1 Abteilung für Anaesthesiologie und Intensivmedizin, LKH Klagenfurt, St. Veiter-Straße 47, A-9020 Klagenfurt, Austria. 2 Klinik für Anaesthesiologie und Operative Intensivmedizin, CharitéUniversitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. 3 Abteilung für Traumatologie, LKH Klagenfurt, Klagenfurt, Austria. 4 Klinik für Anaesthesiologie, Schmerzambulanz, Universität Erlangen-Nürnberg, Erlangen, Germany
* Corresponding author. E-mail: micha.schaefer{at}charite.de
Accepted for publication April 29, 2004.
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Abstract |
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Methods. Following IRB approval and informed consent, patients were randomly assigned to the following i.a. treatments at the end of surgery: group I (n=39), isotonic saline; group II (n=40), 1 mg morphine hydrochloride; group III (n=48), 2 mg morphine hydrochloride; group IV (n=39), 4 mg morphine hydrochloride. Postoperative pain intensity was assessed by the visual analogue scale (VAS), by the time to first analgesic request and by the supplemental piritramide consumption. Synovial specimens from each patient were stained for the presence of inflammatory cells and END and were discriminated into groups with low versus high numbers of these cells. Differences between groups were statistically analyzed by 2, ANOVA and MANCOVA where appropiate.
Results. Patient characteristics and VAS scores did not differ between groups. Total postoperative piritramide consumption decreased and the time to first analgesic request increased significantly with increasing doses of i.a. morphine (P<0.05, ANOVA and linear regression). These doseresponse relationships were not different between patients with low versus high numbers of inflammatory and END-containing synovial cells (P>0.05, MANCOVA).
Conclusions. The doseresponse relationship of i.a. morphine analgesia is not shifted by enhanced inflammation and END expression within synovial tissue. Thus, the presence of END within inflamed synovial tissue does not seem to interfere with i.a. morphine analgesia.
Keywords: analgesia, postoperative ; analgesic techniques, intra-articular ; endorphins ; macrophages ; pain, acute ; pain, postoperative ; pain, mechanisms ; pharmacology, morphine ; receptors, opioid ; surgery, orthopaedic ; T lymphocytes
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Introduction |
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In the periphery, local analgesic effects of opioids are overt following their administration into inflamed tissue.7 8 Apart from experimental studies, this has been shown in patients with acute and chronic inflammatory pain.9 10 The analgesic effect is mediated by opioid receptors on peripheral endings of sensory neurons within inflamed subcutaneous tissue.11 In addition, ß-endorphin (END), an endogenous opioid peptide which is expressed within immune cells in inflamed tissue, can be released upon stressful stimulation and reduce pain by activation of those receptors.12 13 Under experimental conditions, this END release and subsequent analgesia is progressively augmented with increasing duration and magnitude of the inflammatory reaction.13 END has also been identified within inflamed synovial tissue of patients suffering from knee injury or chronic arthritis.1416 In a previous clinical trial, we found a dose-dependency of intra-articular (i.a.) morphine analgesia.17 Here we examined whether enhanced inflammation and END expression within synovial tissue of patients undergoing arthroscopic knee surgery might shift the analgesic doseresponse curve of i.a. morphine.
In a preliminary study using a single dose of i.a. morphine we found no diminution of its analgesic effect in the presence of synovial inflammation.16 This led us to hypothesize that enhanced inflammation and opioid peptide expression within the synovial tissue of surgical patients does not result in cross-tolerance, i.e. a rightward shift in the doseresponse curve, to the analgesic effects of i.a. morphine.
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Materials and methods |
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We examined 200 patients scheduled for arthroscopic knee surgery. They were aged between 19 and 70 yr, weighed between 50 and 90 kg and had American Society of Anaesthesiologists physical status III. Patients were excluded from the study if (i) they had a history of chronic pain or cardiovascular, respiratory, metabolic or neurological disorder, (ii) they had taken any opioids within 12 h before surgery, (iii) they had a history of drug or alcohol abuse or psychiatric disorder or (iv) they were unable to comply with the self-administration of analgesics. Patients were instructed on how to use the visual analogue scale (VAS) for pain rating and the patient-controlled analgesia (PCA) device (Graseby PC-3000).
Patients were given oral midazolam 0.1 mg kg1 45 min before anaesthesia. Anaesthesia was induced with propofol 2 mg kg1, fentanyl 3 µg kg1 and atracurium 0.5 mg kg1 i.v., and maintained with 0.81.2% isoflurane and 65% nitrous oxide in oxygen.
