Download Failure of unilateral carotid artery ligation to affect pressure

Failure of unilateral carotid artery ligation
to affect pressure-induced interruption
of rapid axonal transport in primate
optic nerves
Ronald L. Radius,
Eileen Lerner Schwartz,
and Douglas R.
Anderson
Previous experiments showed that optic nerve axonal transport can be blocked at the level of
the lamina cribrosa by elevated intraocular pressure. In an effort to discover if this blockage
might be secondary to pressure-induced ischemia, we studied the effect of unilateral common
carotid artery ligation upon the pressure-induced interruption of axonal transport. In 13 owl
monkeys (Aotus trivirgatus), the right common carotid artery was ligated within the anterior
cervical triangle. Three days later, ophtalmodynomometry was performed, on all experimental
eyes. In nine of the 13 animals, this estimate of ophthalmic artery pressure was 10 to 20 mm Hg
less in the right compared to the left eye. Optic nerve axonal transport was studied in right and
left eyes during 5 hours of increased intraocular pressure (ocular pressure 35 mm Hg less than
mean femoral artery blood pressure). No significant difference in the extent to which the
transport mechanisms were interrupted could be demonstrated when comparing right and left
eyes of the experimental animals. These observations fail to support a vascular mechanism for
this pressure-induced interruptioii of axonal transport.
Key words: axonal transport, optic nerve, ophthalmodynomometry, glaucoma,
intraocular pressure, carotid artery ligation
I n primate eyes subjected to elevated intraocular pressure, several investigators have
demonstrated an interruption of axonal transport at the level of the lamina cribrosa. 1~9 It
has been suggested that this pressureFrom the William L. McKnight Vision Research Center,
Bascom Palmer Eye Institute, University of Miami
School of Medicine, Miami, Fla., and The Eye Institute, Medical College of Wisconsin, Milwaukee.
Supported in part by PHS Research Grants EY-00031
and EY-0093, and by PHS National Research Service
Award EY-07021, all awarded by the National Eye
Institute, Bethesda, Md. Dr. Anderson is also supported by Research to Prevent Blindness, Inc., as a
William and Mary Greve RPB International Research
Scholar.
Submitted for publication Dec. 1, 1978.
Reprint requests: Ronald L. Radius, The Eye Institute,
8700 West Wisconsin Ave., Milwaukee, Wis. 53226.
induced interruption of the normal transport
mechanism may participate in pathophysiology of glaucomatous optic neuropathy. 1 " 10
Although it is known that brief periods of impaired axonal transport can be well tolerated
with reversibility of the blockade, 2 ' n ~ 13 interruption of this essential neuronal function
leads to failure of neuron function,14' 15 and if
prolonged (perhaps for more than 1 to 2
weeks) it may be lethal to the neuron. 16 " 19
Hypothetically, an irreversible block of normal axonal transport at the lamina cribrosa
could be the mechanism of axon destruction
in eyes with elevated intraocular pressure.
Among other possible factors, impaired
transport may reflect direct mechanical compression of individual axons or neuronal ischemia secondary to vascular compromise. The
0146-0404/80/020153+05$00.50/0 © 1980 Assoc. for Res. in Vis. and Ophthal., Inc.
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153
154 Radius, Schwartz, and Anderson
present study was designed to try to investigate the mechanism by comparing the degree
of impaired axonal transport between the
right and left eyes in primates with unilateral
carotid artery ligation. By contrasting paired
eyes maintained at identical pressure levels,
the extent to which vascular factors (carotid
ligation) aggravate pressure-induced interruption of axonal transport can be studied.
Materials and methods
The right common carotid artery of 13 owl
monkeys (Aotus trivigatus) was ligated within the
anterior cervical triangle. Throughout the operative procedure, these animals were maintained
under deep anesthesia by intraperitoneal injection
of 0.1 cc pentobarbital (Nembutal) and intramuscular injection of 0.05 cc phencyclidine (Sernylan).
The anterior cervical triangle was entered through
a skin and fat incision; the right common carotid
artery was identified deep to the sternocleidomastoid muscle. The artery was isolated from adjacent
structures by blunt dissection, with special care
taken to minimize the surgical trauma to these
tissues (including the cervical sympathetic chain).
The artery was ligated with two interrupted 4-0
black silk sutures. After the skin incision was
closed with interrupted silk sutures, the pupils of
both eyes were dilated with two or three drops of
1% atropine. The animals were returned to their
cages.
Three days after this surgical procedure the
animals were again anesthetized with 0.1 cc pentobarbital (Nembutal 100 mg/cc) and 0.05 cc
phencyclidine (Sernylan, 50 mg/cc). A polyethylene catheter (PE 60, 0176 mm inside diameter)
was inserted into the right femoral artery and connected to a pressure transducer for monitoring
systemic blood pressure. A 25-gauge needle, attached to a fluid reservoir via polyethlene tubing,
was inserted into the anterior chamber of each
eye. The mean artery blood pressure (diastolic
pressure plus one-third the difference between
systolic and diastolic pressures), as well as the systolic and diastolic pressures, were recorded, and
the ophthalmic artery systolic and diastolic pressures were estimated by ophthalmodynomometry.
