Proprioceptive Function Is More Sensitive than MotorFunction to Desflurane Anesthesia

Linda S. Barter, MVSc, PhD*

Laurie O. Mark, BA*

Joseph F. Antognini, MD*†

BACKGROUND: Evaluating the effects of sub-immobilizing anesthetic doses on move-ment will identify target neural circuits for investigation as sites of action foranesthetic-induced immobility.METHODS: Eleven pithed Northern Leopard frogs received 0, 0.4, 0.8, and 1.2 timesthe 50% effective dose for production of immobility (ED50) of desflurane and afurther 7 received 0 and 0.4 ED50 desflurane in random order. An electric stimulusapplied to the forelimb elicited a hindlimb wiping reflex that was captured onvideo for later analysis. Isometric tension developed in the hindlimb during the 30 sstimulus application was measured.RESULTS: Compared to 0 ED50, 0.4 ED50 desflurane significantly increased latency towipe 0.8 (0.1, 4.0) to 17.3 (0.4, 30.0) s (median [min max]), distance traveled by thehindfoot 0.42 (0.09, 1.82) to 0.89 (0.16, 4.82) m, and proximity of the hindfoot tostimulus 1 (0, 5) to 7 (1, 40) mm. It did not alter hindlimb maximum velocity orisometric tension but significantly reduced total hindlimb force 7.3 (1.7, 23.6) to 3.2(1.4, 13.8) N. s proportionate to a reduced number of movements from 12 (3, 28) to8 (2, 14). From 0.4 to 0.8 ED50, motor depressant effects of desflurane becameapparent with significant reductions in maximum tension from 2.0 (0.6, 5.5) to 0.8(0.1, 1.6) N and total force from 3.2 (1.4, 13.8) to 0.9 (0.0, 2.5) N.s.CONCLUSIONS: Proprioceptive function is more sensitive to anesthetic-induceddepression than motor function in frogs. This suggests that the most anesthetic-sensitive component of the spinal neural circuitry underlying movement genera-tion in response to noxious stimulus is prior to the level of the motoneuron.(Anesth Analg 2009;108:867–72)

The spinal cord is capable of generating complexmovement patterns. One example is the wiping reflex ofthe frog: a noxious stimulus placed on the body surfaceis wiped away by a limb. Regardless of stimulus place-ment or body positioning, a spinal frog is able toaccurately wipe away the noxious stimulus.1

Current evidence suggests that volatile anesthetic actionin the spinal cord is important for the production ofimmobility in response to noxious stimulation.2,3 Beyondthis, we do not have a good understanding of where thesedrugs may act to produce immobility. Investigating howstimulus-evoked movement is altered as anesthetic dose isincreased will guide investigation of particular neural cir-cuits as targets for anesthetic-induced immobility.

In rats, increasing the isoflurane dose from 0.6 to 0.9minimum alveolar anesthetic concentration (MAC)

reduces the number but not the force of movements.4

Further increasing the isoflurane dose from 0.9 to 1.1MAC reduces the force of movement or abolishes italtogether. There has been little investigation of theeffects of anesthetics on movement patterns at anes-thetic doses below 0.6 MAC.

We hypothesized that proprioception, the ability toaccurately localize parts of the body in space, wouldbe more sensitive than motor function to depressionby anesthetics. Using the wiping reflex of the frog, weaimed to examine the dose-dependent effects of des-flurane anesthesia on wiping reflex accuracy andhindlimb motor function.

METHODSAnimals

The animal care and use committee of the Univer-sity of California, Davis, approved this study. Eigh-teen adult Northern Leopard frogs (Rana pipiens)weighing 46.6 � 14.3 gm (mean � sd) were obtainedfrom a commercial source (Charles Sullivan, Nash-ville, TN). Frogs were housed in 5 � 3 � 2 foot tubswith free access to water and fed crickets every otherday. The room temperature was maintained between20 and 21°C and a 12:12 h light: dark cycle provided.After a 4-wk acclimatization period, frogs were pithedunder a combination of hypothermia and desflurane

From the Departments of *Anesthesiology and Pain Medicine,and the †Section of Neurobiology, Physiology and Behavior, Uni-versity of California, Davis, California.

