Download Analysis of blood flow in the long posterior ciliary artery of the

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IOVS, March 1999, Vol. 40, No. 3
Reports
Analysis of Blood Flow in the
Long Posterior Ciliary Artery
of the Cat
Michael C. Koss
Experiments were undertaken to use a new technique for direct on-line measurement of blood flow in the
long posterior ciliary artery (LPCA) in cats and to evaluate
possible physiological mechanisms controlling blood flow
in the vascular beds perfused by this artery.
PURPOSE.
METHODS. Blood
flow in the temporal LPCA was measured
on a continuous basis using ultrasonic flowmetry in anesthetized cats. Effects of acute sectioning of the sympathetic nerve and changes in LPCA and cerebral blood
flows in response to altered levels of inspired CO2 and O2
were tested in some animals. In others, the presence of
vascular autoregulatory mechanisms in response to stepwise elevations of intraocular pressure was studied.
Blood flow in the temporal LPCA averaged 0.58 ±
0.03 ml/min in 45 cats anesthetized with pentobarbital.
Basal LPCA blood flow was not altered by acute sectioning
of the sympathetic nerve or by changes in low levels of
inspired CO2 and O2, although 10% CO2 caused a modest
increase. Step wise elevations of intraocular pressure resulted in comparable stepwise decreases of LPCA blood
flow, with perfusion pressure declining in a linear manner
throughout the perfusion-pressure range.
RESULTS.
Ultrasonic flowmetry seems to be a useful
tool for continuous on-line measurement of LPCA blood
flow in the cat eye. Blood flow to vascular beds perfused
by this artery does not seem to be under sympathetic
neural control and is refractory to modest alterations of
blood gas levels of CO2 and O2. Blood vessels perfused by
the LPCA show no clear autoregulatory mechanisms. (Invest Ophthalmol Vis Sci. 1999;40:800-804)
CONCLUSIONS.
any techniques have been used for measurement of uveal
blood flow, including direct cannulation of venous outflow channels, measurement of temperature changes in specific ocular regions, and determination of changes of oxygen
tension by placement of oxygen-sensitive electrodes in the
eye.1'2 Quantitative blood flow determinations have been accomplished using tissue clearance of inert gases.3 Drawbacks
of these techniques include the need to enter the globe for
regional measurements and the limited number of data points
that can be determined in a given time.
M
In the most common noninvasive method for ocular blood
flow determinations, radioactively labeled microspheres are
injected and become trapped in proportion to blood flow in
the capillary beds.4'5 This technique enables investigators to
make quantitative regional determinations of ocular blood flow
without surgical intervention, with the major limitation being
the number of blood flow determinations that can be obtained
in each experimental animal. None of these techniques is
suitable for measurement in a single-conduit artery such as the
long posterior ciliary artery (LPCA).
In this study, an attempt was made to measure LPCA blood
flow directly in a continuous manner in the cat eye in vivo, by
using ultrasonic flowmetry and miniature flow probes specifically designed for flow measurements in very small-diameter
arteries. Cats were chosen because the anatomy and perfusion
region of the two LPCAs in this species are more similar to
those of humans than are those of other nonprimate experimental animals.5'6
Experiments were performed to determine the underlying
physiological mechanisms controlling flow in the blood vessels
supplied by this artery, including the role of sympathetic innervation, responsiveness to alterations of blood gases, and
presence of autoregulatory mechanisms in response to stepwise decreases of perfusion pressure.
MATERIALS AND METHODS
General
Adult cats of either sex were anesthetized with 36 mg/kg
pentobarbital injected intraperitoneally. The trachea was intubated for positive-pressure artificial ventilation. A femoral artery and vein were cannulated with a pressure transducer for
measurement of systemic arterial blood pressure (model P23;
Statham, Hato Roy, Puerto Rico) and for intravenous drug
administration, respectively. The animals were positioned in a
stereotaxic device (David Kopf, Tujunga, CA) to immobilize
the head and were placed on positive artificial ventilation with
room air using a respirator (Harvard Apparatus, South Natick,
MA). Neuromuscular relaxation was achieved with 4 mg/kg
intravenous gallamine triethiodide. End-expiratory CO2 levels
were maintained between 35% and 4% by a capnometer (model 2200; Traverse Medical Monitors, San Luis Obispo, CA) by
adjustments of rate and depth of respiration. Determinations of
arterial pH and blood gas levels were measured (model 1304;
Instrumentation Laboratory, Lexington, MA). Heart rate was
derived from the femoral arterial pulse wave. Rectal temperature was maintained at approximately 37°C with a heating pad
and infrared lamp. All physiological responses were recorded
on a polygraph (model 7; Grass, Quincy, MA). The animals
were treated in accordance with the ARVO Statement for the
Use of Animals in Ophthalmic and Vision Research.
