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Anesth Analg 1999;89:128
© 1999 International Anesthesia Research Society

REGIONAL ANESTHESIA AND PAIN MANAGEMENT

Pre- Versus Postformalin Effects of Ketamine or Large-Dose Alfentanil in the Rat: Discordance Between Pain Behavior and Spinal Fos-Like Immunoreactivity

Ian Gilron, MD, MSc, FRCP(C)*, Rémi Quirion, PhD{dagger}, and Terence J. Coderre, PhD{ddagger},§

*Pain Research Clinic, Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland; {dagger}Department of Psychiatry, McGill University; {ddagger}Pain Mechanisms Research Laboratory, Clinical Research Institute of Montreal; and §Departement de Medecine, Université de Montréal, Montreal, Quebec, Canada

Address correspondence and reprint requests to Ian Gilron, MD, MSc, FRCP(C), Pain Research Clinic, Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 10, Room 3C-434, 9000 Rockville Pike, Bethesda, MD 20892. Address e-mail to igilron{at}yoda.nidr.nih.gov


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The purpose of this animal investigation was to compare behavioral responses with spinal Fos-like immunoreactivity (FLI) after pre-versus postformalin administration of anesthetic doses of IV ketamine or alfentanil. Preformalin and postformalin injection (1.5% subcutaneously) treatment groups included IV saline control (1.5 mL/kg), ketamine (10 mg/kg), and alfentanil (170 µg/kg). In the behavioral study group, nociceptive behavior was evaluated 15–60 min after hindpaw formalin injection. In the spinal FLI study group, rats were perfused 2 h postformalin, and spinal cords were dissected, sliced at 30 µm, and processed by immunoperoxidase staining with an antibody against the Fos protein. Quantification and determination of the laminar distribution of Fos-labeled nuclei were performed at the L4-5 spinal level ipsilateral to formalin injection. Ketamine produced a selective preemptive analgesic effect in behavioral formalin experiments, yet failed to suppress spinal FLI. In contrast, alfentanil failed to demonstrate a selective preemptive analgesia in behavioral experiments, but did produce preemptive suppression of spinal FLI. Together with previous data from our laboratory, we conclude that behavioral analgesia and spinal Fos expression may be uncoupled under certain circumstances.

Implications: In this study, we compared pain reduction produced by IV drugs (ketamine or alfentanil) with the ability to prevent injury-induced spinal cord changes. We measured pain behavior and spinal Fos protein after rats received ketamine or alfentanil before versus after formalin injection. Fos inhibition patterns did not clearly correlate with pain reduction, providing further evidence that Fos inhibition is not always predictive of behavioral analgesia.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Preemptive analgesia—the idea that modulating sensory input before surgery could diminish postoperative pain—is an analgesic strategy thought to be contingent on the prevention or suppression of spinal mechanisms of neuronal sensitization (1). The ability of volatile and GABAergic general anesthetic drugs to produce preemptive analgesia in the laboratory (as evaluated by comparing pre versus postinjury analgesic effects) has been previously studied in the rat formalin model (2–4). Another IV anesthetic, ketamine, is a phencyclidine derivative that produces analgesia in both laboratory and clinical models (5). Ketamine is thought to produce analgesia due to its N-methyl-D-aspartic acid (NMDA) receptor blockade, as well as some opioid µ-receptor activity seen with the S-(+) enantiomer of the racemic mixture of which ketamine is formulated (5). Although large doses of opioids are not thought to reliably produce loss of consciousness (6), they are often used in conjunction with benzodiazepines in administering anesthesia to patients undergoing cardiac surgery (7). Although there is clinical evidence to suggest that large-dose opioids in the setting of cardiac anesthesia (8), or ketamine anesthesia in other types of surgery (9), may exert preemptive analgesic effects, anesthetic doses of these drugs have not been studied in experimental models of preemptive analgesia.

