The pharmacological effects of opioids vary widely among patients because of inherited traits and environmental factors. Because arterial blood pressure status has been linked to pain threshold in humans (1), one such inherited trait could be hypertension. However, although the number of hypertensive patients who experience chronic or postoperative pain is large, few studies have addressed the contribution of hypertension to the antinociceptive effects of opioids. Here we evaluate nociception and opioid antinociception in a widely used animal model of essential hypertension, the spontaneously hypertensive (SHR) rat. At approximately 4 wk of age, arterial blood pressure in SHR increases rapidly, mainly because of increases in cardiac output. As the SHR matures to approximately 3 mo of age, the main hemodynamic factor responsible for hypertension switches to total peripheral resistance. Patients with essential hypertension also present with increased total peripheral resistance throughout their lives, and this points to the utility of the SHR as a model of clinical primary hypertension (2).
Opioid binding sites are upregulated in the brain or spinal cord of SHR (3), and the antinociceptive effects of morphine are greater in SHR rats as compared with their normotensive Wistar-Kyoto (WKY) controls in tests of brief, reflex withdrawal responses, such as the hotplate (4) and tail-flick (5) tests. These studies emphasize linkage between inherited hypertension, the systems that control arterial blood pressure, and opioid inhibition of acute pain. To address linkage with the acute inflammatory pain presented by patients, we tested the hypothesis that opioid inhibition of inflammatory pain is exaggerated in the SHR rat. We chose the formalin test of inflammatory pain because it models both continuing peripheral nerve activity and peripheral sensitization (6–8). In this model, the intraplantar injection of dilute formalin first produces a rapid-onset, 5-min period of peripheral nerve activity, sympathetic activity, and painlike behavior (Phase 1). After a brief quiescent period essentially devoid of nociception (interphase), these responses return and persist for another hour (Phase 2).
Opioids act at peripheral sites to inhibit the innate immune processes initiated by tissue injury (9). Thus, in the setting of inflammation, opioid agonists such as morphine and remifentanil act at small-diameter primary afferent neurons to inhibit substance P-mediated increases in vascular permeability that lead to plasma extravasation and calcitonin gene-related peptide (CGRP)-mediated dilation of blood vessels, leading to increased blood flow and local blood volume (10–12). Because inflammation is differentially regulated in SHR (13), we asked whether opioid inhibition of inflammation is different in this strain. Our experiments compared the antiinflammatory actions of morphine on formalin-evoked edema and paw skin temperature in SHR and normotensive rats.
To evaluate formalin-induced nociception, we monitored not only nociceptive behaviors, but also increases in mean arterial blood pressure (MAP) and heart rate (HR). Our methodology supplements traditional behavioral techniques of pain assessment in rodents with measurement of MAP and HR (7,14,15). These cardiovascular responses increase with formalin concentration, and general anesthesia studies show that they do not indirectly result from the formalin-induced locomotor activity (7). Ness et al. (16) have used a similar strategy in anesthetized rats to demonstrate opioid inhibition of cardiovascular responses to stimulation of nociceptive afferents in visceral organs.
Measurement of MAP not only provided a reliable marker of nociception, but also allowed us to investigate the cardiovascular effects of morphine in SHR rats. Systemic administration of morphine decreases MAP in the rat and evokes orthostatic hypotension in the healthy patient (17). Opioid agonists are routinely used for the induction of anesthesia, stabilization of hemodynamics during cardiovascular anesthesia (18), and treatment of myocardial ischemia. Because little is known of the cardiovascular effects of systemically administered opioid agonists in hypertensive subjects, we designed our studies to evaluate the effects of morphine on MAP and HR in normotensive and SHR rats.
