1Department of Anesthesia and Critical Care,
Yamamura, Hiroyoshi,
Amy Malick,
Nancy L. Chamberlin, and
Rami Burstein.
Reduction of the threshold of cardiovascular and neuronal responses to
facial and intracranial stimulation reflects central sensitization and
cutaneous allodynia in a rat model of migraine. Current
theories propose that migraine pain is caused by chemical activation of
meningeal perivascular fibers. We previously found that chemical
irritation of the dura causes trigeminovascular fibers innervating the
dura and central trigeminal neurons receiving convergent input from the
dura and skin to respond to low-intensity mechanical and thermal
stimuli that previously induced minimal or no responses. One conclusion
of these studies was that when low- and high-intensity stimuli induce
responses of similar magnitude in nociceptive neurons, low-intensity
stimuli must be as painful as the high-intensity stimuli. The present
study investigates in anesthetized rats the significance of the changes
in the responses of central trigeminal neurons (i.e., in nucleus
caudalis) by correlating them with the occurrence and type of the
simultaneously recorded cardiovascular responses. Before chemical
stimulation of the dura, simultaneous increases in neuronal firing
rates and blood pressure were induced by dural indentation with forces
Modern theories on migraine propose that the
initial activation of intracranial perivascular sensory fibers
innervating cephalic blood vessels and the dura is caused by exposure
to endogenous algogenic chemicals (Goadsby and Edvinsson
1993 We showed that these chemicals, when applied topically to the dural
sinuses, cause peripheral nociceptors innervating the dura to become
more sensitive to mechanical dural stimulation (Strassman et al.
1996 Because such electrophysiological studies require that animals be
anesthetized and because studies on motor responses to dural stimulation are lacking, it is not possible to confirm behaviorally which stimuli are nociceptive during the experiment. The goal of the
present study was to determine whether innocuous stimuli such as mild
dural indentation or skin brush are nociceptive when trigeminal brain
stem dura-sensitive neurons become hyperexcitable. One way to address
this issue is to correlate sensory stimuli with the cardiovascular and
neuronal responses they induce. The rationale for measuring
cardiovascular changes such as the pressor response is the notion that
they are correlated with visceral and cutaneous stimuli that cause
tissue damage or pain (Woodworth and Sherrington 1904 In anesthetized animals, pressor responses can be induced by electrical
stimulation of group IV muscle, cutaneous, or tooth dentin afferents
(Allen and Pronych 1997 In the present study, we simultaneously recorded cardiovascular and
nociceptive trigeminal brain stem neuronal responses to mechanical
indentation of the dura and to mechanical and thermal stimulation of
the ophthalmic skin before and after the induction of central
sensitization by chemical stimulation of the dura. To our knowledge,
this is the first attempt to correlate the responses of trigeminal
nociceptive neurons with reflexive cardiovascular changes by recording
them simultaneously.
We report that in anesthetized and paralyzed rats, pressor responses
are induced reliably by noxious stimuli before chemical stimulation of
the dura and by both innocuous and noxious stimuli after chemical
stimulation and that these changes follow the changes in neuronal
responses.
Surgical preparation
Forty-six male Sprague-Dawley rats weighing 350-700 g were
anesthetized with urethan (1.2 g/kg ip) and treated with one low dose
of atropine (0.04 mg ip; this small dose increases heart rate by
~13% for Experimental protocol
To establish physiological (cardiovascular and neuronal)
baselines for each case, nucleus caudalis (Vc) dura-sensitive neurons were identified, their intracranial and extracranial receptive fields
were mapped, and after a 10-min recovery period, MAP, HR and neuronal
spontaneous activity rates were recorded during a 10-min period.
Neuromuscular blocking agents then were infused into the vein, and the
animals were ventilated artificially. Paralyzed and ventilated animals
that failed to maintain baseline cardiovascular and neuronal values
were excluded from the study. Because the effects of the paralytic
agents used in this study lasted ~1 h (see next section), it
was possible to measure withdrawal reflexes hourly and determine the
depth of anesthesia. In the presence of paw or tail withdrawal
reflexes, additional anesthetics were given.
Initial cardiovascular and neuronal response profiles were determined
by recording MAP, HR, and neuronal activity before, during, and after
the application of mechanical and quantitative thermal stimuli to the
dural and facial receptive fields. These stimuli included dural
indentation with a series of calibrated von Frey hairs, brushing,
pressing, and pinching the facial skin and slowly heating it with a
contact thermal stimulator. Each series of stimuli was repeated at
least three times at 5- to 10-min intervals (depending on the time
required for both BP and neuronal activity to return to baseline
level). Only cases in which the responses varied by <10% were
included in the study. The value of the first of the three responses
was used for data analysis. This value was chosen to exclude the
possibility of consequential desensitization. Although the long
interstimulus interval minimized the effect of desensitization (as
evidenced by <10% variability in the three responses), we judged the
first response to be the most reliable one.
Potentially sensitizing agents (low-pH phosphate buffer or a mixture of
histamine, serotonin, bradykinin, 10 After the dura was exposed to both the low-pH phosphate buffer and the
inflammatory soup (IS), receptive fields were remapped and
cardiovascular and neuronal responses were reexamined every 30 min for
as long as reliable recordings were maintained (usually 2-10 h after
chemical stimulation). Receptive field sizes and neuronal and
cardiovascular responses to the different stimuli then were compared
(i.e., before and after chemical stimulation of the dura).
