The effect of arterial PC02 on relative
retinal blood flow in monkeys
M. Tsacopoulos* and Noble J. David
This report describes a method for estimation of relative retinal blood flow in monkeys, based
on dye dilution curves constructed from densitometry measurements on fiuorescing vessels in
fundus angiograms. With this technique, first described by Hickam and Frayser,5 we have
studied the effect of hyper- and hypocapnia. Increased Paco. was found to cause expansion of
the vascular volume and shortening of the mean circulation time, indicating substantial increase
in flow, related to the degree of elevation of this blood gas and paralleling its effect on the
cerebral blood flow. The opposite effect is observed in hypocapnic animals. The changes in
the shape of the dilution curves, in hypercapnia or hypocapnia are analyzed. The final question
of whether the primary change is affected by pH or (HCO*)remains to he answered.
Key words: Retinal blood flow, fluorescein angiography, densitometry, microcirculation,
monkeys, blood gases, hypercapnia, hypocapnia, vasodilation, vasoconstriction.
I
making classic techniques involving arteriovenous differences or clearance curves inapplicable without seriously disturbing the
anatomy of the orbit and the physiologic
conditions of retinal perfusion. The approach described herein attempts to overcome these difficulties by the application
of retinal fluorescein angiography as a refinement of the technique described by
Hickam and Frayser,5 in quantitative
studies of the effect of Pa c02 on the retinal
microcirculation in monkeys. The method
offers new possibilities in the measurement
of relative retinal blood flow in man as
well as in experimental animals, yielding
data which may help to clarify retinal
circulatory changes in a variety of conditions.
n contrast to the well documented observation that arterial PCo2 (PaCo2) is the
principal factor regulating cerebral blood
flow in man and primates,1'2 its effect on
the retinal blood flow has not been clearly
established.:|- ' This lack of information is
most likely the result of the small size
and inaccessibility of the retinal vessels
From the Departments of Ophthalmology and
Neurology, University of Miami School of Medicine, and the Neurology Service, Veterans Administration Hospital, Miami, Fla.
Supported in part by Grant No. 5R01EY00093
Manuscript submitted for publication Oct. 4, 1972;
manuscript accepted for publication Feb. 23,
1973.
Reprint requests: Dr. Noble J. David, Veterans
Administration Hospital, 1201 N.W. 16 St.,
Miami, Fla. 33125.
"Dr. Tsacopoulos' present address is: Clinique
Universitaire d'Ophthalmologie, Hopital Cantonal, Geneva, Switzerland.
Methods
Calculation of relative retinal blood flow. The
blood flow (V) of any vascular system can be determined if one knows the vascular volume (Q)
of the system and the mean circulation time (F):
335
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Investigative Ophthalmology
May 1973
336 Tsacopoulos and David
(1)
Measurement of the total retinal blood volume
in vivo is not currently possible. As a reliable aliquot of this vascular bed, we have measured the
volume of the major retinal vessels proximal to
the second bifurcation, as determined from their
diameter and length on fundus photographs, assuming that we are dealing with a deformed
cylinder. If Dc is the average diameter of this
cylinder (or vessel) in a control condition (c),
then the average cross-sectional area (A) is
AC
= y4 7T
D-
If the length of the cylinder (or vessel) is lc, the
volume (Q) will be
Qe = V4 -
D*
1,
Should the cylinder (or vessel) change by stretching in all directions, it will assume some new
experimental (e) diameter (De) and length (le).
The ratio of the new experimental volume (Qe)
to the control volume (Qc) will be given by
Qe
/De\
Qc ~~ VDc/
2
le
lc
(2)
The mean retinal circulation time (t r ) can be
measured accurately by using densitometric techniques. Thus, on the basis of. these two parameters, one can calculate the relative changes in
the retinal flow from a control condition (Vc) to
an experimental condition (Ve) using Formula 3
which is derived from Formulas 1 and 2:
Ve
tc
Vc
te
(3)
the subscripts, a and v, referring to arterial and
venous.
Mean retinal circulation time (Tr). Hickam
and Frayser 5were the first to use densitometry
based on retinal fluorescein angiograms to determine the mean retinal circulation time in man.
This technique assumes that conventional dye dilution curves can be constructed from serial measurements of optical density (on film negatives
of serial fluorescein angiograms) at given locations
over the major retinal vessels. Mean retinal circulaton time is calculated from these curves. These
workers demonstrated "good linear relationship
between film density and the logarithm of the exposure to light passed through a Wratten No. 57
filter. Linearity was satisfactory between densities
of 0.2 and 1.5," well within their working exposure intensity. Another crucial factor, constancy of
the flash intensity of the fundus camera, was declared to be adequate, with insignificant change
as read by an exposure meter in 30 consecutive
flashes following the somewhat brighter initial
flash. Simultaneous brachial arterial sampling
(serum fluorescence) and fluorescence fundus photography yielded dilution curves of similar shape
and duration following the intravenous injection of
4 ml. of 5 per cent sodium fluorescein.
The specifics of our modification of their technique are as follows: In order to monitor variations in strobe-flash intensity, a fiberoptic bundle
was placed near the flash tube in the lamp housing
and led to the reflex housing where its illumination was focused through a neutral density filter
upon a corner of each frame of film taken by the
fundus camera. Variations in optical density in
any series of strobe flashes ranged from 0.0 to 0.03
units, practically uniform over the 40 to 50 consecutive exposures.
