Intrathecal lidocaine elevates prostaglandin E 2 levels in cerebrospinal fluid: a microdialysis study in freely moving rats

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1 British Journal of Anaesthesia 101 (5): (2008) doi: /bja/aen243 Advance Access publication August 20, 2008 Intrathecal lidocaine elevates prostaglandin E 2 levels in cerebrospinal fluid: a microdialysis study in freely moving rats V. Umbrain 1, L. Shi 2, M.-H. Lauwers 1, I. Smolders 3, Y. Michotte 3 and F. Camu 1 1 Department of Anaesthesiology and Pain Medicine, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, Belgium. 2 J&J Pharmaceutical Research and Development, Beerse, Belgium. 3 Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium Corresponding author. vincent.umbrain@uzbrussel.be Background. In this study, we have investigated whether intrathecal (i.t.) lidocaine administration is accompanied with changes of cerebrospinal fluid (CSF) prostaglandin E 2 (PGE 2 ) levels. Methods. Rats were anaesthetized for i.t. implantation of a triple-lumen spinal loop dialysis catheter. CSF changes in PGE 2 after i.t. injection of saline, 400, or 1000 mg of lidocaine were measured. The impact of i.t. pretreatment with 5 mg MK801(N-methyl-D-aspartate glutamate antagonist) or 10 mg SC76309A (COX-2 inhibitor) was also investigated. CSF dialysates for measurement of PGE 2 were collected for 4 h. During the whole procedure, motor and sensory blocks were evaluated. A separate group receiving i.t. lidocaine 400 mg (without dialysate sampling) was assessed for mechanical (Von Frey) and radiant heat pain. Results. PGE 2 levels increased to 400% of baseline and remained elevated for min after i.t. lidocaine at both doses. Pretreatment with SC76309A and MK801 attenuated this increase. A 40 min period of enhanced pain response was observed after Von Frey filament stimulation during and after sensory and motor block recovery. Conclusions. I.T. lidocaine (400 or 1000 mg) increases PGE 2 levels in the CSF for min along with a transient period of mechanical hyperalgesia after sensory and motor block recovery. Br J Anaesth 2008; 101: Keywords: measurement techniques, microdialysis; pharmacology, lidocaine; pharmacology, prostaglandins Accepted for publication: June 20, 2008 Lidocaine has been used for spinal anaesthesia, acting mainly through inhibition of the voltage-gated sodium channel, for more than yr. But, its use for spinal anaesthesia has been reduced as it is implicated in the appearance of transient neurological symptoms (TNS) such as pain, sensory abnormalities in the lower back, buttock, or lower extremities, or both, which appear within a few hours until approximately 24 h after full recovery from an uneventful spinal anaesthesia. TNS pain intensity varies from light to severe. Remarkably, neurophysiological evaluation during TNS did not reveal abnormalities in somatosensory evoked potential, electromyography, or nerve conduction. 1 Nerve inflammation induced by intrathecal (i.t.) lidocaine injection has been reported 2 with magnetic resonance imaging, and a hypothesis of reversible inflammation induced by i.t. lidocaine with the potential to produce TNS has been proposed. 3 TNS was also interpreted as a sign of possible neurotoxicity of lidocaine, 4 but the exact mechanism is still unknown. Both glutamate and prostaglandin E 2 (PGE 2 ) levels in cerebrospinal fluid (CSF) may be considered as surrogates for assessing central hypersensitivity of the spinal cord. Increased glutamate levels were reported in different animal pain models suggesting their contribution in nociceptive synaptic processing of the central nervous sytem. 56 Alternatively, PGE 2 is a key mediator for peripheral and central pain sensitization. 78 Its direct application to the spinal cord yields mechanical allodynia and thermal hyperalgesia Increased PGE 2 levels were reported in CSF after surgery 15 or peripheral inflammation, or were related to abnormal pain hypersensitivity. 18 Spinal lidocaine administration was recently reported to increase CSF glutamate levels in the absence of peripheral nociceptive activation. 19 Its potential role in the induction This work was partly presented as an oral abstract at the Annual Neuroscience Meeting of Washington on November 14, # The Board of Management and Trustees of the British Journal of Anaesthesia All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 I.T. lidocaine elevates PGE 2 levels in CSF of TNS has also been discussed. 