Chem. Senses 24: 665-669,
1999
© Oxford University Press 1999
Odorants Presented to the Rat Nasal Cavity Increase Cortical Blood Flow
Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA
Correspondence to be sent to: Wayne L. Silver, Department of Biology, Wake Forest University, Post Office Box 7325, Winston-Salem NC 27109, USA. e-mail:silver{at}wfu.edu
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Abstract |
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Complaints about unpleasant environmental odorants, both outdoor and indoor, are increasingly being reported. The main complaints of health symptoms from environmental odorants are eye, nose and throat irritation, headache and drowsiness. Complaints may arise from the stimulation of olfactory receptors or trigeminal chemoreceptors. Stimulation of cerebrovascular nociceptors originating from a branch of the trigeminal nerve may be associated with an increase in cortical blood flow which is thought to be related to headache. Since odorants are reported to elicit headaches, the possibility that odorants may increase cortical blood flow was examined. Cortical blood flow was monitored in rats using a laser-Doppler flowmeter. The flowmeter probe was placed over the left frontal cortex while propionic acid, cyclohexanone, amyl acetate or butanol was delivered to the nasal cavity via an olfactometer. Cortical blood flow increased as the concentration increased for three of the odorants tested. The greatest increase in blood flow occurred to the presentation of propionic acid, followed by cyclohexanone and amyl acetate. There was no response to butanol. These data demonstrate that odorants can alter cerebrovascular blood flow, which may account, in part, for one of the health symptoms reported for odorants.
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Introduction |
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Odorants entering the nasal cavity can stimulate olfactory and/or trigeminal receptors (Silver, 1992
). Chemical stimulation of trigeminal fibers innervating the nasal cavity may lead
to the perception of irritation and produce local as well as systemic responses. These responses tend to
protect the animal from the noxious stimulus and are mediated, in part, by the release (via axon-reflex)
of neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) (Maggi and Meli, 1988).
Trigeminal fibers containing SP and CGRP also innervate vascular structures within the cranium,
including the meningeal arteries and the large arteries forming the circle of Willis (Saito et al., 1987; Tsai et al., 1988). These vessels are the
only pain sensitive structures within the cranium (Ray and Wolff, 1940) and are
collectively referred to as the trigeminovascular system. It is now generally believed that stimulation of
the trigeminovascular system is responsible for the pain associated with vascular headaches
(Moskowitz, 1991; Silberstein, 1992).
Odorants are known to play a role in headaches, such as the precipitation of migraines (Blau and Solomon, 1985). Odorants have also been implicated in what has
been described as the‘sick building syndrome’, one symptom of which is headache
(Hudnell et al., 1992).
Electrical stimulation of trigeminovascular fibers results in an increase in cerebral blood flow (Suzuki et al., 1990a). Sensory trigeminal fibers are sensitive to
capsaicin, which, when presented to the nasal cavity, also alters cerebral blood flow and decreases the
frequency of cluster headaches (Fusco et al., 1994). Since a variety
of odorants are known to stimulate trigeminal sensory nerve fibers (Silver, 1992), the present study was undertaken to determine whether the presentation of odorants to
the nasal cavity of rats would increase cerebral blood flow. If odorants can alter cerebrovascular blood
flow, this may account, in part, for one of the health symptoms reported for these compounds.
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Materials and methods |
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Animals and experimental preparation
Ten male Sprague–Dawley rats weighing between 390 and 750 g were used. Each rat was
anesthetized with urethane (ethyl carbamate, 1.0 g/kg injected i.p.). Two cannulae were inserted into
the trachea of each rat. One cannula was connected to a ventilator and inserted toward the rat's
lungs. The rat was artificially ventilated to eliminate breath holding. This was necessary because, as the
rat holds its breath, arterial CO2 increases resulting in increased cortical blood flow (Nakashima et al., 1995). A second cannula, connected to a vacuum
line, was inserted up into the nasopharynx. This second cannula was used to draw air/stimuli through the
nasal cavity at 250 ml/min. Each rat was then restrained in a head holder. Respiration rate was
monitored by placing a rubber strap, connected to a transducer arm, around the rat's torso.
Heart rate was monitored by placing pin electrodes in the left and right pectoral muscles of the rat.
Appropriate amplifiers were used to amplify respiration and heart rate signals which were then led to an
MP100 data acquisition system running AcqKnowledge software (BIOPAC systems, Goleta, CA) on
a Macintosh computer.
A burr hole ~3 mm in diameter was made over the frontal cortex, ~2 mm rostral to the coronal
suture and 2 mm to the left of midline. This was performed using a drill under a dissecting microscope.
In addition, four of the ten rats had blood flow recorded over the left occipital cortex. This second burr
hole was located ~2 mm to the left of the sagittal suture and 1 mm rostral to the
interparietal–parietal suture. Since opening the dura mater causes the loss of cerebrovascular
autoregulation (Morita et al., 1995), the innermost transparent layer
of bone was left intact.
