British Journal of Anaesthesia 82 (2): 244–9 (1999)
Isoflurane can indirectly depress lumbar dorsal horn activity in
the goat via action within the brain
S. Jinks1, J. F. Antognini2*, E. Carstens1, V. Buzin2 and C. Simons1
1Section
of Neurobiology, Physiology and Behavior and 2Department of Anesthesiology, University of
California, Davis, CA 95616, USA
*To whom correspondence should be addressed at: TB-170, University of California, Davis, CA 95616, USA
We have examined the response of lumbar dorsal horn cells to a noxious mechanical stimulus
during differential delivery of isoflurane to the brain and spinal cord of goats. We hypothesized
that isoflurane, acting in the brain, would depress dorsal horn neuronal responses to a noxious
mechanical stimulus applied to the hindlimb. Eight goats were anaesthetized with isoflurane
and neck dissections performed which allowed cranial bypass. Lumbar laminectomies were
performed to allow measurements of single-unit dorsal horn neuronal activity. Isoflurane 1.3%
was administered before bypass, and during differential delivery it was administered at each of
the following head/torso combinations: 1.3%/1.3%, 0.8%/1.3%, 0.3%/1.3%, 1.3%/0.8%, 0.8%/0.8%
and 0.3%/0.8%. When the torso isoflurane concentration was 1.3%, decreasing cranial isoflurane
from 1.3% to 0.3% did not significantly affect dorsal horn responses (from mean 325 (SD 262)
to 379 (412) impulses min–1; P.0.05). However, when torso isoflurane was 0.8%, decreasing
cranial isoflurane from 1.3% to 0.3% increased mean evoked dorsal horn activity by 42% (388
(359) to 551 (452) impulses min–1; P,0.05). These data suggest that the major effect of
isoflurane on dorsal horn responses to noxious stimuli is direct, but there is an indirect effect
occurring via descending projections from supraspinal regions.
Br J Anaesth 1999; 82: 244–9
Keywords: pain, mechanism; spinal cord; heart, cardiopulmonary bypass; anaesthetics volatile,
isoflurane; goat
Accepted for publication: September 9, 1998
The sites and mechanisms of anaesthesia are largely
unknown. Recent evidence suggests that inhaled anaesthetics, specifically isoflurane, act on the spinal cord to
suppress movement that occurs in response to a noxious
stimulus.1–4 The dorsal horn is a possible site of action as
it is involved in modulating and transmitting noxious stimuli
to other central nervous system sites.5 6 Several studies
have demonstrated that anaesthetics depress dorsal horn
responses to innocuous and noxious stimuli.7–9 Thus anaesthetics may exert their effect by action at the dorsal horn.
Because dorsal horn neurones are influenced by supraspinal
sites, such as the rostroventral medulla,10 it is possible that
anaesthetic effects could occur via direct and indirect
actions. We have reported recently the effect of isoflurane on
dorsal horn responses to noxious stimuli during differential
delivery to the brain and spinal cord.11 In that study,
we examined a wide range of isoflurane concentrations,
including those that were supraclinical (.3%). We found
that 1.3–3% cranial isoflurane tended to decrease the
response, but this did not achieve statistical significance. In
this study, we have examined a more clinically relevant
range of isoflurane concentrations (0.3–1.3%). We hypothes-
ized that isoflurane, via a cerebral action, would indirectly
depress dorsal horn neuronal responses to a noxious mechanical stimulus.
Materials and methods
After obtaining approval from the Local Animal Care and
Use Committee, eight adult female goats, weighing 4468
kg, were anaesthetized with isoflurane by mask and the
trachea intubated. Bilateral neck dissections were performed, the carotid arteries and jugular veins isolated, and
the occipital arteries ligated, as described previously.1 2 11
Lactated Ringer’s solution was infused via a peripheral
i.v. catheter. A carotid arterial catheter was inserted for
measurement of systemic arterial pressure and blood-gas
and packed cell volume analyses. Neuromuscular block was
achieved with pancuronium 0.1–0.2 mg kg–1, repeated
every 1–2 h. Rectal and nasopharyngeal temperatures were
matched closely (37.660.9°C and 37.361.2°C, respectively) and adjusted using a heating lamp, and during bypass,
with the heat exchanger of the oxygenator.
The lumbar spinal cord was exposed via laminectomy.
