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Central and peripheral analgesic properties of local anesthetics : effects of lidocaine on thalamic neurons… Schwarz, Stephan 2002

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Central and Peripheral Analgesic Properties of Local Anesthetics Effects of Lidocaine on Thalamic Neurons and Efficacy of Ropivacaine in Femoral 3-in-1 Nerve Blockade by STEPHAN SCHWARZ M . D . (Ar^tliche Prufung), Georg-August-Universitat Gottingen, 1995 Dr. med., Georg-August-Universitat Gottingen, 1998  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in < T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Pharmacology & Therapeutics)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A March 2002 © Stephan Schwarz, 2002  In  presenting this  degree at the  thesis in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  f ^ ^ t o ^ /  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  /W-S^  ~ZOO^_  j  K'  S. S C H W A R Z i i  Abstract  This thesis is dedicated to the pharmacology & therapeutics of local anesthetics, with a specific focus on their analgesic properties. In the first section, laboratory studies emphasize central mechanisms of the prototype agent, lidocaine, whereas clinical studies in the second section investigate the analgesic efficacy of the recently introduced aminoamide, ropivacaine, when administered peripherally for nerve blockade in knee surgery. The specific objective of the laboratory studies was to define the concentrationdependent effects of lidocaine on the membrane properties and excitability of neurons in the ventral posterior lateral thalamic nucleus (VPL), a major somatosensory and nociceptive relay station that plays a central role in pain states. Lidocaine produces central analgesia and sedation when present in the systemic circulation at low concentrations, and evidence implicates V P L neurons in these actions. Differential interference contrast infrared (DIC-IR) videomicroscopy-guided whole-cell patch clamp techniques were used to record from V P L neurons in rat brain slice preparations. Low, analgesic lidocaine concentrations (10 fiM) significantly decreased neuronal input resistance (ft), which shunted action potentials, increased current thresholds, and reduced tonic firing. The effects were not associated with the classic signs of N a conductance blockade. The +  G A B A A receptor antagonist, bicuculline, had no effect on the lidocaine-induced shunt. Higher lidocaine concentrations (> 300 u M ; clinically CNS-toxic) did not decrease Ri but reversibly unmasked high threshold C a  2 +  spikes (HTSs), susceptible to blockade by C d . 2 +  S. S C H W A R Z iii Extracellular QX-314, a quaternary lidocaine analogue, increased rather than decreased Ri. Similarly, neither procainamide nor bupivacaine reduced Ri. In summary, these studies identified novel actions of lidocaine in the thalamus, distinct from the classic effects on Na  +  conductance. L o w concentrations produced a shunting type of inhibition, likely a  result of interaction with an intracellular target that does not involve G A B A A receptors. These findings serve as an attractive and plausible mechanism for the systemic analgesic and sedative actions of lidocaine in vivo and may provide a basis for the development of novel, selective, and effective agents for the treatment of acute and chronic pain. The observation of an unmasking of high threshold C a  2 +  spikes is in contrast to previous  studies in other tissues showing that lidocaine blocks C a  2 +  conductances, and is of  potential significance for the mechanisms of local anesthetic C N S toxicity. The purpose of the clinical studies was to test i f postoperative pain control in patients undergoing arthroscopic anterior cruciate ligament reconstruction (ACLR) under general anesthesia is improved by addition of a preincisional femoral 3-in-l block with ropivacaine 0.2% to standard intra-articular instillation at the end of surgery. In a prospective, randomized, controlled, double-blind trial (RCT), 44 patients scheduled for inpatient A C L R were studied. Prior to surgery, the treatment group (» = 22) received a femoral 3-in-l block with 40 ml of ropivacaine 0.2%, augmented by additional periincisional infiltrations (20 ml) at the end of the procedure. The control group [n — 22) received saline 0.9% instead of ropivacaine. A l l patients received an intra-articular instillation with 30 ml of ropivacaine 0.2% at the end of surgery. The primary efficacy variable was 24 h morphine consumption postoperatively standardized by weight,  S. S C H W A R Z iv administered intravenously via a patient-controlled analgesia (PCA) pump. There was no significant difference between both groups in the primary efficacy variable. N o difference was found in visual analog scale pain scores, adverse events, or vital signs. More patients in the treatment group did not require any morphine than in the control group, but this difference was not statistically significant. In conclusion, the clinical studies demonstrated no significant effect in an R C T of a femoral 3-in-l block with ropivacaine 0.2% on postoperative analgesic consumption, compared to intra-articular instillation alone, in patients undergoing A C L R under general anesthesia.  S. S C H W A R Z V  Table of Contents  Abstract  ii  Table of Contents  v  List of Tables  ix  List of Figures  x  Acknowledgements  xii  Scope of the Topic and Approach to Research  1  S E C T I O N I: L A B O R A T O R Y S T U D I E S  5  1.1 Introduction  •  6  1.1.1 Overall aim and specific objective 1.1.2 Background. 1.2 Materials and Methods 1.2.1 Ethics  6 6 16 16  1.2.2 Preparation of brain slices  16  1.2.3 Electrophysiological recordings  17  1.2.4 Drugs 1.2.5 Statistical analyses 1.3 Results 1.3.1 Basting membrane properties of VPL neurons  21 22 24 24  1.3.2 Postnatal development of resting membrane properties 1.3.3 Current-voltage relationships  26 28  S. S C H W A R Z v i 1.3.4 Active membrane properties: tonic and burstfiring  29  1.3.5 Effects of lidocaine on resting membrane properties  30  1.3.6 Effects of lidocaine on current-voltage relationships  33  1.3.7 Effects of lidocaine on tonic repetitive firing  35  .  1.3.8 Effects of lidocaine on burstfiring and low threshold spikes  39  1.3.9 Effects of lidocaine on high threshold spikes  39  1.3.10 Effects of GABA.A receptor blockade by bicuculline on lidocaine actions  48  1.3.11 Effects of other local anesthetic agents on membrane properties  51  1.3.11.1 QX-314  ...53  1.3.11.2 Procainamide  56  1.3.11.3 Bupivacaine  62  1.4 Discussion  :  66  1.4.1 Shunting inhibition: a novel effect of low lidocaine concentrations in the CNS 1.4.1.1 Clinical relevance of concentrations  67  1.4.1.2 Previous investigations  68  1.4.1.3 Physiological significance  69  1.4.1.4 Effects of G A B A A receptor blockade  :  74  1.4.1.5 Molecular and cellular actions of lidocaine  76  1.4.1.6 Lidocaine and E E G spindle waves  80  1.4.2 Effects of high lidocaine concentrations 1.4.2.1 Effects on high threshold C a  2 +  spikes  66  81 84  1.4.3 Effects ofprocainamide on membrane properties  86  1.4.4 Limitations andfuture outlook  87  1.4.5 Summary and conclusions  88  S. S C H W A R Z v i i S E C T I O N II: C L I N I C A L S T U D I E S  89  II. 1 Introduction  90  II. 1.1 Overall aim and specific objective  90  II. 1.2 Background  90  II. 1.3 Femoral 3-in-1 block  91  II. 1.4 Choice of local anesthetic.  95  II. 1.5 Primary hypothesis.....  96  11.2 Materials and Methods  97  11.2.1 Ethics 11.2.2 Study design  • ;  97 97  II. 2.3 Inclusion criteria, exclusion criteria, and patient withdrawal. II. 2.4 Treatment interventions II.2.5 Outcome variables II. 2.6 Study drugs and blinding. II.2.7 Statistical analyses 11.3 Results.... II. 3.1 Demographics II. 3.2 Primary efficacy variable  98 99 102 102 ....103 105 105. 106  11.3.3 Secondary efficacy variables II. 3.4 Adverse events 11.4 Discussion II.4.1 Summary and conclusions  108 110 112 119  S. S C H W A R Z viii Overall Conclusion and Closing Remarks  121  Appendix I  125  ,  Appendix II Appendix III  127 '.  131  Abbreviations  132  Bibliography  139  V  S. S C H W A R Z ix  List of Tables  Table 1 Overview of pain syndromes responsive to systemic lidocaine  9  Table 2 Postnatal development of resting membrane properties  28  Table 3 Effects of lidocaine on input resistance and membrane time constant  33  Table 4 Overview of reported molecular and cellular actions of local anesthetics  77  Table 5 Patient demographics, preoperative vital signs, and surgical data  105  Table 6 Timing of treatment interventions  106  Table 7 Postoperative analgesic consumption (intention-to-treat analysis)  107  Table 8 Number of patients not requiring morphine postoperatively  107  Table 9 Postoperative morphine consumption (patients requiring morphine)  107  Table 10 Incidence of common adverse events  Ill  S. S C H W A R Z x  List of Figures Figure 1 Structural formula of lidocaine  7  Figure 2 The path of sensation according to Descartes  10  Figure 3 Location of the thalamus in the mammalian brain  15  Figure 4 Location of the V P L nucleus in the thalamus  15  Figure 5 D I C - I R images of a V P L neuron  21  Figure 6 Resting membrane properties: frequency distributions, and correlations  25  Figure 7 Postnatal development of resting membrane properties  27  Figure 8 Effects of lidocaine on input resistance, membrane time constant, and resting membrane potential....  32  Figure 9 Effects of lidocaine on current-voltage relationships  34  Figure 10 Effects of a low concentration of lidocaine on tonic repetitive firing  36  Figure 11 Effects of a low concentration of lidocaine on injected current  37  Figure 12 Effects of high concentrations of lidocaine on tonic repetitive firing  38  Figure 13 L o w threshold spikes and the effects of lidocaine on burst  40  firing  Figure 14 Time-dependent effects of a high concentration of lidocaine on repetitive spikes  42  Figure 15 Lidocaine unmasks high threshold spikes not blocked by T T X  43  Figure 16 Effects of lidocaine on high threshold spikes in the presence of T T X  45  Figure 17 Effects of C a  2 +  free extracellular solution and C d  spikes unmasked by lidocaine  2 +  on the high threshold 47  S. S C H W A R Z xi Figure 18 Effects of bicuculline on the decrease in tonic firing due to lidocaine  50  Figure 19 Effects of bicuculline on the decrease in input resistance due to lidocaine... 52 Figure 20 Structural formula of QX-314  54  Figure 21 Effects of QX-314 on membrane properties  55  Figure 22 Structural formula of procainamide  57  Figure 23 Effects of procainamide on membrane properties  58  Figure 24 Effects of procainamide on tonic and burst firing  61  Figure 25 Structural formula of bupivacaine  63  Figure 26 Effects of bupivacaine on input resistance  65  Figure 27 Sensory innervation of the lower extremities  92  Figure 28 Structural formula of ropivacaine  96  Figure 29 Treatment interventions  101  Figure 30 Postoperative morphine consumption (patients with no morphine consumption excluded)  108  Figure 31 Postoperative V A S pain scores at rest over time  109  Figure 32 Postoperative blood pressure over time  109  Figure 33 Postoperative heart rate over time  110  S. S C H W A R Z xii  Acknowledgements  Research is always a collaborative effort. I would not have been able to prepare this combined laboratory and clinical research thesis without the many individuals who directly or indirectly gifted me with their inspiration, ideas, ideals, or other forms of help.  First and foremost, I would like to thank my two co-supervisors, Drs. Bernard A . MacLeod and Ernie Puil, for their unfailing support, patience, extraordinary generosity, and outstanding supervision of this thesis, during which they always treated me as a colleague and taught me by providing space and opportunity for independent inquiry and critical thought.  I thank Dr. Morley C. Sutter for serving on my supervisory committee, where he offered his invaluable input and experience in bridging clinical and laboratory research and provided a role model for being a physician-scientist.  I am grateful to Drs. Frank Tennigkeit and Craig Ries for die generous sharing of their expertise in the laboratory and countless discussions, Stefan Reinker for his collaboration in the studies, on procainamide, and Dr. Richard Neuman, Prof. Walter Stiihmer, and my father, D r . Dietrich W. F. Schwarz, for their valuable advice and constructive comments.  Lance Corey, Jens Haeusser, and Patricia Rust provided technical aid and help with computer-related problems, and Silvia F u assisted with some of the experiments.  I owe my gratitude to D r . Michael J. A . Walker for his kind gifts of procainamide and Barolo, time for discussions about statistical analyses, and his sense of humour. D r . Ryan J. Huxtable repeatedly provided financial support for attending the meetings of the Western Pharmacology Society as well as general scholarly guidance, including lessons in English poetry and his perspective on Vinum Columbium Britannicum.  S. S C H W A R Z  xiii  T h e clinical section o f these studies w o u l d not have been possible without the i n v o l v e d nursing staff at U B C H o s p i t a l ( O R suite, P A C U ,  and wards  1C/D),  for  whose  collaboration and patience I am grateful. Furthermore, I w o u l d like to thank L u i Franciosi, D e b b i e C a n n o n , and D r s . A l l e n Bain, Chris Bates, Pat M c C o n k e y , Ross D a v i d s o n , B r i a n D a y , R o b Eger, N a o m i K r o n i t z , D a v i d L e a , M i k e M o u l t , B o b Purdy, B i l l Regan, and T h e o Weideman for their invaluable help.  T h e clinical studies were conducted i n co-operation w i t h Astra Pharma Inc., Canada (Mississauga, O N ) , o f w h o m I w o u l d like to representatively acknowledge R r i s t a N e v i n , Heather Burt, and V a d n a Sime. Sergio Escobedo was helpful i n statistical issues and provided assistance with some analyses using S A S software.  T h e U B C Department o f Anesthesia was instrumental i n the completion o f this thesis through extraordinary and ongoing support. In particular, I w o u l d like to express my sincerest gratitude to D r s . Jamie Renwick, Eleanor Reimer, and D a v i d R. B e v a n (now i n T o r o n t o ) , without w h o m I w o u l d not be where I am today. M y thanks also go to D r . D a v i d Parsons for his kind financial aid for presenting at the Sixth International Conference on Molecular and Basic Mechanisms ofAnesthesia. O n a very special note, I am indebted to D r . Rod  McTaggart for his mentorship i n Anesthesiology, thoughtful comments o n the  manuscript for publication o f the clinical section o f this thesis, and his friendship.  F u n d i n g for this w o r k was provided by a Pharmacia & U p j o h n Special University A w a r d , a British C o l u m b i a Medical Services Foundation ( B C M S F )  Fellowship, a M e d i c a l  Research C o u n c i l o f Canada ( M R C ) Fellowship, and the generosity o f my supervisors through their grants from the M R C / C I H R (Canadian Institutes o f Health Research) for the laboratory studies (Dr. E r n i e Puil) and Astra Pharma Inc., Canada for the clinical studies (Dr. Bernard A . M a c L e o d ) .  T h e most important support o f all came from L i n d a , whose unfailing and tireless patience, tolerance, and love words cannot express.  S. S C H W A R Z 1  S c o p e o f the T o p i c a n d A p p r o a c h to R e s e a r c h  Modern anesthesiology is a discipline that faces tremendous challenges in a time of rapid progress in other medical specialties and our continuously changing society. In order to be able to provide excellence in patient care for a growing elderly and multiethnic population, there is an imminent need to develop novel pharmacological and therapeutic strategies that are effective and overcome the shortcomings, pitfalls, and untoward events of those available at present. A major therapeutic challenge today remains the mastering of acute and chronic pain. Pain is the most frequent symptom encountered in clinical medicine and among the most common causes of disability in industrialized nations. Economically, acute and chronic pain burdens our society more than heart disease and cancer combined, a fact often escaping our awareness (Thompson 1997). More than one out of three adults in North America report pain symptoms (Bonica 1980, 1990). In the United States alone, 70 million people have chronic pain, many of whom are permanently disabled (Osterweis et al. 1987). A prominent study in the United Kingdom estimated that as many as 46.5% of the general population suffer from chronic pain (Elliott et al. 1999). If poorly treated or left untreated, even acute pain (e.g., acute postoperative pain) can quickly turn into chronic pain (Katz et al. 1996). Historically, there has been a sharp discrepancy between the overall magnitude and scope of the problem on the one hand and the degree of progress and innovation in analgesic pharmacology & therapeutics on the other hand. The level of funding and overall support for pain research has been a fraction of that designated for cardiovascular disease, cancer, or other areas of health  S. S C H W A R Z  2  care. O n l y very recently, i n 1996, the U . S . N a t i o n a l Institutes o f Health ( N I H ) established a P a i n Research C o n s o r t i u m under its aegis, some 23 years after J o h n Joseph (originally b o r n as " G i o v a n n i Guiseppe") B o n i c a spearheaded  the creation o f an international  society dedicated to pain research, w h i c h became the International A s s o c i a t i o n o f the Study o f P a i n (IASP). Whereas the Canadian Institutes o f Health Research ( C I H R ) thus far lack an institute or consortium specifically committed to pain or anesthesia research, their co-sponsorship o f the recently introduced " D r . R o n a l d M e l z a c k P a i n Research A w a r d " and one (1) postdoctoral fellowship i n "clinical or basic science as it relates to Anesthesia, Perioperative Medicine, P a i n Medicine a n d / o r  Critical Care M e d i c i n e "  illustrates the growing awareness o f the needs i n this area. Nevertheless, i n the "disease funding table" o f the N I H for the fiscal years 2000—2002, neither pain nor anesthesia are listed as a research initiative/program o f interest, i n contrast to such areas as chronic fatigue syndrome, diagnostic radiology, or topical microbicides ( N I H 2001). T h e most effective analgesic agents today continue to be opioids, non-steroidal anti-inflammatory drugs ( N S A I D s ) , and local anesthetics. A l l o f those have been i n use by ancient societies for as long as 5000 years i n the form o f extracts from o p i u m poppy (Papaver somniferum), w i l l o w  bark  (Salix spp.),  and  coca leaves  (Erythroxylon cocci),  respectively (for reviews o f the history o f analgesia, see Brandt et al. 1997; H a m i l t o n and Baskett 1999, 2000). T h e efficacy o f newer groups o f agents, particularly i n the treatment o f chronic pain, is often modest at best, to the disappointment o f b o t h patients and health care providers (Ashburn and Staats 1999). W h i l e detailed knowledge n o w exists o n the mechanisms that underlie analgesia due to peripheral nerve blockade by local  S. S C H W A R Z 3  anesthetics (reviewed i n Butterworth and Sttichartz 1990), h o w and where precisely drugs such as opioids and N S A I D s act to produce acute pain relief following systemic application  is,  despite  significant  progress  (reviewed  in  Pasternak  1993;  M c C o r m a c k 1994; Cashman 1996; Porreca etal. 1997; Ingram 2000), far from being fully understood. Similarly, the specific sites and mechanisms o f action i n the brain for drugs to alleviate chronic pain remain largely unknown. Hence, there is a vital need to shed more light o n the cellular and molecular actions o f existing agents o n the one hand, and to identify novel therapeutic strategies that are effective, specific, and well tolerated o n the other hand. I n order to successfully face these challenges and to be able to ensure the quality o f life o f our patients, the logical consequence is an integrated research approach that bridges laboratory and clinical science. T h e present thesis has been prepared w i t h the motivation to pursue  this path. W i t h the specific focus o n the pharmacology &  therapeutics o f the analgesic properties o f local anesthetics, this dissertation explores specific questions about both cellular pharmacological action and clinical therapeutic efficacy o f this group o f agents. In approaching the research questions, an effort was made to address diverse aspects within the wide spectrum o f this topic. A t one end o f this spectrum, the first section o f the thesis focuses o n laboratory studies o n the possible mechanisms for the central analgesic actions o f a well-established local anesthetic agent, i.e., lidocaine. A t the other end, the second section concentrates o n clinical investigation to study the analgesic efficacy o f a newly introduced agent, ropivacaine, w h e n administered peripherally for nerve plexus blockade i n knee surgery. Together, the results provide new knowledge about the diverse properties o f local anesthetics and illustrate the needs for  S. S C H W A R Z 4 and benefits o f conducting research i n this area. It is hoped that this w o r k may serve as a small step toward the final goal: to provide the best possible care for our patients today and i n the future.  )  S. S C H W A R Z 5  SECTION I: LABORATORY STUDIES  Effects of lidocaine on membrane properties and excitability of neurons in the ventral posterior lateral thalamic nucleus of the rat  in vitro  Results from this section o f the thesis have appeared i n the following publications: Schwarz S K W , P u i l E : Analgesic and sedative concentrations o f lignocaine shunt tonic and burst firing i n thalamocortical neurones. Br] Pharmacol 1998, 124: 1633-1642. Schwarz S K W , P u i l E : Lidocaine produces a shunt i n thalamocortical unaffected by G A B A A receptor blockade. Neurosci Lett 1999, 269: 2 5 - 2 8 . Schwarz S K W , P u i l E : Lidocaine unmasks high threshold C a neurons. Soc Neurosci Abstr 1999, 25: 723.  2 +  neurons,  spikes i n thalamocortical  Reinker S, Schwarz S K W , P u i l E : Effects o f procainamide o n membrane properties o f thalamocortical neurons. Proc West Pharmacol Soc2001, 44: 89-92.  S. S C H W A R Z 6  1.1 Introduction  1.1.1 Overall aim and specific objective T h i s section o f the thesis focuses o n laboratory studies o n central actions o f the local anesthetic, lidocaine. T h e overall aim o f these studies was to identify the mechanisms by w h i c h lidocaine produces central analgesia and alterations i n consciousness, including sedation and anesthesia. T h e specific objective here was to define the concentrationdependent effects o f lidocaine o n the membrane properties and excitability o f neurons i n a part o f the brain that is crucial for such effects — the thalamus.  1.1.2 Background Lidocaine is the most frequently employed local anesthetic i n clinical medicine and represents the prototype agent o f this class o f drugs (Figure 1). Its well-known peripheral actions to b l o c k the propagation o f action potentials along nerve fibres are widely exploited for surgical regional anesthesia. W h e n present i n the systemic circulation as a result o f local absorption or intravascular injection, however, local anesthetics exhibit effects that imply additional central action (Koppanyi 1962; Garfield and G u g i n o 1987; Biella and Sotgiu 1993). Such effects reflect central nervous system ( C N S ) toxicity as well as valuable therapeutic uses. T h e most frequently observed symptoms o f lidocaine's C N S toxicity are sedation, drowsiness, and alterations i n sensorium (Covino 1987; Wallace et al. 1997b). These are associated with low, "subconvulsive" plasma concentrations, typically  S. S C H W A R Z 7 between 1 and 5 u g / m l . T h e same range o f l o w concentrations produce the therapeutic effects o f lidocaine (see below and de J o n g 1994 for review).  Aromatic head  Amide linkage  Amine tail  Figure 1 Structural formula o f lidocaine  It has been well k n o w n for decades that local anesthetics have central analgesic properties (Peterson 1955). Administered systemically at l o w doses, lidocaine produces analgesia i n acute (inflammatory) pain syndromes such as acute postoperative pain. Lidocaine and other local anesthetics have been used i n the maintenance o f general anesthesia, w i t h analgesic effects comparable to those o f nitrous oxide (de J o n g 1994). M o r e o v e r and perhaps more importantly, systemic lidocaine is effective i n the treatment o f chronic pain syndromes such as neuropathic and central pain, w h i c h are notoriously difficult to treat and largely resistant to conventional analgesic therapy. There even exists evidence that the analgesia resulting from peripheral nerve blockade used for the management o f chronic pain is at least i n part due to the central analgesic properties o f local anesthetics (Arner et al. 1990). In addition to lidocaine, its derivative, mexiletine, administered orally, has been used successfully i n the treatment o f chronic neuropathic  S. S C H W A R Z  8  pain syndromes (Dejgard et al. 1988; Chabal et al. 1992). T h e magnitude o f pain relief from an intravenous (IV) lidocaine infusion provides a tool to predict the response to long-term mexiletine treatment (Galer et al. 1996). Cancer pain has been alleviated w i t h lidocaine and also mexiletine (Sloan et al. 1999). Finally, lidocaine is one o f the few identified agents that are useful for the relief o f tinnitus, w h i c h is viewed by many as a form o f chronic pain (Simpson and Davies 1999; Huxtable 2000). Table 1 provides an overview w i t h corresponding references o f pain syndromes reported to be responsive to systemic lidocaine. F o r neuropathic pain, the effective lidocaine plasma concentrations range between 0.62 and 5.0 u g / m l i n humans (Mao and C h e n 2000). In a recent study o n nonneuropathic  pain  in  human  volunteers,  lidocaine  selectively  reduced  secondary  hyperalgesia i n a heat/capsaicin sensitization m o d e l (Dirks et al. 2000). A n o t h e r group independently made similar observations development  and reported substantial reductions i n the  o f secondary hyperalgesia at lidocaine plasma concentrations  between  1.5 and 3 p g / m l (Holthusen et al. 2000). A n i m a l studies confirm these observations; for example, i n rodent models o f both acute nociception and chronic pain, lidocaine administered systemically produces antinociception (Courteix et al. 1994; R i g o n and Takahashi 1996).  S. S C H W A R Z 9 Table 1 O v e r v i e w o f pain syndromes responsive to systemic lidocaine Acute pain Acute postoperative pain (Bartlett and Hutaserani 1961; Cassuto et al. 1985) And-GD2-ganglioside therapy-induced pain (Wallace et al. 1997a) Chronic pain Neuropathic pain (Boas et al. 1982; Marchettini et al. 1992; Ferrante et al. 1996) Diabetic neuropathy (Kastrup et al. 1987; Bach et al. 1990) Postherpetic neuralgia (Rowbotham et al. 1991) Central pain (Edmondson et al. 1993) Phantom limb pain (Lee and Donovan 1995) Complex regional pain syndrome (Wallace et al. 2000) Migraine (Burke 1989; Lewis 1992;* Maizels et al. 1996t) Chronic daily headache (Kaube et al. 1994; Hand and Stark 2000) Fibromyalgia (Sorensen et al. 1995) Cancer pain (Brose and Cousins 1991) Tinnitus* (Melding et al. 1978; Martin and Colman 1980; Israel et al. 1982; den Hartigh et al. 1993) Rectal administration (Xyloproct® suppositories, not marketed in North America; contain 60 mg lidocaine each) tlntranasal administration *Sce page 8  > I n contrast  to their peripheral effects,  relatively little is k n o w n about  the  mechanisms and sites o f the central actions o f local anesthetics. E x i s t i n g w o r k o n this topic has focused o n the spinal cord and left supraspinal actions largely unexplored (Mao and C h e n 2000). However, several lines o f evidence implicate the thalamus as a crucial supraspinal site i n the C N S where local anesthetics such as lidocaine act to produce their systemic analgesic and sedative effects. Descartes already emphasized the significance o f supraspinal centres i n nociceptive signalling more than three centuries ago, albeit i n terms that since have been modified and updated (Figure 2). C h r o n i c pain per se was first described as the "thalamic syndrome" i n 1906 by Dejerine and Roussy i n their classic paper (1906). Recent studies emphasize the central role o f the thalamus (Figure 3)  S. S C H W A R Z  10  and signal transmission by thalamocortical neurons in acute and chronic pain as well as in analgesia (Di Piero etal. 1991; Jeanmonod etal. 1993; Rosen etal. 1994; Peyron etal. 1998; Brunton and Charpak 1998; Apkarian etal. 2000; for reviews, see Albe-Fessard et al. 1985; Willis 1997; L e n and Dougherty 1997). 