Physiological management
To maintain MAP between 90 and 130 mmHg and HR between 360 and
430 beats per minute (BPM), we replaced blood volume lost during surgery with lactated Ringer solution, administered 5% dextrose (0.2 ml) every hour to prevent hypoglycemia, administered additional anesthetic to eliminate withdrawal reflexes before the induction of
paralysis, maintained artificial ventilation (a mixture of room air and
100% oxygen) at a fast rate (80-95/min) using low tidal volume
(2.5-3.5 ml) and positive pressure (2-5 cm H2O positive end expiratory pressure, as needed), and used a cocktail of
neuromuscular blocking agents (0.5 mg/kg pipecuronium and 0.5 mg/kg
doxacurium in 1.0 ml of 0.9% NaCl) that preserves pressor and
depressor cardiovascular responses when given every 60 min.
In preliminary experiments, the anticholinergic neuromuscular blocking
agents gallamine triethiodide (4 mg/kg) and pancuronium (1 mg/kg) were
used but found to induce severe tachycardia under urethan anesthesia.
Vecuronium (4 mg/kg) and pipecuronium (1 mg/kg) were tested because
they are steroidal neuromuscular blocking agents that are unlikely to
cause tachycardia (Neidhart et al. 1994 Neuronal identification, receptive field mapping, and chemical,
mechanical and thermal stimulation
Detailed descriptions of neuronal identification, receptive
field mapping, chemical and mechanical stimulation of the dura, and
mechanical and thermal stimulation of the skin are provided in a recent
study (Burstein et al. 1998 The dural receptive field was mapped by indenting the dura with
calibrated von Frey hairs. Dural indentations were made at points
separated by 1 mm mediolaterally and 2 mm rostrocaudally, throughout
the exposed dura. At each point, a suprathreshold mechanical force
(usually 4.19 g on the sinuses and 2.35 g away
from the sinuses) was used to indent the dura 10 times. Points at which these forces produced a response in Mechanical thresholds for dural indentation were determined with a
series of calibrated von Frey hairs (Semmes-Weinstein Aesthesiometer, Stoelting; tip shape: flat and round, diameter range = 0.15-0.38 mm) applied for 5 s at 25-s intervals to the most sensitive part of the dural receptive field in ascending order (0.080, 0.176, 0.217, 0.445, 0.745, 0.976, 2.35, and 4.19 g). The threshold value was
considered the lowest strength von Frey hair that elicited at least one
burst of spikes (i.e., Responses to mechanical stimulation of the skin were determined by
applying brief (10 s) innocuous and noxious stimuli to the most
sensitive portion of the cutaneous receptive field. Innocuous stimuli
consisted of slowly passing a soft bristled brush across the cutaneous
receptive field (one 5-s brush stroke from caudal to rostral and one
5-s brush stroke from rostral to caudal) and pressure applied with a
loose arterial clip. Noxious stimuli consisted of pinch with a strong
arterial clip. More intense or prolonged stimuli were not used to avoid
inducing prolonged changes in spontaneous neuronal discharge or
response properties.
Thermal thresholds for cutaneous stimulation were determined by slowly
heating (35-50°C at a rate of 1.0°C/s) the cutaneous receptive
field with a 9 × 9 mm contact thermal stimulator (Yale University).
Thermally conductive paste was applied to the skin, and the stimulated
surface was maintained at 35°C during the periods between stimuli.
Temperatures at which neuronal activity increased by 25% and MAP
changed >4 mmHg over baseline were considered as thresholds.
Restricted, topical chemical stimulation of the dura was ensured by
positioning the head at an angle in which the dorsal surface of the
dura was perfectly horizontal, by forming a wall of agar around the
craniotomy, and by placing a small piece of gelfoam soaked in the
chemicals on the dural receptive field.
Data collection and analysis
Mean arterial pressure was detected by a pressure transducer
(Viggo-spectramed), amplified by a digital blood pressure monitor with
an analog output (Stemtech), collected by computer, and analyzed quantitatively by Neuro-spike software (Pearson Technical Software). Mean arterial pressure was derived from the pulsatile signal. It was
calculated from the integration of the area under the pressure curve
and averaged over time. Heart beats and neuronal spikes were detected
by two chest and one high-impedance recording electrodes, amplified and
sent to separate window discriminators, collected by computer, analyzed
quantitatively with the same Neuro-spike software and presented as
peristimulus time histograms (500-ms binwidth).
In each stimulus trial (e.g., heating the skin from 35 to 50°C),
temporal (latency and duration of responses) and quantitative (response
magnitude) changes in neuronal activity and MAP were studied (Fig.
1). Neuronal and BP response thresholds
were defined as the stimulus intensity at the initiation of the
responses. Measurements of response magnitudes were used to study
quantitative aspects of neuronal and cardiovascular interactions.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
2.35 g and by noxious cutaneous stimuli such as pinching
the skin and warming >46°C. After chemical stimulation, similar
neuronal responses and blood pressure increases were evoked by much
smaller forces for dural indentation and by innocuous cutaneous stimuli
such as brushing the skin and warming it to
43°C. The onsets of
neuronal responses preceded the onsets of depressor responses by
1.7 s and pressor responses by 4.0 s. The duration of
neuronal responses was 15 s, whereas the duration of depressor
responses was shorter (5.8 s) and pressor responses longer (22.7 s)
than the neuronal responses. We conclude that the facilitated
cardiovascular and central trigeminal neuronal responses to innocuous
stimulation of the skin indicate that when dural stimulation induces
central sensitization, innocuous stimuli are as nociceptive as noxious
stimuli had been before dural stimulation and that a similar process
might occur during the development of cutaneous allodynia during
migraine.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Lauritzen 1994
; Moskowitz and
Macfarlane 1993
). Serotonin, bradykinin, histamine, prostaglandins, and potassium and hydrogen ions can activate and sensitize somatic and visceral nociceptors (Davis et al.
