Effects of Breathing O2 or O2 + CO2
and of the Injection of Neurohumors
on the PO2 of Cat Cerebral Cortex
BY WILLIAM J. WHALEN, PH.D., ROGER GANFIELD, PH.D., AND PANKAJAM NAIR, PH.D.*
Abstract:
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Effects of
Breathing Oi
or O, + CO,
and of the
Injection of
Neurohumors
on the PO, of
Cat Cerebral
Cortex
• Tissue PO 2 was measured in the outer 1.7 mm of the brain of the lightly
anesthetized cat by means of a micro O2 electrode introduced through a small
hole in the skull. There was considerable variation from one location to another
and from animal to animal. The interindividual variation was not related to the
level of the arterial pressure or to rectal temperature. Individual values for the
PO2 in 569 locations in the 11 cats ranged from 0 to 90 mm Hg with a mean of
25. There was a significant downward trend in PO2 from the surface layers
inward. When the electrode was left in one location, the PO2 usually fluctuated,
though not often rhythmically as seen in skeletal muscle. Breathing pure O2
( N = 4 1 ) or 95% O 2 - 5 % CO2 (N = 33) caused an equal (50%) increase
in PO2. The intravenous injection of epinephrine or norepinephrine caused a
primary rise in PO 2 usually with a secondary decrease. Acetylcholine caused a
primary decrease and usually a secondary increase.
ADDITIONAL KEY WORDS
tissue PO2
epinephrine
hypercapnia
acetylcholine
hypocapnia
• A number of investigators have measured
oxygen tension on the brain surface or oxygen
availability deep in the cerebral cortex since
the original studies of Davies and Brink in
1942.1 The polarographic electrodes used have
been of three types: (1) a recessed cathode
which gives absolute values for PO2 but can
only be used intermittently and is too large for
intratissue measurements;1 (2) a bare-ended
cathode which can be made small enough for
intratissue use, but whose calibration in tissues
is uncertain;2 (3) Clark-type electrode3 in
which both cathode and anode are covered
with a membrane. The latter electrode gives
absolute values for PO2, but only Silver4 has
made one with a tip (5/x) small enough for
intratissue use. Using this electrode Silver has
made some measurements of the PO2 in the
•St. Vincent Charity Hospital, Cleveland, Ohio,
44115.
This work was supported in part by PHS Grants
# H E 11906 and FR 05631.
194
norepinephrine
brain PO2
brain of the rat and rabbit. Our purpose was to
extend and amplify Silver's observations as
well as to test the response to the breathing of
pure oxygen or 95% oxygen-5% carbon
dioxide and the injection of the neurohumors
epinephrine, norepinephrine and acetylcholine.
/Methods
The electrode we have devised combines some of
the advantages of the separate cathode and the
membrane-covered electrode. The details of its
construction and testing have been published.6
Briefly, it consists of a glass capillary tube drawn
out to a tip of 1 to 2[i. The capillary is filled with
an alloy to within about 20/x of the tip. A few
microns of gold are plated on the alloy, and the
balance of the recess is filled with collodion. An
external reference anode is used. Tests of the
electrode indicate that it is capable of measuring
oxygen tension in tissues.8 Occasionally a largertipped electrode (30/i,) was used for comparison.
This electrode had a slower response time, but
otherwise gave similar results (fig. 1A). Since
there is good evidence that O2 consumption of the
Strokm, Vol. I, May-Juna 1970
P C IN CEREBRAL CORTEX
MJ-1000
800
600
o
w
400
i
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200
FIGURE 1
POi profiles in cat cerebral cortex. Downward-pointing arrows in A and B indicate the moment
of insertion of the electrode tip into the tissue surface. In C the record begins with the electrode tip in the brain surface. Further penetration is indicated by the dashed lines—depth
scale at right. Withdrawal in A is indicated by upward-pointing arrow. Each profile is from
a different animal.
brain is essentially independent of PO2,~ variations
in cortical P0 2 must reflect variations in local
blood flow.
Cats were anesthetized with sodium pentobarbital (30 to 40 mg/kg) given intraperitoneally. In one of the cats urethane anesthesia (1
g/kg) was used and was supplemented with
sodium pentobarbital (10 mg/kg). Since the
results from this latter cat fell within the range of
the others the data were lumped. A tracheal
cannula was inserted, and the femoral artery was
cannulated to monitor arterial pressure. The
femoral vein was cannulated to permit injections.