2  Figure 2 The path of sensation according to Descartes "Iffor examplefire(A.) comes near the foot (B), the minute particles of thisfire,which as you know move with great velocity, have the power to set in motion the spot on the skin of the foot which they touch, and by this means pulling upon the delicate thread CC, which is attached to the spot of the skin, they open up at the same instant the pore, d, e, against which the delicate thread ends, just as by pulling at one end of a rope one makes to strike at the same instant a bell which hangs at the other end." (1664) (Adapted from Melzack and W a l l 1965)  S. S C H W A R Z Thalamocortical neurons relay afferent  11  sensory and nociceptive signals and  encode stimulus intensity into firing frequency and spike patterns. T h e y are endowed with complex membrane electrical properties that serve to fulfill their physiological functions ( M c C o r m i c k 1992). In particular, they exhibit two distinct, voltage-dependent action potential firing modes — tonic repetitive firing at depolarized and burst firing at hyperpolarized membrane potentials (Deschenes et al. 1984; Jahnsen and Llinas 1984a) (cf. 1.3.4, 1.3.8, and 1.4.1.). Thalamocortical neurons contribute to the generation o f conscious states and are critical for the production o f the electroencephalographic ( E E G ) rhythms associated with the different states o f awareness and sleep, e.g., the slow-wave activity and spindles o f drowsiness, sedation, and n o n - R E M sleep (Steriade et al. 1990).* L o c a l anesthetics infused at subconvulsive doses produce spindling and increased slow wave (delta- and theta-) activity i n the E E G o f humans and other mammals, associated w i t h sedation and reduced responsiveness to noxious stimuli (Acheson et al. 195.6; E r i k s s o n and Persson 1966; W a g m a n et al. 1968; Sakabe et al. 197'A; Seo et al. 1982; Shibata et al. 1994). O n the other hand, abnormal thalamic burst firing occurs i n patients suffering from chronic pain syndromes (Lenz et al. 1987, 1989; Tsoukatos et al. 1997), i n w h o m lidocaine is particularly efficacious as a systemic analgesic (cf. Table 1).  *A remarkable clinical illustration o f the thalamus' central role i n the' generation o f spindles and drug-induced synchronized E E G activity is provided by the report o f a 53-ycar-old male with fatal familial insomnia (Lugaresi et al. 1986). In this patient, barbiturates and benzodiazepines failed to produce E E G spindles. Post mortem, the autopsy showed degeneration o f the anterior and dorsomedial thalamic nuclei.  S. S C H W A R Z 12 The ventral posterior lateral nucleus (Nucleus ventralis posterolateralis thalami, V P L ; Le Gros Clark 1930) (Figure 4), located in the ventrobasal complex (VJ3)* of the dorsal thalamus, is the major relay station for somatosensory signals and has a significant role in the transmission of nociceptive signals (reviewed in Jones 1985; Steriade et al. 1997). Specifically, V P L has been associated with the sensory and discriminatory aspects of acute pain perception (Head and Holmes 1911; Melzack and Casey 1968; Albe-Fessard et al. 1985; Lenz et al. 1994a; Apkarian and Shi 1994). Consistent with the former, V P L neurons precisely encode the intensity, location, and time of peripheral stimuli and project  specifically and somatotopically to the primary (SI) and secondary (SH)  somatosensory cortex (Mountcastle et al. 1963; Poggio and Mountcastle 1963; Kenshalo et al. 1980; Casey and Morrow 1983). V P L neurons receive ascending inputs from the dorsal column pathways via the medial lemniscus, and the spinothalamic tract (Ma et al. 1987; Ferrington et al. 1988; Al-Chaer et al. 1996). Additional inputs include descending feedback projections from the cortex, inhibitory GABAergic fibres from the thalamic reticular nucleus, and modulatory afferents (cholinergic),  from the brainstem & basal forebrain  Locus coeruleus (noradrenergic),  raphe  nuclei  (serotoninergic),  and  *VB (also known as ventral posterior nucleus, V P ) is comprised o f V P L and its medial neighbour, V P M (Jones 1985, pp. 47-48), which receives sensory input from the face. In humans, according to old terminology, V P L corresponds to the Nuclei ventro-caudales posterior externus et anterior externus (V.c.p.c. & V.c.a.e.; Hassler Jones 1997), and, combined with V P M , to the vcntrocaudal nucleus (Vc) (Hirai and Jones 1989).  1959;  S. S C H W A R Z  13  hypothalamic tuberomammillary nucleus (histaminergic) ( M c C o r m i c k 1992; Steriade et al. 1997). I n contrast to medial and thalamic nuclei, the receptive fields o f V P L neurons are relatively small and somatotopically organized (for review, see Chudler and B o n i c a 2001). T h e different body regions are represented within V P L mediolaterally by parasaggital lamellae (Lenz and Dougherty 1997). Consistent w i t h this are the observations  that  neurons responding to noxious stimuli are found i n every region o f the V B complex i n the rat (Guilbaud et al. 1980) and the V P L nucleus i n the monkey ( M o r r o w and Casey 1992), and that such neurons may constitute over 50% o f V P L ' s neuronal population (data also from monkey; C h u n g et al. 1986). T h e single-unit and network properties o f nociceptive neurons i n the V P L nucleus have recently been characterized (Apkarian and Shi 1994; A p k a r i a n etal. 2000). Classic evidence for the role o f V P L i n chronic pain comes from lesion studies, where destruction o f tissue i n the lateral thalamus  has been successfully used  for  treatment o f intractable chronic pain syndromes (Talairach et al. 1949; Ramamurthi and Kalyanaraman  1966;  Richardson  1967).  Patients  with  chronic  pain  demonstrate  pathological changes i n V P L , w h i c h i n addition to the abnormal burst firing mentioned earlier includes alterations i n the somatotopic organization (Lenz et al. 1994b). T h e pivotal involvement o f V P L neurons i n chronic pain also is illustrated by in vivo studies o n nociceptive transmission i n rat models o f polyarthritic chronic pain and mechanical hyperalgesia following peripheral nerve injury (Gautron and G u i l b a u d 1982; M i k i et al. 2000).  S. S C H W A R Z 14 A wide variety of analgesic agents from different pharmacological classes have been shown to depress the nociceptive activity of neurons in V B thalamus (Guilbaud et al. 1982; Braga etal. 1985; Carlsson et al. 1988). This section of the thesis considers the possibility that lidocaine could produce its central analgesic effects as well as alterations in sensorium and conscious state at low, subconvulsive concentrations by actions on V P L neurons. However, the cellular effects of local anesthetics on thalamocortical neurons are unknown. Hence, the objective of these studies was to define the concentrationdependent effects of lidocaine on the membrane properties and excitability of V P L neurons in vitro. Here, a novel action of lidocaine in the C N S is reported. Some preliminary results of these studies have been subject of a previous dissertation (Schwarz 1998).  S. S C H W A R Z 15  Figure 3 L o c a t i o n o f the thalamus i n the mammalian brain Shown are schematic illustrations of median-saggital sections through the cerebri of different mammals. The thalamus, also known as the "gateway to scnsorium", emerges as the central (hatched) structure in the diencephalon. Note that the images are scaled differently for the different species. (Modified from Sherman and Guillery 2001)  Figure 4 L o c a t i o n o f the V P L nucleus i n the thalamus (A) Bright-field image of a coronal section of rat thalamus containing the ventrobasal complex with the V P L nucleus laterally and V P M medially (adapted & modified from Desbois and Villanueva 2001). (B) Schematic illustration of a corresponding section from squirrel monkey {Saimiri sciureus). Illustrated is the location of nine neurons (black dots) that responded differentially to noxious mechanical stimulation of the skin in an awake animal (figure & data from Casey and Morrow 1983) (CL, central lateral nucleus; C M , central medial nucleus; L D , lateral dorsal nucleus; L P , lateral posterior nucleus; M D , mediodorsal nucleus; PF, parafascicular nucleus; PO, oral pulvinar nucleus [ = anterior pulvinar nucleus (Burton and Jones 1976)]; V L , ventral lateral nucleus; VPI, ventral posterior inferior nucleus).  S. S C H W A R Z  16  1.2 Materials and Methods  1.2.1 Ethics T h e results presented i n this section o f the thesis were obtained from  experiments  utilizing rat brain slice preparations (see below). T h e experimental p r o t o c o l was approved by the Committee o n A n i m a l Care o f T h e University o f British C o l u m b i a , w h i c h issued an  appropriate  Animal  Care  Certificate  (Protocol N u m b e r A95-027). A l l animal  experiments were conducted i n accordance with the guidelines by the Canadian C o u n c i l o n A n i m a l Care o n the ethical use o f animals ( C C A C 1993), and all efforts were made to minimize the suffering, and, wherever possible, the number o f animals used.  1.2.2 Preparation of brain slices Experiments were conducted with Sprague-Dawley rats aged between postnatal days 10 and 20 (P10-P20), with a majority o f the data obtained from animals aged P 1 4 {of. 1.3.2). T h e animals were decapitated under deep halothane (Wyeth-Ayerst Canada, Inc.; M o n t r e a l , Q C , Canada)  anesthesia.  T h e cerebrum was rapidly removed  and  submerged for 1 m i n i n cold (1-4 °C) artificial cerebrospinal fluid ( A C S F ) . T h e A C S F , prepared freshly o n each experimental day, contained (in m M ) : N a C l , 124; K C 1 , 4; KH2PO4, 1.25; C a C b , 2; M g C k , 2; NaHCC>3, 26; glucose, 10. T h e measured osmolarity was 310 m O s m (Advanced Digimatic Osmometer 3 D I I , A d v a n c e d Instruments,  Inc.,  N e e d h a m Heights, M A , U . S . A . ) ; saturation with 9 5 % O a / 5 % C O 2 for > 1 h yielded a p H  o f 7.4 (measured at 20-26 ° C with a p H meter m o d e l 05669-20, Cole-Parmer Instrument  S. S C H W A R Z  17  Company, V e r n o n Hills, I L , U . S . A . ) . T h e brain was dissected into two blocks, each containing the thalamic tissue o f one cerebral hemisphere. After fixation o f a tissue b l o c k on  a Teflon  stage w i t h  Boucherville, Quebec, 300-500  cyanoacrylate  Canada)  adhesive  (Accu-Flo™  super  and submersion i n cold A C S F ,  glue, Lepage,  coronal slices o f  u m thickness and containing the V P L were prepared w i t h a Vibroslice  (Campden Instruments L t d . , L o n d o n , England). I n the course o f the experimental w o r k for this thesis, the A C S F for above steps was replaced by a solution containing (in m M ) : sucrose, 125; K C 1 , 2.5; N a H P 0 , 1.25; CaCfe, 2; M g C l , 2; N a H C O s , 26; glucose, 25 2  4  2  (Forsythe 1994). Immediately after cutting, the slices were incubated i n normal A C S F , w h i c h , except for some experiments performed i n the initial phase o f the w o r k for this thesis, was heated to 37 ° C (monitored with a Tele-Thermometer, Y e l l o w Springs Instrument C o . , Y e l l o w Springs, O H , U . S . A ) w i t h a Standard Heatblock ( V W R Scientific Products, West Chester, P A , U . S . A . ) . P r i o r to recording, the slices were incubated for at least 1 h i n the A C S F and continuously aerated w i t h 9 5 % Oil5%  CO2.  1.2.3 Electrophysiological recordings Whole-cell patch-clamp recordings (Hamill et al. 1981) were conducted i n the bridge mode using an A x o c l a m p 2 A or 2 B amplifier ( A x o n Instruments, Inc., Foster City, C A , U . S . A . ) , allowing direct current ( D C ) injection to simulate afferent stimulation and test membrane  properties.  The  recording electrodes  were  prepared  from thin-walled  borosilicate glass (World Precision Instruments, Inc., Sarasota, F L , U . S . A . ) using a P P - 8 3 two-stage electrode puller (Narishige Scientific Instrument L a b . , T o k y o , Japan). They  S. S C H W A R Z were  filled  w i t h a solution containing (in m M ) K-gluconate, 140; ethylene  bis((3-aminoethyl ether)-N,N,N',N'-tetraacetic  (disodium  salt),  glycol-  acid, 10; K C L 5; N a C l , 4; M g C b , 3;  N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic adenosine-5'-triphosphate  18  3;  acid]  (free  acid),  10;  guanosine-5'-triphosphate  CaCb,  1;  (sodium  salt), 0.3. T h e solution was titrated to p H 7.3 w i t h 10% gluconic acid and K O H . T h e approximate concentrations o f free C a  2 +  and M g  2 +  ions, calculated for p H 7.3 and 25 ° C  w i t h the use o f W i n M A X C Software, version 1.60 (Chris Patton, Stanford University, H o p k i n s M a r i n e Station, Pacific G r o v e , C A , U . S . A . ) , were 11.2 n M and 354 u M , respectively. T h e estimated electrode resistances were typically ~8 MQ. (range, 6—11 M O ) . F o r recording, the slices were transferred into a submersion type chamber w i t h a volume o f 1.2 or 1.5 m l , covered w i t h polypropylene or n y l o n mesh for fixation, and continuously perfused w i t h oxygenated A C S F (95% 0 2 / 5 %  CO2) at a flow rate o f  1.5-2.5 m l / m i n (controlled by a M A S T E R F L E X ® C / L ™ pump, Cole-Parmer Instrument C o m p a n y , V e r n o n Hills, I L , U . S . A . ) . T h e temperature o f the perfusing m e d i u m i n the chamber was 22-27 ° C . T h e V P L nucleus was identified by aid o f a W i l d 5 M 5 A microscope  (Wild, Heerbrugg,  Switzerland) or a Zeiss A x i o s k o p F S (Carl Zeiss,  Gottingen, Germany) under transmitted light illumination. T h e atlas by Palkovits & Brownstein (1988) was used as a reference. E x c e p t for the initial phase o f experimentation, where whole-cell recording was carried out employing the " b l i n d " technique  (Blanton et al. 1989), the neurons for  recording were visualized using differential interference  contrast infrared  (DIC-IR)  videomicroscopy (Stuart et al. 1993; D o d t and Zieglgansberger 1994) (Figure 5). F o r this  S. S C H W A R Z  19  technique, the slices were transferred into a glass-bottom recording chamber m o u n t e d o n the headstage o f the Zeiss A x i o s k o p F S . T h e microscope was equipped with a high numerical aperture ( N A ) water immersion objective (Zeiss A c h r o p l a n 4 0 x / 0.75 W ; w o r k i n g distance, 1.9 mm), D I C optics, and a 0.9 N A condenser. T h e slices were illuminated w i t h a 12 V / 1 0 0 W halogen lamp powered by a 12 V / 1 0 0 W D C power supply (Carl Zeiss, I n c . / L u d l Electronic Products L t d . , Hawthorne, N Y , U . S . A . ) . Infrared light was obtained by placing a Schott R G 9 glass filter (Schott Glas, M a i n z , Germany;  Xmax = 780 nm) i n the light path. T h e images were recorded w i t h a Hamamatsu  C2400—07ER video camera system (Hamamatsu Photonics K . K . , Hamamatsu, Japan) and  displayed o n  a video monitor  (Black  and  White M o n i t o r  SSM-175,  Sony  Corporation, T o k y o , Japan). T h e electrodes were mounted o n the A x o c l a m p amplifier headstage and advanced w i t h a Nano-Stepper Type B (Scientific Precision Instruments  G m b H , Oppenheim,  Germany) or a low-drift three-dimensional water hydraulic micromanipulator m o d e l M H W - 3 (Narishige Scientific Instrument Lab., T o k y o , Japan). After submersion o f the electrode tip i n the A C S F , the measured potential difference was set to 0 m V w i t h a D C offset  adjustment.  Cell  membranes  were  ruptured  when  high  resistance  ("tight"/"gigaohm-") seals were achieved. After obtaining the whole-cell configuration, access resistance compensation was performed employing bridge balance  techniques.  M e m b r a n e potentials were corrected for a measured liquid junction potential o f 11 m V (cf. H u t c h e o n et al. 1996), and the offset potential measured o n withdrawal o f the electrode  from the neuron. D e p e n d i n g o n the experimental protocol, injection o f  S. S C H W A R Z  20  continuous positive or negative D C was used to manually clamp, hyper-, or depolarize the membrane potential (V ) m  via the A x o c l a m p ' s " D C Current C o m m a n d " . T h e data  were filtered at 10 k H z and recorded o n chart ( G o u l d B r u s h 220 Recorder or B r u s h Recorder M a r k 280, B r u s h Instruments, Cleveland, O h i o , U . S . A . ) , and, after conversion w i t h a L a b Master D M A 40 k H z analog/digital/analog converter (Scientific Solutions, Inc., Solon, O h i o , U . S . A . ) and p C L A M P software, version 5.5 ( A x o n Instruments, Inc., Foster City, C A , U . S . A . ) , o n the hard disk o f an I B M - c o m p a t i b l e personal  computer.  p C L A M P files were imported into C o r e l D R A W software (Corel Corporation, Ottawa, ON,  Canada)  for  creation  o f figures  depicting  experimental  data; for  clarity o f  presentation, spikes were truncated i n some figures. F o r recording o n video cassette ( S L - H F 750 super Beta hi-fi V i d e o Cassette Recorder, Sony Corporation, T o k y o , Japan), the  analog  signal was  digitally converted  by  a  44  k H z P C M - 1 Digital V C R -  Instrumentation Recorder A d a p t o r (Medical Systems Corp., Greenvale, N Y , . U . S . A . ) or a P C M - 5 0 1 E S Digital A u d i o Processor (Sony Corporation, T o k y o , Japan). T h e voltage traces were monitored on-line with a N i c o l e t 310 (Nicolet Instruments Corporation, M a d i s o n , W I , U . S . A . ) or K i k u s u i C O S 5020™ (Kikusui Electronics C o r p . , Y o k o h a m a , Japan) oscilloscope.  S. S C H W A R Z 21  A  B  Figure 5 D I C - I R images o f a V P L neuron Shown is a neuron in the V P L nucleus before (A) and during (B) approach with the recording electrode  for  establishment o f the whole-cell configuration o f the patch-clamp technique. T h e figure depicts photographs taken from the image o n a video monitor obtained by aid o f infrared differential contrast videomicrocopy using an Achroplan 40X/0.75 W water immersion objective (sec text).  1.2.4 Drugs Lidocaine hydrochloride was purchased  from Research Biochemicals International  (Natick, M A , U . S . A . ) . T h e powder was dissolved i n fresh concentrated  ACSF  to prepare a  stock solution o f 5 m M , stored i n aliquots o f 2.2 m l at —22 °C.  Procainamide hydrochloride, bupivacaine hydrochloride, and tetrodotoxin ( T T X ) were obtained from Sigma-Aldrich Canada L t d . (Mississauga, O N , Canada). Q X - 3 1 4 bromide  S. S C H W A R Z  22  was obtained from A l o m o n e Labs (Jerusalem, Israel). Stock solutions for procainamide, bupivacaine, and Q X - 3 1 4 were prepared i n a fashion similar to lidocaine. F r o m the citrate-buffered T T X , a 300 u M stock solution was prepared w i t h distilled water and stored i n 500 p i aliquots at —22 °C. Bicuculline methobromide was obtained from Precision Biochemicals, Inc. (Vancouver, B . C . , Canada) and dissolved i n distilled water to produce a 50 m M stock solution, w h i c h was also stored at —22 °C. P r i o r to application o f the agents, required aliquots were defrosted  and dissolved i n A C S F to obtain the  respective concentrations. D r u g applications i n the bath were performed by switching from  the  control perfusate  (normal A C S F )  to A C S F  containing a desired  drug  concentration. Unless specifically stated otherwise, reported results represent steady-state responses. Results are reported for one neuron per slice subject to drug application only.  1.2.5 Statistical analyses Statistical analyses were carried out with the use o f Student's t tests for comparisons o f two groups and testing for differences from a theoretical mean, and one-way analysis o f variance ( A N O V A ) for multisample analyses. A s post hoc tests, the B o n f e r r o n i test for pairwise multiple comparisons and the Dunnett test for comparisons to control were employed. Differences were considered significant at P < 0.05. A l l data are expressed as mean + S E M , n = sample size (number o f neurons), unless mentioned otherwise. Where feasible,  concentration-response  relationships  were  constructed  using  nonlinear  S. S C H W A R Z  23  regression analyses by means o f fitting the data to a four-parameter logistic equation* using the least sum-of-squares method. T h e data were analyzed using P r i s m version 2.01 and  3.02 software  (GraphPad, San Diego, C A , U . S . A . ) , Microsoft E x c e l 9 7 / 2 0 0 0  software (Microsoft Corporation, R e d m o n d , W A , U . S . A . ) , and the Clampan and Clampfit components o f p C L A M P software version 6 and 8 ( A x o n Instruments, Inc., Foster City, C A , U.S.A.).  Bottom + (Top - Bottom) V = J  7,———,,.„ j _l_ -jQ(logEC50-JC)  , H i l l slope  , where x = logarithm o f concentration and y = response.  J _)_ j Q (log E C s o - X )  H i l l slope  >  t>  F o r normalized data, the value for "Bottom" was fixed at  J  1.0 (100%), reflecting  f  the control (baseline) response.  S. S C H W A R Z 24  1.3 Results  1.3.1 Resting membrane properties of VPL neurons The results reported here are from recordings of 107 neurons in the V P L nucleus. A l l neurons accepted for analysis had overshooting action potentials and stable resting membrane potentials (V ) < —50 mV, lasting for up to 4 hours of recording. The neurons t  had a mean V of —68.0 ± 1 . 0 m V (n — 68), consistent with the results of previous t  investigations on rat V B neurons (cf. Ries and Puil 1999a,b). Input resistances (Ri), determined from the steady-state voltage displacements (AV ) of < —10 m V elicited by m  injection of hyperpolarizing current pulses of 500 ms duration, averaged 263 ± 21 M Q . The mean membrane time constant (x ; see Abbreviations for definition), estimated from m  single exponential fits to the AV , was 34.6 ± 2.0 ms. Input capacitances were calculated m  according to C\ — x / R and averaged 156 ± 8 pF. A l l data were normally distributed. m  Analyses of relative frequency distribution of these parameters were indicative of a uniform population of neurons (Figure 6, A—D), consistent with previous observations that the neuronal population in rodent dorsal thalamic nuclei is almost exclusively comprised of thalamocortical relay neurons and practically devoid of interneurons (Harris and  Hendrickson 1987; Steriade et al. 1997). There was an extremely significant  (P < 0.0001) positive correlation between Ri and % (r = 0.50; Figure 6E), indicating 2  m  behaviour o f the data in accordance with x  — R C N o strong correlation was found  m  between the calculated C\ (see above) and V (r = 0.06; Figure 6E). 2  t  S. S C H W A R Z 25  100  -40-  n  | 50'  • 0.06  -50-  75  r •  -60-| -70 H  r = 0.50 (P< 0.0001) 2  25  250  500  750  -80 -90  1000  0  100  Ri (MO)  200  300  400  500  Q (pF)  Figure 6 Resting membrane properties: frequency distributions and correlations ( A ) - ( D ) Relative frequency distribution o f resting membrane potential {V  [A]), input capacitance (Ci [B]), input  r  resistance (Ri [C]), and membrane time constant (x  m  5 ms.  \D]). B i n width for V , 2 m V ; G , 20 p F ; Ri, 50 Mil; and x ,  (E) A highly significant correlation was observed between Ri and x  correlation was found between Ci and V (r = 0.06; P = 0.04). 2  t  T  m  (r = 0.50; P < 0.0001). 2  m  (F) N o strong  S. S C H W A R Z  26  1.3.2 Postnatal development of resting membrane properties A s mentioned i n 1.2.2, the data presented i n this section o f the thesis largely was obtained from animals i n the age range o f P 1 0 - P 2 0 , with a majority o f the animals aged P14. W h i l e it was not a primary objective o f these studies to perform an in-depth investigation o f postnatal development o f the resting membrane properties o f V P L neurons,  the  available data offered the opportunity for a limited analysis within the above age range and comparison o f the results to the existing literature. Consistent w i t h previously published findings o n thalamocortical neurons i n V B (Perez Velazquez and Carlen 1996) and the medial geniculate body ( M G B ; Tennigkeit et al. 1998b), the input resistance o f the V P L neurons decreased i n the course o f postnatal development. T h i s decrease reached a plateau, such that the difference i n R i between animals aged P 1 4 and P I 5—20 was no longer statistically significant (Bonferroni's multiple comparison test following A N O V A , P > 0.05; Table 2). Input capacitance increased concomitantly, indicative o f an increase i n neuronal membrane surface area associated w i t h cell growth (Figure 7 A ) . Similar to the observations by Tennigkeit et al, there were small decreases i n membrane  time constant with age that d i d not reach statistical  significance (Figure 7B). There were no significant changes i n resting membrane potential w i t h increasing postnatal age (Figure 7C). T h i s observation i n V P L appears i n contrast to M G B but is likely explained by the fact that no animals < P 1 0 were studied here: i n MGB,  for example,  V  t  decreases with increasing age but reaches a plateau  - P 1 0 - P 1 2 (Tennigkeit etal. 1998b).  after  S. S C H W A R Z 27  500-  • 0.20; P <  0.0001  400-  Q.  O  300200100H  •  10  —i— 14  12  — i —  — i —  — i —  16  18  20  Age (d) B 10075-  E  ^ = 0.01  50-  25-  —  : ! 5 »*  . 1•  r  12  10  — i —  14  •• • — i —  16  — i —  20  18  Age (d) Age (d) 10  11  12  13  14  15  16  17  18  19  20  Figure 7 Postnatal development o f resting membrane properties  S. S C H W A R Z  28  Y  n  Table 2 Postnatal development o f resting membrane properties  Ri (Mn)  O (pF)  T (ms)  PI 0-13  389 ± 67  124 ± 1 2  41.6 ± 6 . 0  -68.7 ± 2 . 5  • 15  PH  253 ± 25  147 ± 1 1  33.7 ± 3 . 0  - 6 6 . 2 ± 1.6  29  P15-20  193 ± 2 4  196 ± 1 9  32.2 ± 2.5  -69.3 ± 1 . 4  22  0.0028  0.0052  0.22  0.36  P value*  r  m  (mV)  *One-way A N O V A  1.3.3 Current-voltage relationships Typically, the  I-V  relationships  (i.e., ohmic) at membrane  for the V P L neurons  were  approximately linear  potentials positive to — 9 0 m V (cf. Figure 9). A t more  hyperpolarized voltages, neurons  exhibited some inward (depolarizing) rectification  (cf. Jahnsen and Llinas 1984b; H u t c h e o n et al. 1996; Tennigkeit et al. 1996), consistent w i t h activation o f the inwardly rectifying K  +  current, Ini (Constanti and G a l v a n 1983;  Sutor and H a b l i t z 1993) and the hyperpolarization-activated m i x e d cationic current, Ih ( M c C o r m i c k and Pape 1990b; Pape 1996; Williams et al. 1997b). Similar to other wholecell investigations o f rat thalamocortical neurons (Puil et al. 1994; Tennigkeit et al. 1996; Ries and P u i l 1999a), neurons i n the present study did not routinely exhibit i n the hyperpolarizing  responses  the  depolarizing membrane  potential  "sags"  that  are  characteristic for Ih. Other investigators have observed sags i n intracellular recordings o f thalamocortical neurons  i n the cat and guinea pig ( M c C o r m i c k and Pape  1990b;  S. S C H W A R Z  29  P i r c h i o et al. 1997). Tennigkeit et al. (1996) demonstrated an unmasking o f sags i n rats by application o f B a , a selective blocker o f IIR (Travagli and Gillis 1994). It is possible i n 2 +  the present study that the gluconate i n the electrode (intracellular) solution (cf. 1.2.3) inhibited Jh (Velumian et al. 1997). However, some neurons d i d exhibit sags. Whereas the precise reasons for these discrepancies remain unclear, other factors likely play a role; these include differences  i n recording technique, the species investigated, and  the  developmental state o f the animals.  1.3.4 Active membrane properties: tonic and burst firing All  neurons  exhibited  the  voltage-dependent  firing  patterns  characteristic  for  thalamocortical relay neurons (cf. Deschenes et al. 1984; Jahnsen and Llinas 1984a). Injection o f suprathreshold depolarizing current pulses into neurons near rest elicited repetitive  tonic  firing  hyperpolarizing V  m  (blocked by  600  n M T T X ; cf. Figure  16), whereas  on  (typically to values between —80 and —90 m V ) by D C injection,  depolarization elicited burst firing o n top o f a l o w threshold spike (LTS), a slow envelope o f depolarization (see Figure 13B i n chapter 1.3.8). R e b o u n d L T S bursts also could be evoked from a V  m  near rest by hyperpolarizing current pulses (Figure 13A), k n o w n to de-  inactivate the underlying T-type C a  2 +  current, IT* (Jahnsen  and Llinas 1984a). A s  previously reported by others, the bursts consisted o f ~1—7 high frequency action  *Also known as low threshold- or low voltage-activated ( L V A ) C a  2 +  current  S. S C H W A R Z  30  potentials (here, ~> 80 H z ) that were blocked by T T X (not illustrated), indicating that they were mainly carried by N a . T h e L T S s themselves were not blocked by T T X but +  disappeared w h e n the  superfusing A C S F  contained zero C a , indicative o f their 2 +  generation by IT (cf. 1.3.9). T h e finding by M c C o r m i c k and Pape (1988) that L T S s are absent i n interneurons recorded from cat lateral geniculate nucleus ( L G N ) provided additional  indication that  the  neurons  recorded  here  (cf. 1.3.1,  page  24)  were  thalamocortical relay neurons.  1.3.5 Effects of lidocaine on resting membrane properties A p p l i c a t i o n o f lidocaine decreased the R i o f the V P L neurons i n a reversible manner (Figure 8 A ) . T h e time for an application to produce a steady-state response was typically ~ 5 m i n (range, 2.5—7 min). This effect exhibited a distinct, non-classical concentration dependence. Whereas the maximal decrease i n R i occurred at a l o w concentration (10 u M ; decrease i n R i up to 2 8 % o f control), the magnitude o f this effect decreased w i t h higher concentrations, and no reduction i n R i occurred at 300 pM—1 m M (Figure 8B). These multiphasic relationships implied the presence o f multiple actions o f lidocaine that affect R i i n an overlapping fashion. T h e data did not fit conventional concentrationresponse models (e.g., a four-parameter logistic equation, cf. 1.2.5) for construction o f a meaningful  classic sigmoid  administration  caused  curve. Concomitant with  reductions  in x  with  m  (Figure 8C). T h e m a x i m u m reduction i n x  m  the  a similar  effect  on  concentration  R i , lidocaine dependence  at 10 u M lidocaine was 3 4 % o f control. T h e  respective input capacitances, calculated according to C\ = x / R (cf. 1.3.1) were not m  S. S C H W A R Z  31  significantly different from the control values over the range from 0.6-600 u M (data not shown), indicating a primary effect o f lidocaine o n membrane conductance (of. Figure 8B and Figure 8C). T h e reversal potentials for the increased conductances (1/Ri), determined from current-voltage (I-V) curves (see 1.3.6), were typically between V  r  and ~ - 5 0 m V .  