Evaluating the effects of systemically administered general anesthetics in the rat formalin model requires, in addition to measuring pain behavior, other surrogate measures to address the hypothesis that these drugs work to suppress spinal nociceptive processes (10). In 1987, the spinal expression of Fos protein, which is an immediate early gene product, was first demonstrated in response to both noxious and nonnoxious sensory stimulation (11). Since then, studies measuring the spinal expression of either c-fos mRNA or Fos protein have involved several different pain models (12,13). Fos protein activates DNA transcription, and its noxious stimulus-induced expression has been linked to the increased expression of endogenous spinal opioids such as dynorphin, which may play a role in mechanisms of spinal sensitization (14). The pharmacological suppression of noxious stimulus-induced Fos-like immunoreactivity (FLI) has been demonstrated with opioids such as morphine (12) and NMDA receptor antagonists such as MK-801 (15).

Previous work from our laboratory has correlated the suppression of behavioral hyperalgesia with that of spinal FLI under conditions of pre-versus postformalin treatment with intrathecal lidocaine (16) and, more recently, with IV GABAergic general anesthetics (17). To further complete this characterization with other general anesthetics used in clinical practice, the purpose of this study was to similarly compare behavioral responses with spinal FLI after pre-versus postformalin administration of anesthetic doses of ketamine or alfentanil.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The following experiments were performed under protocols approved by the Institutional Animal Care Committee of the Clinical Research Institute of Montreal and the Canadian Council for Animal Care. The animals studied were male Long Evans rats (Charles River, St. Constant, Québec, Canada) weighing 250–350 g, housed in groups of four, with free access to food and water, and maintained on a 12-h light/dark cycle.

Anesthetics and saline vehicles were administered in a volume of 1–2 mL/kg by tail vein injection over a period of 30–60 s. After immobilizing the awake rat in a cloth restrainer, the tail vein was cannulated with a 25-gauge butterfly infusion set primed with injectate. IV cannulation, as noted by free backflow of blood into the tubing, was followed by syringe attachment and injectate administration. Successful injection was confirmed by again observing blood backflow when the syringe was disconnected after injection. Rats receiving an incomplete injection because of needle dislodgment were excluded from the study.

Phase 2 nociception after formalin injection is thought to be due to two phenomena: noxious stimulus-induced spinal sensitization and a formalin concentration-dependent inflammatory reaction, which results in primary afferent C-fiber activation (18). To more specifically study effects on sensitization (rather than effects on continuing peripheral nociception), rats were injected with 50 µL of 1.5% formalin [a relatively low concentration which produces little inflammation (19)] subcutaneously into the plantar surface of one hindpaw using a tuberculin syringe and a 25-gauge needle.

For the study of anesthetic doses of ketamine and alfentanil, in preliminary experiments, we sought to determine the optimal drug dose that would produce clinical anesthesia (i.e., loss of righting reflex) for at least 5 min (the approximate duration of Phase 1) yet allow sufficient recovery for nociceptive scoring in Phase 2. Vehicle control groups included 0.9% saline for comparison with the ketamine and alfentanil groups. For the treatments described, one set of animal groups was used to study pain behavior and another set to study spinal FLI. In the preformalin groups, rats received IV drugs 30 s (alfentanil 170 µg/kg) and 1 min (ketamine 10 mg/kg) before formalin injection. Rats in the vehicle control group received 0.9% saline IV 1 min before formalin injection. Another control group used to evaluate the basal level of FLI in unstimulated rats included rats who received neither formalin nor IV injections.

To evaluate the specific preemptive effect of the drugs studied, a preinjury group was compared with a postinjury group. In the preinjury groups, rats received treatments before formalin injection so that they were anesthetized during Phase 1 of the formalin test. In the postinjury groups, drugs were administered 5 min after the formalin injection to allow the rats to be untreated during Phase 1 and fully anesthetized in the early part of Phase 2, yet recover fully from anesthesia and allow nociceptive scoring to be initiated 15 min after the formalin injection.

