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BACKGROUND: Evaluating the effects of sub-immobilizing anesthetic doses on movement will identify target neural circuits for investigation as sites of action for
anesthetic-induced immobility.
METHODS: Eleven pithed Northern Leopard frogs received 0, 0.4, 0.8, and 1.2 times
the 50% effective dose for production of immobility (ED50) of desflurane and a
further 7 received 0 and 0.4 ED50 desflurane in random order. An electric stimulus
applied to the forelimb elicited a hindlimb wiping reflex that was captured on
video for later analysis. Isometric tension developed in the hindlimb during the 30 s
stimulus application was measured.
RESULTS: Compared to 0 ED50, 0.4 ED50 desflurane significantly increased latency to
wipe 0.8 (0.1, 4.0) to 17.3 (0.4, 30.0) s (median [min max]), distance traveled by the
hindfoot 0.42 (0.09, 1.82) to 0.89 (0.16, 4.82) m, and proximity of the hindfoot to
stimulus 1 (0, 5) to 7 (1, 40) mm. It did not alter hindlimb maximum velocity or
isometric 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) to
8 (2, 14). From 0.4 to 0.8 ED50, motor depressant effects of desflurane became
apparent 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-induced
depression than motor function in frogs. This suggests that the most anestheticsensitive component of the spinal neural circuitry underlying movement generation in response to noxious stimulus is prior to the level of the motoneuron.
(Anesth Analg 2009;108:86772)
The animal care and use committee of the University of California, Davis, approved this study. Eighteen adult Northern Leopard frogs (Rana pipiens)
weighing 46.6 14.3 gm (mean sd) were obtained
from a commercial source (Charles Sullivan, Nashville, TN). Frogs were housed in 5 3 2 foot tubs
with free access to water and fed crickets every other
day. The room temperature was maintained between
20 and 21C and a 12:12 h light: dark cycle provided.
After a 4-wk acclimatization period, frogs were pithed
under a combination of hypothermia and desflurane
DOI: 10.1213/ane.0b013e318193eabe
METHODS
Animals
867
Anesthesia
Anesthetic doses delivered to the frogs were proportions of the 50% effective dose required to produce
immobility (ED50). The ED50 for immobility in frogs
would be an equivalent anesthetic dose to MAC in
mammals. Eleven frogs received 4 treatments: 0, 0.4,
0.8, and 1.2 ED50 desflurane in random order on
consecutive days. At a separate time, an additional 7
frogs received each of 3 treatments: 0, 0.4 ED50 desflurane or 0.4 ED50 isoflurane in random order on consecutive days. Previous studies in Northern hindlimb
frogs determined population ED50 for immobilization
by desflurane to be 6.78% atm at 21C.5 The isoflurane
data were collected as a preliminary investigation and
are not presented in this article.
Desflurane was delivered from a precision vaporizer in oxygen into a 4 L chamber. The gas outlet of the
chamber had a side port for connection of a gas
sampling line via which anesthetic concentration in
the chamber was monitored (Rascall II, DatexOhmeda, Helsinki, Finland). Frogs were placed in the
chamber up to two at a time and an overpressure
technique used to induce anesthesia to the desired
level.5 In brief, frogs were first exposed to twice the
desired desflurane concentration for a period equivalent to 1 equilibration half-life for that anesthetic (42
min) followed by exposure to the desired concentration for a further two half-lives. The 0 ED50 group
received oxygen only for an equivalent period of time.
At the completion of anesthetic exposure, frogs were
briefly removed from the chamber (no longer than 5
min) and had either the wiping reflex or hindlimb force
evaluated. Frogs were then returned to the anesthetic
chamber for a period of one half-life after which they
were removed for evaluation of the wiping reflex or
hindlimb force, whichever had not previously been
tested. The order of evaluating wiping reflex or force
was alternated such that within each treatment group
half of the animals had each tested first. Anesthetic
uptake and elimination in these animals is reliant upon
cutaneous diffusion. As such, anesthetic kinetics are
markedly slower in frogs when compared to mammals.5
In pilot studies, removal from anesthetic for 15 min did
not alter a frogs response to noxious stimulus. Frogs
were allowed at least 18 h to fully recover between
anesthetic exposures.
Wiping Reflex
Frogs were immobilized in ventral recumbency by
a soft material suit that had been secured to a flat,
smooth plastic surface. The suit was secured around
the body of the frog with Velcro straps such that it did
not impede hindlimb movement. Both forelimbs and
868
Motor Evaluation
Frogs were secured to a flat plate as described
above. The hindlimb to be tested was fully extended at
an angle 45 to the long axis of the body and an
inflexible plastic band secured firmly around the hock.
A calibrated force transducer, positioned perpendicular to the extended limb and vertically level with the
hock, was secured with nonelastic cotton tape to the
hock band. The force transducer was attached to a
Grass DC amplifier (Grass Instruments, Baintree, MA)
and the output displayed and recorded by a personal
computer using a Chart PowerLab interface and
Chart5 software (ADInstruments, CO Springs, CO).
The stimulus as described above was applied for 30 s
and hindlimb isometric tension measured. Force tracings were analyzed off-line and the following variables measured for the 30 s of stimulus application;
maximum isometric tension (N), total force generated
or impulse (N.s), and number of movements.
ANESTHESIA & ANALGESIA
0 ED50
n 18
0.4 ED50
n 18
0.8 ED50
n 11
1.2 ED50
n 11
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 .
