Vol. 94, Issue 3, 860-868, March 2003
Microvascular gas embolization clearance following
perfluorocarbon administration
David M.
Eckmann1 and
Vladimir N.
Lomivorotov2
1 Department of Anesthesia and Institute for
Medicine and Engineering, The University of Pennsylvania, Philadelphia,
Pennsylvania 19104-4283; and 2 Anesthesiology
Department, Research Institute of Circulation Pathology,
Novosibirsk 630055, Russia
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ABSTRACT |
Effective treatment of vascular
gas embolism may be possible with emulsified fluorocarbon
compounds. We tested the hypothesis that a fluorocarbon
emulsion delivered before gas embolization would enhance bubble motion
through the vasculature, favoring more rapid clearance. Air
microbubbles were injected into the rat cremaster microcirculation in
six groups of rats receiving Perftoran, an emulsified fluorocarbon, or
saline immediately before, 2 h before, or after bubble injection.
Embolism dimensions and dynamics were observed by using intravital
microscopy. Surface area at lodging was equal between groups. Bubbles
having smaller volume embolized smaller diameter vessels in the
Perftoran pretreatment groups. A higher incidence of bubble
dislodgement and larger distal displacement occurred in these two
groups, with a 36% decrease in the time to bubble clearance and
restoration of blood flow. Intravascular emulsified fluorocarbon
administration before gas embolization affected initial bubble
conformation, increased bubble dislodgement, and resulted in bubble
displacement further into the periphery of the microcirculation. These
dynamic events did not occur if embolization preceded fluorocarbon administration.
fluorocarbon; bubble; cremaster; microcirculation; reperfusion
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INTRODUCTION |
A BRIEF REVIEW OF
RECENT LITERATURE demonstrates that gas embolism is known to
occur in cardiac surgery, both on-pump from bypass circuit sources
(8) and off-pump (2). It happens in endoscopy
(1), laparoscopic surgery (16), tissue biopsy (21), neurosurgery (30), liver
transplantation (29), during central venous line insertion
and removal (12), in orthopedic surgery (7),
with laser surgery (20), during neuroangiography and
cardiac catheterization procedures (26, 34), in cardiac ablation procedures (17), and in arthroscopy
(14). It has been reported during cardiopulmonary
resuscitation (15), with positive-pressure ventilation
(19), during intravenous antibiotic delivery at home
(24), with the use of ultrasound bubble contrast media
(18), and as a result of iatragenic embolization
(31). Furthermore, it occurs in both recreational and
commercial divers (27). Thus the exposure risk is large,
the true incidence is unknown, and effective treatment remains to be established.
Intravascular administration of emulsified fluorocarbon compounds is
one potential therapy. Fluorocarbon emulsions are stabilized with
surfactants, which have been shown to alter adhesion force at
gas-solid-liquid interfaces (9), promote bubble detachment from the wall in a flowing system (6), and decrease the
time to reperfusion of embolized microvessels (4).
Fluorocarbons are considered to be potential blood substitutes
because they have a high oxygen-carrying capacity compared with
plasma, and, if emulsified, they can be administered directly into the
bloodstream (10, 11). It has been hypothesized that
their presence in circulating blood may also increase the speed of
bubble reabsorption by increasing the solubility of gases in blood
(28).
We hypothesize that patterns of embolization (microbubble gas volume,
diameter of vessel embolized, numbers of bubbles trapped) and
subsequent dynamics (bubble dissolution, detachment and displacement further into the periphery, rate of gas reabsorption, time to reperfusion) can be modified by altering the mechanics at the air-blood
interface with an emulsified fluorocarbon compound. We anticipate that
use of an emulsified fluorocarbon will produce bubble conformations
that favor bubble clearance and speed restoration of blood flow,
minimizing embolism effects on endothelial-mediated responses. We
further hypothesize that timing and order of the administration of the
fluorocarbon relative to gas embolization are critical in generating
effective therapy. We tested these hypotheses in the rat cremaster
circulation pretreated and posttreated with either Perftoran, a
perfluorocarbon emulsion, or saline as control. We used intravital
microscopy to measure bubble dimensions and number after entrapment,
bubble conformational changes postembolization, and effects of
embolization and surfactant treatment on vessel reactivity.
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MATERIALS AND METHODS |
Surfactant characterization.
