Vol. 274, Issue 4, R921-R930, April 1998
Contribution of hypercapnia and trigeminal stimulation to
cerebrovascular dilation during simulated diving
G. P.
Ollenberger and
N. H.
West
Department of Physiology, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada S7N 5E5
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ABSTRACT |
We
investigated the relative contribution of humoral (carbon dioxide) and
neural (trigeminal stimulation) inputs in the cerebrovasodilatory response to simulated diving in the rat. The cerebral hemodynamic profile of rats was determined using the brain blood flow tracer N-[14C]isopropyl-p-iodoamphetamine.
During a simulated dive response, cerebral vascular resistance (CVR)
decreased 63.1%, resulting in a 1.5-fold increase in cerebral blood
flow (CBF). To investigate the contribution of hypercapnia to the
decrease in CVR during simulated diving, we measured CBF during
simulated diving in rats with preexisting hypocapnia. To investigate
the contribution of trigeminal input, we measured CBF during periods of
trigeminal stimulation alone with continued ventilation. Preexisting
hypocapnia abolished the cerebrovasodilatory response to simulated
diving. Trigeminal stimulation alone did not produce a significant
increase in CBF from control values in any brain region, suggesting
that trigeminal input does not contribute to the cerebrovascular
response to simulated diving in rats. These results suggest that the
cerebrovasodilatory response observed during diving in small mammals is
driven primarily by progressive hypercapnia associated with asphyxia.
cerebral blood flow; regional cerebral blood flow; carbon dioxide; bradycardia
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INTRODUCTION |
LABORATORY RATS EXHIBIT a marked redistribution of
cardiac output (CO) in response to both voluntarily initiated diving
(29) and forced head immersion (21). A redistribution of CO during diving preferentially perfuses tissues sensitive to hypoxic challenge, such as the heart and brain, at the expense of decreasing perfusion to
tissues relatively insensitive to hypoxia, such as skeletal muscle and
viscera (17, 18, 41). Although previous studies suggest that the brain
is perfused continuously, few studies have examined the pattern of
blood flow in brain regions during diving. In a recent study, we
determined the cerebral hemodynamic profile of conscious, voluntarily
diving rats (30). We found regional cerebral blood flow (rCBF)
increased markedly during diving due primarily to a corresponding
decrease in cerebral vascular resistance (CVR). Only some regions of
the basal ganglia (caudate putamen and globus pallidus) and limbic
areas (hippocampus and amygdala) did not increase rCBF significantly
during diving. Because some brain regions did not participate in the
intracerebral increase in blood flow, we suggested that the
cerebrovasodilatory response to diving in rats may have both a humoral
component, mediating a global fall in CVR, and a neural component,
mediating differential changes in CVR.
The objective of the present study was to expand on the results of
Ollenberger and West (30) concerning the contribution of humoral and
neural inputs producing the cerebrovasodilatory response to diving in
the rat. We first investigated carbon dioxide (CO2) as a potential humoral
input in cerebrovasodilatory response to diving because
CO2 has been implicated as a
possible mediator of cerebral vasomotion during diving in sea lions
(6), seals (1), and ducks (19, 37). Furthermore, numerous studies have
demonstrated that CBF increases steeply in response to increased CO2 (for review, see Ref. 8).
Therefore, our primary hypothesis was that the progressive increase in
arterial CO2 mediates a global fall in CVR during diving in rats.
Second, we investigated trigeminal stimulation as a potential neural
input to the cerebrovasculature during diving, because this input has
been demonstrated to be necessary in the cardiac response to diving in
small mammals (24) and has been shown to elicit regional alterations in
CBF (10). Moreover, the neural connections of the trigeminal system
with cerebral blood vessels are so numerous that the concept of a
trigeminocerebrovascular system has arisen (27). Therefore, our
secondary hypothesis was that trigeminal afferent input differentially
modulates the global cerebrovasodilatory response to diving in rats.
To investigate these hypotheses, we used a simulated diving model that
enabled us to differentiate between the effects of carbon dioxide and
trigeminal stimulation on the cerebrovasculature during diving in the
rat (25). In the simulated diving model, the diving response was
initiated by flowing water through the nasal passages (trigeminal
stimulation) during expiratory apnea in anesthetized, paralyzed,
artificially ventilated rats. To investigate the first hypothesis, we
measured rCBF during simulated diving in rats with preexisting
hypocapnia to remove the CO2
stimulus (hypocapnic simulated diving). To test the second hypothesis, we measured rCBF during periods of trigeminal stimulation alone with
continued ventilation (trigeminal stimulation). We compared the results
from the hypocapnic simulated diving group and trigeminal stimulation
alone group to the control (anesthetized, paralyzed, artificially
ventilated) and normocapnic simulated diving (nasal water flow plus
apnea) groups.