1993
; Handwerker and Reeh 1991
; Lang et
al. 1990
; Mizumura et al. 1987
; Steen et
al. 1992
, 1995
) and are believed to play a role in the pathogenesis of migraine (Moskowitz and Macfarlane 1993
;
Wolff 1963
).
) and central trigeminal neurons that receive convergent
input from the dura and skin to become more sensitive to mechanical
stimulation of the dura and to mechanical and thermal stimulation of
the skin (Burstein et al. 1998
). On the basis of these
studies, we proposed that while sensitization of both peripheral and
central trigeminovascular neurons could account for the intracranial hypersensitivity (Blau and Dexter 1981
), sensitization
of central but not peripheral neurons could account for the
extracranial hypersensitivity of migraineurs (Drummond
1987
).
).
; Allen et al.
1996
; Johansson 1962
), noxious colorectal or
ureter distension (Ness and Gebhart 1988
; Roza
and Laird 1995
), and noxious chemical irritation of the cornea
or nasal mucosa (Bereiter et al. 1994
; Panneton
and Yavari 1995
). Although the pressor response may be
anesthetic-dependent (Ness and Gebhart 1988
), it has
become a commonly used indicator of nociceptive stimulation.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
2 h) 4 h before the recording. The right femoral artery and vein were cannulated with PE-50 tubing for measuring mean
arterial blood pressure (MAP) and heart rate (HR) and to allow the
infusion of fluid and neuromuscular blocking agents, respectively. To
meet the challenge of keeping the intraarterial line open for many
hours, continuous saline infusion was used. The pressure required to
infuse the saline added 5 mmHg to the blood pressure values.
To minimize surgical bleeding, no heparin sulfate was administered. A
metal tube was inserted into the trachea for artificial ventilation,
and the rat was mounted in a stereotaxic apparatus. End-tidal
CO2 and core temperature were monitored continuously and
kept within physiological range. The ventral half of the occipital bone
and the laminar process of C1 were removed to allow the introduction of
a recording electrode into the dorsal horn at the spinomedullary junction and the trigeminal nucleus caudalis. The dura overlying the
dorsal surface of the brain was exposed carefully using a slow-speed
drill with a blunt bit to remove the frontal and parietal bones (these
methods minimized surgical irritation of the neurons). To prevent the
dura and the exposed brain stem areas from drying, they were surrounded
by agar and covered with warm saline and mineral oil, respectively.
3 M, and
prostaglandin E2, 10
4 M, at pH 5.0) (adapted from
Steen et al. 1992
, 1995
, who termed it "inflammatory
soup") then were applied topically to the dural receptive field for
2 min. To sensitize the dura most effectively, these two chemical
stimuli (low-pH phosphate buffer and inflammatory soup) were applied
for 2 min each at an interval of 10 min. Because we previously found
that this combination of chemical stimuli is most effective at inducing
hypersensitivity in peripheral nociceptors that innervate the dura
(Strassman et al. 1996
) and in central trigeminal brain
stem neurons that receive convergent input from the dura and the
ophthalmic skin (Burstein et al. 1998
), their consecutive application was considered as a single stimulus for the
sensitization study.
; Rathmell
et al. 1993
; Shorten et al. 1995
), but were
found ineffective as recovery occurred within <20 min. To increase the duration of muscular paralysis without inducing tachycardia, we therefore used a mixture of pipecuronium and doxacurium. Doxacurium is
a nondepolarizing (benzylisoquinolinium compound) neuromuscular blocking drug with a longer duration of action (Hunter
1995
). This combination provided ~60 min of muscle paralysis
and therefore was administered repeatedly.
). Briefly, single-unit activity was recorded in the dorsal horn 0-2.5 mm caudal to the obex
with stainless steel epoxy-coated microelectrodes (8-12 M
). To
identify dura-sensitive neurons, the recording microelectrode was
advanced into the ventrolateral dorsal horn while single shocks (0.8 ms, 0.5-4.0 mA, 1 Hz) were delivered repeatedly through a bipolar
stimulating electrode placed on the dura overlying the ipsilateral
transverse sinus. The search area for dura-sensitive neurons was based
on our previous study (Burstein et al. 1998
) and on the
high concentration of these neurons in the ventrolateral area of
nucleus caudalis (Strassman et al. 1994
). This rate is usually sufficient to cause wind-up in sensory spinal cord dorsal horn
neurons (Cook et al. 1987
). To minimize this risk,
search stimuli were delivered for a short period (5 min) and stopped for ~5 min. After a neuron was found to respond to electrical stimuli, the stimulation was turned off and spontaneous activity was
recorded in the absence of stimulation. In most cases, the spontaneous
activity decreased over the following 5-10 min and then returned to
baseline.
50% of the trials were
considered to be within the neuron's dural receptive field. The
cutaneous receptive field was mapped by applying brief innocuous (air
puffs, vibrissae and hair deflection, and brush) and noxious (pinprick, pinch) mechanical stimuli to the nose, vibrissal pad, lips, intraoral mucosa, cornea, ophthalmic, maxillary and mandibular skin areas, limbs,
and tail.