Accepted for publication September 16, 2008.Supported by NIH Grant GM 61283 and GM 47818 (to J.F.A.).Reprints will not be available from the author.Address correspondence to Linda S. Barter, MVSc, PhD, Depart-

ment of Veterinary Surgical and Radiological Sciences, 2112 TupperHall, University of California, Davis, CA 95616. Address e-mail tolsbarter@ucdavis.edu.

Copyright © 2009 International Anesthesia Research SocietyDOI: 10.1213/ane.0b013e318193eabe

Vol. 108, No. 3, March 2009 867

anesthesia then allowed 24 h to recover. Pithingresulted in decerebration and was performed by in-sertion of a needle through the foramen magnumfollowed by rapid vertical and horizontal movementsof the needle tip within the cranial vault.

AnesthesiaAnesthetic doses delivered to the frogs were pro-

portions of the 50% effective dose required to produceimmobility (ED50). The ED50 for immobility in frogswould be an equivalent anesthetic dose to MAC inmammals. Eleven frogs received 4 treatments: 0, 0.4,0.8, and 1.2 ED50 desflurane in random order onconsecutive days. At a separate time, an additional 7frogs received each of 3 treatments: 0, 0.4 ED50 desflu-rane or 0.4 ED50 isoflurane in random order on con-secutive days. Previous studies in Northern hindlimbfrogs determined population ED50 for immobilizationby desflurane to be 6.78% atm at 21°C.5 The isofluranedata were collected as a preliminary investigation andare not presented in this article.

Desflurane was delivered from a precision vapor-izer in oxygen into a 4 L chamber. The gas outlet of thechamber had a side port for connection of a gassampling line via which anesthetic concentration inthe chamber was monitored (Rascall II, Datex-Ohmeda, Helsinki, Finland). Frogs were placed in thechamber up to two at a time and an overpressuretechnique used to induce anesthesia to the desiredlevel.5 In brief, frogs were first exposed to twice thedesired desflurane concentration for a period equiva-lent to 1 equilibration half-life for that anesthetic (42min) followed by exposure to the desired concentra-tion for a further two half-lives. The 0 ED50 groupreceived oxygen only for an equivalent period of time.

At the completion of anesthetic exposure, frogs werebriefly removed from the chamber (no longer than 5min) and had either the wiping reflex or hindlimb forceevaluated. Frogs were then returned to the anestheticchamber for a period of one half-life after which theywere removed for evaluation of the wiping reflex orhindlimb force, whichever had not previously beentested. The order of evaluating wiping reflex or forcewas alternated such that within each treatment grouphalf of the animals had each tested first. Anestheticuptake and elimination in these animals is reliant uponcutaneous diffusion. As such, anesthetic kinetics aremarkedly slower in frogs when compared to mammals.5

In pilot studies, removal from anesthetic for 15 min didnot alter a frog’s response to noxious stimulus. Frogswere allowed at least 18 h to fully recover betweenanesthetic exposures.

Wiping ReflexFrogs were immobilized in ventral recumbency by

a soft material suit that had been secured to a flat,smooth plastic surface. The suit was secured aroundthe body of the frog with Velcro straps such that it didnot impede hindlimb movement. Both forelimbs and

one hindlimb were extended and secured with flatcotton ties. The free hindlimb was positioned with thehip and knee joints at 90° angles and the tarsus at a 45°angle. A stimulus was delivered to the forelimb ipsi-lateral to the free hindlimb for 30 s or until physicallyremoved by the hindlimb. The stimulus was a 0.2 ms50 Hz 10 mA current (Digi Stim II, Neuro Technology,Houston, TX) delivered via two wire electrodes placedon opposing sides of the forelimb. A camera placeddirectly above the back of the frog captured thewiping reflex on video. Video was digitized at 30frames per second and using a web-based free accessvideo analysis program (Video analysis on the Web,http://electron9.phys.utk.edu/video/, M. Breinig andB. Barker, University of TN, last accessed June 9, 2008)the position of the hindfoot relative to the stimuluswas recorded for every frame with minimum resolu-tion of 0.6 mm per pixel. An indicator light on theelectric stimulator shone during current delivery andwas included in the camera frame as a reference fortime of application of stimulus. A metric ruler waspositioned on the table surface next to the frog fordistance calibration of the video image. From thesedata the following variables were measured: timefrom application of the stimulus to first movement ofthe hindfoot (latency to move), time from applicationof the stimulus to accurate wipe (latency to wipe), andthe shortest distance achieved between the hindfootand the stimulus (proximity). An accurate wipe wasdefined as the hindlimb moving within a 5 mm radiusof the stimulus. Distance traveled by the hindfoot wasextrapolated by assuming that the foot traveled astraight line within the horizontal plane betweenframes. From that data, the following variables werecalculated: maximum and average velocity of hindfootmovement, and total distance traveled by the hindfootuntil an accurate wipe was performed or 30 s hadelapsed.

