Full text of DOI:10.1007/s00421-001-0531-y

Eur J Appl Physiol 22002) 86: 209±214
DOI 10.1007/s00421-001-0531-y
ORIGINAL ARTICLE
Vibeke Nossum á Astrid Hjelde á Alf O . Brubakk
Small amounts of venous gas embolism cause delayed
impairment of endothelial function and increase
polymorphonuclear neutrophil in®ltration
Accepted: 14 September 2001 / Published online: 10 November 2001
Ó Springer-Verlag 2001
Abstract Gas bubbles from decompression and gas Eckenho€ et al. 1990). Venous gas bubbles may also
embolization lead to endothelial dysfunction and me- develop following laparoscopy, by accidental injection
chanical injury in the pig, rabbit and lamb. In the study or in cardiopulmonary bypass surgery 2Hoka et al. 1997;
presented here, 0.01 ml air/min/kg was infused through a Johnston et al. 1993; Webb et al. 1997), and they lead to
catheter intothe jugular vein in 12 rabbits for 60 min.
endothelial damage 2Nossum et al. 1999; Philp 1974;
The endothelial response was measured using tension Warren et al. 1973). As the number of gas bubbles
measurements in the blood vessel wall, and morpholog- increases, the likelihood of endothelium damage also
ical changes where quanti®ed using light microscopy and increases, and such damage seems tobe related tothe
image processing. Percent lung water content was cal- amount of gas present 2Nossum et al. 1999).
culated and used to estimate the severity of pulmonary
The amount of endothelial damage resulting from
oedema. The infusion led to a signi®cant decrease in the gaseous microemboli may be important because endo-
acetylcholine-mediated endothelial-dependent vasodila- thelial cells are the source of many vasoactive factors,
tation in the pulmonary artery 6 h after the infusion 26-h including nitric oxide 2NO). NO regulates the diameter
group, n=6). A decrease in substance-P-mediated en- of blood vessels and blood ¯ow and it is an important
dothelial-dependent vasodilatation was also detected. mediator of pulmonary vascular tone 2Busse et al. 1993;
No changes where seen in a group of rabbits examined Stewart and Ba€our 1990). After increased shear stress
1 h after infusion 21-h group, n=6). The impaired en- or binding of vasodilators, such as endothelium-depen-
dothelial-dependent vasodilatation caused by the bub- dent vasodilators, to surface receptors, vascular endo-
bles is probably biochemical in origin, since no visible thelial cells synthesize NO via activation of the enzyme
changes were seen in the endothelial layer. A signi®cant endothelial NO synthase 2eNOS; Mulsch et al. 1989;
increase in polymorphonuclear neutrophils was observed Rubanyi et al. 1986). NO then di€uses intothe under-
in the 6-h group compared to the 1-h group. This study lying vascular smooth muscle cells, activates soluble
demonstrates that small numbers of bubbles, corre- guanylate cyclase and initiates a cascade resulting in
sponding to``silent bubbles'', lead toan impairment of
the endothelial-dependent vasoactive response.
smooth muscle relaxation 2Fiscus 1988). If the endo-
thelial lining becomes disrupted or damaged by gas
emboli, endothelium-dependent vasodilatation could be
depressed. This may cause a reduction in regional per-
fusion 2Helps et al. 1990) and an exaggerated response to
vasoconstrictor agents 2Busse et al. 1993; Ku 1987;
Stewart and Ba€our 1990). A reduced production of
NO, a decrease in the density or number of receptors for
acetylcholine and substance P, or an altered function of
these receptors, can lead to loss of endothelium-depen-
dent pulmonary vasodilatation. Alternatively, there may
be an increased degradation of NO or inhibition of
eNOS activity 2Fineman et al. 1999).