4 spikes) and a MAP change in
50% of the
trials (usually 3 trials). Here and throughout the study a MAP change
was defined as a >4 mmHg shift from baseline. Although mechanical
stimulation with von Frey hairs is applied by hand and thus might not
be as accurate as stated above, it was reliable enough to show changes
in sensitivity.
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Fig. 1.
Measurements of the different types of neuronal and blood pressure
response latencies, magnitudes, and durations to chemical
(top), thermal (middle), and mechanical
(bottom) stimulation of the intracranial dura and facial
skin. Peristimulus time histograms and analogue oscillographic tracings
depict neuronal activity and mean arterial pressure. Horizontal solid
lines illustrate stimulus initiation, intensity and termination.
Vertical dotted lines and open arrowheads indicate the time at which
responses were initiated and terminated. Horizontal dotted lines
indicate blood pressure baseline values and response magnitudes.
Numbers in parentheses depict response magnitudes. Note that although
all types of stimuli were capable of inducing depressor
(I), pressor (II), and mixed
(III) blood pressure responses, chemical and thermal
stimuli (which reach maximal effect slowly) induced depressor and mixed
responses more often than mechanical stimuli, which are quick to reach
maximal effect, and usually caused pressor responses. Br, brush; idr,
initiation of depressor response; inr, initiation of neuronal response;
ipr, initiation of pressor response; IS, inflammatory soup; Pi, pinch;
Pr, pressure; tdr, termination of depressor response; tnr, termination
of neuronal response; tpr, termination of pressor response.
The initiation time of a positive response (see definition in the preceding text) was defined as the earliest time point at which a shift from baseline was detected during a stimulus. The response magnitude was calculated by subtracting the mean ongoing neuronal activity rate or MAP before the onset of the stimulus (10, 25, and 30 s for mechanical skin, mechanical dural, and thermal skin stimulation, respectively) from the peak response value (defined as the maximal increase in mean spikes/s or MAP change during a 2-s period in which mechanical stimuli were applied or during a 5-s period for thermal and chemical stimuli).
Cardiovascular response type
Because experimentally induced cardiovascular responses vary
according to the preparation (e.g., anesthetics, ventilation and
neuromuscular blocking agents), we first sought to determine the
frequencies and characteristics of the different types of cardiovascular responses that could be induced by mechanical, thermal
and chemical stimulation of the intracranial dura and the facial skin
under our experimental conditions. In this pilot study, mean arterial
pressure responses were classified as depressor, pressor or mixed types
(Fig. 1). A depressor response was defined as a decrease in BP from
baseline, a pressor response was defined as an increase in BP over
baseline, and a mixed response was defined as a series of BP changes
which consist of an initial decrease and a subsequent increase over
baseline (the magnitude of a mixed response was calculated by adding
the magnitude of the pressor response to the magnitude of the depressor
response). Mechanical indentation of the dura with a force of 4.19 g induced pressor (66%), depressor (21%), and mixed (13%)
BP responses in 52% of the trials, and no response in 48% of the
trials. Skin pinch induced BP responses (88% pressor and 12% mixed)
in all trials. Skin pressure induced BP responses (83% pressor, 12%
depressor, and 5% mixed) in 48% of the trials. Skin brush induced BP
responses (94% pressor and 6% depressor) in 12% of the trials. Heat
skin stimuli induced BP responses (58% pressor, 13% depressor, and
29% mixed) in almost all (93%) trials, and chemical stimulation of
the dura with IS or phosphate buffer induced pressor (54%), depressor
(10%), and mixed (36%) responses in 51% of the trials. Because of
the predictability and high frequency of pressor responses, their
latencies, durations, and magnitudes were used for data analyses in all
cases where they were present. In their absence (<15%), depressor
response measurements were used. Because heart rate measurements
exhibited spontaneous fluctuations in baseline (12 BPM), we could not
detect changes reliably and therefore did not analyze these responses in the main study.
Anatomic analysis
At the conclusion of each experiment, the recording site was marked with an electrolytic lesion (anodal DC of 25 mA for 20 s). Rats were perfused with 1% potassium ferrocyanide in 10% formalin. The medulla and C1 spinal cord segment were removed, fixed, and reacted for Prussian blue stain of ferric ions. The tissue was cut transversely (50 µm) on a freezing microtome and counterstained with Neutral red. Nonstained and Nissl-stained sections containing the lesions were examined with dark- and bright-field illumination, respectively. Locations of lesions were reconstructed by use of a camera lucida drawing tube.
Statistical analysis
Data are presented as means ± SE for arithmetic averaging
and compared statistically with the parametric Student's
t-test for all but the von Frey hair measurements. Von Frey
hair measurements are compared with the nonparametric Wilcoxon
signed-rank test. The ordinal one-sided confidence interval of
proportion (p 1.64
p(1
p)/n) was used to calculate the lower limits of
the "correlation" between neuronal and BP responses
(Rosner 1995
). Correlations between blood pressure and
neuronal response also were analyzed using linear regression.