A summary o f the numerical data o n the concentration-dependent effects o f lidocaine o n Ri and X m is given i n Table 3. Lidocaine at concentrations < 10 u M d i d not produce consistent changes i n membrane potential except for 2 out o f 11 neurons where application o f 10 u M was associated w i t h depolarizations (7 and 9 m V , respectively). A t 30 or 100 u M , lidocaine depolarized 6 out o f 12 neurons (Figure 8 D ) . T h e time required to reach a maximal depolarization was typically 2 to 3 m i n . T h e depolarizing response observed at 30 u M ranged between 5 and 12 m V i n 2 out o f 6 neurons, and, at 100 u M , between 6 and 9 m V i n 4 out o f 6 neurons. Repolarization to resting values was seen usually after 4—5 m i n o n terminating the application. Higher concentrations (300 uM—1 m M ) again had no consistent effects o n V . t  N o hyperpolarizations were seen as a result o f lidocaine  application. In neurons where application o f T T X (600 n M ) completely abolished action potentials, lidocaine produced small decreases i n Ri that were reversible and associated w i t h variable changes i n V . t  F o r example, 10 u M lidocaine reduced Ri to a level o f  82.0 ± 5.8% o f c o n t r o l values (P = 0.04, n = 5).  S. S C H W A R Z 32  Figure 8 Effects o f lidocaine o n input resistance, membrane time constant, and resting membrane potential (A) A low concentration o f lidocaine (10 u M ; 8 min application) significantly decreased input resistance (Rj), evident as a decreased amplitude o f the steady-state voltage response to hyperpolarizing current injection (pulse duration, 500 ms). T h e magnitude o f the decrease in Rj in the neuron was 51% (400 MCI to 196 M Q ) . T h e effects were completely reversed after 10 min washout.  (B) & (C) Concentration-response curves for the effects o f lidocaine on  Ri and membrane time constant (x ; for each concentration, n = 4—11; cf. Table 3). Note the multiphasic character o f m  the relationships (see text). T h e maximum reduction in Rj occurred with 10 u M lidocaine. (D) Lidocaine application (100 uM) depolarized a V P L neuron from —69 m V to —64 m V . T h e downward deflections in the voltage trace represent responses to hyperpolarizing current pulses (-40 p A , 500 ms); the positive deflections represent rebound L T S bursts (cf. Figure 13). Near the peak response, V  m  was manually clamped at the control (V ) level through D C r  injection for assessment o f a change in input resistance. Close to full recovery was reached after 4 m i n o f washout.  S. S C H W A R Z  33  Table 3 Effects o f lidocaine o n input resistance and membrane time constant  [Lidocaine]  P value  R/  (% of control)  P value  n  (% of control)  0.6  81.1 ± 1 3 . 3  0.21  81.9 ± 1 6 . 4  0.32  6  1  82.6 + 17.0  0.35  81.0 ± 1 7 . 4  0.32  6  ' 3  80.6 ± 1 6 . 9  0.30  81.8 ± 2 3 . 7  0.48  6  10  70.5 ± 7 . 3  0.002  75.3 ± 8 . 3  0.01  11  30  76.6 ± 1 3 . 4  0.14  74.5 ± 6 . 5  0.01  6  100  72.4 ± 7 . 8  0.02  81.1 ± 6 . 9  0.04  6  300  86.1 ± 17.0  0.47  88.7 ± 9.2  0.31  4  600  9,8.2 ± 12.1  0.89  111.7 ± 1 1 . 2  0.34  7  1000  100.4 ± 14.8  0.98  88.7 ± 1 1 . 4  0.39  4  1.3.6 Effects of lidocaine on current-voltage relationships Figure 9 shows the effects o f lidocaine o n the I-V  curves and corresponding voltage  traces o f a representative neuron clamped at —61 m V with D C injection. A l o w concentration o f lidocaine (10 p M ) , w h i c h caused the greatest reduction i n Ri, markedly reduced the slope resistance over a wide voltage range (—125 m V to threshold or —50  m V ) . T h e values for V  m  where control I- V curves intersected w i t h those obtained  under lidocaine application (i.e., the reversal potential for the conductance changes;  V  c  not clamped) typically ranged between V and — 5 0 m V (cf. page 31). F u l l recovery from t  these effects usually was reached —10 m i n after washout w i t h n o r m a l A C S F . Lidocaine had no consistent effect o n the inward rectification o f the neurons (tf. 1.3.1).  S. S C H W A R Z 34  F i g u r e 9 Effects o f lidocaine o n current-voltage relationships (A) Application o f 10 u M lidocaine (cf. Figure 8) for 8 min produced a 72% decrease in Ri, evident as reduction in amplitude o f steady-state voltage responses to D C injection (pulse duration, 500 ms). Consistent with the decrease in Rj, lidocaine revcrsibly shunted rebound low threshold spike bursts (asterisk; see 1.3.8 & Figure 13). Recovery was observed after 10 min washout. differences (AV ) m  (B) I-V  relationships o f the same neuron. Steady-state  membrane potential  were measured at the end o f 500 ms current pulses (A/ ), injected in 10 p A steps (filled and open m  circle in A ) . Lidocaine produced a marked reduction in slope resistance over a wide voltage range (from — 8 5 to spike threshold).  mV  S. S C H W A R Z  35  1.3.7 Effects of lidocaine on tonic repetitive firing Lidocaine, applied at l o w concentrations associated w i t h a decrease i n Ri, shunted tonic repetitive firing o f action potentials elicited by depolarizing D C pulses, producing a marked reduction i n responsiveness to stimulation. Firing frequency (e.g., expressed as number o f spikes/500 ms pulse) and frequency accommodation decreased i n a reversible manner (Figure 10A). There were no concomitant elevations o f the threshold potential for firing or alterations i n spike configuration. T h e maximal rate o f rise determined as "greatest left slope" w i t h Clampfit software  (dV/dtmsa,  [Axon Instruments, Inc.,  Foster City, C A , U.S.A.]) o f spikes remained unchanged. F o r example, the sixth spike i n a train, very sensitive to high lidocaine concentrations i n the range k n o w n to block N a conductance (see below and Figure 12), had a d 7 / d /  m a x  +  o f 49.0 ± 6.2 m V / m s under  control conditions whereas during application o f 10 u M , it was 50.1 ± 4.1 m V / m s (mean ± S D ; P = 0.85; n — 9). F o r the fourth spike i n a train (the first to show a maximal magnitude  o f effect  for 600 p.M lidocaine; cf. Figure 12), the  control value  was  51.8 ± 8.7 m V / m s vs. 45.9 ± 11.8 at 10 u M (P = 0.20; 1 - (3 = 0.8 to detect a difference o f 14.68 m V / m s at a = 0.05; n — 9). In current-frequency relationships, lidocaine caused a parallel shift o f the curve to the right, indicating a relatively unimpaired distribution o f activatable N a  +  channels i n the area o f the spike generator (Figure 10B). Lidocaine  application also diminished a slow component  o f the  spike-afterhyperpolarizations  ( A H P s ) , w h i c h i n isolation w o u l d be expected to increase firing frequency (Figure 10C) (cf. F o e h t i n g et al. 1989). Consistent w i t h a shunt, lidocaine markedly increased current thresholds, i.e., the amplitudes o f current pulses required for spike generation (Figure 11).  S. S C H W A R Z 36 Control  10 uM Lidocaine  Recovery  Control Recovery 10 uM Lidocaine  0  20  40  60  A/  M  80  100  10 uM Lidocaine  20 mV  120  (pA)  fAHP  *  s A H P  100 ms  10 |JM Lidocaine  Control Spike No. 1 2 3 4 5 6  Control  l  1 2 3 4 5 6  Control l 10 | J M Lidocaine  -76 m V J 20 mVl 100pApi0° ms  Figure 10 Effects o f a l o w concentration o f lidocaine o n tonic repetitive firing (A) Lidocaine, administered at a low concentration that produced the maximum decrease in Rj (10 u M ; of. Figure 8), reversibly reduced the spike frequency and inhibited spike accommodation of neurons firing in the tonic pattern. T o n i c firing in the neuron shown was evoked from a V, of—76 m V by D C injection (100 p A / 5 0 0 ms pulse; lower traces). T h e suppression of repetitive firing was not associated with an increase in the threshold potential for firing (arrows) or a decrease in the maximum rate o f rise o f the spikes (dV/dt * ; m  secondary to a shunt rather than N a  +  channel blockade.  x  sec below), consistent with an effect  (B) T h e relationship between the amplitude o f injected  current (AJ ) and the firing frequency (spikes/500 ms) was approximately linear. T h e suppression o f repetitive firing m  by lidocaine was characterized by a shift o f the current-frequency curve to the right, without reducing its slope. (C)  Lidocaine reduced  a slow component  o f the  spikc-afterhyperpolarizations  (sAHP;  fAHP  denotes  fast  aftcrhyperpolarization). T h e two superimposed traces arc matched for spike frequency and were obtained with 60 p A (control) and 110 p A (lidocaine) D C pulses.  (D) Lidocaine increased the current required to elicit a train of  6 spikes (see also Figure 11) but produced no alterations in spike configuration, e.g., dV/dl ^. m  (E) Quantitative  illustration o f the effect o f 10 u M lidocaine on dK/dAnax of spikes in a train of 6 (n = 11 neurons). Lidocaine had no effect o n consecutive  spikes ( A N O V A , P = 0.60; control, P = 0.99; Bonferroni's multiple comparison test,  P > 0.05). Comparisons to control yielded no significant differences for any given spike number (paired / test, P > 0.05; see text for details).  S. S C H W A R Z 37  10 uM Lidocaine  Control  Recovery  x=4  -76 mV20 m V l 40 p A  100 ms  _F  Figure 11 Effects o f a l o w concentration o f lidocaine o n injected current In association with the decreased Rj, lidocaine shunted current injected to stimulate neurons, which manifested as a marked elevation in the threshold D C amplitude required for firing in the tonic pattern. In the neuron shown (cf. Figure 10), 10 u M lidocaine reversibly increased the current pulse amplitude required for spike generation to a level o f 400% o f control (control, 20 p A ; 10 u M lidocaine, 80 pA).  These findings illustrated the predominance o f a shunt as the primary effect at these l o w concentrations.  In  contrast,  higher  concentrations  produced  alterations  configuration conventionally indicative o f a local anesthetic effect o n N a  +  in  spike  permeability.  F o r example, concentrations o f lidocaine ranging from 300 u M to 1 m M decreased the dl/Vd/max o f action potentials and produced a pronounced or complete suppression o f tonic repetitive  firing  (Figure 12). In a concentration-dependent  fashion, lidocaine  application elevated the threshold potential for firing. These concentrations did not significantly shunt the current required for spike generation (cf. Figure 8 and Table 3). Consistent w i t h previous observations o f others (f. Butterworth et al. 1993), recovery was significantly delayed o n terminating an application at a high concentration, usually requiring washout times that exceeded 45 m i n .  S. S C H W A R Z 38 Control  Lidocaine 300 uM  600 uM  1 mM  -56 mV  -66 mV 20 mV 400 pA 200 ms  B  30  E  o o LO  Control 20  Lidocaine 300 uM  CD CL  10  CO  „ „ „ .. ^ 0  0  M  1 mM 100  200  300  A/  Control Spike No.  6  1 2 34 56  m  400  500  600  (pA) Control H3 600 pM Lidocaine  600 uM Lidocaine 123 4  5  6  -66 mV • 20 mVl 400 pA 200 ms 3  4  Spike No.  Figure 12 Effects o f high concentrations o f lidocaine o n tonic repetitive firing (A) High lidocaine concentrations produced concentration-dependent elevations in the voltage threshold for tonic firing (arrows) and alterations in spike configuration without decreasing Ri. Superfusion with 300 uM, 600 uM, or 1 mM lidocaine led to a pronounced reduction infiringproduced by DC injection. At 600 uM and higher, repetitive firing was completely suppressed but could be elicited by current pulses of a markedly increased amplitude (sec C). Note the refractoriness to lidocaine of the first spike in a train of action potentials. (B) Effects of lidocaine on the relationship between the amplitude of injected current (A7 ) and firing frequency (spikes/500 ms; neuron from A). Lidocaine reduced the slope of the current-frequency curve in a concentration-dependent fashion, consistent with a successive increase in the ratio of blocked versus unblocked Na channels at the axon hillock. A saturation effect became evident at 600 uM. (C) A high lidocaine concentration (600 uM) increased the current required to elicit a train of 6 spikes (in the neuron shown, to a level of 450% of control) and successively reduced the dV/d/, of the spikes in the train (cf. Figure 10). (D) Effect of 600 uM lidocaine on d F / d W of 6 consecutive spikes in a train (n = 6 neurons). Lidocaine successively reduced the d F 7 d / (ANOVA, P = 0.01; control, P = 0.61). The asterisks denote comparisons to control for a given spike number (paired t test; *, P < 0.05; * * , P < 0.01). A maximal effect was reached at the fourth spike (Bonferroni's multiple comparison test, P < 0.05). m  +  mx  max  S. S C H W A R Z  39  1.3.8 Effects of lidocaine on burstfiring and low threshold spikes L o w concentrations o f lidocaine associated with a decrease i n R i exerted a depressant influence o n the burst firing mode o f V P L neurons. T h i s was observed o n depolarization from  prepulse-conditioned  (Figure 13A) or tonically D C - m a i n t a i n e d (Figure 13B)  hyperpolarized potentials. Lidocaine (10 u M ) suppressed the rebound bursts elicited by hyperpolarizing current pulses from potentials near rest (Figure 13C & Figure 9 A ) . W h e n hyperpolarization was maintained by D C injection at potentials f r o m w h i c h depolarizing pulses elicited L T S burst firing, a subsequent application o f lidocaine (10 u M ) resulted i n a marked elevation i n the amount o f current required to evoke a spike burst. T h i s action was reversible and similar to its effect o n the tonic firing mode (Figure 13D). T h e effects were associated w i t h a marked decrease i n Ri, implicating a shunting o f input current as a major mechanism for the depression o f the burst activity.  1.3.9 Effects of lidocaine on high threshold spikes A s discussed i n 1.3.7 and shown i n Figure 12, lidocaine, applied at high concentrations that clinically are C N S - t o x i c (300 [ i M - 1 m M ) , reduced or completely suppressed tonic repetitive firing i n a concentration-dependent fashion. T h i s effect, showing a saturation at 600 [0.M, was associated with an elevation o f firing thresholds  and slowing o f the  dV/d/max o f spikes, conventionally indicative o f a local anesthetic effect o n N a  +  channels.  In order to better characterize these actions, the differential effects o f 600 | i M lidocaine o n tonic firing frequency, voltage threshold, and dK/d/max were examined i n relation to the  time  o f administration (n = 2). Lidocaine slowed dV/dt x ma  and reduced  firing  S. S C H W A R Z  Tonic firing  40  Burst firing  LTS 20 mV  200 pA  -64 m V - ^ loo ms J -  1  B  1_ Burst firing  Tonic firing  uuu 20 mV  LTS,  -74 mV-  -88 mV  200 pA 100 ms  Control  10 uM Lidocaine  Recovery  Control  10 uM Lidocaine  Recovery  -66 mV 20 m V 50 p A 100 m s  LTS  LTS^  LTS. -88 mV 20 m V 50 pA  100 ms  Figure 13 L o w threshold spikes and the effects o f lidocaine o n burst firing (A) & (B) Voltage dependent firing patterns evoked by different procedures in two V P L neurons. Injection with depolarizing current pulses from near V evoked repetitive tonicfiring( A l , B l ) . At the same V , hyperpolarizing current pulses of sufficient amplitude to de-inactivate a Trtype C a current resulted in rebound burstfiringafter termination of the pulse, generated by a low threshold spike (LTS). Typically, bursts of 1-7 action potentials fired on top of the LTSs (A2, arrow). When V was hypcrpolarized by D C injection, LTS burst firing also was evoked by depolarizing current (B2, arrow). (C) In a reversible manner, lidocaine (10 uM) shunted rebound LTS bursts evoked from potentials near rest (cf. Figure 9A). (D) In neurons that were hyperpolarizcd to elicit LTS bursts by depolarizing current pulses, lidocaine application produced a reversible elevation in the current threshold for firing. The current pulse magnitude required for burst firing was increased to a level of 233% of control (control, 30 pA; 10 uM lidocaine, 70 pA). Note the decreased Ri in C and D (cf. Figure 11). t  m  2+  m  S. S C H W A R Z  41  frequency i n a time-dependent fashion to the point o f complete suppression o f firing (Figure 14A). T h i s was accompanied by a time-dependent  increase o f the voltage  threshold for firing, consistent with a gradual increase o f effective lidocaine concentration until steady state is reached at the site o f action near the N a channel. T h e relative effect +  o f lidocaine o n firing frequency was significantly more pronounced than o n d T / / d /  m a x  (Figure 14B). T h i s implied that lidocaine at these high concentrations exerted actions o n targets i n addition to N a  +  channels (for w h i c h slowing o f &V/dt * m3  is a sensitive  parameter; of. H u b b a r d et al. 1969) that w o u l d contribute to a suppression o f repetitive firing, well documented i n the literature (cf. Discussion & Table 4 [page 77]). W h e n current pulses o f markedly increased amplitude were injected into neurons i n w h i c h lidocaine had completely suppressed tonic firing, a population o f spikes was triggered that had spike configurations and properties distinct from the " c o n t r o l " discharges i n the tonic mode (Figure 14A, caption labelled ">5:00 min"). These high-threshold spikes (HTSs) were broader w i t h a lower amplitude and significantly slowed d l Z / d / m a x (Figure 14C). These observations raised the possibility that the tonic repetitive spikes suppressed by lidocaine and the H T S s represent distinct spike populations generated by separate mechanisms. I n order to address this hypothesis, T T X (600 n M ) was applied to neurons i n w h i c h H T S s were elicited during lidocaine application as described above. I n all neurons tested this way, the spikes unmasked by lidocaine remained during the application o f T T X (Figure 15; n — 4). T h i s was associated with a further small but significant elevation i n voltage threshold (P < 0.05; mean elevation, 3.1 m V [95% C I , 0.3-5.8 mV]).  S. S C H W A R Z  600 uM Lidocaine 2:00 min  Control  42  2:20 min  2:10 min  -46 mV20 mV 00 ms 100 pAMOC  _r  i 3:00 min  2:30 min  1  B  a  •a -o  =s 2 >• c " o g>  4:00 min  >5:00 min  J~  100-,  12 Firing frequency  75  dV/dt,  600 uM Lidocaine  S  as  CD h8  o > F  50  £  h4  2 £ . 25  <D i_ .c  cn c  c  o ir  CM 0:00  2:00  3:00  4:00  10 mV  5:00  Du ration of lidocaine application (min)  Figure 14 spikes  20 ms  Time-dependent effects o f a high concentration o f lidocaine o n repetitive  (A) Tonic firing was elicited in a neuron from V (-46 mV) with D C injection (100 pA/500 ms pulse). Lidocaine (600 uM) inhibited repetitive spikes in a time-dependent manner (see B) and completely suppressed firing at 4:00 min of application. Note the relative lack of effect on spike frequency accommodation. A current pulse of a markedly increased amplitude (220 pA) injected after 5:00 min of lidocaine application triggered a train of high threshold spikes (HTSs [arrows]) of distinctly altered configuration/slowed dV/dt -ax (cf. Figure 12; sec C for details & asterisks). (B) Shown are the differential effects of lidocaine on tonic firing frequency, voltage threshold, and dV/dtmrn. in relation to the duration of application. Lidocaine decreased firing frequency and dK/d/max (leftj-axis) in a time-dependent manner while increasing the voltage threshold for firing (right j-axis; control, -38 mV). Firing frequency was significantly more sensitive to lidocaine than dl>7d/ ax- The maximum difference between both parameters at a given time was 46% of control. (C) Magnified images of two supcrimposablc spikes from A (* & **). Compared to the control spike, the HTS had a distinctly altered spike configuration with a characteristic high voltage threshold (sec arrows). The findings raised the possibility that the repetitive spikes of the tonic firing mode suppressed by lidocaine and the HTSs represent separate spike populations with distinct underlying mechanisms (sec text). (Note: time zero [0:00] indicates the time of switching from the control perfusate [normal ACSF] to ACSF containing lidocaine. The time to overcome the dead space of the perfusion system was approx. 1:00 min.) t  m  m  S. S C H W A R Z  Control  43  6 0 0 | J M Lidocaine + 6 0 0 nM TTX  6 0 0 | J M Lidocaine HTSs  -37 mV  -48 mV 20  mV  100  pA  100  ms  F i g u r e 15 Lidocaine unmasks high threshold spikes not blocked by T T X Lidocaine, applied at a high concentration (600 uM) that clinically is toxic to the C N S , unmasked high threshold spikes (HTSs) that were elicited by injection o f a D C command with a large amplitude (140 p A ; control, 40 p A ; if. Figure 12 & Figure 14). Application o f T T X (600 nM) did not block the HTSs. In the presence o f lidocaine, T T X produced an elevation o f the H T S voltage threshold o f ~4 m V i n the neuron shown, compared to lidocaine alone. N o other actions o f T T X on H T S configuration were observed. A l l observed effects were reversible and full recovery o f the neuron was observed after 45 min o f washout (superfusion with normal A C S F ; not illustrated).  A l l effects were reversible and the " c o n t r o l " (TTX-sensitive) action potentials o f the tonic firing mode exhibited recovery after washout. W h e n lidocaine was applied to neurons where T T X was present and had completely abolished tonic firing, H T S s could likewise be triggered by D C injection o f a large amplitude (n — 1 out o f 9 neurons; Figure 16). O n e o f these 7 neurons was excluded from further analysis due to loss o f recording prior to recovery. In the remaining neurons, lidocaine application i n the presence o f T T X effectively unmasked H T S s , i.e., current amplitudes that elicited no firing  under  T T X alone  reversibly triggered  HTSs  when  lidocaine was  added  (Figure 16A). T h i s was associated w i t h no consistent changes i n Ri (P > 0.05) as determined  from voltage responses to hyperpolarizing D C injection (see page 24).  Consistent w i t h this finding, there were no significant alterations i n slope resistance or  S. S C H W A R Z overall I-V  44  relationship i n the hyperpolarized range. However, lidocaine increased  amplitudes o f voltage responses to depolarizing current to values up to 190% o f control i n 4 out o f 6 neurons and produced corresponding increases i n slope resistance i n the depolarized range o f the I- V relationships (Figure 16B). Concomitant w i t h these effects, lidocaine decreased H T S voltage thresholds up to 23 m V (« = 4) and current thresholds (cf. 1.3.7, page 35) up to 5 0 % o f control (n - 4) (Figure 16C). N o associated consistent changes i n V were observed. A s i n the experiments w i t h T T X application i n the presence t  o f lidocaine, recovery usually was observed after approx. 20—45 m i n o f washout. T h e data from this series o f experiments w i t h T T X strongly supported the hypothesis that the H T S s unmasked by lidocaine are indeed distinct from die N a  +  dependent action potentials o f the tonic firing mode and generated by a different ionic mechanism. T h e most likely candidate for consideration other than N a  +  to carry such  depolarizing spike potentials as the present H T S s was C a . Others have previously 2 +  demonstrated that thalamocortical neurons express, i n addition to the L V A C a (cf. 1.1.2, 1.3.4, 1.3.8), high voltage-activated ( H V A ) C a  2 +  currents  currents that generate H T S s  2 +  similar to those observed here (Jahnsen and Llinas 1984b; H e r n a n d e z - C r u z and Pape 1989; Coulter et al. 1989; Pfrieger et al. 1992). I n contrast to the former, w h i c h produce L T S s and are critical to the burst firing from hyperpolarized membrane potentials (cf. 1.1.2, 1.3.4), the latter have been implicated i n the regulation o f firing i n the tonic mode at depolarized potentials (Kammermeier and Jones 1997; Z h o u B u d d e et al. 2000). Overall, the modulation o f these C a  2 +  et al. 1997;  currents contributes to the  S. S C H W A R Z 45  Control  600nMTTX  600 nM TTX + 600 uM lidocaine  600 nM TTX (lidocaine  600 nM TTX  Control  Recovery  600 nM TTX + 600 uM lidocaine  i  -26 mV.  HTSs ,  -35 mV.  -73 mV J 20 m V 500 p A  100 m s  Figure 16 Effects o f lidocaine o n high threshold spikes i n the presence o f T T X (A) In the neuron illustrated, repetitive tonic firing was elicited by D C injection and completely blocked by T T X (600 n M ) . A subsequent co-application o f lidocaine (600 u.M) reversibly unmasked a high-threshold spike ( H T S ; cf. Figure 14 & Figure 15). Concomitantly, lidocaine produced an increase in the steady-state voltage responses to depolarizing current (see below). Close to full recovery was observed in the neuron after > 30 min washout. (B) I- V relationships o f the same neuron. Whereas lidocaine had little effect on the I- V curve in the hypcrpolarizcd range, it increased slope resistance in the depolarized range by up to -25%.  (C) Shown are traces from an  experiment with a different neuron. Lidocaine, applied in the presence o f T T X , decreased both voltage and current thresholds for the H T S s . D C injection o f an amplitude that initiated no firing under T T X alone (300 pA) triggered three H T S s when lidocaine was added. Recovery was observed after 27 min o f washout (not shown).  S. S C H W A R Z  46  discharge patterns and synchronized oscillations o f the thalamocortical network that occur physiologically and i n association w i t h various pathological states (Steriade and Llinas 1988; J e a n m o n o d et al. 1996; Shah et al. 2001; cf. Discussion, 1.4.2.1). T o test directly whether the H T S s unmasked by lidocaine i n this study were mediated by C a , two sets o f experiments were conducted using C a - d e p r i v e d A C S F 2 +  and  the  Ca  2 +  2+  channel  blocker, C d , 2 +  respectively.  Cd  2 +  is  a particularly  useful  pharmacological tool for this purpose since it allows one to differentiate H V A and L V A Ca  2 +  currents: i n thalamocortical neurons, 50 p M almost completely blocks H T S s while  leaving L T S s relatively unaffected (Hernandez-Cruz and Pape 1989; Pfrieger et al. 1992; Tennigkeit et al. 1998a). Figure 17 summarizes the results o f these experiments. W h e n neurons i n w h i c h H T S s were triggered as described above (in the presence o f T T X and lidocaine) were disappeared  perfused  w i t h extracellular solution deprived o f C a ,  the  spikes  was fully reversible, w i t h the  HTSs  2 +  completely (n =  4). T h i s effect  reappearing instantly w h e n the superfusing m e d i u m was changed back to the control solution. A p p l i c a t i o n o f C d  2 +  (50 p M ) i n the presence o f T T X and lidocaine completely  blocked the H T S s (n — 4) i n a reversible manner. Neurons exhibited recovery from all effects after washout w i t h normal A C S F . In / - ^ r e l a t i o n s h i p s , C d  2 +  did not b l o c k the  increase i n slope resistance i n the depolarized range produced by lidocaine i n neurons superfused w i t h T T X (cf. Figure 16B; see Discussion [1.4.2.1], page 85). O n the contrary, Cd  2 +  further increased the slope'resistance i n the depolarized range i n 3 out o f 4 neurons  by up to ~ 2 5 % , consistent w i t h a H V A C a  2 +  conductance blockade. C d  2 +  also reversibly  increased slope resistance i n the hyperpolarized range by up to ~ 3 8 % (Figure 17C),  S. S C H W A R Z 47  nM T T X + 6 0 0 | J M lidocaine  OCa  600  Recovery  2  HTSs  -61 mV  40 mV  1_ B  -J  nM T T X + 6 0 0 | J M lidocaine 600  I  1_ 50  |JM Cd  200 ms  400 pA  Recovery  2  HTSs  HTSs  -61 mV  40 mV  1_  200 ms 400 pA  J  200  -200  nM T T X • 6 0 0 nM T T X + 6 0 0 |jM lidocaine - 6 0 0 nM T T X + 6 0 0 | J M lidocaine + 5 0 ^M Cd •600  Figure 17 Effects o f C a free extracellular solution and C d spikes unmasked by lidocaine 2 +  2 +  2+  o n the high threshold  (A) T h e H T S s that were unmasked by lidocaine and remained during the co-application o f T T X revcrsibly disappeared when the extracellular solution 50 u M C d  2 +  (a blocker o f H V A C a  2 +  (ACSF) contained  zero C a . 2 +  (B) Superfusion o f neurons  with  currents; see text) completely blocked the H T S s in a reversible manner. These  features implied that the spikes were carried by C a  2 +  and produced by a H V A C a  2 +  current, revcrsibly unmasked by  lidocaine. N o t e that "Recovery" denotes superfusion with A C S F containing 600 n M T T X and 600 \iM lidocaine; full recovery o f regular tonic and burst firing was observed after 20 min o f washout with normal A C S F illustrated).  (C) Effects o f C d  2 +  (not  on the increase in slope resistance in the depolarizing range produced by lidocaine  in neurons superfused with T T X . C d  2 +  did not block lidocaine's effect but further increased slope resistance over a  wide voltage range. In the neuron shown (cf. Figure 16B; V = —62 m V ) , the increase in apparent resistance occurred T  in a range between — 9 5 m V and spike threshold, amounting to maxima o f ~25% in the depolarized range and ~38% in the hyperpolarized range. Recovery was observed after washout (not illustrated for enhanced clarity).  S. S C H W A R Z  48  likely due to its blockade o f h (Barish and B a u d 1984; N a t h a n 1986; O z a w a et al. 1989) (cf. page 28). In conclusion, the data obtained from these sets o f experiments confirmed the hypothesis that the H T S s unmasked by lidocaine were high threshold C a produced by a H V A C a  2 +  2 +  spikes,  current (Coulter et al. 1989; H e r n a n d e z - C r u z and Pape 1989;  K a m m e r m e i e r and Jones 1997; Tennigkeit etal. 1998a).  