For nociceptive testing, each rat was placed in a 30 x 30 x 30 cm clear plastic box with a mirror below the floor at a 45° angle to allow unobstructed view of the paws. A nociceptive score was determined using the weighted scores method of behavioral rating devised by Dubuisson and Dennis (20). Scoring involved measuring the amount of time spent in each of four behavioral categories: 0 = the injected paw is not favored; 1 = the injected paw has little or no weight on it; 2 = the injected paw is elevated and not in contact with any surface; and 3 = the injected paw is licked, bitten, or shaken. A weighted average nociceptive score (ranging from 0 to 3) was calculated by multiplying the time spent in each category by the category weight, summing these products, then dividing by the total time in each 5-min time block (i.e., 300 s). Because rats in this study were anesthetized during Phase 1 of the formalin test, nociceptive scoring was performed only during Phase 2; that is, 15–60 min after formalin injection.

Rats were killed 2 h after the formalin injection in the following manner: After anesthetizing the rat with 60 mg/kg pentobarbital intraperitoneally, surgery proceeded with sternotomy, transcardiac aortic needle cannulation, and perfusion with 200 mL of phosphate-buffered saline (PBS), followed by 500 mL of 4% paraformaldehyde/PBS. The lumbar spinal cord was subsequently dissected by laminectomy, postfixed for 4 h in 4% paraformaldehyde/PBS, then cryoprotected for 12 h in 30% sucrose/PBS. Extracted spinal cords were labeled with a 1-mm deep lateral, longitudinal incision contralateral to the side of formalin injection, then freeze-mounted in embedding matrix and sliced in 30-µm sections.

Tissue sections were washed in Tris-buffered saline (TBS) and incubated at 4°C for 48 h with a rabbit polyclonal antibody directed against residues 4–17 of the N-terminal region of Fos peptide (Ab-5 solution diluted to 1:75,000; Oncogene Science, Boston, MA) in 0.05% Triton X-100 with 3% normal goat serum. The sections were then rinsed in TBS and incubated at 4°C for 1 h with a biotinylated goat antirabbit immunoglobin G (Vectastain; Vector Laboratories, Burlingame, CA) in 0.05% Triton X-100 with 3% normal goat serum. Sections were then rinsed in TBS and incubated at 4°C for 2 h in an avidin-biotin peroxidase complex (Vectastain). The sections were then rinsed in TBS and 50 mM Tris buffer and rinsed for 10 min in 0.05% 3,3'diaminobenzidine (DAB) in 50 mM Tris. The sections were then incubated for 10 min in DAB/Tris with 0.01% H2O2 added to catalyze the DAB and 8% NiCl2 to color the DAB chromagen blue-black. The sections were then rinsed in TBS to stop the reaction and were subsequently mounted on slides, dehydrated, and coverslipped for image analysis. Negative control experiments were conducted with tissue sections from the formalin-injected saline controls by omitting the primary antibody from the above protocol and adding 2 µg/mL N-terminal Fos peptide to the primary antibody incubation solution. Neither of these controls resulted in any expression of FLI.

Given previous literature suggesting the peak expression of FLI at L4–5 segmental levels (12), only these sections were evaluated, and quantification of FLI neurons was performed only in the gray matter on the side of the cord ipsilateral to the formalin injection. The description by Molander et al. (21) of the characteristic cross-sectional morphological appearance of L4–5 spinal segments allows for the identification of slices from these segmental levels. Thus, tissue sections were first examined using dark-field microscopy to determine the segmental level and gray matter landmarks according to Molander et al. (21). To study the laminar distribution, four regions were defined: superficial dorsal horn (laminae I–II), nucleus proprius (laminae III–IV), neck of the dorsal horn (laminae V–VI), and the ventral gray (VII–X). Sections were then examined by using bright-field microscopy at x6.3 magnification. Each section image was digitized using a Micro-Computer Imaging Device (MCID-Imaging Research Inc., St. Catherines, Ontario, Canada) from which high-resolution printouts were produced and from which FLI neurons were quantified. A predetermined coding system labeled each section image on its underside so as to blind the investigator to treatment group.