Perftoran (OJSC SPC Perftoran, Moscow, Russia) is a proprietary 10%
volume emulsion consisting of two perfluorocarbon compounds, Perfluorodecalin (C10F18, molecular mass = 462 Da) and
Perfluoromethylcyclohexylpiperiden (C12F23N, molecular mass = 595 Da). The solution is stabilized by a surface-active block
copolymer propylene oxide, Proxanol P-268, giving an average particle
size in the range of 0.03-0.15 µm. The solution has a pH in the
range of 7.2-7.8, osmolarity of 280-310 osmol/l, and an
oxygen-carrying capacity of 7.0% volume at 20°C. Perftoran is stored
frozen (between 
4 and 18°C) and can be thawed and refrozen up to
five times. For this study, a 200-ml vial was thawed and partitioned
into 3-ml sterile aliquots, which were immediately refrozen for later use.
A concentration of Perftoran was determined that would not
significantly alter the surface tension at the bubble-blood interface in vivo from the value expected in whole blood. The air-liquid surface
tension of serial dilutions of Perftoran in ultrapure water was
measured repeatedly (n = 6) at 37°C using the
Wilhelmy plate method with a KSV Sigma 703 surface
tensiometer. (4, 6, 9). The surface tension of a 5%
weight BSA solution was also measured repeatedly (n = 6) at 37°C over a range of Perftoran concentrations. BSA was used for
these measurements because protein concentration is a major determinant
of surface tension and eliminates interference from clot formation as
occurs with blood.
Animal experiments.
All experiments were performed by using adult male Wistar rats
(250-325 g). Animals were handled according to National Institutes of Health guidelines and approved by the University of Pennsylvania Animal Care and Use Committee. The intact cremaster surgical protocol and muscle preparation have been described in Branger and Eckmann (3, 4). One important feature of this preparation is the insertion and positioning of a femoral artery microcatheter placed so
that the ipsilateral cremaster microcirculatory bed blanches with
injection of saline. This preparation provides a reproducible method of
gas embolizing the cremaster arteriolar microcirculation.
Experimental protocol.
Anesthesia was induced (5%) and maintained (1.2%) with inhaled
halothane delivered in an air-O2 mixture (inspired
O2 fraction = 0.3). After induction, rats were laid
supine on a Plexiglas tray and intubated through a tracheostomy. The
animals were ventilated by using a positive-pressure, piston-driven
small-animal ventilator. Blood pressure and heart rate were monitored
with a right carotid artery catheter (PE-50). A PE-50 left jugular
venous catheter was placed for intravenous fluid and drug
administration. A PE-10 catheter was inserted into the left femoral
artery for injection of air bubbles directly into the cremaster
circulation. Body temperature was monitored with a rectal thermometer
and maintained at 37°C with a heating pad.
The cremaster muscle was prepared as previously described (3,
4). Briefly, the cremaster muscle was exposed and separated from
the surrounding tissue and organ through a midline scrotal incision and
then through the muscle itself. Loose connective tissue was dissected
away, and the muscle was spread over the transparent pedestal portion
of the Plexiglas tray. Several sutures were attached to keep the muscle
flat on the platform. The cremaster was superfused at 2 ml/min with a
warmed (34°C), gassed (95% N2-5% CO2) Krebs
buffer containing 132 mmol/l NaCl, 25 mmol/l NaHC3, 5 mmol/l KCl, 1.2 mmol/l MgCl2, and 2 mmol/l
CaCl2. The cremaster muscle was allowed to equilibrate for
30 min before any experimentation was started. The cremaster
temperature was monitored with an intramuscular thermocouple placed
away from possible areas of interest. Cremaster temperature was
maintained at 34-36°C by adjusting the temperature of the
superfusate. After surgery and equilibration, a series of clearly
visible consecutive branching arteriolar vessels was selected, and
other nearby arterial vessels were cauterized. This maintained a
physiological environment while creating a more controlled vascular
pathway in which the air embolism could be observed.
To demonstrate the preservation of robust vascular responses after
cremaster muscle preparation, a 0.5-ml bolus of 10
4 mol/l
ACh (Sigma Chemical, St. Louis, MO), diluted in Krebs buffer, was added
topically to the muscle to test for endothelial-mediated vasodilation.