1.3.10 Effects of GABA.A  receptor blockade by bicuculline on lidocaine actions  A recent study demonstrated that local anesthetics enhance the increase i n conductance induced by G A B A i n stretch receptor neurons o f crayfish ( N o r d m a r k and Rydqvist 1997). T h e anticonvulsants, carbamazepine and phenytoin, similarly potentiate  GABA-  induced currents i n cultured rat cortical neurons and i n human embryonic kidney cells expressing the  OC1P2Y2  subtype  o f the  GABAA  receptor  (Granger et al. 1995). I n  thalamocortical neurons, application o f G A B A mediates a rapid increase i n conductance w h i c h is carried by C l (Crunelli et al. 1988; T h o m s o n 1988). I n the present study, the reversal  potential  concentrations  was  for  the  near  increase the  in  conductance  equilibrium  potential  induced for  CF  by  l o w lidocaine  (calculated  EQ\ at  25 ° C = —53 m V ) (cf. page 31). A n increase i n C l conductance due to lidocaine application w o u l d be consistent with the observed decreases i n Rj and depolarizations. I n such a case, the increased conductance produced by lidocaine w o u l d likely represent a major mechanism for inhibition o f firing because the driving potential (V  m  G A B A - i n d u c e d change i n V  m  — Ed) for a  w o u l d be rather small. F o r example, despite depolarizing  S. S C H W A R Z  49  cortical neurons, G A B A still produces inhibition o f firing due to a shunt mechanism (El-Beheiry and P u i l  1990). I n thalamic reticularis neurons, activation o f  GABAA  receptors shunts burst firing (Ulrich and Huguenard 1997). Based o n these findings, a series o f experiments was conducted aimed to address the possibility that the lidocaineinduced shunt observed here i n V P L neurons is mediated by G A B A A receptors. T o test this hypothesis, the effects o n the lidocaine-induced changes i n membrane electrical properties and excitabilities o f the G A B A A  receptor antagonist, bicuculline,* were  investigated. Results o n bicuculline were obtained from n — 6 neurons. O n e o f the neurons was excluded from the analysis due to lack o f recovery. W h e n bicuculline (50 u,M; E l - B e h e i r y and P u i l 1990; v o n K r o s i g k et al. 1993) was applied to neurons following reduction o f D C - e v o k e d tonic firing by 10 U.M lidocaine (cf. 1.3.7, Figure 10), firing rates were increased a n d / o r restored to control values (Figure 18) (Gao et al. 1997), consistent w i t h previous  reports  o n its action  on GABAA  receptors i n thalamocortical neurons  (Lee et al. 1994; M c C o r m i c k et al. 1995; G a o et al. 1997). Bicuculline application per se d i d not elicit firing i n neurons maintained at rest, i n agreement w i t h the absence o f significant effects o n spontaneous activity i n thalamocortical neurons o f anesthetized cats and rats observed by others (Duggan and M c L e n n a n 1971; L e e et al. 1994). H o w e v e r , bicuculline  *Bicuculline is a naturally occurring convulsant breeches),  Adlumiafungosa  alkaloid whose sources include  Dicentra cucullaria (Dutchman's Cordalis spp.  (climbing fumitory/mountain fringe/Alleghany vine), and various  S. S C H W A R Z 50  Control  -76 mV  J  10 | J M Lidocaine  10 uM Lidocaine + 50 uM bicuculline  mMMAUMMM  20 mV| 50 pA  100 ms  + 50 |jM bicuculline  A/ (pA) m  Figure 18 Effects o f bicuculline o n the decrease i n tonic firing due to lidocaine (A)  Following reduction by lidocaine, bicuculline (50 u M ; 8 min application) restored tonic firing evoked by  depolarizing D C injection to the control rate.  (B) Current-frequency relationships o f the same neuron. T h e  lidocainc-induced shunt was evident as a shift o f the current-frequency curve to the right (tf. Figure 10B). Bicuculline application increased tonic firing, and, at D C amplitudes > 90 p A , restored the firing rate to control values (cf. Figure 10A). A J denotes injected current pulse amplitude. m  S. S C H W A R Z  51  did not influence lidocaine's actions to decrease R i (Bonferroni's multiple comparison test following repeated measures A N O V A : lidocaine vs. lidocaine + bicuculline, P > 0.05; control vs. lidocaine, P < 0.05; control vs. bicuculline + lidocaine, P < 0.01; A N O V A overall, P < 0.0001; n — 5). Figure 1 9 A illustrates these findings for a representative neuron. I n I-V  relationships, bicuculline did not antagonize  the decrease i n slope  resistance produced by lidocaine over a wide voltage range (Figure 19B). There also were no  significant changes i n %  m  (Bonferroni's multiple comparison test: lidocaine vs.  lidocaine + bicuculline, P > 0.05; n — 5). Likewise, bicuculline d i d not antagonize the shunt-associated  reductions  of  spike-afterhyperpolarizations  (AHPs)  produced  by  lidocaine (Figure 19C; cf. Figure 10).  1.3.11 Effects of other local anesthetic agents on membrane properties In the final part o f this section o f the thesis, a series o f experiments w i t h a variety o f other agents was conducted to further delineate the observed effects o f l o w lidocaine concentrations o n resting membrane properties, i.e., the lidocaine-induced shunt. In the first set o f these studies, lidocaine's effects were compared w i t h those o f its quaternary analogue, QX-314. These experiments were aimed to test whether the lidocaine-induced decrease i n R i could be due to interaction w i t h an intracellular, as opposed to an extracellular, target. T h i s hypothesis was based o n the findings that (1) lidocaine still produced decreases i n R i i n the presence o f T T X (1.3.5), implying a postsynaptic action; (2) the decreases i n R i were not associated w i t h membrane hyperpolarizations (1.3.5),  S. S C H W A R Z 52  10 uM Lidocaine  Control  10 uM Lidocaine + 50 uM bicuculline  -70 mV 20 m V 100 p A | 100 m s  Figure 19 Effects o f bicuculline o n the decrease i n input resistance due to lidocaine (A) In the neuron shown, lidocaine administration (6.5 min) produced a 20% reduction in input resistance (Rj) and shunted rebound low threshold spike bursts (cf. Figure 9 & Figure 13). Bicuculline had no effect on the decreased Ri (> 9 m i n o f application). T h e shunting o f low threshold spike bursts also remained unaffected during bicuculline application. (B) Corresponding current-voltage-relationships. Membrane potential differences ( A K ) were measured m  at the end o f 500 ms current pulses (A7 ) injected in 20 p A steps (sec arrows in A ) . Bicuculline did not antagonize m  the reduction in slope resistance produced by lidocaine over the voltage range from — 9 0  m V to threshold.  (C) Lidocaine (dotted trace) reduced Ri and spike-afterhyperpolarizations (AHPs) while the neuron fired in the tonic firing pattern (if. Figure 10). W h e n bicuculline was added to the perfusing media, no changes or slight further reductions in A H P s were observed (dashed trace). T h e labels on the right indicate the amplitudes o f the current pulses injected to obtain the respective traces matched for spike rate.  S. S C H W A R Z  pointing  to  mechanisms  distinct  from  an  increase  in  K  +  channel  53  conductance  (cf. Ries and P u i l 1999b); and (3) blockade o f the m a i n extracellular target mediating inhibitory neurotransmission, the G A B A A receptor, had no effect o n the decreases i n R i (1.3.10). In the second set o f these experiments, an effort was made to shed light o n the question whether lidocaine's effects represent a novel action that is specific to this agent, or rather an effect c o m m o n to local anesthetic and related drugs that has previously been u n k n o w n . F o r this, a set o f comparative studies was carried out w i t h procainamide, an aminoalkyl  amide  analog  of  the  aminoester  local  anesthetic,  procaine,  and  the  aminoamide local anesthetic, bupivacaine, neither o f w h i c h are k n o w n f r o m the literature to have clinically useful systemic analgesic properties. T h e drugs were studied over a wide concentration range and concentration-response relationships for their effects o n R i were constructed as feasible.  1.3.11.1 Q X - 3 1 4 QX-314  (lidocaine N - e t h y l bromide; N-[2,6-dhnethylphenylcarbamoylmethyl]triethyl-  ammonium  bromide; M W , 343.3) is a lidocaine derivative whose  sole  structural  difference to the mother c o m p o u n d is i n the presence o f an additional N - e t h y l group (Figure 20). T h i s renders the amino group quaternary, charged. A s a result, the  agent cannot  i.e., permanently positively  readily pass biological membranes.  When  administered to central neurons intracellularly, Q X - 3 1 4 blocks both fast, N a - d e p e n d e n t +  action potentials and voltage-dependent,  noninactivating N a  +  conductances  (Connors  and Prince 1982; M u l l e et al. 1985; cf P u i l and Carlen 1984). Here, a series o f experiments  S. S C H W A R Z Aromatic head  Amide linkage  54  Quarternary amine tail  Figure 20 Structural formula o f Q X - 3 1 4  w i t h Q X - 3 1 4 applied extracellularly was conducted to compare its effects o n membrane properties to those o f lidocaine and determine whether the lidocaine-induced shunt may be result o f interaction w i t h an extracellular (as opposed to intracellular) target. T h e effects o f Q X - 3 1 4 were studied i n n — 6 neurons. O f these, two were excluded from quantitative analyses due to loss o f recording prior to recovery. Q X - 3 1 4 , applied over a wide concentration range (1 uM—1 m M ) , produced no decreases i n R i i n the neurons (P > 0.05) (Figure 21 A ) . Likewise, no significant reductions i n T  m  were  observed. I n contrast, higher concentrations o f Q X - 3 1 4 increased R i i n some neurons (at 1 m M , to a m a x i m u m level o f 169% o f control; mean, 127 + 18%), although this effect was short o f reaching statistical significance ( A N O V A w i t h Dunnett's multiple comparison test, P > 0.05). N o n l i n e a r regression analysis w i t h the use o f a conventional concentration-response R  2  m o d e l (cf. 1.2.5) yielded a curve w i t h a goodness  o f fit o f  = 0.9994 and an E C 5 0 o f 237 u,M for the resistance increase (Figure 21B). Consistent  S. S C H W A R Z 55  10 yM QX-314  1 uM QX-314  Control  100 uM QX-314  -61 mV5 20  mV pA  B  ms  100  1_  150'  125-|  C O O  o  Control  100  v°  EC  W  = 237 p M  10 p M Q X - 3 1 4  Of  100 p M Q X - 3 1 4 75-1 10  100  1000  20  10000  40  Control  100  120  m  10 uM QX-314  1 uM QX-314  100 uM QX-314  JL. II  JL  -61 mV-  80  A/ (pA)  [QX-314] (uM)  D  60  JL  w w w  20 mVl flOO 100pAp°°  ms  Figure 21 Effects o f Q X - 3 1 4 o n membrane properties (A) Unlike lidocaine, extracellular application (~10 min per concentration) o f its quaternary analogue, Q X - 3 1 4 , did not reduce input resistance (R;) over a wide concentration range, here illustrated by the lack o f decreases in amplitude o f the steady-state voltage responses to hyperpolarizing D C injection (cf. Figure 8A). Consistent with this, Q X - 3 1 4 did not exert significant effects on membrane time constants (in the neuron shown, T  M  = 25.2 ms).  (B) Concentration-response relationship for the effect o f Q X - 3 1 4 on Ri. Q X - 3 1 4 exerted no significant effects in the concentration range from 1 u M to 1 m M ( A N O V A , P - 0.54; for each concentration, n = 4). A t 1 m M , Q X - 3 1 4 produced increases in Ri that did not reach statistical significance (Dunnett's multiple comparison test, P > 0.05). However, analysis o f the goodness o f fit for a classic concentration-response curve (cf. 1.2.5) yielded an R o f 0.9994. 2  The  corresponding EC50 was 237 u M . (C) Consistent with the lack o f effect on Rj, Q X - 3 1 4 did not shunt tonic  firing like lidocaine, here illustrated in a representative current-frequency plot (see below) by the absence o f a significant shift o f the curve to the right (cf. Figure 10B). Q X - 3 1 4 did not increase current thresholds for firing (cf. Figure 11), identifiable here as the minimum current pulse amplitude ( A J , abscissa) required for eliciting spikes m  (ordinate).  (D) T o n i c and rebound burst firing responses o f the neuron in C to de- and hyperpolarizing D C  injection (pulse duration, 500 ms). Increasing concentrations o f Q X - 3 1 4 did not greatly affect spike frequency or shunt tonic and L T S burst (arrow in control) firing comparable to lidocaine (cf. Figure 10A & Figure 13C).  S. S C H W A R Z  56  w i t h the lack o f effect o n Ri, Q X - 3 1 4 did not shunt tonic or burst firing i n the neurons (Figure 2 1 C & D ) . O n the contrary, i n some neurons, Q X - 3 1 4 application was associated w i t h small increases i n firing rate (not illustrated). N o consistent significant changes i n V  t  were observed as a result o f Q X - 3 1 4 application.  1.3.11.2 Procainamide Procainamide  (4-amino-N-[2-(diethylamino)ethyl]benzamide;  M W , 271.8;  pK , a  9.2;  Figure 22) is an aminoalkyl amide that, like lidocaine, has cardiac antiarrhythmic properties. It belongs to class 1 A according to the classification o f antiarrhythmic drugs by V a u g h a n Williams (Bigger and H o f f m a n 1990). T h e therapeutic plasma concentration for the antiarrhythmic effects ranges between 4 and 8—10 u g / m l (approx. 14.7—36.8 u M ) i n humans (Zipes 1992; R o d e n 2001). I n contrast to lidocaine, w h i c h i n the same plasma concentration range that is associated w i t h its antiarrhythmic actions (1.5-5 u M ; R o d e n 2001) exerts its systemic analgesic effects (tf. 1.1), no published reports have documented systemic  analgesic  properties  for  procainamide.*  On  the  contrary,  procainamide  (73.5 n M ) microinjections into the Nucleus raphe magnus b l o c k the antinociceptive effects o f nicotine (12.35 n M ) injected i n the pedunculopontine tegmental nucleus (which sends ascending projections to the thalamus; tf. Hallanger et al. 1987) i n rats in vivo, as measured  *An isolated exception is the anecdotal account o f the use o f procainamide in myotonic syndromes associated with "hyperexcitability o f muscle fibres" (Mertens and Lutzenkirchen  1970)  S. S C H W A R Z  Aromatic head  Amide linkage  57  Aminoalkyl tail  F i g u r e 22 Structural formula o f procainamide  by responses to tail-flick and hot-plate tests (Iwamoto 1991). Systemic toxicity due to procainamide typically occurs at plasma concentrations > 10 u g / m l (Roden 2001); its most c o m m o n manifestations include gastrointestinal upset, arterial hypotension, cardiac conduction disturbances,  fever, and a systemic lupus erythematosus-like  syndrome  (Zavisca et al. 1991; Zipes 1992). In ~ 0 . 2 % o f patients, procainamide induces marrow  suppression  and  agranulocytosis,  a  potentially  life-threatening  bone  reaction.  C o m p a r e d to lidocaine, symptoms o f C N S toxicity are less c o m m o n ; they include giddiness, psychosis, and depression. Such central actions are not unexpected because procainamide, despite its hydrophilicity, crosses the blood-brain barrier as demonstrated i n rats (Herken and Rietbrock 1969). W i t h regard to the C N S toxicity, it is noteworthy that the procainamide-induced lupus-like syndrome, unlike primary systemic  lupus  erythematosus, spares the brain. Experiments w i t h procainamide were conducted i n n — 8 neurons, o f w h i c h n — 2 were excluded from statistical analyses due to lack o f recovery. Procainamide produced no significant decreases i n R i over a concentration range from 1 u M to 1 m M . In  S. S C H W A R Z 58  Figure 23 Effects o f procainamide o n membrane properties (A) In a concentration-dependent manner, procainamide application (1 u M - 1 m M ) produced increases and no decreases in Rj, here illustrated by the amplitudes o f the steady-state voltage responses to hyperpolarizing D C injection (cf. Figure 8A) in a representative neuron held at V, (-73 m V ) . Associated with the increases in Rj were concentration-dependent prolongations o f T . Recovery was observed after 15 min washout with A C S F (see next M  Figure).  (B) Concentration-response relationships for the effects o f procainamide on R  and T  M  (for each  concentration, n — 4—5). Fitting o f the respective curves yielded EC50 values for the increases o f 48 u M for Rj and 337 u.M for t  m  (see text for details).  (D) Current-voltage relationships o f the neuron in A . Procainamide produced  concentration-dependent increases in slope resistance and reduced the inward rectification in die hyperpolarized voltage range, here at membrane potentials ~—15 m V negative to  V. T  S. S C H W A R Z  59  contrast, procainamide produced concentration-dependent increases i n R i up to 169% o f control (Figure 2 3 A ) . T h e average increase i n R i was greatest at 1 m M (131.8 + 9.4% o f control; P — 0.04; n — 4). T h e fit o f a corresponding concentration-response (R  =  2  0.9997)  curve  yielded an ECso for the increase i n R i o f 48 u M (Figure 23B).  Concomitantly, procainamide prolonged T Consistent w i t h the results o n R i , the  to maximal values o f 2 1 7 % o f control.  m  3.vcr9.gc  incrc3.se i n  Tm  also was greatest at 1 m M  (157.2 + 24.7% o f control; Dunnett's multiple comparison test, P < 0.05; n = 4); the EC50 was 337 u M (concentration-response curve, R = 0.9985; Figure 23C). 2  T h e difference i n the EC50 for R i and T  m  (P = 0.04; potency ratio, 7.02) likely was  due to procainamide affecting active (i.e., voltage-dependent), i n addition to passive (resting, voltage-independent)  membrane properties. Procainamide increased apparent  resistance i n excess o f what w o u l d be predicted from T  m  = R C (cf. 1.3.1, page 24). Three  lines o f evidence provided confirmation o f this hypothesis. Firstly, the increase i n apparent resistance by 1 m M procainamide was associated w i t h reversible depolarizations of V  t  by up to 7 m V i n 3 out o f 4 neurons (not illustrated), implying blockade o f an  outward (hyperpolarizing) current that contributes to V , i.e., a leak (voltage-independent) t  K  +  current.  component  Secondly, analysis o f I-V to  procainamide's  relationships revealed  concentration-dependent  action  a  voltage-dependent to  increase  slope  resistance, evident as a reduction i n the inward rectification i n the hyperpolarized range (cf. 1.3.3), typically at potentials > 10 m V negative to V  m  (Figure 2 3 D ) . Thirdly, the best  goodness o f fit o f exponential functions to, the voltage responses to hyperpolarizing D C injection (cf. 1.3.1) i n the range where procainamide reduced inward rectification was  S. S C H W A R Z  60  achieved w i t h first order exponential functions under procainamide, as opposed to second order exponential functions under control conditions. F o r example, for the voltage responses o f the neuron shown i n Figure 2 3 A , the S D (5) o f the fitted curve i n the control was 0.221 m V for a first order exponential function and 0.095 for a second order  exponential  function. In contrast,  the  best fit i n the  presence  of 1 m M  procainamide was obtained with a first order exponential function (8 = 0.116 m V ; second order exponential function, 8 = 0.119 m V ) . T h i s implied that the voltage responses under control conditions were a result o f a combination o f passive and active membrane properties whereas i n the presence o f procainamide, the voltage responses were primarily a result o f passive membrane properties alone. I n combination, these data indicated that procainamide reduced both inward rectification and resting membrane  conductance,  giving rise to an increase i n apparent resistance, and, o n the whole, a more ohmic (linear) I - K relationship. Analysis o f procainamide's effects o n tonic and burst firing showed that the agent suppressed the fast, Na -dependent action potentials o f b o t h firing modes, consistent +  w i t h its N a channel blocking actions w e l l - k n o w n from the studies o n the antiarrhythmic +  properties (for review, see Zipes 1992 and R o d e n 2001). In the tonic mode o f firing, this was evident as a concentration-dependent reduction i n firing rate, w i t h 1 m M producing the greatest effect (Figure 24). In particular, procainamide at the higher concentrations preferentially increased the intervals between the initial spikes i n a train, implying a blockade o f spike accommodation. In the burst mode, procainamide similarly decreased the number o f fast spikes o n top o f L T S s (of. 1.3.4 and Figure 13). H o w e v e r , unlike  S. S C H W A R Z 61 lidocaine, procainamide, over a concentration range from 1 u.M—1 m M , d i d not shunt the LTSs.  A Control  Procainamide 10 uM  Recovery 100 uM  1 mM  B  A/ (PA) m  Figure 24 Effects o f procainamide o n tonic and burst firing (A) Shown are tonic and rebound burst firing responses to de- and hyperpolarizing D C injection (pulse duration, 500 ms) o f a neuron manually clamped at V  t  (-73 m V ) . Application o f procainamide (1 u M - 1 m M ; - 8 - 1 2 m i n per  concentration) reduced tonic firing frequency in a concentration-dependent fashion (accompanied by an increase in R;; see previous Figure). Whereas 1-100  u M affected  spike rate comparatively little (response to 1 u M not  illustrated), 1 m M produced a noticeable (38%) suppression o f firing. This was associated with a reduction in spike accommodation, evident as a preferential increase in the intervals between the initial spikes in a train. Unlike lidocaine, procainamide did not shunt rebound L T S s (arrow in control; cf. Figure 9 A & Figure 13C). However, procainamide reduced the frequency o f the fast action potential bursts on top o f the L T S s (asterisk). Full recovery o f tonic firing and partial recovery o f E i were observed after 15 min washout.  (B) Current-frequency relationships  o f the neuron in A . In a concentration-dependent fashion, procainamide shifted the current-frequency curve to the right and decreased its slope (curves for 1 & 100 u M not illustrated for enhanced clarity). Full recovery from the reduction in firing was observed after washout (sec above).  S. S C H W A R Z  62  A l t h o u g h neurons generally exhibited recovery from procainamide's effects, this was m u c h more readily observed for active membrane properties  (e.g., tonic firing;  of. Figure 24B) than for passive ones (e.g., Ri). Partial recovery from increases i n R i was observed i n all neurons included i n the analysis, typically after washout  times o f  — 15 m i n .  1.3.11.3 Bupivacaine Bupivacaine  (l-butyl-N-[2,6-dimemylphenyl]-2-piperidinecarboxamide;  MW,  324.9;  Figure 25) is, like lidocaine, an aminoamide local anesthetic (of. Figure 1). T h e agent was synthesized i n 1957 by Ekenstam as part o f a series o f N-substituted pipecolyl xylidine derivatives  that  also  included  mepivacaine  (l-methyl-N-[2,6-dimethylphenyl]-2-  piperidinecarboxamide) and ropivacaine (l-propyl-N-[2,6-dimethylphenyl]-2-piperidinecarboxamide; see II.1.4 later i n this thesis) (Ekenstam et al. 1957). C o m p a r e d to lidocaine, bupivacaine has a higher lipid solubility, a higher p K value (8.1 vs. 7.7), and is subject to a  more extensive plasma protein binding (~96% vs. ~65%). Clinically, bupivacaine is more potent, has a slower onset o f action, and produces longer lasting blockade than lidocaine. Bupivacaine  is  also  considerably  more  toxic  than  lidocaine, particularly  to  the  myocardium, and is not clinically used for systemic administration (Covino 1987). Bupivacaine plasma concentrations  following application for regional anesthesia vary  considerably depending o n route, dose, concentration, and author. Whereas m a x i m u m concentrations between 0.2 and 4.95 u,g/ml have been reported w i t h peak times between 5 m i n and over 1 h, typical values for most clinical procedures are below 2 u g / m l (see  S. S C H W A R Z  63  Aromatic head Amide linkage Amine tail  CH  3  F i g u r e 25 Structural formula o f bupivacaine  T u c k e r and Mather  1988 for review). W h e n present i n the  systemic circulation,  bupivacaine does not consistently produce the sedation and drowsiness or the E E G slowing and spindling that occur with lidocaine prior to the onset o f C N S excitation and generalized seizure activity. F o r example, there are no distinctive preconvulsive E E G alterations associated w i t h bupivacaine i n monkeys (Covino 1987). Reported bupivacaine b l o o d concentrations  associated w i t h convulsive behaviour are 3.05 p g / m l i n cats,  4.5 p g / m l i n monkeys, and between 5.4 and 74.0 p g / m l i n humans ( M u n s o n et al. 1975; Scott 1975; de J o n g et al. 1980; Agarwal et al. 1992; M c C l o s k e y et al. 1992). O n l y limited data  are  available o n  p l a s m a / C S F concentration  ratios  for  bupivacaine.  In  one  investigation o n patients undergoing ophthalmic surgery w h o received 20 m g bupivacaine for retrobulbar or facial blockade, the C S F / p l a s m a ratio was i n the range o f 0.56—1.33 (Le N o r m a n d et al. 1989). F o r conversion from p g / m l into p M , 1 p g / m l corresponds to 3.08 p M ; conversely, 1 u M corresponds to 0.325 p g / m l (see M W above). Here, i n order to widely cover concentrations relevant to the l o w subconvulsive as well as toxic range, bupivacaine was studied between 0.1 and 100 u M .  S. S C H W A R Z  64  T h e m a i n conclusion from the experiments with bupivacaine was that valid interpretation o f the. results was not reliably possible due to inability to achieve recovery (before cell loss) i n the present setting o f brain slice patch-clamp experimentation, likely a result o f the high lipid solubility o f this agent. Others have previously made similar observations i n in vitro studies (Rossner and Freese 1997). Stable recordings that allowed acquisition o f sequential concentration-response data o n the effects o f bupivacaine o n R i w i t h varying degrees o f recovery were obtained from n — 3 neurons. T h e results are presented qualitatively and no dedicated statistical analysis was performed. N o reversible decreases i n R i due to bupivacaine application were observed i n the concentration range from 0.1-100 u M . Analogous to Q X - 3 1 4 and procainamide, a high concentration o f bupivacaine (100 u M ) produced increases i n R i that exhibited partial recovery. Figure 26 shows the voltage responses o f a neuron that illustrate these findings. T h e fast action potentials o f tonic and burst  firing  also were blocked by 100 u M  bupivacaine (not illustrated); partial recovery was observed only after long washout times (> 30 m i n , similar to the report o f L i u et al. 2001) with normal A C S F .  /  S. S C H W A R Z 65  Control  0.1 uM Bupivacaine  1 uM Bupivacaine  5mV  40 pA  Figure 26 Effects o f bupivacaine o n input resistance Shown are voltage responses to hyperpolarizing D C injection (pulse duration, 500 ms; AV  m  < 10 m V ) o f a neuron  held at V, (—79 m V ) . Application o f bupivacaine (0.1—10 p.M), unlike lidocaine (Figure 8), produced no noteworthy decreases in Ri (in % o f control: 0.1 u M , 106; 1 u M , 102; 10 yM, 97; ~12 m i n o f application each). Application o f 100 u M bupivacaine increased Rj to a level o f 247% o f control. This was associated with a prolongation o f T  M  to  234% o f control (19.8 ms; 100 u M bupivacaine, 46.3 ms). Partial recovery from these effects was observed after 33 m i n o f washout with normal A C S F (in % o f control: Ri, 149; T , 110). Suprathreshold de- and hyperpolarizing M  D C injection at this time elicited fast action potentials o f tonic and burst firing (not illustrated; if. Figure 13).  S. S C H W A R Z  66  1.4 D i s c u s s i o n  1.4.1 Shunting inhibition: a novel effect of low lidocaine concentrations in the CNS T h e results presented i n this section o f the thesis have demonstrated that lidocaine suppressed tonic and burst firing i n thalamocortical relay neurons o f the V P L nucleus, k n o w n to participate i n the transfer o f somatosensory information, nociceptive signals, and i n general, the generation o f conscious states. Here, novel actions o f lidocaine have been identified in vitro that may account for the central analgesic and sedative effects in vivo. These actions occurred at concentrations m u c h lower than those k n o w n to block impulse conduction along peripheral nerve fibres (Boas et al. 1982; Tanelian and Brose 1991; Wallace et al. 1997b). T h e most intriguing finding was an effect that could not be attributed to N a channel blockade: lidocaine markedly reduced neuronal responsiveness +  to electrical stimulation by decreasing input resistance, shunting the current required for spike generation i n the tonic and burst firing mode. This effect predominated at lower concentrations (maximal amplitude at 10 u M ) and not at high concentrations (> 300 p M ) . O t h e r related agents that are structurally similar but clinically not endowed w i t h the systemic analgesic and sedative properties that lidocaine possesses (procainamide, and, under the conditions o f this study, bupivacaine) did not exhibit a similar effect.  S. S C H W A R Z  67  1.4.1.1 Clinical relevance o f concentrations F o r terms o f clinical relevance o f the lidocaine concentrations, 10 u M lidocaine H C I converts to approximately 2.7 [xg/ml (i.e., a 0.00027% solution).* A c c o r d i n g to the classic study by Usubiaga et al. (1967), cerebrospinal fluid (CSF) concentrations o f lidocaine i n humans following intravenous injection correlate to arterial concentrations w i t h a factor between 0.73 and 0.83. I n rabbits receiving a continuous lidocaine infusion intravenously, the correlation factor (with a 10 m i n latency between arterial and C S F sampling) ranges between  ~0.47 and 0.64 (Momota  et al. 2000).t Somewhat i n contrast  to  these  observations are the results o f one single recent study w h i c h found correlation factors between —0.06 and 0.08 i n humans (Tsai et al. 1998). Whereas this discrepancy remains unclear, the existing data imply that a C S F concentration o f 10 p M w o u l d correspond to arterial concentrations between ~0.16 and 2 u g / m l in vivo, w h i c h is precisely i n the range relevant to the subconvulsive C N S effects o f lidocaine (cf. 1.1).  