Pain behavior scores throughout the testing sessions were analyzed by using a repeated-measures analysis of variance. Any group by time interactions were further explored by using simple effects analysis. Post hoc analyses were performed using Newman-Keuls multiple comparisons.

Statistical analysis was performed to compare FLI (in percentage of control, calculated as the number of FLI neurons for a given slice divided by mean total number of FLI neurons for the control group x100%) over each of the specified laminar regions in different treatment groups using a repeated-measures analysis of variance. Any treatment group by laminar group interactions were further explored using simple main effects analysis. Post hoc analyses were performed using Fisher's protected least significant difference multiple comparisons when indicated.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Rats receiving 10 mg/kg ketamine demonstrated loss of righting reflex, which was recovered within 2 min after drug injection. For the 2–8 min after recovery of the righting reflex, rats were hyperactive and ataxic. Figure 1 shows a comparison of nociceptive scores for the following treatment groups: preformalin saline vehicle (NS PRE; n = 6), postformalin saline vehicle (NS POST; n = 5), preformalin ketamine (KETA PRE; n = 6), and postformalin ketamine (KETA POST; n = 7). Ketamine produced significantly lower nociceptive scores compared with vehicle when given either pre- or postformalin. However, ketamine pretreatment resulted in significantly lower scores than posttreatment 20–50 min after the formalin injection (see Fig. 1). To evaluate a possible opioid receptor-mediated analgesic effect of ketamine, in another experiment, we evaluated nociceptive scores in rats receiving ketamine 10 mg/kg co-administered with naloxone 1 mg/kg (a standard dose used to produce nonselective antagonism of all opioid receptors). Figure 2 shows a comparison of nociceptive scores for the following treatment groups: NS PRE (n = 6), KETA PRE (n = 6), and preformalin ketamine with naloxone (KETA/NALOX; n = 5). In this experiment, there were no significant differences between the ketamine and the ketamine/naloxone groups, which suggests that naloxone failed to reverse the analgesia produced by ketamine (Fig. 2).



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  		Figure 1. Comparison of preinjury treatment with postinjury treatment of normal saline vehicle (NS) versus 10 mg/kg ketamine (KETA). This analysis revealed a significant group by time interaction (treatment group: F[3,20] = 13.77, P < 0.01; time period: F[8,160] = 4.64, P < 0.01; group by time interaction: F[24,160] = 1.91, P < 0.05). * KETA PRE was different from KETA POST and also different from NSPRE from 20 to 50 min (P < 0.05, Newman-Keuls post hoc comparison). +KETA POST was different from NS POST from 20 to 35 min. Error bars represent SEM.

 


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  		Figure 2. Comparison of preinjury treatment of normal saline vehicle (NS) with 10 mg/kg ketamine (KETA) and ketamine coadministered with 1 mg/kg naloxone (KETA/NALOX). This analysis revealed a significant group by time interaction (treatment group: F[2,14] = 29.34, P < 0.01; time period: F[8,112] = 3.12, P < 0.01; group by time interaction: F[16,112] = 2.08, P < 0.05). * Although NS PRE was different from both treatment groups throughout the testing session, there were no significant differences between KETA PRE and KETA/NALOX (P > 0.05, Newman-Keuls post hoc comparison). Error bars represent SEM.