Statistical Analysis
Summary data are presented as median (min, max).
For the purposes of comparison, frogs that did not
move, or reach within 5 mm of the stimulus, were
allocated a time of 30 s for latency to move, or latency
to wipe, respectively. Data for most parameters, obtained from the 11 frogs tested under all 4 conditions,
were not normally distributed in the 0.8 and 1.2 ED50
groups. At these higher anesthetic doses, an increasing
number of animals did not wipe or move and so
received 0 for the movement parameters or a maximum time of 30 s for the timed parameters. Friedman
test with Dunns multiple comparisons post hoc tests
were used to assess the effect of anesthetic for all
parameters in the 11 frogs. All data sets from the 0 and
0.4 ED50 treatments met criteria for normality, as
assessed by the Kolmogorov-Smirnov test. Data for 0
and 0.4 ED50 treatments for each parameter from the
two groups of frogs tested at different times were
compared using Students t-test. No significant differences were found, so these data were pooled for
further analysis. Paired Students t-test were then used
to compare effect of desflurane dose (0 and 0.4 ED50)
on all parameters. For all analyses, the significance
level was set at P 0.05.
RESULTS
At an anesthetic dose of 0.8 ED50 or lower, all frogs
moved and at 1.2 ED50 1 of 11 frogs moved. In the
absence of anesthesia, all frogs wiped accurately, that
is the hindfoot reached within 5 mm of the stimulus.
The number of frogs accurately wiping decreased with
increasing anesthetic dose. Nine of 18, 2 of 11, and 0 of
11 frogs wiped accurately at 0.4, 0.8, and 1.2 ED50
Vol. 108, No. 3, March 2009
desflurane, respectively. Summary data for all variables are presented in Table 1.
From 0 to 0.4 ED50, significant increases occurred in
latency to move (0.2 [0.0, 1.5] to 1.5 [0.7, 4.8] s), latency
to 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), and
distance traveled by the hindfoot (0.42 [0.09, 1.82] to
0.89 [0.16, 4.82] m). Average velocity of hindfoot
movement 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 force
generated was significantly reduced (7.3 [1.7, 23.6] to
3.2 [1.4, 13.8] N.s) in part due to a significant reduction
in the number of movements (12 [3, 28] to 8 [2, 14]).
Increasing the desflurane dose from 0.4 to 0.8 and
from 0.8 to 1.2 ED50 both significantly reduced maximum 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 typical
isometric tension tracings recorded from the frog
hindlimb at 0, 0.4, 0.8, and 1.2 ED50 desflurane are
shown in Figure 1.
The minimum distance between the hindfoot and
stimulus (proximity) increased significantly with each
increase in anesthetic dose (Table 1 and Fig. 2). The
path taken by the hindfoot during the wiping reflex
varied with anesthetic dose, with examples of typical
trajectories shown in Figure 2. In the absence of
anesthesia, frogs typically extended the hindlimb followed rapidly by a movement that brought the foot
directly forward to the site of the stimulus and
brushed it laterally away from the body. At 0.4 ED50
desflurane, the first movement was typically forward
towards the stimulus. If the stimulus was not removed
on the first attempt (often the case), the limb was
extended backwards towards its initial position and
the whole movement repeated a further 12 times
before removal of the stimulus or cessation of movement. At 0.8 ED50 desflurane, the first movement of
the foot was directed towards the stimulus typically
2009 International Anesthesia Research Society
869
DISCUSSION
Movement of anesthetized patients in response to
stimulation is a common indicator of anesthetic depth.
It has also provided a descriptor of anesthetic potency
that has been in common use for more than 40 yr. The
MAC of an anesthetic describes the alveolar anesthetic
870
addition to altering their intersegmental coordination.40 Investigation of the effect of volatile anesthetics
on movement elicited by stimulation of the mesencephalic lomomotor region also suggests immobility is
mediated via an action on ventral horn locomotor
networks.41
Depression of motor function seen at and above 0.8
ED50 desflurane in this study was not likely due to an
effect on peripheral motor apparatus. In humans, both
peripheral motor nerve conduction and neuromuscular transmission are unaffected by doses of desflurane
ranging from 0% to 7.4%.19 Instead, direct, or indirect,
effects on the motoneuron may underlie this phenomenon. Anesthetics have been shown to decrease motoneuron excitability as assessed by H-reflex and
F-wave studies.42 44 However, in neither of these
techniques is investigation restricted to intrinsic properties of the motoneuron. An alteration in the balance
of inhibitory and excitatory input to the motoneuron
would also alter H-reflex and F-wave responses. That
is, although motor function becomes depressed at
higher anesthetic doses, it is conceivable that this is the
result of anesthetic effects more proximally in the
neural circuit, reducing the balance of tonic or evoked
input to the motoneuron.
Frogs attempting to wipe away a noxious stimulus
at a desflurane dose of 0.4 ED50 show reduced accuracy and different patterns of movement without
alteration of the force or speed with which the limb
moves. With increase in desflurane dose to 0.8 ED50,
further reductions in movement accuracy occur in
addition to reduction in the force of movement. We
conclude that in the frog a component of the neural
circuit underlying movement generation proximal to
the motor neuron, such as sensory input or interneurons within CPG circuits, is most sensitive to anestheticinduced depression. Whether motoneurons then
become depressed directly by higher anesthetic doses
or whether the reduction in motor function seen
occurs subsequently to continued depression of neural
structures providing input to motoneurons remains to
be evaluated.
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