'Based on the molecular weight o f lidocaine H C I o f 270.81, the 1% (i.e., 10 m g / m l ) solution familiar to clinicians corresponds to a concentration o f 36.93 m M . t i n the same study, whole brain lidocaine concentrations were significantly higher than those in the C S F or blood, indicating that lidocaine accumulates in cerebral tissue. T h e fact that mean whole brain lidocaine concentrations exceeded mean cortical concentrations provides indirect evidence for a preferential accumulation in subcortical tissues, e.g., thalamic nuclei.  S. S C H W A R Z  68  1.4.1.2 Previous investigations This is the first study to show that lidocaine decreases input resistance i n neurons o f the C N S . A l t h o u g h previous investigations demonstrated  that local anesthetics  reduce  neuronal excitability, most have focused o n concentrations i n the high micromolar to millimolar range. O n e exception is the study by K a n e d a et al. (1989) o n isolated C A 1 hippocampal pyramidal neurons, showing that lidocaine blocks voltage-dependent  Na  +  currents w i t h an IC50 around 400 u M but has no effect o n the N a currents below 30 u M , +  i n accordance w i t h the results o f the present studies. A n o t h e r exception and consistent w i t h the former is the report by Fried et al. (1995) that 10 u M lidocaine does not alter the configuration o f evoked population spikes i n hippocampal slices while reducing markers o f anoxic damage (e.g., A T P depletion). Finally, K u n o and Matsuura (1982) showed that lidocaine at l o w concentrations (< 10 u M ) suppresses E P S P s i n frog spinal motoneurons by a postsynaptic  mechanism  and proposed  a direct action o n the  postsynaptic  membrane. N o n e o f these studies, however, have reported effects o n input resistance or conductance. Butterworth et al. (1993) did investigate the effects o f extracellular lidocaine (50 uM—3 m M ) o n input resistance i n hippocampal C A 1 pyramidal neurons and found no decrease or increase. In contrast, the quaternary lidocaine analogues Q X - 2 2 2 (Puil and Carlen  1984), Q X - 5 7 2  (Segal 1988), and Q X - 3 1 4 (Xie and Sastry 1992), applied  intracellularly i n high (millimolar) concentrations, increase R i i n hippocampal neurons. Outside the C N S , i n cultured cardiac myocytes,' N i c k Sperelakis' group reported a "loss o f electrical excitability" associated w i t h depolarization due to local anesthetics (i.e., tetracaine and cocaine) more  than 30 years ago  (Sperelakis and L e h m k u h l 1968;  S. S C H W A R Z 69 H e n n and Sperelakis 1968). T h e millimolar concentrations membrane resistance but produced a blockade o f N a - K +  by the A T P a s e activators, B a Scholz Na  +  et al. (1998)  2 +  observed  studied d i d n o t change  A T P a s e , w h i c h was reversed  +  and S r . I n rat dorsal root ganglion ( D R G ) neurons, 2 +  that lidocaine blocks T T X - s e n s i t i v e  and -resistant  currents w i t h IC50S o f 42 and 210 |u.M, respectively. I n sheep cardiac Purkinje fibres,  A r n s d o r f & Bigger (1972) found that a l o w and clinically antiarrhythmic lidocaine concentration (21.4 uM)* reduces slope resistance as well as membrane time constant, and suggested that this is likely due to an increase i n K  +  conductance. T h i s observation,  coupled w i t h the report that application o f 400 n M lidocaine increases conductance i n isolated somata o f rat superior cervical ganglion neurons (Tabatabai and B o o t h 1990), is consistent w i t h the present findings that the decreased R i occurred only w i t h l o w concentrations o f lidocaine.  1.4.1.3 Physiological significance T h e shunting o f inputs and action potentials represents a powerful mechanism for inhibition i n the C N S . A s a result o f the reduction i n i , the neuron's capability o f m  temporal summation o f synaptic inputs potentially leading to threshold responses w o u l d be diminished — effects representing a form o f "temporal shunting". T h e observed consequences o f the shunt o n neuronal excitability were a marked increase i n the current threshold for action potential generation and a suppression o f tonic repetitive firing.  *It seems likely that the actual concentration was 18.5 u M , since the authors used the M W o f lidocaine (234.33), and not lidocaine H C I (270.81) as used in the experiments, for the conversion o f the concentration (5 ug/ml) studied.  S. S C H W A R Z  70  N o r m a l l y , thalamocortical neurons encode the intensity o f afferent somatosensory signals nearly linearly into firing frequency for "faithful" signal transmission to cortical centres (Mountcastle et al. 1963; Jahnsen and Llinas 1984a; Steriade et al. 1990). T h e findings that lidocaine shunts their tonic firing, and hence, disrupts the faithful transmission o f somatosensory  signals,  are  consistent  with  the  clinical  observations  of  sensory  disturbances and sedation at subconvulsive concentrations. T h e principle o f encoding stimulus intensity into firing frequency and spike patterns also is valid for nociceptive signals, including those transmitted by the V P L neurons (Apkarian and S h i 1994), e.g., via the spinothalamic tract (Simone et al. 1991). Indeed, the vast majority o f V P L nociceptive neurons i n rats or monkeys are o f the "wide-dynamic-range" ( W D R ) type that respond i n a graded fashion to stimuli ranging from innocuous to noxious, with their firing rate increasing w i t h stimulus intensity (Guilbaud et al. 1980; Kenshalo et al. 1980; Casey and M o r r o w 1983; C h u n g et al. 1986; A p k a r i a n and S h i 1994). It is noteworthy that initial studies i n anesthetized animals had failed to reveal V P L nociceptive neurons until experiments were conducted i n awake primates, indicating that such neurons are selectively modified by analgesic and anesthetic drugs (Casey and M o r r o w 1983). G i a n Poggio and V e r n o n Mountcastle wrote i n their classic 1963 paper o n properties o f V B neurons: "The more dynamic aspects of the system — the  temporal cadence of neural activity [...], the quantitative relation of central response to peripheral stimulus — all these appeared to he most severely affected by an anesthetic agent' (Poggio and Mountcastle 1963). Several other groups have since demonstrated that analgesics from a variety o f classes, including salicylates and opioids, depress the nociceptive activity i n V B  S. S C H W A R Z  71  neurons (Guilbaud et al. 1982; Braga et al. 1985; Carlsson et al. 1988). Whereas the effects of N S A I D s  o n membrane  properties o f thalamic neurons  have not been  studied  systematically, Barker & Levitan (1971) showed i n buccal ganglion cells o f the marine mollusc, Navanas inermis, that salicylate decreases membrane resistance and hyperpolarizes neurons. C o u p l e d with the subsequent  observations i n the thalamus that \x. o p i o i d  receptor agonists decrease input resistance o f neurons i n many nuclei including V P L (Brunton and Charpak 1998), these findings are consistent w i t h the hypothesis that a current shunt i n thalamocortical neurons is a c o m m o n mode o f action for drugs to produce analgesia i n mammals. T h e present in vitro findings that concentrations o f lidocaine that produce clinical analgesia effectively suppressed tonic firing i n V P L neurons correspond well to the observations from the in vivo studies and represent an attractive and plausible mechanism contributing to the systemic analgesic properties o f lidocaine.* Likewise, the observed shunting o f burst firing in vitro is consistent w i t h the in vivo efficacy o f lidocaine as a systemic analgesic i n patients suffering from various chronic pain syndromes: a hallmark feature i n such patients is the occurrence o f abnormal thalamic burst firing (Lenz et al. 1987, 1989; Tsoukatos et al. 1997), w h i c h is identical to the L T S burst firing seen i n intracellular and patch clamp recordings. It is conceivable that suppression by a lidocaine-induced shunt o f pathological nociceptive signals i n the form o f such burst discharges and tonic firing could contribute to the alleviation o f neuropathic pain seen w i t h lidocaine i n the clinical setting.  *A reduction o f firing rate in auditory fibres also has been suggested as a mechanism for lidocainc's effect to suppress tinnitus (cf. 1.1) (Huxtable 2000), and it seems possible from the present work that a shunting action in central auditory neurons plays a role.  S. S C H W A R Z  72  Central analgesic properties have also been demonstrated for cocaine, b o t h i n human and animal models (Yang et al. 1982; L i n et al. 1989). In in vivo experiments w i t h adult rats, I V cocaine suppresses nociceptive responses o f neurons i n medial thalamic nuclei (cf. 1.1.2, page 13) (Shyu et al. 1992). In contrast to lidocaine and other local anesthetics,  cocaine  possesses  the  unique  property  o f interacting w i t h  synaptic  norepinephrine and dopamine reuptake mechanisms (for reviews, see Garfield  and  G u g i n o 1987; Leshner 1996), and the results from Shyu et al. indicate that its central analgesic effects are mediated by dopamine D i and D 2 receptors. In contrast to the medial thalamic nuclei, cocaine has no effects o n neurons i n the lateral thalamus (which V P L belongs to), w h i c h may be due to the fact that these do not express either receptor subtype (reviewed i n Steriade et al. 1997, page 151). Nonetheless, these results further emphasize the thalamus' role i n analgesic drug action. Suppression o f tonic repetitive firing also is likely to be a major mechanism by which  a  variety  of  other  agents  exert  their  therapeutic  effects,  anticonvulsants, phenytoin, sodium valproate, and carbamazepine  including  the  (for review, see  M a c d o n a l d and K e l l y 1995). N o n e o f these drugs, however, are k n o w n to  decrease  resistance i n central neurons. Somewhat paradoxically, l o w concentrations o f lidocaine also are effective i n the treatment o f generalized tonic-clonic seizures and Status epilepticus (Taverner and B a i n 1958; Berry et al. 1961; K o p p a n y i 1962; Bernhard and B o h m 1965; L e m m e n et al. 1978), and suppression o f tonic repetitive firing due to a shunt may well be a mechanism critical to these actions. T h e volatile general anesthetic, isoflurane, applied i n clinically relevant concentrations, likewise decreases resistance and shunts tonic and  S. S C H W A R Z  73  burst firing i n V B neurons (Ries and P u i l 1993, 1999a). This observation is most striking i n view o f the clinical similarities between general and local anesthetics. L i k e lidocaine, isoflurane has sedative, analgesic, and anesthetic properties, and, like other general anesthetics, also is effective as an anticonvulsant (Kofke et al. 1985). In 1963, F r a n k and Sanders suggested that general and local anesthetics share a c o m m o n mechanism o f action i n the C N S (Frank and Sanders 1963). However, it is unlikely that the receptor mechanisms o f action o f isoflurane for generating the shunt is identical to lidocaine's. Isoflurane hyperpolarizes thalamocortical neurons by increasing a leak K  +  conductance  (Ries and P u i l 1999b) whereas lidocaine had little effects o n V or depolarized neurons. t  In addition to the reduction i n repetitive firing and the shunting o f input current, l o w lidocaine concentrations exerted a number o f other effects  that also may be  interpreted as being secondary to a decrease i n Rj. These include the observed reduction o f s A H P s and the suppression o f L T S bursts. W i t h regard to the former, an isolated reduction o f s A H P s , likely produced by small conductance Ca -activated K +  +  (SK)  channels (cf. Jahnsen and Llinas 1984a,b), is not compatible w i t h a decrease i n firing frequency (cf. F o e h r i n g et al. 1989). Furthermore, lidocaine (1 m M ) blocks Ca -acrivated +  K  +  channels i n hippocampal neurons (Oda et al. 1992), b u t these channels are o f the high  (or "big") conductance ( B K ) type, possibly similar to those expressed i n V B (Biella et al. 2001). W i t h regard to the latter, there is g o o d evidence that the C a mediate the T-type C a  2 +  2 +  channels that  current (and thus, the L T S s ) i n V P L neurons are primarily  distributed at proximal dendritic locations ( Z h o u et al. 1997; Destexhe et al. 1998;  S. S C H W A R Z  74  Williams and Smart 2000).* F r o m such a distribution, it w o u l d be expected that a p r o n o u n c e d shunting effect w o u l d result from a significantly decreased Ri. O n the other hand, the findings that lidocaine suppressed  L T S bursts even after hyperpolarizing  thalamocortical neurons to a voltage range where one w o u l d anticipate a maximal deinactivation o f IT raise the possibility that lidocaine may directly decrease IT (cf A k a i k e and Takahashi 1992). Since the p r o x i m a l dendritic regions o f thalamocortical neurons receive excitatory synaptic connections from primary afferents, L T S s likely play a role i n the amplification o f sensory inputs (Williams and Stuart 2000). T h i s is supported by experimental evidence that IT amplifies subthreshold E P S P s and IPSPs (von K r o s i g k et al. 1993; Turner et al. 1994; Williams et al. 1997a; K i m and M c C o r m i c k 1998). T h e analgesia, sedation, and sensory disturbances produced by lidocaine in vivo are consistent w i t h an inhibition o f such an amplification o f afferent sensory signals.  1.4.1.4 Effects o f G A B A A receptor blockade I n the literature, pharmacological correction o f a relative G A B A e r g i c hypofunction at thalamic levels has been suggested as a mechanism critical to the treatment o f central pain (Rausell et al. 1992; Canavero and Bonicalzi 1998), i n w h i c h lidocaine is effective (cf. Table 1). Intracerebral injections o f lidocaine obtund the antinociceptive actions o f the G A B A A receptor antagonist, picrotoxin, i n cats (Koyama et al. 1998). In addition to  'There still is some uncertainty about the precise subtype o f C a  2 +  channel(s) that mcdiate(s) IT in V P L neurons. O n l y  recently have three distinct a subunits (termed alG, alH, and all) been cloned that exhibit hallmark characteristics o f native T-type Ca currents and arc designated the gene subfamily, C a y T (Perez-Reyes et al. 1998; Cribbs et al. 2+  1998; Lee et al. 1999; Montcil et al. 2000). O f these, a l G is expressed in the thalamus in relay (including V P L ) and ' intralaminar nuclei whereas a l H and a l l arc found in the reticular nucleus (Talley et al. 1999).  S. S C H W A R Z 75 the central analgesic effects, lidocaine's anticonvulsant properties (tf. page 72) have also been speculated to result from G A B A e r g i c stimulation ( N o r d m a r k and Rydqvist 1997). T h i s is supported by the observation o f Smith et al. (1993) that lidocaine terminates bicuculline-induced seizures i n rats. I n contrast, Stone and J avid (1988)  found  that  lidocaine is mostly ineffective against the convulsant effects o f picrotoxin and bicuculline i n mice. It is well k n o w n from in vivo and in vitro studies that bicuculline increases evoked firing i n thalamocortical neurons (Hicks etal. 1986; V a h l e - H i n z etal. 1994; L e e etal. 1994; M c C o r m i c k et al. 1995; G a o et al. 1997). T h i s is consistent w i t h the present observations that bicuculline reversed the decrease i n firing rate produced by lidocaine. H o w e v e r , the fact that bicuculline d i d not antagonize the lidocaine-induced shunt implies that the effects o f lidocaine and bicuculline o n tonic firing result from interaction with distinct targets. Despite increasing the firing rates, bicuculline did not b l o c k the decrease i n Rj and had n o effect o n the lidocaine-induced shunting o f tonic and burst firing or A H P s . It is possible that bicuculline may have affected a shunt i n regions distal to the recording site, but this remains speculative. I n a recent report, bicuculline (5-40 p M ) blocked the apamin-sensitive A H P component due to JAHP i n thalamic reticular neurons (Debarbieux et al. 1998), similar to observations Johansson  i n other  preparations  (Johnson  and Seutin 1997; Seutin et al. 1997;  et al. 2001). Bicuculline methyl derivatives potently b l o c k b o t h  apamin-  sensitive and -insensitive S K channels, responsible for the A H P s , i n outside-out patches o f Xenopus laevis oocytes (Khawaled etal. 1999). Whereas a similar action o f bicuculline i n  S. S C H W A R Z  76  this study cannot be excluded with certainty (cf. Figure 19C), the observed A H P reductions produced by lidocaine are unlikely due to a predominant blockade o f JAI-IP, because, as discussed earlier, an opposite effect o n tonic firing w o u l d be expected ( F o e h r i n g t f a / . 1989). I n conclusion, the results o f this set o f experiments do not support the hypothesis that the effects produced by l o w lidocaine concentrations i n thalamocortical neurons are mediated by G A B A A receptor activation.  1.4.1.5 Molecular and cellular actions o f lidocaine Lidocaine exerted multiple and overlapping actions that affected resistance. This was evident by the distinct, non-classical concentration-response relationship (cf Figure 8), implying that the local anesthetic had at least one action that decreased Ri at lower concentrations, and at least one action that caused R i to "return" to (and i n some cases, past) c o n t r o l values at high concentrations. It is well k n o w n that local anesthetics are far from being specific agents with regard to their molecular actions and have many effects i n addition to their classic blockade o f N a  +  channels. Lidocaine, for example, has been  found to interact w i t h a wide variety o f other membrane-associated proteins, including K  +  channels, C a  2 +  channels, substance P receptors, and second messengers, to name but  a few (for examples o f selected reviews o n these topics, see Butterworth and Strichartz 1990; Arias 1999; H o l l m a n n and D u r i e u x 2000; H o l l m a n n et al. 2001c). Table 4 gives a comprehensive overview o f the plethora o f the reported molecular and cellular effects o f local anesthetics and illustrates the dichotomy that is associated w i t h many o f them. T h e  S. S C H W A R Z 77  Table 4 O v e r v i e w o f reported molecular and cellular actions o f local anesthetics General neuronal actions Blockade of Na+ channels (Shanes et al. 1959; Taylor 1959; Hillc 1966; Ragsdale et al. 1991) Blockade of K channels (Taylor 1959; Oda et al. 1992; Olschcwski et al. 1998; Komai and McDowell 2001) Blockade of Ca channels (Frelin et al. 1982; Palade and Aimers 1985; Oyama et al. 1988; Sugiyama and Mutcki 1994) Increase and decrease in HVA Ca currents (Liu el al. 2001) Blockade of nicotinic ACh receptors (Stcinbach 1968; Neher and Steinbach 1978; Ruff 1982; Gentry and Lukas 2001) Blockade of muscarinic ACh receptors (Fields et al. 1978; Aguilar et al. 1980; Hollmann el al. 2000) Blockade of opioid receptors (Craviso and Musacchio 1975) Blockade of a adrenoceptors (Fairhurst et al. 1980) Inhibition of substance P binding to NK1 receptors (Li et al. 1995) Excitation of hcat-/capsaicin-sensitivc nociceptors (Vlachova et al. 1999) Increase in synaptic dopamine concentration (Graham et al. 1995) Enhancement of GABAergic neurotransmission (Granger et al. 1995; Nordmark and Rydqvist 1997) Inhibition of glutamate-induced excitation, possibly via action on glycine receptors (Biella and Sotgiu 1993) Inhibition of quisqualate-mediated excitation through a glycinc-likc action (Biclla et al. 1993) Enhancement of NMDA-mcdiatcd excitation (Biella et al. 1993) Inhibition of axonal transport (Fink et al. 1972; Anderson and Edstrom 1973; Kanai et al. 2001) Activation of calmodulin-dependcnt protein kinase II (Kanai et al. 2001) +  2+  2+  Cardiac actions Blockade of Na channels (Weidmann 1955; Hondeghem and Katzung 1977; Grant et al. 1989) Increase in K conductance (Arnsdorf and Bigger 1972) Blockade of K+ channels (Valcnzucla et al. 1995; Olschcwski et al. 1996) Inhibition of Ca currents (Chapman and Leoty 1981; Sanchcz-Chapula 1988; Rossncr and Frccsc 1997) Inhibition of CICR (Endo 1977; Stephenson and Wendt 1986; Zahradnikova and Palade 1993) Blockade of Na+-K+ ATPase (Henn and Sperelakis 1968) +  +  2+  Reduction of diazoxidc-induced mitochondrialflavoproteinoxidation (Tsutsumi el al. 2001) Actions pertinent to neuro- and cardioproleclion Inhibition of cerebral oxygen and glucose consumption (Astrup et al. 1981) Depression of synaptic activity (Schurr et al. 1986) Protection against hypoxic damage and ATP depletion (Lucas et al. 1989; Fried et al. 1995) Delay and reduction of anoxic depolarization (Belousov et al. 1995; Liu et al. 1997; Chen et al. 1998) Depression of anoxic hyperpolarization (Belousov et al. 1995) Inhibition of hypoxia-induced [Ca ]i increase (Liu et al. 1997; Chen et al. 1998) Inhibition of excitatory amino acid release/accumulation (Fujitani et al. 1994; Terada et al. 1999) Scavenging of hydroxyl radicals and singlet oxygen (Das and Misra 1992) 2+  Hematologic & miscellaneous actions Inhibition of platelet aggregation (Fcinstcin et al. 1976; Borg and Modig 1985) Inhibition of thromboxane A2 receptor-mediated signalling (Kohrs et al. 1999) Inhibition of leukocyte phagocytosis, metabolic activity, migration, and superoxide anion production (Cullcn and Haschke 1974; Stewart et al. 1980; Eriksson et al. 1992; Hollmann et al. 2001b) Inhibition of macrophages (Ogata et al. 1993) Reduction in collagen and glycosaminoglycan synthesis (Chvapil et al. 1979) Activation and inhibition of adenylyl cyclase (Gordon et al. 1980; Voeikov and Lcfkowitz 1980) Inhibition of agonist binding to P adrenoceptors (Voeikov and Lcfkowitz 1980) Inhibition of actomyosin motility (Tsuda et al. 1996) Reduction of lysophosphatidatc receptor mediated Ca -activated CI currents (Nietgcn et al. 1997) Inhibition and stimulation of mitochondrial phospholipasc A2 (Waitc and Sisson 1972) Inhibition of lysosomal phospholipasc At & A2 (Waite and Sisson 1972) Inhibition of leukotriene B4 & intcrleukin-loc release (Sinclair el al. 1993) Inhibition of histamine release (Yanagi et al. 1996) Inhibition of PGE2 receptor-mediated signalling (Nollet et al. 2001) Inhibition of GcCq protein function (Hollmann et al. 2001a) Enhancement of Goti protein function (Benkwitz et al. 2001) 2+  Adapted in part and modified from Hollmann and Duricux (2000). F o r abbreviations see appendix.  S. S C H W A R Z 78  latter, i.e., the fact that local anesthetics exert concentration-/dose-dependent multiphasic effects o n the nervous system has been recognized for many decades. Perhaps the most prominent example is represented by the in vivo observation discussed earlier that lidocaine induces generalized (grand mal) seizures w h e n administered at high doses whereas it is an effective anticonvulsant at l o w doses (Taverner and B a i n 1958). M o r e recent examples from in vitro studies include the finding that procaine at concentrations as low as 2 )J.M produces an excitatory inward current i n rat D R G heat-/capsaicin-sensitive nociceptors while blocking N a conductance at higher concentrations (Vlachova et al. +  1999). A l s o i n D R G neurons from newborn rats, ropivacaine (II. 1.4) at 10 and 30 u M markedly increases a high voltage-activated C a  2 +  current ( " H V A - J c a " ) but decreases it at  > 50 u M ( L i u et al. 2001) (cf. 1.4.2.1). T h e phenomenon that systemic lidocaine exerts multiphasic/dichotomous effects has similarly been documented outside the nervous system. F o r example, whereas high concentrations are associated w i t h vasodilatation, hypotension a n d cardiovascular collapse, l o w concentrations (between 10 and 103 n g / m l in  one study) produce vasoconstriction and increase b l o o d pressure  both in vivo  (Blair 1975; A p s and Reynolds 1976; Johns etal. 1985; Wallace etal. 1997b) and in vitro (Gherardini*/*/. 1995). A l t h o u g h it is evident from the results o f the present study that the lidocaineinduced shunt occurs i n a context o f multiple overlapping effects  o n membrane  properties, several inferences about the underlying targets and mechanisms can be made. Firstiy, the observation that lidocaine still produced decreases i n resistance during T T X application implies that a postsynaptic action contributes to the shunt. H o w e v e r , the  S. S C H W A R Z  79  possibility that lidocaine may act o n nerve terminals or neurons presynaptic to the recorded electrode to produce the changes i n membrane properties cannot be excluded w i t h absolute certainty. Secondly, as discussed i n 1.4.1.4, the results obtained i n the experiments w i t h bicuculline do not support the hypothesis that the shunt produced by l o w lidocaine concentrations is mediated by G A B A A receptors. Thirdly, the results w i t h Q X - 3 1 4 indicate that the  decrease i n resistance  due  to lidocaine is mediated  by  interaction w i t h an intracellular target. T h i s w o u l d not represent a surprise, since the bestk n o w n action o f lidocaine, the blockade o f voltage-gated N a enhancement  +  channels through an  o f channel inactivation, is mediated chiefly by its charged cation via an  intracellular ("hydrophilic") pathway (reviewed by Catterall 1987, 1995; Butterworth and Strichartz 1990). H o w e v e r , this action is not compatible w i t h the observed decrease i n resistance, nor is the so-called "open channel b l o c k " o f N a  +  channels (see Butterworth  and Strichartz 1990 for review). It remains a possibility, however, that lidocaine, i n l o w concentrations, may have affected the persistent N a 1996). I n thalamocortical neurons,  JNaP  +  current,  iNaP  (for review, see C r i l l  amplifies depolarizations i n the perithreshold  range (Jahnsen and Llinas 1984b; Parri and Crunelli 1998). Lidocaine (12.5-25 u M ) selectively blocks this current i n cardiac myocytes (Ju et al. 1992). T h e lower slope o f the I-V curve between Vi and — 5 0 m V (Figure 9) (if. Stafstrom et al. 1985), decreased amplitudes o f voltage responses to depolarizing current, and reduction i n tonic firing frequency during application o f 10 u M lidocaine are consistent w i t h a blockade o f I^ v i n a  the subthreshold range. T h e precise molecular basis o f 7NaP is still unclear, however, and the location o f lidocaine's binding site for its blocking effect remains speculative. B e y o n d  S. S C H W A R Z  80  voltage-gated N a channels, lidocaine interacts with a large number o f other intracellular +  targets that include i o n channels, receptors, and enzymes. Whereas documented effects are summarized i n Table 4, there likely exists an abundance o f targets that are yet unidentified. T h e future elucidation o f the latter w i l l shed more light o n the specific molecular mechanisms that mediate the lidocaine-induced decrease i n R i and other actions o f the local anesthetic. A s i n the case o f the definition o f the structure and function o f voltage-gated i o n channels, techniques o f molecular biology w i l l likely be instrumental i n this endeavour.  1.4.1.6 Lidocaine and E E G spindle waves It remains b o t h intriguing and unclear h o w l o w lidocaine concentration could produce the transient spindle waves i n the mammalian E E G (cf. 1.1.2, page 11) by action o n thalamic neurons. Whereas the mechanisms that underlie the generation o f these recurring, waxing and waning synchronized 7—14 H z oscillations by the thalamocortical system (specifically, a complex interplay between thalamic relay, reticular/perigeniculate, and neocortical neurons) have been studied i n detail b o t h in vivo and i n slice preparations in vitro (see reviews i n Steriade and M c C a r l e y 1990; Steriade and M c C a r l e y 1990; Steriade et al. 1997), lidocaine has not been investigated i n these models (see 1.4.4 later i n the Discussion). Critically involved i n spindle generation are the ionic currents, IT (cf. 1.3.8) and Jh (cf. 1.3.3). Physiologically, thalamic spindling involves induction o f rebound L T S s i n relay neurons by repetitive IPSPs from reticular/perigeniculate neurons. T h e L T S s are associated w i t h an increase i n [Ca ]i, w h i c h activates and upregulates K (Luthi and 2+  S. S C H W A R Z  81  M c C o r m i c k 1998). Persistent activation o f Ih i n turn depolarizes the relay neurons such that the I P S P s no longer trigger rebound L T S s , w h i c h causes the spindle waves to wane. It is possible that the disappearance o f the transiently occurring spindles associated w i t h low lidocaine b l o o d concentrations involves a similar mechanism: i n the presence o f a lidocaine-induced shunt as observed i n the present study, IPSPs w o u l d lose their ability to elicit rebound L T S s , w h i c h w o u l d cause the spindle waves to cease. It remains unclear, however, h o w lidocaine could induce the appearance o f the spindles. Possible hypotheses yet to be tested include excitation o f (or removal o f inhibitory influences on) reticular neurons, leading to an enhancement  o f rhythmic IPSPs. A n o t h e r possibility is that  lidocaine acts o n Ih. In thalamocortical neurons, Ih can be modulated directly or indirectly, e.g., via adenylyl cyclase ( M c C o r m i c k and Pape 1990a; Lee and M c C o r m i c k 1996; L u t h i et al. 1998). Whereas its enhancement abolishes spindle waves as discussed above, blockade o f Ih produces continuous spindling by blocking the refractory period after waves (Bal and M c C o r m i c k 1996; L i i t h i et al. 1998). H o w e v e r , a blockade o f Ih w o u l d not produce a decrease i n Ri, and no consistent effect o f lidocaine o n inward rectification was found i n I- Vrelationships  that w o u l d imply such an effect (cf. 1.3.6).  