Keywords Pulmonary artery á Neutrophils á
Endothelial cells á Vascular bubbles á Lung oedema
Introduction
Intravascular gas bubbles occur in the venous system
during most decompressions 2Brubakk et al. 1986;
V. Nossum 2&) á A. Hjelde á A.O. Brubakk
Department of Physiology and Biomedical Engineering,
Norwegian University of Science and Technology,
7489 Trondheim, Norway
E-mail: vibeke.nossum@medisin.ntnu.no
Tel.: +47-73550553
Fax: +47-73598613
To determine whether infusion of air through the
heart and into the pulmonary artery impairs the endo-
thelium-dependent regulation of pulmonary vascular
tone, we studied the response to endothelium-dependent
vasodilators using an in vitro method for isolated

210
Wet-dry weight of the lungs
vessels. We compared the changes in endothelial-de-
pendent vasodilatation to acetylcholine and substance P,
and endothelial-independent vasodilatation to sodium
nitroprusside after 60 min of air infusion in two groups
of animals, 1 and 6 h after infusion. The endothelial
layer was examined using light microscopy to evaluate
possible mechanical damage to the endothelial lining.
The dry weight of the lung tissue was determined from a less than
1-g section of the left lung. The tissue was weighed 2wet weight),
incubated at 120°C for 7 days, and then weighed again 2dry
weight). Percent lung water content [2wet weight ± dry weight)/wet
weight´100] was used to estimate the severity of pulmonary
oedema.
Lung histology
Methods
From all rabbits selected samples from the right and left upper and
lower lung were ®xed in a solution consisting of 70% ethanol, 4%
formaldehyde and 5% acetic acid. Four samples per animal were
taken. On the next day the specimens were transferred to80%
ethanol, before dehydration and embedding in paran for histo-
pathology. Sections were cut at 5 lm and stained with haemat-
oxylin-eosin-safran. An investigator who was blinded to treatment
estimated the accumulation of polymorphonuclear neutrophils
2PMNs) present in the tissue. Four ®elds from each lung section
were examined at ´400 with the aid of a Nikon YS2-H light mi-
croscope. This microscope was equipped with an eyepiece con-
taining a 10´10 graticule grid 20.5´0.5 cm). The number of grid
points falling on the tissue determined the ®eld area. By counting
the total number of PMNs in that ®eld divided by the number of
lung tissue grid points, the number of PMNs per unit lung tissue
was calculated. Each rabbit was represented by a mean value of
eight data points from both the left and right lung. The results are
expressed as mean number of PMNs per unit lung tissue, and one
unit lung tissue=0.25 mm2.
Twelve locally bred New Zealand Black rabbits of both genders,
weighing 2.8±3.4 kg, were used. Nosigns of illness were detected in
these animals during the study period. The experiments were per-
formed in accordance with the Principles of laboratory animal care
2NIH Publication NO 85±23 revised 1985). The experimental pro-
tocol was approved by the Norwegian Committee for Animal
Experiments.
Anaesthesia
The rabbits were tranquillized with an intramuscular injection of
midazolam 25 mg; Dormicum, Ho€mann-La Roche, Basel,
Switzerland) and ¯uanisone 27 mg) + fentanyl 20.22 mg; Hypnorm,
Janssen-Cilag, Saunderton, Buckinhamshire, UK). Within 60 min
the rabbits received an additional intramuscular injection of half of
the above dose of both midazolam and fentanyl/¯uanisone. Thir-
ty minutes before the observation period, the rabbits were given an
intramuscular injection of Buprenor®n 20.02±0.05 mg/kg; Temgesic,
Reckitt and Colman). Body temperature was measured with a
rectal probe and kept at 39.0 20.5)°C by means of a heating blanket.
The animals were allowed to breathe spontaneously throughout the
experiment.
Tension measurements of isolated vessels
A modi®ed tissue-bath technique 2Edvinsson et al. 1974; Hogestatt
et al. 1983) was used, as described previously by 2Nossum et al.
1999, 2000). The pulmonary artery was carefully dissected from the
right lung with the aid of a dissection microscope. The vessels were
cut intocylindrical segments with length ranging from 1.0±1.5 mm
and with a diameter between 1 and 2 mm. Each cylindrical segment
was mounted on two parallel L-shaped metal prongs and immersed
in temperature-controlled 237°C) tissue baths containing a sodium-
Krebs bu€er of the following composition: 119 mM NaCl, 10 mM
NaHCO3, 1.2 mM MgCl2, 4.6 mM KCl, 1 mM NaH2PO4, 1.5 mM
CaCl2, and 11 mM glucose. Air comprising 5% CO2 in O2 was
bubbled continuously through the sodium-Krebs bu€er to keep it
at pH 7.4. The contractile capacity of each vessel segment was
examined by exposure to a potassium-rich 260 mM) Krebs bu€er
solution. The vessels were pre-contracted with cumulative doses of
noradrenaline and the relaxation response was tested with cumu-
Infusion
A catheter 20.36 mm inner diameter) was placed intothe jugular
vein and moved centrally. Air was infused for 60 min by a syringe
in a special build pump. The amount of air infused was 0.01 ml/kg/
min, corresponding to 8±10 bubbles/min. Blood-gases 2oxygen and
carbon dioxide tension, PO2 and PCO2, respectively) and pH were
monitored before, during and 1 and 6 h after air infusion 21-h
group and 6-h group, respectively).