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RESULTS |
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General neuronal properties
In 38/46 attempts we successfully measured BP and HR while
recording from dura-sensitive neurons in Vc. In 24 experiments, both BP
and neuronal activity were stable enough to establish a reliable
physiological baseline and BP measurements were responsive enough to
test for sensitization. In five experiments, recordings were stable
enough to study neuronal sensitization only. For BP, physiological
baseline was defined as stable if it varied by 4 mmHg. For neuronal
activity, physiological baseline was defined as the mean number of
spikes before the beginning of the stimulus, and stable action
potential recording was defined as a consistent
1:3 signal-to-noise
ratio. Although neurons were always responsive to somatosensory
stimuli, BP responses were sometimes lacking. Because a complete lack
of cardiovascular responses to even noxious somatosensory stimuli can
be caused by depth of anesthesia, ventilation parameters or
neuromuscular blocking agents, these data could not be interpreted
reliably.
RECORDING SITES.
Recording sites were identified for 24/29 dura-sensitive neurons in the
trigeminal Vc and first cervical segment (C1). Figure 2A illustrates the recording
sites of 18 neurons that were sensitized () and 6 neurons that were
not sensitized by chemical stimulation of the dura. The sensitized
neurons were recorded in the ventrolateral region of laminae V (83%)
and I (17%) of the medullary (Vc) and cervical (C1) dorsal horn.
|
RECEPTIVE FIELDS. The extracranial and intracranial excitatory receptive fields of the 29 neurons tested for sensitization are illustrated in Fig. 2B (in 5/29 experiments only neuronal sensitization was tested because of plugged intraarterial lines). Intracranial receptive fields were generally small (1-4 mm) and located mainly around the dural sinuses. Cutaneous receptive fields included skin areas innervated by the ophthalmic branch of the trigeminal nerve in all cases. They were most often small and restricted to the ophthalmic region (60%), although in some cases they were large, extending to the maxillary (30%) and mandibular (13%) regions as well. After chemical stimulation of the dura, intracranial receptive fields expanded in almost 50% of the cases (usually within 30 min), and extracranial receptive fields expanded in almost 20% of the cases (usually within 60-120 min).
CHEMOSENSITIVITY. During simultaneous recording of BP and neuronal activity, brief (2-min) chemical stimuli applied topically to the dura induced changes in both BP (>4 mmHg) and neuronal activity in 17/24 neurons and in BP only in 4/24 neurons. Changes in neuronal activity were always accompanied by BP changes. In the cases in which neuronal and BP responses occur simultaneously, increased neuronal activity was accompanied by pressor (56%), mixed (32%), or depressor (12%) responses. In the cases in which BP responses were induced in the absence of neuronal response, only pressor and mixed responses were recorded.
Changes in blood pressure and neuronal responses induced by chemical irritation of the dura
INCREASED SENSITIVITY TO MECHANICAL STIMULATION OF THE DURAL
RECEPTIVE FIELD.
The effects of chemical stimulation of the dura on dural
mechanosensitivity were examined in 16 experiments in which BP and trigeminal brain stem neurons were recorded simultaneously. Before chemical stimulation, mechanical indentation of the dura with von Frey
hairs induced neuronal and BP responses when the applied forces were
2.35 g (median). Twenty minutes after the chemical stimulation with IS, a significant increase in the mechanical sensitivity of the dural receptive field was manifested as a drop in
the minimum force (threshold) required to activate neuronal (2.35-0.445 g) and BP (2.35-0.976 g) responses
(medians, unpaired Wilcoxon signed-rank test, P < 0.0001). Figure 3A illustrates a case in which thresholds for neuronal and BP responses were altered
by chemical stimulation of the dura. As shown in the figure, neuronal
responses were short and rapidly adapting, usually outlasting the
stimulus by 2-3 s, while pressor responses were large (8-14 mmHg) and
longer, usually outlasting the stimulus by 10-15 s. The changes in the
mechanical thresholds required to induce BP (I) and neuronal (II)
responses from the dura in each of the 16 cases are illustrated in Fig.
3B. In 69% of the cases, a drop in the mechanical threshold
for the neuronal response was accompanied by a similar drop in the
mechanical threshold of the BP response; in 19% of the cases, the
mechanical thresholds did not change in either response; and in 12% of
the cases, it changed for neuronal but not BP responses (Table
1, Fig. 3BIII). The point
estimate for the proportion of cases in which the threshold for
mechanical stimulation of the dura changed in both neuronal and BP
responses is 0.69 and the one-sided confidence interval for this
proportion is 0.50. This lower limit confidence interval value is
larger than the critical limit (0.25), which allows us to reject the null hypothesis (of "no correlation") and conclude that the
increased sensitivity of the two responses are correlated. This
significant correlation between BP and neuronal response thresholds was
corroborated by linear regression analysis (Fig.
3BIII). This correlation holds true for all lamina V
neurons [9 wide dynamic range (WDR) and 2 high threshold (HT)], but
not for all lamina I (1 HT and 2 WDR) neurons, where only one WDR
neuron exhibited a drop in threshold for dural indentation. This
intracranial hypersensitivity lasted for 1-7 h. No comments can be
made here regarding the reversal of sensitization because in most cases
stable neuronal recordings were lost before the recovery. After
chemical stimulation of the dura, changes in the magnitude of neuronal
but not BP response were significant (changes in neuronal response
magnitude are not described here further because they were described in
Burstein et al. 1998
).