Motor EvaluationFrogs were secured to a flat plate as described

above. The hindlimb to be tested was fully extended atan angle 45° to the long axis of the body and aninflexible plastic band secured firmly around the hock.A calibrated force transducer, positioned perpendicu-lar to the extended limb and vertically level with thehock, was secured with nonelastic cotton tape to thehock band. The force transducer was attached to aGrass DC amplifier (Grass Instruments, Baintree, MA)and the output displayed and recorded by a personalcomputer using a Chart PowerLab interface andChart5 software (ADInstruments, CO Springs, CO).The stimulus as described above was applied for 30 sand hindlimb isometric tension measured. Force trac-ings were analyzed off-line and the following vari-ables measured for the 30 s of stimulus application;maximum isometric tension (N), total force generatedor impulse (N.s), and number of movements.

868 Effects of Desflurane on Frog Wiping Reflex ANESTHESIA & ANALGESIA

At completion of the experiment, frogs were eutha-nized by overdose of tricaine methanosulfonate(MS222 5 gm/L). Heads were removed and fixed in10% buffered formalin and later dissected to confirmcomplete destruction of forebrain structures.

Statistical AnalysisSummary data are presented as median (min, max).

For the purposes of comparison, frogs that did notmove, or reach within 5 mm of the stimulus, wereallocated a time of 30 s for latency to move, or latencyto wipe, respectively. Data for most parameters, ob-tained from the 11 frogs tested under all 4 conditions,were not normally distributed in the 0.8 and 1.2 ED50groups. At these higher anesthetic doses, an increasingnumber of animals did not wipe or move and soreceived 0 for the movement parameters or a maxi-mum time of 30 s for the timed parameters. Friedmantest with Dunn’s multiple comparisons post hoc testswere used to assess the effect of anesthetic for allparameters in the 11 frogs. All data sets from the 0 and0.4 ED50 treatments met criteria for normality, asassessed by the Kolmogorov-Smirnov test. Data for 0and 0.4 ED50 treatments for each parameter from thetwo groups of frogs tested at different times werecompared using Student’s t-test. No significant differ-ences were found, so these data were pooled forfurther analysis. Paired Student’s t-test were then usedto compare effect of desflurane dose (0 and 0.4 ED50)on all parameters. For all analyses, the significancelevel was set at P � 0.05.

RESULTSAt an anesthetic dose of 0.8 ED50 or lower, all frogs

moved and at 1.2 ED50 1 of 11 frogs moved. In theabsence of anesthesia, all frogs wiped accurately, thatis the hindfoot reached within 5 mm of the stimulus.The number of frogs accurately wiping decreased withincreasing anesthetic dose. Nine of 18, 2 of 11, and 0 of11 frogs wiped accurately at 0.4, 0.8, and 1.2 ED50

desflurane, respectively. Summary data for all vari-ables are presented in Table 1.

From 0 to 0.4 ED50, significant increases occurred inlatency to move (0.2 [0.0, 1.5] to 1.5 [0.7, 4.8] s), latencyto accurately wipe (0.8 [0.1, 4.0] to 17.3 [0.4, 30.0] s),proximity to the stimulus (1 [0, 5] to 7 [1, 40] mm), anddistance traveled by the hindfoot (0.42 [0.09, 1.82] to0.89 [0.16, 4.82] m). Average velocity of hindfootmovement was reduced (0.4 [0.1, 1.2] to 0.3 [0.0, 0.6]m/s) while maximum velocity (2.9 [0.6, 4.8] to 2.1 [0.8,4.7] m/s) and maximum isometric tension (2.1 [0.5,6.2] to 2.0 [0.6, 5.5] N) were unchanged. Total forcegenerated was significantly reduced (7.3 [1.7, 23.6] to3.2 [1.4, 13.8] N.s) in part due to a significant reductionin the number of movements (12 [3, 28] to 8 [2, 14]).