A 0.5-ml bolus of 104 mol/l phenylephrine, (PE; Sigma
Chemicals), diluted in Krebs buffer and placed topically, was used to
confirm smooth-muscle-mediated constriction. At least 10 min passed
between the applications of each vasoactive agent. Tissue responses
were considered intact if the PE elicited at least a 20% decrease in
diameter and the ACh elicited at least a 50% increase in vessel
diameter from baseline. The preparation was not further studied in one
case because these criteria were not met. In the delayed embolization
group defined below, topical application of PE and ACh 10 min after
study compound delivery was used to demonstrate that vessels were
reactive with no change from the prebolus responses elicited.
Pancuronium bromide (1 mg/kg) was administered intravenously 10 min
after vasoreactivity was demonstrated to be intact.
Six groups of animals (n = 6 per group) were studied
for three treatment regimens with either study compound, Perftoran
administration, or saline administration as a control. The three
treatment regimens were immediate pretreatment, immediate
posttreatment, and delayed embolization after pretreatment. For
immediate pretreatment, animals received the treatment compound
(Perftoran or saline), and cremaster embolization followed within 10 min of completion of treatment compound delivery. For immediate
posttreatment, the cremaster muscle was embolized, and the treatment
compound was delivered beginning 2 min later. For delayed embolization,
animals received the treatment compound, and cremaster embolization
followed 2 h after completion of treatment compound delivery.
For treatment compound administration, animals were given an
intravenous bolus of either 0.9% NaCl or undiluted, freshly thawed (second thaw) Perftoran warmed to room temperature. The volume of
saline or undiluted Perftoran delivered was calculated to be 10% of
the individual animal's estimated blood volume (64 ml/kg for rats).
For Perftoran, this resulted in a 1% volume concentration in
circulating blood. The bolus was delivered by syringe pump over 5 min.
For gas embolization, single air bubbles were injected into the femoral
artery ipsilateral to the selected cremaster. Initially, a 3-µl
bubble was injected, and this volume was increased in 1-µl
increments, as necessary, in subsequent injections until a suitably
sized embolism arrived in the cremaster circulation. The maximum bubble
volume required for successful embolization was 5 µl. Once a bubble
of sufficient size embolized the cremaster, no additional experiments
were conducted in that animal. After complete bubble reabsorption or
transarteriolar passage, the tests of vascular reactivity were
conducted in the regions of the vessel in which bubbles had lodged.
Data analysis.
Data analysis was performed by using the videotaped recording of each
experiment with a calibrated video micrometer, as previously published
(3, 4). Two or more bubbles lodging at a given location
were considered to be a single embolism only if the bubbles touched
each other. The total embolism volume was calculated as the sum of the
individual bubble volumes in that case.
The length (L) and average diameter (D) of parent
bubbles were measured at the time of initial entrapment. Initial
embolism volume and aspect ratio (length/radius) were calculated by
using the measured dimensions, assuming that the elongated bubble is approximated by a cylinder with hemispherical end caps, as was done
previously (3, 4). Surface area at the moment of lodging was calculated as the sum of the end cap area
(
D2) and the area of cylindrical central
portion (DL) of each bubble. Predicted absorption times
(Tpredicted) for parent bubbles were computed
based on the initial volume and the aspect ratio measured using our
mathematical model for bubble absorption as described by Branger and
Eckmann (3) and subsequently used by the authors (4,
5). Actual elapsed time (Tobserved)
required for the last remaining observable remnant of parent bubbles to
reabsorb from the embolized vessel was determined from the
videomicroscopy recording. The percent change in observed (actual)
absorption time from the time predicted (
T%) was
calculated as
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(1)
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Bubble entrapment was followed by "stick-and-slip" behavior.
Stick-and-slip behavior generally refers to a lurching phenomenon occurring in the motion of large sliding interfaces, such as a creaky
door hinge, a bowed violin string, or a moving geological fault.
Stick-and-slip behavior is the result of the strong collective interfacial interactions of large numbers of surface molecules. An
increase in stick and slip occurs as the surface interactions become
weaker, indicating a transition between arrested motion and free motion
of the surfaces relative to each other (25). The magnitude
of stick and slip was quantified by measuring the number of episodes of
bubble lodging and subsequent bubble motion during the first 2 min
after initial entrapment and by measuring the distance a bubble
traveled during a slip episode. Only bubble motion lasting >1 s,
during which time the bubble moved at least one vessel diameter
downstream, was considered to be stick and slip. The distance traveled
was normalized to the local vessel diameter in each case for later
statistical analysis and for comparison to the normalized bubble
length, the bubble aspect ratio.
Statistical analysis.
