CO2 asphyxia increases plasma norepinephrine
in rats via sympathetic nerves
VICKY BOROVSKY, MIKE HERMAN, GAIL DUNPHY, ANN CAPLEA, AND DANIEL ELY
Department of Biology, University of Akron, Akron, Ohio 44325-3908
sympathetic nervous system; reserpine; adrenal medulla
in plasma norepinephrine (NE)
levels and CO2 exposure began with the observation in
our laboratory that CO2 euthanasia, which is quick and
inexpensive, produced high levels of plasma NE as
determined from blood taken at termination. This led
to a literature search, which showed that plasma NE
increased in association with hypoxia during the birth
process (10). Also patients with respiratory distress
showed increased tidal volume CO2, which led to increased blood pressure and increased plasma NE as a
result (1, 11, 15). Likewise, plasma NE increased in
patients with sleep apnea syndrome (2), which induces
hypoxemia and periodic hypertension. Also, plasma NE
increased after Sevoflurane anesthesia and other hypoxic conditions in rats (6, 8, 9, 13, 19) and in fetal
sheep (14).
Plasma NE is released primarily by either the adrenal medulla or by spillover from peripheral sympathetic nerve endings. Therefore, our objective was to
investigate the specific source of the NE in rats as they
responded to CO2 and to establish whether the increase
was due to CO2 specifically or asphyxia in general.
OUR INITIAL INTEREST
METHODS AND MATERIALS
The first experiment was designed to measure the relationship between time of CO2 exposure and plasma NE in order to
set standard time for CO2 exposure. Nine groups of male
adult spontaneously hypertensive rats (SHR) were used
(5/group) and were exposed to CO2 for 5–75 s; blood samples
were taken for NE. The second experiment was designed to
measure plasma NE under different treatments. Baseline
plasma NE levels in four strains of rats were measured in
both sexes (n 5 10/group): normal blood pressure or WistarKyoto (WKY) strain, SHR, normal rats with a Y chromosome
from an SHR father (SHR /y), and hypertensive rats with a Y
chromosome from a normotensive father (SHR /a) (5, 18).
Sodium brevital (50 mg/kg ip, Eli Lilly, Indianapolis, IN) was
given, and blood was collected retroorbitally (16). Samples
were centrifuged (5,000 g), and plasma was transferred to a
separate test tube and frozen at 270°C for later NE measurement by high-performance liquid chromatography with electrochemical detection (Waters, Milford, MA) using the technique of Foti et al. (7). The number of animals for each group
that had an adequate blood sample volume for NE measurement ranged from 6 to 10.
One week after this baseline procedure, we determined the
NE response after the rats were exposed to 100% CO2
(asphyxia) in a closed chamber for ,30 s. Blood was collected
immediately afterward and processed for NE.
The next step in our study was to identify the cause of the
NE increase and determine whether it was asphyxia in
general or CO2 specifically that affected the NE level. In order
to do that, 1 wk later we exposed the rats to 100% N2 gas in a
closed chamber for 30 s, and blood was collected and processed for NE as before.
After this, we determined whether blocking the sympathetic nerve release of NE with the use of reserpine, which
depletes the sympathetic nerve endings of NE, would affect
the response to CO2 exposure. If sympathetic nerves were the
major source of NE release in response to CO2, then the NE
level should not increase significantly. The rats were administered reserpine (1.7 g/l, a dose that will lower blood pressure
in hypertensive rats; Sigma, St. Louis, MO) in their drinking
water for 14 days and then they were exposed to CO2. Blood
was collected for NE as before.
Finally, we performed bilateral adrenalectomies on all
animals and evaluated plasma NE levels in response to CO2.
