Sucrose Permeability of the Blood-Retinal and
Blood-Brain Barriers
Effects of Diabetes, Hypertonicity, and lodate
Steven R. Ennis and A. Lorris Berz
The permeabilities of the blood-retinal (BRB) and blood-brain (BBB) barriers to sucrose were determined
simultaneously using an intravenous injection technique in the rat. The method involved direct sampling
of retinal tissue in order to avoid errors caused by sucrose penetration across other components of the
blood-ocular barrier. The permeability X surface area (PS) product for the BRB was approximately
four times greater than for the BBB. Intracarotid infusion of a hypertonic arabinose solution resulted
in a dose-dependent increase in the permeability of both barrier systems. In contrast, 24 hr after treatment
of animals with iodate, the PS product for the BRB but not the BBB was increased. The permeability
of the blood-retinal barrier to sucrose was measured in normal and 2-, 6-, and 20-week streptozocin
diabetic rats. The BRB was unaffected at 2 and 6 weeks of diabetes, and showed only a small increase
in permeability at 20 weeks. Our results suggest that alterations in the blood-ocular barrier in early
diabetes do not result from an increased passive permeability of the BRB. The method described should
permit direct comparison of BRB and BBB permeabilities to a variety of compounds under various
conditions. Invest Ophthalmol Vis Sci 27:1095-1102, 1986
Both the retina and the brain are isolated from the
blood by permeability barriers which greatly restrict
the movement of most polar solutes between plasma
and tissue. The blood-brain barrier (BBB) is formed
by a continuous layer of endothelial cells sealed together
by tight junctions, while the blood-retinal barrier
(BRB) is formed not only by the continuous endothelium and tight junctions of the retinal capillaries, but
also by the retinal pigment epithelium (RPE).1 The
permeability of the BRB and BBB are qualitatively
similar,1"4 however, there have been no direct quantitative comparisons.
In this report, we described a method for simultaneous determination of the sucrose permeability of the
BRB and BBB of the rat. Permeability measurements
were made in control animals and after experimental
modification of the barriers by infusion of hypertonic
arabinose, which is known to affect both the BRB and
BBB, or by administration of iodate, which affects the
BRB but has not been studied at the BBB. We then
measured the permeability of the BRB after 2, 6, and
20 weeks of experimental diabetes.
From the Departments of Pediatrics and Neurology, University
of Michigan, Ann Arbor, Michigan.
Supported in part by NIH Grant EY-03772 and by a research
fellowship from the American Heart Association of Michigan. ALB
is an Established Investigator of the American Heart Association.
Submitted for publication: September 11, 1984.
Reprint requests: Steven R. Ennis, R6060, Kresge Research II,
University of Michigan, Ann Arbor, MI 48109-0570.
Measurement of Sucrose Uptake
Materials and Methods
Experimental Animals
All procedures were conducted in a manner consistent with the ARVO Resolution on the Use of Animals
in Research. Male Long Evans rats weighing between
250 and 400 gm were anesthetized with sodium pentobarbital (50 mg/kg). Following insertion of a tracheostomy tube, catheters of PE-50 tubing filled with heparinized saline were placed in both femoral arteries
and in the left femoral vein. The animal was positioned
with its head in a rodent guillotine, and the arterial
blood pressure continuously recorded through a transducer connected to the left femoral artery. The body
temperature was measured using a rectal thermistor
and maintained between 37 and 38°C with an incandescent lamp. Samples for blood gases were obtained
from the left femoral artery.
The technique used is a modification of the one described for brain.5 The right femoral artery was connected to an 80 cm length of PE-205 tubing, which
was itself connected to a Gilson (Middleton, WI) peristaltic pump. The left femoral vein was used for the
1095
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017
1096
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1986
rapid injection of 25-30 /iCi of l4C-sucrose in 0.2 cc
tissue culture medium containing Earle's salts (saline).
