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Anesth Analg 2004;99:159-165 © 2004 International Anesthesia Research Society doi: 10.1213/01.ANE.0000114552.20268.7F
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Abstract |
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We assessed the objective measurement of central sensitization processes in the awake rat after subcutaneous formalin with cortical somatosensory evoked potentials (CSEPs). Cranial extradural electrodes and intrathecal catheters were implanted in adult male Wistar rats. After 7 days of recovery, CSEPs were induced by electrical stimuli at the tail and recorded before/after the injection of 50 µL of 2% formalin into the hindpaw of rats for 1 h. The drug and tested vehicles were delivered intrathecally 5 min before the injection of formalin. The peak-to-peak amplitude of the P1-N1 (the early positive-negative sequence pair of CSEPs) and the baseline-to-peak amplitude of the N2 (the late negative component of CSEPs) were analyzed. We found that the amplitudes of both signals increased (154.3% ± 10.9% and 168.7% ± 9.8%, respectively) from 10 min after formalin injection to the end of the 60-min test period. Pretreatment with intrathecal ketorolac dose-dependently prevented the increases induced by formalin in both measured variables. Moreover, the increases in P1-N1 and N2 were markedly attenuated either by intrathecal polyethylene-10 tubing or by the solvents used for injection, thus indicating the need for distinguishing an impaired nociceptive signal from antinociception when the effects of drugs are evaluated.
IMPLICATIONS: Variability is the main limitation of cortical somatosensory evoked potential (CSEP) measurements. This study validated CSEPs to examine central sensitization induced by formalin. Testing conditions were found in which CSEP signals could be reliably used to evaluate central sensitization processes, as well as the antinociceptive effect of the drug, in a dose-dependent manner.
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Introduction |
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Because of their relevance in various clinical settings, objective methods for measuring nociception have generated a broad interest among scientists and medical personnel involved in pain research or pain management. Whereas the quantitative assessment of nociception can be addressed by various invasive electrophysiological methods (1,2), the evaluation of pain in unanesthetized animals relies almost exclusively on behavioral testing (3). An electrophysiological method based on a tonic pain model in awake animals would allow a more reliable interpretation of the data and would offer finer pharmacological testing conditions.
Cortical somatosensory evoked potentials (CSEPs) are objective records of activity evoked in neural pathways by a given stimulus. CSEPs correlate with nociception in humans (4–6) and in an anesthetized animal pain model (7). We previously demonstrated the use of CSEPs as an objective measurement of central excitability after subcutaneous (SC) injection of formalin at the base of the tail in the awake rat (8). Two major components of the electrical signal were identified: an early positive-negative (P1-N1) sequence related to the arrival of the afferent volley in the cortex and a more ample late negative (N2) component that presumably arose from associative signal postprocessing. The amplitudes of both CSEP components evoked by nonnoxious stimuli were equally increased after the injection of formalin and were considered to be related to the central sensitization induced by formalin, because the stimuli given to elicit the CSEPs were distant from the site of injection. In attempts to evaluate the effects of intrathecal drugs on formalin-induced central processing by using CSEPs, we found that increased CSEPs were markedly altered by routine polyethylene (PE)-10 catheterization and by the varieties of solvents used for dissolving drugs. Intrathecal injection of the solvents themselves even induced agitation and squeaking in the animals. Although it is reported in some publications (9–12), not much attention has been given to this problem, even though agitation might interfere with the interpretation of the effects of antinociceptive drugs. We therefore investigated the influence of catheterization and drug solvents on formalin-induced nociceptive signals by using CSEPs in awake rats and further validated our method by performing dose-response experiments with the well established antinociceptive drug ketorolac (9).
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Methods |
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The experimental protocols were approved by the University Bioethical Committee for Animal Experimentation in accordance with the guidelines for animal experimentation of the International Association for the Study of Pain. Adult male Wistar rats (250–350 g; B & K Universal Ltd., UK) were housed in groups of 4, with free access to food and water. An automatically controlled photoperiod was set at a 12-h light/dark cycle. The experiments were performed between 8:00 AM and 3:00 PM to minimize animal-dependent variability in the signals.