1.4.2 Effects of high lidocaine concentrations A t high concentrations (300 uM— 1 m M ) , lidocaine produced the expected signs o f N a  +  channel blockade (Weidmann 1955). These concentrations correspond to those i n studies on  in vitro peripheral nerve preparations  (for review, see Strichartz 1976) and  are  associated w i t h generalized C N S / c a r d i o v a s c u l a r depression and death w h e n present i n  S. S C H W A R Z  82  the systemic circulation in vivo. T h e refractoriness to lidocaine o f the first spike i n a train and the increasing depression o f subsequent spikes observed i n the present experiments were similar to findings by others i n hippocampal C A 1 neurons (Capek and E s p l i n 1994). T h i s resilience o f the first spike most likely results from the high density o f N a  +  channels  i n the area o f the axon hillock near the electrode, compared to more distal regions. W h e n high lidocaine concentrations were applied i n the presence o f T T X , a selective increase i n apparent resistance i n the depolarized voltage became evident (Figure 16B). T h i s was indicative o f an effect o n voltage-dependent  conductances and  similar to the "anomalous rectification" described by H o t s o n et al. (1979). O n e possible mechanism for this observation is a blockade o f a voltage-dependent  K  +  (outward)  current. It is well k n o w n that high concentrations o f local anesthetics block voltage-gated K  +  channels (see reviews by Strichartz and Ritchie 1987; Butterworth and Strichartz  1990) (cf. Table 4). A l t h o u g h it w o u l d seem reasonable to assume that lidocaine may exert similar actions i n thalamocortical neurons, no consistent changes i n V  t  (in this case,  depolarizations) occurred concomitantly when T T X was present to indicate a reduction in K  +  conductance.  However, the depolarizations seen i n some neurons  following  application o f 100 p M lidocaine alone (1.3.5) are i n accord w i t h such an effect and resemble  the  findings w i t h procainamide, discussed later (1.4.3). A n o t h e r possible  mechanism is the activation/unmasking by lidocaine o f a subthreshold (inward) booster current that w o u l d amplify depolarizing stimuli. Whereas H o t s o n et al. postulated that a noninactivating  Na /Ca  depolarization, C d  +  2 +  2 +  current  may  contribute  to  a  resistance  increase  with  application i n the present study did not block the observed increase  S. S C H W A R Z  83  i n apparent resistance i n the depolarized range (cf. Figure 17C). A n o t h e r example o f such a booster current is / N a P , discussed earlier (cf. page 79). H o w e v e r , / N a P activation seems unlikely since lidocaine blocks this current Qu et al. 1992). T h e most likely explanation thus is a m i x e d effect that may consist o f a combination o f K  +  channel blockade and  activation o f an unidentified slow inward current. It is noteworthy that high concentrations o f lidocaine have been s h o w n to be neurotoxic (Ready et al. 1985), w h i c h clinically may manifest as "transient neurologic symptoms" ( T N S ) or Cauda equina syndrome following subarachnoidal administration (Rigler et al. 1991; A u r o y et al. 1997; Lambert and Strichartz 1998). Lidocaine 5% (~185 m M ; cf. page 67) produces irreversible conduction blockade i n bullfrog sciatic nerve preparations in vitro (Lambert et al. 1994). Similar results were obtained by others for 40 m M lidocaine (Bainton and Strichartz 1994). In rat D R G in vitro preparations, lidocaine concentrations > 30 m M depolarize neurons irreversibly and induce cell death ( G o l d et al. 1998). In crayfish giant axons, 40 and 80 m M lidocaine irreversibly block action  potentials  (Kanai et al.  1998).  A  recent  study  demonstrated  that  such  concentrations can damage neuronal cell membranes through the generation o f a physical leak (Kanai et al. 2000). A l t h o u g h the precise mechanisms that underlie the lidocaineinduced increases i n membrane conductance i n the present study remain unclear, this effect occurred at significandy lower concentrations, was fully reversible, and at the concentration that produced the greatest magnitude (10 p M ) was not associated w i t h consistent changes i n V . Hence, lidocaine neurotoxicity does not represent a plausible t  explanation for the observed lidocaine-induced shunt i n V P L neurons. O n the other  S. S C H W A R Z  hand, the results o n the effects o f high lidocaine concentrations Ca  2 +  84  o n high threshold  spikes may indeed bear significance for its neurotoxicity. A discussion o f these data  follows below.  1.4.2.1 Effects o n high threshold C a  2 +  spikes  Lidocaine, applied at high (> 300 u M ) , clinically C N S - t o x i c concentrations, reversibly unmasked high threshold C a  2 +  spikes i n the V P L neurons. These novel findings are i n  contrast to previous observations various C a  2 +  conductances  i n other tissue preparations that lidocaine blocks  (see Table 4). O n the other hand, the observations  are  consistent w i t h the results o f M u l l e et al. (1985), w h o reported an unmasking o f fast prepotentials by intracellular Q X - 3 1 4 and concluded that these represent dendritic C a  2 +  spikes. In D R G neurons from newborn rats, ropivacaine (cf. II.1.4) at 10 and 30 u M markedly increases a high voltage-activated C a  2 +  current ( " H V A - J c a " ) but decreases it at  > 50 u M ( L i u eta/. 2001) (cf page 78). What  possible  functional  implications do  these results  have?  The  overall  physiological role o f H T S s i n thalamocortical neurons is incompletely defined above). Some authors have suggested that H V A C a  2 +  thalamic relay neurons by triggering C a - i n d u c e d C a 2+  and subsequent activation o f Ca -dependent K 2+  +  2 +  (see  currents regulate tonic firing i n release from intracellular stores  currents (Ik(Ca)) (Hernandez-Cruz and  Pape 1989; G u y o n and Leresche 1995; Kammermeier and Jones 1997; Z h o u et al. 1997; B u d d e et al. 2000). I n addition to these putative physiological functions, H V A C a channels  2 +  have been implicated i n neurotoxicity. T h e pathogenesis o f neurotoxicity  S. S C H W A R Z  involves several mechanisms that lead to an increase i n [Ca ]i. H V A C a 2+  2 +  85  channels  participate i n this process b o t h directly and indirectly. T h e y directly facilitate C a  2 +  flux  intracellularly, w h i c h , partially due to their slow inactivation, may produce substantial increases i n [Ca ]i (Hernandez-Cruz and Pape 1989; Z h o u et al. 1997). Alternatively, 2+  H V A Ca  2 +  channels, particularly those o f the N-type, may increase [Ca ]i indirectly by 2+  mediating excitatory amino acid ( E A A ) release (Pfrieger et al. 1992; Takahashi and M o m i y a m a 1993) and subsequent E A A - i n d u c e d excitotoxicity (reviewed i n C h o i 1992). Hence, the present findings may have significance for the C N S toxicity o f high lidocaine concentrations seen in vivo. However, the fact that lidocaine unmasked H T S s under conditions  of  presynaptic  transmitter  release  blockade  by T T X renders  indirect  mechanisms o f [Ca ]i release less likely to be relevant for lidocaine's neurotoxicity. 2+  It is noteworthy that the H T S s i n this study, although otherwise similar, had voltage thresholds that were up to ~15—20 m V higher than those reported i n rat M G B by Tennigkeit et al. (1998b). O n the other hand, Ries and P u i l (1999a) showed i n rat V B H T S s w i t h thresholds comparable to those observed here. Whereas these discrepancies remain unclear, it seems possible that H T S s expressed by neurons i n different thalamic nuclei have distinct properties a n d / o r are generated by different C a  2 +  channel subtypes  (see below). Previous studies dedicated to isolate the specific subtypes o f H V A C a currents  that produce H T S s i n thalamocortical neurons  2 +  channels/  have shown that they  are  mediated by a composite o f N-type, L-type, and residual "R"-type currents, w i t h the latter representing the major component (Pfrieger et al. 1992; G u y o n and Leresche 1995).  S. S C H W A R Z  86  In the light o f the lack o f readily available pharmacological tools that are specific for this " R " component, no attempts were made i n this study to duplicate these findings or further characterize the H V A C a  current beyond its well-documented sensitivity to  2 +  Cd . 2 +  1.4.3 Effects ofprocainamide on membrane properties Since there are only few reports i n the literature o n procainamide action i n the nervous system, its observed effects i n the present study are briefly discussed here separate from lidocaine's effects. Previously demonstrated effects o f procainamide o n nervous tissue include inhibition  o f sympathetic  nerve  activity (Dibner-Dunlap et al. 1992)  and  acoustically evoked brainstem potentials (Lenarz et al. 1984). T h e present w o r k represents the first study o f procainamide's effects o n membrane properties o f thalamic neurons. In cardiac  tissue,  procainamide's  ionic mechanism  o f action is well  antiarrhythmic concentrations, it includes blockade o f fast N a contrast to lidocaine) blockade o f K  +  +  established.  At  channel activity and (in  channels, giving rise to an increase i n cardiac action  potential duration (Echt et al. 1989). Consequendy, b o t h agents belong to  different  subgroups i n the Vaughan Williams classification o f antiarrhythmic drugs (procainamide, class I A ; lidocaine, class IB) (reviewed by Bigger and H o f f m a n 1990). A s i n the heart, the observed effects o f procainamide i n thalamic neurons were i n contrast to those o f lidocaine. T h e outstanding feature o f procainamide i n the present study was that it d i d not share lidocaine's effect to decrease R i and shunt tonic and L T S burst firing. I n fact, procainamide's effects i n the thalamus were i n harmony w i t h the observations  from  S. S C H W A R Z  87  cardiac tissue. Whereas its action to decrease tonic firing rate was consistent w i t h the classic N a These  +  channel blockade, the agent also exhibited features o f K  included  decreases  in  resting  conductance  associated  +  channel blockade. with  membrane  depolarizations and a reduction i n inward rectification i n the hyperpolarized potential range. In summary, procainamide affected firing behaviour, membrane potential, and input resistance i n thalamic neurons and produced effects that implied blockade o f N a and K  +  +  channels. Whereas the results obtained here likely have significance for the C N S  effects o f procainamide observed in vivo, the absence o f a shunting action may explain w h y procainamide does not share the analgesic and sedative properties o f lidocaine.  1.4.4 Limitations andfuture outlook A n implicit limitation o f this study results from the fact that it is based o n recordings o f single neurons in vitro. Whereas such experiments yield crucial information about drug action o n membrane properties and firing rates/patterns, and hence, signalling, recent evidence indicates that information coding i n the C N S also is carried out i n terms o f temporal and spatial coordination o f action potentials across groups o f many neurons (Gray et al. 1989; deCharms and Merzenich 1996). T h e properties o f neuronal networks i n turn impact membrane properties o f individual cells, w h i c h for the thalamo-cortical system has been studied and reviewed extensively by Mircea Steriade (2001). T h u s far, the available information about the actions o f lidocaine o n the dynamics and signalling patterns o f whole neuronal populations largely is indirect and comes from in vivo E E G studies {cf. 1.1.2, page 11). Hence, future research o n the effects o f lidocaine (and other  S. S C H W A R Z  88  C N S - a c t i v e agents) o n the (e.g., nociceptive) responses o f entire groups o f neurons recorded simultaneously has the potential to greatly add to the understanding o f its actions i n the brain. F r o m the perspective o f the present study, the recent w o r k by A p k a r i a n and coworkers o n the network properties  o f nociceptive V P L neurons  represents a step i n this direction (Apkarian et al. 2000).  1.4.5 Summary and conclusions T h e major implication o f this section o f the thesis is that lidocaine, at clinically relevant, subconvulsive concentrations, is capable o f inhibiting thalamocortical signal transmission by a shunting mechanism not previously described for local anesthetics i n the C N S . T h e findings provide further support for the hypothesis that thalamocortical neurons are a crucial site o f anesthetic and analgesic drug action (Sugiyama et al. 1992; A n g e l and L e B e a u 1992; Ries and P u i l 1993, 1999a,b; Tennigkeit et al. 1997; D e t s c h et al. 1999; B o n h o m m e et al. 2001).  S. S C H W A R Z  89  S E C T I O N II: C L I N I C A L STUDIES  Analgesic properties of ropivacaine 0.2% in femoral 3-in-1 blockade for arthroscopic anterior cruciate ligament repair  Results from this section o f the thesis have appeared i n the following publication: Schwarz S K W , Franciosi L G , Ries C R , Regan W D , D a v i d s o n R G , N e v i n K , E s c o b e d o S, M a c L e o d B A : A d d i t i o n o f femoral 3 - i n - l blockade to intra-articular ropivacaine 0.2% does not reduce analgesic requirements following arthroscopic knee surgery. Can J A.nesth 1999, 46: 741-747.  S. S C H W A R Z  90  II.l Introduction  II. 1.1 Overall aim and specific objective Whereas the previous section o f the thesis focused o n laboratory studies o n the possible mechanisms for the central analgesic actions o f a well-established local anesthetic agent (i.e., lidocaine), the overall aim o f this section is to explore aspects at the other end o f the spectrum o f research o n analgesic properties o f local anesthetics. Here, clinical studies are directed to investigate the analgesic efficacy o f a newly introduced agent,(i.e., ropivacaine; see below) w h e n administered peripherally. T h e paradigm selected for this purpose was the use o f ropivacaine 0.2 % for "femoral 3 - i n - l " lumbar plexus blockade i n knee surgery. T h e specific objective was to test the hypothesis that the addition o f a preincisional femoral 3 - i n - l b l o c k w i t h ropivacaine 0.2% to standard intra-articular local anesthetic instillation at the end o f surgery improves postoperative pain control i n patients undergoing arthroscopic anterior cruciate ligament reconstruction ( A C L R ) under general anesthesia.  II. 1.2 Background ACLR  is  a  common  procedure  that is  frequently  associated  with  considerable  postoperative pain (Matheny et al. 1993; Reuben et al. 1998). W h e n managed w i t h opioids, pain relief is often unsatisfactory, and associated untoward effects  such as nausea,  vomiting, and urinary retention delay recovery and prolong in-hospital stay (Marks and Sachar 1973; A u s t i n et al. 1980; Matheny et al. 1993). Several reports indicate that postoperative pain following various lower limb procedures including A C L R may be  S. S C H W A R Z 91 reduced by regional anesthetic approaches to the lumbar plexus and femoral nerve (Coad 1991; H o o d etal. 1991; L y n c h etal. 1991; Matheny etal. 1993; Tetzlaff etal. 1997). In a recent uncontrolled study ( E d k i n et al. 1995), the need for administering opioids following A C L R under general anesthesia was eliminated i n 9 2 % o f patients receiving a "femoral 3 - i n - l block", the inguinal paravascular approach to lumbar plexus blockade first described by W i n n i e et al. (1973). However, n o randomized controlled trial had evaluated the efficacy o f a femoral 3 - i n - l block for postoperative analgesia following ACLR.  II. 1.3 Femoral 3-in-1 block T h e sensory innervation o f the lower extremities (Figure 27) consists o f nerve fibres that originate from two principal sources: the lumbar plexus ( T 1 2 / L 1 — L 4 / L 5 ) and the sacral plexus (L4/L5—S2/S3) (Bridenbaugh 1988). These fibres are the provenance o f the four major nerves that supply the leg: the femoral (L2—L4), obturator (L2—L3), and lateral femoral cutaneous (L2—L3) nerves (AT. femoralis, N. obturatorius, and N. cutaneus femoris lateralis) from the lumbar plexus, and the sciatic nerve (N. ischiadicus; L4—S3) from the sacral plexus.  S. S C H W A R Z 92  Figure 27 Sensory innervation o f the lower extremities (Adapted and modified from Bridcnbaugh 1988)  I n 1973, A l o n P . W i n n i e and colleagues described a method to block the three (i.e., the femoral, obturator, and lateral femoral cutaneous) nerves that originate from the lumbar plexus using a single injection o f local anesthetic, and hence termed the technique " 3 - i n - l b l o c k " (Winnie et al. 1973). This inguinal paravascular approach to the lumbar plexus involves placement o f the needle for injection just distal to the inguinal ligament into the fascial sheath o f the femoral nerve.* O w i n g to the proximal extension o f the femoral nerve sheath (situated between the iliacus muscle posteriorly and the psoas major muscle anteriorly) towards the lumbar plexus, the femoral 3 - i n - l block is a result o f  T h i s anatomical approach to the "anterior crural nerve" had already been described in 1922 by Gaston Labat in his seminal book "Regional Anesthesia - Its Technic and Clinical Application", pp. 247-249 (Labat 1922), which was probably largely founded o n the third edition o f the French "LAnesthesie Regionale" published one year prior.  S. S C H W A R Z  93  cephalad spread o f the distally injected local anesthetic solution. Hence, for 3 - i n - l blockade, the needle is angulated pointing proximally, and some authors r e c o m m e n d application o f pressure distal to the injection site (Btidenbaugh 1988), as d i d W i n n i e himself. I n order to achieve blockade o f all three nerves, a m i n i m u m volume o f 20 m l o f local anesthetic solution is administered ("volume anesthesia"). In Winnie's original report, this produced successful blockade o f all three nerves i n all n — 15 patients studied. I n addition, reference i n the paper is made to a larger series i n w h i c h "complete anesthesia o f all three nerves" was achieved i n o f n — 69 out o f 70 patients w h o received > 20 m l o f local anesthetic. M o r e recent studies have found, however, that the femoral 3-in-l  block does not produce complete anesthesia o f all three nerves w i t h such  consistency, particularly with regard to blockade o f the obturator nerve (Lang et al. 1992, 1993; Singelyn et al. 1996; Singer et al. 1998). F o r the latter, success rates as l o w as 3.6% have been reported, w h i c h may be partially due to the fact that investigators have used m o t o r blockade as an endpoint rather than sensory blockade alone i n cognizance o f the large m o t o r component o f the obturator nerve (Lang et al. 1992). There are several methods to facilitate correct placement o f the needle i n the femoral nerve sheath. Perhaps the oldest technique is to advance the needle until a paresthesia is elicited (Labat 1922). I n order to nrinimize the inherent risk o f neuropraxia associated w i t h this approach, newer methods have utilized short bevelled regional block needles. These allow definition o f the penetrated structures with the aid o f tactile criteria: as the needle is advanced through the Fascia lata followed by the Fascia iliaca, a distinct  S. S C H W A R Z 94 "double p o p " is felt, indicate positioning o f the needle tip i n the femoral nerve sheath.* The  most accurate method arguably is the use o f a peripheral nerve stimulator i n  combination w i t h an insulated short bevelled needle (Ford et al. 1984). Here, close proximity o f the needle tip to the femoral nerve is identified by evoking contractions o f the M. quadriceps femoris. T h e standard criterion for correct needle placement and injection advocated by most authorities is the presence o f patellar movement (as a result o f quadriceps muscle twitches) at an injected current magnitude < 0.5 m A (Bridenbaugh and Crews 1998) {cf. II.2.4). Clinically, the techniques o f femoral nerve and femoral 3 - i n - l blockade have been utilized for b o t h analgesia and surgical anesthesia i n a wide variety o f surgical settings. In addition to control o f postoperative pain following lower limb procedures (cf. II. 1), the successful use o f these blocks has been reported for surgical anesthesia i n saphenous vein stripping, knee arthroscopy, muscle biopsies o f the anterior thigh, and split thickness skin grafting  of  the  thigh, to  name  but  a  few  (for  an  overview, see  Moos  and  Cuddeford 1998). T h e reports o n the clinical experience w i t h the technique o f femoral 3in-1 blockade raised the possibility that this block is effective for postoperative analgesia following A C L R surgery. A s mentioned above, however, no controlled trial had been conducted to study the efficacy o f a femoral 3 - i n - l block for this purpose.  'Studying the spread o f 40 m l o f methylene blue injected "into femoral nerves" o f six human cadavers, one isolated study (Rittcr 1995) went so far as to conclude that a femoral nerve sheath capable o f conveying local anesthetic to the lumbar plexus does not exist in human cadavers. This is in sharp contrast to radiographic (Winnie et al. 1973) and the large body o f clinical experience.  evidence in vivo  S. S C H W A R Z  95  II. 1.4 Choice of local anesthetic T h e recently introduced aminoamide, ropivacaine, appeared to be an attractive choice for this application. Ropivacaine (l-propyl-N-[2,6-dimethylphenyl]-2-piperidinecarboxamide; Figure 28) is a long-acting local anesthetic, chemically homologous w i t h bupivacaine (cf. Figure 25) and mepivacaine. T h e sole structural difference between ropivacaine and bupivacaine is that the N - s i d e chain o f the former is one C atom shorter (propyl- vs. butyl-group). H o w e v e r , whereas bupivacaine until very recently has clinically only been available as a racemate, ropivacaine is the first enantiomerically pure local anesthetic marketed commercially and is supplied as the S-isomer. Therapeutically, ropivacaine is characterized by the potential to achieve a higher sensory-motor b l o c k separation (Bader et al. 1989) at a lower level o f systemic toxicity (Scott et al. 1989) compared to racemic bupivacaine. These properties are particularly desirable i n techniques o f regional anesthesia where large volumes and doses o f a local anesthetic are injected, and analgesia rather than complete motor blockade is the desired endpoint. It is hence perhaps not surprising that epidural labour analgesia has been a focus for the clinical study o f ropivacaine (Stienstra et al. 1995; E d d l e s t o n et al. 1996; O w e n et al. 1998; Polley et al. 1999). However, ropivacaine had not been studied i n the context o f the femoral 3 - i n - l block. T h e present investigation represents the  first  controlled trial and the first report i n the literature (Schwarz et al. 1999) o n the use o f ropivacaine i n femoral 3 - i n - l blockade.  S. S C H W A R Z  96  Aromatic head Amide linkage Amine tail  F i g u r e 28 Structural formula o f ropivacaine  II. 1.5 Primary hypothesis T h e primary hypothesis to be tested i n this study was that the addition o f a preincisional femoral 3 - i n - l block with ropivacaine 0.2% (Etches et al. 1997) (augmented by periincisional infiltrations o f the knee) to standard intra-articular local anesthetic instillation at the end o f surgery (Chirwa et al. 1989; K a r l s s o n et al. 1995; Tierney et al. 1995; Brandsson et al. 1996; D e n t i et al. 1997; B r o w n et al. 1997; Reuben et al. 1998) reduces postoperative anesthesia.  analgesic requirements  i n patients  undergoing A C L R  under  general  S. S C H W A R Z  97  II.2 Materials and Methods  11.2.1 Ethics A l l aspects o f the present trial were conducted i n accordance w i t h the principles stated i n the Declaration o f H e l s i n k i (Appendix I). T h e study p r o t o c o l w a s approved by the institutional human research committee (Clinical Screening Committee for Research Involving H u m a n Subjects, T h e University o f British C o l u m b i a ; certificate  number  C96-0047). W r i t t e n informed consent was obtained from each patient prior to inclusion i n the study. E a c h patient was informed i n detail both verbally and i n writing about the nature, purpose, possible risks, and benefits o f the study before signing the consent form (Appendix II) and was given sufficient time and opportunity to ask questions. Particular emphasis was made that a patient's decision not to take part i n the study w o u l d make no difference whatsoever to the quality o f care that the patient w o u l d receive. Patients were free to discontinue their participation i n the study at any time and without prejudice to further treatment.  II. 2.2 Study design T h e study was designed as a single centre (Vancouver H o s p i t a l & Health Sciences Centre, University o f British C o l u m b i a Site),* prospective, randomized,  placebo-controlled,  double-blind, parallel-group trial. A total o f n — 44 patients were to be allocated to the treatment groups i n blocks o f four using a computer generated randomization list.  'Recently renamed to " U B C Hospital"  S. S C H W A R Z  98  II. 2.3 Inclusion criteria, exclusion criteria, andpatient withdrawal T o be eligible for inclusion i n this study, patients had to fulfill all o f the following criteria: M a l e or female scheduled for inpatient A C L R at V a n c o u v e r Hospital, U B C Site under orthopedic surgeons D r s . P . M c C o n k e y , R. D a v i d s o n , B . D a y , or W . Regan A g e d 19 to 45 years A S A physical status I (cf. A m e r i c a n Society o f Anesthesiologists 1963) Written informed consent  Patients were excluded from the study i f they fulfilled any o f the following criteria: History o f sensitivity to local anesthetics o f the amide type, acetaminophen, or opioids Suspected inability to comply with study procedures, e.g., due to language difficulties, medical history, a n d / o r concomitant disease Suspected alcohol, drug, or medication abuse Regular treatment with analgesics, sedatives or any other medication with C N S effects Tendency to bleed easily Inability to rule out pregnancy Previous inclusion i n the study -  Participation i n clinical studies during this study or i n the 14 days prior to admission to this study  S. S C H W A R Z  99  Patients were free to discontinue their participation i n the study at any time as previously mentioned (II.2.1). Patients could be also be withdrawn from the study at any time at the discretion o f the investigator. Specific reasons for patient withdrawal determined a priori included technical failure defined as an inability to verify correct placement o f the needle for  injection o f the femoral 3 - i n - l block (see below) and significant postoperative  drainage from the intra-articular space o f the knee as judged by a treating physician or the investigator.  II. 2.4 Treatment interventions A l l patients received a standardized general anesthetic w i t h midazolam 0.01—0.03 m g / k g I V , fentanyl 1.5 u.g/kg I V total over the duration o f anesthesia, p r o p o f o l 2 - 3 m g / k g I V as required, and nitrous oxide 7 0 % w i t h isoflurane 0.5—2% i n oxygen through a laryngeal mask airway. P r i o r to surgical incision, the treatment group received a femoral 3 - i n - l b l o c k w i t h 40 m l o f ropivacaine 0.2% (Figure 29), using the classic inguinal paravascular approach described by W i n n i e et al. (1973). T h e solution for the b l o c k was injected after correct placement o f the regional block needle (22G X 2.5" insulated short bevel needle; Preferred M e d i c a l Products, T h o r o l d , Ontario, Canada; or 2 2 G IY2" Regional B l o c k Needle; B e c t o n D i c k i n s o n and Company, Franklin Lakes, N J , U . S . A . ) i n the fascial sheath o f the femoral nerve was confirmed by eliciting quadriceps muscle twitches w i t h a peripheral nerve stimulator (Model N S - 2 C A / D X , Life-Tech, Inc., H o u s t o n , T X , U . S . A . ; or N e r v e F i n d e r ® , Regional Master Corp., M i a m i , F L , U . S . A . ) at < 0.5 m A . A l l patients subsequently underwent A C L R utilizing hamstring tendon (semitendinosus and gracilis  S. S C H W A R Z  100  muscle) autografts w i t h a tibial bone tunnel and "over the top" femoral placement. T h e surgical technique was identical for all patients. A tourniquet was used i n all cases. A t the end o f surgery, the femoral 3 - i n - l block was augmented by additional infiltrations o f the lateral surgical incision at the site o f staple insertion over the lateral femoral condyle and the anteromedial incision at the site o f the origin o f the semitendinosus and gracilis tendons (which receive sensory innervation by the sciatic nerve). F o r these peri-incisional infiltrations, a total o f 20 m l (10 + 10 ml) o f ropivacaine 0.2% were administered by the surgeon. T h e control group received saline 0.9% instead o f ropivacaine. A l l n — 44 patients received an intra-articular instillation o f the knee w i t h 30 m l o f ropivacaine 0.2% at the end o f surgery (cf. Figure 29); no drainage tubes were inserted. F o l l o w i n g completion o f the procedure, postoperative pain was assessed i n the postanesthesia  care  unit  (PACU)  using  a  100  mm  visual analog  scale ( V A S ;  no pain = 0 m m , worst pain imaginable = 100 m m [Biomedical Engineering, Flinders M e d i c a l Centre, B e d f o r d Park, S.A., Australia]). A t V A S scores < 50 m m , acetaminophen 300 m g w i t h codeine 30 m g (Carter-Homer Inc., Mississauga, O N , Canada) was given (one to two tablets orally every three to four hours as needed). A t V A S scores > 50 m m (Cepeda et al. 1995), or w h e n pain relief was inadequate as judged by the  patient,  intravenous morphine (Abbott Laboratories, Limited; Saint-Laurent, Q C , Canada) was started, administered via a patient-controlled analgesia ( P C A ) p u m p (LifeCare® P C A Plus II Infuser M o d e l 4100, A b b o t t Laboratories, N o r t h Chicago, I L , U . S . A . ; loading dose [given via syringe], 2—4 m g / 5 m i n ; incremental dose, 1—3 mg; lockout  time,  6-10 m i n ; maximal dose over 4 h, 45 mg). A l l patients received supplemental external  S. S C H W A R Z  cryotherapy to their knee postoperatively by way o f a C r y o / C u f F  M  101  (Aircast, Inc.,  Summit, N J , U . S . A . ) or ice packs ( C o h n et al. 1989; Brandsson et al. 1996; E d w a r d s et al. 1996; R e u b e n et al. 1998). Patients were discharged home according to n o r m a l hospital procedures w h e n they were mentally clear and cooperative, were able to v o i d , were afebrile and had stable vital signs, tolerated oral nutrition, had satisfactory pain c o n t r o l o n oral analgesics, and were able to ambulate with crutches.  