Increasing the desflurane dose from 0.4 to 0.8 andfrom 0.8 to 1.2 ED50 both significantly reduced maxi-mum hindlimb tension from 2.0 (0.6, 5.5) to 0.8 (0.1,1.6) to 0.1 (0.0, 0.6), respectively. An example of typicalisometric tension tracings recorded from the froghindlimb at 0, 0.4, 0.8, and 1.2 ED50 desflurane areshown in Figure 1.

The minimum distance between the hindfoot andstimulus (proximity) increased significantly with eachincrease in anesthetic dose (Table 1 and Fig. 2). Thepath taken by the hindfoot during the wiping reflexvaried with anesthetic dose, with examples of typicaltrajectories shown in Figure 2. In the absence ofanesthesia, frogs typically extended the hindlimb fol-lowed rapidly by a movement that brought the footdirectly forward to the site of the stimulus andbrushed it laterally away from the body. At 0.4 ED50

desflurane, the first movement was typically forwardtowards the stimulus. If the stimulus was not removedon the first attempt (often the case), the limb wasextended backwards towards its initial position andthe whole movement repeated a further 1–2 timesbefore removal of the stimulus or cessation of move-ment. At 0.8 ED50 desflurane, the first movement ofthe foot was directed towards the stimulus typically

Table 1. Effects of Desflurane Dose on the Frog Wiping Reflex

0 ED50n � 18

0.4 ED50n � 18

0.8 ED50n � 11

1.2 ED50n � 11

Latency to move (s) 0.2 (0.0, 1.5) 1.5 (0.7, 4.8)* 1.7 (0.8, 3.3)* 30.0 (1.5, 30.0)*Latency to wipe (s) 0.8 (0.1, 4.0) 17.3 (0.4, 30.0)* 30.0 (1.8, 30.0)*† 30.0 (30.0, 30.0)*†‡Average velocity (m/s) 0.4 (0.1, 1.2) 0.3 (0.0, 0.6)* 0.1 (0.1, 0.5)* 0.0 (0.0, 0.09)*†‡Maximum velocity (m/s) 2.9 (0.6, 4.8) 2.1 (0.8, 4.7) 2.0 (0.6, 3.7) 0.0 (0.0, 2.2)*†‡Proximity to stimulus (mm) 1 (0, 5) 7 (1, 40)* 33 (0, 88)*† 146 (33, 161)*†‡Distance moved (m) 0.42 (0.09, 1.82) 0.89 (0.16, 4.82)* 0.71 (0.11, 6.61) 0.00 (0.00, 1.72)*†‡Maximum tension (N) 2.1 (0.5, 6.2) 2.0 (0.6, 5.5) 0.8 (0.1, 1.6)*† 0.1 (0.0, 0.6)*†‡Impulse (N.s) 7.3 (1.7, 23.6) 3.2 (1.4, 13.8)* 0.9 (0.0, 2.5)* 0.0 (0.0, 0.6)*†‡Number of movements 12 (3, 28) 8 (2, 14)* 6 (0, 13) 0 (0, 8)*†Eleven Northern Leopard frogs received 0, 0.4, 0.8, and 1.2 ED50 of desflurane.An electric stimulus applied to the forelimb was used to elicit a hindlimb wiping reflex.Isometric tension developed in the hindlimb during 30 s application of the same stimulus was measured.Video analysis of the wiping reflex enabled the position of the hindfoot to be tracked relative to the stimulus.Parameters reported in Table 1 pertain to the hindfoot over a period of 30 s or until an accurate wipe was performed (foot within 5 mm of stimulus) and are presented as median (min, max).Within each set of comparisons, significant differences (P � 0.05) from 0 are signified by *, from 0.4 ED50 by †, and from 0.8 ED50 by ‡.

Vol. 108, No. 3, March 2009 © 2009 International Anesthesia Research Society 869

traveling only part of the distance to the stimulus andthen making a series of laterally directed scratchingmovements in space (Fig. 2).

