Hearing Research, 56 (1991) 265-272
0 1991 Elsevier Science Publishers B.V. All rights reserved 0378-5955/91/$03.50
The effects of Carbogen, carbon dioxide, and oxygen on noise-induced
Mark Hatch, Mary Tsai, Michael J. LaRouere, Alfred L. Nuttall and Josef M. Miller
University of Michigan Medical School, Ann Arbor, Michigan, U.S.A.
(Received 16 March 1991; accepted 20 July 1991)
An investigation into the effect of Carbogen (95% 0, /5% CO,), 5% CO, /air, and 100% oxygen on cochlear threshold shifts caused by noise
was undertaken. Five groups of eight pigmented guinea pigs were exposed to 105 dB broad band noise for 6 h per day for five consecutive days
with each group receiving the various gaseous mixtures either during noise exposure or for 1 h immediately after noise exposure. A control group
received the same noise exposure but respired air. Auditory threshold shifts, as measured by the auditory evoked brainstem response, were
measured at 2,4,8,12,16, 20 and 24 kHz. Recordings were taken pre-exposure and at Day 1, 3, 5, and Weeks 2 and 3 after noise exposure.
Carbogen, given during noise exposure, resulted in a trend toward less post noise exposure threshold shift (as compared to controls) which
reached statistical significance by Week 3 at all frequencies except 2 and 20 kHz. Subjects given Carbogen after exposure also showed a general
trend toward decreased noise induced threshold shifts, as compared to controls, but this was not statistically significant. The mixture of 5%
CO, /air given during noise exposure yielded no difference in threshold shifts as compared to controls. When 100% oxygen was administered
during noise exposure, a marked decrease in noise induced threshold shifts could be seen as compared to controls, with differences reaching
statistical significance by day 5 at most frequencies. These results indicate that oxygen (i.e. cochlear-oxygenation) is a more important factor than
CO, (i.e., as a vasodilator) in protection of the cochlea from noise induced damage.
Guinea pig; Auditory brainstem response; Temporary threshold shift; Temperature
It has been suggested that noise induced hearing
loss (Hawkins, 1971; Axelsson and Vertes, 19821, sud-
den hearing loss (Johnson and Hawkins, 1972), and
other inner ear disorders (Vertes et al., 1979; Nuttall et
al., 1981) are attributable to vascular insufficiency in
the inner ear. A vascular theory of noise-induced hear-
ing loss has been supported by several studies in the
past which have demonstrated
vessels (Hawkins et al., 19721, decreased cochlear oxy-
gen tension (Misrahy et al., 1958; Thorne and Nuttall,
1987a), decreased laser Doppler recorded flow (Thorne
and Nuttall, 1987bl and poor penetration
media (Lipscomb et al., 1977) in the cochlea vascula-
ture during sound exposure.
Many manipulations to increase blood flow to the
cochlea have been studied. These include vasoactive
agents (Miller et al., 1983; Suga and Snow, 19691,
hemodilution (Hultcrantz and Nuttall, 1987; Keller-
hals, 19721, sympathectomy
drugs such as Pentoxifylline
carbon dioxide (Witter et al., 1980; Joglekar et al.,
narrowing of cochlear
(Quirk et al., 1988) and
et al., 19771,
Corre~~~de~ce to: Alfred L. Nuttall, University of Michigan Medical
School, Kresge Hearing Research Institute, 1301 East Ann Street,
Ann Arbor, MI 48109-0506, U.S.A.
1977; Brown et al., 1982, 1985) The current investiga-
tion focuses on the effect of the gaseous agent Carbo-
gen (95% O,, 5% CO,> and its components, 5% CO,
and 100% 0,, on threshold shifts caused by noise-in-
duced cochlear damage.