20 However, there is no literature reporting the possible involvement of PGE 2, although non-steroidal anti-inflammatory drugs are considered an optional treatment for TNS in the clinic arena. The objective of the present investigation was therefore to monitor the possible changes in CSF PGE 2 levels after i.t. lidocaine administration in a spinal cord microdialysis model in freely moving rats. We verified the time profile of CSF PGE 2 changes in vivo and the potential relationship between the motor and sensory blocks associated with i.t. lidocaine administration. PGE 2 release after i.t. lidocaine was further investigated with pretreatment of MK801, an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, or of SC76309A, a COX-2 enzyme inhibitor. Moreover, we observed the behavioural changes in response to mechanical and thermal stimuli after i.t. administration of lidocaine. Methods The Bioethical Committee for animal experimentation of the Vrije Universiteit Brussel approved the experimental protocol that complied with the guidelines for animal experimentation of the International Association for the Study of Pain and with the guidelines of the Belgian Ministry of Agriculture. Implantation of the i.t. triple loop microdialysis probe Male Wistar rats (300 g; B&K Universal Limited, UK) were anaesthetized with sodium pentobarbital (60 mg kg 21 ) for i.t. implantation of a triple-loop microdialysis probe (Marsil Scientific, San Diego, CA, USA). This probe consists of i.t tubing with attached inlet and outlet tubes including an active loop dialysis fibre. This triple-lumen catheter permits simultaneous acute i.t. drug delivery and chronic CSF dialysate sampling. The catheter was introduced via the atlanto-occipital membrane as described previously. 21 In short, the loop of the catheter was placed at the rostral margin of the lumbar enlargement and its free ends were externalized through the skin at the top of the skull. Surgery ended with a ml s.c. injection of buprenorphine (Temgesic w 0.3 mg ml 21, Schering-Plough, Brussels, Belgium) for postoperative analgesia. Microdialysis experiment After surgery, the rats were allowed to recover for 5 days. Rats showing neurological dysfunction were discarded. The rats were placed in a microdialysis cage (Freely Moving System BAS/Microdialysis, West Lafayette, IN, USA) in the microdialysis room the evening before the experiment to enable them to acclimatize to their new surrounding. On the day of the experiment, the dialysis probes were connected to a microdialysis pump (CMA 100, CMA/ Microdialysis, Stockholm, Sweden) and perfused with a modified Ringer s solution (NaCl 147 mm, KCl 4 mm, and CaCl mm) at a flow rate of 7.5 ml min 21 for at least 60 min. Lidocaine investigations Baseline measurements were followed by either a 20 ml (Lido 400 mg) or a ml (Lido 1000 mg) i.t. injection of preservative-free lidocaine (Linisol w 2% pro injection, B.Braun, Melsungen, Germany). The control group received ml saline (Saline) i.t. All solutions were injected manually by bolus injection at a rate of approximately 10 ml 40s 21. CSF dialysates after i.t. injection were sampled at 10 min intervals for the first hour and at 30 min for a further 3 h. All samples were collected on ice and stored at 708C for subsequent analysis. MK801 or SC76309A administration before lidocaine In two separate sets of rats, we injected i.t. 10 ml ofeither the NMDA receptor antagonist dizocilpine (5 mg) (MK-801, Sigma w, Germany) or the water-soluble COX-2 enzyme inhibitor SC76309A (10 mg) (Pfizer w, NY, USA) dissolved in saline after 1 h baseline sampling. The doses were chosen based on the previous in vivo studies with MK or COX-2 enzyme inhibitors. 23 A second i.t. injection consisting of 20 ml of lidocaine (Lido 400 mg) was given 10 min later. Dialysate samples were collected at 10 min intervals during the first hour after MK801 or SC76309 and at 30 min intervalsfortheremaining3hoftheexperiment. Assay of CSF PGE 2 concentrations The concentration of PGE 2 in the microdialysate samples was quantified with a commercially available Correlate-EIA PGE 2 (competitive immunoassay) kit in accordance with manufacturer s protocol (Assay Design, Inc., USA). The concentration of PGE 2 was calculated from the measured optical density by means of four-parameter logistic regression. A standard curve was constructed between 39.4 and00pgml 21. Behavioural analysis Sensory and motor function testing Sensory and motor function tests were made immediately before spinal injection, then every 3 min after i.t. injection for the following 45 min. Sensory function was evaluated by seeking an aversive response to pinprick stimulation with a 23-gauge needle, progressing from sacral to cervical dermatomes. The sensory function score was assessed using a three-point grading scale: 2, normal; 1, diminished response is present; and 0, no response is present. Hind-limb motor function was assessed using a fivepoint grading scale proposed by Drummond and Moore: 24 4, normal motor function; 3, ability to draw legs under 717