DISCUSSIONMovement of anesthetized patients in response to

stimulation is a common indicator of anesthetic depth.It has also provided a descriptor of anesthetic potencythat has been in common use for more than 40 yr. TheMAC of an anesthetic describes the alveolar anesthetic

concentration, which suppresses movement in 50% ofanimals subjected to a supramaximal noxious stimu-lus.6 The mechanisms by which anesthetics induceimmobility are not known. When evaluating anesthetic-induced immobility, the focus has typically been onthe presence or absence of gross movement. There hasbeen little investigation of the effects of sub-immobilizing doses of anesthetics on movement. Thecurrent findings, that accuracy and pattern of move-ment are more sensitive to depression by desfluraneanesthesia than motor function, suggest that part (orparts) of the neural circuitry underlying movementgeneration in response to noxious stimulation prior tothe level of the motoneuron are most sensitive toanesthetic depression.

Compared to the anesthetic-free state, 0.4 ED50

desflurane increased latency to wipe, minimum dis-tance (proximity) to stimulus and distance traveled bythe foot. It did not affect the maximum speed ofmovement or tension generated by the limb. Totalforce generated by the hindlimb was reduced but thischange was partly due to the reduction in number ofmovements at 0.4 ED50. It has been reported in ratsthat increasing the halothane or isoflurane dose from0.6 to 0.9 MAC did not significantly depress force permovement in response to foot clamp but did reducethe number of movements and, consequently, totalforce generated during stimulus application.4 This isconsistent with the current findings, although occur-ring at a slightly higher dose range. Motor function ofthe frogs in this study was depressed at 0.8 and 1.2ED50 desflurane. Maximum tension and total forcegenerated by the hindlimb were both reduced. At 0.8ED50, the reduction in total force was proportionatelygreater than the reduction in number of movements.This is also consistent with the previous report in ratsin which increasing halothane or isoflurane dose from0.9 to 1.1 MAC depressed force per movement, totalforce and number of movements of the extremities ofrats in response to foot clamp.4 The depression ofmotor function we describe in frogs occurred at lowerdoses than in the study reported by Antognini et al.,4

although in that study, trends towards reduction inforce per movement were seen in the step from 0.6 to0.9 MAC. Had anesthetic doses lower than 0.6 MACbeen evaluated in that study, a significant change mayhave been seen.

It is hypothesized that complex motor patterns,such as the frog wiping reflex, are constructed byvariably combining small units of motor output con-trolled by intrinsic spinal neuronal circuits (centralpattern generators or CPG).7–10 Sensory input to theseCPG circuits plays a role in initiating and modulatingmovement.8,11–14 Anesthetic depression of sensory af-ferent input into these CPG circuits or direct effects onneurons of the CPGs may underlie the reduction inwiping reflex accuracy and alterations in the pattern ofmovement seen in this study.

Figure 1. Representative examples of force tracings from apithed Northern Leopard frog after receiving 0, 0.4, 0.8, and1.2 ED50 desflurane. An electric stimulus was applied to theforelimb for 30 s and isometric tension development in theipsilateral hindlimb recorded by a force transducer posi-tioned perpendicular and level with the extended hindlimb.

Figure 2. Typical examples of the trajectory of the hindfootduring the wiping reflex in a pithed Northern Leopard frogin the absence of anesthesia (0) or the same frog afterreceiving 0.4 and 0.8 ED50 desflurane. An electric stimuluson the forelimb (black cross) was used to elicit the reflex thatwas captured on video. Analysis of the digitized videoenabled the position of the hindfoot to be tracked in everyframe (closed black circles). For reasons of clarity, where theposition of the hindfoot did not change, a black circle wasnot drawn for every frame.

870 Effects of Desflurane on Frog Wiping Reflex ANESTHESIA & ANALGESIA

Reduced sensory input is not likely to be the resultof anesthetic effects in the periphery. Volatile anes-thetics have been shown to sensitize peripheral noci-ceptors15,16 and to produce hyperalgesia at lowdoses.17,18 In addition, volatile anesthetics do notaffect peripheral nerve conduction.19,20

