Pruritus is one of the most common side effects of intrathecal morphine administration,1 is often difficult to treat, and responds poorly to conventional antihistamine treatments. Opioid receptor antagonists such as naloxone are the most effective treatment for pruritus caused by spinally administered morphine; however, they are not ideal antipruritics because the analgesia effect of opioids could be concurrently reversed.2 Treatment of pruritus induced by opioids remains a challenge for anesthesiologists. Clinical studies have demonstrated that a low dose of propofol (10–20 mg) bolus could alleviate spinal morphine-induced pruritus without disrupting intrathecal morphine analgesia, but its exact mechanism has not been fully understood.3–5
N-arachidonoyl ethanolamine (anandamide; AEA) is an important endogenous cannabinoid that is rapidly degraded predominantly by fatty acid amide hydrolase (FAAH), and it mainly activates cannabinoid-1 (CB) receptor. CB(1) receptors are widely expressed in the brain.6 It has been reported that CB(1) receptor agonists tend to decrease scratching behavior, whereas CB(1) receptor antagonists, such as rimonabant, elicit scratching behavior.7 Therefore, the endocannabinoid system may provide a novel therapeutic target for pruritus.
Using positron emission tomography and functional magnetic resonance imaging (fMRI) brain-imaging techniques, it has been demonstrated that the anterior cingulate cortex (ACC) is an important pruritus perception region in the brain in healthy.8 Propofol may enhance brain AEA by inhibition of FAAH in Sprague-Dawley rats and then indirectly activate the central CB(1) receptor.9 Studies showed that both repetitive scratching and propofol could result in deactivation of the ACC in healthy human subjects.10,11 This suggests that the therapeutic target of propofol for pruritus may be in the ACC. Previous clinical studies have demonstrated that the alleviation of intrathecal morphine-induced pruritus with propofol probably involves the decreased posterior horn transmission in the spinal cord.3–5 To our knowledge, its relation to the expression of CB(1) receptors in the ACC has not yet been prospectively evaluated.
Therefore, in this study, we constructed an intrathecal morphine-induced pruritus model in rats to investigate the effects of propofol on the incidence and severity of intrathecal morphine-induced pruritus. We hypothesized that propofol prevents intrathecal morphine-induced pruritus by upregulating the expression of CB(1) receptors in the ACC of rats.
The study was approved by the Medical Committee on the use and care of animals in the Wenzhou Medical College (China, Zhejiang). Male Sprague-Dawley rats with body weights ranging between 250 and 300 g were studied. The animals were housed in an air-conditioned room with controlled temperatures (24°C ± 2°C) and lights (lights on from 8:00 AM to 8:00 PM). There was free access to food and water. Each rat was used only once in this study.
Intrathecal Catheterization and Jugular Vein Catheterization
After being anesthetized with an intraperitoneal injection of chloral hydrate (5% solution, 350 mg/kg), the rats’ lumbar area was shaved and then washed with 75% ethyl alcohol. A midline incision was made in the skin at the L3-L4 level, and the spinous process of L3 was cut until the spinal cord was exposed. The 22-cm polyethylene intrathecal catheter (PE-10 tube, OD: 0.5 mm, ID: 0.25 mm, AniLab Software and Instruments Co. Ltd, China) was inserted 2.5 cm into the intrathecal space. After entry into the intrathecal space was confirmed by sudden tail movement, a catheter was situated at the lumbar enlargement of the spinal cord. The right jugular vein was catheterized with a vein catheter (PE-50 tube, OD: 0.96 mm, ID: 0.58 mm, AniLab Software and Instruments Co. Ltd, China) filled with heparinized saline.
Both catheters were fixed in the posterior cervical area through subcutaneous tunneling, and the ends of the catheters were plugged. After the rats completely recovered from anesthesia, only those that had normal motor function were chosen for further observation. Three days after surgery, 10 μL of 2% lidocaine was injected through the catheter, and 10 μL normal saline was additionally injected to flush the catheter to observe lower limb paralysis.12 Only the rats showing lower limb paralysis were selected for further observation. Penicillin was continuously injected for 7 days through a jugular vein catheter.
