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Qulian Guoa， Di Fua， Yuhang Aia， Jianqin Yana， Hongwei Cai， Ruping Daia^，b，a

　　The present study investigated the expression patterns of glial cells and interleukin－1β （IL－1b） in the rat spinal cord after a surgical incision which is closely related with clinical postoperative pain. Microglia and astrocytes became activated in the spinal cord following incision. Real－time polymerase chain reaction （PCR） and immunohistochemisty showed that IL－1b mRNA and protein level in the spinal cord was transiently up regulated after surgical incision. The increased IL－1b－immunoreactivity （IR） was mainly localized in neurons but not the activated microglia or astrocytes. Although obvious increase in IL－1b－IR could be observed in the lumbar segments of the spinal cord ipsilateral to a hind paw incision， significant up regulation of IL－1b was not detected in the lumbar segments following thoracic incision. The present study indicated that surgical incision could induce glial activation and segmental up regulation of IL－1b in the spinal cord. The activated glial cells and up regulated IL－1b， in turn， may be involved in the incision－induced pain hypersensitivity.

　　Keywords： Cytokines， postoperative pain， spinal cord， Interleukin－1b （IL－1b）

INTRODUCTION

　　Injuries in peripheral nervous system or peripheral tissue inflammation often result in neuropathic or inflammatory pain hypersensitivity， including hyperalgesia （exaggerating and prolonging the nociceptive pathway to noxious inputs） and allodynia （enabling innocuous inputs to activate nociceptive pathway） （1－3）. Growing evidence has shown that glial cells and interleukin－1b （IL－1b） are activated in the spinal cord in these injuries and contribute to the development of inflammatory and neuropathic pain hypersensitivity （4－6）.

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For example， the injection of inflammatory agents such as zymosan in the hind paw of the rat activated glial cells and increased IL－1b expression in the spinal cord， which correlated with the development of allodynia （7）. It has also been reported that intrathecal administration of IL－1b also produces a significant hyperalgesic effect （8－9） or mechanical allodynia （10）. Furthermore， injecting IL－1b antibody or glial cells inhibitor could attenuate the inflammatory pain hypersensitivity induced by zymosan hind paw injection （11）. However， it is still elusive whether there is a similar expression pattern of the glial cells and IL－1b in other pathological pain states such as postoperative pain.

　　Postoperative incisional pain is a unique and common form of acute pain which can also induce the exaggerated pain states （12）. It has been demonstrated that non－NMDA receptors were linked to the pain hypersensitivity induced by surgical incision whereas NMDA receptors were believed to play critical roles in other types of pathological pain （13， 14）. Early study also suggested that there were distinct neurochemical changes in the spinal cord between incision－induced pain and other types of pathological pain （1）. It remains to be ascertained if IL－1b in the spinal cord， could be involved in pain hypersensitivity induced by surgical incision. Once activated， the glial cells and cytokine may be involved in the incision－induced pain hypersensitivity.

　　Various experimental incision－induced pain models such as abdominal and hind paw incisions have been established to mimic clinical postoperative pain （15， 16）. In the model of hind paw incision， mechanical hypersensitivity to stimuli and non－evoked pain behavior were well correlated with incident and rest pain in clinics.

　　The present study used thoracic surgical incision rat model to investigate the effect of surgical incision on the glial response and the expression of IL－1b in the spinal cord. Once the upreulgaiton of IL－1β was observed， the present study would further determine the distribution and localization of IL－1β following surgical incision.

EXPERIMENTAL PROCEDURE

　　Animals.

　　This study was carried out on male Wistar rats （150? 250 g） obtained from Central South University Animal Services （Changsha， China）.

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The experimental protocol was approved by the Animal Care and Use Committee of Central South University and conformed by IASP guidelines for the study of pain in animals （17）. All efforts were made to minimize the number of rats used and their suffering.

　　Surgical preparation and groups.

