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					Jan. 2008, Volume 5, No.1 (Serial No.38)

Journal of US-China Medical Science, ISSN1548-6648, USA

Expressions of Substance P in Trigeminal Ganglia in Rats with Occlusal Reconstruction∗
LI Xiao-guang1,2, ZHU Sheng-rong1, XIAO Chang-jie2, WANG Yan-xiu3
(1. Department of Stomatology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030; 2. Department of Stomatology, Tai’an City Central Hospital, Tai’an 271000; 3. Department of Anesthesiology, Tai’an City Central Hospital, Tai’an 271000)

Abstract: Objective To study the expressions of substance P (SP) in trigeminal ganglia (TG) in rats with occlusal reconstruction. Methods 30 Wistar male rats were randomly divided into 3 experimental groups and 3 control groups, 5 rats in each group. The molar of right maxillary and mandibular of rats in experimental groups were ground to the gingival level without occlusal contact. The occlusal contact was recovered by stopping grounding molar of the rats. The section of trigeminal ganglia underwent the immunohistological study to evaluate the expressions of SP by using SABC method. Light microscope and microscoic photo analytic software were employed to detect the percentage of SP positive neurons in frozen section of TG in 30 rats. SPSS10.0 statistical software was used for statistical analysis. Results There was no significantly difference in the percentage of SP positive neurons between the early occlusal reconstruction experiment group and control group (P>0.05). There was significantly difference in the percentage of SP positive neurons between the later occlusal reconstruction experiment group and control group (P<0.01, P<0.05). Conclusion The expressions of SP in TG can recover normal in the early occlusal reconstruction and that not in the later occlusal reconstruction. SP might participate in the histopathologic mechanism of temporomandibular disorders. Key words: substance P; trigeminal ganglia; occlusal reconstruction; rats

INTRODUCTION
Temporomandibular disorders are a common ailmen in oromaxillo-facial region. More and more scholars had thought highly of the role of nerves factor during the TMD developing and prognosis. Recently researches have showed that sensory nerve and trigeminal ganglia was nerve ingredient of the TMD, which through released neuropeptide to take part in the genesis and development of the disease. Previous studies also demonstrated that unilateral mastication may cause TMD and changed the SP expression in TG[1-5]. Occlusal reconstruction could recover the pristine joint damage, but there was not report on the expression of SP in TG. In the present study we mainly focused on the expression of SP in TG by the Wistar rats’ occlusal reconstruction, so as to provide with theoretical basis for further studies of TMD.

MATERIALS AND METHODS
*

Acknowledgements: This work was supported by a grant from the Tai’an City Science and Technique Bureau (No. 2003-52). ZHU Sheng-rong (1952- ), male, Ph.D. supervisor, director of Department of Stomatology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology; research field: oral maxillofacial surgery. LI Xiao-guang (1962- ), male, doctor candidate in the Department of Stomatology, Tai’an City Central Hospital. 13

Expressions of Substance P in Trigeminal Ganglia in Rats with Occlusal Reconstruction

1. Animal Grouping 30 Wistar male rats on postnatal days 4 weeks were randomly divided into 3 experimental groups and 3 control groups, 5 rats in each group, and executed in 4, 10, 16 weeks. All the animals were supplied by the Shandong University animal center. The experimental groups were general anaesthesia with 2% sodium pentobarbital through intraperitoneal injection. Then the molar of right maxillary and mandibular of rats were ground to the gingival level without occlusal contact weekly, so as to set up an animal model with unilateral mastication. The occlusal contact was recovered by stopping grounding molar of the rats. The 4 week group animals were executed after 4 weeks and the 10, 16 week group stop stripping the molar after 3, 9 weeks respectively. 2. Specimen Preparing Animals were anesthetized with sodium pentobarbital, transcardially perfused with 0.2% saline and fixed in 40g/ L paraformaldehyde. Then tissues including the hibateral trigeminal ganglia were cryoprotected in 30% sucrose solution for 24 hours, 17μm thickness serial sections were prepared for studying the immunohistochemical study by SABC Method. 3. Immunohistochemistry Sections were incubated sequentially in the following solutions: (1) rabbit anti-SP (1: 50) for 24h at 4℃; (2) biotinylated goat anti-rabbit IgG for 20min at 37℃; (3) avidin-biotin complex for 20min at 37℃; Following each step, the sections were rinsed with 0.01M PBS, 3×5min. For control, some sections were processed as above but with the omission of the first antibody, which resulted in no SP staining. 4. Data Acquisition and Analysis Light microscope was used to detect hibateral TG sectiones and microscoic photo analytic software was employed to detect the percentage of SP positive neurons in frozen section of TG in 30 rats. SPSS10.0 statistical software was used for statistical analysis. Positive neurons were brown.