Rectal temperature was also monitored and
usually held between 36 to 39° C. A dental drill
was used to make a small hole in the skull (0.5 to
1.0 cm diameter) and a slit was made in the dura
for the insertion of the electrode. The brain
surface was continually bathed in warmed
physiological solution (pH 7.2 to 7.3) usually
equilibrated with 25% O2-2% CO2. Occasionally oxygen-free solutions were used, mainly as a
calibration check. A slow flow rate was used,
except during calibration checks, in order to
permit near equilibration with the brain surface.
The anode was placed in the bathing
solution, and under a stereomicroscope the PO 2
Stroke, Vol. I, May-June 1970
electrode was inserted by means of a micro
manipulator into the brain surface. Measurements
of PO2 were made at the surface, and then at each
100 microns into the cortex. The micro-manipulator knob was connected to a potentiometer so that
the depth (down to 1000;n) into the brain could
be recorded simultaneously with the PO2. (In
some experiments deeper penetrations were made
but with far less accurate localization.) At every
third or fourth step the electrode was left in place
while the animal was given pure O2 or 95% O25% CO., to breathe for three to seven minutes. A
planimeter was used to calculate the PO2 from the
area under the PO2 trace.
After three to six penetrations in each animal
injections of epinephrine (5 fig), norepinephrine
(5 fig) or acetylcholine (10 fig) were given
intravenously, in random order, while the electrode remained in one location. The agents were
washed into the circulation with 1 to 2 ml of
saline warmed to body temperature.
Results
It is difficult to describe a typical oxygen
profile, for the variability was extreme. Figure
1 depicts three different profiles which illus195
WHALEN, GANFIELD, NAIR
P02mmHg
44-,
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18-
161412108-
64220O
400
—I—
600
800
Depth
1
1000
1
1200
1
1400
FIGURE 2
Plot of mean PO, ± 5. E. versus depth into the brain.
Each point down to lOOOn represents at least 39 loi •uion\ and a miiimum of nine beyond that depth—
in all, 569 locations in 11 cats.
trate this point. In some areas large gradients
were present (fig. 1C).
In many locations fluctuations in POoccurred at the same frequency as the
respiration (fig. I B ) . Since in many experiments there was perceptible up and down
movement of the cortex with respiration it may
be that the fluctuations are simply movement
artifacts. Indeed, in some locations it was
possible to produce a similar pattern by
moving the electrode up and down with the
manipulator. In other instances, however, the
fluctuations were seen when there was no
perceptible brain movement and when raising
and lowering the electrode did not duplicate
the pattern. Furthermore, when brain movement did occur the fluctuations in PO- were
often out of phase with the movement. We
196
think it is possible that some of the fluctuations
reflect respiratory variation in arterial PO-_» as
found by Purves 8 in the carotid artery, but
much more experimentation will be required
before real evidence is forthcoming.
The values found for POu in 569 locations
in 11 cats ranged from 0 to 90 mm Hg. The
highest value was found near the surface of the
brain when oxygen-free solution was bathing
the surface. The data from the 569 locations
have been plotted and illustrated in figure 2. A
highly significant downward trend from the
surface inward is evident. When the values
from the brain surface to the lOOO^t depth,
inclusive, were averaged, a mean of 25 mm Hg
was obtained. The values from the deeper
locations were not included since (1) there
was less certainty about the depth, and (2) the
electrode tip may have been in white matter.
The average values for tissue PO- for
each animal ranged from 5 to 44 mm Hg.
There was no correlation with arterial pressure.
The only animal with a mean arterial pressure
of less than 75 mm Hg had a mean value for
tissue POj of 35 mm Hg. In this animal the
mean arterial pressure was about 60 mm Hg
throughout the experiments. Likewise,. rectal
temperature was not correlated with tissue
PO:;. As stated earlier, the rectal temperature
of most animals remained between 36 and 39°
C. One animal, however, was inadvertently
heated to 41.2° C, and the PO- values showed
no change. In another animal the temperature
fell to 35° C without noticeably affecting the
POj readings.
The effect of breathing pure O-j or 9 5 %
0^-5% CO- varied considerably. Responses
from three cats are illustrated in figure 3.
Commonly the PO- rose for the first two to
three minutes and then levelled off or declined.
When compared to the first air-breathing
control period, breathing pure O 2 caused an
increase in tissue PO- in 35 trials, no change in
one, and a decrease in five. Breathing 9 5 %
0 - - 5 % CO- caused an increase in 21 trials,
no change in one, and a decrease in 12. The
mean percent of increase was 50% for both
gasses. These results are graphed in figure 4.