Many primary sensory afferent neurons synapseonto neurons in the spinal cord dorsal horn.21,22 Theeffect of desflurane on dorsal horn neurons has notbeen investigated, however, the effects of other vola-tile anesthetics on dorsal horn neuronal activity havebeen reported. Most studies examine effects of anes-thetic doses bracketing the production of immobility,for example, from 0.8 to 1.2 MAC. In this range,halothane, but not isoflurane, reduces evoked dorsalhorn neuronal activity.23–27 There are fewer reportsdescribing the effects of these drugs on evoked neu-ronal responses at lower anesthetic doses. Halothanedelivered at 0.5% to spinal cord transected, decer-ebrate cats reduced noxious heat-evoked responsesfrom dorsal horn neurons.28 In decerebrate rats, atrend towards depression of dorsal horn neuronalwind-up was seen when either halothane or isofluranedoses were increased from 0 to 0.4 and from 0.4 to 0.8MAC.29 These studies provide some evidence thatafferent input into motor circuits may be depressed bydoses of volatile anesthetics in the range of 0.4 ED50.

Afferent input to the spinal cord in the form ofproprioceptive feedback from the wiping limb playsan important role in trajectory control during thewiping reflex.30 Our results suggest that the trajectoryof the hindfoot during execution of the wiping reflex issensitive to anesthesia. Additionally, reduced or ab-sent proprioceptive feedback may account for thereduction in accuracy of the wiping reflex seen withincreasing anesthetic dose. Mammalian propriocep-tive afferents project to both the dorsal and ventralhorn31–34 and populations of spinal neurons are de-scribed that respond exclusively to joint manipulationor deep tissue pressure.35,36 There has been little workdirected specifically at evaluating the effects of generalanesthetics on proprioceptive spinal neurons. Wall35

described severe depression of these neurons inlamina VI of the cat dorsal horn by pentobarbital.Deafferentation, which would abolish proprioceptivefeedback, does not result in loss of movement.37

Anesthetics could, however, modulate these neuronsin a manner different from their total removal fromthe neural circuit, for example, by altering the balanceof excitatory and inhibitory input that proprioceptiveafferent neurons contribute to the motor circuit.

Volatile anesthetics may have direct depressanteffects on CPG neurons. Isoflurane depresses thespontaneous firing rate of ventral horn interneurons incultured spinal cord slices, suggested to reflect re-duced activity of central pattern generating circuits.38

In the lamprey isolated spinal cord, a model forinvestigating vertebrate CPGs,39 isoflurane has beenshown to depress the activity of CPG neurons in

addition to altering their intersegmental coordina-tion.40 Investigation of the effect of volatile anestheticson movement elicited by stimulation of the mesence-phalic lomomotor region also suggests immobility ismediated via an action on ventral horn locomotornetworks.41

Depression of motor function seen at and above 0.8ED50 desflurane in this study was not likely due to aneffect on peripheral motor apparatus. In humans, bothperipheral motor nerve conduction and neuromuscu-lar transmission are unaffected by doses of desfluraneranging from 0% to 7.4%.19 Instead, direct, or indirect,effects on the motoneuron may underlie this phenom-enon. Anesthetics have been shown to decrease mo-toneuron excitability as assessed by H-reflex andF-wave studies.42–44 However, in neither of thesetechniques is investigation restricted to intrinsic prop-erties of the motoneuron. An alteration in the balanceof inhibitory and excitatory input to the motoneuronwould also alter H-reflex and F-wave responses. Thatis, although motor function becomes depressed athigher anesthetic doses, it is conceivable that this is theresult of anesthetic effects more proximally in theneural circuit, reducing the balance of tonic or evokedinput to the motoneuron.

Frogs attempting to wipe away a noxious stimulusat a desflurane dose of 0.4 ED50 show reduced accu-racy and different patterns of movement withoutalteration of the force or speed with which the limbmoves. With increase in desflurane dose to 0.8 ED50,further reductions in movement accuracy occur inaddition to reduction in the force of movement. Weconclude that in the frog a component of the neuralcircuit underlying movement generation proximal tothe motor neuron, such as sensory input or interneu-rons within CPG circuits, is most sensitive to anesthetic-induced depression. Whether motoneurons thenbecome depressed directly by higher anesthetic dosesor whether the reduction in motor function seenoccurs subsequently to continued depression of neuralstructures providing input to motoneurons remains tobe evaluated.

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872 Effects of Desflurane on Frog Wiping Reflex ANESTHESIA & ANALGESIA