台灣疼痛學會雜誌

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膽囊收縮素乙型受體拮抗劑可以預防及逆轉反覆注射嗎啡所誘發之疼痛耐受現象

Cholecystokinin-B Antagonist could Prevent and Reverse Repeated Morphine Injection-induced Antinociceptive Tolerance

關鍵字 Cholecystokinin;CCK antagonist;Morphine tolerance;Tail-flick test;膽囊收縮素;乙型膽囊收縮素（CCK-B）受體拮抗劑;嗎啡耐受性;閃尾試驗
作者 author 劉玉成(Yu-Cheng Liu);黎尚宜(Shang-Yi Lee);歐陽欣漢(Hsin-Han Ouyang);陳坤堡(Kuen Bao Chen);溫永銳(Yeong-Ray Wen)

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Cholecystokinin-B Receptor Antagonist could Attenuate Repeated Morphine Injection-induced Nociceptive Hyperalgesia in the Rats

關鍵字關鍵字：膽囊收縮素、乙型膽囊收縮素(CCK-B)受體拮抗劑、福馬林注射、嗎啡耐受性，類鴉片誘發疼痛過度Introduction Materials and Methods:1. Preparations Studies were performed under the approval of the Animal Care and Use Committee and strictly followed the Guidelines for the Care and Use of Experimental Animals of Shin-Kong Memorial Hospital. Male Sprague-Dawley rats (250-350 g) were housed in groups of two to three at an environment of 22C with a 12-h dark-light cycle and water and food pellets available ad libitum. Total 27 rats in 5 groups were included in this study (Table. 1). 2. Induction of morphine tolerance 3. Behavioral tests Tail-flick test: This test was routinely conducted in all groups to assess the changes of antinociceptive thresholds of the tail in response to an irradiation with a heat bulb source with thermal settings to keep baseline latencies of 3-5 sec and a cutoff time of 10 sec (D’Amour, Smith, 1941; Akil and Mayer, 1972). The percentage of maximal possible antinociceptive effect (%MPAE) was calculated by comparing the test latency before [baseline (BL)] and after a drug injection (TL) using the equation: %MPE = [(TL BL)/(cutoff time BL)] 100%. Formalin test: To determine changes of nociceptive threshold to a suprathreshold nociceptive stimulation, a subcutaneous injection of 50 l of 5% formalin into the mid-plantar surface of the left hindpaw was conducted 1 hr after the last morphine/saline/LY225,910 injection on the 5th day morning. An equal volume of saline was injected in the control group as a sham group. After injection, the rats were transferred to an open iron-wire cage for an 1-h evaluation using a modified weighted pain score method [18,19], and then were euthanized for immunohistochemical analysis. The scores of an early phase (0-15 min), late phase (20-45 min), and total phase (0-60) were recorded and the respective areas under curve (AUC) were separately calculated for group comparison.The experimenter who conducted the above behavioral tests was blind to the group allocation of the rats. 4. Immunohistochemistry and counting of Fos-labeled neurons Under deep anesthesia with an intraperitoneal injection of overdose sodium pentobarbitone, animals were perfused transcardially with 250 ml saline followed by 300 ml of 4 % paraformaldehyde in 0.1 M phosphate buffer (PB, pH=7.4). The spinal cord at the L4-L5 lumbar segments was removed, post-fixed at 4C for 4 h, and cryoprotected in 30% sucrose at 4C for 48-72 h. Frozen sections were cut in a cryostat (30 m) and collected as free-floating sections, then incubated with primary rabbit polyclonal anti-Fos antiserum (Santa Cruz Biotechnology, Inc., CA, USA), diluted 1:1500 with PBS 0.1 M, containing 3 % normal goat serum (NGS) and 0.3 % Triton X-100 at 4C for 48 h. After washing in PBS, sections were incubated with biotinylated second antibody (goat anti-rabbit antiserum; Vector Laboratories, Burlingame, CA, USA) diluted 1: 200 in PBS for 1 h at room temperature, reacted with elite avidin-biotin-peroxidase complex (Vectastain, Vector Laboratories, Inc., CA, USA) diluted 1:50 for 1 h, and then 0.1 % diaminobenzidine (DAB) solution containing 30% H2O2 as substrate was added. All sections were mounted on gelatin-subbed slides, air-dried and protected with a coverslip for light microscopic inspection. For each animal at least ten slices of the Fos-labeled neurons in each segment of the 4th to 5th lumbar spinal dorsal horns were counted. Sections were examined using a dark-field microscope (Axioscope, Zeiss, German) to determine the segmental level according to the gray matter landmarks. The dorsal horn of each section was divided into three regions: (1) the superficial laminae (lamina I/II), (2) the nucleus proprius (lamina III/IV) and (3) the deep laminae (lamina V and VI). Fos-labeled neurons, which showed deep staining distinguishable from background in bright-field illumination, were counted by laminae of layers. 