By trial observations 0.5 ml. of 2 per cent
fluorescein sodium injected into the right atrium
of the monkeys gave optical densities in the fluorescein angiograms not exceeding 1.2 on Kodak
Plus X film, and in most of the experiments ranged
from 0.05 to 1.0 units, measured above base fog
level for unexposed film, within the range for
optical density linearity, with the possible exception of the lowest measurements. The average
weight of the monkeys used was 2.5 Kg., with
the exception of one which weighed 3.5 Kg. (0.7
ml. injected).
Another potential source of error in this technique is in the development of the film. Light intensity (It) or the fluorescein intensity (If) is
proportional to the exponential of the optical density divided by Y:
If a e D/Y
y is a constant which depends upon the condition of the ftlm development, Y must be close
to unity if one is to assume that fluorescence
intensity (i.e., blood fluorescence concentration)
may be calculated as the antilog to the Napierian
base of the optical density. Preliminary testing
revealed that Kodak Plus X film developed in
fresh nondiluted Ethol UFG (ultrafine grain) for
8.5 minutes with continuous agitation at 70° F.
constant temperature yields a Y very close to unity.
Routinely, in these experiments, a bolus of
0.5 ml. 2 per cent sodium fluorescein was injected through a catheter introduced into or
near the right atrium. Fundus photographs were
made at a rate of 4 per second for about 13
seconds. A Baird Atomic Interference Filter (B4
maximum transmission between 465 and 480
m/i) was used as the exciting filter to produce
blue light. A barrier filter, Kodak Wratten No.
13 yellow (which excludes 96 per cent of all
light 460 m/i and below) was inserted at the
film gate to exclude reflected blue light. Control
photographs were virtually unexposed.7 A Nikon
F camera back with a motor driven 250 exposure
load of Kodak Plus X 35 mm. film, was adequate
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Volume 12
Number 5
Effect of arterial PCo2
for all five angiograms done in a single animal
(see below). This film was developed by the
method described above and used for densitometry measurements.
Densitometry measurements were made from
each of the major arteries and veins outside the
disc margin. A Photovolt densitometer was connected to a light microscope fitted with a variable
slit (0 to 0.5 mm.-) with which we measured
average optical density of each vessel in a band
across the total width at the selected location
along the vessel. The zero of the densitometer
was calibrated on the nonexposed part of the
film after the choice of slit width. Stability of
this calibration was checked after every six measurements. The micro-slit is an improvement over
the one point per vessel density reading5 since the
flow is not homogeneous and fluorescence varies,
especially across the major veins. It is also more expedient than scanning microdensitometry,s> 9 while
yielding essentially identical results. The antilog of
the optical density values, which are proportional
to the fluorescence intensity emitted from retinal
vessels, were used to construct dilution curves for
each individual vessel (Fig. 1). The light reflected
from the retinal vessels before the appearance
of fluorescein, with the actual combination of interference filters is near zero. Bulpitt and Dollery,8
in human studies, found that the fluorescent light
was equal to the light emitted from the fluorescein
bolus just injected (I f ) plus light from any
fluorescein still circulating from previous injections (If.i,). With the technique in use in our
laboratory, and for five consecutive injections
during an eight hour interval, we found that
the If.i, was constantly zero.
A computer program was used to match the
experimental fluorescence intensity points to the
best-fitting dilution curve. The area under this
curve
( J^I(t)dt)
is calculated using a least-square fit of the data
to a pair of intersecting exponential functions
and standard numeric integration techniques. Also,
using the same techniques, the time weighted
area
(J4(t):t-dt)
under the curve is calculated. The mean transit
time of the bolus of dye (t) is then given by
the ratio of the weighted area to the unweighted
area:
_
/,', I(t)t dt
(For details see Appendix)
The arterial (Fn) and the venous (t v ) transit
times are calculated independently. When the
on
monkey retinal blood floio 337
10
9
8
7
6
^ 5.
2.
5
Time
, 1 0
(sec)
Fig. 1. Two experimental fluorescein intensitytime curves obtained in normocapnic condition
(semilogrithmic paper). The arterial curve is indicated with dots, the venous curve, with circles.
average arterial (t n ) is subtracted from the
average venous (TV), the retinal mean circulation
time (Fr) is obtained:
t, = tv - ta
(see Fig. 1)
The tr can be introduced into Equation 3 to calculate the relative retinal blood flow.
Vessel diameter and length measurements. During controlled and experimental conditions, photographs were taken especially for the purpose of
estimating changes in vessel diameter. The technique has been described previously0' s ' 1() and
carefully analyzed.11 Photographs were made on
black and "white (Plus X) film or, rarely highcontrast color film (Kodak SO-456) taken in
projections including the disc centered and at the
upper and lower margin of the photographs. Measurements were made with a light microscope
equipped with a micrometer scale in one ocular
and at low magnification. From the photographs
of each eye approximately 10 optimum sites for
diameter measurements were chosen for both
the arterial and venous tree. Identical sites were
used in all comparative measurements which were
usually made double blind. The per cent change
in the vessel diameters from one experimental
condition to another was calculated from the
individual data and averaged. For the blood-flow
calculations the ratio of vessel diameter of the
control to the experimental conditions was used.
