Stop-flow studies of distribution of filtration in rat lungs
W. LIN, E. JACOBS, R. M. SCHAPIRA, K. PRESBERG, AND R. M. EFFROS
Division of Pulmonary and Critical Care Medicine, Department of Medicine,
Medical College of Wisconsin, Milwaukee 53226; and Zablocki Veterans
Affairs Medical Center, Milwaukee, Wisconsin 53295-1000
pulmonary edema; pulmonary vasculature; reflection coefficients; endothelial uptake
INCREASES IN LEFT ATRIAL PRESSURES cause edema accumulation in perivascular and peribronchial tissues of
the lungs (13, 10). Much less fluid appears in the
tissues surrounding the capillaries, and it is not known
whether leakage occurs from alveolar or extra-alveolar
vessels. Weibel and Bachofen (14) suggested that fluid
is lost from the capillary surfaces and then makes its
way along interstitial fibers to lymphatics, which terminate at the respiratory bronchioles. However, leakage
through other vessels is also possible. In the early
studies of Iliff (8), increased alveolar pressures were
associated with leakage from extra-alveolar vessels.
Subsequent studies in which air space pressures were
increased or the arteries or veins were embolized with
polystyrene beads suggested that extra-alveolar filtration is about the same as alveolar filtration (1). Interpretation of these experiments is complicated by the
possibility that high airway pressures or embolism
might alter the permeability of the extra-alveolar vessels.
Filtration in individual venules on the surface of
isolated lungs is significantly greater than that from
arterioles, and the filtration coefficients of the extraalveolar vessels greatly exceed that of the capillaries
(2). Freeze-fracture studies of Schneeberger and Karnovsky (12) indicate that there are discontinuities in the
sealing strands that join adjacent cells at the venous
end of the capillaries, suggesting that increases in
http://www.jap.org
hydrostatic pressure might cause greater filtration
through these regions than through the capillaries. It is
also possible that leakage of fluid may occur through
bronchial vessels.
To obtain information concerning the sites of edema
formation in the pulmonary vasculature, a series of
stop-flow studies was conducted. These experiments
provided information regarding the relative distribution of filtration and diffusion along the pulmonary
vasculature.
METHODS
Twenty-six Sprague-Dawley rats [300 6 25 (SD) g] were
anesthetized with an intraperitoneal injection of 0.7 ml of a
64.8-mg/ml pentobarbital sodium solution. The chest was
opened, and catheters were placed in the trachea, pulmonary
artery, and left atrium. Blood was flushed from the vasculature at 37°C with 5 ml of perfusion fluid that contained (in
mM) 25 NaHCO3, 125 NaCl, 4 KCl, 2.5 CaCl2, and 0.8 MgSO4,
as well as 150 mg/dl glucose, 10 mg/dl urea, and 5 g/dl bovine
serum albumin (Cohn Fraction V, 98–99% albumin, Sigma
Chemical, St. Louis, MO) adjusted to pH 7.4 with 1 N NaOH
when exposed to 5% CO2. The perfusate also contained 2,000
mg/l fluorescein isothiocyanate (FITC)-labeled dextran, with
an average molecular weight (mol wt) of 2,000,000 (Sigma). In
some experiments the albumin was labeled with Evans blue
(100 mg/l, Aldrich, Milwaukee, WI). The lungs were kept
inflated with a 5% CO2-95% O2 mixture at a constant
pressure of 5 cmH2O. Pulmonary vascular pressures were
measured just proximal to the pulmonary artery catheter and
distal to the left atrial catheter.
The apparatus used in these experiments is shown in Fig.
1. The valves were used to 1) begin perfusion of the lungs, 2)
stop flow and expose the left atrium to a pressure of 20
cmH2O, and 3) flush the pulmonary vasculature after the
stop-flow period in an anterograde or retrograde direction.
The dead space volume of the tubing between the left atrium
and the collection site was 1.5 ml. Temperatures were measured with a thermocouple inserted into the arterial line just
proximal to the point at which the catheter entered the
pulmonary artery.