Blood Flow Measurements
From the Department of Cell Biology, University of Oklahoma
Health Sciences Center, Oklahoma City.
Supported by Grant EY09344 from the United States Public Health
Service, Washington, DC.
Submitted for publication June 30, 1998; revised October 28,
1998; accepted November 12, 1998.
Proprietary interest category: N.
Reprint requests: Michael C. Koss, Department of Cell Biology,
University of Oklahoma College of Medicine, PO Box 260901, Oklahoma City, OK 73190.
Blood flow in the temporal LPCA was measured by ultrasonic
flowmetry using a transit time ultrasonic flowmeter (model
T106; Transonic, Ithaca, NY) coupled with a 0.5-mm miniature
flow probe (7.2 mHz). With this technique, after extensive
surgery on the lateral orbit, the vessel is exposed and placed
within the window of the probe, which houses two ultrasonic
transducers and a fixed acoustic reflector. Electrical excitation
causes the transducer to emit ultrasound waves, which inter-
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IOVS, March 1999, Vol. 40, No. 3
Reports
801
LPCA 2.0-,
ml/min
1.0-
0- 1
BP 200-1
mmHg
1000FIGURE 1. A polygraph recording of measurement of blood flow in the lateral LPCA of an anesthetized cat
(upper panel), compared with systemic arterial blood pressure measured from a femoral artery (lower
panel). LPCA blood flow was measured using a 0.5-mm V-style flow probe (Transonic, Ithaca, NY). Traces
were taken at various polygraph paper speeds, with mean LPCA blood flow shown at far right of record
(arrow).
sect the blood vessel, as they travel to and from the acoustic
reflector. The flowmeter analyzes the signals as a measurement
of the transit time of the ultrasound wave from one transducer
to the other. The cycle is repeated in the upstream and downstream directions, the difference in transit times being the
measurement of volume flow. With this instrument, volume
flow is independent of the vessel size and is independent of the
velocity profile of blood flow. For example, ultrasonic beams,
which cross the window without intersecting the vessel, do
not contribute to the volume flow signal, with transit time
sampled at all points across the vessel. For more comprehensive technical details and validation of the technique, see Hartman et al.7 "Zero" ocular blood flow was confirmed in each
preparation after the animal's death at the conclusion of the
experiment.
To compare cerebral with ocular blood flow, in some
experiments blood flow was also measured from the parietal
cerebral cortex using laser Doppler flowmetry (TSI-BPM 403A;
Laserflow, St. Paul, MN). With this technique a small surface
area of the cerebral cortex is exposed to diode-evoked laser
light, which is reflected from stationary tissue and moving
blood cells (up to a depth of approximately 1 mm). Only the
laser light backscattered from moving cells undergoes a Doppler frequency shift, which creates Doppler beat frequencies at
the photodetector. An internal computer processes the Doppler spectrum, which is proportional to total blood flow, which
in tvirn is dependent on the relative concentration of moving
erythrocytes in the tissue and average red blood cell velocity.
Experimental Protocols
In initial experiments, LPCA blood flow was measured before
and after sectioning of the ipsilateral sympathetic nerve at the
midcervical level with stability determined for at least 1 hour.
