Vol. 86, Issue 5, 1497-1504, May 1999
Perfluorocarbon emulsion improves oxygenation of the cat
primary visual cortex
Lissa B.
Padnick1,
Robert A.
Linsenmeier1,3,4, and
Thomas K.
Goldstick1,2,3,4
Departments of 1 Biomedical
Engineering,
2 Chemical
Engineering, and
3 Neurobiology and
Physiology, and
4 Institute for
Neuroscience, Northwestern University, Evanston,
Illinois 60208-3107
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ABSTRACT |
Tissue
PO2 was measured in the primary
visual cortex of anesthetized, artificially ventilated, normovolemic
cats to evaluate the effect of small doses [1 g perfluorocarbon
(PFC)/kg] of a PFC emulsion (1 g PFC/1.1 ml emulsion; Alliance
Pharmaceutical, San Diego, CA) on brain oxygenation. The
change in tissue PO2 (
PO2), resulting from briefly
changing the respiratory gas from room air to 100% oxygen, was
measured before and after intravenous infusion of the emulsion. Before
emulsion, PO2 was 51.1 ± 45.6 Torr (n = 8 cats).
Increases in PO2 of 34.0 ± 26.1 (SD) % (n = 8) and 16.3 ± 8.4% (n = 6) were observed after the
first and second emulsion infusions, respectively. The further increase
in PO2 after the third dose (7.9 ± 10.5%; n = 7) was not
statistically significant. The observed increases in tissue oxygenation
as a result of the PFC infusions appear to be the result of enhanced
oxygen transport to the tissue.
tissue oxygen tension; brain
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INTRODUCTION |
PERFLUOROCARBON (PFC)-based artificial oxygen carriers
have been proposed as a replacement for donor blood as well as a
therapy for minimizing ischemic tissue damage (29, 38). The
administration of a small dose (1 g PFC/kg body wt) of a PFC-based
oxygen carrier (1 g perflubron/ml emulsion; Alliance Pharmaceutical,
San Diego, CA) has been shown to enhance substantially oxygen transport
in the retina of normovolemic cats while the animal was breathing 100%
oxygen (5). This effect was surprising considering that the addition of
such a small amount of PFC to the blood must have increased arterial
oxygen-carrying capacity by 1% or less (APPENDIX A). To be efficacious in certain clinical
applications, PFC emulsions would have to improve oxygenation of organs
other than the retina, including the brain. Although the retina is
often considered representative of the brain, the unique organization of the retinal vasculature (e.g., Ref. 3) may cause tissue oxygenation
to differ in the two neural tissues.
The intravenous infusion of PFC emulsions has been shown to be
beneficial in the treatment of stroke in cat (17, 26) and dog (13)
models. Peerless et al. (26) concluded that the first-generation PFC
emulsion, Fluosol (Green Cross, Osaka, Japan), had a protective effect
on ischemic brain tissue in cats. Macroscopic and histological tissue
evaluations were consistent with the animals' neurological status
after recovery from anesthesia. Fluosol-treated animals (5.25 g PFC/kg)
were in better neurological condition, both histologically and
clinically, than animals given an equal volume of an isotonic saline
solution; however, differences between animals hemodiluted with Fluosol
and animals hemodiluted with a mannitol solution were marginal. Kline
et al. (17) also studied the effect of Fluosol on middle cerebral
artery ligation in cats. After a 2-h ischemic period and a 2-h
reperfusion period, animals were hemodiluted with Fluosol (5.25 g
PFC/kg) or a dextran solution. The delay in treatment was meant to
mimic a human clinical situation. Eight hours after reperfusion,
cytochrome aa3 in
the ischemic penumbra was in a more oxidized state in the animals
treated with Fluosol than in those left untreated or those treated with
dextran. This suggested that the Fluosol-treated cats were in better
condition, metabolically, possibly because of an improved cerebral
oxygen supply. In a canine cerebral ischemia model (13), the
magnitude of the auditory-evoked potential was used as the measure of
brain stem function. Five hours after reperfusion, the animals
pretreated with a PFC emulsion (1.5 g PFC/kg) exhibited an 88%
recovery in evoked potential magnitude, whereas those animals given an
equal volume of saline showed only a 23% recovery (13).