Simultaneous with the injection, the peristaltic pump
was activated to withdraw blood at a constant predetermined rate (approximately 0.1 ml/min). After 2 or
10 min, the animal was decapitated, and the arterial
withdrawal was terminated. The 2-min experiments
were used for determination of tissue blood volume,
while the 10-min period was for calculation of sucrose
permeability. A sample of arterial blood was taken
through the left femoral artery during the last 5 sec of
the experiment. Triplicate 0.05 ml samples of this blood
sample were used to determine the final concentration
of radioactivity in the blood (CT) and the remainder
was used to measure the hematocrit.
The retina was rapidly removed intact from first the
right and then the left eye and placed in pre-weighed
vials containing 1.5 ml of Protosol (E. I. duPont de
Nemours and Co. Inc., Boston, MA). The technique
for retinal removal, which was described previously,6
permitted sampling of the tissue within 5-10 sec of
decapitation. The retina was obtained intact with little
visible contamination by the RPE, but with a variable
small contaminant of posterior vitreous. Much of this
vitreal contamination was removed by briefly blotting
the sample with tissue paper, without touching the retina. Following collection of the retina samples, the
brain was removed and samples from the right and left
cortex were placed into pre-weighed vials containing
2.5 ml of Protosol. The blood and remaining saline in
the PE-205 catheter used for the continuous arterial
withdrawal were expelled into a pre-weighed vial, and
all vials were then reweighed. Triplicate 0.05 ml samples from the arterial withdrawal catheter were counted
for radioactivity and, knowing the weight of this sample, the total amount of l4C-sucrose obtained during
the constant arterial withdrawal (CA) was calculated.
All tissue samples were heated in Protosol at 50°C
for 1-2 hr until dissolved. The blood samples were
dissolved in 1.5 ml of ethanol:Protosol (2:1 v/v) and
then decolorized with 0.05 ml of 30% H 2 O 2 . After the
addition of 10 ml of toluene-based scintillation fluid,
the samples were stored for 48 hr and then counted in
a Beckman (Berkeley, CA) LS-7500 scintillation counter calibrated for automatic quench correction and calculation of dpm.
Arabinose Infusion
The right external carotid artery was isolated and
the small branches in the vicinity of the common carotid bifurcation were ligated. A PE-50 catheter was
inserted retrograde to the origin of the internal carotid
artery. D-arabinose at a concentration of 1.7 M in the
saline solution was infused into the carotid artery at a
rate of 7.6 ml/min using a Harvard (South Natick, MA)
Vol. 27
syringe pump. This rate of infusion completely cleared
the common and internal carotid arteries of blood. The
infusion was continued for 10, 15, or 30 sec. Five minutes after the beginning of the arabinose infusion, 14Csucrose was injected intravenously and the tissues were
sampled 10 min later. The results were compared to
those obtained from animals that had been infused for
30 sec with the saline solution only.
Iodate Treatment
Sodium iodate was dissolved to a concentration of
4% (w/v) in saline. All animals received a dose of 30
mg/kg by intraperitoneal injection.
Experimental Diabetes
Male, pathogen-free Long-Evans rats, weighing 250350 gm, were used for this phase of the study. Streptozocin (65 mg/kg) was administered either by tail vein
or intracardiac injection in anesthetized animals. No
differences in the degree of hyperglycemia were observed between the two routes of administration.
Nonfasting plasma glucose levels were measured 3 days
after streptozocin administration and again at the time
of use.
Calculations
The analysis of the data was similar to that presented
by Ohno et al5 except that the radioactivity in the arterial withdrawal was determined directly, rather than
by an integration technique. The results are expressed
as a rate constant for sucrose uptake, which is analogous
to a clearance value; i.e., the equivalent volume of
plasma which contains the amount of l4C-sucrose taken
up in a unit of time. This rate constant has also been
called a permeability X surface area (PS) product,
since it represents the diffusional permeability of the
solute into the tissue multiplied by the surface of tissue
exposed.5 The PS product was calculated using equation 1.
C e v X F 0 X ( l - Hct)
PS =
(1)
where Cev is the amount of extravascular radioactivity
in the tissue, F o is the rate of withdrawal of arterial
blood, CA is the amount of radioactivity collected during the arterial withdrawal, and Hct is the hematocrit.