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INCREASED SENSITIVITY TO MECHANICAL STIMULATION OF THE CUTANEOUS RECEPTIVE FIELD. The effect of chemical stimulation of the dura on cutaneous mechanosensitivity was examined in 19 experiments in which BP and trigeminal brain stem neurons were recorded simultaneously. Sixty minutes after chemical stimulation of the dura with the IS, a significant increase in the mechanical sensitivity of the skin was manifested as increased neuronal and BP response magnitudes to brush and pressure (P < 0.05). Neuronal responses to brush and pressure increased 2- and 1.6-fold, respectively, while BP responses to brush and pressure increased 3- and 1.5-fold, respectively. Chemical stimulation caused a significant (P < 0.005) increase in the BP (1.3-fold) but not the neuronal response to pinch. Figure 4A illustrates a case in which neuronal and BP responses to mechanical stimulation of the skin were altered by chemical stimulation of the dura. In the illustrated case, neuronal responses to brush and pressure increased twofold, pressor responses to brush occurred only after chemical stimulation, and pressor responses to pressure and pinch increased 1.4-fold. In many experiments, the magnitude of the pressor response to brush after chemical stimulation was similar to the magnitude of the response to pinch before chemical stimulation. The changes in BP (A) and neuronal (B) response magnitude to each of the mechanical stimuli to the skin are summarized in Fig. 4B. The relative incidences of changes in the magnitudes of neuronal and BP responses to brush, pressure and pinch are shown in Table 2. The point estimate for the proportion of cases in which the mechanical stimulation of the skin changed both neuronal and BP response magnitudes is 0.47 for brush, 0.26 for pressure, and 0.21 for pinch; and the one-sided confidence interval for these proportions is 0.28 for brush, 0.1 for pressure, and 0.06 for pinch. These lower limits of the confidence interval indicate that the chemically induced changes in the magnitude of neuronal and BP responses are correlated only when brush is used to stimulate the skin (Fig. 4Bc). This by no means suggests that the magnitudes of neuronal and blood pressure responses are not correlated when stronger stimuli are used. It merely shows their inability to reflect the development of hypersensitivity. Simultaneous increases in neuronal and blood pressure response magnitude were exhibited by 80% of lamina V and 75% of lamina I neurons and by 66% of WDR and 75% of HT neurons. As mentioned above, this cutaneous hypersensitivity lasted at least 1-7 h in the different experiments.
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INCREASED SENSITIVITY TO THERMAL STIMULATION OF THE CUTANEOUS RECEPTIVE FIELD. The effect of chemical stimulation of the dura on cutaneous thermosensitivity was examined in 13 experiments in which BP and trigeminal brain stem neurons were recorded simultaneously. Before chemical stimulation, slow increases in skin temperature (1°C/s) induced neuronal responses at 44.8 ± 0.7°C (mean ± SE) and BP responses ~2 s later, at 46.5 ± 0.7°C. The magnitude of these responses was 30.0 ± 6.4 spikes/s for neuronal activation and 12.1 ± 1.4 mmHg for BP changes. Sixty minutes after chemical stimulation of the dura with IS, increased skin sensitivity to heat was apparent as the lowest temperature capable of initiating neuronal and BP responses dropped significantly (P < 0.0005), by 3.7 and 3.4°C (means), respectively. The neuronal and BP response magnitudes to heat were unchanged after chemical stimulation. Figure 5A illustrates a case in which neuronal and pressor response thresholds to slow heat of the cutaneous receptive field of a dura-sensitive WDR neuron in lamina V dropped by 5-6°C after chemical stimulation of the dura. The 13 cases in which BP and neuronal response thresholds (I-III) and magnitudes (IV-VI) to heat were recorded before and after chemical stimulation are illustrated in Fig. 5B. In 77% of the cases, a drop in the thermal threshold for the neuronal responses was accompanied by a similar drop in the BP response, regardless of the response type (Table 3). In 15% of the cases, the thermal threshold dropped only for the neuronal response and in 8% it dropped for the BP but not neuronal response. The point estimate for the proportion of cases in which the threshold for thermal stimulation of the skin changed in both neuronal and BP responses is 0.77, and the one-sided confidence interval for this proportion is 0.57. This lower limit confidence interval value is larger than the critical limit of 0.25; this allows us to conclude that the increased sensitivity of the two responses is correlated and that the occurrence of one response can predict the occurrence of the other. This significant correlation between BP and neuronal response thresholds was corroborated by linear regression analysis (Fig. 5BIII). This correlation held true for all but one dura-sensitive neuron in lamina V and for the two lamina I neurons. Unlike the correlated change in response threshold, no correlation was found between the neuronal and BP response magnitudes. Small neuronal responses were sometimes accompanied by large changes in BP and vice versa.
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Temporal and quantitative relationship between blood pressure and neuronal responses
TEMPORAL ASPECTS. In >90% of the cases (664 trials), the temporal relationships between trigeminal brain stem neuronal responses and BP changes were as follows: initiation of neuronal responses preceded the initiation of both depressor and pressor responses; peak neuronal responses occurred either after the initiation of the depressor responses and at the onset of the pressor responses (45% of trials) or immediately after the onset of the pressor response (40% of trials); and while depressor responses were 10 s shorter than neuronal responses (5.8 vs. 15.2 s), pressor responses usually outlasted them by ~7 s (22.7 vs. 15.2 s). These temporal relationships are illustrated in Fig. 1 and summarized in Fig. 6. The interval between the initiation of the two responses varied slightly according to the stimulus and the BP response type. When slowly ascending thermal stimuli were applied to the skin, neuronal responses preceded depressor responses by 1.7 ± 0.3 s, and pressor responses by 4.0 ± 0.3 s (Figs. 1, middle, and 6, A and B, top 2 histograms). However, when mechanical stimuli, which are quick to reach maximal effect, were used, neuronal responses preceded depressor and pressor responses by <1 s (Fig. 1, bottom). These temporal relationships between the responses of lamina V dura-sensitive neurons and the changes in BP usually were preserved after the chemical stimulation of the dura and the development of neuronal hypersensitivity. Because of the small sample of lamina I dura-sensitive neurons, their relationship to the BP response is inconclusive.