RESULTS
The SP-positive cells in the trigeminal ganglia were displayed as follows (Fig.1). The percentage of SP positive neurons in TG with Unilateral Chew experimental group was significantly decreased compared with control group (P<0.01, P<0.05). The decreased extent in non-chewing side was much lower than that in the chewing side (P<0.05). There was no significantly difference in the percentage of SP positive neurons between the early occlusal reconstruction experiment group and control group (P>0.05) and there was no significantly difference between the non-chewing side and the chewing side (P>0.05). There was significantly difference in the percentage of SP positive neurons between the later occlusal reconstruction experiment group and control group (P<0.01, P<0.05) and there was significantly difference between the non-chewing side and the chewing side (P<0.05). The percentage of SP positive neurons in frozen section of TG was listed in Table 1.

Fig. 1 The SP positive neurons in the trigeminal ganglia (DAB staining×400)

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Expressions of Substance P in Trigeminal Ganglia in Rats with Occlusal Reconstruction

Table 1 Percentage of SP positive neurons in TG of rats ( X ±s, %, n=5) Group Left TG (chewing side) Right TG (non-chewing side) 4 week experimental groups 21.4±2.2 18.5±1.7* 4 week control groups 10 week experimental groups 10 week control groups 16 week experimental groups 16 week control groups group: P<0.05.
*

24.8±2.1△△ 24.7±2.1 24.9±2.2 22.0±1.7 25.2±1.9△△
△ △△

24.4±2.2△ 24.3±2.0 24.5±2.1 * 19.0±1.8 24.8±1.9△ P<0.05. Compared with the opposite side TG of same

Note: Compared with the same side TG of control group: P<0.01,

DISCUSSION
SP is a neuropeptide substance which contained 11 amino acids[6]. It is a primary neurosensory excitatory transmitter and synthesized by the cell body of the first afferent neuron. Now people have provided that cells of ganglion trigeminale could synthesis SP, then transport it to nucleus sensorius nervi trigemini and temporomandibular joint through central and peripheral process. It takes the role of transmit the nerval message. SP also reside in the oral tissue[7, 8]. Recently researches have showed that sensory nerve could release neuropeptide to take part in the genesis and development of the arthral disease. Data showed that there was no significantly difference between the control groups, which demonstrated that the expression of SP in TG was stabile at normal condition, and the secretary volume almost equivalent for the hibateral TG. The percentage of SP positive neurons in TG with Chew side was significantly decreased compared with non-chewing side for the 4 week experimental groups. WU[9] discovered that the mRNA of PPTA for encoding SP could take place time course changing with nociceptive stimulus. Findings showed that the PPTAmRNA in hibateral TG became up-regulation after the lateral chewing, and there had significant difference between non-chewing side and control group, the same to the chewing side and non-chewing side. Our results showed that unilateral mastication led to the TMD, which could accelerate SP translation from TG to TMJ, and make the expression descend. SP could be precipitated to release from primary afferent termination with nociceptive stimulus[10]. Studies had showed that positive immunity fiber increased in the unilateral mastication group, which illustrated directly the reduction of SP in TG due to the inflammatory stimulation[11]. 10 week experimental groups stopping grounding molar in 3th week, and it could recover occluding relation at 5th week. It contained 4 weeks unilateral mastication and 6 weeks restoration stage in this group. There was no significantly difference in the percentage of SP positive neurons between the early occlusal reconstruction experiment group and control group, which indicated that expressions of SP in TG can recover normal in the early occlusal reconstruction. There were other studies showed that the TMJ could recovery after the occlusal reconstruction in 10th week. All this proved that SP might participate in the histopathologic mechanism of temporomandibular disorders. 16 week experimental groups stopping grounding molar at 9th week, and it could recover occluding relation at 11th week with the eruption. It contained 10 weeks unilateral mastication and 6 weeks restoration stage in this group. We found that the percentage of SP positive neurons in the chewing and non-chewing side was significantly decreased compared to the control group. Above all, we can derive that SP in TG can not recover in the later occlusal reconstruction. Other studies also showed that the TMJ could not recovery at 16th week after 10
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Expressions of Substance P in Trigeminal Ganglia in Rats with Occlusal Reconstruction