Ophthalmodynomometry was performed in a
masked fashion. On instruction from the fundus
observer, an assistant increased or decreased the
intraocular pressure by raising or lowering the
fluid reservoir. Diastolic pressure was recorded as
the level at which small increases in pressure were
reported to produce pulsations of the central reti-
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Invest. Ophthalmol. Vis. Sci.
February 1980
nal artery. The level at which small fluctuations in
pressure eliminated pulsations completely, blanching all disc vasculature, was recorded as the systolic pressure. Each eye was examined individually. The two eyes were then compared simultaneously to minimize error induced by fluctuations
in systemic as well as ophthalmic artery pressure.
The intraocular pressure in these experimental
eyes was then reduced to atmospheric pressure,
and 0.1 cc (100 mCi) of tritiated leucine (L-leucine-4-5-;iH(B); 30 to 50 /u.Ci/mM; New England
Nuclear) was injected intravitreally. The injection
sites were sealed by cyanoacrylate tissue glue. Intraocular pressures were elevated by raising the
fluid reservoir until the ocular perfusion pressure
(the mean femoral artery pressure minus the intraocular pressure) was 35 mm Hg. In one pair of
eyes the perfusion pressure was 25 mm Hg.
Intraocular pressures of all 26 eyes were maintained at this level for 5 hours; specimens were
then fixed in vivo by intra-arterial retrograde (abdominal aorta) perfusion of 100 cc saline, followed
by 200 cc of 10% formalin. An incision in the right
ventricle allowed free egress of blood, saline, and
fixative. The intraocular pressure in all eyes was
reduced to 10 mm Hg immediately prior to this
infusion.
Experimental eyes and attached optic nerves
were removed and tissue specimens containing
the optic nerve head embedded in paraffin. Ten
step sections, every 100 /xm, were taken through
the optic nerve head, placed on a clean glass slide,
and coated with radiosensitive emulsion. These
autoradiographs were developed after 1 week of
exposure as previously described. 1
Each autoradiograph was examined separately
in a masked fashion by each one of the three authors. As in other studies,'~ 3- "~9" 20"~" a quantitation score of 0 to 44- was assigned to each tissue
radiograph. This numerical score reflected the degree of label accumulation seen in the region of
the lamina cribrosa. To judge accumulation, the
observer took into account the overall degree of
label incorporation in the particular specimen by
noting the density of label in the retina, anterior
optic nerve head, and retrolaminar optic nerve.
For example, there was not judged to be any accumulation unless the density of label in the
lamina cribrosa was greater than that of the retina
and prelaminar optic nerve head. Examples of various grades of blockage have been published previously. '~3
An additional eight animals were studied in an
identical fashion except that carotid surgery was
not performed. Perfusion pressure in these eyes
Volume 19
Number 2
IOP, ischemia, optic nerve axonal transport 155
Table I. Pressure-dependent interruption of optic nerve axonal transport in right and
left eyes of normal primates with right common carotid arteiy ligation (perfusion
pressure 35 mm Hg)
Monkey
No.
517
532
553
570
5101
537
540
541
554
563
572
533
549
Ophthalmodynomometry
systolic /diastolic (mm Hg)
Degree of blockade
(a ut ora di ogra p h y)
Femoral artery pressure
(systolic 1 diastolic)
(mm Hg)
Right eye
Left eye
Right eye
Left eye
Eye loith
most blockade
150/120 (130)*
200/170 (180)
190/155 (168)
145/105 (118)
121/97 (105)
170/140 (150)
165/120 (135)
190/124 (146)
205/130 (155)
225/160 (182)
165/120 (135)
170/125 (140)
165/120 (135)
140/121 (127)*
131/112 (118)
137/112 (120)
83/71 (75)
160/170 (140)
146/120 (129)
125/103 (110)
141/121 (128)
179/145 (156)
145/130 (135)
182/156 (165)
166/113 (131)
143/121 (129)*
168/135 (146)
160/130 (140)
108/84 (92)
158/125 (136)
167/137 (147)
152/129 (137)
165/135 (145)
200/165 (176)
155/140 (145)
225/185 (198)
160/112(128)
1.6
2.0
0.0
1.0
0.0
0.1
0.0
0.0
0.1
1.0
2.0
1.1
0.0
0.3
2.0
0.0
1.0
1.9
1.0
0.7
3.6
1.6
2.0
3.0
-
Right
Equal
Equal
Equal
Left
Left
Left
Left
Left
Left
Left
-
* Mean in parentheses.
[Studied at a perftision pressure of 25 mm Hg.
was maintained at 35 mm Hg throughout the experimental period. Paired eyes were compared for
degree of blocked axonal transport following this
period of elevated intraocular pressure.
