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© 1999 International Anesthesia Research Society
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Abstract |
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A population of neurons resident in the caudal ventrolateral medulla are excited by noxious cutaneous and visceral stimuli from large portions of the body. These neurons act as monitors of ascending nociceptive information, and we hypothesized that they would be inhibited by spinally administered analgesics in a clinically relevant fashion. Rats were anesthetized with oxygen/halothane. The caudal medulla was surgically exposed, and a catheter placed into the intrathecal space overlying the lower thoracic spinal cord via the surgical site. Single medullary neurons were characterized for responses to cutaneous and visceral (colorectal distension) stimuli. The effects of IV and intrathecally administered morphine and lidocaine were determined. The intrathecal infusion of morphine for 6 days before testing was also used as a pretreatment. Colorectal distension-evoked responses of medullary nociceptive neurons were inhibited in a dose-dependent, naloxone-reversible fashion by intrathecal and IV morphine (50% effective dose values: 3.5 and 440 µg/kg, respectively). Intrathecal lidocaine abolished responses to colorectal distension and produced a spinal level at doses producing minimal effects when administered systemically. Prior treatment with an infusion of morphine produced tolerance to the effects of subsequent intrathecal morphine administration. These findings support the use of this preparation as a neurophysiologic model of spinal analgesia.
Implications: Neurons in the brainstem, isolated electrophysiologically, were used as whole body monitors of pain-related activity in the rat. As a neurophysiologic model of nociception, this preparation may prove useful for the study of regionally administered analgesics and local anesthetics.
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Introduction |
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Our basic science understanding of the effects of spinally administered analgesics is based primarily on pharmacological studies using behavioral models in animals. Although valuable, these studies do not elucidate precise mechanisms of nociceptive processing and do not fully discriminate between sensory and motor phenomena. Neurophysiologic studies of nociception complement behavioral studies by adding an improved distinction between sensory and motor events and by allowing for an evaluation of intensity coding and summation effects. Neurophysiologic studies of the effects of analgesics on spinal nociceptive processing have been limited to single-unit studies of dorsal horn neurons using iontophoretic application, microinjection, topical application, or the IV administration of drugs in spinally transected, decerebrate preparations (1,2). These studies used preparations in which extensive laminectomies have been performed and other local structures disrupted, such as the dura mater. As a consequence, tonic nociceptive inputs from multiple deep tissues, which have known modulatory effects on nociceptive processing (3,4), are present, and drug delivery may be inconsistent. Spinal dorsal horn neuronal studies have also been quite variable in reported responses to analgesics. In particular, opioids have been demonstrated to produce inhibition of some nociceptive neurons and excitation of others (5).
Yaksh and Rudy (6) made a significant contribution to the pharmacological literature when they introduced the experimental use of an intrathecal (IT) catheter that could administer drugs directly to the lumbar cerebrospinal fluid without significant disruption of the surrounding tissues. Using behavioral responses as indicators of nociception, they were able to demonstrate selective spinal effects of morphine. This effect of morphine was subsequently confirmed and translated into clinical practice. There is no single-unit neurophysiologic correlate to behavioral studies using the delivery system of Yaksh and Rudy, despite the additional information that can be gained from neurophysiologic models.
We previously characterized neurons resident in the caudal ventrolateral medulla that are excited by noxious cutaneous and visceral stimuli from most of the body (7). Located within or immediately adjacent to the medullary lateral reticular nucleus, these neurons encode for ascending nociceptive information in a graded, accelerating fashion and thereby act as global monitors of nociceptive activity within the body. The surgical preparation required for neurophysiologic recording from this area is minimal and nearly identical to the surgical approach used by Yaksh and Rudy to insert their chronic IT catheters. We discovered that it was possible to insert an IT catheter as part of our neurophysiologic preparation. To determine whether this preparation is useful for the study of the effects of spinal analgesics, the effects of morphine and lidocaine when administered IT were examined and compared with the effects of the same drugs when administered IV. Further, to determine whether this model is useful for studies of pharmacological tolerance at the level of the spinal cord, the effect of IT morphine was assessed when the animals had previously received chronic IT infusions of morphine. Portions of these data have been presented in abstract form.