CONTROL GROUP (n = 22)  TREATMENT GROUP (n = 22)  40 ml of saline 0.9%  40 ml of ropivacaine 0.2%  Femoral 3-in-1 block  Surgery  30 ml of ropivacaine 0.2%  Intra-articular instillation  Peri-incisional infiltrations  Figure 29 Treatment interventions  20 ml of saline 0.9%  20 ml of ropivacaine 0.2%  S. S C H W A R Z 102  11.2.5 Outcome variables T h e primary outcome variable was postoperative P C A morphine consumption over 24 h (Joshi et al. 1993) standardized by body weight (initial loading dose administered via syringe included). Secondary outcome variables included postoperative consumption o f acetaminophen w i t h codeine over 24 h as well as V A S pain scores at rest and following mobilization, b l o o d pressure, heart rate, and the incidences o f nausea, vomiting, pruritus, urinary retention, and orthostatic hypotension at 1, 2, 4, 6, 8, 12, 16, 20,' and 24 h following completion o f surgery. T h e reference point (time zero) for these post-operative assessments was the time o f arrival at the P A C U . T h e adverse events, nausea, vomiting, pruritus, and respiratory depression, were assessed as "yes" or " n o " i n response to the question, " H a s the patient experienced any o f the following?" Clinical signs o f orthostatic hypotension i n association with an attempt to mobilize the patient were recorded as "yes" or " n o " . T h e times to readiness for discharge from the P A C U according to the Aldrete scoring system (Aldrete and K r o u l i k 1970; A p p e n d i x III) and the times to discharge from the hospital were recorded and compared between b o t h groups. A l l patients  were  followed up two to eight weeks after hospital discharge by a telephone interview that included questioning o n the occurrence o f neuropraxia.  11.2.6 Study drugs and blinding Ropivacaine 0.2% (2 m g / m l ) and saline 0.9% solutions were manufactured by A s t r a P a i n C o n t r o l A B , Sweden and supplied by Astra Pharma Inc., Canada. T h e solutions were provided i n 50 m l glass vials. Packaging and labelling o f the study drugs was carried out at  S, S C H W A R Z 103 A s t r a P h a r m a Inc., Canada (Mississauga, O N , Canada). A patient-specific b o x containing three 50 m l glass vials was provided for each study patient: one for the femoral 3 - i n - l block, one for the intra-articular instillation, and one for the peri-incisional infiltration (of. Figure 29). T h e appearances o f the vials and solutions for the femoral 3 - i n - l b l o c k and peri-incisional infiltrations were identical for ropivacaine and saline. Strict blinding o f all investigators was maintained throughout the study; all data were recorded by personnel unaware o f the treatment allocation, and patients were not assessed for sensory or motor blockade following the block (Fournier et al. 1998a,b). T h e randomization list was held at the site o f the pharmaceutical company that supplied the study drugs (Astra Pharma Inc., Canada). T h e list was available only to the individuals responsible for drug packaging until all data entry, editing, and validation o f the individual case report forms had been completed. A set o f sealed individual treatment code envelopes indicating the treatment allocation for each randomized patient was kept i n a safe,  accessible  place  at  the  investigational site. A n o t h e r  set  was  held by  the  pharmaceutical company.  II. 2.7 Statistical analyses The  primary outcome  variable for  statistical comparison was postoperative P C A  morphine consumption over 24 h standardized by body weight. T h e primary statistical analysis o f these data (both the dose standardized by body weight i n m g / k g and the total cumulative dose i n mg) and acetaminophen with codeine consumption over 24 h was completed o n an intention-to-treat basis using Student's / test. In order to add power to  S. S C H W A R Z  104  the analysis, secondary comparisons o f P C A morphine consumption were performed following exclusion o f "zero" values (i.e., patients w i t h no morphine consumption) and log  transformation  o f the  data.  This  approach was  based  on  the  premise  that  measurement (unlike count or proportion) data originates from a lognormal distribution. A l l data were tested for normality prior to parametric testing. Postoperative V A S scores at rest and following mobilization were analyzed using repeated measures analysis. Categorical data were analyzed using Fisher's exact test. B l o o d pressure and heart rate were analyzed by repeated measures analysis after replacing values w i t h the last value carried forward method. T h e data were analyzed using P r i s m versions 2.01/3.02 and StatMate version 1.00 software (GraphPad, San Diego, C A , U . S . A . ) , Microsoft E x c e l 97 software (Microsoft Corporation, R e d m o n d , W A , U . S . A . ) , and S A S version 6.12 software (see Acknowledgments) (SAS Institute Inc., Cary, N C , U . S . A . ) . A l l statistical tests were two-tailed and comparisons were declared statistically significant at P < 0.05. Based o n the data from a pilot study conducted prior to the present trial, the target sample size was projected to detect a n d n i m u m important difference o f 20 m g i n total morphine consumption over 24 h between the groups. I n order to have 9 0 % power and a type I error o f 5%, a sample o f n — 22 valid patients per group was required, assuming equal variances and approximate normal distributions o f the groups. I f the assumptions o f the / test o n w h i c h this calculation was performed were shown not to hold, an equivalent non-parametric test w o u l d provide no less than 9 5 % efficiency.  S. S C H W A R Z  105  II.3 Results  II. 3.1 Demographics Twenty two patients were enrolled i n each o f the two groups. A l l n — 44 patients were valid for intention-to-treat analysis. T h e male to female ratio was 13:9 i n the control group and 17:5 i n the treatment group. Other patient demographics were statistically similar i n b o t h groups, as were preoperative baseline vital signs and the duration o f surgery (Table 5). There were no significant differences between the groups i n the doses o f agents used for general anesthesia (data not shown). T h e timing o f relevant treatment interventions (i.e., the times between administration o f the femoral 3 - i n - l block and the start o f the procedure, between the start o f the procedure and intra-articular instillation, and between intra-articular instillation and peri-incisional infiltration) was comparable between the treatment groups (Table 6).  Table 5 Patient demographics, preoperative vital signs, and surgical data  Group  Age (years)  Weight (kg)  Height (cm)  Heart rate (min~ )  Systolic Diastolic Duration blood blood of pressure pressure surgery (mm Hg) (mm Hg) (min)  n  1  Control  28 + 7  74 ± 1 1  174 ± 7  65 + 9  119 ± 14  75 ± 1 1  52 ± 1 3  22  Treatment  31 ± 7  78 + 11  176 ± 8  66 + 10  119 ± 1 1  76 ± 11  55 + 13  22  Data are given as mean ± SD. No statistically significant difference between the groups was seen for any of the variables.  S. S C H W A R Z  106  T a b l e 6 T i m i n g o f treatment interventions  Administration o f femoral 3 - i n - l b l o c k to procedure start (min) Start o f procedure to intra-articular instillation (min) Instillation to peri-incisional infiltrations (min)  Group  Mean  SD  Minimum  Maximum  n  Control  15  4  7  28  22  Treatment  14  5  3  22  22  Control  46  11  17  63  22  Treatment  51  13  28  80  22  Control  2  2  0  7  22  Treatment  2  2  0  10  22  II. 3.2 Primary efficacy variable N o significant difference was found between the control group and the treatment group i n P C A morphine consumption over 24 h (standardized by body weight or expressed as total cumulative dose i n mg) or acetaminophen with codeine consumption over 24 h (intention-to-treat  analysis; for each group, n — 22; Table 7). M o r e patients i n the  treatment group required no P C A morphine postoperatively than i n the control group (cf. II.2.4); however, this difference was not statistically significant (Table 8). W h e n patients w h o required no morphine were excluded from the analysis, no. significant difference i n 24 h morphine consumption was seen (Table 9). Figure 30 gives a graphic representation o f these results showing the log-transformed data. A l t h o u g h not part o f the original a priori hypothesis (cf. 11.1.5, II.2.6), post hoc analysis o f the data also revealed  S. S C H W A R Z 107 T a b l e 7 Postoperative analgesic consumption (intention-to-treat analysis)  Group  PCA morphine PCA morphine Acetaminophen with codeine n consumption over 24 h consumption over 24 h consumption over 24 h standardised by weight (mg) (number of tablets)* (mg/ kg)  Control  0.45 ± 0.44  31.0 + 28.7  6.4 + 3.1  22  Treatment  0.37 + 0.50  27.7 + 38.7  7.6 ± 4 . 8  22  Data are given as mean + S D ; P C A = patient-controlled analgesia. N o statistically significant difference between the groups was seen for any o f the variables. *One tablet contains 300 m g o f acetaminophen and 30 m g o f codeine.  T a b l e 8 N u m b e r o f patients not requiring morphine postoperatively  Group  No morphine required  Morphine required  n  Control  6  16  22  Treatment  10  12  22  N o statistically significant difference between the groups was seen (Fisher's exact test, P = 0.35).  T a b l e 9 Postoperative morphine consumption (patients requiring morphine)  Group  PCA morphine PCA morphine n consumption over 24 h consumption over 24 h standardised by weight (mg) (mg/ kg)  Control  0.62 ± 0.40  42.6 ± 25.0  16  Treatment  0.67 ± 0 . 5 1  50.8 + 39.8  12  Data are given as mean + S D . N o statistically significant difference between the groups was seen for either variable.  S. S C H W A R Z  108  no significant differences i n analgesic consumption earlier i n the postoperative course, e.g., at 6 or 12 h following completion o f surgery (data not shown).  B  a5 cn  0.5-  0.5  0.0-  0.0  c  E  •£ "9» -0.5 8- E E o  £• E -0.5o O) E o  < o n.  < ^  -1.0-1.5-  O CL  Control  Treatment  -1.0 -1.5  Control  Treatment  Figure 30 Postoperative morphine consumption (patients with no morphine consumption excluded) (A) Presentation of the results as "box & whiskers" graph following log transformation (whiskers = range of data; box = 25 percentile, median, 75 percentile). (B) Presentation of the raw data. No statistically significant difference between the groups was seen (Student's / test, P = 0.86). th  th  II. 3.3 Secondary efficacy variables There were no significant differences between the groups i n postoperative V A S pain scores at rest, b l o o d pressure, or heart rate at 1, 2, 4, 6, 8, 12, 16, 20, and 24 h following completion o f surgery (Figure 31—Figure 33). M e d i a n V A S scores at rest were i n the range o f 20—40 m m i n the control group and 2 2 - 4 2 m m i n the treatment group. T h e majority o f patients were mobilized o n postoperative  day one prior to  discharge  according to n o r m a l hospital procedures; as a result, recording o f V A S scores following mobilization was inapplicable i n over 8 0 % o f the cases and this data thus excluded from analysis. T h e times to readiness for discharge from the P A C U according to the Aldrete  S. S C H W A R Z 109  scoring system (Appendix III) were similar for both groups (control group, 19 + 11 m i n ; treatment group, 17 + 10 m i n ; n — 22; P = 0.44), as were the times to discharge from the hospital (control group, 21.5 ± 3.4 h; treatment group, 23.5 ± 7.9 h ; n = 22; P = 0.28).  -too-] oo  Treatment  E E o U co C  CC D. CO ^  Control  s o 40  30  a o  1»  1»  SO  34  Time following completion of surgery (h) F i g u r e 31 Postoperative V A S pain scores at rest over time Shown are median V A S scores; for each group, n — 22. N o statistically significant differences were seen.  ISO -  ^ ^  ISO*  X E E  "•—0k_-* loo  to CO CD Q.  m  Control Treatment  140iao< lOO  SBP  •  so-  DBP  eo •  *oaoo-  Time following completion of surgery (h) F i g u r e 32 Postoperative b l o o d pressure over time Shown are median values for blood pressure; for each group, n — 22; S B P = systolic blood pressure, D B P = diastolic blood pressure. N o statistically significant differences were seen.  S. S C H W A R Z  110  Control Treatment  A  •  a  ia  ia  Time following completion of surgery (h) F i g u r e 33 Postoperative heart rate over time Shown are median values for heart rate; for each group, « = 22. N o statistically significant differences were seen.  II. 3.4 Adverse events T h e most c o m m o n adverse events during the first 24 h following surgery were nausea, pruritus, orthostatic hypotension, vomiting, and urinary retention; their incidences are given i n Table 10. There were no significant differences between the groups i n the incidences o f these adverse events at 1, 2, 4, 6, 8, 12, 16, 20, and 24 h following completion o f surgery (not shown). O n e patient i n the treatment  group reported  prolonged postoperative anesthesia i n the area o f distribution o f the femoral nerve o n the surgical side that lasted for four days but subsided completely. There were no incidents o f persistent neuropraxia. N o signs o f systemic local anesthetic toxicity were observed.  S. S C H W A R Z 111 Table 10 Incidence o f c o m m o n adverse events  Group  Nausea  Pruritus  Control  16  8  6  7  7  22  Treatment  13  9  8  5  2  22  Orthostatic hypotension  Vomiting  Urinary retention  n  Shown are the total cumulative in-hospital incidences during the first 24 h following completion o f surgery. Data are given as numbers o f patients; each adverse event is reported only once for each patient. N o statistically significant difference between the groups was seen for any o f the variables (Fisher's exact test, P > 0.05).  S. S C H W A R Z  112  II.4 D i s c u s s i o n  T h i s section o f the thesis represents the first randomized controlled trial ( R C T ) to assess the efficacy o f a femoral 3 - i n - l block to improve postoperative pain control i n A C L R , and the first report o n the use o f ropivacaine for this purpose (Schwarz et al. 1999). T h e trial found no significant effect o n postoperative morphine consumption o f the addition o f a preincisional femoral 3 - i n - l block (augmented by peri-incisional infiltrations) w i t h ropivacaine 0.2% to intra-articular instillation o f the knee at the end o f surgery w i t h ropivacaine 0.2% i n patients undergoing hamstring tendon autograft A C L R under general anesthesia. There also were no differences between the groups studied i n postoperative V A S pain scores, vital signs, incidence o f adverse events, or times to readiness for discharge from P A C U and hospital discharge. The present results harmonize w i t h those o f Tierney et al. (1987), w h o conducted an R C T to assess the use o f a femoral nerve block with 20 m l o f bupivacaine 0.25% i n patients undergoing open ligament reconstruction o f the knee and found no effect o n the total intramuscular (IM) analgesic dose i n the first 12 h postoperatively. Likewise, H i r s t et al. (1996) saw no effect i n an R C T o f a femoral 3 - i n - l block, either by singleinjection  [20 m l bupivacaine 0.5% w i t h epinephrine 1:200,000] or as a continuous  infusion, o n postoperative P C A morphine requirements after total knee arthroplasty. In a recent report, Fournier et al. (1998a) similarly observed no reduction i n analgesic requirements at 24 and 48 h following prosthetic hip surgery by a preincisional femoral 3 - i n - l b l o c k w i t h 40 m l o f bupivacaine 0.5% w i t h epinephrine 1:200,000. A l s o consistent  S. S C H W A R Z  113  w i t h the results o f the present trial are the findings o f a subsequent study by Rosaeg et al. (2001), w h o saw no effects i n A C L R patients o n verbal pain scores o n postoperative days 1, 3, or 7 o f a pre-emptive regimen that included femoral nerve blockade w i t h 20 m l ropivacaine 0.25%, intra-articular ropivacaine 0.25% with epinephrine 1:200,000 (20 ml) plus morphine 2 m g , and I V ketorolac (30 mg). The  present findings contrast with those o f Ringrose and Cross (1984) w h o  reported a 4 0 % reduction i n I M opioid administration i n the first 24 postoperative hours following a femoral block with 20 m l bupivacaine 0.5% i n patients undergoing "knee joint (anterior cruciate) reconstruction surgery". Nonetheless, this trial was unblinded and patients likely received open knee ligament reconstructions utilizing bone-patellar tendonbone autografts. I n an uncontrolled study with patients undergoing b o t h A C L R and A C L R c o m b i n e d with meniscal procedures, E d k i n et al. (1995) found the femoral 3 - i n - l block useful for the relief o f post-operative pain. In their report, 9 2 % o f patients received no parenteral opioids following administration o f a femoral 3 - i n - l block c o m b i n e d w i t h intra-articular local anesthetic  injection.  However, aside from  the  fact that these  observations were from an uncontrolled study, there were other major differences to the present trial that render direct comparisons difficult. Firstly, the surgical techniques were different. In the study by E d k i n et al, nriddle-third patellar tendon autografts were used, as compared to the present trial, where the considerably less invasive technique utilizing semitendinosus and gracilis muscle tendon autografts  was performed. Secondly, a  different concentration, dose, and type o f local anesthetic was employed. E d k i n et al. used a dose o f 2 - 3 m g / k g o f bupivacaine 0.5%; i n the present study, 1.3-1.5 m g / k g (80 mg)  S. S C H W A R Z  114  o f ropivacaine 0.2% (Etches et al. 1997; Borgeat et al. 2000) were administered for the femoral 3 - i n - l block. Thirdly, E d k i n et al. administered the block postoperatively i n the P A C U , whereas it was performed prior to surgical incision i n the present study. Finally, different protocols for the management o f postoperative pain were used. A l t h o u g h 9 2 % o f the patients o f E d k i n et al. were reported not to have required parenteral opioids, 7 5 % received parenteral ketorolac and oral opioids to control postoperative pain. It is likely that the above differences contributed to differences i n postoperative parenteral o p i o i d consumption, and thus, the difference i n outcome compared to our trial. In a recent hospital database and patient chart review study, a combination o f general anesthesia and femoral  nerve  blockade  was  associated  with  fewer  total  postoperative  nursing  interventions for treatment o f pain, nausea, vomiting, pruritus, and urinary retention compared to other anesthetic techniques (Williams et al. 1998). Similar to the present trial, no differences i n discharge times were noted. Interpretation o f these data is limited, however, by major weaknesses i n study design and data analysis. F o r example, there was no randomization or blinding, no description o f the drug or dosage used for femoral nerve blockade, no direct measure o f analgesic drug consumption or pain scales, and no report o f the actual incidences o f adverse events. Finally, the inclusion o f five different groups and utilization o f multiple / tests for determination o f differences between the individual groups limit the utility o f this report to the generation o f future  hypotheses.  Lastly, i n a prospective randomized study published only i n abstract form at the time o f writing (Auge and G r i f f i n 2000), patients w h o underwent A C L R solely under 3 - i n - l b l o c k w i t h local infiltration (plus a single dose o f propofol), compared to general or  S. S C H W A R Z  115  i  spinal anesthesia, exhibited no requirement for postoperative opioids, no nausea or urinary retention, and a "marked decrease" i n time to achieve discharge criteria. A l t h o u g h no details regarding design and results were reported, it is likely that a high concentration o f local anesthetic was used (see below) and that follow-up was limited to a few hours postoperatively. I n the present study, there were fewer patients i n the treatment group w h o required morphine than i n the control group; however, this difference was not statistically significant. It is possible that differences between the groups may have been detected had higher concentrations  (e.g., 0.5% or 0.75% preparations)  and higher total doses o f  ropivacaine been used (Fanelli et al. 1998; Marhofer et al. 2000). A similar statement could be made about the addition o f a vasoconstrictor to the local anesthetic solution. W i t h regard to the latter, epinephrine 1:200,000 does not p r o l o n g or enhance the effects o f ropivacaine 0.5% i n brachial plexus blockade for upper extremity surgery (Hickey et al. 1990). T h e same is the case for ropivacaine 0 . 2 % / 0 . 5 % i n femoral 3 - i n - l blockade administered via catheter for analgesia after total knee replacement (Weber et al. 2001). W i t h regard to the former, bupivacaine 0.25% was used with promising results i n the pilot study at our centre prior to this trial. A recent clinical trial compared the relative analgesic  potencies  o f bupivacaine  and  ropivacaine i n  epidural labour  analgesia  (Polley et al. 1999). In this study, the analgesic potency o f ropivacaine was found to be significantly less than bupivacaine's (potency ratio, 0.6). T h e results have led to more general speculations whether  the documented  differences  between ropivacaine and  bupivacaine i n terms o f motor blockade and toxicity may be simply a result o f a  S. S C H W A R Z 116 difference i n potency (D'Angelo and James 1999). It seems likely that these findings also bear significance for the results o f the present trial and the differences i n outcome compared to the pilot study with bupivacaine. I n summary, it is evident from these observations  that extensive  future  studies  are required to establish  relationships  for ropivacaine's analgesic effects  dose-response  i n a variety o f regional  anesthetic  techniques, including femoral 3 - i n - l blockade. It has to be emphasized, however, that all patients i n this study (including those i n the control group) received an intra-articular instillation with 30 m l o f ropivacaine 0.2% at the end o f the procedure, i n compliance w i t h our standard institutional m u l t i m o d a l analgesic (Kehlet and D a h l 1993) regimen. Postoperative pain following A C L R originates from a variety o f anatomical sources (Curry et al. 1996), w h i c h include the sites o f the tendon cuts, staple insertion, and surgical incisions. T h e efficacy o f intra-articular local anesthetic instillation o f the knee for postoperative analgesia i n A C L R is very well established (Chirwa etal. 1989; H e a r d etal. 1992; K a r l s s o n etal. 1995; Tierney etal. 1995; Brandsson et al. 1996; Curry et al. 1996; B r o w n  et al. 1997; D e n t i et al.  1997;  Reuben et al. 1998) and n o w standard practice at many institutions, including our centre. In the present study, n o significant subsequent reduction i n analgesic requirements was observed w h e n a femoral 3 - i n - l  block, augmented  by additional local  anesthetic  infiltration o f the lateral and anteromedial incisions (the sites o f staple insertion and p r o x i m a l cut o f the semitendinosus and gracilis tendons, whose sensory supply includes sciatic fibres), was added to the intra-articular local anesthetic instillation. Recently, P e n g et al. (1999) reported  o n a decrease i n morphine consumption i n the first  S. S C H W A R Z postoperative day  following  117  A C L R i n patients receiving a preoperative femoral nerve  b l o c k w i t h 15 m l bupivacaine 0.5% but no intra-articular local anesthetic instillation. These results provide further indirect evidence for the analgesic efficacy o f intra-articular local anesthetics i n patients undergoing knee surgery. O n e study o n patients undergoing diagnostic knee arthroscopy, o n the other hand, failed to show an effect o f intra-articular ropivacaine (20 m l o f a 0.5% solution) o n postoperative V A S pain scores (Rautoma et al. 2000). H o w e v e r , diagnostic arthroscopy is significantly less traumatic than A C L R and easily performed under local anesthesia (Shapiro et al. 1995; Lintner et al. 1996), w h i c h may have prevented the demonstration o f a V A S pain score reduction i n this particular study. It remains possible that a small statistically significant difference may have been detected  had a very large sample size been used. A retrospective  power analysis  (performed after closure o f the database) revealed a level o f (1 — (3) = 0.8 to detect a difference i n 24 h P C A morphine consumption o f 0.41 m g / k g at a = 0.05. Nonetheless, it seems unlikely that such a statistically significant difference w o u l d be o f clinical significance. requirements  The  high variability i n analgesic requirements  specifically  is  a phenomenon  also  observed  i n general by  other  and P C A investigators  (Amanzio et al. 2001) and has been subject o f recent discussion i n the literature about analgesic trial design ( M c Q u a y and M o o r e 2000). O n e possibility w o u l d be to substitute for surrogate markers as outcome variables; i n the present study, however, no differences i n secondary variables were seen.  S. S C H W A R Z  118  Despite the fact that there was no difference between the groups i n postoperative adverse events, their incidence i n the studied patient population was noteworthy; this was particularly the  case for nausea. A l t h o u g h postanesthetic  nausea cannot be easily  separated from nausea specifically triggered by opioids, one may reasonably assume that the high incidence o f nausea i n this study is at least partially attributable to postoperative analgesic medication. Postoperative nausea and vomiting p r o l o n g in-hospital stay and increase costs. It has recently been reported that 58% o f the cost associated w i t h A C L R can be saved w h e n in-hospital stay is shortened and this procedure is performed o n an outpatient  basis ( K a o et al. 1995). These  findings  further illustrate the need  for  optimization o f perioperative care i n A C L R surgery. N o persistent neurologic complications associated with femoral 3 - i n - l blockade and  no signs or symptoms o f systemic local anesthetic toxicity were observed i n this  study. T h e former is consistent w i t h a review o f 882 patients w h o received either femoral 3-in-l  or  femoral  nerve  blocks  without  any  persistent  neurologic  sequelae  (Moos and C u d d e f o r d 1998). W i t h regards to the latter, a shortcoming o f this study is the lack  o f measurement  o f ropivacaine plasma levels, which,  although not directly  addressing the primary hypothesis, w o u l d have provided valuable information. T h e reasons for this are related to issues involving practicality, funding, and study p r o t o c o l negotiations w i t h the supporting pharmaceutical company. In the literature, several studies have measured plasma concentrations o f bupivacaine, lidocaine, and prilocaine following femoral 3 - i n - l / l u m b a r plexus blockade (Dahl et al. 1988; Madej et al. 1989; H o o d et al. 1991). O f these, most relevant for the present trial is the report by Madej et al,  S. S C H W A R Z  119  w h o found that peak concentrations o f b o t h bupivacaine and lidocaine occur at a median o f 37.5 m i n following b l o c k insertion. T h e median peak bupivacaine concentration was 0.67 u.g/ml, w i t h a range o f 0.35—0.80 u,g/ml. A l t h o u g h not studied, it appears reasonable to speculate that similar values w o u l d be observed w i t h ropivacaine. F o r lidocaine, peak plasma concentrations ranged from 1.22—4.42 u g / m l . T h i s is particularly noteworthy i n light o f the results presented i n the first section o f this thesis, as these values clearly are i n the range i n w h i c h lidocaine acts as a systemic analgesic (cf. 1.1). T h u s , the results by Madej et al. raise the possibility that their observed effects o f femoral 3 - i n - l blockade are i n part due to systemic action o f lidocaine. A similar hypothesis cannot easily be projected for the ropivacaine used i n this study's 3 - i n - l block, however, as no additional analgesic effect o f this intervention was observed. O n the other hand, the possibility remains that the ropivacaine injected for the intra-articular instillations (30 m l o f a 0.2% solution) exerted a systemic action, although the corresponding plasma concentrations remain unknown.  