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The Autoradiographic Localization of Urea-C-14 in the Rabbit Eye*
TWIN FLASHES IN ELECTRORETINOGRAPHY
warrant elimination from the diagnosis of the possibility of retinitis pigmentosa, nor does it ensure the definitive intactness of the eyes. SUMMARY
It is shown that the electroretinographic response in cases of tapetoretinal degenera-
THE
tion may vary under various conditions. This variation is chiefly dependent on the intensity of the light stimulus applied. It is no longer possible to maintain that retinitis pigmentosa is characterized by absence of any electroretinographic response. De
AUTORADIOGRAPHIC UREA-C-14 I N
SEYMOUR
B.
GOREN,
M.D.,
THE AND
Chicago,
The concentration of urea in the aqueous humor is less than that in the blood plasma. Moore, Scheie and Adler state that this deficiency is the result of the relatively low permeability of the blood-aqueous barrier to urea as compared to water. Kinsey, Reddy, and Skrentny, studying the intraocular transport of C-14-labelled urea, assumed that the concentration of urea in the secreted fluid was a constant linear function of its concentration in the plasma. 1
2
The direct visualization of urea in the eye would be helpful in confirming these findings. By means of C-14-labelled urea and autoradiographic techniques, the localization of urea in the rabbit eye has been investigated.
63
Pintelaan.
LOCALIZATION RABBIT
FRANK
W.
OF
EYE* NEWELL,
M.D.
Illinois
sodium. The head was then perfused through the aorta with 2,000 cc. of human plasma containing 37.5 mg. percent of nonradioactive urea. Following the perfusion, the eyes were quickly enucleated, processed by a freeze-dry technique and autoradiographs made. A more detailed description of this technique has been described in a previous communication. Paraffin sections, six microns thick, and Kodak No-Screen X-ray film were used for autoradiography. Anterior chamber aqueous humor and vitreous humor paracenteses were performed in other animals, both before and after perfusion, and the ocular fluids obtained were analyzed for radioactivity with a scintillation counter. 3
RESULTS METHOD
Male albino rabbits, weighing 2.0 to 2.2 kg., were given a single dose of 1.0 mc. per kg. of body weight of urea-C-14 (specific activity 2.0 mc. per millimole) into the marginal vein of the ear. T w o hours after the administration of the labelled drug, 20 mg. of heparin was given intravenously and the animal was anesthetized with pentobarbital • F r o m the Department of S u r g e r y ( O p h t h a l m o l o g y ) , T h e U n i v e r s i t y o f Chicago. T h i s investigation w a s supported in part by a research grant f r o m the N a t i o n a l S o c i e t y for the P r e v e n t i o n o f Blindness and by Research Grant N u m b e r B-1515 f r o m the National Institute o f N e u r o l o g i c a l D i s e a s e s and Blindness.
Urea-C-14 was found in all ocular tissues. The endothelial surface of the cornea contained more radioactivity than did the superficial layers while the stroma concentrated lesser amounts (fig. 1 ) . Both the iris and ciliary processes were heavily impregnated with radioactivity (figs. 2 and 3 ) . The inner borders of the ciliary processes and the anterior surface of the iris appeared darker on the autoradiogram s than did the other portions of these structures. The posterior surface of the iris, bathed in posterior chamber aqueous humor, was moderately dark on the autoradiograms. The lens capsule and anterior and posterior cortex concentrated more
64
S E Y M O U R Β. G O R E N A N D F R A N K W .
