MPV-2426 is a novel alpha2-adrenoceptor agonist developed for spinal pain therapy. It has proved to be effective in physiologic and neuropathic conditions. In the current study its effectiveness on mechanical hyperalgesia was assessed in a rat model of postoperative pain.
Rats with intrathecal catheters were anesthetized with pentobarbital, and a 1-cm incision was made in the plantar aspect of the foot and closed. During postoperative days 1 and 2 the antihyperalgesic effects induced by intrathecal MPV-2426, clonidine, and dexmedetomidine were determined by assessing the hind limb withdrawal threshold to calibrated von Frey hairs applied to the skin of the hind paw adjacent to the wound.
MPV-2426 administered into the lumbar spinal cord produced a dose-dependent (0.3-10 microg) attenuation of the mechanical hyperalgesia, and this antihyperalgesic effect was completely reversed by yohimbine (1 mg/kg, subcutaneous), an alpha2-adrenoceptor antagonist. Dexmedetomidine (1-3 microg) produced an equipotent antihyperalgesic effect, whereas the effect of clonidine (1-10 microg) was markedly weaker. MPV-2426 (10 microg in 20 microl) administered adjacent to the wound did not produce any effect. Preoperative treatment with an antihyperalgesic dose of MPV-2426 did not prevent the development of hyperalgesia.
Intrathecal MPV-2426 dose-dependently attenuates postoperative hyperalgesia to mechanical stimulation because of an action on alpha2 adrenoceptors. Its antihyperalgesic action is as effective as that produced by dexmedetomidine and is considerably stronger than that produced by clonidine. However, preoperative treatment with MPV-2426 does not prevent the development of postoperative hyperalgesia.
MPV-2426 or 3-(1H-Imidazol-4-ylmethyl)-indan-5-ol hydrochloride is a novel α2-adrenoceptor agonist developed for spinal pain therapy. It has proved to be a potent antinociceptive agent when administered intrathecally. 1MPV-2426 does not cross the blood–brain barrier and, when injected intrathecally, its pharmacologic effects are more dermatomally restricted than those of dexmedetomidine. 2It is important to test the pain-relieving effects of novel drugs not only during physiologic conditions, but also in different experimental models of pathophysiologic pain. So far, MPV-2426 has already proven to be effective in relieving hyperalgesia in neuropathic animals. 2In the current study, we attempted to find out whether MPV-2426 is effective in a rat model of postoperative hyperalgesia developed recently by Brennan et al. 3In this model, a surgical incision causes reproducible, quantifiable hyperalgesia that parallels the postoperative time course of patients. The most consistent finding in this model is primary hyperalgesia in the immediate vicinity of the wound. 4This model has proved to be sensitive to morphine, 5gabapentin, 6ionotropic non–N -methyl-d-aspartate (NMDA) receptor antagonists, 7a neurokinin-1 (NK1) receptor antagonist, 8to adenosine, 9neostigmine, 9and the prototype α2-adrenoceptor agonist clonidine, 9whereas NMDA receptor antagonists 10and metabotropic glutamate receptor antagonists 11did not have any significant effect in this model. In the current study we compared the antihyperalgesic effect of intrathecally administered MPV-2426 with two older α2-adrenoceptor agonists, clonidine and dexmedetomidine. Moreover, we addressed whether MPV-2426 also could prevent the development of postoperative hyperalgesia.
Materials and Methods
The study was performed with adult male Hannover-Wistar rats (The Finnish National Laboratory Animal Center, Kuopio, Finland; 220–360 g). The study protocol was accepted by the Institutional Animal Care Committee of the University of Helsinki and by the Regional Government of Southern Finland.