The latter factor is used to express the results in terms
of plasma values since sucrose does not enter the
erythrocyte. Extravascular radioactivity was calculated
from the tissue l4C-sucrose content (C), the tissue blood
volume (BV), and the final concentration of radioactivity in the blood (CT).
Cev = C - BV X C T
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017
(2)
1097
BLOOD-RETINAL BARRIER TO SUCROSE / Ennis and Berz
An initial estimate of BV was obtained using the results
from 2-min experiments, with the assumption of no
tissue uptake. This value was then used to obtain an
initial estimate of PS. A better estimate of BV was then
calculated using equation 3.
C-{PSXCA/FO/(1 -Hct)}
CT
(3)
Statistical Analysis
LxJ
Comparisons between groups were made using the
two-tailed Student's t-test. The Bonferoni correction
was used when multiple comparisons to the same mean
were made.7
O
Materials
Long Evans rats were purchased from Charles River
(Portage, MI). l-[l4C]-sucrose (10 mCi/mmole) was
obtained from ICN Radiochemicals (Irvine, CA), and
its purity was checked by paper chromatography. Protosol was produced by New England Nuclear (Boston,
MA). All other chemicals were purchased from Sigma
Chemical (St. Louis, MO). Plasma glucose concentrations were determined by the Michigan Diabetes Research and Training Center Biochemistry Core using
the hexokinase technique.
Results
Preliminary Considerations
The 10-min time period for sucrose uptake was determined based upon preliminary experiments. Equal
amounts of 14C-sucrose were injected into each of six
rats. After 2, 5, 10, 15, 20, or 30 min, a blood sample
for C T was obtained, the animal was decapitated, and
the right and left retinae were removed. The 14C-sucrose
content of each retina was determined and the extravascular radioactivity was calculated using the value
of C T (in dpm/ml of blood) and the average blood volume of the retina from Table 1 (3.79 X 10~4 ml/retina).
The results for retina and plasma 14C-sucrose content
versus time are shown in Figure 1. Between 2 and 20
min, there was a nearly linear increase in retinal I4 Csucrose. This suggests that the sucrose remained predominately in the retina with only minor movement
of sucrose into the vitreous or back to the blood. Assuming that sucrose distributes in only the extracellular
fluid space of the retina which has been reported to be
31%, 8 the concentration of sucrose in the retina at 10
min would be 8.5% of the plasma concentration. The
QL
•o
<
Subsequent iterations between equations 1, 2, and
3 were continued until PS and BV changed by less
than 0.1%.
M RETIf
BV =
8 _
b
(dpm
No. 7
3
•
6
LJ
C/>
O
fT
O
Z>
if)
30tf
200
100
5
10 15 20
TIME(min)
25 30
Fig. 1. Concentration of l4C-sucrose in plasma and retina following
an intravenous injection. 25 \LC\ of 14C-sucrose were injected as a
bolus into the femoral vein of six rats. The animals were sacrificed
at various times and samples of plasma (A) and the right (•) and left
(O) retina were obtained. The extravascular content of l4C-sucrose
in the retina was calculated using the corresponding concentration
of radioactivity in plasma and a blood volume of 3.79 X 10~4 ml/
retina.
value is low enough that backflux to blood should be
small. Thus, an experimental period of 10 min appears
to be an appropriate interval for measurement of unidirectional movement of 14C-sucrose into the retina.
Rapoport et al9 have demonstrated that the same time
period can be used for determination of sucrose permeability at the BBB.
The validity of the conclusion that sucrose remained
predominately in the retina was tested further by measuring its PS product as a function of time. A plot of
the PS product versus time is shown in Figure 2. The
nearly horizontal regression line indicates that the PS
product for sucrose did not vary significantly as a function of time. This is the expected result if there is little
loss of the radioactivity into the vitreous or back into
the blood. It should be noted that the variability at 2
and 5 min of experimental time is due to the difficulty
in measuring the PS product at these short times because only limited amounts of tracer have moved into
the retina, while the plasma concentration is still very
high, as shown in Figure 1. Consequently, the correction for 14C-sucrose remaining trapped within the intravascular space of the retina is much higher relative
to the extra vascular content of the retina than at the
longer time periods.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017
1098
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1986
a:-^.Ore l 1.5
^ £ i.o
11
11
[
1J
1
1
7< »
1
10
15
20
TIME(min)
25
30
Fig. 2. Permeability of sucrose at the BRB as a function of time
after IV injection of radiotracer. The plot shows the mean ± S.D.
for three determinations at each time period. The PS product for left
and right retina were averaged for each rat because no side to side
difference was noted. The line drawn through the data was obtained
by linear regression analysis.