© British Journal of Anaesthesia
Cranial isoflurane affects dorsal horn activity
The spine was secured using four vertebral clamps. The
dura was opened to permit placement of a tungsten recording
microelectrode (resistance µ10 mΩ; F. Haer, Inc.,
Bowdoinham, ME, USA) into the lumbar dorsal horn (at
approximate L5) using a hydraulic microdrive (D. Kopf
Instruments, Tujunga, CA, USA). We sought dorsal horn
units that had mechanical receptive fields on the distal
hind limb (e.g. dew-claws and/or hoof). Action potentials
(extracellular) were amplified, displayed on an oscilloscope
and relayed to a personal computer which constructed
peri-stimulus–time histograms (PSTH, bin width 1 s) of
activity.12 We studied wide dynamic range (WDR) and
nociceptive-specific type neurones that exhibited reproducible responses to a standard noxious mechanical clamp
stimulus applied to the dew-claw or a hoof bulb. The
stimulus consisted of a 25-cm haemostat closed to the first
ratchet and applied for 10 s. The same person always
applied the stimulus to the same site, and was unaware of
the anaesthetic condition. For the control measurement,
end-tidal isoflurane concentration was 1.3%, which is the
minimum alveolar concentration in goats.1 2 Dorsal horn
cell activity was determined for 1 min before, and for 1 min
after, the onset of each stimulus. The stimulus was applied
2–4 times (usually three). At least 5 min elapsed between
applications. Responses were largely consistent in number
(usually within 627% of the mean for each set of three
pinches) and were averaged for each anaesthetic condition.
When control dorsal horn unit activity was determined,
heparin 4 mg kg–1 (repeat doses of 2 mg kg–1 every 1–2 h)
was administered i.v. A cannula was inserted into the carotid
artery and a Y cannula into the jugular vein. Another
cannula was inserted into the other jugular vein to augment
venous return. Blood (500 ml) which had been drained
from the animal was used to prime the bubble oxygenator
(B-10, Bentley, American Edwards, Irvine, CA, USA).
Oxygenator gas flow was 95% oxygen and 5% carbon
dioxide at 5–6 litre min–1. A vaporizer filled with isoflurane
was placed in-line with the gas flow. Isoflurane concentration
in arterial blood perfusing the head and brain was estimated
from the isoflurane concentration in the oxygenator
exhaust,11 13 14 and isoflurane in the torso (and spinal cord)
was determined from end-tidal samples. Oxygenator exhaust
and end-tidal gases were monitored using a calibrated agent
analyser. Cranial bypass was initiated by diverting cranial
venous blood to the oxygenator, with cranial blood flow
initiated at 600–800 ml min–1. Complete bypass was
achieved by clamping the other carotid artery.1 2 9 Glucose
was infused (10–20 mg min–1) into the oxygenator. In
some animals, phenylephrine was infused to maintain mean
arterial pressure greater than 60 mm Hg. Phenylephrine was
chosen because it has no effect on dorsal horn responses.15 16
Additionally, in a few animals, sodium bicarbonate was
administered to treat metabolic acidosis. After a stabilization
period of approximately 15–25 min, spontaneous and
evoked dorsal horn unit activities were recorded with cranial
isoflurane at 1.3%, 0.8% and 0.3%, and with torso isoflurane
Fig 1 Cross-sectional view of a representative section of the lumbar cord
(approximate L5 segment) demonstrating the recording sites. Note that
most sites were in the superficial-to-mid dorsal horn.
at 1.3% and 0.8%. The order was alternated, and the
investigator performing the noxious stimulation was
unaware of the isoflurane concentration. The noxious
mechanical stimulus was applied in the same manner as
described above. At least 15 min elapsed at each new
isoflurane concentration before dorsal horn activity was
recorded. Dorsal horn unit activity was determined again
after bypass in six goats, with 1.3% end-tidal isoflurane.
The spinal recording site was marked with an electrolytic
lesion by passing direct current through the recording
microelectrode. The goat was killed with potassium chloride
and isoflurane. The cord was removed, fixed in formalin,
frozen, cut in 50-µm sections and mounted on microscope
slides. The electrolytic lesions were observed under a light
microscope and plotted onto a camera lucida drawing of
the spinal cord section. When a lesion could not be observed,
the electrode site was estimated from the recording depth.
Statistical analysis
A log transformation was performed on mean responses of
units at each anaesthetic combination.11 17 Repeated measures analysis of variance was used to detect differences
between anaesthetic combinations, followed by the Student–
Newman–Keuls multiple comparisons test.18 Dorsal horn
responses before and after bypass were compared using
an unpaired t test. P,0.05 was considered statistically
significant.
Results
Data were obtained in eight goats (one unit per animal).
Units were localized primarily to the superficial-to-mid
dorsal horn (Fig. 1). Because WDR and nociceptive-specific
cells participate in transmission of noxious stimuli,19 20
responses from both cell types were pooled, as in our
previous study.11
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Jinks et al.
