Download The continuous and quantitative observation of permeability

The continuous and quantitative
observation of permeability changes of the
blood-aqueous barrier in allergic
inflammation of the eye
Mariko Okada and Kohkichi
Shimada
Permeability changes of the blood-aqueous barrier were studied in the eyes of rabbits subjected
to immunologic inflammation. The changes were investigated by slit-lamp microphotometers.
The leakage of fluorescein-labeled. rabbit serum albumin into the anterior chamber was observed, in eyes inflamed by reverse passive Arthus reactions. The permeability of the bloodaqueous barrier changed in biphasic pattern in allergic inflammation; the first phase began 5
min after the antigen challenge and lasted, for 1 hr, followed by the late phase at which the dye
concentration reached a peak 2 hr after the challenge and then gradually decreased.
Key words: continuous observation, permeability, blood-aqueous barrier,
ocular inflammation, allergic inflammation
B
y a variety of agents, the blood-aqueous
barrier is broken down, and serum proteins
flow into the aqueous humor through the
barrier.' The increase of protein concentration in the aqueous humor is associated with
the severity of the ocular inflammation.
Anterior chamber taps have been performed for the measurement of protein concentration in the aqueous humor. 2 ' 3 However, a change of protein concentration cannot continuously be observed. Recently, a
method using a slit-lamp microphotometer
has been developed for the continuous observation of protein concentration in the an-
From the Tokyo Metropolitan Institute of Medical Science, Tokyo and Jichi Medical School, Tochigi-ken,
Japan.
This work was partly presented at the 23rd International
Congress of Ophthalmology, May 1978, Japan.
Submitted for publication Sept. 6, 1978.
Reprint requests: Dr. Kohkichi Shimada, Department of
Ophthalmology, Jichi Medical School, Kawachi-gun,
Tochigi-ken, Japan, 329-04.
terior chamber. Even by this means, the accurate measurement of protein concentration
of the aqueous humor is still very difficult;
corneal opacity often interferes. 4 ' 5
The present paper offers preliminary data
on a new method for the continuous and
quantitative measurement of permeability
changes of the blood-aqueous barrier by slitlamp microphotometry, with the use of fluorescein-labeled rabbit serum albumin (FITCRSA) as an indicator.
Materials and methods
Animals. New Zealand albino rabbits weighing
2.0 to 3.5 kg were employed. Before use, all eyes
were examined for the pre-existing diseases.
Antiserum and antigen. Bovine gamma globulin
(BGG; Miles Laboratories, Inc., Kankakee, 111.)
was dissolved in phosphate-buffered saline (pH
7.2) at the concentration of 2%. Anti-BGG serum
(2.2 ing of antibody N per milliliter) was prepared
by repeated immunization of rabbits with BGG in
complete Freund's adjuvant. A normal rabbit
serum was used as the control. These sera and
the antigen solution were sterilized by passage
0146-0404/80/020169+07S00.70/0 © 1980 Assoc. for Res. in Vis. and Ophthal., Inc.
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169
Invest. Ophthalmol. Vis. Sci.
February 1980
170 Okada and Shimada
R
c
I
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.
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c E
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-esc
L
1
40
u.
0
30
60
90
120
Time a f t e r Injection of Antigen ( minutes )
150
180
Fig. 1. Fluorescence intensity in the anterior chamber of a rabbit during inflammation induced
by an intravenous challenge with antigen 4 days after a single injection with anti-BGG rabbit
serum into left eye (L) and normal rabbit serum into right eye (R). FITC-RSA was also injected
intravenously 30 min before the challenge.
through a 0.22 fjcm Millipore filter (Millipore
Corp., Bedford, Mass.) and stored at -20° C
without preservative.
FITC-RSA. The rabbit serum albumin fraction
(RSA) was obtained by repeated salting out of
pooled normal sera in 50% saturated ammonium
sulfate. RSA was conjugated with fluorescein isothiocyanate (Sigma Chemical Co., St. Louis, Mo.)
according to the method of Riggs et al.6 and stored
at —20° C after sterilization by Millipore filtration.
Antibody injection. An intravitreal injection
was performed after topical anesthesia with 0.5%
proparacaine HC1. The eye was fixed by gently
grasping the superior rectus muscle at its insertion
with forceps; it was rotated downward. A sharp
27-gauge needle (Termo Co., Ltd., Tokyo, Japan)
was then inserted at a slight backward angle 2 mm
behind the corneoscleral junction, avoiding the
lens. A 0.1 ml amount of anti-BGG rabbit serum
was injected into the center of the vitreous. The
initial reaction to the trauma of injection had
completely subsided within 4 days in all eyes. Just
after resolution of the reaction, the challenge was
performed by the intravenous injection of 2.5 ml
of the BGG solution (50 mg).
Fluorescence measurement. The measurement
of fluorescein concentration in the anterior chamber of eyes was carried out using a slit-lamp microphotometer. A 0.5 ml amount of FITC-RSA per
kilogram of body weight was injected intravenously 30 min before the challenge. The intensity
of fluorescence from materials leaking into the anterior chamber was measured by a slit-lamp microphotometer (Hamamatsu I.V. Co., Hamamatsu,
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Japan), constructed according to the methods of
Maurice4 and Mishima.7 Our instrument had two
microphotometers connected with a recorder to
observe both eyes simultaneously and to record
data automatically on one chart. The sensitivities of
the microphotometerfittedon the instrument were
adjusted to the same level with a standard fluorescein solution just before use and checked for their
stability just after each experiment.
