Alteration of P2X3 expression in dorsal root ganglia after sciatic nerve ligation

BACKGROUND： The expressions of P2X3 receptor in dorsal root ganglia （DRG） after different peripheral nerve injuries are diverse. It indicates the different roles of P2X3 in different models-caused neuropathologic pains. OBJECTIVE： To observe the expressions of P2X3 in corresponding DRG after sciatic nerve ligation in rots. 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 rots 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 rots 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. ① 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, ② 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 rots of experimental group were transcardially perfused. L4- 6 corresponding DRG connected to sciatic nerve were taken for preparing transverse sections serially. ③P2X3 expression in L4-6 DRG was detected by immunohistochemistry, immunofluorescence and image analysis techniques. MAIN OUTCOME MEASURES： P2X3 expression in L4-6 DRG of rots in each group. RESULTS： Thirty-five SD rats were involved in the final analysis. ① P2X3 expression in DRG： In normal DRG of rots, 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 ipsilateml L4-6 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. ② P2X3 immuno-positive gray scale in DRG： P2X3 immuno-positive gray scale in ipsilateral side L4-6 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 L4-6 DRG after sciatic nerve ligation. It may exert certain effects in neuropathic pain.

Spatiotemporal expression of inducible nitric oxide synthase and cyclooxygenase 2 in the spinal cord during early stage sciatic nerve crush injury

BACKGROUND： Previous studies have shown that inducible nitric oxide synthase （iNOS） and cyclooxygenase 2 （COX-2） participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury. However, few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury. OBJECTIVE： To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN, TIME AND SETTING： The randomized, controlled, animal experiment was performed at the Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South University, China from September 2006 to September 2007.MATERIALS： Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody （Transduction Laboratory, USA）, as well as biotinylated rabbit anti-mouse lgG and biotinylated rabbit anti-goat IgG （Santa Cruz Biotechnology, USA） were used in the present study.METHODS： A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three groups. In the model group （n = 32）, crush injury to the right sciatic nerve was established using an artery clamp. The model group was further assigned to four subgroups according to survival time （6,12, 24, and 72 hours）, respectively （n = 8）. Sham surgery （n = 8） and normal control （n = 8） groups were also established.MAIN OUTCOME MEASURES： iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the anterior horn of spinal cord, were measured.RESULTS： iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury （P〈0.05） and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side （P〈 0.05）.CONCLUSION： iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush, which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.

Co-expression of calretinin and parvalbumin in the rat facial nucleus

BACKGROUND： Calretinin and parvalbumin are members of the intracellular calcium binding protein family, which transform Ca^2＋ bioinformation into regulation of neuronal and neural network activities. OBJECTIVE： To observe expression and co-expression of calretinin and parvalbumin in rat facial nucleus neurons . DESIGN, TIME AND SETTING： Neuronal morphology experiment was performed at the Research Laboratory of Applied Anatomy, Department Neurobiology and Anatomy, Xiangya Medical College of Central South University from August to October 2007. MATERIALS： Five healthy, adult Sprague Dawley rats were selected. Polyclonal rabbit-anti-parvalbumin and mouse-anti-calretinin were provided by Sigma, USA. METHODS： Rat brains were obtained and cut into coronal slices using a freezing microtome. Slices from the experimental group were immunofluorescent stained with polyclonal rabbit-anti-parvalbumin and mouse-anti-calretinin antibodies. The control group sections were stained with normal rabbit and mouse sera. MAIN OUTCOME MEASURES： Immunofluorescent double-staining was used to detect calretinin and parvalbumin expression. Nissl staining was utilized for facial nucleus localization and neuronal morphology analysis. RESULTS： The majority of facial motor neurons was polygon-shaped, and expressed calretinin and parvalbumin. The calretinin-immunopositive neurons also exhibited parvalbumin immunoreactivity, that is, calretinin and parvalbumin were co-expressed in the same neuron. CONCLUSION： Calretinin and parvalbumin were expressed in facial nucleus neurons, with varied distribution.