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QUANTITATIVE ASPECTS. In >90% of the trials (before and after chemical stimulation of the dura), the magnitude of the trigeminal brain stem neuronal responses to mechanical stimulation of the skin was proportional to the magnitude of the accompanied BP responses. Within the same experiment, stimuli that induced small neuronal responses had the tendency to induce small or no BP responses, and stimuli that induced large neuronal responses had the tendency to induce large BP responses (Fig. 4). A summary of the 19 experiments in which chemical stimulation of the dura induced neuronal and BP responses to mechanical stimulation of the skin is shown in Fig. 7.
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DISCUSSION |
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This study describes the consequences of exposing the dura to
chemical agents associated with inflammation by showing that when
trigeminal brain stem dura-sensitive neurons become more sensitive to
mechanical stimulation of the dura and to mechanical and thermal
stimulation of the skin, this neuronal hypersensitivity is accompanied
by parallel changes in cardiovascular responses associated with
nociception (for further discussion on the sensitization of meningeal
primary afferent fibers by the IS and low-pH low phosphate buffer, and
the sensitizing effects of the electrical search stimuli, the repeated
sensory stimulation and the surgery, see Burstein et al.
1998a). To our knowledge it is the first study that uses
simultaneous monitoring of cardiovascular parameters and nociceptive
trigeminal brain stem neurons to assess the significance of changes in
neuronal responses before and after the induction of central
sensitization. The findings that in the same animal, pressor responses
are induced by noxious stimuli before the neuronal sensitization and by
both innocuous and noxious stimuli after the sensitization suggest that
when this neuronal hypersensitivity develops, innocuous stimuli such as
skin brush and mild warming (<42°C) or dural indentation with small
forces (<1 g) could be as nociceptive as skin pinching and
high heat (>46°C) or dural indentation with maximal forces (>2.35
g) in the absence of neuronal sensitization. Because the
cardiovascular responses that accompanied the neuronal hypersensitivity
are indicative of pain and because the neuronal responses preceded the
cardiovascular responses, we propose that sensitization of these brain
stem trigeminal neurons contributes to the nociceptive response in the
anesthetized animal and similarly to the cutaneous allodynia in awake
patients during migraine (unpublished observations in our clinic).
In the present study, known noxious stimuli such as pinching the skin
induced strong pressor responses in almost all cases in which neuronal
and cardiovascular responses were monitored. Because these results are
in agreement with the notion that noxious stimuli produce pressor
responses (Allen et al. 1996; Bereiter et al.
1994
; Cervero 1982
; Ness and Gebhart
1988
; Roza and Laird 1995
; Sato and
Schmidt 1973
; Woodworth and Sherrington 1904
), their occurrence was used to determine whether a stimulus is
nociceptive or nonnociceptive. However, because innocuous stimuli can
become nociceptive under pathological conditions that induce peripheral or central sensitization (Campbell et al. 1988
;
Neugebauer and Schaible 1990
; Sato and Perl
1991
; Woolf 1983
) and induce pain (Torebjork et al. 1984
), knowing whether peripheral or
central neuronal sensitization existed when a stimulus induces pressor response is critical to the interpretation of the response.
Furthermore, without knowing whether and how the sensory and autonomic
nervous systems are capable of responding to a stimulus or whether the sensory neurons have been sensitized by the surgical procedure, it is
not possible to determine whether the failure to induce a
cardiovascular response has been caused by unresponsive sensory neurons, blocked or saturated autonomic functions, or simply because the stimulus was not nociceptive.
In addition to pressor responses, we also found that depressor
responses were induced by many stimuli. It is believed that although
noxious stimuli induce mainly pressor responses (Allen et al.
1996; Bereiter et al. 1994
; Cervero
1982
; Euchner-Wamser et al. 1994
; Ness
and Gebhart 1988
; Roza and Laird 1995
;
Taylor et al. 1995
), they also can evoke depressor
responses (Brasch and Zetler 1982
; Clark and
Smith 1985
; Lembeck and Skofitsch 1982
; Ness and Gebhart 1988
). In this study, the occurrence of
the two response types in the same preparation under identical
conditions raises the possibility that either could implicate a
stimulus as noxious and a response as nociceptive. Furthermore, the
data suggest that at least two distinct but opposing autonomic
processes may occur in response to pain. Mild noxious stimuli may
increase vagal tone or suppress sympathetic tone, resulting in
depressor or mixed responses; whereas, more severe or sustained stimuli may activate sympathetic efferents. Depressor or mixed responses were
usually apparent when slow stimuli (i.e., heat and chemical) were used
but often were masked by the pressor response during fast stimuli
(i.e., skin pinch). Close examination of all mixed response cases (30%
of the responses) show that the thermal threshold of the two
cardiovascular response types is in the noxious range (i.e., >45°C)
and that the initiation time of the depressor response always precedes
that of the pressor response. We therefore suggest that the noxious
stimuli required to evoke the depressor response are milder than those
required to evoke the pressor response. This conclusion is also in
agreement with Kumada et al. (1975
, 1977
), who showed
that low-frequency electrical stimuli (5-10 Hz) capable of activating
nociceptive fibers in the trigeminal nerve and tract are optimal for
inducing the trigeminal depressor response (TDR) and that
high-frequency stimuli (>50 Hz) are optimal for inducing pressor
responses.