5. Experimental design (Table 1)Rats were randomly allocated into groups receiving morphine alone or morphine with selective CCK-B antagonist, 2-[2-(5-Bromo-1H-indol-3-yl)ethyl]-3-[-(1-methylethoxy- phenyl)-4-(3H)-quinazolinone, LY-225,910 (Tocris-Cookson inc, UK), at two different doses: 0.1 mg/kg and 1 mg/kg. LY225,910 was given subcutaneously 30 min before each morphine injection and this regimen was prepared before each experiment so that the injection volume would be 0.1 ml/kg regardless of the group allocation. Two control groups were conducted with repeated injections of normal saline or LY225,910 (1.0 mg/kg) at the same time points as morphine for 5 days. All groups received formalin injection on the 5th day morning. 6. Statistical analysisAll data were presented as mean standard errors of mean. Data obtained from the tail-flick test were first calculated to yield mean %MPE and the results were shown in previous submission. The data for formalin test by AUCs of 3 patterns and Fos-like immunoreactivities (Fos-LI) by counting the numbers of Fos-labelled neurons were analyzed by using one-way ANOVA to detect overall inter-group differences. When significant main effects were observed, Dunnett post hoc test was performed. A value of p < 0.05 was considered statistically significant. ResultsMorphine antinociceptive tolerance developed at the fifth day after repeatedly subcutaneous injection of morphine (Table 1). Co-administration of CCK-B antagonist LY225,910 dose-dependently attenuated the morphine tolerance (Ref to TJP Submission #ID.205, Fig.1 and 2.). Repeated injection of LY225,910 or normal saline alone did not change the nociceptive threshold and maintained the same tail-flick thresholds for 5 days (Ref to TJP Submission #ID.205, Fig.1 and 2.).Plantar formalin injection resulted in typical biphasic pain patterns in all groups (Fig. 1A). Co-administration of high dose CCK-B antagonist LY225,910 (1.0 mg/kg) with morphine significantly decreased the formalin-induced paradoxical hyperalgesia in the morphine tolerant rats (Fig. 1). The reverse effect was predominantly at the late phase (Fig. 1A and 1D). However, low dose of LY225,910 (0.1 mg/kg) did not show effect on the hyperalgesia in the tolerant rats (Fig. 1A and 1B). In the early phase, there is no statistical differences in AUC among the LY225,910 groups at low or high dose, the saline, the LY225910 control group, and the morphine group (Fig. 1C). It was found that formalin injection induced increases of Fos-LI neurons in the spinal dorsal horns (SDH) at L4 and L5 levels in all groups, predominantly at the superficial laminae at the injection side (Fig. 2). Paralleled with the behavioral hyperalgesic responses in the groups, we found that the M group (morphine tolerant rats without LY225,910 co-injection) showed the highest Fos expression the SDHs indicating a neuronal hyper-reactivities in the SDH, whereas the LY1.0 group (morphine co-administered with 1.0 mg/kg LY225910) had significantly lowered Fos-LI numbers in compression (Fig.2D and Fig. 3). Fos expression at the superficial and deep laminae in the LY1.0 group was markedly suppressed to the level of the saline group and the LY-C group (saline and LY225,910 alone, respectively) (Fig. 3). However, the LY0.1 group showed high Fos expression comparable to the M group, indicating no neuronal suppression by such a low dose co-administration (Fig. 3). Discussion Because we did not administer LY225,910 via an intrathecal route, it is thus difficult to differentiate the suppression of OIH is an effect on the spinal level or on the supraspinal level, i.e. inhibition on RVM and its descending facilitation. However, the evident decrease in Fos expression in the superficial laminae suggesting of the inevitable importance that CCK release in the spinal levels may lead to possible spinal mechanisms in cellular levels and molecular levels. ConclusionWe conclude that CCK-B antagonist LY225,910 could attenuate morphine tolerance-induced behavioral hyperalgesia and spinal Fos reactivity in the rats. The inhibition of morphine-induced hyperalgesia may be mediated through reduction of spinal sensitization. We also postulate that clinical application of LY225,910 or other CCK-B antagonistic drugs may be useful therapeutic agents for management of morphine hyperalgesia and improvement of morphine analgesic quality. AcknowledgementThis study was sponsored by research grants from Ministry of Health and Welfare (formal name “Department of Health”) in Taiwan (DOH92-NNB-1020), and Shin-Kong Memorial Hospital (8302-90-0208-01) to Y.-R. Wen. Key words: Cholecystokinin, CCK antagonist, Formalin test, Morphine tolerance, Opioid-induced hyperalgesia
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