Infusion of PFC emulsions has also been shown to increase oxygen
availability during hyperoxia in the brain of rabbits (9, 33) and cats
(31). These studies measured oxygen availability rather than
PO2 because the investigators used
relatively large cathodes. Large polarographic electrodes consume
enough oxygen so that an artificial
PO2 gradient may be formed within the
tissue, thus altering tissue PO2.
Actual brain PO2 during hyperoxia has
been reported to be increased in dogs hemodiluted with perflubron
emulsion (1). Batra et al. (1) measured the average
PO2 in a fairly large region of the
parietal cortex with a commercial oxygen electrode system (Eppendorf,
Madison, WI).
The purpose of the present research was to directly examine the
efficacy of a PFC emulsion (AF0104, 1 g perflubron/1.1 ml emulsion;
Alliance Pharmaceutical) in enhancing brain
PO2 by recording locally from the
visual cortex of the cat. Cortical tissue
PO2 was recorded during 2-min
episodes of ventilation with 100% oxygen (hyperoxia), both before and
after small doses of the PFC emulsion (1 g PFC/kg body wt) were
delivered intravenously. By using small (5-10 µm) polarographic
microelectrodes, we were able to obtain more local measurements than
did most previous investigators. A unique aspect of this study was that
visual-evoked potentials, recorded from the same site as oxygen
measurements, were used to examine cortical activity and to verify
electrode penetration of the tissue (24).
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METHODS |
Animal preparation.
The surgical procedures and experimental setup have been described
previously (25). Three of the cats used in the previous study (25) were
also used in this one, along with six additional cats for a total of
nine animals. All measurements were made with double-barreled
oxygen-voltage electrodes. Polarographic oxygen electrodes are not
influenced by the presence of PFC emulsions (5, 6).
Experimental protocol.
Tissue PO2 transients were measured
during 2-min episodes in which the animal breathed 100% oxygen
(hyperoxia). Two-minute periods were used to avoid any physiological
changes that may be induced by long-term hyperoxia. These periods of
hyperoxia were at least 10 min apart to allow the animal to completely
return to its basal state. Each PFC dose consisted of 1.1 ml
emulsion/kg. This dose resulted in the addition of 1 g PFC/kg. Each of
the three emulsion doses contributed an incremental, theoretical
fluorocrit (volume %PFC in blood) of 0.7% (assuming 70 ml blood/kg).
All doses of the PFC emulsion were administered over ~3 min during ventilation with room air.
Before the PFC was administered, three control episodes of hyperoxia
were used to check for reproducibility of the tissue PO2 transients and stability of the
baseline. When the control transient responses were too variable,
negative, or when only an immeasurably small
PO2 change
(PO2) could be detected during
hyperoxia, the electrode was repositioned in the brain. In some
cortical locations, a positive-going oxygen transient in response to
hyperoxia was not observed. Of all the intracortical locations sampled
for a response to hyperoxia, approximately one-third of them had a
negative-going oxygen transient or showed no measurable
PO2. These locations were not used to test the effect of the PFC emulsion on brain oxygenation. When no
satisfactory intracortical location could be found, the electrode was
positioned 50-150 µm above the brain surface, and the experiment was performed with the electrode in the chamber fluid
(n = 4).
Data analysis.
The effect of the first dose of the perflubron emulsion was determined
by calculating the percent increase in the difference between tissue
PO2 during ventilation with 100%
oxygen and room air (PO2). The
transient immediately before and the transient immediately after
emulsion infusion were used to calculate the percent
PO2. To determine whether inherent
brain variability was influencing the results, the two values of
PO2 immediately before emulsion
infusion were compared with each other in the same manner. All changes between consecutive transients were compared to zero with a two-tailed, paired t-test. Data were excluded from
analysis in one cat because of baseline instabilities, and in another
cat (cat 169) an outlying result for
the second emulsion infusion (2.35 SD away from mean), in which the
recording became unstable during or just after the recording of the
postinfusion transient, was excluded. In addition, a computer error
resulted in loss of data for the third PFC infusion in one cat
(cat 165).