Perfusate was initially recirculated through the lungs at 6
ml/min for 10 min from a reservoir containing 100 ml of
perfusate at 37°C. After this initial period of recirculation, the
lungs were perfused without recirculation at 6 ml/min for 1
min (see Fig. 1) from the pulmonary artery to left atrium, and
baseline samples were collected. Left atrial pressures were
kept at zero during these intervals. Perfusion was then
stopped for 10 min, during which time the left atrial outflow
was connected to an upper reservoir in which the top of the
fluid column was kept 20 cm above the lungs. At the end of the
10-min period, 0.5 ml of perfusate containing 9 µCi of 3HOH
(DuPont, Boston, MA) was instilled into the air spaces. Then,
the perfusate in the lungs was flushed with either anterograde (pulmonary artery to left atrium) or retrograde (left
atrium to pulmonary artery) perfusion at 3 ml/min for 4 min.
Outflow samples were collected by hand at 10-s intervals, and
0161-7567/98 $5.00 Copyright r 1998 the American Physiological Society
47
Downloaded from http://jap.physiology.org/ by 10.220.33.5 on June 18, 2017
Lin, W., E. Jacobs, R. M. Schapira, K. Presberg, and
R. M. Effros. Stop-flow studies of distribution of filtration in
rat lungs. J. Appl. Physiol. 84(1): 47–52, 1998.—The stopflow approach was used to investigate where filtration occurs
in the pulmonary vasculature after elevation of left atrial
pressure and aspiration of HCl. Rat lungs were perfused for
11 min at zero left atrial pressures, and then flow was stopped
for 10 min and left atrial pressures were increased to 20
cmH2O. Thereafter, 3HOH was instilled into the air spaces,
and the pulmonary vasculature was flushed by perfusing it
from the pulmonary artery to left atrium (anterograde flush)
or in the opposite direction (retrograde flush). Increases in
fluorescein isothiocyanate (FITC)-dextran (molecular weight
2,000,000) indicated filtration, and these preceded increases
in 3HOH after anterograde but not retrograde flushes. This
suggests that some filtration occurred through vessels that
were relatively venous compared with those through which
3HOH exchange had occurred. Filtration increased fivefold
after instillation of 0.1 N HCl in isotonic saline into the air
spaces before perfusion. Increases in Evans blue-labeled
albumin concentrations were ,40% those of FITC-dextran,
indicating loss from the vasculature, but increases in unlabeled albumin and FITC-albumin were comparable.
48
STOP-FLOW STUDIES OF PULMONARY FILTRATION
Fig. 1. Stop-flow apparatus. Lungs were initially
perfused for 10 min with recirculation (not shown).
Thereafter, lungs were perfused for 1 min without recirculation, and samples were collected
from outflow (initial perfusion). Flow was then
stopped, and lungs were exposed to 20-cmH2O
pressure for 10 min (stop-flow). Flow was resumed from pulmonary artery (pul art) to left
atrium (atr; anterograde flush) or from left atrium
to pulmonary artery (retrograde flush), and
samples were again collected. Perfusate and lungs
were kept warm with fluid recirculated from a
water bath around lower reservoir, tubing, and
lungs. Fluid was placed in upper reservoir just
before stop-flow period to ensure temperature
was also kept close to 37°C.
Table 1. Experimental protocols
Group
n
Perfusate Indicators, Intervention
Direction of Flush
1
2
3
4
6
6
5
6
FITC-dextran, Evans blue
FITC-dextran, Evans blue
FITC-dextran, Evans blue, HCl
FITC-dextran
Anterograde
Retrograde
Anterograde
Anterograde
n, No. of animals; FITC, fluorescein isothiocyanate.
Concentrations of protein (only albumin was present in the
perfusate) were measured by the Bradford technique (3) in
five experiments that did not include Evans blue.