The length of retinal arteries between two clearly identifiable points was measured by projecting the negative fundus photographs on a screen
and tracing the vessels with a precise map curvimeter. These measurements were performed on
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338
Investigative Ophthalmology
May 1973
Tsacopoulos and David
connected through a Statham pressure transducer
to a Grass polygraph. Blood gases (Pao2 and
Paoo.) and pH were measured immediately in
anaerobically taken arterial blood samples using
microelectrodes operated at 37° C. (Radiometer,
Copenhagen, Denmark). The values were corrected for body temperature, which was continuously monitored, and pH values were referred to standard phosphate buffers. Hemoglobin
was measured six times during the experiment
and these values introduced into the calculation
of the acid-base balance.12
Often the monkeys developed some degree of
metabolic acidosis (about -4 mEq. per liter, presumably because of the production of lactates
during the surgical procedure. This disturbance
was corrected with injection of sodium bicarbonate
(milliequivalents of NaHCOa equals base deficit
(milliequivalents per liter) x 0.3 x weight [Kilograms]). When a steady state of acid-base balance had been attained, the control fundus photographs and fluorescein angiograms were done. In
the first six experiments only one level of hypercapnia (7.7 per cent carbon dioxide in inspired
T I M E
(t«c)
Fig. 2. Three experimental fluorescence intensitytime curves obtained in (a) normocapnia, (b)
hypercapnia, and (c) hypocapnia. (Semilogrithmic paper. For description see text.)
the same photographs used for the vessel diameter estimation. Several measurements were
made on the same vessel and the values obtained
were averaged. The ratio
Ka)e
was calculated and
introduced into the flow equation.
Other details of the experimental protocol. We
studied 16 animals for relative retinal blood-flow
changes in response to variations in Paco.jMonkeys (pigtail macaca or stumptail macaca)
weighing between 2.5 and 3.5 Kg. were used.
After tranquilization with Sernylan (Phencyclidine
hydrochloride), tracheostomy was performed and
a catheter was introduced 3 to 4 cm. into the
right femoral artery for continuous monitoring
of blood pressure. Another catheter was inserted
into the right femoral vein and advanced (24
to 25 cm.) into the right atrium. Paralyzed with
intravenous injection of 1 ml. of Flaxedil (gallamine triethiode), the animals were artificially
ventilated with a continuous flow of 75 per cent
N2O plus 25 per cent O2. This technique yields
a superficial anesthesia which has very little effect on systemic blood pressure. The stroke
volume was carefully adjusted in order to maintain a Paco2 at the normal range for monkeys
35 to 42 mm. Hg). The intraocular pressure
(IOP) was also recorded through a 28 gauge
needle introduced into the anterior chamber and
air and average PACO, =
66.0 mm. Hg)
and
one
level of hypocapnia (50 per cent increase in respiratory stroke volume and Pace-., average 22.5
mm. Hg) were studied. Because a wider range
of observations was desired, the next 10 animals
were studied as follows. Hypercapnia was attained with (a) 7.7 per cent carbon dioxide
(average = 59.0 mm. Hg Paco.j) and (b) 13.8
per cent, carbon dioxide (average Paco. = 92.0
mm. Hg) in the inspired gas mixture. Hypocapnia
was produced (a) by a 50 per cent increase in
stroke volume (average PaCo, = 28.0 mm. Hg)
and (b) by a 100 per cent increase in stroke
volume ( P a c , = 20.0 mm. Hg) (See Table II).
The same sequence was followed in 8 of the 10
animals. In the other two animals hypocapnia
was produced after control studies and before
the hypercapnia trials. A total of six hours was
usually required for the five photographic experiments. Between the second level of hypercapnia and artificial hyperventilation the animal
rested for at least 2 hours breathing a normal
gas mixture. Additional Flaxedil was injected before the onset of hypercapnia or hyperventilation.
No effect of this drug on the blood pressure has
been noted to date. The photographic studies
were repeated after each stage of the experiment,
10 minutes after onset of CO* breathing, and
15 to 40 minutes after the start of hyperventilation. A blood sample for acide-base and Pao,
determination was taken just before each injection
of fluorescein dye.
During all studies a contact lens was placed
on the cornea. This precaution combined with
small amounts of normal saline preserved corneal
transparency during the eight hour experiment.
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Volume 12
Number 5
Effect of arterial PCo2 on monkey retinal blood flow
339
150
% A PnC 0 .
-4 0
5 0
r4 0
I50
% A P„ C 0 .
Figs. 3, A and B. Relationship between changes in Paoo2 (APaoo..) and vessel diameter (ADIA).
Graph depicts (a) arterial, and (b) venous changes in units of per cent A from control
diameter.
Results
Retinal perfusion pressure. Both systemic
mean blood pressure (MBP) and IOP increased after CO2 was introduced into the
inhaled gas mixture. This rise of MBP and
IOP began about two minutes after the
onset of C(X breathing and reached its
maximum in about five minutes. Subsequently, both decreased, but in different
patterns. The MBP returned to control
levels after 10 minutes of C(X breathing
while IOP remained elevated. Further
increase in the inhaled COL. concentration induced an additional rise, especially
in the IOP. The retinal perfusion pressure (P.P.) as it was calculated in the
conventional formula (PP = MPB - IOP)
shows changes in spite of a similar direction of increase of both MBP and IOP.