Different catheters were used: PE-10 (Becton Dickinson; 0.023-in. outer diameter x 0.0105-in. inner diameter; n = 8), stretched PE-10 (calibrated PE-10 stretched to twice its original length; n = 5), and 32-gauge polyurethane (PU-32; Micro, Allison Park; 0.0107-in. outer diameter x 0.005-in. inner diameter; n = 8). Two suggested solvents (11,13) for dissolving specific cyclooxygenase (COX) inhibitors were tested: 1) dimethyl sulfoxide (DMSO; Sigma-Aldrich, Steinheim, Germany) diluted 1:1 with 50% 2-hydroxy-propyl-ß-cyclodextrin (Roquette, Lestrem, France) and 2) 50% polyethylene glycol (PEG) 200 (Sigma-Aldrich). Ketorolac tromethamine (Lot No. CC99020001, injectable pharmaceutical grade; Roche, Basel, Switzerland) was dissolved with saline (B/Braun, Melsungen, Germany).
The osmotic pressures of the final injected solutions were measured by a Fiske 2400 (Fiske Associates, MA) osmometer, a device based on cryoscopy. Mannitol (Sigma-Aldrich) was used to control the effect of the osmolality of the solution on CSEPs.
Animals were previously habituated to test conditions on every other day a week before surgery. After anesthesia with intraperitoneal injection of ketamine and xylazine (100 and 10 mg/kg, respectively), the rats underwent surgery for implantation of both cranial extradural electrodes and an intrathecal catheter. A right parietal craniotomy was performed 2 mm lateral to the midline, exposing the sensorimotor cortex corresponding to the tail of the animal (8). A row of gold-plated electrical connector pins spaced 2.5 mm apart was positioned on the dura mater and fixed with clinical dental cement. A reference electrode was positioned and fixed in the temporooccipital ridge.
At the same time, the intrathecal catheter was introduced subarachnoidally through the atlantooccipital membrane by using the modified method of Yaksh and Rudy (14). After the study of the different catheters, 32-gauge PU tubes were used for the subsequent experiments. The 8.5-cm length served to place the tip of the catheter at the rostral margin of the lumbar enlargement. Animals were allowed at least 1 wk of recovery from surgery before testing started. Only rats with no neurological deficits were used for the experiments (90% of all operated rats).
For the somesthetic stimulation, two 27-gauge sterile stainless-steel needles were placed in the middle-third of the tail on the side contralateral to the cortical electrodes. These needles were connected to a stimulating voltage source. Stimuli were 1-ms square-wave pulses at a rate of 0.5 Hz. The stimulus intensity was adjusted to the lowest or threshold level necessary to evoke a distinct signal. The measured electroencephalogram was digitized at 5 kHz and stored on a hard disk by using a custom-developed interface (LabView®; National Instruments). Each analyzed record represents the average of 20 300-ms sampling periods. The peak-to-peak amplitude of the P1-N1 signal and the baseline-to-peak amplitude of the N2 signal were measured. The increased amplitudes of CSEPs induced by formalin in rats without intrathecal catheterization were chosen as a positive control.
After three control measurements, inflammation was obtained by the SC injection of 50 µL of a 2% formalin solution into the plantar area of the hindpaw 5 min after the intrathecal delivery of 10 µL of study vehicles or drug. CSEPs were further recorded every 10 min for 1 h after the injection of the formalin. In control animals, the same amount of saline was injected into the paw.
Data are presented as the percentage of the preformalin control amplitudes. The time-response curves of the different test groups were compared by using analysis of variance for repeated measurements. The individual dose-response curves obtained with ketorolac were generated by the gravimetric integration of the sensitization responses versus the baseline controls. Dunnett multiple comparison tests were used for post hoc comparison of time-points within a group. Results are expressed as mean ± SE, and P < 0.05 was considered statistically significant.
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Results |
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The electrophysiological characteristics (shape, relative amplitudes, and time course) of the two major CSEP components induced by electrical stimulation were not altered by intrathecal catheterization. The unfiltered electrical recordings (Fig. 1) demonstrating recording traces of the experiment setting showed the P1-N1 followed by an N2.
