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Veterans Affairs Palo Alto Health Care System and Stanford University Department of Anesthesiology, Palo Alto, California
Address correspondence and reprint requests to J. David Clark, MD, PhD, Veterans Affairs Palo Alto Health Care System, Anesthesiology, 112A, 3801 Miranda Ave., Palo Alto, CA 94304. Address e-mail to djclark{at}stanford.edu
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Abstract |
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IMPLICATIONS: Opioids are a mainstay of treatment for many types of chronic pain. These studies provide evidence that the hyperalgesia induced by chronic opioid administration may be in part to spinal neuroplastic changes.
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Introduction |
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In addition to the experiments documenting the induction of OAH after the administration of intrathecal opioids, other evidence points to the spinal cord as playing some role in OAH. For example, Vanderah et al. (5) have shown that tonic descending spinal cord facilitation from the rostral ventromedial medulla during chronic exposure to opioids may be, at least in part, responsible for the etiology of OAH. Likewise, after chronic exposure to opioids, noxious stimulation of a rat’s hind paw with formalin leads to increased expression of spinal cord dorsal horn Fos in opioid-treated compared with control animals (14). The nuclear expression of Fos is often used as an index of spinal cord neuron activation after noxious stimulation. These data were interpreted to indicate that opioid receptor-expressing neurons in the spinal cord—especially in the superficial dorsal horn cells, where a great deal of sensory processing takes place—become sensitized to noxious input.
This series of studies attempts to extend our understanding of the role of spinal sensitization in OAH. The first hypothesis tested was that mice with OAH would manifest increased pain behavior in response to the intrathecal application of glutamate and substance P, substances involved in nociceptive signal transduction within the spinal cord. Morphine reduces the nociceptive behaviors and hyperalgesia caused by the intrathecal application of substance P and glutamate receptor agonists (15–17). Thus, the suggestion has been made that the substance P/glutamate receptor systems interact with opioid receptor systems at the level of the spinal cord dorsal horn. The second hypothesis was that Fos expression in superficial dorsal horn neurons would be induced to a greater extent in the spinal cords of the OAH mice than in control mice in response to intrathecal application of nociceptive neurotransmitters.
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Methods |
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Generating mice with OAH was accomplished according to our recently described method (1). For these experiments, a 75-mg morphine pellet (NIDA) was first coated on one surface with acrylic nail polish, which prevented the disintegration of the tablet with time in the moist subcutaneous environment. Mice to be rendered hyperalgesic (OAH group) were briefly anesthetized with isoflurane, and a small subcutaneous skin pocket was made on the animal’s back into which a morphine tablet was placed, uncoated side down, followed by closure with surgical staples. Animals in the control group had a skin pocket made and had their incisions closed. Intermittent morphine abstinence for animals in the OAH group was accomplished by injecting naloxone 1 mg/kg subcutaneously on Days 2, 4, and 6 after pellet implantation. Control animals received saline at these times. On Day 6, morphine pellets were removed from animals in the OAH group. Control animals underwent reopening followed by reclosure of the skin pocket on Day 6. Behavioral and biochemical testing were performed beginning 18 h after the removal of pellets. Thus, the majority of studies presented here used two groups of mice: one group of control animals and another group with 18 h of abstinence from morphine.
Substances involved in nociceptive neurotransmission were injected intrathecally into mice according to the method of Hylden and Wilcox (18). For these injections, a small patch of fur over the lumbar area of the mice was shaved the day before experimentation. To assess nociceptive responses, mice were briefly restrained with a gloved hand, and a 30-gauge, 1/2-in. needle was inserted intrathecally at approximately the L5-6 level; appropriate positioning was signaled by advancement of the needle after walking the tip off the adjacent lamina and, often, by a brief twitch of the tail. Then, 5 µL of drug solution was injected with a 25-µL microsyringe (Hamilton, Las Vegas, NV). A preliminary series of experiments using motor block with 5% lidocaine as a test of appropriate intrathecal injection documented a success rate of approximately 95%. Mice were then placed on a glass surface in a clear plastic cylinder with a 20-cm diameter for observation of behaviors. The substance P and sodium glutamate used for these experiments were purchased from Sigma (St. Louis, MO). These were made in 0.9% saline with the pH titrated to 7.0–7.4 before the injections.