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Methods |
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These studies were approved by the local institutional review board for animal studies. All studies were performed in male Sprague-Dawley rats (290–360 g) that underwent an inhaled induction using a tight-fitting mask and high concentrations of halothane (4%–5%). This was subsequently reduced to 1%–2% halothane after placing an intratracheal cannula and maintained during surgery. Venous (external jugular) and arterial (femoral) cannulae were placed. Skin and muscles were retracted, and the atlanto-occipital membrane was identified and incised, exposing the caudal medulla at the level of the obex. A flexible polyethylene catheter (PE-10; Clay Adams, Parsippany, NJ) was threaded 50 cm caudad through the IT space in a method similar to that of Yaksh and Rudy (6). After surgery, the anesthetic was lowered to 0.8%–1%, and the rats' lungs were artificially ventilated. As demonstrated by multiple experiments in our laboratory, at this level of anesthesia, rats have no spontaneous movements or evidence of tonic sympathetic nervous system activation (e.g., piloerection). However, they do maintain cardiovascular and motor reflex responses to cutaneous and visceral stimuli (8), which allows for the performance of parallel neurophysiologic and reflex experiments. The level and type of anesthetic used have a significant effect on brainstem neuronal responses (unpublished observations); therefore, a functional measure of anesthetic depth was used (presence of a flexion reflex response to hindpaw pinch). All rats were subsequently paralyzed with pancuronium (0.1-mg boluses IV as needed).
Visceral stimulation consisted of distension of the descending colon and rectum of the rat (colorectal distension; CRD) by air inflation of a 7- to 8-cm flexible latex balloon, which provided a maximal distension stimulus to the entire descending colon and rectum. Pressure within the balloon was measured by using an in-line, low-volume pressure transducer. The balloon assembly was inserted via the anus and kept in position (end of balloon 1 cm from the anus) by taping the connecting catheter to the tail. The pressure within the balloon was controlled using a pressure-control device described elsewhere (9). Cutaneous stimuli consisted of nonnoxious light touch (brush, light pressure, blowing on field) and noxious pinch with fine forceps.
Tungsten microelectrodes (1.2–1.8 M
; Micro Probe, Clarksburg, MD) were used for conventional extracellular single-unit recording. The area 2 mm lateral to obex and 2.0–3.0 mm below the dorsal surface was searched for neurons responsive to CRD. All neurons were characterized for responses to repeated CRD (80 mm Hg, 20s, every 4 min). If a consistent response (±20% on three trials) was obtained, then neurons were selected for additional study. All neurons were also characterized for excitatory/inhibitory responses to cutaneous stimuli presented throughout the body. Only neurons with large (half to whole body), bilateral, cutaneous receptive fields excited by noxious stimuli were selected for pharmacological study. In an extensive characterization of the neurons of this region (7), it was found that these neurons were common in the area near the medullary lateral reticular nucleus.
To quantify neuronal responses, units were displayed oscillographically for continuous monitoring, discriminated conventionally from background, converted into uniform pulses, and counted and saved by the computer. The total number of unit discharges were counted during a preselected interval (20 or 40 s) starting with the onset of the stimulus. Evoked activity was defined as the number of unit discharges during this preselected interval minus spontaneous activity determined during the 10 s immediately preceding the onset of CRD. Because responses of different neurons to the same distending stimulus naturally vary in maximal response and total number of unit discharges, each unit's response was normalized to that produced by the 80-mm Hg stimulus for the purposes of within- and between-group comparisons and illustrations. Changes in spontaneous activity were limited to a twofold increase.