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) and the amplitudes of P1-N1 (peak to peak) and N2 (peak to basal level) are identified. Negative amplitudes are represented as upward deflection. P1-N1 = the early positive-negative sequence pair of CSEPs; N2 = the late negative component of CSEPs.The size of the intrathecal catheter affected the amplitude of the CSEP response after the injection of formalin (Fig. 2). With regard to controls, there was only a slight measurable increase in amplitude after formalin when the standard PE-10 catheter was used. The signal-to-noise ratio of the method markedly decreased. Conversely, the PU-32, the smallest catheter, left the increased signals almost unaltered. No significant difference was found between the group catheterized with PU-32 and the group without catheterization (CSEPs increased after formalin by 154.3% ± 10.9% and 168.7% ± 9.8% for P1-N1 and N2, respectively, versus 155.0% ± 9.7% and 164.8% ± 8.2%; P > 0.05). Changes in amplitude of the P1-N1 and N2 signals were reduced to, respectively, 119.5% ± 7.5% and 133.6% ± 15.9% with the PE-10 catheter (P < 0.01 versus the PU-32 group). Stretching this catheter to make it thinner resulted in CSEP signals of intermediate magnitude (137.4% ± 11.9% for P1-N1 and 144.3% ± 15.1% for N2; P < 0.05 versus the PE-10 group; Fig. 2). After the animals were killed, a thick fibrosis of the intrathecal space was induced with the PE-10 catheters (Fig. 3), whereas the thinner PU-32 catheter left the spinal cord morphologically intact.
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In 70% of the rats that received either 10 µL of DMSO mixed 1:1 with 50% cyclodextrin or 50% PEG, a transient allodynia with shriek was observed during the intrathecal injection. The amplitudes of P1-N1 and N2 were markedly decreased by intrathecal DMSO/cyclodextrin or PEG injection when compared with saline control (P < 0.01 versus the saline control group; Fig. 4). No increased CSEPs were elicited by the injection of formalin. Because the tonicity of the solution could lead to a transient local osmotic stress in the spinal cord at the tip of the catheter and thus alter the responses of CSEPs, we measured the osmotic pressure of all solutions used. When compared with nontoxic mannitol at 160 or 320 mM, the decrease of CSEPs seemed to correlate well with the increased osmotic concentration of the solutions between 300 and 1300 mOsm (correlation coefficient, –0.92; P < 0.001 with regression analysis).
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Pretreatment of the animals with intrathecal ketorolac alone did not change the baseline amplitudes of CSEPs but prevented the increases of both acquisition variables (P1-N1 and N2) induced by formalin. The effects of ketorolac were dose dependent (Fig. 5A), and a dose-response curve could be established (Fig. 5C). The maximal inhibitory effect of the drug was seen at approximately 10 µg (1 µg corresponds to 2.7 nmol), and the 50% inhibitory concentration was 4.5 ± 1.8 µg. Ketorolac—at 1 µg—displayed the least effect on formalin-induced increases of CSEPs.
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Unexpectedly, at a supramaximal dose of 50 µg of intrathecal ketorolac, we recorded a large increase in the amplitudes of the CSEP variables (234.6% for P1-N1 and 213.8% for N2; P < 0.01 versus baseline; Fig. 5B) instead of an inhibition. This dose also elicited vocalization and aggressive behavior in three of the five treated animals. Because this paradoxical effect might have been due to a local—intrathecal—toxicity of the drug as well as to pH or osmotic stress, we then performed control experiments to assess the effects of these variables on the CSEPs.
The pH values of the ketorolac solutions with the doses used were stable between 6.0 and 5.8 and were not different from those of saline (one-way analysis of variance; P = 0.7). The rat cerebrospinal fluid is neutral (7.58 ± 0.05; n = 10). No differences in osmotic pressure were found within our ketorolac concentration range. The highest value for 50 µg of ketorolac was 309 mOsm, which is similar to that of saline.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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With this work, we demonstrated that CSEPs provide an objective measurement of formalin-induced central sensitization in unanesthetized rats. We also refined this method for intrathecal pharmacological application by performing dose-response experiments with a well established antinociceptive drug (ketorolac). The most notable findings, besides validation of the method itself, were that intrathecal catheterization with PE-10 and the vehicles used widely for dissolving poorly soluble drugs seriously impaired the increased CSEPs induced by formalin. When recording reduced evoked potentials, it is important to distinguish between a true antinociceptive effect (e.g., induced by a drug) and damage to the conduction path (produced by a catheter or a damaging drug).