Bubble detection
Gas bubbles were detected in the heart using a 5-MHz transducer
connected to an ultrasonic scanner 2750 Vingmed, Horten, Nor-
way). The number of gas bubbles was evaluated using a grading
system from 0 to 5 2Eftedal et al. 1994): grade 0 is no bubbles, 1
represents an occasional bubble, 2 represents at least one bubble
every fourth heart cycle, 3 is at last one bubble every heart cycle, 4
is continuous bubbling, and 5 is massive bubbling. This scoring
system is approximately exponential compared with the number of
bubbles in the right ventricle 2Eftedal et al. 1994). The grades ob-
served were converted to bubbles/cm2 using the conversion table
given by Eftedal et al. 21998).
lative doses of acetylcholine 210±9±10±4 M) and substance P 210±12
±
10±7 M). The response depended upon how much of the endothelial
layer was damaged by the bubbles. The maximum relaxation re-
sponse 2Imax) was de®ned as the maximal dilatory response re-
gardless of the concentration induced by an agonist, and is
expressed as a percentage of the pre-contraction induced by a pre-
contracting agent. The performance of the vascular smooth muscle
cells was evaluated with cumulative doses of sodium nitroprusside
210±9±10±5 M). In addition, dose-response curves for all agonists
were calculated.
Observation period
Silver nitrate staining
The rabbits were divided into two groups of six animals, the 1-h The segments were cut open in strip form and mounted carefully
group was observed for 1 h after air infusion, and the 6-h group with needles on a Para®lm-covered cork plate with the vessel lu-
was observed for 6 h after air infusion. After the observation pe- men-side 2endothelial layer) up. The mounted segments were
riod, the animals were given a lethal intravenous dose of potassium stained with silver nitrate using a method described by 2Abrol et al.
chloride, under anaesthesia 2midazolam 5 mg and ¯uanisone 7 mg 1984). The stained segments were transferred toan object glass and
+ fentanyl 0.22 mg). The lungs were immediately harvested.
mounted using an aqueous mounting medium.

211
the 1-h and 6-h groups: 0.08 20.03) bubbles/cm2 21-h
Microscopy and photography
group) and 0.09 20.02) bubbles/cm2 26-h group). All
rabbits survived the infusion and observation period.
The measurement of blood-gases showed no change in
PCO2 for the animals during the air infusion compared
to that observed prior to and after infusion.
Each segment was examined by light microscopy 2Nikon Micro-
photo-FXA ¯uorescence microscopy) and photographed 2Nikon
FX-35DX ) at ´250 2Fuji®lm ISO 100). Since each photograph only
partly covered the segment, several photographs of each segment
were taken. In order to claim reproducible and reliable results, the
photographs were taken in the same manner for each segment
2three or ®ve pictures). The photographs were also taken in the
same pattern in order to be as representative as possible, usually
three pictures from each segment.
Relaxation response
The 6-h group showed a lower 2P=0.04) Imax response
[48.3 222.4)%] to acetylcholine compared to the 1-h
group [74.5 215.5)%; Table 1]. From the dose-response
curves, it appears that the dose-related relaxation re-
sponse was lower at 10±7 M for the 6-h group compared
tothe 1-h group 2 P=0.003; Fig. 1). Although the dif-
ference was not signi®cant, a lower response was seen for
all other concentrations of acetylcholine in the 6-h group
compared to the 1-h group.
There was a lower Imax in the 6-h group compared to
the 1-h group for substance P 2Table 1). However, the
di€erence between the 6-h group [42.9 216.8)%] and the
1-h group [60.4 210.)7%] was not statistically signi®cant.