All subsequent experiments were undertaken after initial sectioning of the vagosympathetic nerve trunk. Blood gas levels
were altered by connecting the respirator with gases contain-
ing 5% CO2 or 10% CO2 in air or with 8% or 100% O2. The time
of exposure was approximately 5 minutes, with no more than
two gas challenges administered in any one experimental animal. For alteration of ocular perfusion pressure, the anterior
chamber was cannulated with a 30-gauge needle connected to
a mercury manometer. Intraocular pressure GOP) was measured from a side arm by means of a pressure transducer
(Statham). The blood pressure transducer and IOP transducer
were placed at the level of the eye. Intraocular pressure was
elevated by 10- to 15-mm Hg steps and held constant until
LPCA blood flow stabilized.
Statistical Analysis
Results are expressed as means ± SEM. Perfusion pressures
were calculated as mean arterial pressure (MAP) — IOP. Comparison of single time points within groups was achieved by
paired Student's /-test, with P < 0.05 considered to indicate
statistical significance.
RESULTS
Measurement of LPCA Blood Flow
A typical recording of blood flow in the LPCA of a pentobarbital-anesthetized cat, viewed at a variety of display speeds, is
illustrated in Figure 1. The initial LPCA blood flow was 0.58 ±
0.03 ml/min in 45 anesthetized cats. In 33 animals, the diameter of the LPCA was measured (under magnification) at the
site of the recording probe. In these, the average arterial
diameter was 0.38 ± 0.01 mm.
The first series of experiments was designed to determine
the role of sympathetic neuronal tone and the relative stability
of LPCA blood flow. In the 16 animals studied, there were no
significant alterations of blood flow levels after sectioning of
the vagosympathetic nerve trunk at the midcervical level. In
these preparations, LPCA blood flow was 0.54 ± 0.09 ml/min
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IOVS, March 1999, Vol. 40, No. 3
1. Effects of Alteration of Paco2 and Pao2 on LPCA and Cerebral Blood Flow, Cardiovascular Parameters, and
Blood Gases
TABLE
10% CO2 (n = 17)
5% CO2 (Cn = 9 )
LPCA
(ml/min)
CBF (%
control)
MAP
(mm Hg)
HR
(beats/min)
Paco 2
(mm Hg)
Pao 2
(mm Hg)
pH
8% O 2 (n = 10)
Before
After
Before
After
Before
After
0.46 ± 0.07
0.46 ± 0.07
0.46 ± 0.05
0.52 ± 0.07*
0.43 ± 0.05
0.48 ± 0.07
130 ± 9-5*
187 ± 25*
142 ± 21.9t
166 ± 6.0
159 ± 5.5
159 ± 5.0
167 ± 4.0*
168 ± 5.0
158 ± 5.0
183 ± 5.4
177 ± 5.1t
195 ± 7.0
190 ± 7.0
202 ± 9.0
216 ± 10*
35 ± 1.9
52 ± 1.7*
34 ± 1.0
66 ± 2.2*
38 ± 1.3
36 ± l . l f
130 ± 8.7
7.30 ± 0.02
124 ± 4.5
7.15 ± 0.01*
129 ± 3.9
7.26 ± 0.01
130 ± 5.2
7.04 ± .008*
131 ± 6.8
7.22 ± 0.02
31 ± 0.82*
7.27 ± 0.02*
Values represent means ± SEM; n = number of determinations. CBF, cerebral blood flow; HR, heart rate.
*P<0.0l.
iP< 0.05.
before and 0.58 ± 0 . 1 ml/min 5 to 10 minutes after acute
sympathetic denervation. LPCA blood flow was also stable over
time. In 12 of the studied animals, LPCA blood flow was 0.68 ±
0.07 ml/min immediately before nerve sectioning and was
0.67 ± 0.06 ml/min and 0.66 ± 0.09 ml/min after 30 and 60
minutes, respectively.
LPCA blood flow. Table 1 is a composite representation of the
effects of 5% CO2, 10% CO 2 , and 8% O 2 on LPCA and cerebral
blood flows. Also shown are corresponding cardiovascular
parameters and arterial blood gas changes. In seven animals, a
5-minute period of respiration with 100% O 2 did not alter LPCA
or cerebral blood flow significantly (data not included).
Blood Gas Alterations
Effect of Decreased Perfusion Pressure
Effects of switching the inhaled gas mixture from room air to
an air mixture containing 5% CO 2 and 10% CO 2 was studied.