Changes in cortical PO- following injection of neurohumors were followed in eight
experiments performed on five cats. In one
experiment there was no response to any of the
agents. Since the control PO- in this instance
Stroke, Vol. I, Moy-June 1970
PO., IN CEREBRAL CORTEX
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FIGURE 3
Records from three experiments which show the variations in cortical tissue PO, during
breathing of 100% O* (100), 95% O2-5% C0t (95-5), or the control gas, air.
was 0, it is possible that the electrode tip was
in an avascular location. In the remaining
seven experiments epinephrine caused an initial
increase in PO^ (sometimes slight) in all trials,
and a delayed decrease in five. Norepinephrine
also caused an increase in all trials, but a
secondary decrease in only two. Acetylcholine
caused a decrease in six, an increase in one,
and a secondary increase above the control
level in four trials. In these seven trials the
control POj ranged from 5 to 30 mm Hg.
Typical responses are shown in figure 5. Note
in this illustration that breathing 100% O^ did
not increase the PO2. This lack of response was
also found in three other of the eight locations.
Although the small number of trials makes the
evaluation uncertain, there was no obvious
relationship between the response to O^ and
that to the neurohumors.
Discuss/on
The range of values we report here for PO-; in
cat brain cortex are similar to those found by
Silver4 in rat and rabbit brain cortex. The
highest value he found was 97 and the lowest 2
mm Hg. Silver noted, as others had before him,
Stroke, Vol. I, May-June 1970
that the high values were found near blood
vessels and the low values approximately
midway between vessels. Silver did not report a
mean value or a plot of the POL> versus depth.
However, the PO^ profile he showed was not
dissimilar from several we saw in the cat brain.
He made no mention of a downward trend in
PO 2 from the surface inward, and we infer that
there was none. Likewise, in another similar
study, but with a bare-ended platinum electrode, no such trend was reported." It is
possible that the PO 2 in the outer 100 microns
of the surface was affected by the PO 2 of the
solution bathing the brain surface but not
deeper layers, for we have shown that when
exposed to a PO2 of 380 mm Hg at the surface
the critical depth in an isolated slice of the cat
cortex is only about 110 microns (to be
published). In the present study the POL. of the
bathing solution was never more than 190 mm
Hg. Thus we are inclined to believe that the
downward trend is real and may reflect loss of
oxygen in the vessels leading down from the
pia. In line with this view, we previously noted
that even large arteries leak oxygen," and
Duling has found a substantial longitudinal O-_.
197
WHALEN, GANFIELD, NAIR
P02
mrnHg
30-,
25-
20-
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15-
10-
1
Control
I
Gas
1
Post - control
FIGURE 4
Effect of breathing 100% O, (0
0) or 95%
Or5% CO, (0
0) as compared to air breathing before (control) and after (postcontrol). Each
point represents the mean ± S. E. of at least 34 trials
in 11 cats.
gradient along arterioles (personal communication). Furthermore, Staub 10 has shown that
in the lung oxygen exchange occurs in vessels
much larger than the capillaries.
Our study indicates that cortical PO^ is
considerably above that in white skeletal
muscle, where we found mean values of about
4 and 6 mm Hg for the guinea pig6 and cat 11
respectively. In the studies on skeletal muscle
we were quite certain that the electrode tip was
seldom if ever in a blood vessel and, therefore,
the values for PO 2 represented tissue PO 2 . In
the present study the electrode tip may at times
have been in a blood vessel so that the mean
value is higher than the true mean for tissue
PO 2 . However, it was nearly always possible to
return to a particular location and obtain the
198
original value—an unlikely possibility if the
electrode had damaged the blood vessel. In
addition, there was no significant change with
time in any one location as might be expected
if the electrode interfered with the blood
supply. Finally, Silver4 reported that even with
the larger-tipped electrode he used spontaneous
electrical activity of neurones recorded by his
electrode continued for hours. Thus, it is likely
that the mean values are representative of
mean tissue PO 2 . Whether all cells are at times
exposed to the mean POL» is problematical. The
relative stability of the values at one particular
location would suggest that they are not, but
rather that cells near blood vessels are always
exposed to a higher oxygen tension, and one
population of cells remote from blood vessels
may always exist on the brink of hypoxia.
More work is necessary to resolve this
question.
The results of the experiments in which
100% O 2 was breathed were not unexpected.