Concentrations of dye and unlabeled albumin in samples
flushed from the lungs after the stop-flow period were divided
by the concentrations in the control outflow, and these were
plotted against the volume of fluid recovered from the left
atrium. The volume (DV) of fluid not containing FITC-dextran
that was lost from the ith sample of perfusate was calculated
with use of the equation
DV 5 Vin 2 Vout 5
[dex]in 2 [dex]out
[dex]in
Vout
(1)
where [dex]in designates the concentration of FITC-dextran
in the perfusate fluid entering the lungs, [dex]out refers to the
concentration of FITC-dextran in the ith sample of the
perfusate flushed out of the lung after the stop-flow period,
Vout is the volume of fluid in this collection sample, and Vin is
the volume of fluid that would have been in the sample had
none been lost by filtration from the perfusate.
The mean outflow volumes (V) of the increases in FITCdextran and 3HOH above baseline were calculated from the
equation
n
o (c 2 c )V
i
V5
0
i51
n
o (c 2 c )
i
i
2 VD
(2)
0
i51
where ci is the concentration of FITC-dextran or 3HOH in the
ith sample, c0 is the concentration of the indicator in the
baseline period just before the stop-flow interval (this is zero
for 3HOH), Vi is the volume of the ith sample, and VD is the
dead space of the tubing.
Mean values of Vdex/V3HOH for each of the sets of experiments were compared by a one-way analysis of variance and a
Neuman-Keuls test of differences between individual mean
values (SigmaStat I, Jandel, San Rafael, CA).
Downloaded from http://jap.physiology.org/ by 10.220.33.5 on June 18, 2017
the volume of fluid collected in these samples was determined
from the weight of the collection cups. The volume of fluid
remaining in the upper reservoir was continuously monitored
from the weight of a hollow 10-cm-long, 0.5-cm-diameter
glass cylinder that was suspended in the reservoir from a
strain gauge.
Four sets of experiments were conducted (see Table 1). In
group 1, the fluid remaining within the lungs was flushed
from the vasculature after the stop-flow period from the
pulmonary artery to the left atrium (anterograde flush). In
group 2, the fluid was flushed from the left atrium to the
pulmonary artery (retrograde flush). In group 3, 1.0 ml of 0.1
N HCl in 154 mM NaCl was instilled into the air spaces before
the lung was perfused, and the lung was flushed in an
anterograde direction. Three control experiments (not shown
in Table 1) were also conducted in which 1.0 ml of 154 mM
NaCl was instilled into the lungs without HCl. In group 4,
Evans blue was not incorporated in the perfusate and the
lungs were perfused in an anterograde direction.
Samples were diluted 1:9 and centrifuged at 13,000 g for 10
s in a Fisher Scientific Marathon Centrifuge-13K (Pittsburgh,
PA), and optical densities were measured at 495 nm (FITC)
and 620 nm (Evans blue) in a Milton Roy 1001 Plus spectrophotometer (St. Louis, MO). In addition, 0.1-ml aliquots from
the collection vials were added to 3 ml of Biosafe II (Research
Products International, Mount Prospect, IL). Radioactivity of
the sample was determined with an automated beta scintillation counter and corrected for background counts. Radioactivity of the samples was divided by that in the solution instilled
into the lungs and plotted separately against the volume of
fluid recovered from the left atrium.
STOP-FLOW STUDIES OF PULMONARY FILTRATION
49
To test binding of Evans blue to albumin, samples of the
original perfusate containing Evans blue were filtered by
centrifugation at 1,000 g at room temperature through a
30,000-mol-wt-cutoff polysulfonated filter (Microcon, Beverly,
MA). No blue dye was detected in the filtrate. As a further test
of binding, 1 ml of perfusate without dye was placed in a
10,000-mol-wt-cutoff dialysis chamber (Slide-A-Lyzer dialysis
cassette, Pierce, Rockford, IL) and immersed in a flask
containing 200 ml of the perfusate with Evans blue that was
slowly stirred overnight at room temperature. None of the
Evans blue dye was found in the dialysis chamber. Samples of
the perfusate containing FITC-dextran (mol wt 2,000,000)
were also filtered by centrifugation at 1,000 g at room
temperature through a 100,000-mol-wt-cutoff polysulfonated
filter (Microcon). None of the FITC-dextran was found in the
filtrate.
RESULTS
Fig. 3. Group 2 experiments. After stop-flow, pulmonary vasculature
was flushed from left atrium to pulmonary artery (retrograde flush).