Dose-response curves showed lower relaxation responses
for the 6-h group compared to the 1-h group at two
Quanti®cation of endothelial damage
Endothelial damage was evaluated using an image-processing
program 2Adobe Photoshop 5.0). All photographs from each seg-
ment were scanned into the computer. The area containing dam-
aged endothelial layer was coloured using the paint bucket function
to increase the contrast and to simplify the quanti®cation. The level
of damage was calculated from the pixel dimensions of the marked
and stained area 2re¯ecting the endothelial rupture) and compared
to the pixel dimension of the whole picture 2expressed as a per-
centage). This procedure was followed for all of the photographs
from each segment. The mean value for each animal was calculated
from the mean value of every segment. The value is a result of at
least 12 photographs and represents the ®nal percentage of endo-
thelial damage for this vessel.
Drugs
2‹)Noradrenaline[+]-hydrogen-tartrate, substance P, acetylcho-
line and sodium nitroprusside-dihydrate 2all Sigma) were dissolved
in saline or small amounts of distilled water. All concentrations
given are the ®nal molar concentration in the tissue bath during the
experiments.
Statistics
Data were subjected toanalysis using the Mann-Whitney U and
Wilcoxon signed-rank tests for unpaired and paired data, as ap-
propriate. The level of statistical signi®cance was set at P<0.05.
The results are expressed as mean 2SD).
Results
Pulmonary artery bubbles
Fig. 1 Dose-response curves for the responses to acetylcholine
2ACh, solid lines), substance P 2SP, dotted lines) and sodium
nitroprusside 2SNP, dashed lines) in the animals observed 1 h after
the infusion of air into the jugular vein 2the 1-h group, n=6) and in
those observed 6 h after the infusion 2the 6-h group, n=6)
Eight to10 bubbles/min were infused intothe superior
caval vein and corresponded to grade 1±2 when detected
with a ultrasonic scanner in the pulmonary artery. There
was nosigni®cant di€erence in this parameter between
Table 1 Comparison of values for animals observed 1 h after the prusside, wet weight, polymorphonuclear leucocyte 2PMN) in®l-
infusion of air into the jugular vein 21-h group) and for those ob- tration and mechanical damage for the 1-h and 6-h groups. Values
served 6 h after the infusion 26-h group). Maximum %-relaxation are presented as the mean 2SD) in each group
values 2Imax) for acetylcholine, substance P and sodium nitro-
Experimental group Imax 2%)
Acetylcholine
Wet weight 2%) PMN/unit lung
tissue
Mechanical
damage 2%)
Substance P
Sodium nitroprusside
1-h group 2n=6)
6-h group 2n=6)
74.5 215.5)
48.3 222.4)*
60.4 210.7)
42.9 216.8)
90.7 25.9)
83.9 29.5)
80.55 22.65)
79.87 21.00)
0.0597
0.1202**
1.9 21.2)
1.6 20.5)
*P=0.04
**P=0.0004

212
concentrations 2P=0.003; Fig. 1). Lower responses were between the 1-h and 6-h groups. There were no signs of
seen for every concentration in the 6-h group compared mechanical damage in the pulmonary endothelial layer,
tothe 1-h group. as assessed by light microscopy, indicating a biochemical
Application of the endothelial-independent agonist, disruption to the endothelial layer.
sodium nitroprusside resulted in no signi®cant di€er-
The change in the vasoactive response occurred 6 h
ences in Imax between the two groups. The dose-response after the infusion, while the response seems to have been
curves did not show any di€erences between the two una€ected after 1 h. The loss of endothelium-dependent
groups 2Fig. 1).
vasoactivity was reduced for the Imax toacetylcholine,
while the response to substance P was reduced at some
concentrations 2dose-response). The endothelium-inde-
pendent response to sodium nitroprusside seems to have
been una€ected by air bubbles in both groups, and
Wet weight and PMN in®ltration
The pulmonary water content was 80.6 22.7)% in the 1-h con®rms that the change in vasoactive response is only
group and 79.9 21.0)% in the 6-h group 2Table 1). The related to the endothelial function and not to function in
di€erence was not signi®cant. the vascular smooth muscle layer. Albertine et al. 21984)
The results of the histological examination 2left and and Berner et al. 21989) showed that with air emboli-
right lung) from the 1-h and the 6-h group are given in zation, the pulmonary vascular endothelium is the site of
Fig. 2. Nosigni®cant di€erence was found between the injury.
