Currently α2-adrenoceptor agonists are receiving attention as adjuncts to anesthesia, analgesia, and sedation/anxiolysis in the pediatric population (1–4). The efficacy of these drugs is known in adults but little information is available at younger ages and extrapolating findings in adults to the pediatric population is likely to be misleading. Pain processing pathways differ between immature and mature animals; notably, neonates lack functional descending inhibitory neurons (5,6), and consequently analgesic drugs that rely on supraspinal mechanisms and connectivity to the spinal cord via descending inhibitory neurons are ineffective, as we have demonstrated with nitrous oxide (7). This may have profound clinical implications because it is likely that ineffective analgesia in neonates can result in long-term neuroplastic changes producing a hyperalgesic state with low threshold for subsequent painful stimuli (8–11). Thus, a prudent strategy to optimize analgesia in this age group may be with the use of drugs that directly target spinal sites of action, thereby bypassing the need for functional connectivity between supraspinal and spinal sites via the descending inhibitory neurons.
Alpha adrenoceptors are present in the spinal cord at birth (12,13) and we have previously shown antinociceptive efficacy using an ex vivo paradigm of the isolated perfused neonatal rat spinal cord (14,15). In this study we investigated the hypothesis that because of the immature development of connections between the spinal and supraspinal regions dexmedetomidine (Dex), an α2A-adrenoceptor specific agonist, would have no in vivo antinociceptive in rats aged <21 days. We also sought to determine any dose-dependent sedative effect of Dex because sedation may confound the interpretation of the behavioral assessments of pain.
The study protocol was approved by the Home Office (UK), and all efforts were made to minimize animal suffering and the number of animals used. Fischer rats were used for the entire study (B&K Universal, Grimston Aldbrough Hull, UK). The rats were provided ad libitum food and water, and artificial lighting between 6 am and 6 pm. The date of birth for the animals was defined as 0 day-old. Experiments were performed on rat pups of 7, 15, 19, 23, 28 days of age and on adult rats (11–12 wk-old); these ages are equivalent to neonate, toddler, child (prefunctional descending inhibition), child (postfunctional descending inhibitory neurons), adolescent, and adult (16).
Within each age group, there were 3 cohorts administered either saline (control) or Dex at one of 2 doses (10 or 50 μg/kg, administered subcutaneously). Twenty minutes after drug administration 5% formalin was injected into the plantar surface of the animal’s left hindpaw subcutaneously. The volumes of formalin or saline injected were adjusted for each age group as previously reported (7) and were as follows: 10 μL for 7 days old, 15 μL for 19 days old, 20 μL for 28 days old, 50 μL for adults.
Immediately after injection of formalin, behavior was recorded for 60 min with a video camera (MegaPixel, Digital Handycam, Sony, Tokyo, Japan) positioned approximately 50 cm beneath the floor of the chamber to allow an unobstructed view of the paws (visible via a video monitor) and to facilitate recording of animal behavior. The chamber and holding area for pups waiting to be tested were maintained at room temperature throughout the experiment. Pups were only separated from their dams for the duration of the experiment.
Nociceptive behavior was assessed in the 7-day old pups for the presence (score = 1) or absence (score = 0) of flexion, shaking, and whole body jerking per epoch of time (17) and calculated as a nociceptive score = T/300, where T is the duration (s) of nociceptive behavior exhibited during consecutive 300 s postinjection epochs.
Older rat pups were scored across four categories of pain behavior: no pain (the injected paw was in continuous contact with floor = 0), favoring (the injected paw rested lightly on the floor = 1), lifting (the injected paw was elevated all the time = 2), and licking (licking, biting, or shaking of the injected paw = 3) (17) and calculated as nociceptive score = (T1+ [T2 × 2] + [T3 × 3])/300, where T1, T2, and T3 are the durations (s) spent in categories 1, 2, or 3 per 300-s epoch.
At each age we investigated the sedative effect Dex by using a loss of righting reflex (LORR) end-point, defined as the inability of animals to right themselves when positioned in a supine position in a warm environment. The percentage of animals with LORR at Dex 0.1, 0.5, 1, 5, 10, 25, 50, and 250 μg/kg were used to establish dose response curves (n = 6–11).
Ninety minutes after the formalin injection, animals were deeply anesthetized with pentobarbital (100 mg/kg, IP) and perfused with 4% paraformaldehyde. The entire spinal cord was removed. The lumber enlargement was sectioned transversely at 30 μm and stained for c-Fos as previously described (7). Briefly, sections were incubated for 30 min in 0.3% H2O2 in methanol and thereafter washed 3 times in 0.1 M phosphate-buffered saline. The sections were then incubated for 1 h in a “blocking solution” consisting of 3% donkey serum and 0.3% Triton X in phosphate-buffered saline (PBT) and subsequently incubated overnight at 4°C in 1:5,000 goat anti-c-Fos antibody (sc-52-G, Santa Cruz Biotechnology, Santa Cruz, CA) in PBT with 1% donkey serum. The sections were then rinsed 3 times with PBT and incubated with 1:200 donkey anti-goat immunoglobulin G (Vector Laboratories, Burlingame, CA) in PBT with 1% donkey serum for 1 h. The sections were washed again with PBT and incubated with avidin-biotin-peroxidase complex (Vector Laboratories) in PBT for 1 h. The sections were rinsed 3 times with phosphate-buffered saline and stained with 3,3′-diaminobenzidine (DAB) with nickel ammonium sulfate in which hydrogen peroxide was added (DAB kit, Vector Laboratories). After the staining was completed, the sections were rinsed in phosphate-buffered saline followed by distilled water and mounted, dehydrated with 100% ethanol, cleaned with 100% xylene, and covered with cover slips.