Application of both CO 2 concentrations (and hypoxia) caused
a graded increase of blood flow in the cerebral cortex, whereas
only the 10% CO 2 concentration caused a slight increase of
The final set of experiments was undertaken to investigate
what effects stepwise elevations of IOP would have on LPCA
blood flow. In six animals, the initial IOP and mean systemic
arterial blood pressure values were 20 ± 3 mm Hg and 174 ±
7 mm Hg, respectively. LPCA blood flow was 0.53 ± 0.09
~ 0.5
1 min
100
-.200
f
E
FIGURE 2. Example of reduction of blood flow in the lateral LPCA in response to stepwise elevations of
IOP in a cat anesthetized with pentobarbital. Lower tracing shows systemic arterial blood pressure (BP)
measured from a femoral artery. Numbers in parentheses represent calculated perfusion pressure (MAP IOP). Note stepwise reduction of LPCA blood flow at each step of IOP elevation.
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10VS, March 1999, Vol. 40, No. 3
A
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DISCUSSION
0.75 T
The LPCA represents an important pathway in the arterial
circulation of the anterior aspect of the eye. In cats and humans, the two LPCAs emerge from the ophthalmic artery and
travel nasally and temporally without further branching until
they enter the root of the iris.5'6 The LPCAs provide the major
supply of blood to the iris and ciliary body, with additional
perfusion of the nasal and temporal peripheral choroid.5
Although there are many techniques and studies of regional ocular blood flows,5 none of these is readily adaptable to
measurement of blood flow in a single ophthalmic conduit
artery such as the LPCA. Because of its small size and relative
0.00
inaccessibility within the orbit, there have been few successful
10
30 50 70 90 110 130 150
170
attempts to measure LPCA blood flow directly with convenPerfusion Pressure
tionalflowprobes. We are aware of only one such report in the
literature, in which evidence supporting myogenic autoregulation in the cat eye was presented.8 The recent introduction of
miniature flow probes coupled with a transit time ultrasound
technology provide the opportunity to make continuous measurements of blood flows in small arteries that are independent
•£
100 • •
of vessel size or probe orientation.7
o
o
Because of the continuous nature of our measurement
/
B 75technique, we were able to study the relative stability of LPCA
/
blood flow and to investigate the possible influence of sympathetic neuronal tone on basal LPCA blood flow. LPCA blood
SOOQ
flow was remarkably stable over time and was not affected by
/
TD
sectioning of the cervical sympathetic nerve. We do not know,
CD
£
25 +
however, the extent to which pentobarbital might have re/
duced basal ocular sympathetic tone.
£
It is the consensus that vascular regions of the eye vasoo
i—i—i—i—i—i—|—i—|—i—h
dilate in response to increased arterial carbon dioxide tenZ
0
20 40 60 80 100 120 140
sions.4'91 ' In contrast, only vessels perfusing the retina seem
Perfusion Pressure
to show strong responses to altered oxygen tensions.5 In the
FIGURE 3. Effects of reduction of perfusion pressure (PP) on blood
present study we found no alteration of LPCA blood flow when
flow in a LPCA of pentobarbital anesthetized cats. Perfusion pressure
cats were exposed to 5% CO2, 8% O2, or 100% O2. Higher
(MAP — IOP) was lowered by stepwise increase in IOP through a
levels of CO2 (10%) produced only a modest elevation of LPCA
cannula inserted into the anterior chamber of the eye. (A) Individual
blood flow of less than 15%, whereas cerebral bloodflowwas
responses for six cats. (B) Composite representation of the pressureincreased by approximately 85%. These minimal LPCA blood
flow correlation normalized by conversion of values to percentage of
flow alterations in response to changes in blood gas levels may
the initial control blood flows (n — 6). Data were grouped in 20-mm
relate to the very high levels of uveal blood flow and the
Hg bins and represent mean responses ± SEM. Note the linear correconsequent low oxygen-extraction ratio.10 The elevation of
lation throughout the pressure-flow range.
LPCA blood flow may also be a result of the systemic blood
pressure elevation seen in these experiments.