The results from the groups are presented as arithmetical means ± SD. Variances of the groups were examined by using the
F-test. If the variances were equal, statistical
significance between groups was established by using ANOVA, with
P < 0.05 considered statistically significant by using
the Bonferroni correction. Nine separate comparisons were applied: the
six comparisons made between groups receiving the same compound at
different times relative to the embolization; and the three comparisons
made between groups for the same embolization timing but receiving
different compounds. Changes within the same group at different time
points were considered statistically significant for P < 0.05, calculated by using the paired Student's t-test.
The variances were not equal for analysis of the distance bubbles
traveled in stick-and-slip behavior. In this case, the
t-test for unequal variances was used to compare specific
group means.
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RESULTS |
Surface tension.
The surface tension of the 5% weight BSA solution was 50.6 ± 0.4 mN/m, compared with 50 mN/m for blood (33). The surface tension was 45.3 ± 0.5 mN/m for undiluted (10%) Perftoran,
50.4 ± 0.3 mN/m for 1% Perftoran diluted in ultrapure water, and
50.7 ± 0.8 mN/m for 1% Perftoran diluted in 5% weight BSA.
Hemodynamic stability and baseline vessel tone.
Heart rate and blood pressure data before, midway through, and
5 min after infusion of the study compound or control injectate are
presented in Table 1. There were no
significant changes within or between groups (P > 0.65). The administration of either Perftoran or saline had no
significant effect on arteriolar tone or reactivity. There was no
appreciable change in second- or third-order arteriole diameter
(<5.2% change in all cases, P > 0.79) 1 min after
bolus delivery compared with the prebolus diameter.
Initial bubble lodging and bubble dynamics.
Air emboli (range 4.8-9.9 nl) lodged in the cremaster
microvasculature within 5 s of injection. The number of injections
required to embolize successfully was the same in all groups, ranging
from one to three in all experiments. Either a single bubble or two adjacent bubbles (two cases) formed the embolism. There was a linear
correlation between embolism volume and vessel diameter, as shown in
Fig. 1. Data segregated into two
clusters. The Perftoran pretreatment and delayed embolization data
formed one cluster, and the remaining four groups formed the other.
Mean values and bidirectional error bars for each cluster are also
plotted. Bubbles in the two Perftoran pretreatment groups had smaller
volumes (P < 0.001) and lodged in smaller vessels
(P < 0.001) than in the other four experiments.
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Fig. 1.
Entrapping vessel diameter in relation to the gas
embolism bubble volume. Symbols signify the emboli groups of animals
receiving saline pretreatment ( ), saline posttreatment
( ), saline with delayed embolization
( ), Perftoran pretreatment ( ),
Perftoran posttreatment ( ), and Perftoran with delayed
embolization ( ). Linear regression of all data is
shown. Ensemble means with bidirectional SD error bars are shown for
clusters of Perftoran pretreatment and delayed embolization data
( ) and the other 4 groups ( ).
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The means bubble volumes, embolized vessel diameters, initial bubble
length, and initial aspect ratio are shown in Table
2 for each group. Values were not
different among the three saline treatment groups. Bubble length and
aspect ratio were not different among the Perftoran treatment groups.
Volumes were smaller in both the Perftoran pretreatment and delayed
embolization groups compared with their saline controls
(P < 0.0056). Bubble volume was also smaller after
Perftoran pretreatment compared with Perftoran posttreatment
(P < 0.0056). The mean diameter of emboli was smaller for both Perftoran pretreatment and delayed embolization compared with
saline controls (P < 0.0056). Bubble volume was also
smaller after Perftoran pretreatment compared with Perftoran delayed
embolization (P < 0.0056). The initial bubble aspect
ratio was larger for Perftoran pretreatment compared with saline
pretreatment (P < 0.0056).
Bubble surface area after initial lodging is illustrated in Fig.
2. There were no differences between
groups (P > 0.14 for all comparisons). End-cap surface
area was smaller for both the Perftoran pretreatment and delayed
embolization groups compared with their respective saline controls and
compared with Perftoran posttreatment (P < 0.0056 for
all comparisons) but not compared with each other (P > 0.37). Surface area of the cylindrical bubble portion was not different
between groups (P > 0.18 in all cases).
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Fig. 2.
Initial total bubble surface area comprising bubble
end-cap surface area (open bars) and bubble cylindrical core surface
area (solid bars).