Previous experiments have been performed on Car-
bogen’s effect of temporary and permanent
shifts (TTS and PTS1. Witter et al. (1980) demon-
strated a reduced maximal TTS and a more rapid rate
of recovery in chinchillas pretreated
while Joglekar et al. (1977) showed a decrease in the
TTS in both chinchillas and humans given either Car-
0:. ,. ,, ,.,
,, ,. , , , ,. . . , , , ,, ,,
Fig. 1. Sound pressure levels expressed in dB SPL measured at three
different locations within the sound chamber. Levels are noted to be
similar at each area across all frequencies (l-30 kHz).
bogen or 100% oxygen, during or after noise exposure.
The greatest effect in threshold shifts was seen in the
Carbogen group. Brown et al. (1982, 1985) showed a
decrease in PTS in guinea pigs given Carbogen during
120 dB broad band noise exposure. They also found
significantly less outer hair cell loss in the Carbogen
group as compared to controls.
These studies are consistent with the view that Car-
bogen provides a protective function from hypoxic in-
jury that may result from a noise induced vascular
compromise. It is reasonable to hypothesize that the
CO] in Carbogen is the active factor producing an
increased blood flow to the cochlea, thus increasing
A study by Hultcrantz et al. (1980) compared in-
creases in cochlear blood CBF flow between Carbogen
and CO,/air using microsphere technique. There was
a 115% increase in CBF after 5 min in the CO,/air
group as compared to 29% increase in the Carbogen
group. Moreover, 30 min after exposure there was still
improved blood flow to the cochlea in the COz/air
group as compared to the Carbogen group (52% vs.
44%). It would seem that CO,/air might also provide
an equivalent or even enhanced protective effect on
noise-induced cochlear damage. With this in mind, a
study was performed to compare the effect of both
Carbogen and its components on noise-induced
Materials and Methods
Male and female albino guinea pigs weighing be-
tween 200 and 250 grams with normal Preyer reflexes
were used in the study. The study was performed
two phases. In phase 1, two groups of animals (each
with N = 8) received Carbogen gas (95% 02, 5% CO,);
one group receiving Carbogen during noise exposure,
and one group receiving Carbogen for a period of 1 h
immediately after noise exposure. A third group (N =
4) were room air breathing controls. In phase 2, one
group of animals (N = 8) received 5% CO, in air
during noise exposure, and a second group (N = 8)
received 100% 0, during noise exposure, and an addi-
tional control group (N = 8) breathed room air while
exposed to noise. In all cases the animals were awake
POBT E-E WEEK 12
Fig. 2. Mean threshold
(N = 8). from 2 to 24 kHz. An improvement
at Weeks 2 and 3. * denotes
shifts in animals (N = 8) breathing Carbogen
levels can readily be seen in the Carbogen
P 5 0.05. Vertical bars indicate
during noise exposure [Carbogen CD)] as compared
group as compared
above and below the mean.
especially in the hearing
one standard deviation
and freely respired
All animals were subjected to 105 dB SPL broad-
band noise for 6 h per day on five consecutive days. A
sound chamber was used fitted with speakers driven by
a noise generator (band width = l-30 kHz) and power
amplifier. The chamber was lighted and ventilated.
Animals were placed in separate cages and positioned
in different areas daily to insure equal exposure. Sound
levels were measured at 3 different locations within the
sound chamber using a flat weighted sound level me-
ter. Sound spectrum levels (determined
analyzer) were similar at each location (Fig. 1). The
concentration of administered
and kept constant with the aid of CO, and 0, analyz-
ers which sampled the gas at a central level in the
chamber. Measurements made at various locations
within the chamber showed that the gas concentration
distribution was uniform. The delivered gases and that
exhaled by the animals escaped from the chamber
through ventilation holes.