 
Rats receiving 170 µg/kg alfentanil demonstrated loss of righting reflex and profound muscle rigidity, which resolved by 9 min after drug injection. Despite this profound rigidity, rats maintained slow shallow respiration and did not become cyanotic. Figure 3 shows a comparison of nociceptive scores for the following treatment groups: NS PRE (n = 6), NS POST (n = 5), preformalin alfentanil (ALFENT PRE n = 5), and postformalin alfentanil (ALFENT POST; n = 5). In both the pre- and postformalin alfentanil groups, nociceptive scores were significantly lower than their respective controls 15–35 min after the formalin injection. The timing of alfentanil administration relative to formalin injection had no effect on the intensity or temporal profile of nociceptive scores (Fig. 3).



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  		Figure 3. Comparison of preinjury and postinjury treatment of vehicle and alfentanil (170 µg/kg). This analysis revealed a significant group by time interaction (treatment group: F[3,17] = 6.07, P < 0.01; time period: F[8,136] = 6.02, P < 0.01; group by time interaction: F[24,136] = 8.34, P < 0.01). * Although not different from each other, ALFENT PRE and ALFENT POST were different from both controls from 15 to 35 min (P < 0.05, Newman-Keuls post hoc comparison). Error bars represent SEM.

 
FLI neurons were distinctly recognized by their densely stained blue-black nuclei and sparsely stained cytoplasm. The saline control group exhibited most spinal cord gray matter FLI on the side ipsilateral to the site of formalin injection; quantification of FLI was performed on this side only. Figure 4 is a photomicrograph of representative tissue sections from the saline control (formalin) and basal (no formalin) FLI groups. Table 1 (absolute numbers of Fos-immunoreactive neurons) and Figure 5 (FLI as a percentage of control) show mean FLI across laminar regions for the basal FLI (n = 3) and saline control (n = 4) groups. Formalin injection produced significant increases of FLI across all spinal cord laminar regions (Fig. 5).



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  		Figure 4. Photomicrographs of representative saline control (formalin) and basal (no formalin) Fos-like immunoreactivity (FLI) lumbar spinal cord sections. Photomicrographs of 30-µm thick lumbar spinal cord sections. A, Saline control 2 h postsubcutaneous hindpaw formalin injection. Arrow = incision made contralateral to the side of formalin injection. B, Basal FLI (no formalin injection). Scale bar = 50 µm. C, Magnification of section from A illustrating characteristic distribution of Fos-labeled neurons. D, Magnification of section from B. Scale bar = 50 µm.

 

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  		Table 1. Basal FLI and FLI for the Groups
 


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  		Figure 5. The effect of drug pretreatment of formalin-induced Fos-like immunoreactivity (FLI). Histograms plotting the number of FLI neurons across laminar regions for basal FLI, saline control, and drug pretreatment groups. Analysis of variance revealed significant main effects of treatment group (treatment group: F[1,6] = 145.8, P < 0.05) and laminar distribution (F[3,18] = 30.1, P < 0.05), as well as a group by region interaction (F[3,18] = 28.6, P < 0.05). ** Fisher's protected least significant difference post hoc comparisons demonstrated significant increases in FLI in all laminar groups of saline controls (P < 0.05) compared with the basal FLI group. Analysis of variance revealed significant main effects of treatment group (F[2,9] = 6.2, P < 0.05), and laminar distribution (F[3,27] = 40.7, P < 0.05), as well as group by region interaction (F[6,27] = 5.3, P < 0.05). * Fisher's protected least significant difference post hoc comparisons with control demonstrated a significant increase of FLI in laminae I–II of the ketamine-treated group (P < 0.05) and significant reductions in FLI in laminae I–II and laminae V–VI of the alfentanil-treated group (P < 0.05). Error bars represent SEM.

 
Table 1 (absolute numbers of Fos-immunoreactive neurons) and Figure 5 (FLI as a percentage of control) show mean FLI across laminar regions for the groups receiving preformalin administration of saline (CONTROL; n = 4), KETA PRE (n = 4), and ALFENT PRE (n = 4). Ketamine pretreatment produced a significant increase in FLI in spinal laminae I–II, whereas alfentanil pretreatment produced a significant decrease in this same region, as well as in laminae V–VI (Fig. 5). Compared with the mean total number of FLI neurons in the control group, a reduction in FLI of 60% was observed with alfentanil, and an increase of 35% was observed with ketamine.