Before the operations, the rats were well-habituated to the experimental conditions by allowing them to spend 1 or 2 h daily in the laboratory for 2 days. An intrathecal catheter (PE-10) for drug administration in the lumbar level of the spinal cord was inserted during pentobarbital anesthesia (50 mg/kg), as described in detail elsewhere. 12After recovery from anesthesia, the correct placing of the catheter was verified by administering lidocaine (5 μl, 4%, followed by 10 μl saline for flushing). Only those rats, that had no motor impairment before lidocaine administration and a bilateral paralysis of hind limbs after intrathecal lidocaine were studied further. One or 2 days after the lidocaine test, the rats were again anesthetized with pentobarbital (50 mg/kg). Then, a 1-cm longitudinal incision was made in the plantar aspect of the foot and closed, as described in detail elsewhere. 3After surgery, the rats were allowed to recover in cages.
Assessment of Mechanical Hypersensitivity
For behavioral assessment of mechanical hyperalgesia, the rats were placed on an elevated metal grid. Hind limb withdrawal thresholds to mechanical stimulation of the paw were determined with a calibrated series of von Frey hairs (range, 0.1–46.6 g; Stoelting, Wood Dale, IL). The hairs were applied to the paw at an increasing force until the rat withdrew its hind limb. This was repeated three times. The median of these three threshold determinations was used in further calculations. The site of stimulation was in the heel, adjacent to the wound (the area of primary hyperalgesia). 4In each condition, the threshold determination was performed 5 min before drug administration and at various intervals (5–90 min; except in groups 6 and 7, in which the testing was performed until time point 60 min) after drug administration. The effects of intrathecally administered drugs on postoperative hyperalgesia were tested in the following experimental groups: (1) saline control; (2) 0.3 μg MPV-2426; (3) 1 μg MPV-2426; (4) 3 μg MPV-2426; (5) 10 μg MPV-2426; (6) 1 mg/kg subcutaneous yohimbine; (7) 3 μg MPV-2426 + 1 mg/kg subcutaneous yohimbine; (8) 1 μg dexmedetomidine; (9) 3 μg dexmedetomidine; (10) 1 μg clonidine; (11) 3 μg clonidine; and (12) 10 μg clonidine. The order of testing various drug doses and drugs was varied between the animals to avoid serial effects.
Assessment of Preemptive Effects
In a group of the animals, 10 μg intrathecal MPV-2426 or physiologic saline was administered 60 min before operation to study preemptive effect of α2-adrenoceptor agonists on the development of postoperative hyperalgesia. The effect of drug treatment on the development of postoperative hyperalgesia was assessed on the first postoperative day.
Assessment of Possible Periwound Effects
In a group of animals, MPV-2426 (10 μg in 20 μl) was administered intraplantarly adjacent to the wound, and the antihyperalgesic effect was assessed before drug treatment and 5 min and 15 min after the intraplantar injection.
Assessment of Motor Impairment
In a group of animals that were not operated on and with intrathecal catheters, MPV-2426 was administered at the dose of 10 μg, and the effect on locomotor behavior was assessed using the Rotarod test at various intervals for 90 min after the injection. In this test, the rat was placed on a revolving drum (12 rpm) and the time until drop from the drum was measured. The cutoff latency was 60 s.
The α2-adrenoceptor agonists MPV-2426 or 3-(1H-Imidazol-4-ylmethyl)-indan-5-ol hydrochloride (Orion Pharma, Turku, Finland), 1dexmedetomidine 13,14(Orion Pharma, Turku), and clonidine (Sigma, St. Louis, MO) were dissolved in sterile water to obtain the volume of 5 μl. Physiologic saline (Orion Pharma, Espoo, Finland) was used as a control. Each drug injection was followed by 10 μl saline for flushing the catheter. Yohimbine, an α2-adrenoceptor antagonist (Sigma), was administered subcutaneously in an attempt to reverse the MPV-2426–induced effects.
Nonparametric statistics were used to assess the statistical significance of withdrawal threshold changes within (Friedman test) and between (Kruskal-Wallis test) groups. Post hoc testing was performed using the Dunn test. The Mann-Whitney U test was used to assess differences in withdrawal thresholds between two groups. Parametric one-way analysis of variance (ANOVA) with repeated measures was used to assess drug-induced changes in Rotarod latencies. P < 0.05 was considered to represent a significant difference.
