摘要：实验在SD大鼠上应用脑室微量注射和辐射热测痛的方法,研究了脑内微量注射白介素-1β 对大鼠痛阈的影响。实验大鼠分为给药组和对照组, 在给药组大鼠脑室注射不同剂量的白介素-1β(5、50和500 pg/kg), 对照组大鼠脑室注射配药液。白介素-1受体拮抗剂 (IL-1ra, 50 ng/kg)在脑室注射白介素-1β 前20 min给予。实验以大鼠对光热刺激引起的缩爪反射潜伏期为痛阈指标。结果表明, 脑室注射白介素-1β 可显著缩短大鼠对光热刺激的缩爪反射潜伏期, 并具有剂量依赖性关系。脑室给予500 pg/kg的白介素-1β 20 min后, 大鼠对光热刺激的缩爪反射潜伏期显著缩短, 40 min时达峰值, 然后逐渐恢复。该作用可被白介素-1β 受体拮抗剂阻断。结果提示脑中白介素-1β可通过作用于白介素-1受体引起热痛敏作用。1 MATERIALS AND mETHODS

1.1 Animals The study was conducted in concordance with the guidelines of the Ethical Standards of the International Association for the Study of Pain[15]. Male Sprague-Dawley rats (220±20 g) were provided by the Department of Experimental Animals of medical Center of Fudan University, and were kept at a constant ambient temperature (22±1℃) under a 12 h light and dark cycle with free access to food and water.

1.2 Cannula implantation The cannula (o.d. 0.8 mm) for i.c.v. injection, was implanted stereotaxically into the lateral cerebral ventricle (P 0.8 mm, L 1.5, and H3.5 mm) of rat anesthetized with pentobarbital (40 mg/kg i.p.), and fixed on the skull with dental cement. Animals were allowed to recover for 4 d before experimental observation.

1.3 Measurement of the nociceptive threshold According to the previously described method[16,17], the paw withdrawal latency (PWL) to the noxious thermal stimulation was measured by using an analgesiameter (IITC/Life Science Instruments, USA). The room temperature was kept at 22±1℃. Each rat was placed in a clear plastic chamber (20 cm × 20 cm× 40 cm) on the glass floor. After a 20 min habituation period, the plantar surface of the paw was exposed to a beam of radiant heat applied through the glass floor. Paw withdrawal was taken as a behavioral index of the nociceptive threshold. Therefore, significant reduction of PWL indicated hyperalgesia. A cut-off time of 20 s for the thermal stimulus was set to assure that no tissue damage would occur to the paw.

the PWL was determined four times at 10 min intervals before i.c.v. injection, and the average values of the last three times were taken as the baseline. After the i.c.v. injection, the PWL was measured repeatedly over 120 min with intervals of 10 min.

1.4 Experimental procedures Rats were divided into control (n=7) and drug-administration groups. Each animal of control group was given a vehicle solution (10 µl, 0.1 mol/L PBS, containing 0.1% bovine serum albumin) via i.c.v. injection. In the drug administered group IL-1β were given in different doses via i.c.v. injection, i.e., 5 pg/kg (n=7), 50 pg/kg (n=10) and 500 pg/kg (n=7). In the interleukin-1 receptor antagonist (IL-1ra) treated groups, IL-1ra (50 ng/kg) was injected 20 min before the injection of IL-1β or the vehicle solution. IL-1β(recombinant rat interleukin-1) and IL-1ra were purchased from R&D Systems (USA). Drugs were dissolved in 5 µl PBS and administered in 10 s via the catheter and followed by5 µl vehicle solution for flushing. After the experiment, the brain of each rat was removed and the location of the cannula was verified.

1.5 Statistics Data were expressed as mean±SE. The statistical comparison between groups was performed by using analysis of variance (ANOVA) followed by Tukey test. differences were considered to be significant if P＜0.05.2 RESULTS

2.1 Effects of i.c.v. injection of IL-1β on PWL

fig.1. Time courses of the effects of i.c.v. injection of IL-1β on PWL in rats. Symbols represent the level of significance vs control group. *P＜0.05, **P＜0.01, ***P＜0.001.

rats of control group responded to the thermal-stimulation with fairly constant latency during the post-injection period, which did not differ significantly from the baseline (ANOVA, P＞0.05, Fig.1). Icv injection of IL-1β at the doses of 5, 50, and 500 pg/kg significantly shortened PWL when compared with vehicle control (ANOVA, P＜0.05 to P＜0.001, Fig.1). IL-1β displays a dose-dependent shortening in PWL (ANOVA, F=4.775, P＜0.01, Fig.2). At the dose of 500 pg/kg, the shortening of the PWL occurred at 20 min, reached a peak within 40 min and lasted for 100 min after injection, then the PWL gradually returned to the baseline level (Fig.1).

fig.2. Dose-response curve for IL-1β on PWL after i.c.v. injection in rats. Each point and vertical bar represents the mean and SE at the 40 min after i.c.v. injection from 7 rats (10 rats for50 pg/kg). *P＜0.05, **P＜0.01 compared with vehicle control group.

2.2 Effects of iL-1ra on IL-1βinduced changes in PWL

fig.3. Effects of pretreatment with IL-1ra on IL-1β induced changes in PWL in rats. Symbols represent the levels of significance vs control group. *P＜0.05, **P＜0.01, ***P＜0.001．

in order to assess whether the effects of IL-1β on the nociceptive threshold is mediated by IL-1 receptor, IL-1ra (50 ng/kg) was given via i.c.v. cannula 20 min before i.c.v. injection of IL-1β (500 pg/kg), and the PWL to thermal stimulation was measured within 120 min after i.c.v. injection of IL-1β. pretreatment with IL-1ra completely blocked the IL-1β-induced shortening of PWL; IL-1ra alone exhibited no effect on the nociceptive threshold(Fig.3).

3 DISCUSSION

the present study demonstrates that i.c.v. injection of IL-1β shortens the PWL on thermal stimulation, suggesting a superspinally IL-1β-induced thermal hyperalgesia in rats. This result confirms and extends the previous results assessed by tail-flick test[14], hot-plate test[13], and mechanical stimulation test[11]. However, it has also been reported that IL-1β shows an analgesic effect in rats[11, 12], being contrary to our results and those of others. The reason for the conflicting results is not yet clear.

iL-1ra was recently cloned and was shown to competitively block the binding of IL-1β and IL-1α to their receptors[18]. The present results demonstrate that the IL-1ra (50 ng/kg), which showed no obvious effect on thermal nociception, could block the hyperalgesic effect of IL-1β, indicating the involvement of interleukin-1 receptor (IL-1R) in this processing. two types of IL-1Rs are presently recognized[7, 8, 19]. The type I receptor is the major IL-1 receptor on cell surface and transmits some signals, but the type II receptor does not. Furthermore, several reports have indicated that type I IL-1R mRNA was detected in mouse and rat brain, mainly in the hippocampus and hypothalamus[3~8], but type II IL-1R mRNA was not detected in normal rat brain[20]. These findings suggest that the effect of IL-1β on thermal nociception may be mediated by type I receptor in the brain.

It has been considered that as one of the cytokines, IL-1β plays an important role in immune and inflammatory systems. The thermal hyperalgesia induced by i.c.v. injection of IL-1βsupports the notion that IL-1β acts as a modulator of the nociception processing in the central nervous system.

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