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MATERIALS AND METHODS |
Experiments were performed on 30 male Sprague-Dawley rats (434.9 ± 16.1 g). All experimental interventions were approved by the Animal
Care Committee of the University of Saskatchewan and were performed in
accordance with guidelines of the Canadian Council on Animal Care.
Surgical preparation.
A similar surgical preparation has been described previously (25).
Briefly, rats were anesthetized with fentanyl-droperidol (Innovar-Vet,
MTC Pharmaceuticals, Cambridge, Ontario; 0.15-0.2 ml/kg im,
diluted to a 10% solution in saline), after initial inhalation
induction with methoxyflurane (Metofane, MTC Pharmaceuticals). One-half-dose injections were given hourly to maintain anesthesia. For
experiments lasting longer than 2 h, oxymorphone (Numorphan, Du Pont
Merck Pharmaceuticals; 0.1 ml im, diluted to a 33% solution in saline)
was given to ensure analgesia was maintained. Both femoral arteries
were cannulated with polypropylene tubing (PE-50, Clay Adams,
Parsippany, NJ) that was advanced 2.0 cm toward the abdominal aorta.
The right and left femoral veins were cannulated with silicone tubing
(Baxter Scientific, McGaw Park, IL; ID 0.635 mm, OD 1.194 mm) that was
advanced 6 cm into the inferior vena cava. All the cannulas were filled
with heparinized saline (Hepalean, Organon Teknika, Toronto, Ontario;
10 IU/ml). Copper leads were inserted under the skin to record the
electrocardiogram (ECG) and monitor heart rate (HR). One arterial
cannula was attached to a pressure transducer (type 4-327-C,
Beckman Instruments, Schiller Park, IL) and recorded on a chart
recorder writing on rectilinear coordinates (Beckman R511A). The second
arterial cannula was connected to a preweighed heparinized 5.0-ml
syringe attached to an infusion/withdrawal pump (Harvard Syringe
infusion pump 22, Ealing Scientific, St. Laurent, Quebec; 0.4 ml/min).
One venous cannula was connected to a 1.0-ml syringe containing the
radioactive blood flow tracer and the other to a syringe containing a
paralytic agent (D-tubocurare, Sigma, St. Louis, MO; 2 mg/kg iv). Body temperature was maintained at
37 ± 1°C with a heating pad (Harvard animal temperature control unit, Ealing Scientific). In preparation for artificial ventilation, the rat was placed in a supine position and the trachea was exposed through a midline incision along the length of the neck. Rostral- and
caudal-facing tracheal cannulas (PE-205, Clay Adams) were inserted. The
caudal-facing cannula was attached to a ventilator (model CTP-930, CWE,
Ardmore, PA), and the rat was paralyzed with D-tubocurare (Sigma). The
oral-facing cannula was attached to a pump (model 501U/R,
Watson-Marlowe, Falmouth, UK) that was used to withdraw water from the
cannula. Two cannulas (PE-50, Clay Adams) were inserted ~1.0 cm into
the nares. Room temperature water was infused into the nares through
the two cannulas using a syringe pump (Harvard Apparatus
Infusion/Withdrawal Pump, 1.8 ml/min). As the water flowed through the
nares, it was withdrawn from the rostral-facing cannula at a rate
comparable to the infusion rate.
Experimental protocol.
Cardiovascular and cerebrovascular variables were determined in four
groups of rats. The control group (n = 10) consisted of anesthetized, paralyzed, artificially ventilated rats
[respiratory frequency (f) = 70 min
1, tidal volume
(Vt) = 2.6-3.8 ml].
The normocapnic simulated dive group
(n = 7) consisted of anesthetized,
paralyzed, artificially ventilated rats in which the dive response was
elicited by flowing water into the nares (trigeminal stimulation)
during concurrent apnea. We compared the results from the above groups
to the results from flowing water into the nares with continuous
ventilation (trigeminal stimulation alone group,
n = 6) and to simulated diving after
the arterial partial pressure of
CO2
(PaCO2) was reduced predive by
hyperventilation (hypocapnic simulated dive,
n = 7, f = 90, Vt = 4.5-5.8 ml). Arterial
blood gases were determined before measurement of rCBF in all
experimental groups (Table 1) to ensure
blood gases were within the physiological range (BGM200 blood gas meter
and BC202 blood gas cell; Cameron Instrument, Port Aransas, TX).
Measurement of rCBF.
The brain blood flow tracer
N-[14C]isopropyl-p-iodoamphetamine
(IMP) (NEN, Boston, MA) was used to quantify rCBF. The specific activity was 44.7 mCi/mmol. IMP is extracted 100% during first pass in
the brain capillaries, with a time to one-half brain washout (t1/2) of 318 s
(40). Therefore, the experiment was performed using a modification of
the indicator-fractionation technique first described by Goldman and
Sapirstein (11). In all protocols a reference blood sample was
withdrawn at a steady rate of 0.4 ml/min, which provided a reference
flow rate (R in Eq. 2) that is
necessary to determine rCBF.
