Background

Objective

To observe the expressions of P2X3 in corresponding DRG after sciatic nerve ligation in rats.

Design

Controlled observation experiment.

Setting

Department of Morphology, Hunan Traditional Chinese Medical College; Department of Human Anatomy and Neurobiology, Xiangya Medical College, Central South University.

Materials

Thirty-five healthy adult SD rats of clean grade an d either gender, weighing (200±20)g, were involved. According to the random digits table, the involved rats were randomized into 3 groups: normal group (n =5), sham-operated group (n =5) and experimental group (n =25). The experimental group were subdivided into 3,7,14,21,28 days groups according to different surviving time after operation, 5 rats at each time point. Polyclonal rabbit anti-P2X3 antibody (ABCAM company); biotinylated goat anti-rabbit IgG (Zhongshanjingqiao Biotechnical Co., Ltd., Beijing); Motic fluorescence microscope (Motic, Germany).

Methods

The experiments were carried out in the Department of Human Anatomy and Neurobiology, Xiangya Medical College, Central South University from June to December 2006. Rats of experimental group were created into models by ligation of right sciatic nerve according to the method of Seltzer et al. Left sciatic nerve was used as self-control. As for rats in the sham-operated group, ligation of sciatic nerve was omitted, but other procedures were the same as those in the experimental group. Rats of normal group were untouched. Rats of the normal group and sham-operated group survived for 14 days separately, and those of experimental group survived for corresponding time. After being deeply anesthetized by intraperitoneal injection of over-dose sodium pentobarbital, the rats of experimental group were transcardially perfused. L_4-6 corresponding DRG connected to sciatic nerve were taken for preparing transverse sections serially. P2X3 expression in L_4-6 DRG was detected by immunohistochemistry, immunofluorescence and image analysis techniques.

Main outcome measures

P2X3 expression in L_4-6 DRG of rats in each group.

Results

Thirty-five SD rats were involved in the final analysis. P2X3 expression in DRG: In normal DRG of rats, there were abundant P2X3 immuno-positive small- and medium-sized primary sensory neurons, especially the small ones, which mostly received the input from C fibers. There were only a few large neurons expressing P2X3. The immuno-positive products mostly were located in the cytoplasm and processes. The expression of P2X3 had a slight but significant decrease in ipsilateral L_4-6 DRG 3 days after sciatic nerve ligation, and a decreasing tendency was observed with the elongation of time. At 28 days, the expression had not returned to base line, and still maintained at a low level. P2X3 immuno-positive gray scale in DRG: P2X3 immuno-positive gray scale in ipsilateral side L_4-6 DRG was 117.74±2.38,129.12±4.86,133.56±3.79,148.75±6.90 and 150.49±5.15, respectively at 3,7,14, 21 and 28 days after sciatic nerve ligation, which was significantly higher than that in the normal group and sham-operated group (105.11±3.52,104.22±5.41,F =78.861,P < 0.05), also significantly higher than that in the contralateral side (105.53±5.85,108.54±3.70,104.07±4.16,106.55±2.02,106.29±5.19,t =3.48-13.95,P < 0.05); There were no significant differences when comparing sham-operated group or contralateral side at each time point with normal group (P > 0.05)

Conclusion

P2X3 is significantly down regulated in L_4-6 DRG after sciatic nerve ligation. It may exert certain effects in neuropathic pain.