Spinal Carbonic Anhydrase Contributes to Nociceptive Reflex

Anesthesiology 2003; 98:921–7 © 2003 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Spinal Carbonic Anhydrase Contributes to Nociceptive Reflex Enhancement by Midazolam, Pentobarbital, and Propofol Bing Wang, M.D., M.Sc.,* Naaznin Samanani, B.Sc.,† Sheldon H. Roth, Ph.D.,‡ David P. Archer, M.D., M.Sc.§ Background: Systemic administration of acetazolamide flex enhancement by pentobarbital may be due to exci- blocks nociceptive hyperreflexia induced by pentobarbital. The tatory effects of the drug that are mediated by bicarbon- authors assessed the effect of intrathecal carbonic anhydrase ate ion and dependent on carbonic anhydrase (CA). One inhibitors (CAIs) on nociceptive reflex enhancement by pentobarbital, propofol, and midazolam. of the pharmacological actions of pentobarbital is to Methods: Twenty-seven rats with chronic indwelling sub- prolong the open time of the ␥-aminobutyric acid type A Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/98/4/921/405881/0000542-200304000-00019.pdf by guest on 02 October 2021 arachnoid catheters were studied. Nociceptive paw reflex la- 8 (GABAA) receptor ionophore. Neuronal excitation by tency (PWL) for paw withdrawal from radiant heat was measured activation of GABAA receptors has been described in in forelimbs and hind limbs. Measurements were obtained under central synapses.9–11 Briefly, the mechanisms for the control conditions, 15 min after lumbar intrathecal injection of 12 10 ␮l artificial cerebrospinal fluid containing the CAIs acetazol- latter phenomenon proposed by Kaila and Staley et 13 Ϫ Ϫ13,14 amide or ethoxyzolamide, and during the 55 min after intra- al. involve an anion shift from Cl to HCO3 ions. peritoneal injection of three sedative drugs: 30 mg/kg pento- This shift is thought to be caused by a prolongation of barbital, 50 mg/kg propofol, or 1.9 mg/kg midazolam. the open time of the GABAA ionophore either by high Results: Control values of PWL averaged 10.9 ؎ 1.5 s in the 13 local concentrations of GABA or by drugs such as the ؍ ؎ forelimbs and 11.1 1.6 s in the hind limbs (P 0.18). Intra- 15 Ϫ thecal injection of 50 ␮M ethoxyzolamide reduced PWL by 8% barbiturates. By catalyzing the conversion of HCO3 to -CO2, CA plays an essential role in the excitatory mech ;(0.01 ؍ and 4% in the forelimbs and hind limbs, respectively (P all other CAI injections had no effect on PWL. Following anes- 13 anisms of GABAA receptor activation. thetic injection, PWL in the forelimbs was reduced by approx- In the current study, we sought further evidence for a imately 35–40% of control values; in the hind limbs, CAI treat- role for CA in anesthetic-induced nociceptive hyperre- ment decreased the PWL reduction to 8–16% for pentobarbital (P < 0.001), 30–32% for propofol (P < 0.02), and 9–16% for flexia. The current study focused on two objectives: (1) midazolam (P < 0.001). The hind limb reduction of hyperre- to examine the role of spinal CA on nociceptive hyper- flexia by CAI was less for propofol than for midazolam or reflexia induced by pentobarbital; and (2) to compare pentobarbital (P < 0.002). the effects of intrathecal CAI on nociceptive hyperre- Conclusion: Spinal carbonic anhydrase contributes to noci- flexia induced by pentobarbital with that observed with ceptive hyperreflexia induced by pentobarbital and midazolam and to a lesser extent with propofol. These findings are consis- two other drugs, propofol and midazolam. tent with a role for carbonic anhydrase in nociceptive signal enhancement by these drugs. Materials and Methods LOW concentrations of many general anesthetics de- The study protocols, approved by the Faculty of Med- crease the threshold for perception of painful stimula- icine Animal Care Committee (University of Calgary, 1–3 tion in humans and the threshold for nociceptive Calgary, Alberta, Canada), were designed to comply with 4–6 withdrawal reflexes in rats. We previously reported the guidelines of the International Association for the that pentobarbital-induced nociceptive hyperreflexia in Study of Pain and the Canadian Council for Animal Care. rats can be blocked by systemic administration of acetazolamide (a carbonic anhydrase inhibitor [CAI]), amilo- Chronic Intrathecal Catheter Preparation ride (which blocks cellular hydrogen ion transport), and Male Sprague-Dawley rats (weight, 300–500 g) were 7 inhibitors of nitric oxide synthase. On the basis of the instrumented with chronic indwelling intrathecal cathe- 7 latter findings, we have proposed that nociceptive re- ters, with the distal catheter ports located in the lumbar region as previously described.16,17 Briefly, under gen- * Assistant Clinical Professor, † Research Assistant, § Associate Professor, De- eral anesthesia with halothane (2–3% in oxygen), rats partment of Anesthesiology, ‡ Professor, Departments of Anesthesiology and were positioned prone in a stereotactic frame. A length Pharmacology and Therapeutics, University of Calgary. of polyethylene-10 catheter (Becton Dickinson