weeks unilateral mastication[1]. This proved that the expression of SP in TG was coincident to the damage level of TMJ. It is obvious that SP participate in the mechanism of temporomandibular disorders.

REFERENCES
LI Xiao-guang, YU Xiao-peng, WANG Yan-xiu, et al. Changes of temporomandibular joint in occlusal reconstruction for unilateral chewing rats. Journal of Comprehensive Stomatology, 2005, 21(6): 625-627. [2] LI Xiao-guang, LIU Tai-sheng, GAO De-an, et al. Changes of masticatory muscles after occlusal recovery in rats with unilateral chew. Shanghai Journal of Stomatology, 2005, 14(5): 499-503. [3] WANG Yan-xiu, ZHAO Hai-lan, LU Hui-qing, LI Xiao-guang, JING Wei, CAO Ling-ming. Changes of temporomandibular joint in unilateral chew in rats. Journal of Taishan Medical College, 2005, 26(2): 104-107. [4] WANG Yan-xiu, MA Gui-fen, WANG Chun, LI Xiao-guang, REN Xu-hua, WEI Shu-ming. Changes of mastication muscles in unilateral chew in rats. Journal of Taishan Medical College, 2005, 26(3): 218-221. [5] HE Yu-ling, FANG Xiu-bin, ZHENG Xiao-hui, LIU Chun-yi. The expression of PPTA mRNA, CGRP mRNA and their proteins in trigeminal ganglia of rats with unilateral chewing. Progress of Anatomical Sciences, 2004, 10(1): 5-8. [6] HUA Xiao-ying. The neuropeptide and its periph function in esthesioneure. Progress in Physiological Sciences, 1988, 19: 304-309. [7] Holmlund, A., Ekblom, A., Hansson, P., et al. Concentrations of neuropeptides substance P, neurokinin A, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide in synovial fluid of the human temporomandibular joint. Int J Oral Maxillofac Surg, 1991, 20: 228. [8] Aghabeigi, B., Henderson, B., Hopper, C., et al. Temporomandibular joint synovial fluid analysis. Br J Maxillofac Surg, 1993, 31(1): 15. [9] WU Shen-xi, WANG Ya-yun, LI Yun-qing, SHI Ji-wu. Temporal changes of PPTA, CGRP and PPEmRNAs and their derived peptides in rat caudal spinal trigeminal nucleus and trigeminal ganglion during adjuvant-induced inflammation. Chinese Journal of Neuroanatomy, 1998, 14(3): 222. [10] Noguchi, K., Morita, Y., Kiyama, H., et al. A noxious stimulus induce the preprotachykinin A gene expression in the rat dorsal root ganglion: a quantitative study using in situ hybridization histochemistry. Mol Brain Res, 1988, 4: 31-35. [11] HE Yu-ling, ZHAO Yong-kang, FANG Xiu-bin, DENG Yan. Changes of expressions of substance P and calcitonin gene-related peptide in rats with unilateral postern teeth loss. Journal of China Medical University, 2002, 31(4): 282-284. [1]

(Edited by Jane Chen, Joesmile)

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