In all groups the arterial withdrawal was started first, followed by
injection of IMP into the femoral vein cannula. In trigeminal stimulation and simulated dive groups, the injection of IMP occurred within 2-3 s of a visible bradycardia on the ECG tracing (Figs. 3
and 4). In the control group, the IMP was allowed to circulate for 35 s, after which the rat was decapitated to stop the circulation of the
tracer. In the other three experimental groups, IMP circulation time
was extended to 50 s to ensure that the peak of the tracer concentration in the arterial blood had passed during the decreased CO
associated with bradycardia. These tracer distribution times were
chosen based on arterial radioisotope-dilution curves that were
determined previously in control and simulated diving rats (Fig.
1). Briefly, we measured IMP concentration
in arterial blood by dripping an open arterial cannula into
scintillation vials during injection of IMP. The peak of the arterial
curve occurred ~10-20 s after injection in the control rat (HR = 427 beats/min), whereas the peak did not appear until ~25-35 s
after injection in simulated diving rats (HR = 104 beats/min).
Therefore, we adjusted the circulation time of IMP in both simulated
diving groups from 35 to 50 s to reflect the cardiovascular changes
associated with diving bradycardia. We also adjusted the circulation
time of IMP to 50 s in the trigeminal stimulation alone group due to
the occurrence of a significant bradycardia (Table 1). Based on the
above arterial circulation times for IMP, rCBF values in the current
experiments reflect a limited time frame after injection of IMP and not
a "smeared" measurement of the entire experimental period. For
the control group, the values in the current experiment are indicative of rCBF in the time-frame of ~10-20 s after injection, whereas for the dive groups, the rCBF values are indicative of rCBF in the time
frame of ~25-35 s into the dive.
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Fig. 1.
Arterial radioisotope dilution curves for control and normocapnic
simulated diving rats. Radioisotope was injected at
time 0 in both rats. During
normocapnic simulated diving, initial uptake of radioisotope was
delayed ~12 s due to decreased cardiac output (CO) associated with
diving bradycardia. This shifted the peak of the concentration curve
from ~10-20 s in control to ~25-35 s in simulated dive.
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A detailed account of the determination of rCBF has been described in
detail elsewhere (3, 37). Briefly, rCBF is calculated according to the
following relationship
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(1)
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where
Cb is concentration of the tracer
in a particular brain region, and
Ca is concentration of the tracer
in arterial blood at any time t. The
evaluation of the numerator in Eq. 1
is determined by quantitative autoradiography, and the denominator can
be determined by measuring the tracer contained in the withdrawn blood
during the experimental period (T)
(11, 34, 38). It is important that the arterial blood withdrawal is
terminated at the same time the animal is killed. However, timing
errors are minimized by injecting IMP as a bolus, because the arterial
concentration is very low toward the end of the experimental period
(31). The integrated arterial blood sample can be expressed in terms of rate of blood withdrawal (R) and the total tracer activity in the
withdrawal blood sample (QA)
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(2)
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QA
was determined by analyzing four 20-µl aliquots of blood from the
reference blood sample that were weighed, solubilized (NCS II tissue
solubilizer, Amersham, Oakville, Ontario), decolorized with 30%
hydrogen peroxide, and counted in a liquid scintillation counter
(Beckman LS 9800). QA was obtained
as follows
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(3)
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where
Cs is the quantity of tracer in an
aliquot of blood, Ms is the mass
of the aliquot, and Mr is the mass
of the entire reference blood sample. A mean value of the four
estimates was used for subsequent calculations.
At the end of the experimental period, the brain was removed from the
skull and rapidly frozen in isopentane (2-methyl-butane) at
50°C. The brains were cut at 20-µm thicknesses and placed in contact with autoradiographic film (Kodak TMS-1 RA, Eastman Kodak,
Rochester, NY; 18 × 24 cm) in a light-tight cassette. After a
short exposure period (5-10 days), the film was developed and a
gray level brain image was produced. Densitometry was performed on
autoradiographic images by a computer-based image analysis system
(Image 1, Universal Imaging, West Chester, PA). The autoradiograph gray
level density was converted to tissue tracer concentration using
calibrated 14C standards (American
Radiolabeled Chemicals, St. Louis, MO) that were packed with the brain
slices. rCBF was calculated in absolute terms
(ml · min1 · 100 g1), and rates of blood
flow were pseudo-color coded and displayed as a color image. To
positively identify brain nuclei, the 20-µm brain slices were stained
with neutral red, which stains for Nissl bodies in neurons. The stained
slides were then compared with a stereotaxic brain atlas (32) to
identify specific brain regions.