Injection of formalin into the rat paw induces a typical biphasic response (15,16). The initial phase is due to the direct stimulation of nerve fibers by formalin, whereas the second phase is due to subsequent inflammation and central sensitization (17,18). One of the external signs of central sensitization is the appearance of secondary hyperalgesia into an area of the body distant from the site of injection. Central sensitization also manifests as facilitation of responses to innocuous stimuli, reduction of mechanical and thermal thresholds, and spatially extended receptive fields of convergent neurons, indicating that the degree of central sensitization can be measured by applying nonnoxious stimuli outside the formalin-inflamed zone, as we have done. This formalin/CSEP model is distinct from the classic behavior formalin test. The biphasic response of the formalin test is related to spontaneous activity in A and C afferent nerve fibers. We used evoked potential to measure central sensitization alone (8), which correlated with the increased amplitudes of P1-N1 and N2. This technique can thus be used to measure the antinociceptive effects of drugs active on various states of central hyperexcitability.
Because the variability of CSEPs is one of the major problems in its application both in humans and in animals, the experimental conditions should be precisely defined. PE-10 tubing is widely used for intrathecal delivery of drugs in rats. However, a 32-gauge PU intrathecal catheter improved motor coordination and decreased paralysis when compared with PE-10 tubing (19). In our study, the sensitization-related increase of CSEP amplitude was very small in animals with intrathecal PE-10 when compared with control animals that received formalin without catheterization. PE-10 intrathecal catheters thus impaired the CSEP signals of intermediate magnitude. This effect was related to the catheter size, as evidenced by the improved results obtained with stretched PE-10 tubing. Only the PU-32 intrathecal catheter conserved the CSEP response elicited by the formalin injection and gave us enough resolution to perform the subsequent pharmacological experiments. Gross observation of the animal’s spinal cord showed cord compression signs and fibrosis with the PE-10 catheter only. This is consistent with the reported benefits of using smaller tubing (20). The loss of the sensitization-induced increases of CSEP amplitude with the PE-10 tubing highlights the sensitivity of our method and confirms the drawback of PE-10 tubing, particularly when it is used in an electrophysiological study.
A further observation of this study was that the CSEP amplitudes decreased when the osmolality of the injected solution was more than 320 mOsm. At approximately 640 mOsm, the CSEP amplitudes were decreased by half (a perfect linear decrease), and a lower plateau value (approximately 40% of control) was reached with tonicities between 1300 and 2000 mOsm. This can be partly explained by the depression of central nervous system function induced by intrathecal hypertonic solutions (21). DMSO is a popular solvent for drugs even though it induces neural side effects. Aggressive behavior of animals after intrathecal DMSO delivery was reported in the literature (10,11), but it was ignored when the biphasic responses of formalin were still elicited behaviorally (13,22). We found, using CSEPs, that DMSO markedly impaired the formalin-induced sensitized signals and decreased the signal-to-noise ratio of the method although it was diluted with 50% cyclodextrin. The loss of CSEP response might be due to the tonicity of the intrathecal solutions (>2000 mOsm), low pH (4.6), and the neural side effect itself. It is necessary to point out that DMSO is not suitable as an intrathecal adjuvant for the delivery of drugs including the specific COX inhibitors.
Working with the thinner catheter PU-32 allowed us to use the CSEP method for analyzing the pharmacological effects of ketorolac. The intrathecal administration of ketorolac resulted in a dose-dependent inhibition of the formalin-induced CSEP response from 10 to 60 minutes, which is similar to findings from behavioral tests (9,23). A maximal effective dose (approximately 80%) inhibition in preventing central sensitization (10 µg), the 50% inhibitory dose (4.5 µg), and even a toxic dose (50 µg) were also defined. Further, by controlling the pH and the tonicity of the saline control solution, the ketorolac solutions, and various other solutions, we can ascribe the paradoxical effects of the toxic dose of ketorolac to the molecule itself and not to eventual pH or osmotic stresses that occur at the spinal level. It seems that our refined model is a reliable tool for studying the pharmacological effects of antinociceptive drugs on central sensitization.
In conclusion, the CSEP/formalin model provides a reliable tool to objectively measure formalin-induced central sensitization in unanesthetized rats when the testing conditions are precisely defined. In the context of pharmacological experiments with intrathecal drug delivery, only small PU 32-gauge intrathecal tubing offered a working resolution of the CSEP signal. DMSO is an unsuitable solvent for intrathecal delivery of drugs, particularly when the effect of antinociceptive drugs is being assessed.
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Acknowledgments |
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This work was supported by the Research Grant Program of the Society for Anesthesia and Resuscitation of Belgium (LS) and Grant RSTD-WB-03 from the Department of Defense. LS is supported by a Free University Brussels fellowship. MZ is supported by the Belgian Department of Defense.
The authors thank Fernand Vereecke and Willy Smets for their reliable technical support.
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