Vol. 27
Table 1. There were no significant side-to-side differences for either measure in either tissue. Intracarotid
infusion of saline for 30 sec did not change the blood
volume or the PS product for the cortex; however, the
PS product for the retina was increased slightly on both
the infused and non-infused sides. This suggests that
the carotid catheterization and/or infusion alone have
a small effect on BRB permeability. A much larger
effect on both BBB and BRB permeability was observed
when 1.7 M arabinose was included in the infusion
solution. Figure 4 shows the effect of arabinose infusion
on sucrose permeability of the retina relative to the
cerebral cortex. For both tissues, the degree of barrier
opening is approximately proportional to the length of
infusion of the hypertonic solution. A 30-sec infusion
2.0
The PS product for brain is generally expressed as
the rate of uptake per unit of tissue mass. However, as
mentioned previously, the retina sample that we obtain
is contaminated by a small amount of posterior vitreous. Nevertheless, when the retina is sampled early
enough, the contaminant should not contain a significant amount of tracer. A plot of the PS product calculated per mass of retina sampled would then be expected to vary inversely with the sample weight. This
relationship was confirmed for our data, and is shown
in Figure 3A. Since the sample contains the entire retina
and the variation in weight is due to vitreous containing
little radioactivity, then the PS product calculated per
whole retina should not vary with the weight of the
sample. This relationship is confirmed in Figure 3B,
where the PS product has been calculated for the same
data in Figure 3A per retina rather than per gm of
retina. The same relationship was observed when the
tissue blood volume was calculated per gm of retina
versus per retina (data not shown).
In all further analysis, the BV and PS product for
the retina are calculated per retina. We believe that this
is justified by the results shown in Figure 3, by the
linear uptake of l4C-sucrose over a 10-min period
shown in Figure 1, and by the constant PS product
between 2 and 30 min shown in Figure 2. We have
also determined that the dry weight of retinal tissue
samples obtained with the method described here is
the same (2.07 ±0.17 mg, N = 37) for animals weighing
from 250-450 gm. This indicates that similarly sized
retina samples can be obtained from animals within
this range of weights.
Control and Arabinose Infusion
The blood volume and PS products for each side of
the brain and retina of control animals are shown in
KP
oX
c
1.5
1.0
E
o
cr
o
E 0.5
E
0
3/5
C
<r
O
»-
o
B
2.0
3
Q
O
a:
Q_
a.
^^
g
^x
1.5
c
E 1.0
o
0.5
E
0.010
0.020
0.030
SAMPLE WEIGHT (gm)
Fig. 3. Permeability of sucrose at the BRB. The PS product for
sucrose movement into the retina was determined and the results
were calculated either on a weight or whole retina basis. The results
are plotted versus the weight of the sample which contains the entire
retina and a variable amount of contamination from the posterior
vitreous. The data used in this analysis are taken from the control
and 1 hr iodate treated groups presented in Table 2. The lines drawn
through each set of data were obtained by linear regression analysis.
The correlation coefficient was 0.852 for the data in A and 0.0695
for the data in B.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017
No. 7
1099
BLOOD-RETINAL BARRIER TO SUCROSE / Ennis and Berz
Table 1. Summary of results for control and saline infused experiments
Cortex
Retina
Right
Right
Left
Left
2
Blood Volume
Control
Saline infused
ml/retina (X10")
3.57 ± 0.70
4.02 ± 0.66
3.53 ±0.99
4.00 ±1.82
ml/gm(X10 )
1.25 ±0.07
1.29 ±0.16
1.40 ±0.1
1.26 ±0.09
PS Product
Control
Saline infused
ml/retina/min (X105)
1.32 ±0.38
1.13 ±0.33
2.08 ±0.36*
1.84 ±0.42
ml/gm/min (X104)
3.34 ± 1.01
2.84 ± 0.68
3.15 ±0.60
2.89 ± 0.24
Differences between right and left sides were not significant.
generally resulted in a greater barrier dysfunction than
did 10 or 15 sec. Although the fold increase in BBB
permeability is greater than that of the BRB, there does
not appear to be any difference in threshold for barrier
opening, since both tissues are affected proportionately.