Arterial blood gases during simulated diving.
We were unable to determine the status of arterial blood gases during
the injection of the radioisotope (IMP). Therefore, separate
experiments were performed (n = 5) to
demonstrate that PaCO2 was successfully
eliminated as a potential humoral input on the cerebrovasculature
during hypocapnic simulated diving. Arterial blood was withdrawn and
analyzed [arterial partial pressure of
O2
(PaO2),
PaCO2, and
pHa] predive and during a
10-s interval (25-35 s) into a dive response in normocapnic and
hypocapnic rats (Fig. 2). In Fig. 2, 30 s
was chosen to reflect the midpoint in the arterial withdrawal interval.
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Fig. 2.
Arterial partial pressure of O2
and CO2
(PaO2 and
PaCO2, respectively) and pH predive
(inset) and 25-35 s into dive response in
normocapnic-normoxic and hypocapnic-normoxic rats.
PaCO2 was successfully eliminated as a
potential afferent input during simulated diving in hypocapnic group.
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Estimation of CO.
CO was determined using the "reference sample" technique. Blood
was withdrawn from the femoral artery at a rate of 0.4 ml/min during
the experimental protocol. CO was determined from the equation
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(4)
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where
Ct is the total counts of tracer
injected, determined by the principle described in Eq. 3. Therefore, CO was determined by dividing the
withdrawal rate by the fraction of injected tracer in the reference
sample. A potential source of error in the determination of CO is the
possibility that IMP is not extracted during first pass in perpheral
tissues, resulting in an overestimation of CO.
Statistical analysis.
All values reported in the text and figures are grand means ± SE;
HR (beats/min) and mean arterial blood pressure (MABP, mmHg) were
measured for each animal in all protocols. HR and MABP were determined
by calculating HR and MABP at 5-s intervals and then averaging the
values for the entire experimental period. MABP was calculated from
pulsatile blood pressure traces (diastolic plus 1/3 pulse pressure).
Stroke volume (SV, ml) and total peripheral resistance (TPR,
mmHg · ml1 · min)
were calculated by substituting MABP, CO, and HR into the equations
MABP = CO × TPR and CO = HR × SV. rCBF
(ml · min1 · 100 g wet brain tissue1) was
determined in 32 brain regions. Grand means were calculated by
averaging the rCBF values in brain regions from all animals in a
protocol. Brain regions were grouped into divisions based on function
(basal ganglia and thalamus, limbic system, and primary cortical
regions) except for the hindbrain, which was grouped according to
anatomic location, because the hindbrain consists of a multitude of
smaller functional regions. Global CBF (ml/min) was estimated by using
the equation CBF = average rCBF × brain weight, where average
rCBF represents the nonweighted average of all 32 brain regions
measured in each animal. We assumed an average of the 32 brain regions
reflected flow throughout the whole brain, because the regions spanned
from posterior to anterior. Because the brain had to be rapidly frozen
on removal, brain weight was calculated by using a correlation
(r2 = 0.98)
between rat body weight and rat brain weight from previously published
data (41). CVR
(mmHg · ml1 · min)
was determined by substituting CBF and MABP into the equation MABP = CBF × CVR. Statistical analyses were performed with a computer package (Systat, Evanston, IL). The data were analyzed with one-way analysis of variance with significance reached when
P < 0.05 (42). In the case of
significant F values, Tukey's honest
significant difference a posteriori tests were performed to determine
differences among group means.
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RESULTS |
Cardiovascular responses to normocapnic simulated diving.
Normocapnic simulated diving resulted in an immediate bradycardia that
was maintained throughout the entire 50-s stimulation period (Table 1,
Fig. 3). The physiological variables in the four groups of rats are presented in Table 1. HR decreased an average
of 74.9% during the normocapnic simulated dive period compared with
control values (427.6 ± 19.0 to 107.3 ± 7.3 beats/min). Bradycardia resulted in a significant 55.9% decrease in CO compared with control values (164.7 ± 14.1 to 64.8 ± 5.4 ml/min). All
estimated CO values were moderately higher than values previously
determined by microspheres or thermodilution techniques (4). During
normocapnic simulated diving, MABP decreased below control values and
was maintained at that level throughout the experimental period (Table 1, Fig. 3). We found a significant increase in SV (0.36 ± 0.03 to
0.64 ± 0.02 ml) during normocapnic simulated diving compared with
control values. Blood gases, determined before normocapnic simulated
diving, were not significantly different from either the control or
trigeminal stimulation groups (Table 1).
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Fig. 3.
Original chart recording of simulated diving response. Simulated diving
resulted in immediate bradycardia and hypotension that was maintained
throughout entire 50-s stimulation period.