3 Umbrain et al. body and hop, but not normally; 2, some lower-extremity function with good antigravity strength, but inability to draw legs under body; 1, poor lower-extremity motor function, weak antigravity movement only; and 0, paraplegic with no lower-extremity motor function. Von Frey and radiant heat testing Rats were subjected to Von Frey and radiant heat tests 25 before and after i.t injection of 400 mg lidocaine. For the Von Frey test, unrestricted rats were placed in a clear plastic chamber on an elevated wire grid and allowed to acclimatize. Von Frey monofilament fibres with forces of g (Stoelting w, IL, USA) were applied in ascending force order to the frontal portion of the plantar hind paw of the injected side until the animal withdrew its paw. The withdrawal threshold was determined as the lowest force (withdrawal threshold in grams) that evoked a clear withdrawal response at least twice in 10 applications. Thermal sensitivity was assessed using an infrared noxious heat stimulus. In short, animals were placed in a clear plexiglass box ( cm) with a dry glass floor and allowed to acclimatize for 15 min or until exploratory behaviour ceased. A focused beam of radiant heat at a constant temperature of 468C and a wavelength of nm was applied to the plantar surface of the paw. The hind paw withdrawal latency (s) to this stimulus was tested. The device has an automatic cut-off at 22 s to avoid the risk of thermal injury to the skin. Baseline values of either the heat withdraw latency time (s) or the Von Frey mechanical withdraw latency (g) were taken 60, 40, and 20 min before administration of 20 ml (400 mg) i.t lidocaine. Further changes were investigated at 20, 40, 60, 90, 120, 1, or 180 min after injection. Verification of probe positioning The animals were killed after each experiment with an overdose of pentobarbital. That part of the spinal cord containing the i.t. catheter membrane was dissected and the position confirmed by injecting methylene blue. Data analysis Data were analysed with SPSS 12 for Windows (SPSS w Lead Tools LT, Inc.). To evaluate the effects of lidocaine injection, we averaged PGE 2 baseline responses and set this average to 100%. Drug effects were expressed as per cent change of baseline values. PGE 2 data are presented as per cent change (SEM). Statistical significance of any differences was accepted at P,0.05. Tests used for analysing the within-group differences of the PGE 2 data were repeated-measures general linear model with post hoc analysis using the Bonferroni corrected test at each time interval. Group differences of PGE 2 between Lido 1000 mg, Lido 400 mg, saline, MK801, and SC76309A pretreatment were analysed using one-way analysis of variance. Mann Whitney test was performed to detect the between-group differences at fixed time intervals and also at selected intervals. Kruskal Wallis test was used to assess the between-group differences for the sensory and motor block data. Wilcoxon test was used to assess the within-group differences of blocks at the different intervals and of mechanical pain and radiant heat stimulation. Results Thirty-six rats without procedural neurological sequelae were selected for lidocaine microdialysis (n¼8 per group, 400 and 1000 mg lidocaine and saline control) and the MK801 or SC76309A (n¼6 per group) investigations. An additional 11 rats were included for Von Frey and radiant heat testing. Baseline levels Baseline CSF dialysate PGE 2 concentrations were 112 (6) pg ml 21 in all 36 rats tested [mean (SE)]. As the individual baseline value varied from one rat to another ( pg ml 21 ), we opted to express our results in percentages of the mean baseline for further analysis. Changes in CSF PGE 2 concentrations PGE 2 concentrations after i.t. injection of saline did not change. In both Lido 400 mg and Lido 1000 mg groups, spinal PGE 2 levels increased reaching a peak value of approximately 400% at 20 min (Fig. 1). PGE 2 concentrations gradually decreased to baseline values within the following 90 and 120 min in Lido 400 mg and Lido 1000 mg groups, respectively. No significant differences were observed in PGE 2 responses when comparing these two groups. PGE 2 % of baseline Time (min) l.t. injection Lido 1000 mg Lido 400 mg Saline Fig 1 PGE 2 concentrations in CSF dialysates after i.t. injection of saline ( ml), 400 mg lidocaine (20 ml), or 1000 mg lidocaine ( ml). Data are reported as mean (SEM) per cent change of baseline. P,0.05 vs baseline for Lido 1000 mg; P,0.05 for Lido 400 mg. 718