Increases in concentrations of FITC-dextran and Evans-blue albumin occur at about same time as those in 3HOH. This suggests that
relatively little filtration occurred from vessels that are more arterial
than from sites of 3HOH exchange from air spaces.
Fig. 2. Group 1 experiments. After stop-flow interval, lung was
flushed from artery to vein (anterograde flush). Note that increases in
outflow concentrations (Conc) of both fluorescein isothiocyanate
(FITC)-dextran and Evans blue-albumin precede those in 3HOH.
This is consistent with filtration from vessels that are more venous
than those that were responsible for 3HOH exchange. Increases in
Evans blue-albumin concentrations are less than one-half of those in
FITC-dextran, indicating losses of Evans-blue albumin from pulmonary vasculature during stop-flow period. Dotted line here and in
Figs. 3 and 4 indicates the position of 3HOH curve shown below.
Maximal values of this curve were set to those of Dextran to facilitate
comparison.
concentrations of FITC-dextran were attributed to transudation of fluid that contained relatively low concentrations of FITC-dextran from the vasculature during the
stop-flow period. If it is assumed that none of the
FITC-dextran is lost from the capillaries when edema
formation is occurring, then the amount of fluid that
entered the interstitium of the lungs during the stopflow period averaged 0.22 6 0.02 (SE) ml in the
anterograde experiments and 0.19 6 0.04 ml in the
retrograde experiments.
Instillation of 0.1 M HCl into the lungs before the
stop-flow period significantly increased the rise in
FITC-dextran concentrations (compare Figs. 2 and 4,
noting difference in ordinate scaling). This corresponds
to a loss of 1.01 6 0.31 ml (P , 0.05) from the
vasculature to the interstitium.
Increases in concentrations of Evans blue-albumin
were considerably less than those of FITC-dextran
(Figs. 2–4), suggesting more of the labeled albumin was
lost from the vasculature than was FITC-dextran.
Increases in Evans blue-albumin averaged only 38 6
8% of those of FITC-dextran in the anterograde and
retrograde experiments (no differences were noted in
this regard between the anterograde and retrograde
experiments). After instillation of HCl into the lungs,
increases in Evans blue-albumin fell to 5.4 6 3.0% of
Downloaded from http://jap.physiology.org/ by 10.220.33.5 on June 18, 2017
Elevation of left atrial pressure to 20 cmH2O during
the stop-flow period resulted in increases in the concentrations of FITC-dextran, (mol wt 2,000,000) in the
perfusate flushed from the pulmonary vasculature,
regardless of whether an anterograde or retrograde
flush was used (Figs. 2 and 3). These increases in the
50
STOP-FLOW STUDIES OF PULMONARY FILTRATION
those in FITC-dextran, indicating a rise in the fraction
of Evans blue-albumin lost from the pulmonary vasculature. Instillation of 1 ml of isotonic saline without
HCl (in 3 experiments) did not cause any increase in
FITC-dextran concentrations and did not influence
increases in Evans blue-albumin or 3HOH concentrations.
No evidence was found for any detectable dissociation of Evans blue from albumin in the infusion solution by either ultrafiltration or dialysis (see METHODS ).
It is possible that dissociation of Evans blue from
albumin was due to metabolism or uptake in the
pulmonary vasculature during the stop-flow period.
However, increases in unlabeled albumin approximated those in FITC-dextran during the stop-flow
period (see Fig. 5), suggesting that there was no
metabolism or uptake of unlabeled albumin during this
interval.
When the fluid remaining in the pulmonary vasculature was flushed out of the lungs in an anterograde
fashion, increases in FITC-dextran concentrations occurred before those in 3HOH (Fig. 2), which had been
instilled into the air spaces [Vdex/V3HOH 5 0.56 6 0.04
(SE), n 5 6]. This observation is consistent with the
hypothesis that filtration had occurred at more-venous
sites than those through which the 3HOH had ex-
Fig. 5. Group 4 experiments. Evans blue was not incorporated in
perfusate of these experiments (anterograde flush). Increases in
unlabeled albumin were comparable to those in FITC-dextran,
suggesting little loss of unlabeled albumin.