However, after 10 minutes of CO., breathing, the PP was found to be 2 mm. Hg
lower than the prehypercapnic state which
would not have contributed to increased
blood flow. When the normal gas mixture
was resumed, the MBP returned to a slightly lower level (3 mm. Hg) than control
levels, while the IOP remained at hypercapnic levels. Hyperventilation had practically
no measurable effect on the MBP and we
were unable to detect a consistent effect of
hypocapnia on the IOP.
Blood measurements. It was difficult to
obtain a normal acid-base balance in the
experimental conditions described above.
As is shown in Table I, the calculation of
acid-base balance in 10 experiments (see
Methods) often revealed a metabolic acidosis which in one experiment reached
(-4.0 mEq. per liter). In the same experiment, even in the hypocapnic state, the
animal was in acidosis. In 4 of 10 experiments hyperventilation did not produce a
base excess in spite of an increase in pH
level.
Table II shows the average values of
PaC0:!, pH, and Pa().,. As is shown, the Paoj
was maintained within normal limits. In
one experiment, hyperventilation produced
a drop in the Pa o . (84.1 mm. Hg) probably
due to bronchospasm, but correction for
temperature and pH gave a per cent saturation of 97.3 and a corrected PaoL. of 95
mm. Hg.
Dilution curves and Tr. The arterial
fluorescein dilution curves obtained in this
study (Fig. 1) are similar in shape to the
conventional dye dilution curves in use
for the estimation of cardiac output. The
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Investigative Ophthalmology
May 1973
340 Tsacopoulos and David
Table I. Values of acid-base excess in milliequivalents per liter found in 10
experiments. The acid-base excess was calculated from Siggard-Anderson
normogram using the actual values of Paco2, pH, and hemoglobin
Experiment
No.
1
2
3
4
5
6
7
8
9
10
Normocapnia
0
-1.0
-4.0
-2.0
-2.0
-1.0
-0.5
0
0
0
Acid-base excess (mEq./L.)
Hypercapnia (1) \ Hypercapnia (2) Hypocapnia (1) Hypocapnia (2)
-9.0
-8.5
-9.0
-8.0
-7.5
-4.0
-3.5
-7.0
-3.0
-6.0
recirculation curve is always obvious but
appears in the downslope of the curve (disappearance slope"; there were enough
points so that extrapolation of the curve
to zero could be accurately performed (see
Appendix). The venous dilution curves
had the same shape as the arterial dilution
curves, but the peak concentration appeared lower, and, in general, the over-all
curve is wider than the arterial curve.
Finally, recirculation is also evident in the
venous curve but starts at a higher level
of fluorescence intensity than in the arterial
curve.
Hypercapnia (Fig. 2) produced some
changes in the shape of the fluorescein dilution curves. Peak arterial concentration remains at about the same level as in the
curves obtained in normocapnic conditions,
but the over-all curves in hypercapnia were
always narrower than in control curves.
Onset of the recirculation curve can be
easily identified in the disappearance slope
and is much more apparent than in normocapnia. In the same condition it was again
observed that the height of the venous
curve is lower and the over-all curve definitely smaller than in normocapnic venous
curves. In spite of a fast recirculation time
extrapolation of the venous curves to zero
could be accurately performed in most of
the experiments because of the high frame
speed (4 per second) of the recording
camera.
-14.0
-16.0
-10.0
-11.5
-11.0
- 8.0
- 7.0
- 8.0
- 4.5
-11.0
+3.5
+2.5
-7.0
+2.0
-2.0
-3.0
+4.0
+2.0
+3.0
+4.0
+3.0
+3.0
-3.0
0
0
0
+4.0
+5.0
+7.0
+1.0
Hypocapnia also produces changes in
the intensity-time curves (Fig. 2). In
general, these curves were wider than
the normal curve, especially the venous
curves and recirculation was less striking.
Table II shows the average values of
the arterial, venous, and retinal mean circulation times measured on the basis of
the dilution curves by the technique described in Methods. The first interesting
observation is that the tr in the control
situation was quite consistent (mean 1.5
± 0.4 seconds) with the exception of one
experiment.
The first degree of hypercapnia consistently provoked a small but statistically
significant shortening of the tr (mean =
1.2 ± 0.3 seconds p < 0.001.) This shortening of the Ir has been observed to be more
pronounced at higher levels of hypercapnia
(mean = 1.00 ± 0.2 seconds (p < 0.001.)
Finally, hypocapnia produced a constant
and statistically significant increase of the fr
(mean = 1.7 ± 0:5 seconds, p < 0.001)
which was most obvious at the lowest
PaC02 levels (mean 2.0 ± 0.5 seconds, p <
0.001.)