The hotplate assay was performed as previously described (19). Equipment for this assay was obtained from IITC (Woodland Hills, CA). The hotplate was set thermostatically at 52°C ± 0.1°C. Mice were placed in a square clear enclosure on the hotplate, and the time to licking of a hind paw, the end point of the assay, was measured with a stopwatch. Animals were left on the hotplate no longer than 60 s to prevent permanent tissue damage.
Mechanical allodynia was assayed with nylon von Frey filaments according to the algorithm described by Chaplan et al. (20), as we have used previously (1). In these experiments, mice were placed on mesh platforms in plastic enclosures. After 20 min of acclimation, fibers of sequentially increasing stiffness (n = 7; 0.2–2 g) were applied to the center of the plantar surface of the hind paw just distal to the first set of foot pads and left in place for 5 s. Purposeful withdrawal of the hind paw from the fiber was scored as a response. When no response was obtained, the next stiffest fiber in the series was applied; if a response was obtained, a less stiff fiber was next applied. Testing proceeded in this manner until four fibers had been applied after the first one that caused a withdrawal response, allowing the estimation of the mechanical withdrawal threshold.
Response latencies to noxious thermal stimulation were measured with the method of Hargreaves et al. (21) as we have modified it for use with mice (1). In this assay, mice were placed on a glass platform (23.5°C–24.0°C) in a plastic enclosure as described previously. After 20 min of acclimation, a beam of focused light was directed toward the same area of the hind paw as described for the von Frey assay. The time to purposeful withdrawal of the foot from the beam of light was measured. A 20-s cutoff was used to prevent tissue damage. The light beam intensity was adjusted to provide a 9- to 10-s baseline, thus facilitating the detection of thermal hyperalgesia. Two measurements were made per animal per test session.
Animals that undergo intrathecal injection of glutamate or substance P exhibit pain-related behavior characterized by licking, biting, and scratching the tail and hind limbs (22–24). For both of these substances, the majority of pain-related behavior occurs within the first 10 min after intrathecal injection. Thus, after animals were injected, they were observed for this pain-related behavior for the first 10 min after injection while the amount of time spent on the behavior was measured with a stopwatch.
Studies performed to quantify Fos expression in lumbar spinal cord tissue were performed as recently described (25,26). Briefly, animals were asphyxiated with CO2 60 min after intrathecal injection and then subjected to intracardiac perfusion with 10 mL of 0.9% NaCl. This was followed by perfusion with 20 mL of 4% paraformaldehyde in 0.1 M phosphate-buffered saline. The spinal cord was then extruded onto a chilled surface and carefully transected in the upper sacral and lower thoracic regions. This segment of tissue was postfixed for 4 h in paraformaldehyde, followed by incubation overnight in 30% sucrose at 4°C. After embedding in freezing medium, 40-µm sections were made at -20°C by using a refrigerated microtome (Leica, Germany) in a caudal to proximal manner. Sections were collected in groups of 12, which represented approximately 0.5 mm of spinal cord tissue as measured along the spinal cord’s longitudinal axis. Two to three sections of each group were subjected to toluidine blue staining to determine the spinal cord level to which they corresponded (25).
Sections corresponding to the L4 and L5 levels were processed for visualization of nuclear Fos expression by using a floating technique (26). Briefly, blocking took place overnight at 4°C with 5% dry milk, followed by overnight exposure to the primary antibody anti-Fos 1:10,000 (Santa Cruz Biotechnology, Santa Cruz, CA). After rinsing, avidin-biotin-complex reagents (Vector Laboratories, Burlingame, CA) were used to visualize immunoreactive structures. Examination of slides was performed with standard light microscopy by using an Olympus (Tokyo, Japan) BH-2 microscope coupled to a digital camera. Later, images were digitally overlaid with templates for the various spinal cord laminae (Photoshop software; Adobe, San Jose, CA), and Fos-positive nuclei were counted. Tabulation of Fos expression was performed by an investigator other than the person who collected the images. The images were digitally magnified when necessary to allow resolution of closely apposed nuclear profiles in the superficial dorsal horn. This procedure was performed for five sections per animal, and at least four animals were used for each experimental condition.