Seven days after the operation, a scratching behavior response to intrathecal morphine was observed, and the number of hindlimb scratching episodes were counted. These episodes were recorded by camera. Before intrathecal morphine injection, a rat was adapted to an individual transparent chamber (24 × 20 × 40 cm) for 2 hours, which was also used as the observation chamber after the injection. One scratch was defined as 1 short-duration episode of scraping with a hindpaw ending with licking or biting of the toes and/or placement of the hindpaw on the floor.13 The scratching response was recorded from 30 minutes before the intrathecal injection to 60 minutes after the injection in an isolated environment without the observer present. Only rats with >2 scratching episodes in the first 5 minutes after intrathecal morphine injection continued to be observed. The video was played back to evaluate the scratching behavior and calculate the scratching times. The scratching response was scored by a trained individual who was blinded to experimental conditions. Hindpaw scratching behavior was recorded every 5 minutes.
The first part of this study was designed to create an intrathecal morphine-induced pruritus model in rats. Sample size was based on our pilot data, as well as on the sample sizes used in previous studies.14–16 A total of 48 rats were enrolled, and, after the intrathecal catheterization surgery, 4 rats were excluded due to the failure of the intrathecal catheterization. Before the intrathecal morphine injection stage, 8 rats were excluded because of lack of bilateral motor blockade or catheter dislodgement. Thirty-six rats were randomly divided into 4 groups using the random number generator in Microsoft Excel (Microsoft Corporation, US): a control group (n = 6) and 3 morphine groups of 20, 40, 80 μg/kg (n = 10). We obtained full data from 6 animals in each group. The dose-response study was conducted to investigate whether intrathecal administration of morphine could induce a scratching response in rats and whether it had a dose-dependent effect. Morphine sulfate (NO. 120403-1, Yichang Humanwell Pharmaceutical Co. Ltd, China) was dissolved in normal saline for intrathecal administration. Morphine and normal saline were intrathecally injected in a volume of 10 μL using a microinjection syringe. An additional 10 μL normal saline was administered to flush the catheter. After the injection, the end of the catheter was plugged. After the dose-response curves of intrathecal morphine were established in the 4 groups, the morphine dose that elicited maximal scratching response was chosen for the next part of the study.
The second part of this study was to evaluate the effects of propofol on intrathecal morphine-induced pruritus. The procedure was in accordance with the first part of the study. Eighty-two rats were enrolled, and 60 rats completed the evaluation for a scratching behavior response to intrathecal morphine. They were then randomly divided into 4 groups using the random number generator tool in Microsoft Excel: control, normal saline, intralipid, and propofol. In the control group, rats were intrathecally injected with 10 μL normal saline, while normal saline, intralipid, and propofol groups were intrathecally injected with 40 μg/kg morphine to successfully create the pruritus model as described above. An additional 10 μL normal saline was administered to flush the catheter. Propofol (2%, Fresenius Kabi, Germany) was dissolved in intralipid containing 10% soybean oil. Intralipid® (10%, Huarui Pharmaceutical Co. Ltd, China) was used as the control solution. Normal saline 80 μL/kg, intralipid 80 μL/kg, and propofol 0.8 mg/kg were administered via the jugular vein 10 minutes after intrathecal administration of morphine to the 4 groups, respectively. All venous injections were completed within 10 seconds. Rats were randomly assigned to pruritus behavior observation (n = 6) or killed at 8 minutes after venous injection, and brain tissues were collected for the next part of the study (n = 9).
The third part of this study was to investigate the receptor mechanism of the effect of IV propofol on attenuating intrathecal morphine-induced pruritus (n = 9). Immunohistochemistry was performed to identify the expression of CB(1) receptor in ACC (n = 6), and the concentration of CB(1) receptor in ACC was determined by Western blot analysis (n =3).