Fig 2 Population response. Mean (SD) responses are shown (n58). The log-transformed data (impulses min–1) were distributed normally at each
anaesthetic condition (range 2.3660.40 to 2.6060.39). The upper and lower 95% confidence intervals for the evoked responses are shown in each
column. Note that while torso isoflurane is 0.8%, decreasing cranial isoflurane from 1.3% to 0.3% resulted in a significant increase in the evoked
response. However, when torso isoflurane was 1.3%, changing cranial isoflurane had no significant effect. *P,0.05 compared with the 0.3%–0.8%
combination. The dominant direct spinal action of isoflurane was demonstrated by comparison of the 0.8%–0.8% and 0.3%–1.3% responses. When
cranial isoflurane was increased to 0.8% (from 0.3%) and torso isoflurane was decreased to 0.8% (from 1.3%), the response was unchanged. If any
depressant cranial action of isoflurane were predominant, we would have expected the response to decrease.
Figure 2 shows averaged spontaneous and evoked activity
for the eight units at each combination of anaesthetic to the
head and torso. With torso isoflurane at 0.8%, the mean
evoked response increased significantly as the head isoflurane concentration was decreased from 1.3% to 0.3% (from
388 (SD 359) to 551 (452) impulses min–1; P,0.05). When
torso isoflurane was 1.3%, decreasing cranial isoflurane
concentration from 1.3% to 0.3% had no statistically significant effect (from 325 (262) to 379 (412) impulses min–1;
ns). Spontaneous activity was numerically higher when
torso isoflurane was 0.8% than when it was 1.3%, but this
was not statistically significant. An individual example of
a WDR neurone is shown in Figure 3. The upper row of
peri-stimulus time histograms (PSTH) shows that the unit’s
response did not change appreciably when cranial isoflurane
concentration was reduced progressively from 1.3% to 0.3%
with torso concentration held at 1.3–1.4%. However, the
lower row of PSTH shows that the response increased
markedly with decreasing cranial isoflurane concentration
when the torso concentration was held constant at 0.8%.
Blood-gas, glucose and packed cell volume data are
shown in Table 1. As expected in this bypass model,11 there
was a mild decrease in packed cell volume, and moderate
metabolic acidosis. Systemic mean arterial pressure was
103 (34) mm Hg before bypass, 89 (23) mm Hg during
bypass and 76 (16) mm Hg after bypass. Four goats
developed facial swelling on the side of the head where
arterial blood was infused; this appeared to be associated
with the higher bypass blood flow rates. However, we do
not believe that this significantly affected the data, as the
mean unit responses in these goats increased with decreasing
isoflurane concentration to the brain in a manner that
parallelled the mean responses recorded in the goats with
lower cranial perfusion rates.
Discussion
The main finding in this study was that when the anaesthetic
concentration was selectively decreased to the brain, while
maintaining the torso isoflurane concentration constant at
0.8%, evoked responses of nociceptive dorsal horn neurones
increased. When the torso isoflurane concentration was
1.3%, there was no significant change in neuronal responses
with decreasing cranial isoflurane concentration, indicating
that the direct spinal action of isoflurane may have masked
any descending influences from the brain.
It is of interest to compare the present results with those
of our previous study.11 In Figure 4, we have combined
data from the previous study with data from the present
study to demonstrate the effect of changing cranial isoflurane
concentration from 0.3% to 3% while maintaining torso
isoflurane concentration at 1.3%. Within this range, the
cranial action of isoflurane had essentially no effect on the
evoked neuronal response. This suggests that the cranial
action of isoflurane is manifested only when the torso
isoflurane concentration is 0.8% or less. Also, Figure
246
Cranial isoflurane affects dorsal horn activity
Fig 3 Individual response of a wide dynamic range cell. These peri-stimulus time histograms (PSTH, bin width 1 s) show the response to changing
cranial and torso isoflurane concentrations. In the top row of PSTH, torso isoflurane was kept at 1.3–1.4%, and as the cranial isoflurane concentration
was decreased, the evoked response was unchanged. In the bottom row of PSTH, torso (and hence spinal cord) isoflurane was 0.8%, and decreasing
cranial isoflurane from 1.3% to 0.3% increased the evoked response.