Vessel diameters and vessel length
changes. The increase of the PaC02 from
38 ± 2 mm. Hg to 59 ± 6 mm. Hg produced
vasodilatation of +21 per cent ± 6 for the
arteries and +18 ± 1 for the veins. When
the PaCoj level rose to 92 ± 6 mm. Hg the
vasodilatation was more pronounced: +34
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Volume 12
Number 5
Effect of arterial PCOz on monkey retinal blood flow
341
Table II. Range, mean, and standard deviation of Paooa, pH, and Pao2 values
with corresponding mean arterial (t a ), venous (t v ), and retinal (t r ) circulation
times. At right are shown results of the statistical analysis concerning tr
experimental changes
Paco,
Experiment
Nofmocapnia:"
Range
Mean
S.D.
n = 16
Hypercapnia (1):°
Range
Mean
S.D.
n = 16
Hypercapnia (2):
Range
Mean
S.D.
n = 10
Hypocapnia (1):°
Range
Mean
S.D.
n = 16
Hypocapnia (2):
Range
Mean
S.D.
n = 10
(mm. Hg)
35.3- 41.7
38
± 2.0
Pao,
pH
(mm. Hg)
7.365-7.419 99.0-139.2
7.400
120.
0.018
11.1
t
(sec.)
(sec.)
4.0
0.5
5.5
0.7
(sec.)
1.5
0.40
51.5- 68.8
59
± 6.0
7.145-7.238 101.6-141.3
119.
7.190
0.026
12.6
3.1
5
4.3
0.6
1.2
0.3
80.5-100.0
92
± 6.0
6.943-7.092 99.0-132.2
111
7.026
12.3
0.050
3.2
0.8
4.2
0.8
1.0
0.2
23.0- 31.6
28
± 3.3
7.415-7.562 93.8-148.5
116
7.515
16.5
0.040
5.1
1.2
6.8
1.6
1.7
0.5
16.2- 24.3 7.567-7.684 97.3-150.4
20
116.
7.637
18.0
± 2.2
0.033
4.9
0.9
6.9
1.4
2.0
0.5
t t TT
nI < 0.001
|
f
"The preliminary experimental results for tr are included in these groups (see text).
fThe p values were obtained from a matched-pair t test of the data from each animal, not from the means of each experimental condition.
± 6 for the arteries and +29 ± 7 for the
veins. In contrast, hypocapnia (PaCo2 —
28 ± 3 mm. Hg) regularly produced vasoconstriction of -13 per cent ± 4 for the
arteries and -13 per cent ± 5 for the veins.
In deeper hypocapnia (Paco- — 20 ± 2 mm.
Hg) the vasoconstriction observed was
more pronounced: -22 ± 4 for the arteries
and -18 ± 5 for the veins. The change of
vessel diameters from the normocapnic
state to the experimental state, as well as
from one experimental level to another is
statistically significant (p < 0.001).
Fig. 3, A and B demonstrate the consistent effect of rising Paco^ o n vessel
diameters. The regression coefficients for
each experiment were calculated using a
classical method1' and the slopes of all
the experiments were found to be homogeneous.
We found a small but statistically significant (p < 0.01) increase of the vessel
lengths (+2.6 per cent) in the first level
of hypercapnia. In the higher hypercapnia
(92 mm. Hg) the increase of the length
was +3.6 per cent (p < 0.01). The change
of length in hypocapnia was inconsistent;
the average per cent change showed a
decrease for both levels of low Paco- (-2.5
per cent and -2.4 per cent). The "t" test
showed that this decrease is statistically
significant (p < 0.05).
Fundus photographs reveal striking
changes in tortuosity of the vessels as related to the Paco2 (Fig. 4). In hypercapnia
even straight vessels became tortuous. This
interesting phenomenon will be analyzed
in another paper.
Relative blood flow. Fig. 5 illustrates the
consistent response of relative retinal blood
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Investigative Ophthalmology
May 1973
342 Tsacopoulos and David
Fig. 4, Three fundus photographs taken in the same animal under different experimental conditions: A, normocapnia; B, hypercapnia; and C, hypocapnia. Vasodilatation and tortuosity of
the vessels (more obvious in the veins) in hypercapnia and vasoconstriction in hypocapnia.
flow to change in Paco^ a relationship approaching linearity. The ratio of retinal
blood flow^ was calculated as described
in Methods.
Discussion
The technique described has yielded reliable and reproducible results in the study
of retinal blood flow as related to Paco2Determination of the different values of
mean retinal circulation time and changes
in vessel diameter and length have been
introduced into the flow equation (see
Methods) allowing the calculation of experimental flow vs. control flow. Values
obtained from this calculation are not absolute (milliliters per 100 Gm, tissue per
minute) but relative, with unity indicating
no change in flow. A regression line may
be drawn describing the relationship between retinal blood flow and Paco- in
monkeys. As PaCo- increases, retinal blood
flow is also increased, the reverse occurring
with Paco- reduction. For technical reasons
{limited number of fluorescein injections)
it was not possible to explore the full range
of responsiveness of retinal blood flow to
changes in PacO:> as Reivich- has done for
cerebral blood flow in monkeys.
We chose to study the Paoon range in
which the monkey's cerebral blood flow is
reported to show linear response.2
The question arises as to why such minor
changes were found in the tT in spite of
the large change in dilution curves. The
experimental hypercapnia in monkeys produced impressive narrowing of the retinal
arterial and venous dilution curves, but the
corresponding values of tr were only slightly shortened. At highest levels of Pacoa, a
condition in which retinal blood flow should
be greatly increased, the tr was definitely
shorter than the corresponding tr in normocapnia. The converse was found (small increase in tr) in the hypocapnic state in
spite of widening of the dilution curve.
Interpreting these observations one must
recall that the mean circulation time (t)
of any vascular system varies with the
vascular volume (Q) and the blood flow
(V).
