Spinal Action of Ketorolac, S(+)- and R(-)-ibuprofen on Non-noxious Activation of the Cathechol Oxidation in the Rat Locus Coeruleus : Evidence for a Central Role of Prostaglandins in the Strychnine Model of Allodynia

All surgical procedures and experiments were performed in compliance of the rules set forth by the Animal Care Committee of Queen's University. Animals were housed in group cages on a 12-h light-dark cycle with a room temperature of 22 [degree sign]C. Food and water were provided ad libitum.

Adult male Sprague-Dawley rats weighing 300 to 400 g (Charles River; St-Constant, Quebec, Canada) were anesthetized using halothane and oxygen and were intubated for artificial respiration (Harvard Apparatus [Montreal, Quebec, Canada] rodent respirator, set at f = 50 strokes/min; induction with 4% halothane and a maintenance dose of 1% to 1.25% halothane). Rectal temperature was maintained at 37 +/− 0.5 [degree sign]C by placing the rats on a warming blanket connected to a temperature controller (Yellow Springs Instruments, Yellow Springs, OH). To administer drugs intravenously, a polyethylene catheter (PE-50) was placed in the jugular vein and, in all experiments, saline (0.9% NaCl) was infused through the catheter (5 ml [middle dot] kg^-1[middle dot] h^-1) using a Harvard minipump for the rest of the experiment. Blood pressure and heart rate were monitored using a Grass model 7 physiograph (Quincy, MA) via a polyethylene catheter (PE-50) placed in the carotid artery and coupled to a Statham blood pressure transducer. Carotid cannulation was used, which kept hair deflected away from the surgical field. To produce muscle relaxation, 400 [micro sign]g/kg vecuronium was administered intravenously to the animals after an adequate level of anesthesia to nociceptive stimulation was ensured. After the animals were stabilized, halothane anesthesia was maintained at 1% to 1.25% and in vivo voltammetric experiments were done.

The metabolic activity of catecholaminergic neurons can be monitored in vivo by using the electrochemical technique of DNPV in combination with treated carbon fiber electrodes (for a review, see Buda et al. [14]). Voltammetry is based on the susceptibility of molecules to oxidation at specific voltage potentials where the application of a brief double potential pulse and the differential display of sampled current responses provide a peak-shaped current-potential response (voltammetric signal). The voltammetric signal (CA [middle dot] OC) recorded from the locus coeruleus corresponds to the faradaic current arising from the oxidation of the deaminated metabolite of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC). Manufactured carbon fiber electrodes were treated electrically in vitro [30,31]and tested in a standard phosphate-buffered saline (pH = 7.4) solution containing ascorbic acid (200 [micro sign]M; Sigma Chemical Co., St. Louis, MO) and DOPAC (20 [micro sign]M; Sigma Chemical Co.). The CA [middle dot] OC was identified as a voltammetric peak occurring at +55 mV in vitro and in vivo. Electrically treated carbon fiber (8 to 10 [micro sign]m diameter x 500 [micro sign]m length) microelectrodes were stereotaxically implanted in the locus coeruleus. [13,18] 

Anesthetized rats were placed prone in a stereotaxic frame with an incisor bar placed 10 mm below the interaural line. An intrathecal catheter made from PE-10 tubing stretched to approximately 1.5 times its original length [6,18]was filled with saline and inserted through a slit in the atlantooccipital membrane of the cisterna magna. The catheter was guided through the spinal subarachnoid space (approximately 8.5 cm caudally) with the tip terminating near the L1 spinal segment. The externalized end of the intrathecal catheter was secured to the musculature to prevent movement.