In contrast to the infused side, the PS product for
the retina on the non-infused side of the arabinose
group did not differ significantly from the non-infused
retina from the saline controls. For the cortex, the PS
product of the non-infused side was significantly (P
<0.01) elevated 6.3-, 4.5-, and 3.3-fold in the 30-,
15-, and 10-sec arabinose infusions respectively (data
not shown). This suggests some cross-over of infused
arabinose into the left cerebral but not the retinal circulation.
Iodate Treatment
Animals treated with intraperitoneal iodate were
studied after either 1 or 24 hr. Since no significant sideto-side differences were noted in either the control or
the iodate-treated animals, the data shown in Table 2
were calculated using the averages of the right and left
samples. For the retina, neither the blood volume nor
the PS product were significantly different from control
at 1 hr. However, after 24 hr the PS product was increased over eightfold. Because of this high permeability, it was not possible to accurately determine the
blood volume. For the cortex, neither the BV nor the
PS product were significantly different from control at
either 1 or 24 hr. Thus, iodate appears to have a selective effect on the BRB, but not the BBB.
Experimental Diabetes
The average plasma glucose concentrations for control animals and for diabetic animals 3 days (initial)
after induction of diabetes are given in Table 3. Also
shown are the plasma glucose concentrations for each
group just prior to measurement of sucrose permeability. The values for diabetic animals at all time periods studied are significantly greater (P < 0.001) than
* P < 0.02 compared to control.
control values, and represent a severity of hyperglycemia similar to that which has been reported previously.4'6 Final blood pH is presented for control and
diabetic rats at 2, 6, and 20 weeks. No significant differences from the control values were noted in our diabetic rats in blood pH, mean arterial blood pressure,
temperature, PCO2, or pC>2 (data not shown).
The blood volumes for brain and retina in normal
and diabetic rats at 2, 6, and 20 weeks of diabetes are
shown in Table 4. The blood volume for the retina in
the 20 week diabetic group is significantly different (P
< 0.05) from normal values. The PS products for sucrose at the BBB and BRB are also shown in Table 4.
The BRB showed a difference in PS to sucrose only
i~ 8r
UJ —
O
I
H o
O §
a- £
2
4
6
8
10
PS PRODUCT FOR CORTEX
ml/gm/min (xlO 3 )
Fig. 4. PS products for the retina and cortex following osmotic
opening of the barriers. A solution containing 1.7 M arabinose was
infused into the right internal carotid artery for 10 (A) 15 (O) or 30
(•) sec and the PS products for sucrose in the ipsilateral retina and
cortex were determined during the interval of 5-15 min following
the infusion. Because of the degree of barrier opening, it was not
possible to accurately determine the blood volume following arabinose
infusion;10 therefore, the data shown were calculated using the blood
volumes for saline infused animals (Table 1). The average PS products
for 30 sec saline infused animals are also shown (X). The line drawn
through the data was calculated by linear regression analysis.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1986
1100
Table 4. Summary of results for normal
and diabetic animals
Table 2. PS products for iodate-treated animals
Retina
ml/ret/min (X103)
Cortex
ml/gm/min (X104)
n
1.22 ±0.34
1.22 ±0.36
9.72 ± 1.86*
3.09 ± 0.83
2.24 ± 0.60
3.21 ±0.32
7
4
4
Control
1 hr
24 hr
Vol. 27
Values are the averages ± S.D. of the average between the right and left
tissue samples. The blood volumes for the 1 hr iodate animals were not from
controls (data not shown).
* P < 0.0005 compared to control.