Photomicrographs of three sections per each animal were scored in a blinded fashion for c-Fos positive neurons by an observer who was blinded as to the experimental treatment. For the purpose of localizing the c-Fos positive cells to functional regions of the spinal cord, each section was divided into A/B (laminae I-II or the superficial area), C (laminae II-IV or nucleus proprius area), D (laminae V-VI or the neck area, and E (laminae VII-X or the ventral area) (18).
The mean of c-Fos positive neurons for three representative sections in each region as described above was the aggregate score for each animal. The results of nociceptive intensity or c-Fos positive neurons are reported as means ± sem. The statistical analysis was performed by one-way analysis of variance, followed by Newman-Keuls test. A P value < 0.05 was regarded as statistically significant.
LORR dose response data were fitted according to the method of Waud (19) to a logistic equation of the form:
where P is the percent of the population anesthetized, D is the drug dose, n is the slope parameter, and ED50 is the drug dose for half a maximal effect.
The time course of the nociceptive response of each cohort in each age category is presented in Figures 1 and 2. Intraplantar formalin injection produced a typical biphasic response. Dex exhibited a dose-dependent effect; Dex 10 μg/kg had little effect at all ages but 50 μg/kg produced a profound antinociceptive effect (Fig. 1). Saline injection caused minimal nociceptive behavior.
During the preinjection period, all animals were awake and active. After injection with formalin, the 7-day-old animals exhibited intense nociceptive behavior. Administration of 10 μg/kg had little effect on pain behavior. The 7 day-old rats exposed to Dex 50 μg/kg exhibited only mild nociceptive behavior for approximately the first 3 min after formalin injection followed by no further movement for the rest of the 60-min observation period.
Injection of formalin in the 15 day-old group led to intense nociceptive behavior; however, nociceptive behavior was decreased with administration of Dex 50 μg/kg, this same effect was observed at each age group (19, 23, 28 day-old and adult), although this larger dose induced hypnosis in all ages (Fig. 3).
Formalin-induced c-Fos expression at the lumbar level of the spinal cord was increased ipsilateral to the site of injection in all age groups in the outer laminae of the dorsal horn (laminae A/B). At all ages tested, exposure to Dex 50 μg/kg, but not Dex 10 μg/kg, significantly suppressed c-Fos expression in this region, indicating suppression of nociceptive transmission (Fig. 2). Plantar saline injection also caused c-Fos expression ipsilateral to the injection; however, this was much less intense than that induced by formalin injection (data not shown).
In the 7-day-old pups 50 μg/kg Dex administration reduced c-Fos expression in response to formalin by 38% in laminae A/B (P < 0.05). In the 15-day-old rats 50 μg/kg Dex suppressed mean c-Fos expression in response to formalin by 68% (P < 0.05); at 15 days old Dex 10 μg/kg also inhibited c-Fos expression in laminae A/B of the spinal cord with non-noxious stimulation. Dex (50 μg/kg) depressed c-Fos expression in 19-day-old rats, in response to formalin by 35% in laminae A/B (P < 0.05). In the 23-day-old rats, 50 μg/kg Dex altered c-Fos expression in response to formalin by 34% in laminae A/B (P < 0.05). In the 29-day-old rats, 50 μg/kg Dex altered c-Fos expression in response to formalin by 38% in laminae A/B (P < 0.05); at 29 days old c-Fos was reduced contralaterally in laminae A/B after 50 μg/kg Dex. Similarly, in adult rats Dex 50 μg/kg reduced c-Fos expression by 59% in laminae A/B (P < 0.05).
Dex produced a dose-dependent sedative effect with the neonate showing marked sensitivity compared with animals 15 days old and older. Dex produced LORR with an ED50 (95% confidence interval) of 0.8 (0.6–1.0) μg/kg for 7-day-old rats; this significantly increased to 10 (7–13) μg/kg by 15 days old (P < 0.05). Thereafter increasing age was not associated with a significantly increasing ED50 (Fig. 3).
Dex, by systemic application, exerts an antinociceptive against formalin injection in Fischer rats at each of the 6 developmental stages tested, i.e., at 7, 15, 19, 23, and 28 days and in adults. Interestingly, the neonate shows increased sensitivity to sedation/hypnosis (Fig. 3), with a 10-fold increased sensitivity to Dex compared with more mature animals, unlike volatile anesthetics to which neonates are relatively less sensitive (as assessed by minimum alveolar concentration) (20,21). These data support the potential clinical application and study of Dex use in the pediatric population (2–4) because Dex exerts antinociceptive efficacy at an age when connectivity between spinal and supraspinal sites are undeveloped and produces enhanced sedative potency in the neonate.