After demonstrating a reliable, reproducible response to CRD (80 mm Hg, 20 s), three doses of isotonic sodium chloride solution (20 µL) or morphine sulfate (2.5, 5, and 10 µg/kg cumulative doses in 10-µL volumes, followed by a 10-µL isotonic sodium chloride solution flush) were administered IT at 16-min intervals. Doses of morphine were followed by IV naloxone (0.4 mg/kg) and by IT lidocaine (20 µL/kg of a solution of 5% lidocaine hydrochloride [1 mg/kg] in dextrose, followed by a 10-µL isotonic sodium chloride solution flush). During drug dosing, responses to CRD were determined at 4-min intervals. After the IT administration of lidocaine, responses to pinch and CRD were determined every 15 min.
After demonstrating a reliable, reproducible response to CRD (80 mm Hg, 20 s), sequential doses of isotonic sodium chloride solution (1 mL/kg) or morphine sulfate (0.5 and 2 mg/kg cumulative doses in 0.5-mL/kg volumes, followed by a 0.5-mL/kg isotonic sodium chloride solution flush) or lidocaine hydrochloride (0.25, 1, 4, and 8 mg/kg cumulative doses, followed by 0.5-mL/kg isotonic sodium chloride solution flush) were administered IV at 16-min intervals. IV naloxone (0.4 mg/kg) was subsequently administered in experiments using morphine. Responses to CRD were determined throughout at 4-min intervals.
In 10 rats, chronic IT catheters (gas-sterilized versions of the same catheters used in acute experiments) were placed 1 wk before further testing. Rats were briefly anesthetized with a halothane/oxygen mixture delivered via a mask, and the catheters were placed using the method of Yaksh and Rudy (6). These catheters were connected to osmotic minipumps (Alzet® 2001; ALZA, Palo Alto, CA) and secured subcutaneously between the scapula. Five rats received a 1-µL/h infusion of 20 nmoL/h morphine sulfate, and the other five rats received a similar infusion of isotonic sodium chloride solution. One day before testing, the rats were again briefly anesthetized, and the catheter was removed along with the minipump. The following day, the rats underwent the same surgical preparation for neurophysiologic study and followed the same IT drug protocol for morphine as the other rats. This protocol for chronic morphine administration has been demonstrated to result in profound tolerance to the effects of morphine after 6 days of infusion in behavioral studies (10).
All drugs were commercial grade. Halothane was obtained from Halocarbon Laboratories, River Edge, NJ; naloxone hydrochloride was obtained from Astra Pharmaceutical Products, Westborough, MA; pancuronium bromide was obtained from Gensia Laboratories, Ltd, Irvine, CA. The morphine sulfate (Infumorph®, Elkins-Sinn, Cherry Hill, NJ) was saline-diluted and preservative-free, as was the lidocaine hydrochloride (Astra Pharmaceutical Products).
Data are presented as the mean ± SEM unless otherwise stated. Statistical comparisons were made by using Student's paired and unpaired t-tests and analysis of variance for repeated-measures analysis and post hoc analysis using Tukey's honest significant difference test. 
and P 
0.05 were considered significant. The 50% effective dose (ED50) values were calculated using log-linear regression analysis with 95% confidence intervals (GB-STAT; Dynamic Microsystems, Inc., Silver Springs, MD).
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Results |
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A total of 52 neurons located in the caudal ventrolateral medulla were studied. All were excited by noxious pinch with large bilateral cutaneous receptive fields. The response characteristics of these neurons are similar to those of a larger sample of neurons described elsewhere (7). All neurons had continuing spontaneous activity (18.5 ± 2.4 Hz), and all were excited by CRD. An 80-mm Hg, 20-s distension produced evoked activity of 392 ± 45 neuronal discharges.