Co., Par- Received from the Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. Submitted for publication August 1, 2002. Accepted for publication sippany, NJ) was inserted through an incision in the December 9, 2002. Supported by the Canadian Anesthesiologists Society–Abbott atlantooccipital membrane to a position of 8 cm caudal Laboratories, Toronto, Ontario, Canada, and the Canadian Institutes for Health Research, Ottawa, Ontario, Canada. Presented in part at the annual meeting of to the cisterna magna at the level of the lumbar subarach- the American Society of Anesthesiologists, New Orleans, Louisiana, October 15, noid space and secured with dental cement. Postopera- 2001. Address reprint requests to Dr. Archer: Department of Anesthesiology, Foot- tively, animals free from motor deficits were allowed to hills Medical Center, 1403 29th Street Northwest, Calgary, Alberta, Canada, recover from surgery for a week before testing. Animals T2N 2T9. Address electronic mail to: david.archer@calgaryhealthregion.ca. Indi- vidual article reprints may be purchased through the Journal Web site, were housed in groups of two or three, with free access www.anesthesiology.org. to food and water and a 12-h light–dark cycle. Each Anesthesiology, V 98, No 4, Apr 2003 921 922 WANG ET AL. Table 1. Paw Withdrawal Latencies: Effects of Limb Selection and Intrathecal Carbonic Anhydrase Inhibitor Injection Condition n Forelimb Hindlimb P Control 76 10.9 Ϯ 1.5 11.1 Ϯ 1.6 0.18 Intrathecal CAI Control CAI P Control CAI P 20 ␮M acetazolamide 24 10.8 Ϯ 1.8 10.7 Ϯ 1.5 0.99 10.4 Ϯ 1.4 10.5 Ϯ 1.5 0.76 50 ␮M ethoxyzolamide 20 11.8 Ϯ 1.4 10.8 Ϯ 0.9 0.01 11.1 Ϯ 1.3 10.6 Ϯ 1.1 0.01 Data are shown as mean (s) Ϯ SD. P values for paired t test. CAI ϭ carbonic anhydrase inhibitor. animal was used for up to three experiments with a rest drawal latencies can be determined within 0.1 s. Paw of at least 1 week between each experiment. Correct withdrawal latencies in untreated animals range from Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/98/4/921/405881/0000542-200304000-00019.pdf by guest on 02 October 2021 location of catheters was confirmed by injection of 9–12 s, with a cutoff time of 14 s. Measurements were methylene blue dye at postmortem examination. made sequentially in the four limbs at each measurement time described in the next section. Study Hypotheses and Design Each rat was allowed to acclimatize in the test cham- The studies were designed to test three hypotheses. ber until it was resting quietly, which usually took 10–15 First, we sought evidence that intrathecal pretreatment min. After control measurements of PWL were recorded, with CAI would block the nociceptive reflex enhance- the intrathecal catheters were injected with 10 ␮l artifi- ment associated with systemic anesthetic administration. cial cerebrospinal fluid (aCSF), either plain or containing Second, by administering the CAI into the lumbar cere- acetazolamide or ethoxyzolamide. Fifteen minutes after brospinal fluid (CSF), we examined whether the block of intrathecal drug injection, animals received an intraperi- nociceptive reflex enhancement would occur in the toneal injection of one of the three sedative drugs: pen- hind limbs but not the forelimbs, suggesting a drug tobarbital (30 mg/kg), propofol (50 mg/kg), or midazo- action localized to the lumbar cord or nerves. Finally, we lam (1.9 mg/kg). The effects of sedative drug injection in evaluated whether pretreatment with intrathecal CAI each animal were represented by the mean value of the produced a similar degree of blockade of the hind limb paw withdrawal latency and the mean sedation score hyperreflexia in animals sedated with pentobarbital, mi- calculated from measurements made 5, 15, 25, 35, 45, dazolam, and propofol. In testing the first hypothesis, and 55 min after the intraperitoneal injection. For each the role of CA was evaluated as suggested by Maren18 animal, one mean value for the PWL following intraperi- using two CAIs, acetazolamide and ethoxyzolamide. toneal drug injection was calculated by averaging the Dose–response relations were evaluated in concentra- measurements obtained during the 55 min after intra- tion ranges that are thought to be specific for CAI with peritoneal drug injection. For comparisons between few if any other effects.18 Acetazolamide was used in drugs, the individual PWL measurements were ex- concentrations of 0.2, 2, 20, and 200 ␮M, and ethoxyzo- pressed as a percentage of the predrug injection value in lamide was used in concentrations of 5 and 50 ␮M. each animal and averaged over the 55-min postinjection time. Paw Withdrawal Reflex Latency The observer, blinded to the nature of the intrathecal Sedation drug injected, measured paw withdrawal latency (PWL) After each measurement of PWL, the observer deter- in the forelimbs and hind limbs using a custom-made mined the presence or absence of the righting reflex and Hargreaves box19 as previously described.7 The testing assigned a sedation score.

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