Top to
bottom: time marker, electrocardiogram
(ECG), pulsatile arterial blood pressure, ventilation
(up, inspiration;
down, expiration), and event marker
[down, bolus injection of
N-[14C]isopropyl-p-iodoamphetamine
(IMP); up, decapitation].
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Cardiovascular responses to trigeminal stimulation alone.
Trigeminal stimulation alone produced an immediate and intense
bradycardia that decreased in magnitude toward the end of the stimulation period (Fig. 4). During the
trigeminal stimulation period, HR significantly decreased by 39.8%
(427.6 ± 20.0 to 257.3 ± 40.4 beats/min), producing a
significant 37.8% decrease in CO (164.7 ± 14.1 to 102.4 ± 15.4 ml/min) compared with control values. To counteract a decreased CO, TPR
increased significantly (0.6 ± 0.1 to 1.3 ± 0.2 mmHg · ml1 · min)
that maintained MABP significantly higher than control values during
the trigeminal stimulation period (94.3 ± 4.0 to 117.0 ± 6.4 mmHg). There was no change in SV during the trigeminal stimulation
period. Blood gases, determined before trigeminal stimulation, were not
significantly different from either the control or simulated diving
groups (Table 1).
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Fig. 4.
Original chart recording of trigeminal stimulation alone. Trigeminal
stimulation produced immediate and intense bradycardia that subsided
toward the end of the stimulation period. Top to
bottom: ECG, pulsatile arterial blood pressure, ventilation
(up, inspiration; down, expiration), and event
marker (down, bolus injection of IMP; up,
decapitation).
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Cardiovascular responses to hypocapnic simulated diving.
Hyperventilation before simulated diving resulted in a significant
increase in pHa (7.57 ± 0.02) and decrease in PaCO2 (20.5 ± 0.7 mmHg) from control values (Table 1). During hypocapnic simulated diving, HR (427.6 ± 19.0 to 141.5 ± 15.6 beats/min) and CO (164.7 ± 14.1 to 60.5 ± 4.9 ml/min) decreased
significantly from control values, similarly to normocapnic simulated
diving. A significant increase in TPR occurred during hypocapnic diving (0.6 ± 0.1 to 1.0 ± 0.1 mmHg · ml1 · min)
compared with control values, that maintained MABP at a slightly higher
level than during normocapnic simulated diving (Table 1). There was no
change in SV during the hypocapnic dive period (Table 1).
Arterial blood gases during simulated diving.
Figure 2 shows the results of separate experiments to determine
arterial blood gas changes during simulated diving in the normocapnic
and hypocapnic dive groups. During simulated diving in the normocapnic
group, PaCO2 rose from a predive level
of 38.6 ± 1.0 to 48.7 ± 2.2 mmHg, 25-35 s into the dive
response. During a dive response in the hypocapnic group,
PaCO2 rose from a predive level of 24.8 ± 0.8 to 35.7 ± 1.0 mmHg 25-35 s into the dive response. The changes in PaCO2 during simulated
diving were accompanied by corresponding changes in
pHa.
PaO2 was maintained within the normal
physiological range predive in both simulated diving protocols and
decreased to approximately the same level during simulated diving (Fig.
2).
Global cerebrovascular response to normocapnic simulated diving.
Figure 5 shows the global cerebrovascular
effects of normocapnic simulated diving. During normocapnic simulated
diving, CBF increased a significant 1.5-fold from control values (3.4 ± 0.3 to 5.0 ± 0.5 ml/min), whereas CVR decreased significantly
compared with control values (30.1 ± 3.4 to 11.1 ± 0.8 mmHg · ml1 · min).
This indicates that the increase in CBF during normocapnic simulated
diving is due primarily to a corresponding 63.1% decrease in CVR. The
percentage of CO directed toward the brain increased >2.8-fold during
normocapnic simulated diving compared with control values (2.4 ± 0.2 to 6.9 ± 0.7%).
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Fig. 5.
Cerebral blood flow (CBF, A),
%cardiac output to brain (% of CO,
B), and cerebrovascular resistance
(CVR, C) in control, trigeminal
stimulation alone, hypocapnic dive, and simulated dive groups. Values
are means ± SE. During normocapnic simulated diving, CBF
(A), %CO to brain
(B), and CVR
(C) were all significantly different
from control values. Preexisting hypocapnia abolished increase in CBF
that was observed during normocapnic simulated diving
(A). ** Response significantly
different from all other groups, * response significantly
different from control values; P < 0.05.
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Global cerebrovascular response to trigeminal stimulation and
hypocapnic simulated diving.
CBF did not change from control values during either trigeminal
stimulation and hypocapnic simulated diving (Fig. 5). During hypocapnic
simulated diving the brain's share of CO increased significantly
compared with control values (2.4 ± 0.2 to 5.6 ± 0.4%). This
corresponded to a significant reduction in CVR during hypocapnic
simulated diving compared with control values (30.1 ± 3.4 to 18.4 ± 1.3 mmHg · ml1 · min).
rCBF during normocapnic simulated diving.
rCBF was determined in 32 brain regions (Figs. 6-9). During
normocapnic simulated diving, rCBF increased significantly in 16 of the
32 brain regions examined, compared with control values (Figs.