To implant the carbon fiber electrode, a small opening was made in the skull penetrating the dura mater, and the treated electrode was lowered through the cerebellar surface toward the locus coeruleus using these coordinates: 0.7 mm posterior from the interaural line, 1.33 mm lateral from the midline, and a depth of 5.5 to 6.5 mm from the cerebellar surface. Electrode placement in the locus coeruleus was identified by gradually lowering the electrode until the height of the CA [middle dot] OC peak signal recorded in the locus coeruleus began to diminish. The electrode was raised to the position where the peak signal was recorded. An auxiliary electrode (platinum wire) and Ag-AgCl reference electrode were placed on the skull surface using a semiliquid contact. The response of noradrenergic neurons was recorded by measuring the CA [middle dot] OC at 3-min intervals using DNPV (Biopulse, Tacussel, Villerurbanne, France; the parameters were linear sweep potential from -0.25 to 0.15 V, scan rate of 4 mV and 0.4 s, pulse amplitude of 30 to 40 mV, pulse duration of 40 to 60 ms, and prepulse duration of 80 to 100 ms).

After catheter insertion and electrode implantation, the halothane concentration was reduced incrementally to zero, and anesthesia was maintained with intravenous urethane (1 to 1.2 g/kg infused during 10 min; Sigma Chemical Co.). After a stable anesthetic state was achieved, the catechol oxidation current was allowed to stabilize for 1 h before the experiments were performed.

All drugs for intrathecal administration were injected using a 50-[micro sign]l Hamilton syringe. Intrathecal doses were delivered in a total volume of 5 [micro sign]l, followed by 10 [micro sign]l saline to flush the catheter. The following drugs were used: strychnine sulfate (molecular weight = 766.9; Sigma Chemical Co., St. Louis, MO); ketorolac (ketorolac tromethamine; molecular weight = 376; SYNTEX, Ireland); cyclodextrin (2-Hydroxypropyl-[small beta, Greek]-cyclodextrin; molecular weight = 1,423; Research Biochemicals International, Natick, MA); and S(+)- and R(-)-ibuprofen (molecular weight = 206.27; Research Biochemicals). Ketorolac was diluted in physiologic saline (0.9% w/v). Both S(+)- and R(-)-ibuprofen were dissolved in 5% cyclodextrin (Research Biochemicals). After the CA [middle dot] OC signal was stabilize, normal saline or strychnine (40 [micro sign]g; Sigma Chemical Co.) was injected intrathecally in a 5-[micro sign]l volume and flushed with 10 [micro sign]l saline. After injection, animals underwent hair deflection, which involved repeated brushing with a cotton-tipped applicator, held bilaterally to the legs, flanks, and lower back. Brushing was done with no more force than needed to move through the hair, and only the pelage was disturbed. Sensitive dermatomes, identified by hair deflection-induced increases in blood pressure and heart rate (with or without a motor withdrawal response), were brushed with an oscillating motion (at a rate of 1 or 2 s^-1for 5 min) at 5-min intervals for 1 h. The maximum evoked increase in CA [middle dot] OC, mean arterial pressure (MAP), and heart rate for each stimulus period was determined.

To evaluate the spinal action of NSAIDs, groups of animals were pretreated with either intrathecal ketorolac (30 [micro sign]g), S(+)-ibuprofen, or R(-)-ibuprofen (10 [micro sign]g) 20 min before strychnine was administered (40 [micro sign]g). At the end of each experiment, the monoamine oxidase inhibitor pargyline (75 mg/kg; Sigma Chemical Co.) was administered intraperitoneally. Subsequent decay of the voltammetric peak confirmed that the signal was caused by catechol oxidation in the locus coeruleus. [15] 

All voltammetric data (CA [middle dot] OC peak height) were expressed as a percentage of the mean baseline value calculated by averaging the four consecutive catechol oxidation peak heights measured before intrathecal strychnine or saline administration. The MAP was calculated from the following equation:(diastolic + 1/3 [systolic - diastolic]). The evoked change in MAP after hair deflection was expressed as a percentage of the mean baseline response relative to the immediate prestimulus control for each point in the time course. The maximum evoked increase in MAP recorded in each 5-min period was used. Statistical analyses within each group were performed using a one-way repeated-measures analysis of variance. Significant (P < 0.05) differences were identified using the post-hoc Newman-Keul's test. To determine a treatment effect, the peak catechol oxidation (CA [middle dot] OC) and mean arterial pressure response were compared using a one-way analysis of variance among the strychnine, saline, and ketorolac groups. Significant differences (P < 0.05) were identified using the Newman-Keul's test. Statistical analysis of the peak CA [middle dot] OC and MAP response between the stereoisomers of ibuprofen was performed using an unpaired Student's t test, and significance was assessed at P < 0.05. Variability associated with single measurements concerning CA [middle dot] OC and MAP were expressed as the mean +/− SEM.