The effectiveness of the second and third doses (each 1 g PFC/kg) was
evaluated in the same manner as described above for the first dose.
Because the same recording site could not always be used for all
recordings, the cumulative effect of the second and third doses could
not be obtained from direct comparison with the control
PO2 transient recorded before the
first emulsion infusion. Consequently, the hypothetical cumulative dose
effects had to be calculated from the individual effects as described in APPENDIX B.
Statistics.
Statistical significance was determined by a two-tailed, paired
Student's t-test except in the case
in which the results from the present study and a separate retinal
study were compared. Here, a two-tailed, unpaired Student's
t-test was performed. A P value of <0.05 was used as the
criterion for statistical significance.
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RESULTS |
Response in tissue PO2 to
ventilation with 100% oxygen (hyperoxia).
Local responses in cortical PO2 to
brief (2-min) episodes of hyperoxia were examined. As in previous
studies (18, 19, 21, 23, 36), a variable response was observed.
Different locations showed an increase, decrease, or no measurable
PO2 after the respiratory gas was
changed from room air to 100% oxygen. In three of the cats, all three
types of responses were observed in the same animal with the same electrode.
Figure
1A shows
two responses from different locations in the same cat recorded at a
similar depth 2 h apart. As is demonstrated, intracortical areas that
had a higher baseline PO2 and a
positive-going transient in response to hyperoxia tended to have a
larger transient amplitude (PO2)
than did areas with smaller baseline
PO2 values. A significant correlation
between baseline PO2 and
PO2 was found for intracortical
(r2 = 0.665, P <0.0001,
n = 43 transients) and extracortical
(r2 = 0.187, P = 0.009, n = 36 transients) data (Fig.
1B).
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Fig. 1.
Hyperoxic transient amplitudes [change in
PO2
(PO2)] generally increased
with baseline PO2.
A: 2 transients recorded in same cat
in different locations at similar depths 2 h apart.
B:
PO2 as function of baseline
PO2 for positive-going responses.
There was a significant correlation between baseline
PO2 and magnitude of positive-going
oxygen transient for intracortical
(r2= 0.665, n = 43 transients) and extracortical
(r2 = 0.187, n = 36 transients) recordings. Only 7 of these transients, all collected before any perfluorocarbon (PFC) was
infused, were used as control transients for PFC measurements. Control
transient in 1 cat had a baseline of 0.1 Torr and
PO2 of 136.2 Torr, which was an
outlier for this relationship, and so it has been omitted from this
figure.
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Effect of the PFC emulsion during ventilation with room air
(normoxia).
After the first 1 g PFC/kg infusion, while the animal was still
breathing room air, the average increase in tissue
PO2 was 8.2 ± 18.5 Torr
(n = 7;
PO2 during infusion not recorded in 1 cat). This increase was not statistically significantly different from
zero (P = 0.29), probably due to the
variability of the response (3 increases, 2 decreases, 3 no change in
PO2). The
top trace in Fig.
2 shows a slight increase in
PO2 during emulsion infusion; the
bottom trace shows a slight decrease
in PO2 during emulsion infusion. The
average changes in normoxic PO2
during the second and third infusions were 0.9 ± 2.7 and 1.9 ± 3.3 Torr, respectively. Neither of these changes was significantly
different from zero.
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Fig. 2.
Consecutive oxygen transients separated by first PFC infusion in 2 cats. A: data from
cat 169 were recorded intracortically
at an electrode depth of 500 µm. B:
data from cat 174 were recorded 150 µm above surface of brain.
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Infusion of the first emulsion dose seemed to affect the systemic blood
pressure slightly. On infusion of the first dose, the blood pressure
sometimes transiently decreased 5-10 mmHg (while the animal was
still breathing room air). This decrease lasted for only a few minutes.