6-9). Most regions of the hindbrain and thalamus increased rCBF
during normocapnic simulated diving compared with all other groups
(Figs. 6 and
7). Regional CBF did not increase
significantly during normocapnic simulated diving to any region of the
basal ganglia or limbic system (Figs. 7 and
8). Most primary cortical regions increased
rCBF significantly from control values during normocapnic simulated
diving (Fig. 9). The largest absolute
difference in rCBF (normocapnic simulated diving and control) occurred
in the habenular complex in the dorsomedial aspect of the thalamus (133 ml · min1 · 100 g1), whereas the largest
decrease in rCBF occurred in the anteroventral thalamic nucleus (63 ml · min1 · 100 g1).
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Fig. 6.
Regional cerebral blood flow (rCBF) values in hindbrain regions of
control, trigeminal stimulation alone, hypocapnic dive, and simulated
dive groups. Values are means ± SE. During normocapnic simulated
diving, rCBF increased significantly from all other groups in most
brain regions. Preexisting hypocapnia abolished increase in rCBF that
was observed during normocapnic simulated diving. Sp5C, spinal
trigeminal nucleus, caudal part; Sp5I, spinal trigeminal nucleus,
interpolaris part; Sp5O, spinal trigeminal nucleus, oral part; Gr,
gracile nucleus; Cu, cuneate nucleus; Sol, nucleus of the solitary
tract; 12, hypoglossal nucleus; Lrt, lateral reticular nucleus; IO,
inferior olive; 7, facial nucleus; Cb, cerebellum. ** Response
significantly different from all other groups;
P < 0.05.
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Fig. 7.
rCBF values in basal ganglia [caudate putamen, posterior (CPu-P);
caudate putamen, anterior (CPu-A), and globus pallidus (GP)] and
thalamic nuclei [ventral posteromedial and ventral posterolateral
thalamic nuclei (VPM/VPL), habenular nucleus (Hb), laterodorsal
thalamic nucleus (LD), mediodorsal thalamic nucleus (MD), and
anteroventral thalamic nucleus (AVVL)] regions of control,
trigeminal stimulation alone, hypocapnic dive, and simulated dive
groups. Values are means ± SE. During normocapnic simulated diving,
rCBF did not increase significantly to any region of basal ganglia but
increased significantly to most regions of thalamus. ** Response
significantly different from all other groups;
P < 0.05.
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Fig. 8.
rCBF values in reticular formation [medullary reticular nucleus,
ventral part (MdV) and medullary reticular nucleus, dorsal part
(MdD)] and limbic brain regions [entorhinal cortex (Ent),
hippocampus (Hi), hypothalamus (H), amygdala (A), and cingulate cortex
(Cg)] of control, trigeminal stimulation alone, hypocapnic dive,
and simulated dive groups. Values are means ± SE. Blood flow did
not increase significantly to any brain region of reticular formation
or limbic system in both diving groups.
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Fig. 9.
rCBF values in motor and somatosensory cortex [ frontal cortex
(Fr), hindlimb area of cortex (HL), and parietal cortex (Par)],
visual cortex [occipital cortex (Oc)], and auditory cortex
[dorsal cochlear nucleus (DC) and inferior colliculus (IC)]
regions of control, trigeminal stimulation alone, hypocapnic dive, and
simulated dive groups. Values are means ± SE. During normocapnic
simulated diving, rCBF increased significantly from control values in
all regions of motor and somatosensory cortex. ** Response
significantly different from all other groups, * response
significantly different from control values,
# response significantly
different from trigeminal stimulation and hypocapnic dive groups;
P < 0.05.
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rCBF during trigeminal stimulation and hypocapnic simulated diving.
Trigeminal stimulation alone did not produce a significant increase in
rCBF from control values in any brain region, including the trigeminal
nuclei themselves (Figs. 6-9). During simulated diving after
preexisting hypocapnia, rCBF did not increase significantly in any of
the brain regions examined (Figs. 6-9).
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DISCUSSION |
This is the first study to investigate the relative contribution of
humoral and neural inputs on the cerebrovasculature during a dive
response in a small mammal. During simulated diving, CVR decreased by
63.1%, resulting in a 1.5-fold increase in CBF. Preexisting hypocapnia
abolished the cerebrovasodilatory response to simulated diving.
Trigeminal stimulation alone did not produce a significant increase in
rCBF from control values in any brain region. These results support the
primary hypothesis that the progressive rise in arterial
CO2 produces a global fall in CVR
during diving in rats. However, these results do not support a role for
trigeminal input in the cerebrovascular response to simulated diving in
rats.