After being anesthetized with chloral hydrate (350 mg/kg) intraperitoneal injection, rats were perfused transcardially with saline solution (0.9% NaCl) and 4% paraformaldehyde fixative. The brains were kept in the fixative for 24 hours at 4°C and then transferred to 30% sucrose in phosphate buffered saline (PBS) for 24 hours at 4°C. The brains were frozen in cryostat embedding medium (Thermo Scientific Microm HM 525, Germany). Serial 10 μm sections of the brain were cut using a cryostat and thaw-mounted onto glass slides. After being washed in PBS, nonspecific antibody binding was inhibited by incubation for 30 minutes at 37°C in blocking solution (5% bovine serum albumin, 0.5% Triton-X 100 in PBS). Primary antibodies were diluted in PBS blocking buffer, and slides were incubated overnight at 4°C in primary rabbit polyclonal antibodies against CB(1) receptor (1:300 dilution, Abcam). The slides were washed 3 times with 10 mM PBS and incubated with fluorescent-labeled secondary antibodies (1:100; Alexa Fluor 488, Molecular Probe, Invitrogen, Carlsbad, CA) at 37°C for 1 hour. Fluorescently labeled sections were viewed with a fluorescence microscope (Nikon, Japan) to locate the cells and identify the area of the brain. Three high-power fields of ACC areas were counted in each slide. Six slides were counted, and the average numbers of positive cells were calculated. The average number of neurons was compared using a completely randomized analysis of variance (ANOVA).
Samples of tissue were collected from ACC of rats’ brains and flash frozen in liquid nitrogen and then stored at −80°C until the measurement. The frozen tissues were rapidly thawed and homogenized at 4°C in 200 μL cold radio immunoprecipitation assay (RIPA, Beyotime, China) and sonicated to dissolve the tissue completely. The homogenates were centrifuged at 12000g for 10 minutes at 4°C, and the supernatants were collected. Protein concentration was determined by bicinchoninic acid assay (BCA) kit (Beyotime). A total of 30 μg proteins from each sample were loaded per lane for SDS-PAGE (10% SDS gel). Protein samples were denatured and resolved in 10% SDS-PAGE, then transferred to a Millipore (Bedford, MA) Immobilon-P polyvinylidene fluoride (PVDF) membrane at 4°C, 300 mA for 70 minutes. The transfer buffer contained 20% methanol, 48 mM Tris Ph9.2, and 39 mM glycine. The membranes were blocked with 5% nonfat milk in TTBS (20 mmol/L Tris-Cl, PH7.5, containing 0.15 mol/L NaCl, and 0.1% Tween-20) for 2 hours and then incubated overnight at 4°C with primary rabbit polyclonal antibodies against CB(1) (1:200 dilution, Abcam) and mouse monoclonal antibody against β-actin (1:1000 dilution, ZSGB-BIO). On the following day, membranes were washed and exposed to horseradish peroxide (HRP) conjugated goat anti-rabbit (1:5000 dilution, Jackson ImmunoResearch Laboratories, Inc., PA) or mouse (1:10000 dilution, Jackson ImmunoResearch Laboratories, Inc.,) IgG as second antibodies at room temperature for 1 hour. The intensities of bands obtained from Western blot were estimated with ImageQuant LAS4000 mini (GE Healthcare, Sweden).
Values are presented as mean ± standard deviation (SD) or number as appropriate. Behavior data were made by repeated measures 2-way analysis of variance (RM-ANOVA) with group and time as variables and Dunnett multiple (post hoc) comparisons (SPSS 17.0, SPSS Inc, Chicago, IL). Western blot and immunohistochemical data were compared using ANOVA. The post hoc comparisons were performed by Newman-Keuls tests and Bonferonni corrections. A P value of <0.05 was considered statistically significant.