In a previous study of choroidal circulation of rabbits and
cats,
Bill9 showed no effect in uveal vascular resistance in
ml/min before and 0.48 ± 0.07 ml/min after cannulation of the
response to changing the inspired oxygen concentration to
anterior chamber.
10% or 100%. The effect of 6% CO2 was inconsistent, whereas
In Figure 2, a polygraph recording shows the initial poradministration of 12% CO2 reduced uveal vascular resistance
tion of an experiment in which IOP was progressively elevated
by 10% to 15% in rabbits and by 20% to 50% in cats. When
from a basal level of 26 mm Hg to 114 mm Hg. With each
these experiments were repeated, using the microsphere techstepwise increase of IOP, there was a corresponding lowering
nique, inhalation of 10% CO2 resulted in a 2.5- to 3-fold inof LPCA blood flow.
crease in blood flow in all ocular tissues.4 Other than differThis relatively linear correlation throughout the entire
ences in measurement technique, the greater responsiveness
pressure-flow curve is illustrated in Figure 3. Figure 3A shows
to CO2 in the later study may have been caused by the longer
the pressure-flow curve in each of the six cats (LPCAflowin exposure to CO2 (15 versus 5 minutes) and the fact that the
milliliters per minute). In Figure 3B, the data were normalized
animals exposed to CO2 had significantly higher blood presto the percentage of initial control values, with calculated
sures than did the control animals. Finally, the absolute
changes in Paco2 (from 26 mm Hg to 81 mm Hg) were fatvalues for perfusion pressure grouped into ±10-mm Hg bins.
greater than those in the present study (see Table 1).
With both types of representation, there was an essentially
linear correlation between LPCA blood flow and the correIn past years, there have been numerous studies in which
sponding calculated perfusion pressure.
the question of whether the ocular vasculature has the ability
B
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Reports
to autoregulate (i.e., maintain blood flow in the face of changing perfusion pressure) has been investigated.5 The general
consensus is that there is a linear correlation between choroidal blood flow and perfusion pressure (MAP — IOP) when IOP
is sequentially elevated.'~'s'10'12"14 However, there is experimental evidence suggesting that autoregulation may be present
in the iris and ciliary body that is masked by the relatively
higher choroidal blood flow when total uveal blood flow is
measured.4 Others have presented evidence for autoregulatory
mechanisms in the cat LPCA and in the rabbit posterior choroid, especially when blood pressure rather than IOP is the
controlling variable.815
With direct measurement of LPCA blood flow, we found
no evidence for autoregulatory control mechanisms; there was
a linear drop in blood flow with every stepwise increase of
IOP. Although it is possible that autoregulation would be seen
if the blood pressure was used as the controlling variable as
observed by others (see earlier). However, other investigators
found that the choroid does not autoregulate if the arterial
blood pressure is reduced by hemorrhage.1416
In conclusion, we have shown that blood flow can be
continuously measured in the cat LPCA by means of ultrasonic
flowmetry. Blood flow in this conduit artery is approximately
0.6 mm/min and, under controlled conditions, is stable for
periods of several hours. Vascular beds supplied by the LPCA
are not reactive to alterations of arterial oxygen tensions and
respond only modestly to large increases of arterial carbon
dioxide levels. The linear pressure-response correlations that
occur when IOP is elevated suggest an absence of autoregulatory mechanisms in vascular beds supplied by the long posterior ciliary arteries.
Acknowledgments
The author thanks Linda Hess for technical support.
References
1. Bill A. Intraocular pressure and blood flow through the uvea. Arch
Ophthalmol. 1962;67:336-348.
Metabolic Acidosis—Induced
Retinopathy in the Neonatal Rat
Jonathan M. Holmes,x Shuichen Zhang,x
David A. Leske,1 and William L Lanier2
Carbon dioxide (CO2)-induced retinopathy
(CDIR) in the neonatal rat, analogous to human retinopathy of prematurity (ROP), was previously described by our
group. In this model, it is possible that CO2-associated
acidosis provides a biochemical mechanism for CDIR.
Therefore, the effect of pure metabolic acidosis on the
developing retinal vasculature of the neonatal rat was
investigated.
PURPOSE.