If the adrenal glands were a major source of NE in response to
CO2 asphyxia, the NE level should not significantly increase
in adrenalectomized animals after CO2 exposure. The rats
were anesthetized with sodium brevital (50 mg/kg ip); the
abdominal area was shaved and disinfected with an iodine
solution. A midline incision was used, and each adrenal gland
was removed. Ethicon (4–0) was used to suture the peritoneum, and surgical staples closed the skin. Animals were
placed on 0.9% saline, final exposure to CO2 was conducted 2
days later, and blood was collected for NE analysis. In
another group of adult males of the same four strains [SHR,
SHR /y, SHR /a, and WKY (n 5 5–6/group)], aortic telemetry
transmitters were implanted (Data Sciences, St. Paul, MN)
(4), and, after recovery (1 wk), systolic blood pressure responses to CO2 were measured. The following statistics were
performed: one-way and two-way analysis of variance, followed by Newman-Keuls t-tests where appropriate, Pearson
correlation (Sigma Stat, Jandel Scientific, San Rafael, CA),
and significance was assumed if P , 0.05.
0363-6119/98 $5.00 Copyright r 1998 the American Physiological Society
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Borovsky, Vicky, Mike Herman, Gail Dunphy, Ann
Caplea, and Daniel Ely. CO2 asphyxia increases plasma
norepinephrine in rats via sympathetic nerves. Am. J. Physiol.
274 (Regulatory Integrative Comp. Physiol. 43): R19–R22,
1998.—The objective of this study was to determine whether
the plasma norepinephrine (NE) increase in rats exposed to
CO2 asphyxia was due to adrenal gland release or sympathetic nerve ending (SNS) release. Plasma NE was measured
by high-performance liquid chromatography in hypertensive
and normotensive rats using the following protocol: control
session, CO2 exposure, N2 exposure, reserpine 1 CO2, and
adrenalectomy 1 CO2. Four strains of male and female rats
were used: spontaneously hypertensive rats, Wistar-Kyoto
rats, and two congenic strains with different Y chromosomes.
The same rats were used throughout the experiment (n 5 80).
Blood pressure measured by aortic telemetry increased ,50–
60 mmHg in response to CO2 in all strains. CO2 increased NE
6–103 in all strains and both genders. N2 produced a
significant increase in NE (73% of CO2 response). Reserpine
significantly decreased (67%) plasma NE after CO2. Adrenalectomy did not significantly reduce the NE response to CO2.
In conclusion, the increase in plasma NE after CO2 was
associated with SNS release and not adrenal medullary
release, was mainly due to hypoxia, and was not a specific
response to CO2.
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CARBON DIOXIDE AND NOREPINEPHRINE
RESULTS
Figure 1 shows the significant positive correlation
between CO2 exposure time and plasma NE (r 5 0.93,
P , 0.001) in the group of SHR males.
Figure 2 represents plasma NE responses for both
males and females of the SHR/y and WKY strains and
of the SHR/a and SHR strains. For the NE level after
CO2 exposure, NE increased 6- to 12-fold compared
with baseline (significant in all strains and both genders, P , 0.001). The results for the NE level after N2
exposure ranged from 59 to 94% of the NE response to
asphyxia with the average N2 response being 73% of the
asphyxia response. The values for the NE level after
asphyxia exposure in rats treated with reserpine were
about the same as baseline level (67% decrease, P ,
0.01–0.001 compared with CO2 response for all strains
and both genders). The NE response to asphyxia in the
Fig. 2. Plasma norepinephrine levels
by strain and treatment. A: WistarKyoto (WKY) rats with a Y chromosome
from a SHR father (SHR /y) and WistarKyoto rats (WKY). B: SHR and SHR
with a Y chromosome from a normotensive father (SHR/a). Values are means 6
SE. ** P , 0.01, *** P , 0.001, CO2 vs.
baseline, N2 vs. baseline, baseline vs.
adrenalectomy, and CO2 vs. reserpine.
1 P , 0.05 and 111 P , 0.001, N2 vs.
CO2.
Fig. 3. Change in systolic blood pressure taken by telemetry after
30 s of CO2 in males by strain. Values are means 6 SE. ** P , 0.01,
*** P , 0.001, compared with respective baseline.
adrenalectomized group was about the same as in the
rats with an adrenal gland: the range of NE increase
was 91–107% of the baseline values (P , 0.01–0.001
compared with baseline for all groups and both genders).