The evoked activities of 11 medullary neurons were inhibited in a dose-dependent, naloxone-reversible fashion by IT morphine (Figure 1.) The ED50 value (95% CI) for the effect of IT morphine on evoked activity was 3.53 (3.07–4.00) µg/kg. Spontaneous activity was significantly increased after the first administration of morphine but returned to normal within 13 min. The subsequent administration of morphine IT lidocaine abolished the evoked activity and produced a spinal level in the cutaneous receptive fields of the medullary neurons. Excitatory responses to thoracic, lumbar, and sacral cutaneous stimulation were abolished, whereas cervical and/or trigeminal cutaneous stimuli still excited in 10 of the 10 neurons tested (Figure 2). This effect lasted for 1.75–3.25 h. IT saline produced no significant change in the evoked activity of six neurons but, similar to IT morphine, produced a transient increase in spontaneous activity (Figure 1).
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) and spontaneous activity (
) for the 20 s before CRD. Evoked activity is the difference between the two. Dotted lines indicate drug administration. F and G, Similar representation of grouped data of medullary neurons in rats receiving IT isotonic sodium chloride solution (NS; n = 6; 
) or IT MS (n = 11; •). IT MS produced dose-dependent, naloxone-reversible inhibition of CRD-evoked activity but had no statistically significant effects on spontaneous activity. The subsequent administration of IT lidocaine (Lido; 20 µL/kg of a 5% solution in dextrose) abolished evoked activity. IT saline had minimal effects. Data in F and G were normalized to the mean of two baseline (predrug) activities and are presented as the mean ± SEM. *P < 0.05 and **P < 0.01 between measure and baseline activities.
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The spontaneous activities and evoked activities of eight medullary neurons were inhibited in a dose-dependent, naloxone-reversible fashion by IV morphine. The ED50 value for the effect of IV morphine on spontaneous activity was 0.65 (0.29–0.71) mg/kg and on evoked activity was 0.44 (0.35–0.64) mg/kg. IV lidocaine produced transient effects on the spontaneous and CRD-evoked activities of five neurons (Figure 3, closed symbols), but IV saline produced no significant change in the evoked or spontaneous activities of five other neurons (Figure 3, open symbols).
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The effect of IT morphine on the CRD-evoked activity of caudal medullary neurons was reduced in the five rats that had received a 6-day infusion of morphine sulfate before testing. Evoked activity was reduced to 50.4% ± 8.9% of baseline levels 13 min after the third (maximal) dose of IT morphine sulfate (cumulative dose 10 µg/kg). In contrast, evoked activity was reduced to 30.2% ± 8.0% of baseline levels 13 min after the first dose of IT morphine sulfate (2.5 µg/kg) in five rats treated with chronic saline infusions and to 10.1% ± 5.4% of baseline levels after the third dose. Differences between the morphine- and saline-pretreated groups were statistically significant (P < 0.001.)
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Discussion |
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The goal of the present study was to determine whether a neurophysiologic preparation used to examine caudal ventrolateral medullary neurons is useful as a model of spinal anesthesia/analgesia. In a previous study, we extensively characterized neurons in the area of the lateral reticular nucleus and determined that those neurons excited by noxious pinch with large, bilateral cutaneous receptive fields had activity representative of ascending visceral nociceptive information (7). In this previous study, we demonstrated that the activity of these neurons was not secondary to hemodynamic alterations and was graded in relation to the intensity of the noxious stimulus. In the present study, we demonstrated that morphine produced dose-dependent inhibition of those nociceptive neurons in a fashion similar to its clinical actions. IT administered morphine was approximately 100 times more potent than systemically administered morphine, and prior exposure to morphine led to a decreased responsiveness to subsequent morphine (i.e., tolerance). Regional delivery of drugs was demonstrated by the effects of IT lidocaine, which provided a long-lasting spinal level at doses that had few effects when given systemically. These data support using this neurophysiologic preparation as a model of spinal analgesia. An important advantage of this neurophysiologic preparation over behavioral models is its ability to dissociate sensory from motor phenomena. Behavioral responses would have been abolished by muscle relaxants, but vigorous neuronal responses were maintained. Although not formally examined in the present study, the ability to examine intensity coding in this neurophysiologic model allows analgesic effects on low-intensity threshold responses to be differentiated from effects on responses to high-intensity stimuli. In this way, weak analgesics can be differentiated from those with greater analgesic efficacy. Additional benefits of this model include the ability to examine summation effects whereby time-dependent (i.e., tonic pain) or area-dependent (i.e., multisite pain) variables may be manipulated. Using this model may allow for a more precise determination of the mechanisms of nociceptive processing and analgesic actions within the spinal cord.