All hyperoxic episodes were imposed after the blood pressure had
recovered. The second and third emulsion infusions had no significant
effect on the blood pressure.
Effects of the PFC emulsion during ventilation with 100% oxygen
(hyperoxia).
Figure 2 shows examples of oxygen transients recorded immediately
before and after infusion of 1 g PFC/kg body wt. Figure 2A was recorded intracortically, and
Fig. 2B was recorded slightly above
the brain in the artificial cerebrospinal fluid. Often a trace with a
stable baseline showed increased fluctuations during hyperoxia, as
shown in Fig. 2A.
Table 1 gives the effect of the first
emulsion dose for each cat. Recording depths ranged from 150 µm above
the cortex (negative) to 500 µm within the cortex (positive). The
first PFC emulsion infusion always resulted in an increase in
PO2
(n = 8), but it was of variable
amplitude. The mean increase in
PO2 was 34.0 ± 26.1 (SD) % (P = 0.008). On the other hand, the
average PO2 between the two
transients taken immediately before emulsion infusion was not
significantly different from zero. For some unexplained reason, there
was, however, a nearly significant (P = 0.054) decrease in
PO2
of 17.8 ± 21.6%
(n = 8) between the first and second
transients after the first emulsion infusion.
Table 2 provides the baseline
PO2 and average transient amplitudes,
both before and after PFC infusion, for the second and third doses. The
second PFC infusion (1 g PFC/kg) resulted in an increase in
PO2 of 13.7 ± 10.2%
(n = 7, P = 0.004). The third emulsion
infusion (1 g PFC/kg) resulted in an increased
PO2 of 7.8 ± 10.5%
(n = 7), but this effect was not
statistically significant (P = 0.074).
Figure 3 shows the effects of all three
individual doses.
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Fig. 3.
Individual effects of each PFC dose. Individual cat results (solid
symbols) as well as mean effect (open symbols) are shown together.
Error bars represent 1 SD from mean. PFC effects for 1st, 2nd, and 3rd
individual doses were 34.0 ± 26.1, 16.3 ± 8.4, and 7.9 ± 10.5%, respectively.
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It is important to note that the mean baseline
PO2, reported in Tables 1 and 2, is
not representative of average cortical tissue
PO2. Because it was necessary to
collect data at sites selected for sizable positive-going hyperoxic
PO2 transients, our baseline
PO2 was skewed toward a higher value
(Fig. 1B). Average cortical
PO2 has been reported to be 12.8 Torr
(25). In addition, approximately one-half of the baseline values
averaged for Tables 1 and 2 were obtained extracortically.
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DISCUSSION |
Measurement of brain oxygen enhancement.
The present study demonstrates the ability of a PFC emulsion
(AF0104, Alliance Pharmaceutical) to enhance cortical oxygenation during transient hyperoxic conditions. Previous studies examining the
effect of PFC infusions have used large electrodes to obtain a relative
measurement of cortical oxygenation (19, 31, 33). The present study is
one of the first to use small (5- to 8-µm tip) recessed-cathode
microelectrodes inside of a sealed skull chamber. The smaller
electrodes have several advantages. Polarographic electrodes consume
oxygen in the process of measuring it, and the larger the electrode,
the greater the oxygen consumption. Because of this phenomenon, larger
electrodes create greater artificial oxygen sinks, and therefore
gradients, making the PO2 in the
presence and absence of the electrode different. For this reason,
investigators using larger electrodes are forced to report oxygen
availability rather than PO2. With
recessed cathode microelectrodes, such a small amount of oxygen is
consumed that tissue PO2 is not
affected (30). In addition, a smaller electrode should compress the
tissue and blood vessels less than a larger one. Also, smaller
electrodes give very local measurements of
PO2, whereas larger electrodes give
spatial averages of tissue oxygenation. We attempted to make all
measurements within the brain to exploit these advantages of our
microelectrodes, but this was not always possible. Fortunately,
measurements in the chamber fluid near the brain gave very similar
results to intracortical ones. It is possible that the PFC effect may
not be uniform at all cortical locations, and it would be worth
investigating this with a technique in which
PO2 could be measured in multiple
cortical locations simultaneously.