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A small decrease in vessel diameter occurred in the first 5-8 s
after bubble entrapment in each experiment. Vessel constriction did not
exceed 6.5% in any experiment, with group means ranging from 2.7 ± 1.9% (saline-delayed embolization) to 3.9 ± 2.2% (Perftoran pretreatment). A slight increase in bubble length, ranging from 6.7 ± 6.1% (saline-delayed embolization) to 7.5 ± 5.8%
(Perftoran pretreatment) accompanied the decrease in bubble diameter.
The aspect ratio increased ~10-13% per group [range 9.9 ± 12.8% (saline-delayed embolization) to 13.1 ± 10.0%
(Perftoran pretreatment)]. These changes in bubble diameter, length,
and aspect ratio were not significant (P > 0.81 in all cases).
Minimal embolism breakup, or parent bubble dissolution into multiple
smaller bubbles, followed bubble lodging. In five cases (one saline
pretreatment, one saline-delayed embolization, two Perftoran
pretreatment, one Perftoran-delayed embolization), breakup resulted in
formation of two additional bubbles. Stick-and-slip events postlodging
were accentuated in the two Perftoran pretreatment and delayed
embolization groups. Bubbles in these groups moved appreciably further
distally in the microcirculation, whereas bubbles in the other four
groups essentially remained fixed once they lodged. Table
3 shows the average number of
stick-and-slip events occurring in the first 2 min after embolism
bubble lodging. Stick-and-slip events were rare in the saline-treated
animals and in the Perftoran-delayed embolization group. Only two or
three episodes occurred in these groups compared with 35 and 29 total episodes occurring after Perftoran pretreatment and delayed
embolization, respectively (P < 0.0056). In the saline
groups and the Perftoran posttreatment group, bubbles traveled
approximately one bubble length (with a bubble aspect ratio of
~40-50) per slip event (Fig. 3).
The normalized slip distance traveled was more than twice as far for
both the Perftoran pretreatment and delayed embolization groups by
comparison to their saline controls (P < 0.0167) and for Perftoran pretreatment compared with posttreatment
(P < 0.0046). Coupled with the greater absolute number
of slip events, bubbles in the Perftoran pretreatment and delayed
embolization groups dislodged and moved 10 times further into the
periphery of the microvasculature during the first 2 min. This
corresponds to a displacement of ~1.5 cm compared with only 1.5 mm
for the other four groups.
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Fig. 3.
Normalized bubble displacement distances traveled during
slip events (n). Statistical analysis showed
# P < 0.167 compared with saline
pretreatment; § P < 0.01 compared with
Perftoran posttreatment; and ¥ P < 0.0046 compared with saline-delayed embolization.
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Bubble reabsorption and reperfusion.
Predicted and observed reabsorption times for the six embolization
experiments are presented in Fig. 4. The
data fell into two distinct populations. One data cluster contains the
three saline groups and the Perftoran posttreatment group. The other data cluster contains the Perftoran pretreatment and delayed
embolization groups. Linear regression analysis was performed on each
cluster. The regression line was forced through the origin because
extremely small bubbles should reabsorb rapidly (3). The
regression line slope of 0.994 for the data cluster from the four
experimental groups is essentially the line of identity. The associated
large correlation coefficient (R = 0.933) indicates
that the observed reabsorption times measured collectively were closely
predicted individually. This is further supported by the separate
measure of the percent deviation between the two values calculated by Eq. 1 for each experiment. The values are presented in Table
3.
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Fig. 4.
Relationship between the actual elapsed time
(Tobserved) and the predicted time
(Tpredicted) required for the last remaining
observable remnant of parent bubbles to clear from the embolized
vessel. Linear regressions are forced through the origin. Symbols are
as defined in Fig. 1 legend.
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The slopes of the two regression lines in Fig. 4 were different
(P < 0.05), with the slope of the regression line for
the data cluster from the Perftoran pretreatment and delayed
embolization groups being 1.557 and R = 0.920. This
means that the observed reabsorption time in these two groups was
35.8% faster than predicted. This correlates well with the measures of
the percent deviation between the two values calculated by Eq. 1 for these two groups (Table 3). For Perftoran pretreatment,
reabsorption was significantly faster than was predicted, with the
measured value deviating from the predicted value by 38.3 ± 3.9%
(range 31.7-42.1%, P < 0.0056 compared with
saline pretreatment and Perftoran posttreatment). In the case of
Perftoran-delayed embolization, the measured reabsorption time was
faster than predicted by 33.8 ± 5.2% (range 26.3-39.2%, P < 0.0056 compared with saline-delayed embolization
and Perftoran posttreatment). An ensemble average for these two groups
gives a measured reabsorption time that is 36.0 ± 4.9% faster
than predicted, similar to the correlation analysis result.