Blood flow measurements
Blood flow was recorded using a Moor dual probe laser-Doppler blood flow monitor (MBF3D).
Laser-Doppler flowmetry provides a convenient, non-invasive method for recording relative blood flow
changes. The probes were positioned using a micromanipulator so that the tip rested on the bone at the
bottom of the burr hole. Since blood flow in the bone is negligible and flow in the dura is small, the
registered blood flow represents mainly cortical flow. Mineral oil was added to the burr hole to prevent
desiccation. The principles of laser-Doppler flowmetry are described elsewhere (Skarphedinsson et al., 1988).
The laser-Doppler flowmeter calculates the volume and velocity of moving particles within the recording area. These two values are multiplied together to give an arbitrary flux value. Flux was recorded using the MP100 acquisition system and AcqKnowledge software.
Odorant presentation
Odorants were presented using a computer-controlled, airdilution olfactometer. The design and
operation of the olfactometer have been thoroughly described by Silver et al. (Silver et al., 1990). Briefly, the apparatus works by mixing a clean air stream and
an air stream saturated with an odorant. By combining the two air streams in different proportions,
different concentrations of odorants can be presented. The two air streams combine for a final flow rate
of 2 l/min. The odorants used were propionic acid (1242, 392 and 124 ppm), cyclohexanone (1416,
448 and 142 ppm), amyl acetate (3020, 955 and 302 ppm) and butanol (6546, 1306 and 260 ppm).
These odorants, at the concentrations used, have been shown previously to stimulate the ethmoid
branch of the trigeminal nerve (Silver, 1992). All concentrations of each
odorant were presented to all ten rats. Stimulus duration was 90 s. Clean air was presented as a
control. Each odorant was presented in a series of increasing concentrations while the order of the
odorants used was randomized for each rat.
Data analysis
Cortical blood flow, respiration rate and heart rate were recorded for 90 s before, during and after odorant presentation. For each trial, the blood flow values recorded during the 90 s prior to odorant presentation were averaged from 13 500 data points. This mean then served as a baseline for the entire trial (270 s). The blood flow for each 10 s block during a trial was recorded as a percentage change from this averaged baseline. Blood flow, respiration rate and heart rate were then analyzed using a 4 (concentration) x 18 (time) repeated measures analysis of variance. Significance was examined using Tukey's comparison of means at P < 0.05.
To determine if the cortical blood flow during an individual trial increased significantly above controls, the mean blood flow for all clean air trials was calculated to be –0.74% (±2.12 SEM). A significant increase for an individual trial was then set at two standard deviations (+8.96%) above the calculated control mean.
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Results |
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Three of the four odorants presented to the rats resulted in a significant increase in cortical blood flow. Propionic acid at 1242 and 392 ppm caused a significant increase in blood flow above that recorded during the clean air trials (Figure 1). These values reached significance ~50 s after stimulus onset for 1242 ppm and 60 s after stimulus onset for 392 ppm (Figure 1). Cortical blood flow remained significantly higher than controls for the remainder of the trial (Figure 1). The lowest concentration of propionic acid (124 ppm) did not result in a significant increase in cortical blood flow (Figure 1).
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Cyclohexanone at 1416 ppm caused a significant increase in cortical blood flow, beginning ~30 s after stimulus onset and continuing to remain significantly higher than clean air trials until ~20 s after the stimulus was turned off (Figure 1). No other concentration of cyclohexanone elicited a significant increase in cortical blood flow above controls.
Amyl acetate at 3020 ppm resulted in an average blood flow increase significantly higher than air, starting ~30 s after stimulus onset and continuing until ~30 s after the stimulus was terminated (Figure 1). In addition, amyl acetate at 955 ppm caused a significant increase in blood flow above clean air trials ~60, 100 and 110 s after the stimulus was turned on (Figure 1). The lowest concentration of amyl acetate (302 ppm) caused no significant increase in the blood flow above controls. No concentration of butanol presented to the animals resulted in a significant increase in blood flow above air trials.
The average heart rate of the 10 animals remained constant throughout the experiment, regardless of the odorant presented. In addition, the respiration rate remained constant throughout all experiments for all the animals.
Blood flow was recorded simultaneously from the frontal and occipital cortices through two different burr holes in four of the animals tested. Odorant-elicited blood flow changes in the frontal cortex were highly correlated with changes in the occipital cortex (r = 0.562, P < 0.001).
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Discussion |
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Cerebral blood flow is mainly dependent on vascular resistance when autoregulation is intact. Vessel tone is regulated by parenchymal factors (such as metabolites), endothelial factors and perivascular nerves (Wahl and Schilling, 1993
). In the present study, the
odorant-induced blood flow changes recorded in the frontal and occipital cortices were highly
correlated. This finding suggests that blood flow increases were not mediated by local parenchymal or
endothelial factors but rather by perivascular nerves.