All protocols were approved by Institutional Animal Care and Use Committees at both the University of Missouri and Tulane University and were in accordance with the guidelines set forth by the International Association for the Study of Pain. Male WKY, SHR, and Sprague-Dawley (SD) rats were weight-matched at the time of ordering (225–236 g) from Charles River Laboratories (Hollister, CA) and were allowed to acclimate to our facility for 6–7 days before surgery. They were individually housed at 20°C ±1°C on a 12-h light-dark cycle (7:00 AM lights on), with food and water provided ad libitum. The WKY rat is the strain from which the SHR was originally derived. Because of evidence for genetic heterogeneity in the WKY rat control strain (19), we also evaluated the effects of morphine on formalin-evoked responses in the SD rat. Throughout the study, our protocol generally adhered to a balanced design, and animals were usually tested in sets of 6 (3 strains [WKY, SHR, and SD] × 2 treatments [saline and morphine]).
Femoral arterial and venous jugular catheters were constructed and placed as previously described (7,20). Briefly, with rats under isoflurane anesthesia, we isolated the left femoral artery, jugular vein, or both by blunt dissection and advanced a polyethylene catheter prefilled with heparinized saline (100 IU/mL) within the vessel. It was secured and tunneled under the skin and then was exteriorized at the nape. After rats recovered from anesthesia, we returned them to their cages and allowed them to recover for 2–5 days before experimentation.
As previously described (7,21), each animal was transferred to a bedded Plexiglas box in a quiet testing room on a 12-h light/dark cycle for at least 15 h before testing. After rats recovered from the handling that is required to connect arterial catheters to the pressure transducer, baseline readings were recorded (AD Instruments, Castle Hill, Australia). MAP and HR data were averaged in 1- to 2-min bins for analysis and presentation. Resting MAP was calculated as a mean of measurements taken during the 5 min just before saline or opioid injection.
Morphine (3 mg/kg) or saline (2 mL/kg body weight) was administered subcutaneously 20 min before the midplantar injection of formalin (50 μ L of 5% buffered formalin in distilled water). We purposely chose a systemic dose of morphine (3.0 mg/kg) that produces only a partial antinociceptive effect in the formalin test (8). This minimized our chances of producing a floor effect in every strain (i.e., complete analgesia), thus masking any interaction between strain and morphine antinociception. Remifentanil (30 μ g/kg bolus 15 s before formalin, followed 90 s later by a 10 μ g · kg−1 · min−1 infusion for 13.5 min) or saline was administered through the jugular venous catheter (14).
The number of flinches and time spent licking during the second, third, fourth, and fifth minute after formalin injection were counted during Phase 1 (time points 1–5). After Phase 1, flinches and time spent licking were counted in 2-min intervals, as previously described (7,15). Each animal received a single formalin injection and a single drug treatment.
Swelling and paw temperature were assessed as previously described (12). Briefly, by using gentle restraint, paw thickness and paw skin temperature were measured with microcalipers and a contact surface probe and thermometer (YSI Inc.). Triplicate measurements were taken just before MAP recording and immediately after completion of the formalin test.
Differences between the means were analyzed by three-way analysis of variance (ANOVA), with strain and drug treatment as the between-subjects factors and time as the repeated measure. After significant main effects of the factors were determined, the flinching data were further analyzed by expressing it as area under the curve for Phase 1 (sum of time points 1–5) and Phase 2 (sum of time points 20–65), followed by one-way ANOVAs. When F values reached statistical significance (P < 0.05), the ANOVA was followed by post hoc comparisons with unpaired Student's t-tests (Systat software; SPSS Inc., Chicago, IL).
Isoflurane (99.9%) was obtained from Henry Schein (Melville, NY). Formalin was obtained from Fisher Scientific (Formalde-Fresh; Houston, TX). Morphine and remifentanil were provided by the National Institute on Drug Abuse Drug Supply Program and Glaxo-Wellcome, respectively.
Before administering saline or morphine, we confirmed the hypertensive state in awake, unrestrained SHR rats by using femoral arterial catheters. As illustrated in Table 1, baseline MAP was higher in SHR rats when compared with normotensive strains (P < 0.05). For each strain, we observed no significant difference in pretreatment values between the saline and morphine groups (P > 0.05).