Downloaded from http://jap.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 4. Group 3 experiments. Before perfusion was commenced, 1.0
ml of 0.1 N HCl was instilled into lungs (anterograde flush). Increases
in concentrations of FITC-dextran were ,5 times greater than in
control experiments (e.g., compare with Fig. 2, noting difference in
scale of ordinate). Increases in Evans-blue albumin were ,10% those
of FITC-dextran, indicating increased losses of Evans-blue from
vasculature.
changed. When the lungs were flushed in a retrograde
direction, increases in FITC-dextran and 3HOH were
observed in the outflow at about the same time
(Vdex/V3HOH 5 0.97 6 0.05, n 5 6) (Fig. 3), suggesting
that there had been relatively little ultrafiltration from
vessels that were more arterial than those through
which exchange of 3HOH had occurred. Differences in
Vdex/V3HOH between the retrograde and each of the
anterograde groups were significant (P , 0.05).
In the lungs that were flushed in an anterograde
fashion after exposure to 0.1 N HCl, increases in
FITC-dextran concentrations occurred before those in
3HOH (V
dex/V3HOH5 0.80 6 0.06, n 5 5). This ratio was
significantly more than that observed in the control
anterograde study, but it was still less than that
observed when retrograde flow was used to flush the
lungs (P , 0.05). It can therefore be concluded that
overall filtration was increased after exposure to HCl,
but the increase in prevenous filtration may have
exceeded that lost from the veins. Evidence for venous
filtration was also noted when an anterior flush was
used in the lungs that were perfused with a solution
that did not contain Evans blue (Vdex/V3HOH 5 0.76 6
0.02, n 5 6).
More fluid was lost from the upper reservoir during
the stop-flow period than could be accounted for by
increases in concentrations of FITC-dextran (see Fig.
6). An average of 4.2 6 0.1 ml was lost from the
reservoir in the seven anterograde and retrograde
STOP-FLOW STUDIES OF PULMONARY FILTRATION
experiments in which this parameter was measured, or
,20 times as much as that calculated from the FITCdextran data. Even more fluid (12.0 6 2.3 ml) was lost
from the pulmonary vasculature during the stop-flow
period after exposure of the air spaces to 0.1 N HCl in
isotonic saline.
DISCUSSION
Stop-flow experiments have been used for many
years to determine the sites of solute and water exchange in the nephron (9). In the latter experiments (9),
urine flow was arrested for a period of time and then
resumed, allowing serial samples to be analyzed to
determine whether solute and fluid movement occurred
more proximally or distally. Application of this approach to the lungs has been much more limited,
despite the fact that there are potentially some intrinsic advantages to using stop-flow studies in the lungs.
Because access to both the upstream and downstream
compartments is available, it is possible to use either
anterograde or retrograde flows to flush the contents of
the vasculature from the pulmonary circulation.
The observation that, when the lungs are flushed in
an anterograde direction after the stop-flow period,
increases in FITC-dextran and albumin precede those
in 3HOH is consistent with the conclusion that some
filtration occurs at sites which are more venous than
the site at which 3HOH enters the pulmonary vasculature after instillation into the lungs. On the other hand,
the failure to observe a similar pattern when the lungs
are flushed from the venous to arterial vessels suggests
that relatively little filtration occurred through vessels
more proximal than the capillaries. Even in lungs
injured with instillation of 0.1 N HCl in isotonic saline,
filtration, which was greatly augmented, appeared to
occur in more-venous vessels, although Vdex/ V3HOH was
not as low in the group 3 or group 4 experiments as it
was in the initial series of anterograde flush studies.