Auditory evoked brainstem responses (ABR) were
measured prior to noise exposure and on Days 1,3,5
the gas mixtures under normal
with a wave
gases was monitored
and Weeks 2 and 3 after noise exposure. Animals were
anesthetized with a mixture of Xylazine (5 mg/kg) and
Ketamine (30 mg/kg) given intramuscularly
ABR. Differential active needle electrodes were placed
subcutaneously at the vertex and left ear. A ground
electrode was positioned near the contralateral
The sound stimulus consisted of a 15 ms tone burst
with a rise-fall time of one millisecond, at frequencies
2,4,8,12,16,20 and 24 kHz. Two hundred fifty-six tone
presentations were averaged using a microcomputer
and custom software to obtain a waveform. ABR
threshold was defined in this study as a sound level
producing a distinctly noticeable wave V on the ABR
(i.e. a peak waveform deflection of approximately 1 pv
referred to the animal).
The mean ABR threshold shifts observed in animals
breathing Carbogen during and after noise exposure
compared to a control (air breathing) group are shown
in the bar graphs in Figs. 2 and 3, respectively. The
Fig. 3. Mean threshold
controls (N = 8). Although
shifts in animals
there is a slight improvement
Vertical bars indicate
(N = 8) breathing Carbogen
in this group
after noise exposure
for 1 h [Carbogen
above and below the mean.
(P)] as compared
difference no statistically exists.
y-axis of each graph represents
(post-exposure A.B.R. - pre-exposure
axis gives the frequencies tested. Threshold shifts ob-
served on Days 1 and 5 and Weeks 2 and 3 following
exposure are shown. In Fig. 2 one can see that at Day 1
post exposure, when Carbogen was given during noise
exposure, there is a smaller threshold shift as com-
pared to controls over all frequencies
During the following 3 weeks, an improvement in the
hearing levels can be seen in both the Carbogen (D)
and control group with the greatest recovery seen in
the Carbogen (D) group. This is most evident at Week
2 and 3 post exposure, whereby the difference in the
two groups is statistically significant for 4 of the 7
frequencies tested. When Carbogen was given post
exposure (Fig. 31, although there is a trend toward
improvement in the threshold shifts in the Carbogen
(P) group, no statistically significant difference
pared to controls was demonstrated
even when followed over time.
An examination of three individual frequencies (Fig.
4), demonstrated a trend toward continued improve-
ment over time to near normal levels in the group
given Carbogen during noise exposure as compared to
the other two groups.
The results of the second phase of the study are
shown in Figs. 5-8. A separate group of animals was
exposed to either 5% CO,/air
noise exposure and were compared to a separate group
of controls. As seen in Fig. 5, 5% CO,/air
during noise exposure showed only small and statisti-
cally insignificant differences
compared to the control group.
In contrast to the CO/air
group (Fig. 6) had significantly less threshold
than controls, this being readily evident by Day 5 post
noise exposure. These results were clearly demon-
strated in a graph of the temporal pattern of change
for the individual frequencies (Fig. 7). Again, a marked
improvement in threshold shifts can be seen in the
100% oxygen group as compared to either the CO/air
or control group when followed over the weeks of
Fig. 8 compares the differences in threshold shift
between the controls and the individual gases across
the duration of the study. Seen at Weeks 2 and 3
post-exposure, there is a greater than 50 dB difference
between the control and oxygen group. This compares
to a 22 dB difference in the Carbogen (D) group and a
14 dB difference in the CO,/air
difference in the Carbogen (P) group at Week 3.
A.B.R). The X-
except 2 kHz.
at any frequency,
or 100% oxygen during
in threshold shifts as
group, the oxygen-treated
group and a 7dB
The results of this investigation confirm the benefi-
cial effect of oxygen (both as 100% O2 and Carbogen)
Fig. 4. Mean threshold
to Week 3 post-exposure)
threshold shift, can be seen in the Carbogen
to the Carbogen
shifts in the Carbogen
against time (Day 1 post-exposure
for frequencies 12, 16, and 20 kHz. A
(D) group as compared
(P) group and control group.
groups (N = 8 each)
group (N = 8) plotted
continued an overall improved
on noise-induced hearing loss as reported in the previ-
ous studies by Brown et al., (1982, 1985), Witter et al.