The data in Fig. 4 have been recast in Fig.
5 to demonstrate the relationship between
initial bubble surface area and the observed clearance time, as was
done in Ref. 3. Linear regression analysis forced through
the origin is included for the two resultant data clusters as were
identified in Figs. 1 and 4. The two regression line slopes were
different (P < 0.05). The slope from the three saline
groups and the Perftoran posttreatment group was nearly double the
slope derived from the Perftoran pretreatment and delayed embolization
data. Thus for fixed bubble surface area, clearance took nearly twice
as long if Perftoran was not given before embolization.
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Fig. 5.
Relationship between initial bubble surface area and
Tobserved required for the last remaining
observable remnant of parent bubbles to clear from the embolized
vessel. Linear regressions are forced through the origin. Symbols are
as defined in Fig. 1 legend.
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DISCUSSION |
Gas embolization can obstruct blood flow, cause ischemia,
initiate thromboinflammatory events, and injure or denude the
endothelium, all of which can be extremely detrimental, particularly in
the cerebral circulation (13, 22). Fluorocarbon emulsions
have been thought to enhance the rate of bubble reabsorption by
increasing the solubility of gas in the fluorocarbon-laden blood. The
surfactants used to stabilize the emulsion have also been thought to
lower surface tension and thereby promote bubble entrapment in more distal regions of the vasculature. Determining the effects of timing of
delivery of such compounds in relation to the timing of embolization in
the treatment of gas embolism is also important. Neither the duration
of effect of any pretreatment nor the effectiveness of delivery of a
treatment compound to the site of embolization after bubble deposition
has occurred has been characterized. In these in vivo experiments, we
have used intravital microscopy to quantify the effects of timing of
delivery of a fluorocarbon emulsion, Perftoran, on air embolism bubble
deposition into, and clearance from, the arterial microcirculation.
Initial bubble lodging and bubble dynamics.
The use of Perftoran as a pretreatment was expected to promote bubble
deposition, with smaller bubbles lodging in more distal vessels, and to
accelerate reabsorption, whereas fluorocarbon emulsion instillation
after embolization was expected to have no effect (4, 23).
Indeed, treatment delivered postembolization was not different between
the Perftoran and saline control groups in any aspect of the study.
This most likely indicates that the treatment compound did not reach
the site of embolization. This would be expected, because the Perftoran
would not be convected into those regions of tissue in which blood flow
was obstructed. Neither did the emulsion appear to have diffused
through stagnant blood to achieve any appreciable concentration at the
gas-liquid interface or the sites of adhesion between the bubble and
the vessel wall. Thus there would be no measurable effect of Perftoran if it were administered after a gas embolism that had already occurred.
If, however, the emulsion were already circulating, it would have the
following effects: decreased volume but equal surface area of the
bubbles that lodged (Figs. 1 and 2, Table 2); decreased diameter of the
vessel that was embolized (Fig. 1, Table 2); shorter length of time
that they stayed lodged in one location (Fig. 3, Table 3);
greater distance that they moved each time they dislodged (Fig. 3,
Table 3); and shorter elapsed time until complete reperfusion had
occurred (Figs. 4 and 5, Table 3).
When gas embolization was preceded by Perftoran administration
immediately or 2 h before, the gas volume lodging was 25-35% less than if saline were administered or if Perftoran were given postembolization. Although this could in part be the result of increased gas solubility within the blood because of the nonzero fluorocrit, the very rapid transit time from bubble injection to
intravessel lodging is not consistent with the time that would be
required for gas transport out of the bubble and into the blood. Thus
it is more likely a primary effect of "snap off" of small bubbles from the injected bubble, as was predicted by Tsai and Miksis
(32) and seen in vivo by Branger and Eckmann
(4). In the presence of the circulating fluorocarbon
emulsion, which has some surfactant properties, it is possible that
smaller bubbles split free from the injected embolism and subsequently
enter the cremaster circulation.