Evans blue has been used for many years as a
convenient tracer for albumin in physiological experiments. In single-pass studies with this dye, intravenous injections of hypertonic solutions resulted in
comparable dilution of Evans blue-labeled protein and
hemoglobin, suggesting that little had leaked out of the
vasculature (5). However, recent studies of Evans bluelabeled albumin and 125I-labeled albumin conducted in
perfused rat lungs showed considerably greater pulmonary uptake of Evans blue than 125I-albumin over a
3-min period (4). No significant dissociation of dye from
albumin was found in the present studies when the
perfusate was ultrafiltered through a membrane with a
cutoff of 30,000 mol wt or when it was dialyzed against
a protein-free solution. The unlabeled albumin was
placed in the dialysis bag in the latter experiments so
that any free Evans blue in the large volume of fluid in
the external solution would have the opportunity to
equilibrate with the small volume of fluid in the
dialysis bag and then bind with albumin in this compartment. This would appear to argue against significant
dissociation of Evans blue from albumin before it
reached the lungs.
It is possible that exposure of the lungs to an albumin
solution containing Evans blue increased the permeability of the lungs to albumin. However, increases in the
concentrations of FITC-dextran (mol wt 2,000,000) in
experiments in which Evans blue was included were
similar to those in which unlabeled albumin was used.
This suggests that the Evans blue did not increase the
filtration coefficient of the pulmonary vasculature.
The similarity of outflow emergence of unlabeled
protein (albumin, mol wt 68,000) and FITC-dextran
(mol wt 2,000,000) from the lungs suggests that these
molecules provide a reasonable index of filtration in
undamaged lungs. Ideally, a comparison with a cellular
element such as red blood cells would permit assessment of how many of these macromolecules may have
been lost through the capillary walls (11, 15). Unfortunately, settling of red blood cells during the stop-flow
period made it difficult to utilize this approach. Nevertheless, the close correspondence of these curves suggests that neither macromolecule crosses normal capillary walls during a 10-min stop-flow period. More
albumin than FITC-dextran would probably be lost if
the lungs were sufficiently injured: it is likely that the
failure of Evans blue concentrations to increase as
much as FITC-dextran after instillation of HCl into the
air spaces reflects increased loss of albumin molecules.
Excision of the lungs from the chest inevitably resulted in tearing of bronchial vessels, and much of the
fluid from the upper reservoir was presumably lost
through disrupted vessels. Instillation of HCl into the
air spaces before perfusion increased losses of perfusate from the upper reservoir.
Most studies of filtration in the lungs have been
made by collecting lymphatic fluid or measuring changes
in organ weight. Interpretation of lymphatic experiments has been complicated by uncertainty concerning
the tissues drained by the lymphatics and the effect of
the nodes themselves on lymph constituents. In gravi-
Downloaded from http://jap.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 6. Volume of fluid lost from upper reservoir during stop-flow
interval. Instillation of 0.1 N HCl into the air spaces before start of
experiments (group 3 experiments) accelerated losses of perfusate
from upper reservoir compared with experiments without HCl exposure (groups 1 and 2 experiments).
51
52
STOP-FLOW STUDIES OF PULMONARY FILTRATION
This study was supported by National Heart, Lung, and Blood
Institute Grant HL-18606 and Department of Veterans Affairs Grant
7731–05.
Address for reprint requests: R. M. Effros, 9200 Wisconsin Ave.,
Milwaukee, WI 53226 (E-mail: [email protected]).
Received 17 April 1997; accepted in final form 21 August 1997.
REFERENCES
1. Albert, R. K., S. Lakshminarayan, N. B. Charan, W. Kirk,
and J. Butler. Extra-alveolar vessel contribution to hydrostatic
pulmonary edema in in situ dog lungs. J. Appl. Physiol. 54:
1010–1017, 1983.
2. Bhattacharya, J. Hydraulic conductivity of lung venules determined by split-drop technique. J. Appl. Physiol. 64: 2562–2567,
1988.
3. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 72: 248–254, 1976.
4. Dallal, M. M., and S. W. Chang. Evans blue dye in the
assessment of permeability-surface area product in perfused rat
lungs. J. Appl. Physiol. 77: 1030–1035, 1994.
5. Effros, R. M. Osmotic extraction of hypotonic fluid from the
lungs. J. Clin. Invest. 54: 935–947, 1974.
6. Effros, R. M., A. Hacker, E. Jacobs, S. Audi, and C. Murphy.
Continuous measurements of changes in pulmonary capillary
surface area with 201Tl infusions. J. Appl. Physiol. 77: 2093–
2103, 1994.