Braun et al. (5) observed a substantial increase in retinal oxygenation
after the intravenous administration of 1 g PFC/kg. The present study
was done to determine whether this enhancement was similar in other
neural tissue. To compare the PFC effect on the brain and the retina,
we had to utilize similar analyses. Considering that in the preretinal
vitreous only positive values of
PO2 were observed, we could only
examine cortical locations with a positive
PO2 so that a fair comparison between the brain and the retina could be made.
Mechanism of the PFC-induced tissue oxygenation enhancement.
After a single dose of 1 g PFC/kg, the brain, like the retina,
exhibited an increase in tissue oxygenation that was much greater than
expected from the minute increase in the oxygen-carrying capacity of
the blood. Also, arterial blood PO2
does not change after emulsion infusions. In a previous experimental series, in which the effect of the emulsion on the retina was examined
under virtually identical systemic conditions (5), no changes in
normoxic or hyperoxic arterial PO2
were observed after emulsion administration. There was also no
measurable effect of the infusion on hematocrit with the use of the
standard evaluation methods (5). Apparently, the blood volume is
regulated to be constant, by either renal mechanisms or blood and/or
extracellular fluid shifts, so that the increase in oxygen-carrying
capacity of the blood after the addition of 1 g PFC/kg to the blood is only 1.2% under hyperoxic conditions (APPENDIX
A). If this regulation of blood volume did not occur,
blood volume would increase by at most 1.66%, and the oxygen content
in 100 ml of hyperoxic arterial blood would decrease by ~0.4% after
the first PFC infusion. The observed enhancement of tissue
PO2 must have resulted from something
more than simply a change in the oxygen-carrying capacity of the blood.
Either a PFC-mediated increase in cerebral blood flow or a decrease in
brain oxygen consumption could explain the enhancement. Experimental
data suggest that cerebral blood flow and metabolism are not affected
by the specific PFC emulsion used in this study. No experimental
evidence has been published that shows vasoactivity of small doses of
the PFC emulsion used here. Systemic hemodynamics do not change after
intravenous infusion of the emulsion (e.g., Refs. 4, 20). Furthermore,
it should be noted that increased oxygen transport has been observed in
vitro in oxygenators, where flow was experimentally maintained at a
constant rate (34). In addition, while the animal was breathing room
air, neither brain PO2 nor
electrophysiological activity (flash visual-evoked potential) was
altered by the administration of the PFC, suggesting that metabolism
was not affected. A previous study that used the same emulsion
formulation as examined in the present work did not show any decrements
in brain stem activity after emulsion infusion (13). Because systemic
hemodynamics, cerebral blood flow, and cortical electrical activity
appear not to be changed by the presence of small amounts of PFC,
alternative explanations have been postulated.
Other possible mechanisms involve an increase in the overall mass
transfer coefficient of oxygen from blood to tissue. Whereas it is not
possible to quantify the contributions of these mechanisms, the basic
ideas can be discussed. It is important to note that PFC particles
essentially do not enter brain tissue. The PFC leaves the bloodstream
unchanged through excretion by the lungs (29, 38). Only the transport
within the blood will be discussed, as it seems unlikely that transport
characteristics of the tissue would be changed by the intravascular
infusion of the emulsion.
The dominant resistance to oxygen transport from blood to tissue is
thought to be in the plasma. There is comparatively little resistance
to oxygen transport present in the red blood cell (RBC) (22). For
vessels that are 100-200 µm in diameter, the identical PFC
droplets as used in the present study (~0.3 µm in diameter) have
been found to be concentrated in an erythrocyte-free annulus near the
wall (32). This phenomenon effectively decreases the overall resistance
of the plasma phase to oxygen transport (8) by creating an annulus of
PFC droplets that may act as "stepping stones" for oxygen (11).