Blood volume
Normal
Diabetic (2 wk)
Diabetic (6 wk)
Diabetic (20 wk)
Retina
Cortex
ml/retina (XI0 4 )
3.40 ± 0.48
2.67 ± 0.69
3.58 ± 0.49
2.07 ± 0.37*
ml/gm(X10 2 )
n
1.22 ±0.08
10
7
4
ml/retina/min
(X105)
ml/gm/min
(X104)
PS products
after 20 weeks of diabetes. This late change represented
a 25% increase in the permeability of the BRB (P
< 0.001, one-tailed). We noted no significant increases
in the PS product to sucrose at the BBB.
Normal
Diabetic (2 wk)
Diabetic (6 wk)
Diabetic (20 wk)
1.35
1.23
1.56
1.79
±0.32
±0.36
±0.29
±0.26*
1.20 ±0.16
1.36 ±0.09
1.26 ±0.12
4.13
4.03
2.50
3.27
±0.97
± 0.43
± 0.40*
± 0.52
8
n
9
8
5
8
Values are the averages ± S.D.
* P < 0.05 compared to control
Discussion
The BRB and the BBB are formed by morphologically similar capillary endothelial cells. In addition, the
RPE of the retina plays an important role in formation
of the BRB. In the anterior segment of the eye, the
blood-aqueous barrier (BAB) is formed by the nonpigmented epithelium of the cilary body and by the
epithelium covering the iris in the posterior chamber.
In addition to this epithelial barrier, the BAB is also
formed by the endothelial cells of the vessels of the iris.
Collectively, the blood-ocular barriers serve as selective
permeability barriers to regulate the microenvironment
of the retina and the composition of the intra-ocular
fluids.' The barrier systems of both the brain and the
eye are similarly tight to large protein tracers such as
horseradish peroxidase;110 however, the relative permeabilities of the barriers to small polar molecules is
less well defined. It has been established that the extraction of sodium across the RPE3 and retinal capillaries4 is of the same order of magnitude as that of the
BBB. Recently, Aim and Tornquist2 used the single
pass, intracarotid bolus injection technique to simultaneously measure the extraction by retina and brain
Table 3. Plasma glucose and blood pH values
for normal and diabetic animals
Normal
Diabetic (2 wk)
Diabetic (6 wk)
Diabetic (20 wk)
N
Plasma glucose
Initial
19
15
9
16
436 ± 80
518 ± 92
501 ± 70
(mg/dl)
Final
144
410
430
446
±
±
±
±
14*
30f
24f
64f
Blood pH
Final
7.30
7.35
7.28
7.32
±
±
±
±
0.06
0.04J
0.08$
0.09$
Values are averages ± S.D. for N determinations unless otherwise noted
* N = 10
t P < 0.001 compared to normal
% not significantly different from normal
of several solutes relative to 3H2O. The method was
originally developed by Oldendorf to study BBB permeability, and has been extensively used for that purpose. '' While Aim and Tornquist found 4-5-fold higher
relative uptakes of sucrose and L-glucose for retina
compared to brain, the authors believed that direct
comparisons could not be made because of contamination by tracer outside of the barrier systems and the
markedly different wash-out of tracers from the two
tissues. In addition, because the vitreous body is situated between the BRB and the BAB, substances in the
vitreous have free access to the posterior chamber as
well as the extracellular space of the retina.12 Therefore,
movement of solutes into the vitreous provides a good
measure of the permeability of the blood-ocular barrier, but does not exclusively measure permeability of
the BRB.