_ Q
Increase in vascular volume without
change in blood flow should produce an
increase of the I. Conversely, in the face of
an expanding vascular volume, blood flow
must increase substantially in order to yield
a shortening of the t. Thus it is evident that
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Volume 12
Number 5
Effect of arterial PCo2
on
monkey retinal blood floio
343
3_
i_
'.V
—i
1
PnCO,
r
100
50
(mm Hg )
Fig. 5. Graph illustrating the relationship between PACO2 and relative retinal blood flow calculated as described in Methods.
the direction and extent of the t change
depends on change in blood flow with respect to change in vascular volume.
Thus it is possible to speculate that the
small but constant changes of the tr at
moderate levels of hypercapnia or hypocapnia are due to a respective increase or
decrease in retinal vascular volume. When
the retinal blood flow was greatly increased
(hypercapnia) or decreased (hypocapnia)
the t,. change was more pronounced, in
spite of the respective canceling effects of
vascular volume. Another explanation for
this relatively small change in the value
of the tr is that the method may underestimate it. The method of Hickam and
Frayser"1 was applied in determining mean
retinal circulation time as the difference between mean venous time and mean arterial
time. Obviously, when both values, venous
and arterial, change in the same direction,
change in retinal circulation time will be
small. On the other hand, if the arterial
dilution curve from which the arterial time
is calculated remains unchanged and retinal vascular resistance changes, this should
be reflected primarily in a change in the
mean venous circulation time, and consequently, a larger alteration in tr. In these
experiments the integral under the arterial
fluorescein dilution curves was 24.0 per
cent smaller in hypercapnia and only 1.2
per cent larger in hypocapnia than in the
normocapnic condition. If fluorescence intensity is strictly proportional to dye concentration, the integral under the retinal
arterial dilution curve measures cardiac
output. Change in the integral under the
arterial curves during experimental, respiratory acidosis or alkalosis should indicate an increase or decrease of the cardiac
output, respectively. It is known that cardiac output and heart rate show linear increase in response to a rising Paco^ m the
range of 20 to 80 mm. Hg in anesthetized
man.15 The same probably holds true for
superficially anesthetized monkeys. It is
clear that these changes in the arterial dilution curves will affect the value of the
mean retinal circulation time, while the
tr reflects important changes on the venous
side. The dye disappearance slope in hypercapnia rapidly fell to zero, indicating
a low vascular resistance, whereas in hypocapnia the decay of this slope was very
gradual suggesting a marked increase in
retinal vascular resistance. Calculation of
the mean venous circulation time from
these curves revealed definite changes as
compared to the normal curve (Table II).
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Investigative Ophthalmology
May 1973
344 Tsacopoulos and David
The lower level of the peak density observed in hypercapnia, more pronounced in
the venous curves, can be explained on the
basis of the pH changes occurring in this
condition. It is well known10 that the fluorescence of fluorescein conjugates is 50
per cent lower at pH 6.0 than at pH 8.0
and that the light absorption at 495 m^ is
similarly reduced. Although, in hypercapnia, the pH did not drop below 6.9, even
this decrease may explain this phenomenon.
A possible source of underestimation in
the relative retinal blood flow calculation
is the vessel diameter measurement. First,
it is not possible with techniques now in
use to measure the arteriolar diameters
(25/.i) at which level caliber changes critically affect the peripheral vascular resistance. Second, Bulpitt, Dollery, and Kohner11 demonstrated that in hypercapnia the
arterial and venous marginal plasma zones
increase to a greater degree than does the
diameter of the retinal vessels. Since the
fundus photographs from which our measurements were made do not show the
plasma zone, the tendency is to underestimate increases in vessel diameter in hypercapnia. Fluorescein angiograms would be
the ideal material for measuring the vessel
diameters, even in small arterioles, but
since vessel filling is not homogeneous,
such measurements are not uniformly reliable.
In spite of these reservations these studies demonstrate that arterial Paco- plays an
important role in the regulation of retinal blood flow as well as in the brain. This
is not surprising considering the embryologic and anatomic similarities between the
brain and retina. This conclusion is in contrast to the findings of other authors0-17
who, using similar techniques, found that
the Paco- has very little effect on the retinal
circulation in man. The reason for this discrepancy probably lies in the difficulty in
inducing significant hypercapnia in conscious man. Sechzer and co-workers18 reported some of the dramatic symptoms experienced by subjects during mild experi-
mental acute hypercapnia. Since Frayser
and Hickam17 did not measure the PaCoduring their experiments, one wonders
whether the level of hypercapnia reached
was sufficient to affect retinal vascular resistance. However, the effect of hypocapnia
after hyperventilation (easier to produce in
man) was more obvious.
Few experimental studies have been performed in other animals which help to
clarify the retinal circulatory effects of
19
PACOS- Spalter, TenEick, and Nahas were
the first to quantitatively study the effect
of experimental hypercapnia (about 80
mm. Hg of PaooL.) on the retinal vessel diameters under constant intracranial pressure (ICP) in dogs. They performed these
experiments in order to study the possible
role of elevated ICP in explaining the retinal vasodilatation which they consistently
observed. In these experiments the ICP
was not measured, but the IOP was continuously recorded. Hypercapnia produces
a significant increase in the IOP, confirming similar findings in rabbits.20 The most
probable cause of the increased IOP in the
hypercapnic state is the increase of the
intraocular vascular volume as related to
PaCo- The systemic blood pressure increases simultaneously with the IOP in
hypercapnia and may be a factor affecting
the rise in IOP. However, the net reduction of retinal perfusion pressure was only
2 mm. Hg in hypercapnia.