This radial separation of large and small particles with blood flow in
a 200-µm tube was first shown with small latex beads that were 2.5 µm in diameter (10) and is known as the "near-wall excess" phenomenon.
The near-wall excess phenomenon, as it has been studied to date,
applies only to arteriolar-sized vessels, but the brain is mostly
oxygenated by a three-dimensional capillary mesh (27). It is,
therefore, important to consider also mechanisms in capillaries that
may be improving tissue oxygenation.
Capillary oxygen transport can be separated into two types. Some
capillaries may be perfused only with plasma, preventing them from
significantly contributing to tissue oxygenation. Assuming that a 1 g
PFC/kg dose is evenly distributed in the plasma, the plasma would
undergo a 28.0% increase in oxygen
solubility (hematocrit = 43.7, the average in 8 cats).
Most of the oxygen would still be carried to the tissue by the RBCs,
however, with the amount of oxygen dissolved in the plasma phase
(plasma and PFC oxygen) only comprising 4.8% of the total amount of
oxygen in the blood. In addition, only 3.6% of capillaries within the
first 250 µm of the rat cortex were found to be cell free in an in
vivo confocal laser microscopy study (35), so this mechanism is not
likely to be important.
Most capillaries contain RBCs that flow in single file with spaces
between single cells or groups of stacked cells (rouleaux). In such a
case, the cell-free gaps probably contribute little to oxygen
transport. By adding PFC droplets to the circulation, however, oxygen
transport can take place over the entire capillary surface area,
increasing overall oxygen delivery to the tissues. This can be viewed
as a "gap excess" of PFC droplets. In these capillaries, oxygen
may first diffuse from the RBC to the PFC droplets within the plasma
before diffusing to the tissue. Although some capillaries are smaller
than RBCs, a cell-free annulus next to the vessel wall still exists
where the tiny PFC droplets (0.3 µm in diameter) may create a
near-wall excess, even at the microcirculatory level. Hochmuth et al.
(14) have shown that flowing RBCs in 4- to 10-µm-diameter glass tubes
display a cell-free annulus 0.6-1.9 µm thick.
Faithfull and Cain (11) believe that the PFC droplets also facilitate
oxygen transport through the capillary endothelium, because PFC
particles have been found residing within endothelial cells of both
ischemic and healthy tissue. The trapped droplets may enhance oxygen
transport by giving it a pathway of decreased resistance across the
endothelium. Other mechanisms, unknown to us at this time, may also be operating.
Data from successive PFC doses further support a mechanism that lowers
plasma oxygen transport resistance. Under control conditions, when
there is no PFC in the bloodstream, the plasma is the dominant resistance to oxygen transport (22). Each dose of PFC lowers this
resistance. At the point when the plasma oxygen transport resistance
becomes approximately the same as other resistances to oxygen
transport, further doses of PFC would show little effect. A calculation
of the cumulative effect of multiple doses (APPENDIX B) is shown in Fig. 4.
Saturation of the enhancement of transport was observed to occur
between 2 and 3 g PFC/kg in the present study.
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Fig. 4.
Calculated cumulative effects of each dose of PFC. Individual cat
results (solid symbols) as well as mean effect (open symbols) are shown
together. Error bars represent 1 SD from mean. Calculated cumulative
effects for 2nd and 3rd doses were 47.0 ± 21.3%
(n = 7) and 51.7 ± 17.2%
(n = 6), respectively.
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All of the above-mentioned mechanisms would also affect cortical
PO2 during normoxia (ventilation with
room air); however, the magnitude of the effect would be substantially
lower than that observed under hyperoxic conditions. In the present study, a statistically insignificant increase of 8.2 ± 18.5 Torr (n = 7 cats) was observed during the
first 1 g PFC/kg infusion only. We believe that this increase reflects
a slight PFC effect and that mechanisms that lower the plasma
resistance to oxygen transport are operating. The variability among
cats resulted in the statistical insignificance of the increase in
normoxic cortical PO2. As with the
hyperoxic results, the source of this variability is unknown. Again, a
technique that measures brain PO2 at
multiple sites simultaneously would be valuable in determining the
effect of PFC during normoxia.