The rats had a significant scratching response after intrathecal morphine injection, which is shown in Fig. 1. The scratching times progressively increased; the peak presented at 10 to 15 minutes after morphine injection and almost subsided at 55 to 60 minutes after morphine injection. RM-ANOVA analysis revealed a significant morphine effect (F[3, 20] = 15.33, P < 0.001) and group × time interaction (F[33, 220] = 1.57, P = 0.031). The average peak scratching times in the 20, 40, 80 μg/kg morphine groups were 13, 19, 18 times per 5 minutes, respectively. The continuous data of scratching times were not normally distributed (Kolmogorov–Smirnov test of normality). Compared with the control group, 20, 40, 80 μg/kg morphine groups had higher mean scratching response rates after intrathecal morphine injection (P = 0.020, 0.005, and 0.002, respectively; Dunnett test). There was a statistical difference between 20 and 40 μg/kg morphine groups at 10 to 15 and 15 to 20 timepoints after intrathecal morphine injection (P = 0.049 and 0.017, respectively; Dunnett test). Rats in the 20, 40 μg/kg morphine groups did not experience sedation, but the 80 μg/kg morphine group experienced sedation (e.g., eyes closing, less movement).
RM-ANOVA revealed a significant effect of propofol and group × time interaction (F[(2, 15] = 46.87, P < 0.001; F[22, 165] = 2.37, P = 0.001); compared with intralipid and normal saline groups, the scratching behavior was significantly attenuated in the propofol group (P < 0.001, Dunnett test) (Fig. 2). The continuous data of scratching times were not normally distributed (Kolmogorov–Smirnov test of normality). There was no sign of sedation or motor impairment (e.g., eyes closing, erratic movements, general behavioral depression), and all rats retained their righting reflex in the propofol group.
Compared with the control, normal saline and intralipid groups, the protein expression of CB(1) receptor in ACC (Western blot) in the propofol group increased (0.86 ± 0.21, 0.94 ± 0.18, 0.86 ± 0.13, and 1.34 ± 0.32, respectively, P < 0.001) (Fig. 3, A and B). There was no significant difference among the normal saline, control, and intralipid groups.
Immunopositive reactions of CB(1) receptors were detected in the area of ACC (Fig. 4, A and B). Compared with control, normal saline, and intralipid groups, the average number of neurons of CB(1) receptors in the ACC area through immunoreactivity (appeared in green fluorescence) was higher in the propofol group (21.0 ± 1.4, 19.3 ± 1.8, 24.8 ± 7.7, and 37.2 ± 3.3, respectively, P < 0.001).
In this study, we demonstrated that morphine-elicited scratching responses followed intrathecal injection in rats, and there was a dose dependent only between 20 and 40 μg/kg morphine groups at 10 to 15 and 15 to 20 timepoints after intrathecal morphine injection. Morphine intrathecal injection-induced scratching responses were reversed by low-dose propofol, while the protein expression of CB(1) receptors in ACC was increased in the propofol group, which would suggest that the therapeutic target of propofol for pruritus may be in the ACC.
The doses of intrathecal morphine injection in this study that could have caused the antinociceptive effect were selected based on previous studies.17,18 It has been demonstrated that 10 μL is the most adequate volume for the spinal space of rats, which is better for drug spreading uniformly within the spinal space and also for safety reasons.19 From the first part of the study, we found that the dose of 80 μg/kg morphine had a sedative effect (eyes closing, less movements), so the dose of 40 μg/kg was given in the second part of the study. Furthermore, it has been reported that the anesthetic dose of propofol in rats is 8 mg/kg,20 but one-tenth the anesthetic dose (0.8 mg/kg) of propofol was administered in this study because this is the dose used clinically for treating pruritus.
Thomas and Hammond14 reported that microinjection of morphine into rats’ medullary dorsal horn (MDH) produced a dose-dependent increase of facial scratching. Both intracisternal and intrathecal morphine injection can also elicit a dose-dependent increase of scratching in mice.21 Similarly, in monkeys, the injection of morphine into the MDH and intrathecal space also produced dose-dependent facial and body scratching.22,23 However, 1 study indicated that intrathecal morphine injection in rats produced body scratching only at a very high dose, and this scratching behavior was associated with agitation and allodynia.14 Ko and Naughton23 demonstrated that morphine produced a dose-dependent scratching response in lower dose ranges and seemed to reach a plateau at higher doses. Intrathecal administration of 0 to 0.5 mg morphine in cesarean delivery patients showed a significant trend toward increasing pruritus only at a lower dose (0–0.2 mg). There was no significant change from 0.2 to 0.5 mg.24 It appears, therefore, that intrathecal morphine has only a limited dose-response effect. The present study showed that intrathecal administration of morphine-induced scratching responses in the 20 and 40 μg/kg morphine groups at 10 to 15 and 15 to 20 timepoints after intrathecal morphine injection, possible because the selected doses of morphine were beyond the dose-response range or that the sample size was fairly small.