(19801, and Joglekar et al. (1977), when Carbogen was
given during noise exposure. In contrast to Joglekar et
al. (19771, however, we obtained no statistical signifi-
cant difference between the control group and the
group given Carbogen after noise exposure, although
there certainly seems to be a trend toward improved
threshold shift. This may, in part, be related to the
insufficient time of exposure to the gaseous mixture
after the acoustic trauma or the exact conditions of the
noise exposure itself. Carbogen was administered for a
6-h period, throughout noise exposure in the ‘during’
group; the ‘post-exposure’ group breathed the mixture
for 1 h.
The theory of Carbogen’s beneficial effect is that
the CO,, a known vasodilator, acts Synergistica~Iy with
oxygen in Carbogen to produce increased o~genation
of cochlear tissues and reduce cochlear damage. A
study by Hultcrantz et al. (1980) did show a marked
increase in blood flow to the cochlea when 7% CO,/air
mixture was given using the microsphere
Previous work has also shown elevated oxygen tension
in the endol~ph (Prazma et al., 1978) and periiymph
(Fisch et al., 1976) with inhalation of 5-N%
Kallinen et al. (1991) have shown that Carbogen does
not decrease the apparent vascular resistance of the
cochlea (i.e. give vasoditation)
effects of CO, are countered by high arteriaf Ievets of
because the dilative
Until now, however, no investigations
undertaken to determine the beneficial effects of the
individual components of Carbogen.
that each component might also provide a protective
function to the cochlea. Or perhaps the high oxygen
component wil1 counteract
effect of CO,. As seen in Fig. 5, however, 5% CO, in
air given during noise exposure showed no beneficial
effect on the threshold shifts through the time course
of this study.
When 100% oxygen was administered
noise exposure, however, a marked improvement
acoustic damage, as measured by ABR, was seen. The
oxygen-exposed animals returning to near normal hear-
ing levels by Day 5 post-exposure.
fects of Q, alone were even greater than when the
Carbogen was given. This would seem to suggest that it
is the oxygen and not the GO, in Carbogen
provides the protective effect from acoustic injury
within the cochlea.
Studies of cochrear bIood fIow have indicated that
high levels of sound (> 110 dB SPL) cause increased
It would seem
the supposed vasodilative
The beneficial ef-
Fig. 5. Mean thresboid shifts in the 5% CO, [air/
two groups exists over al1 frequencies tested. Vertical bars indicate one standard deviation above and below the mean.
breathing group (IV = 81 compared to controfs fN = 41. No significant difference between the
flow (eg. Prazma et al., 1983; Quirk et al., 1990) but the
lower levels used in the current study actually decrease
the flow (eg. Thorne 1987b) as well as the intracochlear
PO, (eg. Thorne, 1987a). Blood flow changes may also
be associated with local metabolic demand as has been
strongly suggested by flow increases observed for more
moderate sound levels (< 100 dB SPL) (see Ryan et
al., 1982; Quirk et al., 1991).
Kaliinen et al. (1991) have shown that guinea pigs
freely respiring Carbogen do not have elevated blood
arterial PCO, but do have greatly elevated PO,. That
elevated arterial PO, was associated with vasoconstric-
tion in the cochlea. All of this evidence would suggest
that, for the sound intensity used in the current work,
the physiological effects of vasoconstriction
cochlea are offset by improved intracochlear
The current study was conducted
interval. The first group of animals tested was the
Carbogen group with controls and the second group of
over a 6-month
DAY l i l
animals included the CO,/air
controls. Despite constant monitoring of sound pres-
sure levels within the sound chamber, there are evident
differences in threshold shifts between the two control
groups. Post hoc examination of our experiment sug-
gest that a potential variable between these two groups
was the temperature in the sound chamber. The tem-
perature in the sound chamber of the Carbogen groups
was approximately 5-10 degrees (20-25” vs 15’) Cel-
cius cooler than the CO/air
was due to seasonal differences in the temperature
the building area where the experiment was conducted.