METHODS. A
preliminary saidy of arterial blood pH was performed to confirm acidosis in our model. In neonatal rats
with preplaced left carotid artery catheters, acvite blood gas
IOVS, March 1999, Vol. 40, No. 3
2. Aim A, Bill A. The oxygen supply to the retina, I: effects of changes
in intraocular and arterial blood pressures, and in arterial Po2 and
Pco2 on the oxygen tension in the vitreous body of the cat. Ada
Physiol Scand. 1972;84:26l-274.
3. Yu D-Y, Alder VA, Cringle SJ, Brown MJ. Choroidal blood flow
measured in the dog eye in vivo and in vitro by local hydrogen
clearance. Polarography: validation of a technique and response to
raised intraocular pressure. Exp Eye Res. 1988;46:289-303.
4. Aim A, 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. Ada Physiol Scand.
1972;84:306-319.
5. Aim A. Ocular circulation. In: Hart WM, ed. Adler's Physiology of
the Eye: Clinical Application. St. Louis: Mosby-Year Book; 1992;
198-227.
6. Wong VG, Macri FJ. Vasculature of the cat eye. Arch Ophthalmol.
1964;72:351-358.
7. Hartman JC, Olszanski DA, Hullinger TG, Brunden MN. In vivo
validation of a transit time ultrasonic volume flow meter. / Pharmacol Toxicol Methods. 1994;31:153-l60.
8. Best M, Gerstein D, Wald N, Rabinovitz AZ, Hiller GH. Autoregulation of ocular blood flow. Arch Ophthalmol. 1973;89:l43-l48.
9. Bill A. Aspects of physiological and pharmacological regulation of
uveal blood flow. Ada Soc Med Upsaliensis. 1962;67:122-134.
10. Aim A, Bill A. Blood flow and oxygen extraction in the cat uvea at
normal and high intraocular pressures. Ada Physiol Scand. 1970;
80:19-28.
11. Friedman E, Chandra SR. Choroidal blood flow III. Effects of
oxygen and carbon dioxide. Arch Ophthalmol. 1972;87:70-71.
12. Armaly MF, Araki M. Effect of ocular pressure on choroidal circulation in the cat and Rhesus monkey. Invest Ophthalmol. 1975;
14:584-591.
13. Duijm HFA, Rulo AHF, Astin M, Maepea O, Van Den Berg TJTP,
Greve EL. Study of choroidal blood flow by comparison of SLO
fluorescein angiography and microspheres. Exp Eye Res. 1996;63:
693-704.
14. Friedman E. Choroidal blood flow pressure-flow relationships.
Arch Ophthalmol. 1970;83:95-99.
15. Kiel JW, Shepherd AP. Autoregulation of choroidal blood flow in
the rabbit. Invest Ophthalmol Vis Sci. 1992;33:2399-24l0.
16. Bill A. Effects of acute hemorrhage on the blood flow in the eye
and some other tissues in the rabbit: role of sympathetic nerves.
Klin Monatsbl Augenheilkd. 1984; 184:305-307.
samples were taken 1 to 24 hours after gavage with either
NH4C1 1 miIlimole/100 g body weight or saline. In the subsequent formal retinopathy study, 150 newborn SpragueDawley rats were raised in litters of 25 and randomly assigned to be gavaged twice daily with either NH4C1 1
millimole/100 g body weight (n — 75) or saline (n = 75)
from day 2 to day 7. After 5 days of recovery, rats were killed,
and retinal vasculature was assessed using fluorescein perfusion and ADPase staining techniques.
RESULTS. In
the preliminary pH study, the minimum pH
after NH4C1 gavage was 7.10 ± 0.10 at 3 hours (versus
7.37 ± 0.03 in controls, mean ± SD, P < 0.01). In the
formal retinopathy study, preretinal neovascularization
occurred in 36% of acidotic rats versus 5% of controls
(P < 0.001). Acidotic rats showed growth retardation
(final weight 16.5 ± 3.0 g versus 20.2 ± 2.6 g, P <
0.001). The ratio of vascularized to total retinal area was
smaller in acidotic rats (94% ± 4% versus 96% ± 2%,
P < 0.001).
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