Main Effect of Time on Mechanical Hyperalgesia
The incision produced a marked ipsilateral decrease in the hind limb withdrawal threshold;i.e. , mechanical hyperalgesia or allodynia. The median withdrawal thresholds determined on postoperative days 1 and 2 (before drug administrations) were in all experimental groups less than 5 g, whereas, preoperatively, the withdrawal threshold was always more than 20 g and usually more than 40 g.
The main effect of time after intrathecal saline was not significant for hind limb withdrawal threshold evoked by mechanical stimulation adjacent to the wound (n = 11;fig. 1A). MPV-2426 dose-dependently (0.3–10 μg intrathecal) attenuated hyperalgesia. The onset of significant antihyperalgesia after intrathecally administered MPV-2426 was quite long (> 15 min). The main effect of time was statistically significant for MPV-2426 at the dose of 1 μg (n = 10;P < 0.0005), 3 μg (n = 14;P < 0.0001;fig. 1B), and 10 μg (n = 7;P < 0.001;fig. 1C), whereas the antihyperalgesic effect at the dose of 0.3 μg was not significant (n = 5).
Dexmedetomidine had a dose-related antihyperalgesic effect of short onset (≥ 15 min). The main effect of time on withdrawal thresholds after dexmedetomidine was significant at the dose of 3 μg (n = 5;P < 0.01;fig. 1D) but not at the dose of 1 μg (n = 5). Intrathecal clonidine at the dose range used (1–10 μg) had only very weak effects on mechanical hypersensitivity. The main effect of time on withdrawal threshold after clonidine reached significance only at the dose of 3 μg (n = 6;P < 0.02) but not at the dose of 10 μg (n = 6;fig. 1E) or the dose of 1 μg (n = 5). Yohimbine at the dose (1 mg/kg subcutaneous) that alone had no significant effects (n = 4) completely reversed the antihyperalgesic effect induced by MPV-2426 (n = 5;fig. 2A).
The Main Effect of Drug Dose on Mechanical Hyperalgesia
The main effect of drug dose on mechanical hyperalgesia was assessed at a time of the peak effect of each drug. The antihyperalgesic effect of MPV-2426 significantly increased with an increase of the drug dose (up to 10 μg;P < 0.001;fig. 2B). At a time of peak effect (90 min) after administration of MPV-2426 at the doses of 3 and 10 μg, the withdrawal threshold reached the cutoff value of 46.6 g in 36 and 42% of the animals. This is likely to distort the linearity of the dose–response curve within the dose range (3–10 μg) of MPV-2426.
Intraplantar Injection of MPV-2426
In five rats, MPV-2426 was microinjected (10 μg in 20 μl) intraplantarly adjacent to the wound to find out whether the long onset of the antihyperalgesic action after intrathecal administrations could be explained by a slow spread of the drug from the central site to the skin;i.e. , whether the antihyperalgesia was caused by a periwound action. No antihyperalgesic effect was observed after intraplantar injection of MPV-2426 during the 15-min observation period (fig. 1F).
Preemptive Effect on Development of Hyperalgesia
When 10 μg MPV-2426 (n = 5) was administered intrathecally 60 min before skin incision, the wound hyperalgesia determined on the first postoperative day was not significantly different from that observed in rats (n = 14) that received saline preoperatively (fig. 2E).
Rotarod Test in Nonhyperalgesic Animals
To exlude a possibility that the increase of hind limb withdrawal thresholds was caused by suppression of motor responses, intrathecal MPV-2426 (n = 4) was administered at a dose of 10 μg and the motor behavior of nonoperated rats was observed at various intervals for 90 min during the Rotarod test. No significant MPV-2426–induced effect was observed during the Rotarod test (fig. 2F).