4 I.T. lidocaine elevates PGE 2 levels in CSF PGE 2 % of baseline l.t. MK801 l.t. Lido Lido 400 MK801 Lido Radiant heat test and Von Frey test After Von Frey filaments stimulation, the mechanical withdraw latency (g) was decreased in the lidocaine 400 mg group at 20, 40, and 60 min compared with the saline group (Fig. 4A). After radiant heat application, the lidocaine 400 mg group had a shorter withdraw latency time compared with the saline group at 40 min (11 s compared with 15 s) (Fig. 4B) Time (min) Fig 2 Effect of pretreatment with i.t. MK801 (5 mg) before i.t lidocaine 400 mg on dialysate CSF PGE 2 concentrations. Data are reported as mean (SEM) per cent change of baseline. P,0.05 vs baseline. Peak CSF PGE 2 concentrations were attenuated after pretreatment with MK801 (2%) (Fig. 2) or SC76309A (260%) (Fig. 3) compared with the lidocaine 400 mg group (400%). PGE 2 levels after pretreatment with SC76309A tended to be lower than after pretreatment with MK801. Behavioural testing Sensory and motor function tests Sensory blocks after i.t. injection and before total recovery lasted 19 min in the lidocaine 400 mg and 23 min in the lidocaine 1000 mg groups, respectively. Rats began to move after 10 min, but the effects lasted 23 min in the lidocaine 400 mg and 26 min in the lidocaine 1000 mg groups before total recovery (data not shown). In both lidocaine groups, spinal PGE 2 increases clearly outlasted the duration of sensory and motor block. PGE 2 % of baseline l.t. Lido l.t. SC76309A Lido 400 SC7639 Lido Time (min) 180 Fig 3 Effect of pretreatment with i.t. SC76309A (10 mg) before i.t. lidocaine 400 mg on dialysate CSF PGE 2 concentrations. Data are reported as mean (SEM) per cent change of baseline. P,0.05 vs baseline. Discussion We have demonstrated that i.t. lidocaine in rats is accompanied by a min MK801 and SC76309A sensitive increase in CSF PGE 2 levels. In addition, there was a transient period of mechanical hyperalgesia and increased sensitivity to radiant heat during the recovery period. Lidocaine-mediated increases in PGE 2 initially coincided with the onset of lidocaine-induced motor and sensory block, but the PGE 2 elevation outlived both sensory and motor block duration, implicating no direct causal relationship. Increased CSF glutamate has previously been observed after i.t. administration of several local anaesthetics, including lidocaine. To further elucidate the consequences of this elevation, we focused on a possible downstream mechanism of action, and in particular on its relationship to PGE 2 release. We found that i.t. lidocaine induces the activation of dorsal horn neuronal circuitry in which PGE 2 is implicated. A possible explanation linking glutamate and PGE 2 is as follows: glutamate released by i.t. lidocaine injection induces a postsynaptic depolarization leading indirectly to an increase in intracellular calcium, which in turn results in the activation of a number of intracellular enzymes, including phospholipase A 2 (PLA 2 ). PLA 2 activation then induces an increase in cytosolic arachidonic acid, which will enter the cyclooxygenase cascade leading to the synthesis of a variety of prostaglandins that gain access to the extracellular space. Prostanoids then affect presynaptic prostanoid E receptors that further increase intracellular calcium in sensory afferents and depolarize dorsal horn neurones and increase spinal excitability 8. In agreement with this view, we found that i.t. MK801 decreased CSF PGE 2 levels. Another explanation is that i.t. lidocaine may directly increase PGE 2. It is possible that PGE 2 increases could be the consequence of a direct biochemical effect of lidocaine on the dorsal horn neuronal cell membrane, as recently suggested 27 with a cell culture investigation, 28 which demonstrated increased calcium levels after lidocaine (.5 mg ml 21 ) administration. The calcium increase and its intensity were dose-related lasting 5 min with 300 mg lidocaine and being sustained for more than 60 min with 10 mg lidocaine. Johnson and colleagues 29 speculated that the observed calcium increase might initiate a period of enhanced electrical responsiveness by the neurone and cause hyperalgesia or transient nerve irritation. Alternatively, this calcium 719