A systemic change in vessel caliber could be induced by the odorants entering the blood stream. If
this were the case, there would be a corresponding decrease in systemic blood pressure which could
affect blood flow. Since blood pressure was not measured in this study, this possibility cannot be ruled
out. However, the time-course for cortical blood flow increase was very similar to that seen during
electrical stimulation of fibers innervating cerebral blood vessels (Suzuki et al.,
1990a, 1990b), suggesting perivascular nerve stimulation.
Nerves responsible for regulating cerebral vessel caliber include sympathetic fibers,
parasympathetic fibers and sensory fibers of the trigeminovascular system (Wahl and
Schilling, 1993).
Stimulation of sympathetic fibers results in minor to modest decreases in cerebral blood flow
(Baumbach and Heistad, 1983), while stimulation of parasympathetic fibers or
trigeminal fibers results
in increases in cerebral blood flow (Suzuki et al., 1990a,b). The significant increase in blood flow caused by three of the four odorants presented
suggests that parasympathetic and/or trigeminal fibers were stimulated resulting in cerebral vessel
dilation.
Propionic acid, cyclohexanone, amyl acetate and butanol are known to stimulate both olfactory and
trigeminal nerves at the concentrations used in the present study (Silver, 1992).
Since olfactory stimuli can elicit changes in autonomic function (Cassel and Roberts, 1991), the blood flow increases elicited by these odorants could have been caused by
parasympathetic stimulation via the olfactory nerve. However, odorant stimulation of the trigeminal
nerve may be more likely to lead to increases in cerebral blood flow. Trigeminal ganglion stimulation in
the cat results in increased cerebral blood flow (Lambert et al., 1984). Electrical stimulation of the nasociliary nerve in rat also increases cerebral flow (Suzuki et al., 1990a). In addition, significant increases in blood flow required relatively
high concentrations of odorants. Typically, the odorants used in this study stimulate olfactory receptors
at much lower concentrations and trigeminal nerve stimulation requires higher concentrations (Silver, 1992).
It is not clear why butanol did not elicit changes in cerebral blood flow in the present study, since the concentrations used should have been high enough to stimulate trigeminal nerve fibers. However, previous work in our laboratory has shown that butanol, at the concentrations tested, elicits the weakest ethmoid nerve response compared with the concentrations of the other three odorants used in this study.
Blood flow increases due to trigeminal nerve stimulation may be mediated by parasympathetic
fibers. The parasympathetic nerves that innervate cerebral blood vessels arise from the sphenopalatine
ganglion, which is known to be innervated by trigeminal fibers (Suzuki et al., 1989). The trigeminal nerve may also mediate cerebral blood flow changes directly. Trigeminal
fibers that innervate cerebral vessels may be collaterals from the same fibers that innervate the nasal
cavity, since trigeminal fibers innervating the nasal cavity have been shown to project collaterals back
into the cranium (Finger and Böttger, 1993). Additional evidence to
support a connection between nasal trigeminal and cerebrovascular trigeminal fibers involves capsaicin
application to the nasal mucosa as a treatment for cluster headache. Cluster headaches are severe
unilateral attacks of head pain that occur in a series of episodes. Symptoms include blockage of nasal
passages and rhinorrhea. Studies by Fusco et al. (Fusco et al., 1994) have shown that capsaicin applied to the nasal mucosa of humans causes changes in
cerebral blood flow in addition to significantly decreasing the number of headaches in patients suffering
from cluster headache. These findings tend to suggest that the desensitizing actions of capsaicin may
reduce the frequency of cluster headache because collaterals of the same trigeminal nerve may innervate
both the nasal cavity and cerebral blood vessels.
The results of the present study indicate that odorants presented to the nasal cavity can elicit
significant increases in cortical blood flow. Clearly, the underlying pathological mechanisms for
headache are complex and not completely understood; however, since odorants can trigger vascular
headaches (Blau and Solomon, 1985; Hudnell et al.,
1992) and stimulation of the trigeminovascular system results in both head pain and
increased cortical blood flow (Ray and Wolff, 1940; Lambert et al., 1984; Suzuki et al., 1990a), the headache
elicited by odorants may be due to activating the trigeminovascular system. Further experiments are
needed to determined whether this activation is via the olfactory or trigeminal systems.
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Acknowledgments |
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The authors wish to thank Drs Michael Morykwas and William Conner for their support in this research and Drs Thomas Smith and Linda Porrino for their assistance in the preparation of this manuscript. We would also like to acknowledge Dr Mark Leary and Dr Derek Zelmer for their help with statistical calculations. Finally, we would like to thank the Department of Plastic and Reconstructive Surgery of the Wake Forest University School of Medicine for the use of their laser-Doppler flowmeter.
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Accepted June 15, 1999
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