The current results indicate that lumbar intrathecal injection of MPV-2426 produces a dose-dependent attenuation of mechanical hyperalgesia in a rat model of postoperative pain. The antihyperalgesic action was completely reversed by yohimbine, an α2-adrenoceptor antagonist with a nonimidazoline structure, indicating that the antihyperalgesia induced by MPV-2426 was mediated by α2adrenoceptors. This is in line with the receptor-binding studies that indicate that MPV-2426 and dexmedetomidine are potent full agonists on all three subtypes of α2adrenoceptors. 1The current results also indicate that the antihyperalgesic potency of MPV-2426 is equal to that of dexmedetomidine and considerably stronger than that produced by clonidine. In line with results of a recent study, 9clonidine at the dose range up to 10 μg had only a very weak effect on mechanical hypersensitivity. However, within this dose range clonidine has a significant antinociceptive effect in a test of thermal nociception. 9Thus, thermal nociception appears to be more sensitive to α2–adrenoceptor-induced effects than mechanical hypersensitivity. In line with this interpretation also, MPV-2426 tended to have a more potent influence on heat-induced tail-flick response than on mechanical hypersensitivity in neuropathic animals. 2
The maximal attenuation of mechanical hyperalgesia by MPV-2426 was reached at a longer latency (up to 60–90 min) than that produced by dexmedetomidine (≥ 15 min). Because intraplantar injection of MPV-2426 adjacent to the wound did not produce any significant antihyperalgesia within 15 min, it seems improbable that a direct periwound action played a major role in the α2-adrenergic antihyperalgesia. This finding also suggests that the slow onset of action in a test of mechanical hyperalgesia after intrathecal administration of MPV-2426 could not be explained by a slow diffusion of the drug from the central nervous system to produce its action on peripheral α2adrenoceptors in the skin. 16This interpretation is further supported by the previous physiologic evidence indicating that MPV-2426 only poorly, if at all, penetrates the blood–brain barrier. 2A drug (MPV-2426) that only slowly diffuses within the central nervous system is likely to act with a longer latency than a more soluble drug such as dexmedetomidine. Whether this or some other mechanism contributed to differences observed in the onset of antihyperalgesic action between MPV-2426 and dexmedetomidine remains to be studied.
In behavioral pain tests, a suppression of motor activity could produce an increase of withdrawal thresholds, mimicking antihyperalgesic action. However, because an intrathecal administration of MPV-2426 at an antihyperalgesic dose (10 μg) did not produce any significant suppression of motor activity in the Rotarod test, it seems that the drug-induced increases in withdrawal thresholds were not caused by this type of an artifact.
Preoperative intrathecal treatment of rats with a single dose of MPV-2426 did not have any significant effect on the development of postoperative hyperalgesia. Therefore, it seems that α2-adrenoceptor agonists do not have any marked preemptive effects on the development of postoperative hypersensitivity, although these compounds effectively attenuate maintained hyperalgesia. This result is in accordance with some previous findings in other models of pathophysiologic pain that showed that a preemptive treatment with α2-adrenoceptor agonists did not prevent the development of hyperalgesia. 17–19However, there is at least one study that reported that clonidine administered preoperatively prevented development of mechanical hyperalgesia in a model of mononeuropathy. 20
Intrathecally administered MPV-2426 proved to attenuate mechanical hyperalgesia, caused by action on α2adrenoceptors, in a rat model of postoperative pain. Because of its pharmacokinetic properties, MPV-2426 provides a more targeted and at least as potent treatment of dermatomally restricted postoperative hyperalgesic conditions than some older α2-adrenoceptor agonists, such as dexmedetomidine or clonidine. Moreover, it was recently shown that intrathecally administered MPV-2426 at an antinociceptive dose has considerably less cardiovascular side effects than does clonidine. 21
The authors thank Dr. Ernst Mecke and Dr. Hong Wei, Department of Physiology, Institute of Biomedicine, University of Helsinki, Helsinki, Finland, for their help in the experiments.