Role of carbon dioxide in the cerebrovasodilatory response to
simulated diving.
The major finding of this study is that the progressive rise in
arterial CO2 decreases CVR and
increases CBF during simulated diving in the rat. Numerous studies have
clearly demonstrated that hypercapnia elicits a marked vasodilation in
the cerebral circulation (for review, see Ref. 8). In almost all
studies, CBF increases steeply in response to increased
CO2. This suggests that
progressive hypercapnia during diving possibly produces widespread cerebrovasodilation. The reactivity of CBF in response to
PaCO2 has been described by a sigmoid
curve with the linear portion ranging from ~25 to 70 mmHg (14, 28,
33). Therefore, as PaCO2 increases
during asphyxia associated with the dive response, CBF increases
linearly. Other investigators have also suggested that
CO2 is the primary stimulus that
increases CBF during diving in both mammals (1, 6) and ducks (19, 37).
In this study, PaCO2 increased from a
predive level of 36.8 ± 1.0 to ~48.7 ± 2.2 mmHg during
normocapnic simulated diving, a level within the linear portion of the
CBF-CO2 curve. With use of a
previously published equation to estimate CBF reactivity in response to
PaCO2, the increase in
PaCO2 during normocapnic simulated
diving is predicted to increase CBF by ~33% (15). We report here
that CBF increased by nearly 50% during simulated diving in the
regions we studied. However, our estimate for CBF included brain
regions composed primarily of gray matter. Perfusion to white matter
has been shown to be significantly less than perfusion to gray matter
(8).
During asphyxic diving, however, as
CO2 is increasing, arterial oxygen
is also decreasing. Therefore, it is possible that hypoxia also
stimulates a decrease in CVR. The reactivity of the cerebrovasculature
in response to hypoxia is significantly less than to hypercapnia (26).
In fact, CBF has been shown to be virtually unchanged over a
PaO2 range of 55 to 140 mmHg, at
constant PaCO2 (26, 23). Below ~55
mmHg, however, CBF increases sharply. Therefore, the effect of
hypoxemia on the cerebrovasculature most likely occurs later in the
dive period, only after PaO2 has fallen considerably below 55 mmHg. Furthermore, although
PaO2 decreased to the same level in both
simulated dive groups, rCBF did not increase in the hypocapnic
simulated dive group to the same magnitude as in the simulated dive
group. This suggests that the majority of the increase in rCBF during
simulated diving is due to hypercapnia during the dive period and not
hypoxemia. Jones et al. (19) proposed that the effects of hypoxemia and
hypercapnia summate to produce the marked increase in CBF observed
during diving. If the additive effect of hypoxemia on CBF occurs after
hypercapnia, the ability of hypoxemia to increase CBF is possibly
reduced, because cerebral blood vessels may already be near maximal
dilation in response to hypercapnia. Therefore, our results support
hypercapnia as the primary stimulus that increases CBF during diving in
the rat.
Blood pressure effect on CBF during simulated diving.
Simulated diving resulted in an immediate bradycardia that was
maintained throughout the entire stimulation period. However, the
decrease in CO was not matched by an increase in peripheral resistance,
resulting in a significant hypotension during simulated diving (Fig. 3,
Table 1). Blood pressure is maintained above control levels in
conscious, voluntarily diving rats (30). The hypotension is likely due
to the effect of the paralytic agent d-tubocurare on sympathetic ganglions
(13). During a mammalian diving response, an increase in sympathetic
outflow produces peripheral vasoconstriction that maintains arterial
blood pressure (for a review, see Ref. 2). After curare administration,
vasoconstriction could be partially blocked, resulting in hypotension
during simulated diving. Hypotension has been shown to diminish the
responsiveness of the cerebrovasculature to hypercapnia (14, 39). The
decreased responsiveness is likely due to the autoregulatory mechanism
in the cerebral circulation. Autoregulation keeps CBF constant by vasodilating in response to decreased arterial pressure and
vasoconstricting in response to increased arterial pressure (8).
Therefore, cerebral vessels may have lost some of their capacity to
dilate in response to hypercapnia if cerebral vessels were previously dilated in response to hypotension. This may explain why CBF did not
increase in the same magnitude as during conscious diving in the rat
(30).
Role of trigeminal stimulation in the cerebrovasodilatory response
to simulated diving.
We hypothesized that trigeminal input differentially modulated the
global cerebrovasodilatory response to simulated diving in rats.
Trigeminal stimulation alone produced profound cardiovascular changes
that resulted in a decreased CO (Fig. 4, Table 1). However, trigeminal
stimulation alone did not produce a significant increase in rCBF from
control values in any brain region. Therefore, these results do not
support a role for trigeminal input in the cerebrovascular response to
simulated diving in rats. This result was somewhat unexpected due to a
significant amount of evidence demonstrating that stimulation of the
trigeminal nerve or a ganglion associated with the nerve produces
regional variation in CBF (for review, see Ref. 16).