Figure 3 shows the peak blood pressure change
measured by telemetry in males by strain after they
were exposed to asphyxia. An ,60-mmHg increase in
blood pressure was demonstrated with no significant
strain difference.
DISCUSSION
Our results showed a 6- to 10-fold increase in plasma
NE after asphyxia and a 6.8-fold increase to N2 gas
exposure, which suggests a generalized response to the
lack of oxygen, not specifically because of CO2 or
gender. Both genders were examined since we have
seen gender effects in blood pressure and NE responses
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Fig. 1. Relationship between CO2 exposure time and plasma norepinephrine in spontaneously hypertensive rat males. Values are
means 6 SE; r 5 0.93 and P , 0.001 (error bars at time 0 5 6200 and
at 20 s 5 6500 but are too small to be seen).
CARBON DIOXIDE AND NOREPINEPHRINE
than those with a Y chromosome and from a normotensive father (WKY, SHR/a) (3).
In conclusion, acute exposure to 100% CO2 (asphyxia)
increased plasma NE 6- to 12-fold, mainly through
increased release at sympathetic nerve endings and not
from adrenal medullary release. This response appears
to be a generalized one that is not gender or strain
specific or CO2 specific, but rather is due to a lack of
oxygen. This finding may be important to know when
using CO2 to euthanize animals, since such increased
SNS arousal could interfere with other physiological or
biochemical variables that may be collected at that
time. Also, this response could be used as a stressor to
examine maximal SNS responses without causing euthanasia (i.e., 30 s).
The authors acknowledge the following people who assisted with
the research and with the preparation of this manuscript: Fieke
Bryson, Hamid Daneshvar, and Sarah Francis.
Financial support from the Department of Biology of The University of Akron, the National Heart, Lung, and Blood Institute (HL48072–04), and the Ohio Board of Regents-Hypertension Center is
greatly appreciated for grants related to this research.
Address for reprint requests: D. L. Ely, Dept. of Biology, Univ. of
Akron, Akron, OH 44325-3908.
Received 23 June 1997; accepted in final form 22 September 1997.
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to acute stress. Similar results were found where
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Because reserpine decreased the amount of plasma
NE produced in response to asphyxia, it appears that
sympathetic nerve release of NE plays a major role in
increasing plasma NE. Because adrenalectomy did not
reduce the NE response to asphyxia, but in seveneighths of the groups actually increased the response,
the majority of NE appeared to be released from the
sympathetic nerve spillover and not from the adrenal
medulla. Although there are genetic differences in
ventilation rate, minute ventilation, and oxygen consumption in different rat strains (17), all of our strains
of rats and both genders reacted to asphyxia in a
similar way, which suggests a generalized response.
The blood pressure increase of 50–60 mmHg after
CO2 exposure in the males was most likely attributable
to the tremendous elevation of NE, acting as a vasoconstrictor. The animals were conscious when the gases
were administered and with the CO2 became unconscious at the end of the 30 s, which could account for
some of the rise in NE. However, studies in our
laboratory using ether, which agitates the animals even
more than CO2, only doubled plasma NE compared
with a 6–12 times increase in the current study.
Therefore, the agitation and handling factor does increase NE but not to the same extent as the sympathetic nervous system (SNS) response from asphyxia.
Also taking blood retroorbitally even under anesthetic
will produce higher NE values than via catheter collection, but, nevertheless, our results show tremendous
NE elevations over baseline values. These results have
important implications for studies using CO2 for euthanasia, especially when final blood samples are collected
for various assays. The tremendous SNS activation for
survival could mask or elevate other hormones or
signals being assayed. Female blood pressure was not
examined because the NE data did not show any
differential response. With milder stimuli, we have
shown that the strains with the Y chromosome from a
hypertensive father (SHR, SHR /y) had higher acute
and chronic indexes of SNS activity and blood pressure
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