Berndt and Wagner (1981) have shown that lowering
of the ambient temperature
significantly lessens noise damage to the cochlea in
guinea pigs. A similar effect on TI’S has been reported
in humans (Wright et al., 1981). These studies would
be consistent with our data. The control/Carbogen
group (exposed when the ambient air was cool) had
less threshold shift than the control (CO, and 02)
and oxygen group with
and oxygen groups. This
during noise exposure
POST EXPOSURE DAY #5
POST EXPOSURE WEEK (12
Fig. 6. Mean threshold
shifts in the 100% oxygen breathing
in the threshold shifts with the oxygen treated
bars indicate one stand- .d deviation
group f 4 = 8) as compared
above and below the mean.
(N = 4). As can be seen, by Day 5 there is a
to near normal. * denotes hearing P $0.05. Vertical
group exposed when the ambient air was warmer.
Although there were differences in the control groups
for the Carbogen treated animals and the CO, and 0,
treated animals, it is still obvious that there is a marked
improvement in hearing levels in the Carbogen and
100% oxygen group when comparing them to either
Further studies need to be performed on the effect
of oxygen given after noise exposure, as these results
12 K Hz
Fig. 7. Mean threshold
old shifts are clearly charmed
shifts in the 100% oxygen group (N = 8), 5%
(N = 8) and controls (N = 8) plotted
12, 16, and 20 kHz. The oxygen-treated
to the other two erouns.
Berndt, H. and Wagner,
the set-up and recovery
Brown, J.J., Meikle, M.B. and Lee, C.A. (1985) Reduction
tally induced auditory impairment
H. (1981) Influence
of body temperature
by inhalation of carbogen gas
COMPARISON OF MEANS
(CONTROLS - GASES)
DAY 3 DAY 5 WEEK 2 WEEK 3
Fig. 8. The mean
against time. At especially
be seen in the oxygen-treated
gases (at 12, 16, and 20 kHzJ plotted
Weeks 2 and 3, a marked
group as compared
indicate one standard
below the mean.
the threshold shifts of the
to the other
bars deviation and
may hold clinical relevance. Perhaps in industry, mili-
tary, or other occupations where there are excessive
amounts of noise exposure, oxygen administration may
be of benefit.
Carbogen favorably affects threshold shifts that re-
sult from noise-induced cochlear damage, with the
greatest effect being when Carbogen is given during
5% CO, in air has no benefit on threshold shifts
caused from noise exposure
100% oxygen, however, shows a marked improve-
ment in threshold shifts when given during noise
oxygen (i.e. cochlear oxygenation) is a more impor-
tant factor than CO, (as a vasodilator) in protection
of the cochlea from noise-induced cochlear damage.
This work was supported by NIH grant DC-00105,
ROl-AG8885, and DRF Fellowship for M. Tsai.
vessels after noise. In: R.P. Hamernik,
(Eds.), New Perspectives
Press, New York, 49-67.
A. and Vertes, 0. (1982) Histological findings in cochlear
and R. Saki
Hearing on Noise-Induced
gen gas (permanent
Fisch, V., Murata,
tension in human perilymph.
Hawkins, J.E., Johnson,
Hawkins, J.E. (1971) The role of vasoconstriction
hearing loss. Ann. Otol. Rhinol.
Hultcrantz, E., Linder,
effects on cochlear blood flow at different
Inserm. 68, 271-278.
Hultcrantz, E., Larsen, H.C. and Angelberg,
COz breathing on cochlear
Hulcrantz, E. and Nuttall,
cochlear blood flow measured
Otolaryngol. 8, 16-22.
Joglekar, S.S., Lipscomb,
of oxygen inhalation
and chinchillas. Arch. Otolatyngol.
Johnson, L.C. and Hawkins,
human inner ear associated
gol. 81, 364-37.5.
Kallinen, J., Didier, A., Miller, J.M. and Nuttall,
effect of CO, and Oz gas mixtures
cochlear and skin hlood
Kellerhals, B. (1972) Acoustic
an experimental and clinical study on pathogenesis
of inner ear lesions after acute noise exposure.
nol. Laryngol. 18, 91-168.
