| Pain is one of the most serious problems affecting people's health, but until now there's no effective therapy to relieve patient's pain. As the development of molecular neurobiology, it's very popular to investigate the molecular and genetic mechanism of pain[1]. Basing on the formalin induced inflammatory pain model and L5 Spinal nerve ligation (SNL) induced neuropathic pain model, we observed the nociceptive behavioral change and the expression of c-fos, after intrathecal administration of c-fos antisense oligodeoxynucleotides(ASO), and thus may supply some theoretical evidence for the clinical treatment of chronic pain.AIM:The present research was to investigate the effects of intrathecal (i. t.) administration of c-fos ASO on inflammatory and neuropathic pain of rats, and thus may supply some theoretical evidence for the clinical treatment of chronic pain.METHODS:Basing on the formalin induced inflammatory pain model (formalin 5%, 50μl) and SNL induced neuropathic pain model, we observe the nociceptive behavioral change and the expression of Fos positive neurons after intrathecal (i.t.) administration of c-fos ASO. 30 adult male Sprague–Dawley rats (body weight 180–220 g) , assigned to 3 groups at random: formalin (n=8), SNL (n=16), control group (n=6). After polyethylene tube (PE10) was introduced into the subarachnoid space of the lumbar enlargement[2], the rats were allowed to recover for a period of 3–5 days before further use[3]. Only the animals judged as neurologically normal were used for the following experiments.. 1. Formalin Test: Animals were received a 50μl injection of 5% formalin solution to the plantar of the left hind paw 4 h prior to the formalin injection, the rats were randomly assigned to three groups: (1) rats with i.t c-fos ASO(5'-GAACAT CATGGTCGT-3')at a dose of 50μg in 10μl saline(n=4); (2) rats with i.t. c-fos sense oligodeoxynucleotide (SO, 5'-ACG ACC ATG ATG TTC-3') at a dose of 50μg in 10μl saline(n=4),(3) rats with i.t. saline (10μl, 0.9%, n=2). Behavioral test was performed immediately after formalin injection and the incidences of spontaneous flinching were counted, per 5 min in 1 h.2. Neuropathic Pain Model: Rats were anesthetized with pentobarbital (40 mg/kg), and nerve injury was produced by tight ligation of the L5 spinal nerve[4]. The rats were randomly assigned to two groups: one was the pretreatment group(drugs were injected 4 h prior to SNL) and divided into 3 subgroups: (1) rats with i.t c-fos ASO(50μg /10μl, n=4),(2) rats with i.t.c-fos SO (50μg /10 μl,n=4),(3) rats with i.t. saline (10μl, 0.9%, n=2). Behavioral test was performed 4 h prior to and after i.t, last for 3 days. The other was the post-treatment group (drugs were injected 3 d after SNL) and was also divided into 3 subgroups: (1) rats with i.t c-fos ASO(50μg /10μl,n=4),(2) rats with i.t.c-fos SO (50μg /10μl,n=4), (3) rats with i.t. saline (10μl, 0.9%, n=2). Behavioral test was performed daily and lasted for 2 w[5].3. Behavioral test: Mechanical withdrawal threshold(MWT)was tested using von Frey filaments (Stoelting, Kiel, WI, USA) in a blinded manner. The hind paw of the rat was pressed with one of a series of von Frey filaments with increasing stiffness (2, 4, 6, 8, 10, 15, and 26 g), presented to the plantar surface (5-6 s for each filament)[6]. The paw withdrawal threshold (PWT)was determined using Dixon's up-down method. Thermal withdrawal latency(TWL)was tested according Hargreaves test[7].4. Immunofluorescent histochemistry was used after behavioral tests to detect the Fos expression after drugs administration on SNL rats.RESULTS:Behavioral results: Injection of formalin into the plantar aspect of the hindpaw in rats induced tonic monophasic acute pain behaviors (such as biting, licking, and limb flinching) and lasted for more than 1-2 h[7]. Pretreatment of c-fos ASO significantly diminished the formalin induced spontaneous flinches(P<0.01)compared to the saline or SO control.L5 SNL significantly induced long term mechanical allodynia and thermal hyperalgysia[4]. Compared to the SO or saline group, pretreatment of c-fos ASO 4 h prior to SNL significantly prevented SNL induced neuropathic pain. But post treatment of ASO 3 d after SNL has no evident effects on mechanical allodynia or thermal hyperalgysia induced by SNL. Immunofluorescent histochemistry: A large number of Fos positive neurons were observed in the superficial laminae of the ipsilatera dorsal horn 2 h after injection of formalin into the plantar aspect of the hindpaw. Most of the Fos-positive neurons were observed concentrated in laminae I and II with a few in laminae III and IV(Fig. 1). Pretreatment of c-fos ASO significantly decreased the Fos expression induced by formalin injection(P<0.01).SNL induced a significant up-regulation of Fos expression in the ipsilateral dorsal horn compared with the contralateral side .The expression of Fos positive neurons induced by SNL were significantly reduced after c-fos ASO administration.CONCLUSIONS:C-fos activation in the spinal dorsal horn is of great importance in inflammatory pain and neuropathic pain development. And it is more meaningful to suppress its activation in the preventing than the treating stage. Pretreatment of c-fos ASO significantly diminished the mechanical allodynia induced by SNL, but c-fos ASO could not reverse the neuropathic pain once the allodynia developed. The present research indicates that c-fos probably only take part in the forming of neuropathic pain but did not participate in the maintenance of neuropathic pain.
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