Test solutions of different concentrations of morphine (0.01, 0.02 and 0.04%) and isotonic saline were prepared and coded by the hospital pharmacy. At the end of the procedure, 10 ml of test solution was given through the arthroscope into the patient's knee joint. The thigh tourniquet remained in place for another 10 min and was then released. If drains were placed into the knee joint, they were clamped for the first 60 min to prevent immediate loss of the injected test solution. Using a random number table, patients were assigned to one of four i.a. treatment groups in a double-blinded manner: group I received 10 ml saline, group II received 1 mg morphine (10 ml of 0.01% solution), group III received 2 mg morphine (10 ml of 0.02% solution) and group IV received 4 mg morphine (10 ml of 0.04% solution). The morphine doses were based on a previous study which had demonstrated a dose-dependent analgesic effect within this range.17
Pain intensity was evaluated using a VAS extending from 0 (no pain) to 100 (unbearable pain). VAS scores were recorded by a blinded observer at 1, 2, 3, 6, 9, 12, 18 and 24 h after the end of surgery. In addition, the time that elapsed until patients asked for the first supplemental analgesic was recorded. At the patients' request, they received an i.v. bolus of 0.1 mg kg1 piritramide, an opioid agonist which is nearly (0.75 times) equipotent with morphine and very often used in Germany for postoperative pain therapy. Patients were then connected to a PCA device (Graseby PC-3000) which was set to deliver an i.v. bolus of 2 mg piritramide with a lockout time of 8 min. At the end of the 24 h assessment period the total consumption of piritramide was documented for each group.
The degree of sedation was assessed using a categorical five-point scale extending from 1 (awake) to 5 (asleep). In addition, patients were asked if they experienced side effects such as nausea, vomiting, pruritus or urinary retention. The were asked to grade these symptoms from 0 to 3 (0, no symptoms; 1, mild; 2, moderate; 3, severe). The duration of anaesthesia was determined as the time from induction until the first response to a verbal command. The duration of surgery was documented as the time from the first incision until the final wound closure.
During surgery two synovial biopsies of 510 mm2 were obtained from each patient for further histological and immunohistochemical examination. The tissue was immediately fixed in formalin, dehydrated in ethanol and embedded in paraffin. Paraffin blocks were cut serially at 4 µm thickness. For characterization of inflammatory cells, serial tissue sections were stained with haematoxylineosin and giemsa to detect granulocytes, plasma cells and mast cells and with anti-CD3 antibody (Dako, Glostrup, Denmark; dilution 1:300) and anti-CD68 antibody (Dako, Glostrup, Denmark; dilution 1:200) to identify T lymphocytes and monocytes/macrophages, respectively. A blinded observer counted the total number of inflammatory cells per square millimetre in 15 squares per section and five sections per patient using a Zeiss microscope (objective 40x). The median number of inflammatory cells per square millimetre was calculated and patients were then categorized into two subgroups with numbers below and above the median.
The abundance of END-containing cells was examined in two or three representative samples of each category (i.e. patients with less than and with more than than the median number of inflammatory cells per square millimetre). The number of samples was limited by the amount of available synovial specimens but we have previously shown that there is a linear correlation between the number of END-containing cells and the number of inflammatory cells.13 According to previously published protocols,9 15 the synovial specimens were immediately fixed in 4% (w/v) paraformaldehyde with 0.2% (v/v) picric acid in 0.16 M phosphate buffer solution (pH 6.9) for 5 h, and then placed in 15% (w/v) sucrose solution at 4°C overnight. Tissues were then embedded in OCT compound (Miles Inc., Elkhart, IN). Serial tissue sections (4 µm thick) were prepared using a cryostat and were mounted onto gelatin-coated slides. Unless otherwise stated, all incubations were done at room temperature and phosphate buffered saline (PBS) was used for washing (three times for 10 min) after each step. The sections were incubated with PBS, 0.3% H2O2 and 10% methanol for 45 min to block endogenous peroxidase. To prevent non-specific binding, the sections were incubated for 60 min in PBS containing 0.3% Triton X-100, 1% bovine serum albumin (BSA), 4% goat serum and 4% horse serum (block solution). The sections were then incubated overnight at 4°C with a polyclonal rabbit END antibody (Peninsula Laboratories, Belmont, CA; dilution 1:1000) in PBS containing 1% BSA, 3% goat serum and 0.3% Triton X-100. After washing, sections were incubated for 90 min with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) (goat anti-rabbit) in PBS containing 1% BSA, 3% goat serum and 0.3% Triton X-100. After washing, sections were incubated with avidinbiotin-conjugated peroxidase for 90 min. Finally, sections were stained with 3',3'-diaminobenzidine tetrahydrochloride (Sigma, Deisenhofen, Germany) containing 0.01% H2O2 in 0.05% Tris-buffered saline pH 7.6 for 35 min. After the enzyme reaction, the sections were washed in tapwater, dehydrated in alcohol, cleared in xylene and mounted in DPX (Merck, Darmstadt, Germany). To demonstrate specificity of staining the following controls were included: (i) preabsorption of diluted antibody against END with 5 µg ml1 purified END (Peninsula Laboratories) for 24 h at 4°C; (ii) omission of either the primary or secondary antibody or the avidinbiotin complex.
