Cardiovascular and Neuronal Responses to Head Stimulation Reflect Central Sensitization and Cutaneous Allodynia in a Rat Model of Migraine

Hiroyoshi Yamamura,1 Amy Malick,2,3 Nancy L. Chamberlin,1 and Rami Burstein1,2,3

 1Department of Anesthesia and Critical Care, Beth Israel Deaconess Medical Center,  2Department of Neurobiology, and the  3Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 >= 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
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Abstract
Introduction
Methods
Results
Discussion
References

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; 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).

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) 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).

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; 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.

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.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 <= 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.

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, 10-3 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.

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; 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.

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). 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 MOmega ). 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.

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 >= 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.

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., >= 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.

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.



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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 radical 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.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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.



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Fig. 2. Locations of recording sites (A) and receptive fields (B). A: locations of lesions marking the recording sites of 24 dura-sensitive neurons in the upper cervical dorsal horn and the trigeminal nucleus caudalis. Locations of 5 neurons were not recovered. , recording sites of individual sensitized neurons with firing rates that exhibited temporal or quantitative correlation with cardiovascular responses; open circle , recording sites of individual neurons that did not sensitize. B: intracranial (dural) and extracranial (skin) receptive fields of 20 neurons that were sensitized and 9 neurons (marked by * in the ear) that were not sensitized by topical application of inflammatory agents or low-pH phosphate buffer to the dura. On the skin, black areas indicate receptive fields regions where stimuli induced the largest neuronal responses and dark gray areas indicate regions where stimuli induced smaller responses. On the dura, black areas depict the size of the mechanical receptive field before the application of chemical stimulation to the dura. Light gray areas on the skin and the dura show the expanded area of the receptive fields after chemical stimulation. Neuronal classification and locations of recording sites are indicated below each case. Note that both WDR and HT neurons exhibited expanded dural receptive fields but only WDR neurons in lamina V exhibited expanded cutaneous receptive fields. C1, first cervical segment; HT, high threshold; IS, inflammatory soup; lat, lateral dorsal horn; LRN, lateral reticular nucleus; PB, low pH phosphate buffer; SRD, subnucleus reticularis dorsalis; SRV, subnucleus reticularis ventralis; Vc, nucleus caudalis; vlm, ventrolateral medulla; WDR, wide dynamic range; i-v, gray matter laminae.

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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Fig. 3. Increased sensitivity to mechanical stimulation of the dura after chemical stimulation. A: example of the changes in the minimum force required to induce blood pressure and neuronal responses before and after the chemical stimulation of the dura. Note that before chemical stimulation of the dura, neuronal and blood pressure responses were induced only by indenting the dura with a force >4 g and that 20 min after chemical stimulation, similar neuronal and blood pressure responses were induced by smaller (<1 g) forces as well. B: summary of the 16 experiments in which thresholds of blood pressure (I) and neuronal (II) responses to dural indentation were determined before and after sensitization. Scatter plots (III) illustrate the linear correlation between the neuronal and cardiovascular responses before (open circle , r = 0.625, P < 0.01) and after (, r = 0.826, P < 0.0001) the neuronal sensitization. --- and - - -, regression lines before and after sensitization, respectively. Note that in ~70% of the cases, a drop in the minimum force required to induce a neuronal response (2.35-0.445 g) was accompanied by a drop in the minimum force required to induce blood pressure response (2.35-0.976 g). Lines above histograms indicate stimulus duration and numbers above these lines depict the force used to indent the dura. Boxes depict the mechanical threshold for eliciting the neuronal response. Numbers in parentheses indicate the magnitude of blood pressure change.


                              
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Table 1. Relative incidence of neuronal and BP threshold changes to dural indentation

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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Fig. 4. Increased sensitivity to mechanical stimulation of the facial skin after chemical stimulation of the dura. A: example of simultaneous recording of neuronal and blood pressure responses to mechanical skin stimulation showing that before chemical stimulation of the dura, only pinch and pressure induced neuronal and blood pressure responses and that 60 min after the chemical stimulation similar responses were induced by brushing the skin. B: summary of the 19 experiments in which the magnitude of blood pressure and neuronal responses to mechanical skin stimulation was determined before and after the chemical stimulation of the dura. As: plots of changes in the blood pressure response magnitude to brush pressure and pinch. Bs: plots of changes in the neuronal response magnitude to brush pressure and pinch. Cs: scatter plots illustrating the lack of correlation between the magnitudes of the neuronal and cardiovascular responses to skin stimulation before (open circle ) and after () the neuronal sensitization: brush (r < 0.32, P > 0.15), pressure (r < 0.25, P > 0.3), and pinch (r < 0.2, P > 0.4). Bold number in B (As, Bs) indicate mean ± SE of the response magnitudes. --- and - - - in Cs represent the regression lines before and after sensitization, respectively.


                              
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Table 2. Relative incidence of neuronal and BP response magnitude changes to mechanical skin stimulation

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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Fig. 5. Increased sensitivity to thermal stimulation of the facial skin after chemical stimulation of the dura. A: example of simultaneous recording of neuronal and blood pressure responses to thermal skin stimulation showing that before chemical stimulation of the dura neuronal and blood pressure responses were induced at 51 and 54°C and that afterward they were induced by 46 and 48°C, respectively. ···, initiation times (vertical) and the thresholds (horizontal) of the responses. Note that the initiation of the neuronal responses preceded the initiation of the pressor responses by 2-3 s and that the correlation between peak neuronal response and the initiation of the pressor response is preserved before and after the sensitization. B: summary of the 13 experiments in which the thresholds of blood pressure and neuronal responses to thermal skin stimulation were determined before and after the chemical stimulation of the dura. I: plots of changes in the blood pressure response thresholds. II: plots of changes in the neuronal response thresholds. III: scatter plots illustrating the linear correlation between the neuronal and cardiovascular response thresholds before (open circle , r = 0.762, P < 0.003) and after (, r = 0.686, P < 0.01) the neuronal sensitization. IV: plots of changes in blood pressure response magnitude. V: plots of changes in neuronal response magnitude. VI: scatter plots illustrating the lack of correlation between the magnitudes of neuronal and cardiovascular response to thermal skin stimulation before the neuronal sensitization (open circle , r = 0.295, P > 0.3) but the significant correlation after sensitization (, r = 0.67, P < 0.02). --- and - - - in III and VI represent the regression lines before and after sensitization, respectively. Note that in ~77% of the cases, a drop in the minimum temperature required to induce a neuronal response (44.8-41.1°C) was accompanied by a similar drop in the minimum force required to induce a blood pressure response (46.5-43.1°C).


                              
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Table 3. Relative incidence of neuronal and BP threshold changes to skin heating

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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Fig. 6. Neuronal and blood pressure response latencies and duration. A: horizontal lines illustrate the onset and duration of neuronal (black), depressor (dark gray), and pressor (light gray) responses to heat in 49 individual trials. B: histograms showing the incidence of temporal relationship between the onsets of the neuronal and depressor responses, the onsets of the neuronal and pressor responses, and the duration of depressor, pressor and neuronal responses to thermal stimulation of the facial skin. Note the correlation among the onset of the responses and the dissociation among their durations.

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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Fig. 7. Scatter plots illustrating mean (n = 19) and standard errors of neuronal and pressor responses to brush (, open circle ), pressure (black-triangle, triangle ), and pinch (, ) before (open circle . triangle , and ) and after (, black-triangle, and  closed symbols) chemical stimulation of the dura. Note that the graded increase in neuronal and blood pressure response magnitudes reflects the intensity of stimulus and that the relative change in the magnitude of the 2 responses is proportional.


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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