During the1 preparation of this manuscript Aim and Bill,21 using autoradiographic techniques, have reported that hypercapnia produces an increase of the retinal blood flow in cats.
These experiments in monkeys have demonstrated that the retinal vascular reactivity
is not related to species as previously postulated3- 4 but to differences in experimental
conditions. The anatomy of the monkey's
retinal vessels and their relationship to the
glial cells is identical to that found in
man.22 Our monkeys were lightly anesthetized with N2O as demonstrated by the
blood pressure rise with the Pacoj increase.
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Volume 12
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Effect of arterial Pc02 on monkey retinal blood flow 345
In addition, the body temperature was kept
within the normal range during the experiment and the hematocrit was reduced only
3 to 4 units per cent by the end of the experiment. Furthermore, special care was
taken to obtain an appropriate normocapnic
control state, with a near zero acid-base
excess or deficit, and to maintain the arterial Pao2 within the normal limits
throughout the entire experimental procedure. Finally, techniques were used which
are applicable to human studies and which
permit quantitative measurement of the
relative retinal blood flow in the intact eye
under physiologic conditions.
The physiologic mechanism of the effect
of the Pacou on the retinal vascular resistance remains to be analyzed. When experimental PaooL- changes are induced, proportionate and parallel changes occur in [H+],
raising the question as to which of these
is the prime factor regulating blood flow
in this tissue. The same question remains
to be answered in the regulation of cerebral
blood flow. It is reasonable to assume that
CO2 diffuses very quickly through the retinal or brain barrier and into the surrounding cells and tissue. There the chemical
reaction
CO, + H,0 ^ H+ + HC03
occurs very quickly in the presence of the
enzyme carbonic anhydrase with a consequent fall in tissue pH. We would speculate that this increase of H+ affects arteriolar smooth muscle and produces the change
in vascular volume.23 This theory is not universally accepted. Further studies will be
necessary to accomplish the difficult task of
separating these factors in an appropriate
experimental situation.
The authors wish to thank the following for
their help: Mr. Melvin Johnson for his technical
assistance in these experiments, Mr. Layman
Hazelton who prepared the computer programs to
match the dilution curves, M. Jean Marie Parel
who designed the fiberoptics, and Miss Rita E.
Stein who prepared this manuscript, and Gerard
Eperon who assisted in reviewing the manuscript.
REFERENCES
1. Kety, S. S., and Schmidt, C. F.: Effects of
altered arterial tensions of carbon dioxide
and oxygen on cerebral blood flow and cerebral oxygen consumption of normal young
men, J. Clin. Invest. 27: 484, 1948.
2. Reivich, M.: Arterial Pco: and cerebral hemodynamics, Am. J. Physiol. 206: 25, 1964.
3. Duke-Elder, S.: System of Ophthalmology,
The Physiology of The Eye And of Vision,
St. Louis, 1968, The C. V. Mosby Company,
Vol. IX, p. 92.
4. Wise, C , Dollery, C. T., and Henkind, P.:
The retinal circulation. New York, 1971,
Harper and Row, Publishers, Inc.
5. Hickam, J. B., and Frayser, R.: A photographic method for measuring the mean
retinal circulation time using fluorescein, INVEST. OPHTHALMOL. 4: 876, 1965.
6. Hickam, J. B., and Frayser, R.: Studies of
the retinal circulation in man. Observations
on vessel diameter, arteriovenous oxygen difference, and mean circulation time, Circulation 33: 302, 1966.
7. Kulvin, S. M., and David, N. J.: Experimental retinal embolism, Arch. Ophthalmol.
78: 774, 1967.
8. Bulpitt, J. C , and Dollery, C. T.: Estimation of retinal blood flow by measurement of
the mean circulation time, Cardiovasc. Res.
5: 406, 1971.
9. Tsacopoulos, M., Girardier, L., and Vuagnat,
P.: Essai de mesure du debit sanguin r6tinien
relatif par l'angiographie fluoresceinique. Presented at the Swiss Ophthalmological Society, Sept. 1971, (To be published in
Ophthalmologica.)
10. Dollery, C. T., Hill, D. W., and Hodge,
J. V.: The response of normal retinal blood
vessels to angiotensin and noradrenalin, J.
Physiol. 165: 500, 1963.
11. Bulpitt, C. J., Dollery, C. T., and Kohner,
E. M.: The marginal plasma zone in the
retinal microcirculation, Cardiovasc. Res. 4:
207, 1970.
12. Siggaard-Andersen, O.: An acid-base chart
for arterial blood with normal and pathophysiological reference areas, Scand. J. Clin.
Lab. Invest. 27: 239, 1971.
13. Wood, E. H., and Swan, H. J. C : Definition
of terms and symbols for description of circulatory indicator-dilution curves, J. Appl.
Physiol. 6: 797, 1954.
14. Sokal, R. R., and Rohlf, J.: Biometry, San
Francisco, 1969, W. H. Freeman and Company.