Our findings are consistent with previous studies that have demonstrated dose-dependent inhibition of spinal dorsal horn neuronal responses to CRD (1,2), as well as effects of spinally administered opioids on behavioral responses to CRD (10–12). As in the present study, others have found nociceptive neurons to be easily located in the caudal medulla (13–15), and these neurons are inhibited by IV morphine (16). Others have demonstrated effects of IT morphine on supraspinal neurons but have used electrical (nonphysiologic) stimuli that were only partially inhibited (17) or have examined effects indirectly in the context of pain modulatory systems (18,19). Some of the studies examined only the effects of morphine on spontaneous activity (16). Therefore, it is notable that we demonstrated that the effects of spinally administered analgesics on spontaneous activity were not always the same as their effects on activity evoked by a noxious stimulus. Systemically administered morphine reliably produced inhibition of both the spontaneous and evoked activity of medullary neurons, whereas the IT injection of morphine generally produced a transient increase in spontaneous activity with a clear decrease in evoked activity. The source of this increase in spontaneous activity is likely a nonpharmacologic (i.e., physical) action of the drug before its diffusion to spinal cord opioid receptors, but precise mechanisms have yet to be elucidated.
The region of the medulla we examined (the area in or adjacent to the lateral reticular nucleus) has been implicated in the regulation of autonomic function (20,21), as well as pain modulation (22). It receives afferent input predominantly from fibers ascending in the ventrolateral quadrant of the spinal cord (the spinothalamic tract) (23) and serves as a central site of sensory integration with known relays to and modulation by the cerebellum and other central nervous system sites (24,25). For purposes of the present discussion, the precise function of the neurons of this region is not as important as their reliability as monitors of nociceptive activity within the body. Responses to CRD were reliable and reproducible over time, as evidenced by the saline treatment data. IV lidocaine produced only transient effects on nociceptive processing, whereas the IT administration of the drug had long-lasting and potent effects. This demonstration of the long-lasting effects of IT administered lidocaine is also supportive of regional actions of the drug. Because the neurons we examined have large bilateral, excitatory cutaneous receptive fields, it may be possible to examine the effects of peripheral nerve blocks on central nociceptive processing using a similar model. For such studies, the data related to the effects of IV lidocaine can act as control data, accounting for systemic effects of the local anesthetic.
In summary, we demonstrated effects of IV and IT morphine and lidocaine on medullary nociceptive neurons that are similar to the clinical effects of these drugs in humans. These neurons have characteristics that make them monitors of nociception within most of the body. By exploiting these characteristics, it is possible to demonstrate the effects of spinally administered drugs, and it may be possible to study the effects of more peripherally administered drugs. As a consequence, this preparation should find utility as a neurophysiologic model of regional anesthesia/analgesia.
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Acknowledgments |
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This work was supported by a Young Investigator's Grant from the American Society for Regional Anesthesia, University of Alabama at Birmingham Cancer Center institutional mini-grants sponsored by the American Cancer Society, and departmental funds at the University of Alabama at Birmingham and University of Iowa. TJN is supported by National Institute of Diabetes and Digestive and Kidney Diseases (DK51413).
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Footnotes |
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This work has appeared in part as an abstract in Regional Anesthesia 1995;20(Suppl 2):3.
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References |
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