Voltammograms of spiked solutions in vitro (Figure 1B) resulted in two distinct peaks at -89 mV (peak 1) and +55 mV (peak 2) that corresponded to the oxidation of ascorbic acid and DOPAC, respectively (Figure 1B). No oxidation peaks were detected in phosphate-buffered saline alone (Figure 1A). Implantation of the carbon fiber electrode in the locus coeruleus yielded an oxidation current at +55 mV representing the baseline oxidation of DOPAC (Figure 1D, peak 2). To confirm that the source of this oxidation peak was DOPAC, the monoamine oxidase inhibitor pargyline (75 mg/kg) was administered intraperitoneally at the end of each experiment. Pargyline treatment resulted in a gradual and complete decay of the oxidation peak detected at +55 mV. The ascorbic acid peak, which occurred at a potential of -89 mV (Figure 1D), remained unchanged.

Repeated brushing of the caudal dermatomes after intrathecally administered strychnine evoked an immediate and significant increase in the CA [middle dot] OC signal recorded from the locus coeruleus (Figure 2A) The maximum effect (peak height = 149.7 +/− 7.2% of baseline) occurred 24 min after the onset of stimulation, which was significantly different when compared with the peak saline response (peak height = 106.6 +/− 0.9% of baseline) and remained significantly elevated for an additional 22 min (Figure 2A). In contrast, hair deflection applied to the same dermatomes of rats treated intrathecally with saline (n = 4) evoked no changes in either the CA [middle dot] OC (Figure 2A) or MAP (Figure 2B). After pretreatment with intrathecal ketorolac (30 [micro sign]g), 20 min before strychnine, there was no significant increase in the CA [middle dot] OC response induced by innocuous hair deflection (Figure 2A). The maximum evoked increase in the CA [middle dot] OC (peak height = 18.7 +/− 7.4% of baseline) in animals pretreated with ketorolac was significantly attenuated when compared with the maximum peak effect in the strychnine control group (Figure 2A). In animals receiving S(+)-ibuprofen (10 [micro sign]g), 20 min before strychnine there was no significant change in the CA [middle dot] OC response (Figure 3A). In contrast, pretreatment with R(-)-ibuprofen (10 [micro sign]g) failed to attenuate the strychnine-induced CA [middle dot] OC response, with a significant increase occurring 6 to 60 min after strychnine administration. Comparison of the peak CA [middle dot] OC response between the stereoisomers of ibuprofen revealed a significant difference. The peak CA [middle dot] OC response in the S(+)-ibuprofen treatment group was 112.3 +/− 2.2% of baseline, but after R(-)-ibuprofen pretreatment the peak CA [middle dot] OC response was 153.8 +/− 8.2% of baseline (Figure 3A).

A significant increase in MAP elicited repeated brushing of sensitive caudal dermatomes after administration of intrathecal strychnine was also observed (peak height = 125.3 +/− 5.2% of the baseline MAP [93.6 +/− 8.6 mmHg];Figure 2B). Blood pressure partially recovered between stimulus trains (hair deflection) yet remained elevated and eventually reached a plateau after 15 min with a maximum increase of 127.5 +/− 3.8% of baseline, which was significant when compared with the peak MAP response in saline-treated animals (peak height = 104.4 +/− 6.6% of baseline MAP [99.2 +/− 3.5 mmHg];Figure 2B). After pretreatment with intrathecal ketorolac, 20 min before strychnine, no significant change in MAP was observed. Ketorolac was effective in attenuating the peak brushing-evoked MAP response when compared with the maximum MAP response in the strychnine control group (Figure 2B). The maximum evoked increase in MAP in animals receiving ketorolac (30 [micro sign]g) was 106.9 +/− 2.4% of baseline MAP (108.2 +/− 16 mmHg;Figure 2B). Pretreatment with 10 [micro sign]g intrathecally administered S(+)-ibuprofen significantly blocked the peak brushing-evoked MAP response (peak height = 103.7 +/− 3.4% of baseline MAP [113.3 +/− 6.2 mmHg]) after intrathecal administration of strychnine when compared with 10 [micro sign]g R(-)-ibuprofen (peak height = 127 +/− 7.2% of baseline MAP [102.9 +/− 5.4 mmHg];Figure 3B). Hair deflection of sensitive caudal dermatomes after intrathecal strychnine or saline had no effect on heart rate within each of the five groups (data not shown).