Table 1 (absolute numbers of Fos-immunoreactive neurons) and Figure 6 (FLI as a percentage control) show mean FLI across laminar regions for the groups receiving preformalin administration of saline (CONTROL; n = 4), ALFENT PRE (n = 4), and ALFENT POST (n = 3). Although alfentanil pretreatment reduced FLI in laminae I–II and V–VI, alfentanil posttreatment only reduced FLI in laminae I–II. In laminae I–II, alfentanil pretreatment reduced FLI more than alfentanil posttreatment (Fig. 6).



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  		Figure 6. The effect of pre- versus postformalin alfentanil (ALFENT) administration on formalin-induced Fos-like immunoreactivity (FLI). Histograms plotting the number of FLI neurons across laminar regions for saline control, alfentanil pretreatment, and alfentanil posttreatment groups. Analysis of variance revealed significant main effects of treatment group (F[2,33] = 20.5, P < 0.05) and laminar distribution (F[3,99] = 100.5, P < 0.05), as well as group by region interaction (F[6,99] = 10.0, P < 0.05). * Fisher's protected least significant difference post hoc comparisons demonstrated significant reductions in FLI in laminae I–II and V–VI of ALFENTpre and in laminae I–II of ALFENTpost compared with vehicle controls (P < 0.05). +In laminae I–II, FLI in the ALFENTpost group was significantly higher than that in the ALFENTpre group (P < 0.05). Error bars represent SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, general anesthesia with IV ketamine produced more analgesia during Phase II of the formalin test when active during Phase I compared with that after Phase I. The fact that ketamine produced preemptive analgesia suggests that it acts to prevent central hyperexcitability during Phase I, the period thought to be critical for the development of noxious stimulus-induced spinal sensitization (22). This result is supported by clinical data suggesting preemptive analgesia with ketamine (9), by electrophysiological animal data demonstrating inhibition of dorsal horn neuronal firing in response to noxious stimulation after the administration of ketamine (23), and by a behavioral study of intrathecal ketamine in a different experimental model of persistent pain (24).

Although preemptive analgesia with ketamine may be due to antagonism of spinal NMDA receptor sites, ketamine exerts several other pharmacological actions, including blockade of voltage-sensitive calcium channels (25), depression of sodium channels (26), and agonism of opioid receptors (27). Despite evidence that ketamine may exert a weak opioid receptor-mediated effect, the failure of naloxone to reverse the analgesic effects of ketamine rules out opioid receptor-mediated analgesia with ketamine in this model. In fact, Maurset et al. (28) similarly showed that naloxone failed to reverse ketamine analgesia in human trials of experimental ischemic pain and postoperative oral surgery pain, thus arguing for a phenyl cyclohexyl piperidine receptor-mediated blockade of the NMDA receptor-operated ion channel.

Because previous evidence, including the above behavioral data, has suggested that ketamine has antinociceptive effects in both clinical (9) and laboratory studies (23,24), we hypothesized that it would decrease noxious stimulus-induced spinal FLI. However, quite to the contrary, ketamine was observed to increase FLI. If analgesic drugs have been previously shown to suppress formalin-induced FLI, why then do we not observe such suppression with ketamine, which demonstrated a clear preemptive effect in behavioral studies? Given limited data that ketamine may act at {varsigma}-receptor binding sites (29) in the spinal cord (30), recent evidence of Fos induction by a selective {varsigma}-receptor ligand could be a possible explanation for increased spinal FLI with ketamine (31). From these data, we conclude that ketamine does not suppress noxious stimulus-induced FLI, which is consistent with data from two previous studies failing to demonstrate suppression of noxious stimulus-induced spinal FLI with ketamine (32,33). Unlike these results with ketamine, another NMDA antagonist, MK-801, has been shown to suppress noxious stimulus-induced FLI (15). Our rationale for studying ketamine, rather than a more selective NMDA antagonist (such as MK-801), was to evaluate the effects of a drug used in clinical practice. Further studies to elucidate the effects of ketamine alone on spinal FLI would be of value.