The maximal morphine MDH-induced scratching response in rats was reported to occur at 30 to 40 minutes after the injection,14 whereas after intracisternal and intrathecal morphine injection in mice, it occurred approximately 10 minutes after injection.21,25 Facial scratching peaked during the initial 20-minute period after intracisternal morphine in mice.26 In our study, the maximal scratching response caused by intrathecal administration of morphine occurred 10 to 15 minutes after the injection. One explanation for this discrepancy could be that multiple brain regions are involved in the scratch-inducing effects of morphine.
Compared with normal saline and intralipid, the second part of the study showed that 0.8 mg/kg propofol significantly attenuated the morphine-induced scratching response and also had no sedative effect. Our findings were consistent with clinical studies reporting that propofol was effective for the treatment of intrathecal morphine-induced pruritus.3,27,28 Gent et al.29 reported that intrathecal morphine-induced pruritus in cats that was successfully treated with low-dose propofol. However, other studies reported that propofol did not relieve intrathecal morphine-induced pruritus in women after cesarean delivery; the reasons that were considered included a smaller dose of propofol and the vulnerability to spinal morphine-induced pruritus for parturient patients.30,31
The endocannabinoid system consists of CB(1) and CB(2) receptors and several endogenous ligands including AEA and 2-arachidonoyl glycerol (2-AG). CB(1) receptors were found in most brain areas, generally located in presynapse and played an important role in controlling neurotransmitter release.32 As the inhibitor of FAAH, propofol could increase the AEA concentration and indirectly stimulate the CB(1) receptor.9 This study demonstrated that the expression of CB(1) receptors in ACC of the propofol group was significantly higher than other groups; therefore, the effect of propofol to relieve intrathecal morphine-induced pruritus might be related to the upregulation of CB(1) receptors. Unlike CB(1) receptor agonists with prominent central side effects such as motor suppression, dependence, hypothermia, and hyperphagia, FAAH inhibitors have fewer adverse effects.33 Therefore, propofol could be developed as a new therapy for pruritus. The ACC (Brodman area 24) is the rostral part of the cingulated gyrus on the medial surface of each hemisphere, overlaying the corpus callosum, and the density of the presynaptically localized CB(1) receptors in the ACC is moderately high in humans.34 During the past decade, there has been a growing interest in the role of the ACC in pruritus. A previous study using fMRI and positron emission tomography has demonstrated that pruritus could cause the activation of ACC.8 Also, ACC was deactivated during scratching behavior that inhibited pruritus.10 From above, the change of the CB(1) receptors in ACC might closely relate to the antipruritic efficacy of propofol. There were several limitations in our study. First, the AEA concentration in ACC was not determined. Second, we did not study the effect of a CB(1) antagonist to elucidate the role of this receptor.
In summary, we demonstrated that low-dose IV propofol could relieve intrathecal morphine-induced pruritus in rats, and increased protein expression of CB(1) receptors in ACC may contribute the reversal of intrathecal morphine-induced scratching.
Name: Xiulan Liu, MD.
Contribution: This author helped conduct the study, analyze the data, and write the manuscript.
Attestation: Xiulan Liu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Jing Zhang, MD.
Contribution: This author helped design and conduct the study and analyze the data.
Attestation: Jing Zhang has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Hongyan Zhao, MD.
Contribution: This author helped conduct the study.
Attestation: Hongyan Zhao has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Hongxia Mei, MD.
Contribution: This author helped conduct the study.
Attestation: Hongxia Mei has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Qingquan Lian, MD.
Contribution: This author helped design the study.
Attestation: Qingquan Lian has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: WangNing ShangGuan, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript
Attestation: WangNing ShangGuan has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
The authors thank Yun Xia, MD, Department of Anesthesiology, The Ohio State University Medical Center, Columbus, Ohio, for his critical review.
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