Table 1 Packed cell volume (PCV), glucose and blood-gas values (mean (SD)). PO25partial pressure of oxygen; PCO25partial pressure of carbon dioxide;
BE5base excess
PCV
Glucose (mg dl–1)
pH
PO2 (kPa)
PCO2 (kPa)
BE (mEq litre–1)
Pre-bypass arterial
Bypass-body arterial
Bypass-oxygenerator-arterial
Bypass-oxygenerator-venous
Post-bypass arterial
29 (5)
101 (54)
7.39 (0.08)
64.4 (8.4)
4.5 (0.9)
–3 (3)
22 (4)
96 (42)
7.39 (0.08)
65.2 (6.1)
4.7 (1.3)
–3 (3)
23 (5)
145 (94)
7.24 (0.10)
63.2 (6.7)
5.7 (1.6)
–8 (3)
23 (5)
145 (96)
7.23 (0.08)
37.8 (16.0)
6.0 (1.5)
–8 (4)
18 (2)
71 (19)
7.35 (0.06)
64.1 (6.0)
5.1 (0.9)
–4 (4)
4 plots mean responses to decreasing concentrations of
isoflurane to the torso when cranial isoflurane concentration
was maintained at 1.3%. This shows that the increased
activity occurred with the change in torso concentration
from 0.8% to 0.2%, which again suggests a direct spinal
action of isoflurane on dorsal horn cells at higher concentrations. None the less, our data also revealed a descending
influence that manifested at lower cranial and torso isoflurane concentrations. Peripheral actions of isoflurane cannot
be excluded. However, isoflurane excites peripheral nociceptors21 which would tend to increase dorsal horn responses
with increasing isoflurane, which is opposite to our observations.
What descending pathways might mediate the effect of
cranial isoflurane on spinal dorsal horn neurones? Numerous
studies have shown that pathways activated from the mid-
brain periaqueductal gray and rostral ventromedial medulla
descend via the spinal dorsolateral funiculi to inhibit spinal
nociceptive transmission.6 22–24 Anaesthetics depress neurones in the midbrain reticular formation25 and rostral
ventromedial medulla, including ON- and OFF-cells,26
which are probably involved in descending inhibition and
facilitation, respectively, of spinal nociceptive reflexes.6 We
propose a model to explain our results, based on the
following assumptions: (1) the response of a spinal neurone
to a noxious stimulus depends, in part, on the balance of
descending inhibitory and facilitatory influences, and (2)
OFF-cells are less susceptible to inhibition by anaesthetics
than ON-cells. In this model, increased anaesthetic concentration to the brain would inhibit ON-cells more than OFFcells, thereby increasing descending inhibition. This is
parsimonious with our previous observation that increased
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Jinks et al.
0.8% spinal cord isoflurane was equivalent to 1 MAC
(i.e. movement was suppressed in response to noxious
stimulation). Thus we do not believe that the animals in
our study were conscious at any time.
In summary, we found that cranial isoflurane affected
dorsal horn responses to noxious stimulation, but only when
the spinal cord concentration was 0.8%. Thus the indirect
action of isoflurane in the brain was masked by the direct
action in the spinal cord.
Acknowledgements
Fig 4 Data from our previous study11 were compared and combined with
data from the present study to demonstrate a wider range of isoflurane
concentrations. Because the evoked responses with the 1.3%–1.3%
combination were similar between the two studies, these data were
combined. Note that with the torso concentration at 1.3%, changing the
cranial concentration from 0.3% to 3% had no effect on the evoked
response. However, when the cranial isoflurane was constant at 1.3%,
changing the torso isoflurane from 1.3% to 0.2–0.3% had a significant
effect only at the change from 0.8% to 0.2%. This suggests that the direct
action of isoflurane on dorsal horn responses occurs at concentrations less
than 0.8%.
isoflurane concentrations in the brain significantly depressed
dorsal horn neurones.11 Decreased anaesthetic concentration
to the brain is proposed to disinhibit ON-cells more than
OFF-cells, shifting the balance towards stronger descending
facilitation, again consistent with the present data. An
advantage of the proposed model is that it can be tested.
Data from Leung and Mason26 suggest that firing of some
OFF-cells may be depressed less compared with ON-cells
when isoflurane concentration is increased. However, a
larger database is needed to determine if general anaesthetics
affect OFF-cells less than ON-cells.
Some investigators have hypothesized that the supraspinal
action of inhaled anaesthetics could antagonize the direct
depressive effect on the spinal cord.27 This theory is supported
by data which indicate that γ-amino butyric acid (GABA)
agonist action in the periaqueductal gray or raphe magnus
results in an increased response to noxious stimulation, presumably by inhibiting neurones that, via descending tracts,
suppress nociception.28 29 Inhaled anaesthetics, such as halothane, potentiate the action of GABA, and could therefore
indirectly enhance nociception.30 The present data suggest the
opposite (i.e. that isoflurane has an indirect antinociceptive
effect). None the less, the combination of 0.3% isoflurane to
the head–0.8% to the torso suppresses movement in response
to noxious stimulation,2 and therefore a supraspinal nociceptive action of inhaled anaesthetics could occur as a direct
action on the motor system, and not on the dorsal horn.
The low cranial isoflurane concentration used in our
study (0.3%, with torso isoflurane 0.8–1.3%) was probably
sufficient to prevent awareness. In a previous study,2 we
found that the combination of 0.3% cranial isoflurane and
This work was supported in part by the Foundation for Anesthesia
Education and Research with a grant from Abbott Laboratories, and by
NIH RO1 GM57970–01.
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