To test the hypothesis that opioids produce more intense analgesia in SHR during inflammatory pain, we evaluated the effect of morphine on formalin-induced behavioral responses (Figs. 1 and 2). Significantly more flinches were observed in SHR after saline as compared with WKY. Similarly, in the SHR, morphine produced a more pronounced effect in reducing the number of flinches.
We also evaluated the cardiovascular effects of subcutaneous morphine. As illustrated in Figure 3, morphine slightly and transiently reduced MAP and HR in normotensive rats. By contrast, morphine robustly decreased MAP and HR in SHR. Significant strain × drug interactions indicate that these cardiovascular effects were potentiated in the SHR compared with WKY or SD rats. Figure 3 also illustrates that morphine significantly reduced formalin-associated changes in MAP and HR in SHR and SD, but not WKY, rats. Further comparisons between WKY and SHR rats revealed strain × drug interactions, indicating a potentiated effect of morphine on formalin-associated cardiovascular variables in the SHR.
Because antiinflammatory effects could contribute to the antinociceptive effects of morphine, we evaluated edema and local increases in skin temperature. Figure 4A demonstrates that morphine reduced formalin-induced swelling in SHR and SD rats, and further statistical analysis indicated that swelling in the presence of morphine was less in SHR than in WKY (see the legend to Fig. 4). Figure 4B demonstrates that morphine reduced formalin-induced increases in paw skin surface temperature in WKY but not SD and SHR rats, although a marginally significant effect was found in SHR rats (P = 0.052). Thus, whereas morphine decreased paw temperature in the WKY group, it tended to increase paw temperature in the SHR group.
Opioids not only inhibit the magnitude of formalin-induced inflammation, but, when administered at early time points, also delay the onset of inflammation. This is reflected by a delay in the time course of formalin-induced nociception (12,14,20). We next tested the hypothesis that this delay is altered in the setting of high MAP. We used the ultra-short-acting properties of remifentanil to restrict opioid antinociception to the first 15–20 min of the formalin test and then observed recovery of the Phase 2 response in WKY, SHR, and SD rats. As in previous studies with SD rats (12,14,20), we found that remifentanil dramatically delayed the time course of formalin-induced persistent nociception in SHR and WKY rats (Fig. 5). We now report that this delay occurred in WKY and SHR rats as well (P < 0.001; drug × time).
These studies are the first to compare the effects of systemic morphine on MAP and HR in awake SHR and normotensive rats. We now report that morphine dramatically reduces MAP and HR in the SHR rat. Our studies were not designed to delineate the central nervous system (CNS) or peripheral sites involved; however, the exaggerated hypotension in the SHR may merely reflect higher resting MAP. Alternatively, it could result from differences in opioid-induced histamine release, inhibition of sympathetic outflow, or activation of vagovagal reflexes (22–24). In the brain, μ- and/or κ-opioid receptors are upregulated in the hypothalamus, hippocampus, and midbrain of SHR rats (25), pointing to the possibility that an upregulation of opioid receptors in central cardiovascular centers of the SHR strain might contribute to exaggerated opioid inhibition of MAP and HR.
As reported previously, we found that SHR, as compared with WKY, are particularly sensitive in models of inflammatory nociception (21,26). Here we report for the first time that this difference was not significant in morphine-treated animals. Thus, morphine produced a greater reduction in the number of flinches in SHR as compared with WKY. Similarly, morphine produced greater inhibition of formalin-evoked pressor and tachycardia responses in the SHR than in WKY. It must be kept in mind, however, that the relatively long-lasting effects of morphine on MAP and HR in the SHR rat complicate formalin-induced changes in these variables. We conclude that morphine attenuates the hypersensitivity to formalin pain in the SHR.
Because opioid antinociception also varied between outbred SD and inbred WKY rats, genetic factors unrelated to MAP may contribute to morphine antinociception in the formalin test. This appears to hold true with regard to transient thermal nociception as well. For example, Plesan et al. (4) recently reported that the antinociceptive effects of morphine in the hotplate test varied among these three strains in the order SD > SHR > WKY. If certain genes pleiotropically affect both hypertension and nociception, then quantitative trait locus mapping techniques of loci involved in both pain and hypertension could yield valuable information regarding differential nociceptive processing in hypertensive individuals.
