE. (B) Injury score for colon and bladder tissues (n = 5). (C) Units of MPO activity per gram of the colon and bladder (n = 5). (D) CK20 immunohistochemistry in the bladder. **P < 0.01.

Figure 2

The expression of CXCR4 was increased in the colon and bladder after TNBS-induced colitis. (A) Immunofluorescence staining of CXCR4 in the colon and fusion picture of DAPI and CXCR4. (B) Immunofluorescence staining bladder sections, CXCR4 (red), DAPI (blue) are shown. Quantification of the fluorescence intensity in the colon (C) and bladder (D) tissue (n = 5). (E) Western blot results of CXCR4 in the colon and (F) quantitative analysis of Western blot analysis normalized to GAPDH (n = 6). (G) Western blot results of CXCR4 in the bladder and (H) statistical analysis of Western blotting results (n = 6). **P < 0.01.

Figure 3

Colonic inflammation increased CXCR4 expression level in the DRG and SDH. (A) Sections of L6-S1 DRG and spine were stained with anti-CXCR4 antibody. (B) Percentage of DRG neurons that were positive for CXCR4 expression (n = 5). (C) Quantification of the fluorescence intensity in L6-S1 segments of the spine (n = 5). (D) CXCR4 expression levels of L6-S1 DRG were detected by Western blotting. (E) Gray-scale analysis of Figure 3D (n = 6). (F) Western blot analysis of CXCR4 protein in the spine, and (G) gray-scale analysis of Western blot analysis results (n = 6). **P < 0.01.

Figure 4

Intrathecal AMD3100 improves bladder voiding function in colitis rats. (A) Pressure waveform during urination of the CON group, CON+AMD group, TNBS group, and TNBS+AMD group. (B) Data analysis of the intercontraction interval (ICI) (n = 5). (C) Data analysis of the maximum bladder pressure (MBP) (n = 5). *P < 0.05.

Figure 5

Intrathecal AMD3100 reduces the number of nociceptive behavior and bladder mast cells. (A) The number of licking events caused by resiniferatoxin (RTX) (n = 6). (B) The number of RTX-induced freezing events during 15 min (n = 6). (C) Representative toluidine-blue staining images of the rat bladder. Black arrows indicate mast cells. (D) Total bladder mast cells were counted in each group (n = 5). *P < 0.05, **P < 0.01.

Figure 6

Intrathecal AMD3100 decreases colitis-induced p-ERK1/2 in the spinal dorsal horn (SDH) but not DRG. (A) Representative Western blot results of p-ERK1/2 expression level and (B) quantification of Western blot analysis in SDH (n = 6). (C) Western blot results of p-ERK1/2 expression level and (D) quantification of Western blot analysis in DRG (n = 6). (E) p-ERK1/2 expression in L6-S1 SDH and DRG. **P < 0.01.

Figure 7

ERK antagonist decreases TNBS-induced bladder overactivity and hyperalgesia. (A) Western blot analysis of p-ERK1/2 expression level and (B) quantification of Western blot analysis in the spinal cord (n = 6). The effects of PD98059 on RTX-induced nociceptive behaviors: licking events (C) and freezing events (D) (n = 6). (E) Pressure waveform during urination of the CON group, CON+PD group, TNBS group, and TNBS+PD group. (F–G) Statistical analysis of the ICI and MBP (n = 5). *P < 0.05, **P < 0.01.