15. Nunn, J. F.: Applied respiratory physiology,
Washington, D. C , 1969.
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346 Tsacopoulos and David
16. Nairn, R. C : Fluorescent protein tracing.
Edinburgh and London, 1969, E. and S.
Livingstone, Ltd.
17. Frayser, R., and Hickam, J. B.: Retinal vascular response to breathing, increased carbon
dioxide, and oxygen concentrations, Invest.
Ophthalmol. 3: 427, 1964.
18. Sechzer, P. H., Egbert, L. D., Linde, H. W.,
et al.: Effect of CO2 inhalation on arterial
pressure, ECG and plasma catecholamines,
and 17-OH-corticosteroids in normal man, J.
Appl. Physiol. 15: 454, 1960.
19. Spalter. H. F., TenEick, R. E., and. Nahas,
G. G.: Effect of hypercapnia on retinal vessel size at constant intracranial pressure, Am.
J. Ophthalmol. 57: 741, 1964.
20. Kaskel, D., and Neumann, J.: Investigations
on the influence of CO : and O- partial pressure upon the intraocular pressure in rabbits,
Ophthalmol. Res. 2: 211, 1971.
21. Aim, A., and Bill, A.: The oxygen supply to
the retina. II. Effects of high intraocular pressure and of increased arterial carbon dioxide
tension on uveal and retinal blood flow in
cats. A study with radioactively labelled microspheres including flow determinations in
brain and some other tissues, Acta Physiol.
Scand. 84: 306, 1972.
22. Hogan, N. J., and Feeney, L.: The ultrastructure of the retinal blood vessels. I. The
large vessels, J. Ultrastruct. Res. 9: 10, 1963.
23. Wahl, M, Deetjen, P., Thuran, K., et al.:
Micropuncture evaluation of the importance
of perivascular pH of the arteriolar diameter
on the brain surface, Pflugers Arch. 316: 152,
1970.
Appendix
The intensity of light (I) is related to the optical density (D) by the well-known equation
I = A (e"/T - 1)
(1)
being equal to unity within the linear region of
the H & D curve, and e = 2.71828 . . . is the
base of the Napierian logarithm.
From the general theory of molecular diffusion we find that if the fluorescein were diffusing
without turbulence, the intensity of fluorescence
(which is directly proportional to the molecular
density of fluorescein at the intensity of illumination used) should both rise and fall exponentially,
but not necessarily with the same rate of rise as
the rate of fall. If this were perfectly correct, then
the optical density as a function of time would
consist of a pair of intersecting straight lines (Fig.
6), the first of positive slope, the second of negative slope. In the experimental state, due to a
host of reasons, the measured data meets this criterion only to the first order.
We wish to avoid the inclusion of the curve due
Investigative Ophthalmology
May 1973
Fig. 6. Typical densitometer data and associated
linear approximations.
to recirculation of dye in our analysis, so we excluded all data to the right of the first minimum
in the optical density function.
We can then, using standard variational methods, find those straight lines which best fit the
measured density values. Thus we obtain the four
coefficients a, b, V, and I of the linear functions
D t = at + b
,t < T
(2)
Da = 7t + £
,t > T
where r is the time at which maximum fluorescence intensity occurred. A problem arises in that,
since the data was taken at discrete times instead
of continuously, we do not know T precisely.
Physically, T is the time when the intensity of
fluorescence stops rising and begins to fall, i.e.,
the time corresponding to the intersection of the
functions described by Equation 2. Solution of
these equations obtain t:
(3)
t =
Finally, substitution of (2) into (1) yields a
first order best fit to the intensity of fluorescence.
This produces two intersecting experimental functions.
From a physiologic standpoint, we may wish to
calculate the mean venous and arterial circulation times (Fv, n). To this end, we calculate the
integral (S: area under the curve), and various
moments of the time:
s=jvi(t)dt
f T I (t) t dt
f(v „ , = i 2o
U.
J V I (t) dt
_
la
fj
JY
i (t) t» dt
i (t) t dt
(4)
(5)
(6)
where t2 is the second moment of the elapsed
time. The upper integration limit, T, is the time
when the extrapolated best-fit intensity curve
reaches zero. Finding T is quite straightforward.
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Volume 12
Number 5
Effect of arterial PCo2
The optical density to the right of the maximum is
D2 = i?t + £
and the intensity will therefore be
I = A(e'/? <# + «> - 1)
Obviously, I = o when the exponent is zero, or
when D2 = O. This leads immediately to
T = - If one wishes to calculate the mean retinal circulation time (tr) one should use Tv, a, not ti,
as Hickam and Frayser did. If, on the other hand,
one wishes to differentiate between different physiologic conditions (hypercapnia, etc.), the second
moment is more indicative. This is because the
rise times for the various conditions are probably
on
monkey retinal blood flow 347
similar, and the second moment is a better indication of the resistance of the system which can
determine TV.
All calculations were performed on a Univac
1106 computer, and the curves were plotted on
a Calcomp drum plotter. All integrations were
performed using a 512 point Simpson's Rule integrating routine. The entire program was written
using Univac Fortran V with as little use of
system resident mathematical routines as possible
to facilitate its use at other installations. A second
form is also available which plots histograms on
a line printer and uses no system resident routines
whatsoever.
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