Statistics
The primary outcome parameter was to detect significant differences in the postoperative supplemental analgesic consumption between four different i.a. treatment groups. According to a previous study,17 assuming an type I error of 0.05 and a ß type II error of 0.2, the minimum number of patients required per group in order to detect significant differences would be 20. Patient characteristics, sedation scores and the occurrence of side effects were analyzed by analysis of variance (ANOVA) or
2 test, respectively. Pain intensity, determined as VAS score, was analyzed by a two-factorial analysis of variance with repeated measurement design (RM-ANOVA). To test for dose-dependent effects of VAS scores, the area under the curve (AUC) for each dose of i.a. morphine was calculated using the trapezoid rule, and a subsequent linear regression ANOVA was performed.17 To test for differences in the total piritramide consumption and the time to first analgesic request, data were analyzed by ANOVA and post hoc by linear regression for dose-dependent effects. Following immunohistochemistry, we investigated whether the number of inflammatory cells per square millimetre contributes as a covariate to the differences in pain intensity of the four different treatment groups. To this end patients were grouped into those below versus those above the median number of inflammatory cells per square millimetre synovial tissue section. Subsequently, VAS scores (AUC), total piritramide consumption and the time to first analgesic request were displayed in relation to each patient's individual number of inflammatory cells per square millimetre. They were then analyzed by two-factorial multivariate analysis of covariance (MANCOVA) with repeated measurement design. Probability P<0.05 was considered significant.
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Results |
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Discussion |
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The second and most important finding of this study is that patients with high and low synovial inflammation showed similar doseresponse relationships in i.a. morphine analgesia. In particular, there was neither a right- nor leftward shift of doseresponse curves, i.e. neither an increase nor a decrease in the potency of i.a. morphine. This is demonstrated by the similar doseresponse curves for both supplemental opioid consumption and time to first medication request, except for the 4 mg morphine dose with respect to the latter. In addition, the individual number of each patient's inflammatory cells had no influence on the patient's corresponding pain scores or analgesic intake within each i.a. treatment group. Also, we found more END-containing cells in high degrees of synovial inflammation and less in low degrees. This is consistent with our experimental studies which indicate that both the higher number of END-producing cells and the content of END within inflamed tissue increase with the duration and development of inflammation.13 15 Upon stressful stimulation, END is released from these cells and acts on opioid receptors of peripheral sensory nerve endings which have also been shown within synovial tissue.12 16 22 The functional consequence of this enhanced opioid peptide expression within inflamed tissue is progressively augmented pain relief.13 15 However, this clinical trial does not show a potentiation in pain relief in the group with a high degree of synovial inflammation which may be due to the clinical circumstances that synovial inflammation is not as well controlled as under experimental conditions.
On the other side, the local expression of END within inflammatory cells of synovial tissue does not result in any alterations of the doseresponse relationship of i.a. morphine analgesia. Since endogenous and exogenous opioids exert their analgesic effects through the same opioid receptors,23 the increased expression of endogenous END in marked synovial inflammation may produce cross-tolerance, i.e. a rightward shift in the analgesic doseresponse curve of i.a. morphine. In fact, this has been described for central opioid analgesic actions, which appear to be exquisitely susceptible to the development of tolerance, i.e. a decrease in potency with prolonged exposure to exogenous or elevated levels of endogenous opioid agonists.1 4 5 In the present study, however, the enhanced presence of END-positive cells within inflamed synovial tissue did not result in the development of such cross-tolerance. This might be also due to changes in the functional activity of opioid receptors on peripheral sensory nerve endings which have not been investigated in this trial. While this can be done in experimental studies,24 25 it is very difficult to perform such investigations in humans.
The present results confirm a doseresponse dependency of i.a. morphine analgesia. Interestingly, this can be observed in patients with both high and low degrees of synovial inflammation. Apparently, an increased number of inflammatory cells and an enhanced expression of endogenous opioids within synovial tissue do not seem to alter this dose dependency. Our findings might also be of interest for the local opioid treatment of chronic arthritic pain patients. Further studies are needed, particularly with repeated injections of i.a. morphine in such patients, to gain more insight into alterations of the effectiveness of such treatment during inflammation.
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Acknowledgments |
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Footnotes |
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References |
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