Magnitude of oxygen transport enhancement.
Although the enhanced tissue oxygenation attributable to the PFC tended
to be smaller in the cortex than in the retina (Fig. 5), the two results were not statistically
significantly different. Retinal data in this figure were obtained from
a previous study by Braun et al. (5). The tendency for a somewhat
smaller effect in the cortex may be due to differences in the vascular
anatomy of the two organs. The retina is oxygenated by both arterioles and capillaries, as is apparent from the capillary-free zones surrounding retinal arterioles (e.g., Ref. 28). Also, the retina frequently has metarteriolar branches, which feed into the capillary network, that leave arterioles at a 90° angle. Plasma skimming may
be facilitated by this branching pattern, and, as a result, it has been
theorized that retinal capillaries have a lower hematocrit than do
other capillaries (2), an idea supported by experimental observations
(15). This lower capillary hematocrit, and, therefore, lower
oxygen-carrying capacity, in the retinal capillaries may be the
underlying reason for the somewhat larger enhancement of oxygenation by
small doses of intravenous PFC in the retina compared with the brain.
Another possibility may be that preretinal
PO2 was influenced by oxygen
diffusing through the vitreous humor from distant arteries and
arterioles (7), in which the PFC effect may have been larger than in
capillaries.
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Fig. 5.
Comparison of effect of first 1 g PFC/kg infusion in a previous retinal
study (5) and the present cortical study. Both individual cat results
(solid symbols) and mean effect (open symbols) are shown. Error bars
represent 1 SD from mean. Difference in mean %increase of
PO2 was not statistically
significantly different in the two studies
(P = 0.083).
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Whereas the mean effect on the brain was not statistically
significantly different from the mean effect on the retina, the responses to the PFC emulsion in the brain were more variable based on
the coefficient of variation (0.77 in brain vs. 0.45 in retina). This
may simply be a result of inherent brain variability. On the other
hand, it may be that there is variability in the effect of PFC in
different locations in each brain and that, if multiple locations were
sampled simultaneously, the average effect would be more similar among
cats. We could not detect a basis for variability, because the
magnitude of the PFC effect was not correlated with recording depth,
control transient variation, or hematocrit. The age of the emulsion was
also considered because the small PFC droplets can enlarge over time.
This also did not appear to influence the effect of PFC on tissue oxygenation.
The observed effect of the PFC on cat brain
PO2 was less than the comparable
effect on rabbit brain oxygen availability (33). In that study, van
Rossem et al. (33), using the same PFC emulsion as used here, measured
the difference between oxygen availability during ventilation with
100% oxygen and room air in a manner similar to the present study. The
change in oxygen availability nearly doubled with a single, larger 5.4 g PFC/kg dose. It is doubtful that the higher dose completely explains the larger effect observed, because the PFC effect appears to be
nonlinear with dose (Fig. 4). In the present study, the calculated cumulative effect nearly saturated at 52% with a dose of 3 g PFC/kg, and it is unlikely that additional doses of PFC would have increased the effect. Two previous studies that used an earlier PFC emulsion (Fluosol), one in rabbits (9) and the other in cats (31), showed a very
similar difference in emulsion effect (100 vs. 33%). Both of those
studies (9, 31) measured oxygen availability and used similar PFC
doses. Another study that used the same PFC emulsion as the present
work, with a hemodiluted dog model, showed an increase in hyperoxic
brain PO2 of 33% after emulsion infusion (1). It is, therefore, reasonable to conclude that the
discrepancy between rabbits and higher mammals might simply be a
species effect.
Treatment with a PFC before or after experimentally induced stroke has
proven beneficial in dogs (12) and cats (9, 26). The increased
oxygenation of the brain with the addition of small amounts of a PFC
could explain the protective effects that PFC emulsions have been
observed to have during stroke.
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APPENDIX A |
Oxygen-Carrying Capacity of the Blood and the Perflubron Emulsion
Natural blood oxygen-carrying capacity.