Morphine reduced formalin-induced swelling to a greater degree in SHR as compared with WKY rats (but not SD rats). This pattern remarkably resembles the strain differences in formalin-induced behavioral and cardiovascular nociceptive responses. By contrast, the inhibition of paw skin surface temperature (an indirect measure of local blood flow) was smaller in SHR compared with WKY rats. Indeed, for reasons that we cannot yet explain, the evoked skin surface temperature after morphine was greater in SHR than WKY rats. Regardless, our data argue that exaggerated morphine inhibition of swelling in the SHR rat is unrelated to the mechanisms that regulate paw skin temperature, such as CGRP-mediated vasodilation (12). Instead, it is more likely that morphine inhibits formalin-induced increases in vascular permeability to a greater extent in the SHR rat. Of course, further studies evaluating the effects of morphine on plasma extravasation in SHR and normotensive rats will be required to test this hypothesis.
In the formalin test, we have reported that early administration of remifentanil inhibits initial inflammation during Phase 1 and also delays the onset of persistent inflammation associated with Phase 2 in SD rats (12,20). Here we show similar effects in SHR and WKY. By demonstrating this phenomenon in multiple rat strains, these results support our contention that early opioid administration does not produce a preemptive effect in this model of postoperative pain (27). Rather, we have proposed that preemptive analgesia may actually produce an undesirable extension of the time course of persistent pain (14,20). As illustrated by time points 25–30 in Figure 4, complex three-way interactions possibly reflected a relatively earlier onset of Phase 2 behavior after remifentanil in the SHR group. We speculate that preemptive analgesia may actually increase the duration of persistent pain in hypertensive subjects, but further studies are necessary to test this hypothesis.
One might argue that a decreased metabolism of morphine in SHR would yield enhanced opioid receptor-mediated antinociception. However, systemic administration of morphine to WKY and SHR rats yielded similar values for the area under the serum morphine concentration-time curve, half-life, apparent volume of distribution at steady-state, terminal elimination rate constant, and total body clearance (5). Furthermore, close inspection of Figure 1 suggests that, in the presence of morphine, residual Phase 2 responses are initiated relatively quickly in SHR as compared with WKY rats. Also, as shown in Figure 4, the effects of remifentanil appear to wear off more quickly in the SHR. These data indicate that the opioid metabolism is faster, rather than slower, in the SHR and thus do not explain our results.
However, previous studies suggest that morphine may distribute to the CNS to a greater extent in SHR than in WKY, because the concentration of morphine in the brain and spinal cord of the SHR is much larger than that of the WKY after systemic administration (28). This may be particularly relevant because SHR rats exhibit a relatively high density of opioid binding sites in the CNS. These sites include areas that contribute to pain control, such as the anterior cingulate cortex and hypothalamus (3). Indeed, opioid injection directly into the hypothalamus produces greater antinociceptive effects in SHR than in WKY rats. In conclusion, it would be valuable to test the hypothesis that CNS mechanisms contribute to exaggerated morphine analgesia during persistent nociception in the SHR.
The current data indicate that genetic factors and/or the mechanisms that contribute to sustained increases in MAP potentiate the effect of morphine pain inhibition, edema, hypotension, and bradycardia. The implications of our results to the anesthe-siologist are twofold. First, they suggest that morphine-induced decreases in cardiovascular activity are greater in the SHR. If this holds true in hypertensive humans, then MAP status should be considered when opioid regimens are planned during surgical procedures. Second, the opioid dose may need to be adjusted in patients with genetic hypertension to achieve optimum analgesia during the postoperative period.
Future studies evaluating the antihyperalgesic and antiallodynic actions of opioids in hypertensive subjects by using models of chronic inflammatory or neuropathic pain would yield further clinically relevant information. Such studies would aid in the development of a rational therapy for the large population of hypertensive patients who also experience persistent or chronic pain.
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© 2004 International Anesthesia Research Society
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