5 Umbrain et al. A 60 Lidocaine 400 mg Saline Mechanical withdraw latency (g) B Thermal withdraw latency (s) Baseline 20 min 40 min 60 min 90 min 120 min 1 min 180 min 0 Baseline 20 min 40 min 60 min 90 min 120 min 1 min 180 min Fig 4 Radiant heat sensitivity and Von Frey testing after i.t. lidocaine 400 mg. Data are reported as mean (SEM) per cent change of baseline. P,0.05 vs baseline. increase might induce COX-2 m-rna transcription 30 and subsequently PGE 2 increases in CSF. Prostanoid induction is observed when the integrity of the cell membrane is jeopardized, as in cases of inflammation or trauma affecting the cell. 31 The amphipathic character of local anaesthetics affecting cell membrane lipid bi-layer permeability 32 when given i.t. might explain the PGE 2 increases. Recent reports showing a direct effect of lidocaine on the cell membrane of spinal cells and ending with a direct induction of prostanoids is therefore plausible. Whether the source of this lidocaine-induced spinal prostanoid induction relies on neuronal or glial expression could not be established in the present study. In agreement with this, we showed that the water-soluble COX-2 enzyme inhibitor SC76309A given before i.t. lidocaine attenuated PGE 2 release. Both pretreatments attenuated PGE 2 release when given before i.t. lidocaine. On the contrary, the lack of complete block of the PGE 2 may be related to COX-1 up-regulation. Interestingly, the Von Frey and to a lesser extent the radiant heat test demonstrated that rats had a transient increased mechanical and thermal hyperalgesia sensitivity after i.t. injection. Together with the observed transient increases of PGE 2 values, this demonstrates that i.t. lidocaine induces a temporary state of spinal cord sensitization. 720

6 I.T. lidocaine elevates PGE 2 levels in CSF We believe that these surprising changes of spinal PGE 2 levels after i.t. injection of lidocaine merit further investigations. The observed changes might simply reflect the reaction of the spinal cord to a foreign chemical substance such as our preservative-free lidocaine. But alternatively, i.t lidocaine may trigger a lidocaine-induced physicochemical reaction. As these changes were not observed after i.t. saline, we could exclude injection pressure and volumerelated changes. In vitro lidocaine produces conduction failure, membrane damage and loss of membrane potential, enzyme leakage, intracellular calcium release, growth cone collapse, and neurone degeneration and cell death. Recently, in rat dorsal root ganglion cell lines, lidocaine has been shown to produce a dose-dependent sodium-channel-independent mitochondrial dysfunction leading to the induction of apoptopic pathways. 33 We speculate that the observed PGE 2 increases in the CSF in our study reflect a lidocaine-induced effect on the spinal cell. PGE 2 increases could be an initiating signal of the effect of lidocaine on the spinal cell before it further affects the mitochondria of glial cells, astrocytes, or neurones. The previously established role of PGE 2 in the CSF as a central pain sensitizer and the observed short-lived dose-independent changes in PGE 2 levels, together with our observed behavioural changes suggest a relationship with the TNS observed in the clinical arena after i.t. lidocaine anaesthesia. However, the observed phenomena in rats were of shorter duration. Clinical confirmation with other species/gender studies is therefore suggested before human extrapolation. Our data contrast with the current knowledge in the field, as some authors report that local anaesthetics inhibit NMDA receptors. For instance, lidocaine inhibits NMDA receptor signalling, but these investigations were obtained either in vitro with recombinant human NMDA-receptors in Xenopus laevis oocytes or using ventral root potential recordings in hemisected spinal cord preparations. Therefore, they might not be applicable to this in vivo study in rat spinal cord. Also, in contrast to our findings is a recent report 36 where i.t. lidocaine reversed tactile allodynia after an established spinal nerve injury. It was speculated that lidocaine down-regulated prostagladin (PG) systems either by inhibition of the production or the release of PGs or by EP1 receptor inhibition. In contrast to their investigation, our rats were not hypersensitive before lidocaine administration, which may explain the differences. One shortcoming of our study is that we only investigated two doses of lidocaine. One small prospective study reported that TNS incidence after lidocaine spinal anaesthesia did not change after the modest lidocaine dose reduction from 75 to 60 mg. 37 Similarly, diluting spinal lidocaine from 5% to 2% 38 or from 2% to 0.5% 39 also failed to alter the incidence of TNS. In our study, however, we observed a prolonged duration of spinal PGE 2 increases in the lidocaine 1000 mg group. This may imply that the observed PGE 2 changes are dose-dependent. Further studies with other lidocaine doses therefore seem justified before linking spinal PGE 2 increases to TNS. Funding Fonds voor Wetenschappelijk Onderzoek (FWO). Acknowledgements We thank Professor Martin Zizi of the Department of Neurophysiology (VUB) for allowing us to perform rat surgery and PGE 2 measurements in his department. I.S. is a postdoctoral research fellow of the FWO Vlaanderen. 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