The smaller bubbles in the Perftoran pretreatment and delayed
embolization groups also lodged further out into the periphery of the
microvasculature, resulting in bubble deformation, yielding a total
surface area profile that was not different from that determined for
the other groups. The fact that the surface area of the bubble end caps
was smaller for these two treatment groups is indicative of the smaller
vessel diameter, because the end-cap surface area depends only on
diameter. The end-cap surface area contributes only ~5% of the total
surface area, leaving the central cylindrical portion of the bubble to
contribute ~95% of the total surface area (Fig. 2). With equal
surface areas but smaller volumes, it is expected that bubbles in the
Perftoran pretreatment and delayed embolization groups should reabsorb
faster by diffusion of a smaller volume of gas across an equal surface
area, as we have found (Fig. 5). But this finding does not account for
the continued motion of bubbles further into the periphery of the vascular tree precipitated by preembolism administration of the study compound.
The finding that stick-and-slip events are enhanced in the present
study suggests that a smaller adhesion force develops between the
bubble surface and the vessel wall in the two groups receiving Perftoran before embolization. Although the exact nature of the adhesion force between the bubble surface and the vessel wall has not
been identified, there is likely to be some adhesion interaction between elements of the endothelial surface (e.g., the glycocalyx or
endothelial surface layer) and plasma-borne molecules that have
adsorbed to the bubble surface (e.g., proteins). The circulating emulsion can also adsorb to the bubble surface. The surface-active components can compete with plasma-borne molecules to occupy the bubble
interface, and this may lower the surface concentration of plasma-borne
molecules, which could potentially decrease adhesion to the endothelial
surface. Also, despite the fact that they are considered to be
biologically and chemically inert (nonreactive), the molecular
components of the emulsion may also have some interaction with the
endothelial surface that reduces its adhesive interaction with adsorbed
molecules on the bubble surface.
We limited our observation and analysis of continuing bubble detachment
and downstream displacement to the first 2 min after embolization
because this permitted comparison of events that were occurring in
similar-diameter vessels with bubbles of similar volumes. The greater
incidence of stick-and-slip events (Fig. 3, Table 3) and the larger
distance traveled (Fig. 3) in the two fluorocarbon-pretreated groups
suggest that the adhesion force between the bubble and the vessel wall
was smaller in these two groups. It is the force of adhesion made
between the bubble surface in contact with the vessel wall that retards
bubble motion. The pressure difference across the bubble multiplied by
the bubble's cross-sectional area perpendicular to the axis of the
vessel is the sole driving force for bubble displacement in the absence of blood flow. Systemic blood pressure did not change in the experiment (Table 1) and neither did vessel diameter after bubble lodging. Thus,
while the driving pressure and cross-sectional area remain constant, it
must be a reduction in the force of adhesion developed per unit surface
area, a decrease in the available bubble surface area exposed to the
vessel wall, or a combination of the two that leads to a net reduction
in the adhesion force. The pattern of bubble lodging after initial
embolization is probably mediated by both of these effects and can be
explained mechanistically. Bubbles having smaller volumes but equal
surface areas lodged after instillation of Perftoran (Figs. 2 and 5),
and these bubbles progressed initially into vessels having diameters
~25% smaller than those that were embolized in the other four groups
(Table 2). The aspect ratio (ratio of bubble length to radius) of
bubbles lodging in the two fluorocarbon-pretreated groups was longer
than in the other groups, indicating that bubbles had to deform to a
more slender and elongated shape, effectively increasing in surface
area before they lodged. As the bubble moves distally, the rapid
dilation of interfacial area during bubble deformation lowers the
surface concentration of any adsorbed species, thus increasing
intermolecular distances on the interface. This accentuates the effect
of having a smaller initial surface area, due to smaller initial volume
and spherical shape before lodging, available for adsorption.
Furthermore, as the bubble progresses into the periphery, both the
local blood pressure and the bubble cross-sectional area decrease,
diminishing the net driving force for bubble movement until arrest of
motion occurs. Such behavior could also result from effects on the
bubble surface due to competition between blood-borne molecules and
components of the emulsion for interfacial adsorption or from an
interaction between the fluorocarbon emulsion and the vessel wall that
lowers adhesiveness for the bubble. The net effect is to permit more
distal gas embolization.