Tissue preparation. Animals were killed by intravenous air injection. The eyes were removed
and fixed with 10% formaldehyde. Paraffin sections were cut at 3 fxm and stained with hematoxylin and eosin (H&E).
Results
The experiments were designed to observe
continuously and quantitatively the permeability changes of the blood-aqueous barrier in allergic inflammation of the eye by
slit-lamp microphotometry with FITC-RSA
as an indicator.
Concentration of FITC-RSA in the anterior chamber. Nine animals were employed. Anti-BGG rabbit serum was injected
into an eye on one side, and the control eye
on the opposite side received the same volume of normal rabbit serum. Just after resolution of the initial reaction to the trauma of
intraocular injection, a challenge was performed by the intravenous injection of antigen. FITC-RSA was also injected intravenously 30 min before the challenge.
Volume 19
Number 2
Permeability changes of blood-aqueous barrier
171
z
100
so
60
50 "
30
60
90
120
150
180
210
210
TIME AFTER FITC-RSA INJECTION ( MINUTES )
Fig. 2. Relative concentrations of FITC-RSA in the circulating blood of two rabbits after the
intravenous injection of FITC-RSA. The dye intensity was measured by a slit-lamp microphotometer, and ratios between concentrations observed at 30 min and at other time
intervals were calculated.
Fluorescence was not demonstrable in the
anterior chamber of either eye until 5 min
after the antigen stimuli. In the anti-BGGinjected eye, the dye appeared in the anterior chamber through the pupil about 5 min
after the challenge. The fluorescein intensity
increased over the next 15 min but soon
reached a plateau. The dye concentration was
maintained for about 50 min, after which it
again began to increase remarkably and then
gradually reached a peak 2 hr after the challenge (Fig. 1). After the passage of the peak,
the concentration gradually decreased, and
the dye had completely disappeared from the
anterior chamber by the next day. Meanwhile, FITC-RSA was undetectable in the
anterior chamber of control eyes throughout
the experiment.
Concentration of FITC-RSA in circulating
blood. Two animals were bled from an auricular vein at 30 min intervals after intravenous injection of FITC-RSA. The sera were
then separated by centrifugation at 2500 x g
for 10 min. The fluorescein intensity of these
sera was measured by a slit-lamp microphotometer. Ratios between dye concentrations
at 30 min and those observed at other times
were calculated. Fig. 2 shows changes of the
ratios in two animals. The dye concentration
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of blood in peripheral vein decreased steeply
during the first hour, moderately during the
next hour, and then gradually thereafter.
Ratio of FITC-RSA concentration in the
anterior chamber and to that in circulating
blood. The amount of FITC-RSA that leaks
into the aqueous humor depends on the concentration of the dye in the circulating blood,
which varies as shown in Fig. 2. Therefore
the ratio of dye concentration found in the
anterior chamber to that found in the circulating blood was considered to be a more appropriate indicator for permeability changes
of blood-aqueous barrier than the concentration of the dye in the anterior chamber itself.
In nine animals, the dye concentration in
the circulating blood was measured 90 min
after the FITC-RSA injection, and concentrations for other intervals were calculated
with the above values and the standard curve
(Fig. 2). The time course of the ratio of dye
concentration in the anterior chamber to that
observed in the circulating blood is shown in
Fig. 3.
Pathological observation. Histopathological examination was performed on intraocular
tissues 30 min after the antigen challenge.
In the antibody-injected eyes, marked
edema was observed in the interstitial tissues
Invest. Ophthalmol. Vis. Sci.
February 1980
172 Okada and Shimada
u.
O
ANT 1 SERUM
•
CONTROL EYE
20
60
90
120
150
180
TIME ( MINUTES )
Fig. 3. Relative concentrations of FITC-RSA in the anterior chamber of eyes during allergic
inflammation in rabbits. The zero time indicates the antigenie challenge. The points plotted
represent mean values of nine rabbits.
of the ciliary and indie processes. The ciliary
body and the proximal part of the iris were
slightly inflamed. A few polymorphonuclear
leukocytes were found at the filtration angle
and in the vitreous humor near the ciliary
body. There were no pathological findings in
other intraocular tissues. The control eyes
were normal. This fact indicates that the sites
of immune reactions in our experiments were
restricted mainly to the ciliary and iridic
processes.
Discussion
The purpose of our experiments was to develop a method which would allow us to observe continuously and quantitatively the
fluorescein intensity in the anterior chamber
by a slit-lamp microphotometer, placing emphasis on the following three points: (1) simultaneous observation of both eyes of a
rabbit, (2) induction of standardized inflammation in one eye, and (3) use of a stable
indicator which enters the anterior chamber
of normal rabbit eyes only in trace quantities
and is not irritating.