If noxious stimuli cause increased sympathetic and parasympathetic
output, whether these changes are manifested as pressor, depressor, or
both will likely depend on the baseline physiology of the animal.
Different combinations of anesthetics and neuromuscular blocking agents
may favor either pressor or depressor responses; previous studies
showed that different anesthetics selectively block pressor responses
(Farber et al. 1995; Ness and Gebhart 1988
; Samso et al. 1994
), that steroidal and
nonsteroidal anticholinergic neuromuscular blocking agents induce
different levels of tachycardia and increased BP by altering vagal tone
in different ways (Appadu and Lambert 1994
;
Hunter 1995
; Neidhart et al. 1994
;
Rathmell et al. 1993
; Shorten et al.
1995
), and that animals of different sources can display
differences in cardiovascular responses to sensory stimuli
(Abdeen et al. 1995
; Meller et al. 1992
;
Taylor et al. 1995
).
In most experiments in which recordings were made from lamina
V-nociceptive trigeminal brain stem neurons that receive convergent input from the dura and facial skin, the initiation of the neuronal response to thermal and mechanical stimulation preceded the initiation of the depressor and pressor responses. More important, however, is the
observation that these relationships were preserved after the induction
of central sensitization in these neurons. When sensitized trigeminal
brain stem neurons responded earlier to thermal stimuli (i.e., lower
temperature), depressor and pressor responses followed them by also
responding earlier. These temporal relationships suggest that
facilitated responses of sensitized trigeminal brain stem neurons can
initiate the reflexive depressor and pressor responses, possibly
through anatomic projections (Hazlett et al. 1972;
Mehler et al. 1960
; Menetrey and Basbaum
1987
; Zemlan et al. 1978
) to brain stem
depressor and pressor areas such as the rostral ventrolateral medulla
(Dampney et al. 1982
; Neil and Loewy
1982
; Ross et al. 1984
; Sun et al.
1988
), periaqueductal gray (Bandler and Shipley
1994
; Lovick 1993
) the nucleus of the solitary
tract (Doba and Reis 1973
; Snyder et al.
1978
). and the caudal ventrolateral medulla (Masuda et
al. 1992
; Willette et al. 1984
).
While the initiation of these BP responses may be dependent on the
responses of the trigeminal brain stem neurons, their length seems
independent, as depressor responses are usually shorter and pressor
responses longer than the neuronal response. This observation may be
explained by the involvement of multiple hormonal and neural circuits
in the regulation of heart rate, cardiac output, arterial pressure, and
regional blood flow (Blessing 1997; Saper 1995
). For example, Ross et al. (1984)
proposed
that the pressor response consists of a short-latency, neuronally
mediated ("stimulus-locked") component and a long component
mediated by catecholamine release from the adrenal medulla and
vasopressin release from the pituitary gland.
Previous studies (Ness and Gebhart 1988; Roza and
Laird 1995
) showed linear correlation between the intensity of
a stimulus and the magnitude of the pressor response. In this study, a
similar correlation was found between the peak (i.e., maximal increase) neuronal and pressor responses when mechanical stimuli were used. The
mechanism that underlies such a correlation is a matter of speculation.
Theoretically, the consequence of evoking large numbers of spikes in
nociceptive lamina V neurons could be either a proportionally large
activation of the same number of neurons that mediate the pressor
response or a recruitment of additional neurons.
In summary, the present study better defines the relationship between
cardiovascular and trigeminal brain stem neuronal responses to
somatosensory stimuli during the development of central sensitization. It supports the concept that chemical irritation of the dura activates and sensitizes trigeminovascular nociceptors (Strassman et al. 1996), that this peripheral sensitization leads to brief
activation and long sensitization of central trigeminal neurons
(Burstein et al. 1998
), and that the sensitization that
develops in the final common trigeminovascular pathway might play a
role in patient perception of intracranial and extracranial
hypersensitivity during migraine (Drummond 1987
).
Because innocuous stimuli such as brush usually are perceived as
nonpainful when applied to control patients but often as painful (i.e.,
mechanical tactile allodynia) when applied to chronic pain patients
suffering from inflammatory or neuropathic pain (Bennett
1994
; Dubner and Basbaum 1994
), we suggest that
after dural irritation, changes in neuronal and cardiovascular responses to mild dural indentation, skin brushing or low temperatures (<43°C) reflect the development of allodynia and represent a shift in the perception of these stimuli from nonnociceptive to nociceptive.
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ACKNOWLEDGMENTS |
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We thank Drs. C. J. Woolf, C. B. Saper, A. M. Strassman, and G. M. Bove for critical comments on the manuscript and J. J. Fink for editorial comments.
This work was supported by National Institutes of Health Grants DE-10904 and NS-35611-01 and by gifts from the Boston Foundation, and the Goldfarb and Fink families.
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FOOTNOTES |
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Address for reprint requests: R. Burstein, Dept. Anesthesia and Critical Care, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 July 1998; accepted in final form 16 October 1998.
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REFERENCES |
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