There are two possible explanations why our results suggest that
trigeminal stimulation does not influence CBF. First, it is possible
that during trigeminal stimulation alone, the neural pathway that
increases CBF is potentially inhibited at the brain stem level. The
trigeminal stimulation alone protocol required continuous ventilation
of the rat, indicating that afferent input from pulmonary stretch
receptors (PSR) was feeding back to an integration site within the
medulla during the stimulatory period (9). Absence of PSR inputs, as
during apnea, potentially "gates" information in the medulla
during bradycardia (22). Therefore, it has been suggested that
reduction of PSR afferent input is an important factor in the
development of diving bradycardia (5). The neural pathway that
increases CBF during trigeminal ganglion stimulation occurs via a
reflex that traverses the brain stem (20). Therefore, during trigeminal
stimulation alone, the neural pathway from the trigeminal ganglion that
increases CBF is possibly inhibited at the brain stem level by PSR
feedback. However, although trigeminal afferent input may be inhibited
at the brain stem, this input would presumably increase local metabolic
activity. Because neural metabolism and rCBF are normally tightly
coupled, it would be expected that a metabolically driven increase in
rCBF would occur to the trigeminal nuclei (23). Regional CBF did not
increase significantly in any of the trigeminal nuclei during trigeminal stimulation. This raises the possibility that either metabolic activity in the trigeminal nuclei did not increase, appreciably increasing rCBF, or that the IMP tracer technique is
insensitive to small metabolically driven changes in rCBF.
A second explanation why trigeminal stimulation did produce CBF changes
is that the trigeminocerebrovascular pathway was not elicited in
response to water flow (trigeminal stimulation). Substance P and
calcitonin gene-related peptide are both vasoactive neuropeptides in
the trigeminocerebrovascular system and produce cerebrovasodilation (7). These peptides coexist in capsaicin-sensitive unmyelinated C
fibers innervating the nasal mucosa that may be nociceptive in nature
(36). If water flow through the nasal cavity was not sensed as a
painful stimulus, then it is possible the trigeminocerebrovascular pathway was not activated during the trigeminal stimulation protocol.
In summary, this study examined the contribution of humoral (carbon
dioxide) and neural (trigeminal stimulation) inputs in the
cerebrovasodilatory response to simulated diving in the rat. These
results provide evidence to suggest that the decrease in CVR during
diving in small mammals is driven primarily by progressive hypercapnia
associated with asphyxia. Although trigeminal stimulation is necessary
for the cardiac component of the mammalian dive response, we have found
no evidence to suggest that this input has any role in differentially
modulating the cerebrovascular response to diving.
Perspectives
There is little doubt that the brain is preferentially perfused during
asphyxic diving in all vertebrates. Increased CBF maintains oxygen and
substrate delivery to this crucial organ despite a reduced CO and a
progressively falling arterial oxygen content. This study is the first
attempt to investigate the mechanisms underlying the increase in CBF
during the diving response in a small mammal. We used a simulated dive
protocol that allowed us to investigate the role of the neural and
humoral stimuli thought to be important in diving. Under circumstances
in which ventilatory activity can acquire oxygen from the environment,
rCBF is very sensitive to local changes in cerebral metabolism. Such
local changes might reasonably be expected in simulated diving, because the central integration of input from cardiorespiratory receptors presumably results in some brain regions, such as the nucleus of the
solitary tract and trigeminal nucleus, showing increased neural
activity and metabolic rate. Carbon dioxide is a potent vasodilator in
the cerebral circulation, suggesting that locally produced
CO2 might be one important factor
in the coupling of rCBF to local changes in aerobic metabolism in the
brain. However, the largely homogeneous nature of the increase in rCBF
during simulated diving suggests that any matching of rCBF to local
metabolic activity, by whatever mechanism, is uncoupled by an
overwhelming hypercapnic signal of systemic origin. The physiological
outcome of this CO2-driven
cerebrovasodilation is a prolonged maintenance of oxygen and glucose
delivery, substrates critical for brain survival and therefore
underwater endurance.
| |
ACKNOWLEDGEMENTS |
We thank B. Matushewski and Dr. P. F. McCulloch for assistance.
| |
FOOTNOTES |
The research was supported by a research grant from the Natural
Sciences and Engineering Research Council of Canada to N. H. West. G. P. Ollenberger was supported by a Heart and Stroke Foundation of Canada
Research Traineeship.
Address for reprint requests: G. P. Ollenberger, Dept. of Physiology,
Univ. of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan,
Canada S7N 5E5.
Received 21 August 1997; accepted in final form 19 November 1997.
| |
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Abstract
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