In order to meet the first requirement, two
microphotometers were used, connected so
as to record data for both eyes on a chart.
With this instrument, both eyes can be observed simultaneously. Ocular inflammation
was induced by an immune reaction: the so-
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called reverse passive Arthus reaction. Eyes
were sensitized by an intravitreal injection of
anti-BGG rabbit serum. After resolution of
the inflammation that resulted from the
trauma of injection, an intravenous challenge
of antigen caused a moderate and easily standardized inflammation in eyes which had received antibody at an earlier time. As an indicator, we used FITC-RSA. Although fluorescein sodium itself passes through the
blood-aqueous barrier of a normal rabbit, 8
FITC-RSA does not. It was not detected in
the anterior chamber of eyes injected with
rabbit serum.
In all, permeability changes of the bloodaqueous barrier were observed in nine rabbits. The fluorescence intensity in the anterior chamber of inflamed eyes appears to
change in two steps as shown in Fig. 3. Five
minutes after the intravenous challenge of
antigen, the dye appeared in the anterior
chamber through the pupil. The increase of
dye concentration was steep during the first
30 min and gradual during the following 30
min, after which the fluorescence was again
increased markedly in the anterior chamber.
The concentration reached a peak 2 to 3 hr
after the stimuli and then gradually decreased. The results suggest that permeability changes of the blood-aqueous barrier of
the rabbit may be biphasic in the case of
Volume 19
Number 2
Permeability changes of blood-aqueous barrier 173
Fig. 4. Eye injected with anti-serum (upper), taken 30 min after an intravenous challenge with
antigen. Marked edema was observed in the stroma of the ciliary and indie processes. The
ciliary body and the proximal part of iris were slightly inflamed. The control eye (lower)
appeared completely normal. (H&E; X32.)
inflammation produced by reverse passive
Arthus reactions.
Di Rosa and colleagues9 have shown that
the vascular permeability in rat feet by injection of carrageenin also changes in a biphasic
pattern. The immediate phase of the increased permeability is observed during the
first hour, and is considered to be mediated
by histamine and serotonin. Therefore we
examined effects of antihistamines. Promethazine (8.5 mg/kg), an H-l receptor blocker,
was administered intravenously in eight
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rabbits 5 min before and 60 min after the
antigen challenge. As shown in Fig. 5, the
drug suppressed dye leakage into the anterior chamber throughout the period of experiments. After 60 min, however, dye permeated the blood-aqueous barrier. As shown
in Table I, there was no significant difference
of the increase of dye concentration in the
anterior chamber after 90 min between the
two groups of animals. The drug markedly
suppressed vascular responses during the
first hour, but the later responses were not
Invest. Ophthalmol. Vis. Sci.
February 1980
174 Okada and Shimada
O
ANT I SERUM
•
CONTROL EYE
60
90
120
150
180
TIME ( MINUTES )
Fig. 5. Effects of promethazine on permeability changes of the blood-aqueous barrier in
allergic inflammation of eyes. Promethazine was administered as described in the text. The
antigen was injected intravenously at zero time. All values are the mean ± S.E. The p values
were calculated by Welch's t test.
Table I. Effect of promethazine on the permeability change during the
late phase of ocular inflammation of our model
AF
Untreated (n=8)
Promethazine-treated (n=7)
p value*
90 min
120 min
180 min
17.9 ± 3.9
9.2 ± 2.1
<0.05
23.0 ± 5.0
13.5 ± 3.7
27.3 ± 4.6
22.9 ± 5.3
n.s.
U.S.
AF = dillerence ol relative dye concentration between indicated times and 60 min alter the antigen challenge; n.s. = not significant;
n = number ol animals.
*p values were calculated by Student's t test.
affected at all. This result indicates that permeability changes of the blood-aqueous barrier are biphasic and that the first phase of
the inflammation may be partly mediated by
histamine.
Wilhelm and Manson10 also reported biphasic changes of vascular permeability after
thermal inflammation of rabbit skin. In their
experiments, the first phase began a few
minutes after the stimulus and lasted for 30
min. The time course of vascular leakage is
the same in the two experiments, but there is
a lag in the first response in ours. The lag is
due to the delay in observing the dye concentration at the center of the anterior chamber.
Since it takes several minutes for dye to appear in the anterior chamber after the leakage through small blood vessels in ciliary and
iridic processes, the increased vascular permeability may well have occurred a few minutes after the intravenous challenge. Finally,
after ultraviolet light injury, vascular permeability also changes biphasically.11 However,
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the late phase slowly develops, reaching a
peak in 23 to 26 hr. On the contrary, the peak
of the last phase in our experiments occurred
in 2 to 3 hr. This suggests that the change in
vascular permeability during the late phase of
ultraviolet light injury differs from that in allergic and thermal inflammation.
By the method described above, continuous and quantitative observation of permeability change in the blood-aqueous barrier is
possible. As a result, effects of pharmacological agents on ocular inflammation are
being examined in detail by this technique.
We thank Dr. Richard G. O'Connor from the Proctor
Foundation, San Francisco, Calif., for his helpful suggestions and encouragement in the preparation of the
manuscript and Ms. Mariko Asoh for help in preparing
the manuscript.
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