The results of the current study support the hypothesis that the blockade of spinal glycine receptors with intrathecal strychnine induces an allodynia-like state in urethane-anesthetized rats. [6,18]Thus, hair deflection applied to the caudal dermatomes sensitized by spinally administered strychnine triggered the activation of noradrenergic neurons in the locus coeruleus (as measured by in vivo voltammetry) and a significant increase in arterial blood pressure. These responses were comparable to those evoked by noxious mechanical and chemical stimulation in the absence of intrathecally administered strychnine. [13]The increase in the locus coeruleus catechol oxidation evoked by hair deflection in strychnine-treated animals was not secondary to the concurrent cardiovascular response. There is no consistent relation between locus coeruleus firing and changes in blood pressure. [32] 

The metabolic activity of catecholaminergic neurons can be monitored in vivo by using the electrochemical technique of DNPV in combination with treated carbon fiber electrodes (for a review, see Buda et al. [14]). Voltammetry is based on the susceptibility of molecules to oxidation at specific voltage potentials, but the chief shortcoming is limited chemical resolution. Detection of neurotransmitters that can be oxidized and their metabolites becomes difficult because of the presence of high levels of ascorbic acid in the brain, which oxidizes at the same potential as certain catechols (i.e., DOPAC), thus interfering with their detection on untreated carbon fiber electrodes. Consequently, the advent of an electrochemical treatment protocol, resulting in the modification of the active surface state of the electrode, allows a valid and reproducible separation of ascorbic acid from DOPAC, which therefore improves the selectivity of carbon fiber electrodes. The concentrations of electroactive species used to test the electrode in vitro should be similar to those found in vivo and should replicate conditions in the brain. Manufactured carbon fiber electrodes are electrochemically pretreated and then tested in vitro before being used in vivo. This test ensures that the standard electrochemical pretreatment applied to the electrode produces valid voltammograms that exhibit well resolved peaks at -89mV and +55mV corresponding to the redox potentials of ascorbic acid and DOPAC, respectively, and that both peaks increase during successive scans until an equilibrium is achieved. This allows us to relate the current and concentration of the electroactive species (ascorbic acid and DOPAC) detected and determines the temporal response of the electrode.