The overall blood oxygen-carrying capacity
(O2 blood) is the sum of
the oxygen that is held by hemoglobin (Hb;
O2 Hb), the oxygen that is
dissolved in the RBC
(O2 RBC), and the oxygen
that is dissolved in the plasma
(O2 plasma) (36)
Hemoglobin fractional saturation,
Y, is modeled by the Hill equation
(12)
where
P50 is partial pressure of oxygen
(in Torr) at which the Hb is 50% saturated (38.8 Torr for cat Hb)
(16), P is oxygen partial pressure of the blood (in Torr), and
n is the value of the exponent that
most closely fits experimental data (2.95 for cat Hb) (16).
The amount of oxygen dissolved in the RBC and the plasma is determined
by the oxygen solubility (4.7 × 10
3 ml
O2 · 100 ml
RBC1 · mmHg1
and 2.9 × 103
ml
O2 · 100 ml
plasma1 · mmHg1,
respectively) and the PO2 of the
blood (12, 36).
The total amount of oxygen in 100 ml of blood is therefore
where
O2 max is maximum oxygen
carrying capacity of Hb (100% saturation) (in ml
O2/100 ml blood) and equals 0.45 × %hematocrit (5), VRBC is
the fractional volume of the RBC, and
Vplasma is the fractional volume
of the plasma.
Under hyperoxic conditions (PO2 = 500 Torr), the amount of oxygen carried by a 4-kg animal (70 ml/kg blood)
with a hematocrit of 43.7 is 60 ml
O2.
PFC emulsion oxygen-carrying capacity.
The oxygen-carrying capacity of a PFC emulsion is determined by its
solubility for oxygen. The emulsion used in the present work is able to
hold 32 × 105 ml
O2 · ml
emulsion1 · mmHg1.
When a 1 g PFC/kg (1.1 ml emulsion/kg) dose is added to the blood of a
4-kg cat, the extra amount of oxygen carried by the blood at arterial
PO2 = 500 Torr is 0.7 ml
O2. Assuming total blood volume
remains constant, the increase in the amount of oxygen carried by 100 ml of blood is only 1.2%. The increase in the amount of oxygen carried
by the plasma is increased to 4.8% from 3.7% of the total oxygen in
the blood (increase of 30%).
If total blood volume does not remain constant, the volume of the
emulsion would simply add to the initial blood volume of 280 ml, so the
oxygen-carrying capacity of the blood would change to 60.7 ml
O2 in 284.4 ml blood, a 0.4%
decrease in arterial oxygen-carrying capacity during hyperoxia.
| |
APPENDIX B |
Calculations to Arrive at Cumulative Effects for the Second and
Third PFC Doses
The following equations were used to obtain the hypothetical cumulative
effects after the second and third doses from the data available. Let
where
pre(n) is
PO2
before the
nth dose and
post(n) is
PO2 after the
nth dose of 1 g
PFC/kg body wt.
Assuming that post(1) = pre(2), the hypothetical cumulative dose
effects can be calculated from
where
cumulative(2) and cumulative(3) are the hypothetical cumulative effects
after the second and third doses, respectively. Because the electrode
was sometimes moved between doses, the additional data manipulation is
necessary to arrive at the above equations.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Jameel Ahmed, Dr. Monique McRipley, and Jennifer Kang
for assistance during experiments and Dr. David Ferster for advice
regarding animal preparation and useful discussion. We also thank Dr.
Peter Keipert for encouragement, help in obtaining PFC emulsion, and
assistance in editing this manuscript.
| |
FOOTNOTES |
This work was supported by Alliance Pharmaceutical (San Diego, CA) and
by National Eye Institute Grant EY-05034.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. A. Linsenmeier, Northwestern Univ., Dept. of Biomedical Engineering,
2145 Sheridan Rd., Evanston, IL 60208-3107 (E-mail:
r-linsenmeier{at}nwu.edu).
Received 27 January 1998; accepted in final form 6 January 1999.
| |
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