Statistical analysis of possible differences in pain behaviors or Fos expression was performed with Student’s t-tests. Our cutoff for significance was P < 0.05 or P < 0.01. Data are presented as mean ± SEM.
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Results |
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To evaluate the hypothesis that animals with OAH would demonstrate increased pain behaviors in response to the intrathecal administration of specific substances known to be involved in spinal nociceptive neurotransmission, we measured spontaneous pain behaviors after the injection of glutamate and substance P (Fig. 1). In these experiments, animals injected with saline alone demonstrated very little pain behavior (<10 s) over the 10 min after injection. Both glutamate and substance P caused vigorous caudally directed licking, biting, and scratching in a dose-dependent manner. In OAH mice, we observed increased licking behaviors in response to glutamate (Fig. 1A) or substance P (Fig. 1B).
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Discussion |
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Other reports have suggested that the chronic exposure of rodents to opioids might lead to neuroplastic events in the spinal cord. For example, several articles document hyperalgesia to peripherally delivered noxious stimuli after the selective intrathecal administration of opioids (5–8). This altered sensitivity to noxious stimuli might involve the activation of NMDA receptors (3,6), which has led to the comparison of OAH to neuropathic pain (4). Also, Vanderah et al. (5) have reported on a series of experiments looking at the alteration of descending spinal cord pathways in rats treated chronically with morphine, suggesting that at least part of the apparent alteration of spinal cord nociceptive processing in OAH has to do with tonic descending facilitation from the rostral ventromedial medulla. Other investigators have demonstrated that morphine metabolites such as morphine-3-glucur-onide can cause mechanical allodynia when injected intrathecally (28). We did not attempt to quantify these metabolites in our study. Although not specifically limited to spinal cord neurons, the studies of Crain and Shen (29,30) have clearly documented opioid receptor-mediated excitatory phenomena and hyperalgesia in sensory neurons.
Our studies perhaps build most directly on that of Rohde et al. (14), who measured increases in spinal cord Fos expression after hind paw formalin administration in rats chronically treated with morphine. They believed that perhaps opioid receptor-expressing neurons within the spinal cord were in some way sensitized to noxious input from the periphery. Our studies extend the findings of Rohde et al. by demonstrating that at least two of the principal spinal cord nociceptive neurotransmitters, glutamate and substance P, do have an enhanced ability to cause pain behaviors and Fos expression in the setting of OAH.
Finally, it should be recognized that hyperalgesia caused by the chronic administration of opioids is a clinically important issue. Both thermal hyperalgesia and mechanical allodynia have been observed in humans in whom therapeutically administered opioids have been rapidly withdrawn (11–13). Patients maintained on once-daily administration of methadone show thermal hyperalgesia in the cold pressor test without other evidence of opioid withdrawal (9,10). Others have suggested that short courses of opioids given during anesthesia provide enough exposure that some degree of hyperalgesia develops in the postoperative period (31,32). Furthermore, as an analgesic class, opioids are second only to nonsteroidal antiinflammatory drugs in terms of the number of prescriptions written for the treatment of chronic pain (33). Thus, a very large number of patients are exposed to opioids, and the analgesic benefit they derive may be compromised by the emergence of hyperalgesia. Further investigation in this area may help to devise strategies for opioid administration that would tend to avoid OAH or perhaps to provide treatment strategies for OAH once it is established.
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  V. Fernandez-Duenas, O. Pol, P. Garcia-Nogales, L. Hernandez, E. Planas, and M. M. Puig Tolerance to the Antinociceptive and Antiexudative Effects of Morphine in a Murine Model of Peripheral Inflammation J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 360 - 368. [Abstract] [Full Text] [PDF]
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 M. C. Borras, L. Becerra, A. Ploghaus, J. M. Gostic, A. DaSilva, R. G. Gonzalez, and D. Borsook FMRI Measurement of CNS Responses to Naloxone Infusion and Subsequent Mild Noxious Thermal Stimuli in Healthy Volunteers J Neurophysiol, June 1, 2004; 91(6): 2723 - 2733. [Abstract] [Full Text] [PDF]
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