　　An incision model in the rats was established through a deep thoracic incision. In brief， adult male rats were anesthetized with diethyl ether. Under sterile condition， a skin incision about <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />1 cm was made in the midline of the thorax， starting from the sternal angle and running parallel to the sternum. The incision wound was deepened until the sternum was exposed but without breaking it. After that， the thoracic musculature and skin incision were closed by 3/0 suture. The sham－operated animals underwent the same procedure except that the incision was not carried out. In an independent experimental group， the animals were employed with an incision on its left hind paw， a well established pain model （18）.

For RNA extraction， the experimental rats following a deep thoracic incision were randomly divided into groups and sacrificed at 1 hour， 6 hours， 1 day and 3 days （n＝5 for each group）. Age matched， sham－operated rats were sacrificed at the indicative time and pooled together as controls （n＝2 at each time point）.  In a separate setup， the sham－operated and various groups of experimental rats （n＝3 for each group） were perfused using 2％ paraformaldehyde for immunohistochemistry.

　　RNA extraction and reverse transcription.

　　The first and second segments of thoracic spinal cord （T1－2 segments） were collected at indicated time intervals and frozen in the liquid nitrogen and kept in a －80?C freezer until use. Total RNA was isolated from the tissue using TRIzol? reagent based on the company protocol （Invitrogen， USA）. RNA concentration was determined by spectrophotometry at 260nm. RNA integrity was electrophoretically verified.  For reverse transcription， the reaction mixture containing 2 ?g of RNA， 2.5 ?M of oligo（dT） primer， and 5 units of Molony Murine Leukemia Virus Reverse Transcriptase （M－MLV， Promega， USA） in a total volume of 25 ?l， was incubated for 1 h at 42 °C and stopped by heating for 5 min at 95 °C. The cDNA was quantified using spectrophotometry and diluted to 10－50 mg/ml for polymerase chain reaction （PCR） or Real－Time PCR assay.

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GFAP－IR becomes more intense in the dorsal horn of the spinal cord at 6 hours after incision （d） compared to that of the sham－operated rat s（c）. Scale bars， 200?m in （a） and （b）； 100?m in （c－f）.

Fig 2 Relative IL－1b gene expression in the rats after thoracic incision and age－matched sham－operated rats. Note that significant increase of IL－1b mRNA level in the thoracic segments of the spinal cord at 1 hour， 6 hours and 1day after operation compared with the sham－operation. Values are Mean±S.E.M （n＝5 for each experimental group and n＝8 control group）.

Fig 3 Representative photomicrographs of IL－1b－IR in the dorsal （a） and ventral （b） horns of the T1－2 segments. The upregulation of IL－1b－IR is observed in the dorsal （c） and ventral （d） horns at 1 hour after operation. The staining becomes more intense in both the dorsal （e） and ventral （f） horns at 6 hours following incision. Scale bar， 100um.

Fig 4 Double labeling of IL－1b with OX－42 （a－c）， GFAP （d－f） or NeuN （g－i） in the dorsal horn of the T1－2 segments in rats at 6 hours following thoracic incision. Note that numerous co－localizations of IL－1b－IR and NeuN－IR in the dorsal horns （g－i）. Arrows in these photos indicate that some IL－1b immunoreactive cells are also labeled with NeuN antisera （g－i）. Scale bar， <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />50mm.

Fig 5 Representative photomicrographs of IL－1b－IR in the lumbar enlargement in the sham－operated rats （a and e）； rats following paw incision （b－d）； rats following thoracic incision （f）. Increased IL－1b－IR is observed and most prominent in the superficial dorsal horn and laminae VIII and IX （arrows in b） in the ventral horn of the ipsilateral segments in response to paw incision （b）； in the superficial dorsal horns， numerous small diameter neurons are intensively stained by IL－1b whereas the staining of IL－1b is very mild in the contralateral side to paw incision （d）； The staining of IL－1b is also weak in the sham－operated rats （e） and rats following thoracic incision （f）. Scale bar， 200mm in （a and b）； 100mm in （c－f）.