An important aspect of our method is the ability to
rapidly remove the retina from the rat eye. This is essential to avoid diffusion of tracer into adjacent tissue
not included in the sample. Use of pigmented rats allows visual inspection for RPE contamination; however, a variable amount of posterior vitreous is unavoidably attached to the retina sample. Fortunately,
the tracer content of this material appears small, as
indicated by the lack of association between the radioactivity in the sample and the amount of vitreal contamination (Fig. 3). In order to have an accurate measure of the PS-product, it is important that very little
sucrose enter the posterior vitreous from the retina
during the 10 min of experimental time. The constant
PS product over a 30-min time period supports this
experimental constraint (Fig. 2). This does not mean
that tracer will not eventually move into the vitreous
from the retina, but simply that its appearance in pos-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017
No. 7
DLOOD-RETINAL BARRIER, TO SUCROSE / Ennis and Derz
1101
terior vitreous is delayed as compared to its appearance
in the retina. More direct proof that 14C-sucrose does
not appreciably enter the vitreous from the retina during the 10-min experimental period would require direct measurement of the 14C-sncrose concentration
profile in the posterior vitreous. However, techniques
are not currently available to do this. Furthermore,
determination of the 14C-sucrose content of the entire
vitreous cannot be used to reject our hypothesis because
it has been repeatedly demonstrated that diffusional
tracers move from the posterior chamber into the anterior and central vitreous more rapidly than they move
into the posterior vitreous.1
Using a technique similar to the one described here
except for sampling of vitreous rather than retina,
DiMattio and Zadunaisky13 obtained a rate constant
for sucrose penetration into the vitreous of 4.6 X 10~3/
min. Assuming that the specific gravity of the vitreous
is 1.0, and that a rat retina weighs 3 mg, this value is
equal to 3.68 X 10~5 ml/retina/min, which is 3.2 times
higher than the value we obtained when sampling the
retina (Table 2). These results suggest that many tracers,
including sucrose, may actually penetrate more freely
into the vitreous from the ciliary body or aqueous than
oxidase tracer technique combined with electron microscopy.17 Other investigators were unable to detect
fluorescein in the retina of diabetic rats when examined
by fluorescence microscopy, even though fluorescein
was detected in the retinal pigment epithelium.18 In
addition, the recent report by Maepea et al19 during
streptozoticin diabetes found no increase in uptake of
L-glucose into either brain or retina, expressed as a
percentage of the plasma equivalent space. Consequently, we believe that there is no increase in the passive permeability of the BRB in short-term experimental diabetes.
The method described here should prove to be a
useful tool for studies of BRB permeability. This intravenous injection technique has been used to quantitate BBB permeability, not only to poorly permeable
molecules such as sucrose, but also to more permeable
compounds, such as D-glucose, which cross the barrier
by carrier-mediated transport.20 The method can be
used under a variety of conditions, for example to study
the effects of anesthesia,21 ischemia,22 and experimental
diabetes.23 It should now be possible to quantitate BRB
permeability to a variety of tracers under various conditions.
from the retina. Therefore, in the present study of BRB
Key words: blood-retinal barrier, blood-brain barrier, permeability, capillary, retinal pigment epithelium, hypertonic
arabinose, iodate, sucrose, streptozocin, experimental diabetes
permeability, we made every effort to avoid contamination of the retina sample by anterior vitreous.
Osmotic modification of the BBB has become a
standard method of barrier opening that may be a clinically useful tool for delivery of polar drugs to the
brain. 914 Laties and Rapoport have shown that intracarotid infusion of hypertonic solutions open the BRB
as well.15 Although BRB opening was not quantitated,
they were able to observe a leak of fluorescein across
both the retinal capillaries and the RPE. Our results
confirm the effect of hypertonic arabinose of the BRB
and show that the degree of BRB opening is proportional to the degree of BBB opening.
In contrast to the results obtained with hypertonic
arabinose infusions, the BRB, but not the BBB, showed
an increase sucrose permeability 24 hr after intraperitoneal iodate injection. The selectivity of this toxin in
damaging the RPE was first described by Noell,16 and
has since been confirmed by a number of investigators.
It seems reasonable to speculate that the increase in
BRB permeability is due to RPE and not retinal capillary breakdown, since the capillary barrier in the brain
is not affected. However, we are not aware of studies
that directly examined the integrity of the retinal capillaries after iodate.
The PS product for sucrose penetration across the
BRB at 2 and 6 weeks of experimental diabetes was
not significantly increased. This result is in agreement
with the studies of Wallow using the horseradish per-
References
1. Cunha-Vaz JG: The blood-retinal barrier. Doc Ophthalmol 41:
287, 1976.