II.4.1 Summary and conclusions I n a randomized controlled trial, a femoral 3 - i n - l block w i t h ropivacaine 0.2% had no significant effect o n postoperative analgesic consumption, V A S pain scores, vital signs, or adverse events i n patients undergoing A C L R under general anesthesia, compared to intraarticular instillation with ropivacaine 0.2% alone. T h e data do not support the routine addition o f a preincisional femoral 3 - i n - l block with ropivacaine 0.2% to the standard anesthetic management o f these patients. However, this study provides further evidence  S. S C H W A R Z 120 that intra-articular local anesthetic instillation o f the knee is effective for postoperative pain control and supports the routine use o f this intervention as standard o f care. P a i n control and prevention o f adverse events following A C L R remain issues o f clinical and pharmacoeconomical significance, and future studies w i l l aid to further i m p r o v e the management o f these patients (cf. page 122).  S. S C H W A R Z  121  Overall Conclusion and Closing Remarks  T h i s thesis was dedicated to the study o f specific questions about the pharmacology & therapeutics o f local anesthetics. W i t h a focus o n the analgesic properties o f this group o f agents, the investigations combined research o n cellular pharmacological actions and clinical therapeutic efficacy, addressing diverse aspects within the wide spectrum o f this topic. I n conducting the research, an attempt was made to apply the same high methodological standards to both components. In the first section o f the thesis, laboratory investigations examined cellular effects o f the prototype agent, lidocaine, taking advantage  o f state-of-the-art  experimental  techniques. T h e experiments identified novel actions that potentially play a critical role i n its central analgesic and sedative actions as well as toxicity. T h e results may represent a first step toward the creation o f a basis for the future development o f new therapeutic agents based o n precise knowledge o f the desired cellular effect, and not o n empiricism or structural single-target specificity. T h e second section was dedicated to the study o f the analgesic efficacy o f ropivacaine, the most recendy introduced agent i n clinical practice, using the "gold standard"  for experimental design i n clinical research — the R C T . W h i l e the  trial  demonstrated no additional analgesic effect o f ropivacaine administered for femoral 3-in-l  blockade i n A C L R  beyond that produced by standard  intra-articular local  anesthetic infiltration, the results emphasize the needs for and merits o f evidence-based practice i n anesthesiology. It is difficult to determine w i t h certainty the global effect o n  S. S C H W A R Z  122  clinical practice o f an R C T following publication, and such an analysis for the trial presented here is beyond the scope o f this work. F o r the purpose o f this thesis, however, it is noteworthy that the results o f the present study have had a significant local institutional impact i n several ways. Firstly, the reporting o f the data has dramatically changed clinical practice w i t h i n the Department o f Anesthesia at U B C Hospital, w h i c h is the primary centre  for  arthroscopic knee surgery outside private practice i n British Columbia. Since 1998/1999, femoral 3 - i n - l blockade has no longer been utilized i n the routine anesthetic management o f patients undergoing A C L R (personal communication). Secondly, the results and conclusions drawn from this study have led to the design and completion o f a subsequent R C T targeted to improve postoperative pain control following A C L R (Butterfield et al. 2001). In this trial, a combination o f specific pre- and postincisional local infiltrations o f the knee  (bupivacaine 0.25% w i t h  epinephrine  1:200,000; 40 + 15 ml) reduced analgesic requirements by more than 57% (P - 0.008), compared to the standard intra-articular instillation alone. In addition, patients i n the treatment group fulfilled standard hospital discharge criteria o n average 37 m i n earlier than those i n the control group (P < 0.05) and had a zero incidence o f postoperative adverse events, including P O N V . Thirdly, the present study was highlighted i n an extensive dissertation o n the evaluation o f clinical trials using a novel Clinical Trial Evaluation System ( C T E S ; Franciosi 1998). I n this dissertation, the C T E S was employed to compare six clinical trials w i t h the use o f a scale consisting o f five categories that c o m m o n l y define Good Clinical Practices  S. S C H W A R Z  123  (i.e., "ethics", "design", "question", "statistics", and "standard operating procedures"), w i t h a total o f 85 items. T h e study found that o f the trials tested, the present R C T reached the highest C T E S score (82/85) w i t h regard to representing a "Best Possible Trial". These examples also serve to illustrate the vital importance o f communicating the findings o f clinical trials w i t h "negative" results, w h i c h c o m m o n l y remain unreported as a result o f publication bias (Easterbrook et al. 1991). T h e consequences o f such bias, often investigator-based (Dickersin and M i n 1993), are far-reaching. "Negative" studies, i n addition to their potential clinical and academic impact exemplified above, may affect our society, business, and governments (Miller and M o u l d e r 1998). Preferential publication o f "positive" results may lead to overestimation o f the effects o f therapeutic interventions, limit the validity o f meta-analyses and review articles, waste valuable resources, and unnecessarily expose patients  to ineffective or intolerable treatments (Menger and  V o l l m a r 2000). T h e D a n i s h gastroenterologist, R C T expert, and co-ordinating editor o f the Cochrane Hepato-Biliary G r o u p , Christian G l u u d , addresses these issues i n a recent editorial, where he writes: "'Negative trials' are positive! They must be published and we should not refer to them as 'negative trials''''' (Gluud 1998).' Today, A C L R is routinely performed o n an ambulatory basis at U B C Hospital, w h i c h is i n sharp contrast to the period before 1995/1996, w h e n the planning phase for the femoral 3 - i n - l b l o c k trial presented i n this dissertation was initiated. A l t h o u g h there are multiple reasons for this development, there exists no doubt that it w o u l d not have been sustainable without i m p r o v e d anesthesia care and its underlying scientific basis.  S. S C H W A R Z  124  Hence, above examples illustrate the potential impact o n clinical anesthesia practice o f R C T s and clinical research i n general. Together with laboratory research, the latter w i l l continue to serve as the crucial basis for the creation o f the scientific evidence required to reach the ultimate goal: to provide the best possible care for our patients today and i n the future.  S. S C H W A R Z 125  Appendix I  World Medical Association Recommendations Guiding Physicians in Biomedical Research Involving Human Subjects [Declaration of Helsinki] Adopted  by  the  18  World  th  Medical  Assembly  Helsinki, Finland, June 1964  th  World  Assembly,  Tokyo,  Japan,  W o r l d Medical Assembly, Venice,  th  Italy, October 1983; 41  W o r l d Medical Assembly,  st  th  used  for  research  must  be  Because it is essential that the results o f laboratory experiments be applied to human beings to further scientific knowledge and to help suffering humanity, the  World Medical Association recommendations  has  as  prepared  a guide  to  the  every  subjects. They should be kept under review in the future. It must be stressed that the  standards  as  drafted are only a guide to physicians all over the world. Physicians are not relieved from criminal, civil  H o n g K o n g , September 1989; and the 48  animals  physician in biomedical research involving human  Medical  October 1975; 3 5  of  respected.  following  and amended by the 29  welfare  General Assembly, Somerset West, Republic o f  and ethical responsibilities  South Africa, October 1996  own countries.  INTRODUCTION  I. B A S I C P R I N C I P L E S  It is the mission o f the physician to safeguard the  1.  health o f the people.  must  His or her knowledge  and  Biomedical research conform  conscience are dedicated to the fulfillment o f this  principles  mission.  performed  T h e Declaration o f Geneva o f the W o r l d Medical Association binds the physician with the words, "The  and  on  to  and  under the laws o f their  involving human  generally  should  be  based  knowledge  scientific  on  laboratory and animal  a thorough  subjects  accepted  adequately  experimentation of  the  scientific  literature.  Health o f my patient will be my first consideration,"  2. T h e design and performance o f each experimental  and  procedure  the  International  declares  that,  Code  of  Medical  Ethics  " A physician shall act only in  the  involving  human  subjects  should  patient's interest when providing medical care which  should be transmitted for consideration,  might have the effect o f weakening the physical and  and guidance  mental condition o f the patient."  independent  T h e purpose o f biomedical research involving human subjects must be to improve diagnostic, therapeutic and prophylactic procedures and the understanding o f the aetiology and pathogenesis o f disease. In  current  therapeutic hazards.  medical or  This  practice  prophylactic applies  most  diagnostic,  procedures  especially  to  involve  biomedical  research.  be  clearly formulated in an experimental protocol which  -provided  to  a specially  o f the  that  this  investigator independent  conformity with the country  in  which  appointed  comment committee  and the  sponsor  committee  laws and regulations the  research  is of  experiment  in the is  performed. 3. Biomedical research  involving human  subjects  should be conducted only by scientifically qualified persons  and under the  supervision o f a clinically  competent medical person. T h e responsibility for the human subject must always rest with a medically  Medical  progress  ultimately  must  is  based  on  research  rest in part on  which  qualified person and never rest o n the subject o f the  experimentation  research, even though the subject has given his or her  involving human subjects.  consent.  In the field o f biomedical research a fundamental  4.  distinction  cannot  must  be  recognized  between  medical  Biomedical research legitimately  involving human  be  carried  out  subjects  unless  the  research in which the aim is essentially diagnostic or  importance o f the objective is in proportion to the  therapeutic for a patient, and medical research, the  inherent risk to the subject.  essential object without  o f which is purely scientific  implying direct diagnostic  or  and  therapeutic  value to the person subjected to the research.  5.  Every  human  biomedical  subjects  research  should  be  project  preceded  involving by  careful  assessment o f predictable risks in comparison with  Special caution must be exercised in the conduct o f  foreseeable benefits  research which may affect the environment, and the  Concern for the interests o f the subject must always  to  the  subject  or to  others.  prevail over the interests o f science and society.  S. S C H W A R Z 126 6. T h e right o f the research subject to safeguard his  II. M E D I C A L R E S E A R C H C O M B I N E D  or  P R O F E S S I O N A L C A R E (Clinical Research)  her integrity must always be respected.  Every  precaution should be taken to respect the privacy o f the subject and to minimize the impact o f the study on the subject's physical and mental integrity and o n the personality o f the subject. 7.  Physicians  should  from  engaging  in  research projects involving human subjects unless they  are  satisfied  that  the  hazards involved  are  believed to be predictable. Physicians should cease any  investigation  if  the  hazards  are  found  to  outweigh the potential benefits. 8. In publication o f the results o f his or her research, the physician is obliged to preserve the accuracy o f the  results.  Reports  accordance with the  of  1. In the treatment o f the sick person, the physician must be free to use a new diagnostic and therapeutic measure, if in his or her judgement it offers hope o f saving  abstain  experimentation  not  in  principles laid down in this  Declaration should not be accepted for publication.  WITH  life,  reestablishing  health  or  alleviating  suffering. 2. T h e potential benefits, hazards and discomfort o f a new  method  advantages  should  of  the  be  best  weighed current  against  the  diagnostic  and  therapeutic methods. 3. In any medical study, every patient — including those o f a control group, i f any - should be assured of  the  best  proven  diagnostic  method. This does not  and  exclude  the  therapeutic use  o f inert  placebo in studies where no proven diagnostic or therapeutic method exists.  9. In any research o n human beings, each potential subject must be adequately informed o f the aims, methods, anticipated benefits and potential hazards o f the study and the discomfort it may entail. H e or  4. T h e refusal o f the patient to participate in a study must  never  interfere  with  the  physician-patient  relationship.  she should be informed that he or she is at liberty to  5. If the physician considers it essential not to obtain  abstain from participation in the study and that he or  informed  consent,  she  to  proposal  should  participation at any time. T h e physician should then  protocol  for  obtain the subject's freely-given informed consent,  committee (I, 2).  is  free  to  withdraw his  or her consent  preferably in writing.  the be  specific  stated  transmission  reasons  for  this  in  the  experimental  to  the  independent  6. T h e physician can combine medical research with the  professional care, the objective being the acquisition  research project the physician should be particularly  o f new medical knowledge, only to the extent that  cautious i f the subject is in a dependent relationship  medical research is justified by its potential diagnostic  to h i m or her or may consent under duress. In that  or therapeutic value for the patient.  10.  When  obtaining  informed  consent  for  case the informed consent should be obtained by a physician who is not engaged in the investigation and  III.  who  RESEARCH  is  completely  independent  of  this  official  NON-THERAPEUTIC INVOLVING  BIOMEDICAL  HUMAN  SUBJECTS  (Non-Clinical Biomedical Research)  relationship. 11. In case o f legal incompetence, informed consent  1. In the  should  guardian in  research carried out on a human being, it is the duty  accordance with national legislation. Where physical  o f the physician to remain the protector o f the life  be  obtained  from  the  legal  purely scientific  or mental incapacity makes it impossible to obtain  and  informed consent, or when the subject is a minor,  research is being carried out.  permission from the responsible relative replaces that o f the subject in accordance with national legislation. Whenever the minor child is in fact able to give a consent,  the minor's consent must be obtained in  addition to the consent o f the minor's legal guardian.  health o f  application o f medical  that person  on  whom  biomedical  2. T h e subject should be volunteers — cither healthy persons  or patients  for  whom  the  experimental  design is not related to the patient's illness. 3. T h e investigator or the investigating team should discontinue  the  research  if  in  his/her  or  their  12. T h e research protocol should always contain a  judgement it may, if continued, be harmful to the  statement o f the ethical considerations involved and  individual.  should indicate that the principles enunciated in the present Declaration are complied with.  4. In research on man, the interest o f science and society  should  never  take  precedence  over  considerations related to the wcllbeing o f the subject.  S. S C H W A R Z  127  Appendix II  PATIENT I N F O R M A T I O N SHEET A N D CONSENT F O R M PATIENT I N F O R M A T I O N Study Title:  Femoral 3-in-l Block Combined with Local Infiltrations in Patients Undergoing Arthroscopic Cruciate Ligament Repair: A Double Blind Comparison Between Ropivacaine 2 mg/ml and Saline (0.9%) for PostOperative Pain Relief  Local Study Number:  DC-ROA-0001  Study Site:  Vancouver Hospital: U B C site  Dear Patients: We would like to invite you to take part in a research study being conducted at this hospital. You will be given the opportunity to speak to one of our doctors or his staff and be able to ask questions that you feel are relevant. If you decide you do not want to take part in the study, this will make no difference whatsoever to the quality of treatment you will receive. If, however, you do decide to take part, then we ask you to do everything you can to follow all instructions given to you. Approximately 44 patients will be taking part in this study.  The drug being tested Arthroscopic cruciate ligament repair (ACLR) is a procedure routinely used in the treatment of knee injuries at this hospital. Pain of varying intensity is normally experienced after A C L R surgery (postoperative pain). This postoperative pain is cornmonly treated with intravenous injections of strong analgesics (pain killers) such as morphine. Unfortunately, morphine is associated with unwanted effects such as nausea, itching, drowsiness or breathing problems. Another method of postoperative pain relief is to perform a local anaesthetic block (femoral 3-in-l block combined with local infiltrations into the area of your knee) to "freeze" the nerves that carry pain from the surgical area. Ropivacaine is the local anaesthetic to be used for this nerve block. Ropivacaine is a new long acting local anaesthetic which has been shown to be similar to other well known anaesthetics. Ropivacaine is not approved or marketed in Canada, however, it has been registered in Sweden, Finland, the Netherlands, and Australia since 1995. More than 2800 patients have received ropivacaine in clinical trials. From these trials there is sufficient information to show that ropivacaine is an effective local anaesthetic with few side effects. Consequently there should be no unusual risks brought about by the drug being tested should you decide to participate in this study. The purpose of this study is to test the ability of a ropivacaine femoral 3-in-l block combined with local infiltrations, as compared to placebo (an identical looking solution that has no active ingredients), to provide relief for pain following A C L R .  S. S C H W A R Z  128  What will happen? Prior to the study: Within four weeks before surgery a physical examination including pulse rate and blood pressure will be performed. This would be performed whether or not you enter into the study. Your medical history will also be recorded and you will be asked to complete a discomfort questionnaire. Exclusion from the study You may be excluded from the study if you have had a previous reaction to amide local anaesthetics, acetaminophen or narcotics; are unable to comply with the study procedures or have significant alcohol, drug or medication abuse. You will not be included in the study if you are being treated regularly with analgesics, sedatives, or other central nervous system medications, or have a tendency to bleed. If you are a female in childbearing age and are pregnant or not practicing contraception, you may not be included in the study. Also, if you participated in another study in the last 14 days you are not eligible for this study. During the study: Surgery: Your operation will take place according to normal hospital procedures and will only differ from this routine as follows: once you have been put to sleep under general anaesthesia and before starting surgery, the anaesthetist will insert a needle into your groin region and inject the study drug into the area about the femoral nerve (femoral 3-in-l block). This injection will contain either ropivacaine or a saline solution (placebo). Neither you nor your doctor will know which treatment you are receiving. According to normal hospital routine, local anaesthetic will be injected into the knee joint space after the surgery. Following this procedure, you will receive a further injection of either ropivacaine or saline placebo in your knee area where the staples have been inserted and where the tendon has been cut. These procedures are all done under general anaesthesia. After surgery: You will be assessed according to hospital procedures, only more frequently for measurement of your pulse rate and blood pressure. We also would like to kindly ask you to verbally assess the amount of pain you are experiencing, the quality of pain management you are receiving and to tell us if you experience any side effects, e.g. nausea, itching. These assessments will be done regularly during the first 24 hours after the surgery is completed. Prior to surgery and for the first three days following surgery you will be asked to complete a discomfort questionnaire. For the first week following surgery we would like you to keep track of the pain you are experiencing and medication you are taking in a diary. We will also ask you to indicate your satisfaction with the pain treatment at 24 hours and one week after the surgery. Postoperative medication: If the pain relief is not sufficient during the first 24 hours after surgery, you will be given oral Tylenol 3 (acetaminophen with codeine). If this medication is still not sufficient, you will be given intravenous morphine through a patient controlled analgesia device. The morphine will be delivered directly into your vein when you press a button.  S. S C H W A R Z  130  CONSENT F O R M Study Title:  Femoral 3-in-l Block Combined with Local Infiltrations in Patients Undergoing Arthroscopic Cruciate Ligament Repair: A Double Blind Comparison Between Ropivacaine 2 mg/ml and Saline (0.9%) for Postoperative Pain Relief.  Local Study Number:  DC-ROA-0001  Study Site:  Vancouver Hospital - UBC  I, (Name, b l o c k letters) have discussed the study it concerns with Dr. (Name, b l o c k letters) involves.  site  have read the attached explanation, and understand what the study  a) I am willing to participate in the study.  Signed  Date  Witness  Date (Signature)  (Name,  block  letters)  I, (Name o f i n v e s t i g a t o r , block the nature of the study to: (Name, b l o c k letters)  (Telephone  letters)  Signed  have explained  Date (Investigator's  signature)  number)  S. S C H W A R Z  131  Appendix III Aldrete scoring system of criteria for discharge from PACU (Aldrete and K r o u l i k 1970) T h e following parameters were recorded every 15 minutes after arrival at the P A C U . Patients were deemed ready for discharge w h e n a total score o f > 9 was reached. 1. Respiration 2 = A b l e to breath and cough freely 1 = D y s p n e a or limited breathing 0 = Apnea 2.  Circulation 2 = Systolic B l o o d Pressure + 2 0 % o f pre-anesthetic level 1 = Systolic B l o o d Pressure ± 21 - 4 9 % o f pre-anesthetic level 0 = Systolic B l o o d Pressure ± 5 0 % o f pre-anesthetic level  3. Skin colour 2 = Pink 1 = Pale, dusky, blotchy, other 0 = Cyanotic 4. Consciousness 2 = Fully awake 1 = A r o u s a l o n calling 0 = N o t responding 5. Nausea 2 = None 1 = Present, but responding to treatment 0 = Severe, i.e., not responding to treatment 6. P a i n (in surgical area) 2 = None 1 = Present (adequate ongoing pain treatment) 0 = Severe (morphine o n request)  S. S C H W A R Z  Abbreviations  A  ampere; SI base unit for electric current (!)  ACh  acetylcholine  ACLR  arthroscopic anterior cruciate ligament reconstruction  ACSF  artificial cerebrospinal fluid  A/D  analog-to-digital  AHP  afterhyperpolarization  ANOVA  analysis o f variance  AP  action potential  ASA  A m e r i c a n Society o f Anesthesiologists  ATP  adenosine-5'-triphosphate  B K (channels)  large ("big")-conductance Ca -activated K  C  electric capacitance; unit F  a  input capacitance  Ca  2+  ionized calcium  2 +  [Ca* ]i  intracellular calcium concentration  cAMP  cyclic  3',5'-adenosine-monophosphate  cGMP  cyclic  3',5'-guanosine-monophosphate  CI  confidence interval  CICR  Ca -induced C a  CIHR  Canadian Institutes o f Health Research  +  2+  2 +  release  +  (channels)  132  S. S C H W A R Z  133  CNS  central nervous system  d  mathematical symbol for differential  A  mathematical symbol for difference  DC  direct current  DIC-IR  differential interference contrast infrared videomicroscopy  D/A  digital-to-analog  DBP  diastolic b l o o d pressure  DRG  dorsal root ganglion  e  ~2,7182818; base o f the Eogarithmus naturalis  E  equilibrium potential (also reversal potential or Nernst potential)  Ea  equilibrium potential for CI  EAA  excitatory amino acid (e.g., glutamate, aspartate)  EC50  concentration response  EEG  electroencephalogram  EGTA  ethylene glycol-bis(p-arninoethyl e t h e r ) - N , N , N ' , N ' -  o f a drug that produces  a half-maximal  tetraacetic acid EPSP  excitatory postsynaptic potential  fMRI  functional magnetic resonance imaging  F  farad; unit for capacitance (C; m • kg  fAHP  fast afterhyperpolarization  g  gram; SI base unit for weight  ' g  2  conductance; unit S  _ 1  •s  - 4  • A ) 2  S. S C H W A R Z 134 G  giga; 109  Gi  input conductance  G i (protein) a  inhibitory guanosine-5'-triphosphate-binding protein (a subunit)  GABA GTP  y-aminobutyric acid guanosine-5'-triphosphate  h  hour; unit for time  HEPES  N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]  HVA  high voltage-activated  Hz  hertz; unit for frequency (s )  I  current; unit A  IAI-IP  (apamin-sensitive) afterhyperpolarization-generating current  Jh  hyperpolarization-activated mixed cationic current; also  -1  J ("queer current") or If ("funny current") (of. Pape 1996) q  IIR  inwardly rectifying ( K ) current  ik(Ca)  Ca  Jm  membrane current  /NaP  persistent N a current; cf. (Grill 1996)  /NaT  transient N a  IT  l o w threshold ("transient"/"tiny") C a  +  2 +  dependent K  +  current  +  "T-type C a  2 +  +  current 2 +  current; also  current" (of. Huguenard 1996)  IM  intramusculardy)  IPSP  inhibitory postsynaptic potential  IV  intravenous (ly)  S. S C H W A R Z K+  ionized potassium  1  liter; unit for volume  LGN  lateral geniculate nucleus (visual thalamus)  LVA  low voltage-activated  LTS  low threshold spike  m  milli; 10~ ; also meter; SI basis unit for length  M  molar; m o l • 1 ; unit for molarity (concentration o f a chemical substance; see mol); also: mega; 10  135  3  _1  6  MW  molecular weight  MGB(v)  (ventral partition o f the) medial geniculate body (auditory thalamus)  mol  mole; SI basis unit for the quantity o f a chemical substance (1 m o l = 6.02214 • 1 0 molecules) 23  min  minute; unit for time micro; 10~  6  MW  molecular weight; unit g / m o l  n  nano; 1 0 "  n  sample size  NA  numerical aperture  Na  ionized sodium  +  9  NIH  (U.S.) N a t i o n a l Institutes o f Health  NK  neurokinin  NMDA  N-methyl-D-aspartate  S: S C H W A R Z 136 NSAIDs  non-steroidal anti-inflammatory drugs  Q.  o h m ; unit for electric resistance (R)  Osm  O s m o l • l " ; unit for osmolarity; 1  quantity o f molecules i n 1 1 solution expressed i n m o l p  piko; 1 0 ~  P  postnatal day (e.g., P 2 = postnatal day two)  P  probability value; level o f statistical significance  PACU  postanesthesia care unit  PCA  patient-controlled analgesia  PET  positron emission tomography  P G E 2  prostaglandin  pH  hydrogen i o n concentration; —log [ H ]  pK  12  E 2 +  dissociation constant; p H — log ([base]/[cation])  a  PSP  postsynaptic potential  R  electric resistance; unit Q  r  "r squared"; coefficient o f determination  2  R  2  -  measure o f goodness o f fit; fraction o f the total variance that is explained by a m o d e l (equation)  Rei  electrode resistance  Ri  input resistance  rCBF  regional cerebral b l o o d flow  RCT  randomized controlled trial  REM  rapid eye movement  ofy  S. S C H W A R Z s  second; SI base unit for time  S  Siemens; unit for electric conductance (g); reciprocal value o f electric resistance; 1 S = 1 Q  sAHP  slow afterhyperpolarization;  SI  primary somatosensory cortex  SII  secondary somatosensory cortex  SBP  systolic b l o o d pressure  SD  standard deviation  SEM  standard error o f the mean  S K (channels)  small-conductance Ca -activated K  t  time; units s, m i n , h  Xm  membrane time constant; unit s;  2+  time required for AV  m  +  - 1  (channels)  to reach (1 - 1/e) (~ 63.21%)  o f its steady-state value as described by  AV  m  (t) = Aim R i (1 -  TEA  telraethylammonium; K  TTX  tetrodotoxin; poison o f the Japanese puffer fish,  +  channel blocker  Sphaeroides rubripes; specific N a channel blocker +  V  volt; unit for electric potential (V)  V  voltage; electric potential; unit V  VAS  visual analog scale  V  membrane potential; unit V  V  resting membrane potential; unit V  VB  ventrobasal complex o f the thalamus  m  t  137  S. 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