At a dose of 170 µg/kg alfentanil produced unresponsiveness and loss of righting reflex in all study rats. In this study, the timing of alfentanil administration had no influence on the temporal profile of formalin-induced behavioral pain scores; that is, the analgesic profile of alfentanil administered preformalin was identical to that of postformalin alfentanil administration. Our interpretation of this result is that alfentanil did not produce a selective preemptive analgesia. This failure of an opioid to produce preemptive analgesia has been suggested by other studies of subcutaneous alfentanil (34) and IV remifentanil (35). However, previous studies have shown that intrathecal morphine suppresses spinal sensitization in both behavioral and electrophysiological models (36,37).

This study shows that, although preformalin alfentanil administration suppressed FLI in both laminae I–II and laminae V–VI [consistent with previous studies of morphine (12)], postformalin alfentanil suppressed FLI only in laminae I–II and to a significantly smaller degree. This suggests that alfentanil produces preemptive spinal FLI suppression. Why is postformalin alfentanil less effective than preformalin alfentanil in suppressing FLI when a selective preemptive effect was not observed in behavioral studies? There may be two possible explanations for this. First, our studies of FLI reflect a 2-hour period after formalin injection, whereas the behavioral studies only investigated the first hour postformalin. This time period difference could explain such discrepancies. Second, this demonstration of preemptive analgesia with intrathecal, but not systemic, opioids may suggest that, with systemic opioid administration, supraspinal effects predominate and have a similar effect in both preinjury and postinjury treatment groups. Thus, it is possible that systemic opioids do exert a preemptive effect at the spinal level (as supported by intrathecal studies and our FLI data), but that a predominant supraspinal effect of systemic opioids masks any behavioral difference between the pre- and postinjury groups.

A previous study from our laboratory has shown that intrathecal lidocaine suppresses Phase II behavioral hyperalgesia after preformalin, but not postformalin, treatment (16). Although lidocaine pretreatment resulted in significantly greater spinal FLI suppression compared with lidocaine posttreatment, the fact that lidocaine posttreatment suppressed spinal FLI but not Phase II pain behavior suggests some degree of uncoupling between behavioral and Fos protein responses (16). In another study by Hammond et al. (38), the intrathecal administration of the {delta}2 opioid receptor agonist (D-Ala2,Glu4) deltorphin suppressed formalin-induced pain behavior, yet failed to suppress spinal FLI. Hammond et al. (38) explain this finding by suggesting that because {delta} opioid agonists inhibit synaptic transmission primarily through presynaptic mechanisms (and thus do not act to hyperpolarize second-order neurons), other excitatory inputs may go unchecked and thus continue to induce Fos in the presence of this drug. This explanation would not apply to our findings with ketamine, which likely does act more directly on dorsal horn neurons. In the present study, systemically administered ketamine failed to suppress spinal FLI despite a clear preemptive analgesic effect based on behavioral responses.

From this and other studies, we conclude that, although preemptive analgesia is thought to be contingent on the prevention of noxious stimulus-induced spinal neuroplasticity, spinal neuron Fos induction may not always be an appropriate marker of the pharmacological modulation of such changes.


   Acknowledgments
 
This study was supported by MRC(C) funding.

We thank Drs. Satyabrata Kar, Jim Pfaus, and Jean-Guy Chabot for their valuable assistance with this study.


   Footnotes
 
Presented, in part, at the annual meeting of the Society for Neuroscience, San Diego, CA, 1995, and the annual meeting of the Society for Neuroscience, New Orleans, LA, 1997.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication March 30, 1999.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 1999 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press