The distribution of animals with carotid ligation
within each of three possible groupings, i.e., right
eye blocked more than left, left eye blocked more
than right, and both eyes blocked equally, was
compared with that of the control animals without
carotid ligation. Differences between these two
populations were tested for statistical significance
by chi-square analysis. A paired Student's t-test
analysis was used to test the difference between
right and left eyes of the graded values of blocked
transport.
Table II. Pressure-dependent interruption
of optic nerve axonal transport in right and
left eyes of normal primates (perfusion
pressure 35 mm Hg)
Degree of blockade
Monkey No.
Right eye
Left eye
Eye with
most blockade
439
440
445
449
538
441
443
527
2.5
1.5
2.0
0.0
1.3
1.0
1.0
0.8
1.0
0.3
2.0
0.0
1.3
3.5
2.5
1.4
Right
Right
Equal
Equal
Equal
Left
Left
Left
Results
All 13 animals survived ligation of the right
common carotid arteiy without clinical neurologic deficit. In nine of the 13 animals,
right ophthalmic artery pressure was 10 to 20
mm Hg less than that of the left (Table I). In
one animal, reliable readings were not possible because of poor needle placement and
corneal clouding. In the three remaining
animals, the difference in ophthalmic arteiy
pressures was less than 5 mm Hg. The left
eyes in two of the 13 experimental animals
were damaged during tissue processing to an
extent preventing adequate microscopic examination. Data from the remaining 11 ani-
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mals were used to compare the degree of
transport blockage seen in right vs. left eyes.
In 15 of the 22 eyes examined, there was
accumulation of labeled material at the level
of the lamina cribrosa. As judged by qualitative scoring of tissue autoradiographs, seven
animals showed less blocked transport in the
right experimental eye, three of the animals
had equal degrees of disruption in both eyes,
and one animal experienced less block in the
right eye. The average score and standard
error given to right eyes was 0.71 ± 0.84;
that of the left eyes was 1.5 ± 1.1. Although
the mean score for right eyes fell below that
Invest. Ophthalmol. Vis. Sci.
February 1980
156 Radius, Schwartz, and Anderson
for left eyes, this difference is not statistically
significant (0.1 < p < 0.2, t = 1.75).
Table II presents the results from eight
pairs of eyes in animals without carotid surgery. In two animals the right eye had
greater block. In three animals there was no
difference between eyes, and in three animals there was more block in the left eye
than in the right eye. No difference between
this distribution and that seen in animals with
carotid ligation was established statistically
(p < 0.1, X 2 = 2.29).
Discussion
In previous studies of pressure-induced interruption of axonal transport in the optic
nerve it has been noted that, corresponding
with decreasing perfusion pressure (defined
as mean femoral arteiy blood pressure minus
intraocular pressure), there is a greater degree of the transport abnormality at the
lamina cribrosa.' This observation is compatible with the premise that elevated intraocular pressure compromises bloodflowto
regions of the optic nerve head, specifically
within the lamina cribrosa, and that secondary axonal ischemia results in blocked axonal
transport. However, decreased perfiision
pressures necessarily implies increased intraocular pressures. In these previous experiments1 it was therefore impossible to
define whether increased interruption in
transport resulted from some direct mechanical pressure effect upon optic neurons
as a result of these higher pressures.
The present study was designed to investigate the degree to which differences in
ophthalmic artery blood pressure might aggravate pressure-induced blockage of axonal
transport in paired eyes studied at equivalent
intraocular presures. If greater block had
been demonstrated in the right eye (the side
with a ligated carotid artery), a vascular
mechanism for the pressure-induced block
would have been demonstrated. As it is,
these experimental results are inconclusive.
The amount of transport block seen in right
eyes was not significantly (p > 0.1) different
from that seen in left eyes. When comparing
paired eyes, the distribution of animals into
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three groups—right eye more blocked,
equal block between eyes, or left eye more
blocked—suggested that the arterial ligation
protected the eye from pressure insult. This
observation was especially true if only eyes
with unequal ophthalmic artery pressures are
considered in the analysis. However, by chisquare testing, this distribution is not significantly (p > 0.1) different from that seen
when eyes of normal exeprimental animals
are studied.
Unfortunately, even this negative result is
not definitive. Although ophthalmodynamometry measurements are sensitive enough
to demonstrate reduced intra-arterial pressure in the ophthalmic artery,23"24 we cannot
be certain that the carotid ligation produced a
reduction in blood flow at the nerve head.
Autoregulatory mechanisms in the optic
nerve must be considered. These mechanisms cannot be directly quantified, but they
may be a significant factor in the dynamics of
blood flow, oxygenation, and tissue physiology (including axonal transport) in eyes with
reduced perfiision pressure.
Thus, as in a previous study altering
carotid artery pressure.22 we can say only that
the present experiments fail to support the
hypothesis that pressure-induced block of
axonal transport is produced via local tissue
ischemia. These results are consistent with
either of two possible theories: transport abnormalities may be a consequence of direct
mechanical compression of neurons or they
may be caused by axonal ischemia due to reduced nerve head perfusion.
We were most fortunate to have the skillful assistance
of Mr. E. Barry Davis.
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