NEWELL
F i g . 1 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a m s h o w i n g m o r e radioactivity o n the inner and outer surfaces of the cornea than in the stroma. T h e anterior surface of the iris ( I ) appears darker than the rest of the iris. Clumps of radioactivity are present in the anterior chamber.
urea than did the inner layers of the lens (fig. 4 ) . The retina demonstrated a homogeneous distribution. Minimal amounts of radioactivity were found in the choroid. Hematoxylin-eosin stained sections revealed that the blood vessels of the choroid and iris were free from erythrocytes after perfusion. The sclera was diffusely stained with ureaC-14 (fig. 5 ) . The superficial layers of the conjunctiva contained more radioactivity than did the deeper portions. The concentration of C-14-labelled urea in the blood plasma remained fairly constant throughout the course of the experiment. After two hours, the anterior chamber aqueous humor contained more radioactivity than did the vitreous humor and the amounts of radioactivity in these two chambers appeared to be unaffected by the perfusion. DISCUSSION
F i g . 2 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a m demonstrating that the anterior surface of the iris ( A ) concentrates more urea-C-14 than does the posterior surface ( B ) , while the stroma contains lesser amounts.
The finding that the inner and outer surfaces of the cornea contained more radioactivity than did the stroma is probably the result of both surfaces being bathed with ureaC-14 and diffusion taking place in both directions. Urea-C-14 is present in tear fluid ; this would also explain why the superficial layers of the conjunctiva contained more radioactivity than did the deeper layers. The ciliary processes and anterior surface of the
AUTORADIOGRAPHIC LOCALIZATION OF
UREA
65
F i g . 3 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a p h of an oblique section through the angle demonstrating the relatively high concentration of C-14-labelled urea in the ciliary processes and anterior surface of the iris as compared with surrounding structures.
iris contained large amounts of radioactivity, which is in agreement with the assumption that diffusional exchange takes place between the blood and posterior chamber across the ciliary epithelium and between the blood and anterior chamber across the anterior surface
F i g . 4 ( G o r e n and N e w e l l ) . A u toradiogram of the equatorial region o f the lens. T h e lens capsule, cortex, and nucleus contain u r e a - C 14 in a gradient decreasing f r o m the periphery to the center o f the lens. Distribution is similar in the a n terior, equatorial, and posterior p o r tions.
of the iris. The lens capsule, cortex, and nucleus contained urea in a gradient decreasing from the periphery to the center of the lens. Distribution was similar in the anterior, equatorial and posterior portions. It appears that urea diffuses into the lens
66
S E Y M O U R Β. G O R E N A N D F R A N K W .
NEWELL
F i g . 5 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a m s h o w i n g h o m o g e n e o u s distribution in the retina ( R ) , minimal infiltration of the choroid ( C ) and diffuse staining of the sclera ( S ) .
from both the posterior chamber aqueous humor and the vitreous humor in equal amounts two hours after its intravenous administration. After two hours, the retina was homogeneously infiltrated with radioactivity while minimal amounts were found in the choroid. The level in the choroid was difficult to determine because of the irregular shrinkage of the posterior uveal tract during freezing and dehydration. The concentration of the C-14-labelled urea in the plasma and anterior chamber aqueous humor was similar to that reported by Kinsey, et al. After two hours, the concentration in relative units to the plasma was 61 percent in the anterior chamber and 36 percent in the vitreous cavity. 2
SUMMARY
The autoradiographic localization of ureaC-14 in the rabbit eye has been investigated. Urea-C-14 was found in all ocular tissues two hours after the intravenous administration of the compound. High concentrations were present in the ciliary processes and anterior surface of the iris which is in agreement with the generally accepted theory of the sites of diffusional exchange. Urea entered the cornea via the anterior chamber aqueous humor and tear fluid. Equal amounts diffused into the lens from the posterior chamber aqueous humor and vitreous humor. The relationships between blood, anterior chamber aqueous humor and vitreous humor concentrations have also been studied. 950 East 59th Street
(37).