Lipscomb, D.M.. Axelsson.
sound on hearing sensitivity,
and vasculature of the chinchilla.
Miller, J.M.. Marks, N.J. and Goodwin,
measurements of cochlear
Misrahy. G.A., Shinabarger,
of cochlear action po-
J.A. and Fenwick,
J.A. (1982) Reduction
impairment by inhalation
K. and Hessli, $I. (1976) Measurement
L.G. and Preston,
in normal and damaged
81, 278. (Stockh.)
R.E. (1972) Cochlear
ears. Laryngoscope 82,
C. (1977) Sympathetic
J. and Angelborg,
C. (1980) The effect of
blood flow. Arch Otolaryngol. 228.
A.L. (1987) Effect
by laser Doppler
D.M. and Shambaugh,
G.E. (1977) Effects
threshold shifts in humans
J.E. (1972) Vascular
with aging. Ann. Otol. Rhinol. Laryn-
changes in the
A.L. (1991) The
on laser Doppler
flow in guinea Res. 55,
trauma and cochlear microcirculation:
Adv. Otol. Rhi-
A. et al. (1977) The effects of high level
P.C. (1983) Laser Doppler
blood flow. Hear. Res. 11, 385.
E.W. and Arnold.
J.E. (1958) Changes
Prazma, J., Fischer,
nia. Ann. Otolaryngol.
Prazma, J., Rogers,
flow. Arch. Otolaryngol.
Quirk, W.S., Avinash.
Quirk. W.S.. Dengerink,
Wright, J.W. (1988) The effects
blood flow in normotensive
Hear. Res. 36, 175-180.
Quirk, W.S., Nuttall,
changes in red blood
lateral wall of the rat cochlea.
ter Beach, FL. 1990.
Ryan. A.F., Goodwin,
tion alters the pattern
Brain Res. 234, 213-225.
Suga, F. and Snow, J.B. (1969) Andrenergic
flow. Ann Otol Rhino1 Laryngol
Thorne, P.R. and Nuttall,
gol. (Stockh.) 107, 71-79.
Thorne, P.R. and Nuttall, A.L. (1987bI Laser Doppler
of cochlear blood flow during loud sound exposure
pig. Hear. Rea. 27, I-10.
Vertes, D.. Axelsson, A. and Lipscomb,
effects of noise exposure
laryngol. 224. 97-101.
Witter, H.L., Deka, R.C., Lipscomb,
(1980) Effects of prestimulatoty
duced temporary threshold
J. Otol. 1, 227-232.
Wright. J.W., Dengerink, H.A., Thompson,
Plasma angiotensin II changes
of ambient temperature.
availability, action potential
during asphxia, hypoxia, and
to loud sounds. J. Acoust.
potentials after short anoxia.
G.K. and Pillsbury.
Sot. Am. 30, 701.
W.P. and Ascher,
D. (1978) The
H.C. (1983) Cochlear blood
109, 61 l-615.
G., Nuttall, A.L. and Miller, J.M. (1991) The
of loud sound on red blood cell velocity and capillary
in lateral wall vessels of the guinea pig cochlea.
St. Petersburg Beach. FL, 1991.
M.J.. Hall, K.W. and
A.L. and Miller,
J.M. (1990) Noise-induced
and vessel diameter
Assoc. Res. Otolaryngol.,
P., Woolf, N.K. et al. (1982) Auditory
uptake in the inner ear.
control of cochlear
A.L. (1987a) Alterations
during loud sound exposure.
in the guinea
D.M. (1979) Some vascular
in the chinchilla cochlea. Arch. Oto-
D.M. and Shambaugh,
shifts in humans
and chinchillas. Am.
P. and Morseth, S. (1981)
with noise exposure
J. Acoust. Sot. Am. 70, 1353-1356.
at three levels