Cross and Silver9 reported that they occasionally found areas where breathing pure O2 did not
increase the cortical tissue PO 2 . However, they
interpreted this finding as an indication either
that the electrode was not working properly or
that the tip (1-10/x bare-ended) was in
unhealthy brain tissue. We prefer to interpret
our results to mean that in some areas
constriction of blood vessels reduced blood
flow sufficiently to maintain tissue PO 2 constant or even to decrease it. The constrictor
effect of oxygen on brain blood vessels is well
known.12 Furthermore, Cooper et a!.13 sometimes saw evidence of a fall in blood flow in the
area around their O 2 electrode when their
patients breathed 100% O 2 . We considered but
rejected the possibility that the locations which
showed no change or decrease in PO 2 in our
studies were those too far from open blood
vessels to be affected—presumably those areas
having low PO 2 values. One of the locationswhich showed a decrease (slight) in PO 2 when
100% O 2 was breathed had a control value of
52. The largest decrease of PO 2 of 8 mm Hg
occurred in a location having a control value of
27. On the other hand, locations with control
values as low as 0 mm Hg responded to O2
with an increase in PO 2 , although with a delay
of about one minute.
The surprising finding that breathing 9 5 %
O 2 -5% CO2 caused only the same increase in
tissue PO 2 and, in fact, no change or decrease
Strok; Vol. I. May-Jun* 1970
PO., IN CEREBRAL CORTEX
-,•-,-
r
.
i - . -
MinuUi
FIGURE 5
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Effect of the injection (I.V.) of 5 ng epinephrine (E), 5 tig norepinephrine (NE), or 10 /ig
acetylcholine (Ach); or breathing 100% O, (100) on cortical tissue PO, (lower trace left-hand
scale) and arterial pressure (right-hand scale). Record begins with insertion of the electrode
at arrow. Subsequent depth indicated by dashed lines (right-hand scale).
in more locations, is difficult to explain in view
of the dilator action of carbon dioxide on
cerebral blood vessels.12 In other studies we
found that administering the gas to cats in the
same manner caused a rise in arterial PO 2 to
480 mm Hg and a drop in arterial pH of about
0.1 (to be published). Assuming this pH
change occurred in these experiments, it should
have been adequate to increase cerebral blood
flow12'14 and tissue PO2. As with pure O 2
breathing the control level of tissue PO 2 was
not correlated with the direction or magnitude
of the response. In 12 of 28 locations, where
paired comparison was possible, the response
to the two gasses was in opposite directions. In
explanation, we can only suggest that some
blood vessels respond to carbon dioxide by
dilating, others by constricting.
The results of the injections of the
neurohumors resemble those obtained by
others4' "• 1 5 ' 1 0 using a bare-ended electrode in
the cerebral cortex. The injections of the
catecholamines consistently caused a rise in
cortical PO 2 . The rise was greatest and more
prolonged with norepinephrine, in association
with the prolonged arterial pressure response.
However, the delayed decrease in PO 2 which
occurred most often with epinephrine often
took place in advance of the return of the
arterial pressure to the control level. This
response may be due to a stimulating effect of
catecholamines on cerebral metabolism,17 or it
Stroke. Vol. I, May-June 1970
may simply be a manifestation of autoregulation. Further studies in which arterial pressure
or blood flow are controlled and blood gasses
monitored will be required to reveal the
mechanism. The same comment applies to the
overshoot in PO 2 which we commonly saw
after acetylcholine injection; also, before the
arterial pressure reached the control level.
References
1.
2.
3.
4.
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6.
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Davies PW, Brink R: Microelectrodes for
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Davies PW: The oxygen cathode. I n : Physical
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Clark LC Jr: Monitor and control of blood and
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Silver IA: Some observations on the cerebral
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Whalen WJ, Riley J, Nair P: A microelectrode
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WHALEN, GANFIELD, NAIR
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Purves M J : Fluctuations of arterial oxygen
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Cross BA, Silver IA: Some factors affecting
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Staub NC: Microcirculation of the lung utilizing very rapid freezing. Angiology 12: 469472, 1961
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Whalen WJ, Nair P: Skeletal muscle PO 2 :
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Kety SS, Schmidt CF: Effects of altered
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Aquilonius SM: Relationship between druginduced changes in blood pressure and cerebral oxygen availability. Acta Physiol Scand
7 3 : 2 2 0 - 2 2 5 , 1968
Cater DB, Garattini S, Marina F, et a l : Changes
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man. J Clin Invest 3 1 : 273-279, 1952
Stroke, Vol. I, May-June 1970
Effects of Breathing O2 or O2 + CO2 and of the Injection of Neurohumors on the PO
2 of Cat Cerebral Cortex
WILLIAM J. WHALEN, ROGER GANFIELD and PANKAJAM NAIR
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Stroke. 1970;1:194-200
doi: 10.1161/01.STR.1.3.194
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