Perftoran has a lower oxygen-carrying capacity than does arterial
blood. At a 10-fold dilution, as was used, there may still be a
sufficient decrease in local oxygen tension so that hypoxemia-induced tissue-mediated events contribute to earlier reperfusion. One possible
mechanism is hypoxia-mediated changes in endothelial surface layer
structure, leading to a less adhesive surface. It is not possible to
separate this effect from a molecular binding interaction, as described
above, but the stick-and-slip phenomena (Fig. 3, Table 3) do reflect a
decreased adhesiveness in the two groups receiving Perftoran before
embolization. Bubbles detached and moved many more times in those
groups. As the bubbles reabsorbed, the surface area in contact with the
vessel wall shrank until a critical surface area was reached, at which
point the bubble detached. This happened significantly more often in
these two groups, suggesting that the strength of adhesion was reduced
by the Perftoran. This stick-and-slip behavior induced by a circulating surface-active compound and influenced by the available interfacial surface area has also been observed in vitro by Cavanagh and Eckmann (6). The distance traveled with each dislodging episode
was also higher in those same two groups, indicating that the bubble surface and the vessel wall did not form sufficient adhesion
interactions until the bubble had displaced further downstream.
Bubble reabsorption and clearance.
Bubbles in the two groups receiving Perftoran before gas embolism not
only moved further out to the periphery, but they disappeared from the
microcirculation sooner with more rapid restoration of blood flow than
that which occurred in the other groups (Figs. 4 and 5). The influence
of the fluorocarbon on the rate of gas reabsorption was not assessed
independently but is not believed to have been the major reason for
this finding. The compound studied was selected in part because the
oxygen-carrying capacity of Perftoran is actually less than that of
whole blood. Thus it was not expected that a change in gas-carrying
capacity would be the major mechanism responsible for more rapid
reperfusion. Rather, it was by a combination of gas reabsorption and
bubble movement out to the periphery. As bubbles moved further into the
periphery, they elongated and became more slender, so that the surface
area available for gas transport out of the bubble increased, as seen
in Fig. 5. Bubbles in the Perftoran pretreatment and delayed
embolization group did not cease to stick and slip after 2 min and thus
continually displaced further into the periphery until small bubbles
disappeared from view or appeared to pass transcapillary and enter
directly into the venous circulation. As a result, blood flow was
reestablished in less than two-thirds of the time predicted, whereas in
the other four groups the actual restoration of blood flow occurred at
the time predicted (Figs. 4 and 5, Table 3).
After embolization, there were no differences between treatment groups
in the amount of vasoconstriction elicited or the degree of bubble
elongation that developed. This is hypothesized to be a
surface-tension-mediated effect (4). In these experiments, although the fluorocarbon emulsion does have the potential to lower
surface tension, it was dosed so as not to alter surface tension. We
found that increasing the fluorocarbon emulsion concentration reduced
surface tension of both ultrapure water and 5% weight BSA solution in
a concentration-dependent fashion in vitro. We used a Perftoran
concentration of 10% of the estimated rat blood volume as the target
dose in vivo. We assumed this concentration would not alter the
air-blood interfacial tension from its native value, based on the BSA
and water results. In addition, this dosing was well tolerated by the
animals, with no discernable effects on heart rate, blood pressure, or
microvascular tone (Table 1). Thus it is not surprising that the
vasoconstriction and bubble breakup reported previously with a
different surface-active compound (4) were not observed in
this study.
The net effect of the phenomena that we recorded and analyzed was that
bubbles traveled further into the periphery initially, continued to
move further distal in quantum events, and cleared from the circulation
faster than predicted only when Perftoran was given in advance of the
gas embolism. Although no measurements of tissue ischemia,
endothelial injury, inflammatory response, or thrombus formation
precipitated by gas embolism were performed in this study, the 36%
reduction in the duration of blood flow obstruction, coupled with the
continued distal movement of bubbles, could limit the degree of injury
developed. Targeting the mechanism of bubble adhesion to the
vasculature provides a potential pharmacological approach to
therapy for gas embolism.
| |
ACKNOWLEDGEMENTS |
The authors thank Lytal Kaufman-Grob and Stephen Armstead for
experimental assistance and Dr. Dmitri Guvakov for help with procurement of the Perftoran. Dr. E. Andrew Ochroch provided
statistical support.
| |
FOOTNOTES |
This study was supported by National Heart, Lung, and Blood Institute
Grant R01 HL-60230.
Address for reprint requests and other correspondence:
D. M. Eckmann, Dept. Of Anesthesia, Univ. Of Pennsylvania, 7 Dulles/HUP, 3400 Spruce St., Philadelphia, PA 19104 (E-mail:
eckmanndm{at}uphs.upenn.edu).
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. Section 1734 solely to indicate this fact.
First published November 15, 2002;10.1152/japplphysiol.00719.2002
Received 5 August 2002; accepted in final form 14 November 2002.
| |
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ABSTRACT
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