The effects of acupuncture on bladder interstitial cells of cajal excitability in rats with overactive bladder

Abstract

It is well known that acupuncture treatment has an effect on patients with an overactive bladder, but the mechanism of its action remains to be clarified. This study was aimed to investigate the effects of acupuncture on bladder overactivity, and the excitability of interstitial cells of Cajal of the bladder in a rat model of partial bladder outlet obstruction. Electroacupuncture (continuous wave, 30 Hz, 1 mA) was applied to stimulate the Ciliao point (BL32) and the Huiyang point (BL35) of rats for 20 min, 3 days. Results showed that acupuncture suppressed detrusor unstable contraction frequency and decreased detrusor maximum pressure in the bladder filling period. Compared with the normal control rats, HCN2 mRNA and protein expression within the bladder were upregulated and were reversed by electroacupuncture in overactive bladder rats as determined by RT-PCR, western blotting and immunohistochemistry. Moreover, in-vitro cell-cultured OAB rats bladder interstitial cells of Cajal intracellular Ca(2+) concentration were higher than normal control rats, which were lowered after acupuncture treatment. These findings suggest that acupuncture stimulation can suppress bladder overactivity, and regulate the excitability of bladder interstitial cells of Cajal in treatment of overactive bladder myogenic mechanism.

Figure 1

The original tracing of cystometrogram after treatment. (a) Normal control group (b) model group, and (c) acupuncture group; (d): Glivec group.

Figure 2

The detrusor unstable contraction frequency in bladder filling period before and after treatment for normal control, model, acupuncture, and Glivec groups. ▲ after treatment versus before treatment P < 0.05. ∗ versus normal control group P < 0.05. △ versus model group P < 0.05.

Figure 3

The detrusor unstable contraction maximum pressure in bladder filling period before and after treatment for normal control, model, acupuncture, and Glivec groups. ▲ after treatment versus before treatment P < 0.05. ∗ versus normal control group P < 0.05. △ versus model group P < 0.05.

Figure 4

Relative expression level of the HCN2 mRNA in bladder determined by RT-PCR. ∗ versus normal control group P < 0.05. △ versus model group P < 0.05.

Figure 5

Relative expression level of the HCN2 protein in bladder determined by Western blotting. ∗ versus normal control group P < 0.05. △ versus model group P < 0.05.

Figure 6

Changes of distribution and quantity in bladder ICCs. ((a), (b), (c), and (d)) Immunofluorescence of c-Kit in bladder. Nucleus stained by DAPI (blue), c-Kit (red) in urothelium, suburothelia, and muscle layer. c-Kit-positive ICCs (↑). (a) Normal control group, (b) model group, (c) acupuncture group, and (d) Glivec group. Scale bar is 50 μm. (e) Comparisons of relative quantity of ICCs in bladder after treatment. ∗ versus normal control group P < 0.05.

Figure 7

The quantity of HCN2 channel in bladder ICCs. ((a), (b), (c), and (d)) Double-labeled immunofluorescence of HCN2 and c-Kit in bladder. Nucleus stained by DAPI (blue), c-Kit (green), and HCN2 (red) staining in urothelium, suburothelium, and muscle layer. Some ICCs were c-kit positive (↑), some were HCN2 positive (◆), and some were double labeled (▲). (a) Normal control group, (b) model group, (c) acupuncture group, (d) Glivec group. Scale bar is 50 μm. (e) Comparisons of relative quantity of HCN2 channel in bladder ICCs after treatment. ∗ versus normal control group P < 0.05. △ versus model group P < 0.05.

Figure 8

The morphology of ICCs (↑) and detrusor cell (▲) under inverted microscope (×400).

Figure 9

The c-Kit immunofluorescence of ICCs under Inverted Fluorescence Microscope (×400). (a) c-Kit-positive ICCs (green). (b) Nucleus stained by DAPI (blue). (c) Merge.

Figure 10

Comparisons of relative bladder ICCs intracellular-free Ca²⁺ concentration among four groups after treatment. ∗ versus normal control group P < 0.005, △ versus model group P < 0.005.