2. Aim A and Tornquist P: The uptake index method applied to
studies on the blood-retinal barrier. Acta Physiol Scand 113:73,
1981.
3. Tornquist P: Capillary permeability in cat choroid, studied with
the single injection technique (III). Acta Physiol Scand 106:425,
1979.
4. Tornquist P, Aim A, and Bill A: Studies on ocular blood flow
and retinal capillary permeability to sodium in pigs. Acta Physiol
Scand 106:343, 1979.
5. Ohno K, Pettigrew KD, and Rapoport SI: Lower limits of cerebrovascular permeability to nonelectrolytes in the conscious
rat. Am J Physiol 235:H299, 1978.
6. Ennis SR, Johnson JE, and Pautler EL: In situ kinetics of glucose
transport across the blood-retinal barrier in normal rats and rats
with streptozocin-induced diabetes. Invest Ophthalmol Vis Sci
23:447, 1982.
7. Wallenstein S, Zucker CL, and Fleiss JL: Some statistical methods
useful in circulation research. Circ Res 47:1, 1980.
8. Ames A and Nesbett FB: Intracellular and extracellular compartments of mammalian central nervous tissue. J Physiol 184:
215, 1966.
9. Rapoport SI, Fredericks WR, Ohno K, and Pettigrew K.D:
Quantitative aspects of reversible osmotic opening of the bloodbrain barrier. Am J Physiol 238:R421, 1980.
10. Ra viola G: The structural basis of the blood-ocular barriers. Exp
Eye Res 25(Suppl):27, 1977.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017
1102
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1986
11. Oldendorf WH: Brain uptake of radiolabeled amino acids, amines
and hexoses after arterial injection. Am J Physiol 221:1629, 1971.
12. Cunha-Vaz J: Sites and functions of the blood-retinal barriers.
In The Blood Retinal Barriers. Cunha-Vaz JG, editor. New York,
Plenum Publishing Co. 1979, p. 101.
13. DiMattio J and Zadunaisky JA: Glucose transport into the ocular
compartments of the rat. Exp Eye Res 32:517, 1981.
14. Neuwelt EA, Hill SA, Frenkel EP, Diehl JT, Maravilla KR, Vu
LH, Clark WK, Rapoport SI, Barnett PA, Lewis SE, Ehle AL,
and Beyer CW: Osmotic blood-retinal barrier disruption: pharmacaodynamic studies in dogs and a clinical phase I trial in
patients with malignant brain tumors. Cancer Treat Rev 65:39,
1981.
15. Laties AM and Rapoport SI: The blood-ocular barriers under
osmotic stress: studies on the freeze-dried eye. Arch Ophthalmol
94:1086, 1976.
16. Noell WK: Experimentally induced toxic effects on structure
and function of visual cells and pigment epithelium. Am J
Ophthalmol 36:103, 1953.
Vol. 27
17. Wallow IHL: Posterior and anterior permeability defects? Morphologic observations on streptozocin-treated rats. Invest
Ophthalmol Vis Sci 24:1259, 1983.
18. Kirber WM, Nichols CW, Grimes PA, Winegard AL, and Laties
AM: A permeability deficit of the retinal pigment epithelium:
Occurrence in early streptozocin diabaetes. Arch Ophthalmol
98:725, 1980.
19. Maepea O, Karlsson C, and Aim A: Blood-ocular and bloodbrain barrier function in streptozocin-induced diabetes in rats.
Arch Ophthalmol 55:1366, 1984.
20. Gjedde A: Rapid steady-state analysis of blood-brain glucose
transfer in rat. Acta Physiol Scand 108:331, 1980.
21. Gjedde A and Rasmussen M: Pentobarbital anesthesia reduces
blood-brain glucose transfer in the rat. J Neurochem 35(6): 1382,
1980.
22. Sage JI, Van Uitert RL, and Duffy TE: Early changes in bloodbrain barrier permeability to small molecules after transient cerebral ischemia. Stroke 15:46, 1984.
23. Gjedde A and Crone C: Blood-brain glucose transfer: repression
in chronic hyperglycemia. Science 214:456, 1981.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933359/ on 06/17/2017