These data are qualitatively different from those that we reported with nitrous oxide (7) in which no antinociceptive effect (neither behaviorally nor immunohistochemically) was noted in animals younger than 23 days old. In contrast, we have found xenon to be effective at all ages in this paradigm (22) based on its ability to act at the level of the spinal cord. Similarly we have found Dex was effective at 50 μg/kg at all ages, reducing c-Fos expression in the outer laminae of the dorsal horn (Fig. 2) and reducing nociceptive behavior (Figs. 1 and 2) (although it is a potent hypnotic at this dose), suggesting it is an appropriate antinociceptive for pediatric populations. To circumvent the problem of using behavior to assess the antinocieptive effect of a sedative drug, we have applied dual modality analysis of nociception applying immunohistochemical and behavioral methods. Indeed this confound is clearly observed (Fig. 3).
There are substantial data available commenting on the analgesic effects of α2-adrenoceptor agonists (23); indeed their efficacy has been previously demonstrated in the formalin pain model (24), which provides a validated model of inflammatory pain. In the clinical situation inflammation contributes to both postoperative pain and hyperalgesia and is therefore an important target for analgesia; it should be noted, however, that this model is not as analogous a model of postoperative pain as the Brennan surgical incision model. Nonetheless this is the first report of Dex’s effect in immature animals against formalin-induced pain. In the neonate the dorsal horn of the spinal cord is the obligate site of action for antinociception as measured by our immunohistochemical data and this provides evidence that Dex is effective at this target, in agreement with previous reports that have suggested that the spinal cord is the prime site of analgesic action for α2-adrenoceptor agonists (25,26). Dex mediates its effects via α2A adrenoceptors that are present in the neonatal rat spinal cord (12,13) associated with primary afferent nociceptive neurons (27) and are thought to reduce excitatory neurotransmission by modulating neurotransmission; for example, by reducing glutamate release (28). Formalin induces a biphasic pain response (Fig. 1): acute pain, induced by activation of peripheral small afferent fibers, followed by secondary hyperalgesia that reflects central sensitization and nociceptive stimulation by inflammatory mediators. Interestingly, Dex had a more marked effect on the second phase of the formalin test (Fig. 1), indicating that it is potent at combating hyperalgesic states; this is not surprising as Dex has a potent effect on decreasing intracellular cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity (29,30), both of which are required for maintenance of hyperalgesia (31,32). Galeotti et al. (30) have suggested that clonidine-induced spinal antinociception is mediated by PKA-sensitive potassium channels. We have previously shown that the administration of a cAMP phosphodiesterase inhibitor did not inhibit Dex’s antinociceptive efficacy against acute thermal pain (33), but we did not assess the effect of this perturbation on inflammatory pain states such as that produced by formalin injection.
Dex also exerts an effect supraspinally in the locus ceruleus (34), contributing to spinal antinociception at ages when there is functional connectivity between descending inhibitory neurons and higher centers (i.e., after 23 days of age in rats). Antagonism of Dex’s action in the locus ceruleus reduced, but did not attenuate, Dex’s antinociceptive effect in the adult (34); consistent with this observation, we have shown that Dex provides antinociception when there is absence of functional connectivity between the locus ceruleus and the dorsal horn.
Dex demonstrates a strong sedative profile at all ages but especially in the neonate; enhanced neonatal sensitivity to anesthesia, measured by minimum alveolar concentration, is not noted with volatile anesthetics (20,21). Although the increased sensitivity to Dex has been clinically exploited (4), the mechanisms underlying this effect, whether pharmacodynamic or pharmacokinetic, have yet to be elucidated.
Dex has been used safely in pediatric anesthesia with applications including sedation for mechanical ventilation and gastroduodenoscopy, intraoperative controlled hypotension, anxiolysis, analgesia, and reduction of postanesthesia delirium (2–4). No adverse hemodynamic or respiratory events occurred, except for one infant who developed bradycardia with concomitant administration of digoxin (35), although the investigators found Dex to be ineffective as the sole drug for gastroduodenoscopy (2). Indeed many of these reports herald from use in the pediatric intensive care unit where sedation with Dex has advantages over the use of opiates and benzodiazepines (2,3); e.g., in the treatment of postoperative agitation (3,4) where midazolam was ineffective (3), in postoperative shivering, drug withdrawal, and sedation for the spontaneously ventilating infants (3).
In summary, Dex shows a formidable analgesic and sedative profile at 50 μg/kg based on our behavioral and immunohistochemical data. Recent use in pediatric patients showed both safety and efficacy at IV doses of 30 μg/kg (4); whether this will apply at other doses remains to be determined in humans. In addition it will be necessary to define the pharmacodynamic and pharmacokinetic responses to Dex in children. Nonetheless Dex demonstrates potential use for analgesia and sedation in the pediatric population.
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