As reported in several previous studies, [13,33-35]DNPV, an in vivo electrochemical detection technique, allows the catecholamine metabolism to be monitored in the locus coeruleus, a major noradrenergic nucleus in the pons. Differential normal pulse voltammetry is based on the application of a brief double potential pulse and the differential display of sampled current responses to provide a peak-shaped current-potential response (voltammetric signal). This technique is a hybrid of differential pulse and normal pulse waveforms and has the advantage of high current sensitivity because of the short duration of the double pulses and its ability to allow the background nonfaradaic events the working electrode to be negligible. The voltammetric signal (CA [middle dot] OC) recorded in the locus coeruleus corresponds to the faradaic current arising from the oxidation of the deaminated metabolite of dopamine, DOPAC. The amplitude of this peak changes in parallel with changes in metabolic activity of noradrenergic neurons. [15]The CA [middle dot] OC recorded in this study satisfied all of the criteria for the oxidation of DOPAC in the locus coeruleus. [15]Specifically, the current peak occurred at an oxidation potential of +55 mV, was restricted to a brain area (5.5 to 6.5 mm below the cerebellar surface) corresponding to the locus coeruleus, and was abolished by the systemic administration of pargyline, a monoamine oxidase inhibitor, thereby distinguishing if from the ascorbate signal at -89 mV, which was not affected by this treatment. Importantly, none of the NSAIDs used in this study interfered with the catechol signal recorded in vitro, indicating that the attenuation of the evoked signal was not due to a physicochemical interaction. Changes in CA [middle dot] OC are known to be a useful index of functional activity of catecholaminergic neurons. [15-17,36]In the current study, repeated brushing of the hair at circumscribed sites having no measurable effect on CA [middle dot] OC in the locus coeruleus of saline-treated rats evoked a sustained increase in activity after intrathecal administration of strychnine. This exaggerated effect to an otherwise innocuous stimulus corresponds to previous studies using this model of allodynia, [5,6,9,18]The dose and route of strychnine administration, and the restricted caudal sites at which hair deflection evoked the neurochemical and cardiovascular responses are consistent with a spinal mechanism of allodynia. That this mechanism involves the central production of prostaglandins, probably within the spinal cord itself, is strongly supported by our results. Thus, intrathecal ketorolac and S(+)-ibuprofen blocked the peak responses evoked by hair deflection in strychnine-treated rats. In contrast, the inactive R(-)-isomer of ibuprofen had no effect. The stereospecificity of this blockade is consistent with earlier reports that only the S(+)-enantiomers of NSAIDs inhibit prostaglandin synthesis in vitro. [37] 

It is unlikely that the inhibitory effect observed with intrathecal NSAIDs resulted from their redistribution to peripheral sites. The doses of intrathecal NSAIDs used were 100 to 1,000 times less than those needed to produce antinociception after systemic administration, and both ketorolac and S(+)-ibuprofen were effective within minutes of intrathecal injection, displaying a similar time-effect relation. Furthermore, strychnine-induced allodynia is centrally induced induced and achieved without injury or inflammation to the peripheral nervous system.

The involvement of spinal prostaglandins in the abnormal processing of low-threshold afferent input is supported by earlier studies showing a direct allodynic effect of intrathecal prostaglandins in rodents. In an extensive series of studies, intrathecal prostaglandin E²or prostaglandin F²[small alpha, Greek] was shown to induce behavioral allodynia in conscious mice and rats. [38-41]Interestingly, the time course and spinal pharmacology of intrathecally administered strychnine and prostaglandin E^2-inducedallodynia are remarkably similar. The report that prostaglandin E^2-inducedallodynia was dose dependently reversed by the glycinoceptor agonist taurine, but not by a glycine-binding site antagonist at the N-methyl-D-aspartate receptor (7-CI-KYNA), [42]indicates a close link between prostaglandin E^2-inducedallodynia and glycine receptor blockade, an effect unrelated to the strychnine-insensitive glycine coagonist site on the N-methyl-D-aspartate-ion channel complex. The similarity between the prostaglandin-induced and spinal disinhibitory models is intriguing and directly relevant to the results of the current study. Nonsteroidal antiinflammatory drugs reduce prostaglandin-induced hyperalgesia by inhibiting cyclooxygenase, the key enzyme that catalyses the conversion of arachidonic acid to prostaglandins. [43-45]In addition, there is evidence that these drugs have a central antinociceptive action involving the inhibition of prostaglandin synthesis in the spinal cord. [29,46-49]Whether prostaglandins are synthesized and released during allodynia has not been determined.

In conclusion, somatosensory processing in the spinal cord of urethane-anesthetized rats is altered because of the removal of glycinergic inhibition such that normally innocuous stimuli results in a nociceptive-like activation of the locus coeruleus and cardiovascular responses. The ability of intrathecal ketorolac and S(+)-ibuprofen, but not R(-)-ibuprofen, to suppress strychnine-dependent tactile evoked responses provide pharmacologic evidence that central prostaglandins play an important role in the abnormal sensory processing of strychnine-induced allodynia.