left and right lung, for both the 1-h and the 6-h group. The water content of the lungs was not di€erence
However, an increase in PMNs was observed in the 6-h between the groups, while there was a greater in®ltration
group compared to the 1-h group 2P=0.004; Fig. 2).
of leucocytes in the animals that survived for 6 h after
the infusion of air. Hjelde et al. 21999) found a con-
nection between the duration of observation and PMN
accumulation in animals exposed to many bubbles.
Thus, it seems that PMN in®ltration increases with time.
Mechanical damage
Evaluation of the endothelial layer by light microscopy The number of PMNs observed after 1 h was similar to
did not reveal any mechanical damage for the two the number of PMNs seen in control animals observed
groups 2Fig. 3). The percent damage for the 1-h group for 2 h without exposure to bubbles 2Hjelde et al. 1999).
was 1.9 21.2)%, and did not di€er signi®cantly from that From the light microscope analysis, it appears that in
of the 6-h group [1.6 20.5)% mechanical damage; the present study the infusion of air did not result in
Table 1].
damage to the endothelial layer. However, following
exposure to more bubbles 2Nossum et al. 1999, 2000),
mechanical injury to the endothelium does occur, in
addition to a decreased response to endothelium-
dependent vasodilators. Yet any mechanical damage
would probably have an acute e€ect and would also be
observed in the 1-h group.
The dose-response curves obtained for both acetyl-
choline and substance P revealed a large di€erence be-
tween the 6-h group and the 1-h group. Normally,
Discussion
The results of this study show that the endothelium-
dependent response to vasoactive substances in the
pulmonary artery would change 6 h after the infusion of
small amounts of air bubbles. Furthermore, there was an
increase in PMN in®ltration at that time. However, no
di€erence in oedema formation 2wet weight) was found
Fig. 2 Number of polymorphonuclear leucocytes 2PMN) per unit
of lung tissue for the left 2open bars) and right lungs 2solid bars) Fig. 3 Photomicrograph of an intact endothelial layer from an
from animals in the 1-h 2n=6) and 6-h 2n=6) groups. Values are animal in the 6-h group that exhibited a decreased relaxation
mean ‹SD
response 2magni®cation ´ 250)

213
PMNs circulate within the vasculature as unstimulated that small numbers of gas bubbles would a€ect the en-
cells and donodamage tothe vascular endothelium.
dothelium and lead to increased PMN in®ltration in the
However, these cells can become activated and dra- lungs. Contrary to ®ndings by 2Nossum et al. 1999,
matically increase oxygen uptake, resulting in the pro- 2000), the small number of gas bubbles did not lead to
duction of oxygen metabolites, lysosomal enzyme release mechanical disruptions in the endothelial layer, as eval-
and subsequent endothelial damage 2Fantone and Ward uated by light microscopy, suggesting that the changes in
1982; Roberts 1988). The surface of the bubbles acts as a endothelial function is biochemical in origin.
foreign substance and is capable of activating the alter-
Acknowledgements This work was supported by the Norwegian
Petroleum Directorate, Norsk Hydro, Esso Norge and Statoil
under the ``Dive contingency contract'' 2No 4600002328) with
Norwegian Underwater Intervention 2NUI). The help of Anne-Lise
Ustad, Arn®nn Sira and Tove Svartkjùnnli is gratefully acknowl-
edged.
native complement pathway in vitro 2Hjelde et al. 1995;
Ward et al. 1986, 1987). During activation of the com-
plement pathway, three anaphylactic peptides are re-
leased intothe ¯uid phase, with C5a being the most
important. Intravascular complement activation leads to
acute lung injury, and PMNs play a key role in this
development 2Czermak et al. 1998; Till et al. 1982).
Complement-activated PMNs, when in close contact
with lung vascular endothelium, may release toxic oxy-
gen metabolites that can destroy the endothelium 2Sacks
et al. 1978; Tofukuji et al. 1998). Gaseous microemboli
can cause direct vascular injury as a result of transient
capillary obstruction 2Feinstein et al. 1984).
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