7. Effros, R. M., E. R. Jacobs, C. Berger, A. Hacker, and S. K.
Ozker. Measurements of 201thallium uptake by lungs and
pulmonary capillary volume with a new stop-flow procedure
(Abstract). Am. Rev. Respir. Dis. 143: A790, 1991.
8. Iliff, L. D. Extra-alveolar vessels and edema development in
excised dog lungs. Circ. Res. 28: 524–532, 1971.
9. Malvin, R. L., and W. S. Wilde. Stop-flow technique. In:
Handbook of Physiology. Renal Physiology. Washington, DC: Am.
Physiol. Soc., 1973, sect. 8, chapt. 5, p. 119–143.
10. Nicolaysen, G., and N. C. Staub. Time course of albumin
equilibration in interstitium and lymph of normal mouse lungs.
Microvasc. Res. 9: 29–37, 1975.
11. Pilati, C. F., and M. B. Maron. A technique to measure the
reflection coefficient using endogenous vascular indicators. Microvasc. Res. 32: 255–260, 1986.
12. Schneeberger, E. E., and M. J. Karnovsky. Substructure of
intercellular junctions in freeze-fractured alveolar-capillary membranes of mouse lung. Circ. Res. 38: 401–411, 1976.
13. Staub, N. C., H. Nagano, and M. L. Pearce. Pulmonary edema
in dogs, especially the sequence of fluid accumulation in lungs. J.
Appl. Physiol. 22: 227–240, 1967.
14. Weibel, E. R., and H. Bachofen. Structural design of the
alveolar septum and fluid exchange. In: Pulmonary Edema,
edited by A. P. Fishman and E. M. Renkin. Bethesda, MD: Am.
Physiol. Soc., 1979, p. 1–20. (Clin. Physiol. Ser.)
15. White, P., Jr., R. Brower, J. T. Sylvester, T. Permutt, and S.
Permutt. Factors influencing measurement of protein reflection
coefficient by filtered volume technique. J. Appl. Physiol. 74:
1374–1380, 1993.
Downloaded from http://jap.physiology.org/ by 10.220.33.5 on June 18, 2017
metric studies, it is difficult to distinguish alterations
in the vascular, interstitial, and cellular compartments
from increases in lung weight when venous pressures
are increased. Measurements of filtration in continuously perfused preparations require a delay before
changes in indicator concentration can be detected
because of the large amount of perfusate needed to fill
the reservoirs and tubing used in these experiments.
The stop-flow approach may prove useful because it
permits measurement of filtration out of the circulation
over a 10-min interval from a relatively small volume of
perfusate, i.e., that which is within the vasculature
during the stop-flow period. Furthermore, it can potentially provide information regarding the relative arterial and venous sites of filtration, diffusion, and metabolism in the intact lung, which would be difficult to
obtain by other experimental approaches.
There are a number of modifications that might be
made in the stop-flow approach that could provide
additional information regarding the distribution of
diffusion and filtration in the pulmonary vasculature.
Rather than the use of instillation of 3HOH into the air
spaces to mark the microvascular environment, potassium analogs such as 201Tl1 or 86Rb could be included in
the perfusate (6, 7). Because these indicators become
concentrated in lung cells, concentrations within the
vasculature fall to very low levels, and the site of solute
exchange would be indicated by a fall in the concentrations of these indicators. This would avoid any possible
alterations in local flow induced by instilling small
volumes of fluid into the air spaces. The fact that
increases in outflow concentrations of 3HOH and FITCdextran occurred in the same samples in the retrograde
experiments suggests that instillation of small volumes
of fluid does not have a significant effect on regional
flow in these experiments. Although the sites of diffusion of 3HOH on the one hand and 201Tl1 on the other
cannot be precisely defined, the fact that the capillary
surface area greatly exceeds that of the arterial and
venous vessels makes it likely that most of these
indicators diffuse across capillary membranes. The use
of a particulate indicator that remains in suspension
during the stop-flow period represents another potentially valuable modification in the stop-flow approach
because it would make it possible to detect losses of
high-mol-wt molecules such as FITC-dextran and albumin.