REFERENCES
1. Moore, E . , Scheie, H . G., and Adler, F. H . : Chemical equilibrium b e t w e e n blood and a q u e o u s humor: Further studies. A r c h . Ophth., 2 3 : 1 7 0 , 1939. 2. Kinsey, V . E., Reddy, D . V . N . , and Skrentny, Β . Α . : Intraocular transport of C-14-labelled urea and the influence of D i a m o x on its rate of accumulation in aqueous humors. A m . J. Ophth., 5 0 : 1 1 3 0 , 1960. 3. Goren, S. Β., N e w e l l , F . W . , and O T o o l e , J. J.: T h e localization of D i a m o x - S - 3 5 in the rabbit eye. A m . J. Ophth., 5 1 : 8 7 , 1961.
warrant elimination from the diagnosis of the possibility of retinitis pigmentosa, nor does it ensure the definitive intactness of the eyes. SUMMARY
It is shown that the electroretinographic response in cases of tapetoretinal degenera-
THE
tion may vary under various conditions. This variation is chiefly dependent on the intensity of the light stimulus applied. It is no longer possible to maintain that retinitis pigmentosa is characterized by absence of any electroretinographic response. De
AUTORADIOGRAPHIC UREA-C-14 I N
SEYMOUR
B.
GOREN,
M.D.,
THE AND
Chicago,
The concentration of urea in the aqueous humor is less than that in the blood plasma. Moore, Scheie and Adler state that this deficiency is the result of the relatively low permeability of the blood-aqueous barrier to urea as compared to water. Kinsey, Reddy, and Skrentny, studying the intraocular transport of C-14-labelled urea, assumed that the concentration of urea in the secreted fluid was a constant linear function of its concentration in the plasma. 1
2
The direct visualization of urea in the eye would be helpful in confirming these findings. By means of C-14-labelled urea and autoradiographic techniques, the localization of urea in the rabbit eye has been investigated.
63
Pintelaan.
LOCALIZATION RABBIT
FRANK
W.
OF
EYE* NEWELL,
M.D.
Illinois
sodium. The head was then perfused through the aorta with 2,000 cc. of human plasma containing 37.5 mg. percent of nonradioactive urea. Following the perfusion, the eyes were quickly enucleated, processed by a freeze-dry technique and autoradiographs made. A more detailed description of this technique has been described in a previous communication. Paraffin sections, six microns thick, and Kodak No-Screen X-ray film were used for autoradiography. Anterior chamber aqueous humor and vitreous humor paracenteses were performed in other animals, both before and after perfusion, and the ocular fluids obtained were analyzed for radioactivity with a scintillation counter. 3
RESULTS METHOD
Male albino rabbits, weighing 2.0 to 2.2 kg., were given a single dose of 1.0 mc. per kg. of body weight of urea-C-14 (specific activity 2.0 mc. per millimole) into the marginal vein of the ear. T w o hours after the administration of the labelled drug, 20 mg. of heparin was given intravenously and the animal was anesthetized with pentobarbital • F r o m the Department of S u r g e r y ( O p h t h a l m o l o g y ) , T h e U n i v e r s i t y o f Chicago. T h i s investigation w a s supported in part by a research grant f r o m the N a t i o n a l S o c i e t y for the P r e v e n t i o n o f Blindness and by Research Grant N u m b e r B-1515 f r o m the National Institute o f N e u r o l o g i c a l D i s e a s e s and Blindness.
Urea-C-14 was found in all ocular tissues. The endothelial surface of the cornea contained more radioactivity than did the superficial layers while the stroma concentrated lesser amounts (fig. 1 ) . Both the iris and ciliary processes were heavily impregnated with radioactivity (figs. 2 and 3 ) . The inner borders of the ciliary processes and the anterior surface of the iris appeared darker on the autoradiogram s than did the other portions of these structures. The posterior surface of the iris, bathed in posterior chamber aqueous humor, was moderately dark on the autoradiograms. The lens capsule and anterior and posterior cortex concentrated more
64
S E Y M O U R Β. G O R E N A N D F R A N K W .
NEWELL
F i g . 1 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a m s h o w i n g m o r e radioactivity o n the inner and outer surfaces of the cornea than in the stroma. T h e anterior surface of the iris ( I ) appears darker than the rest of the iris. Clumps of radioactivity are present in the anterior chamber.
urea than did the inner layers of the lens (fig. 4 ) . The retina demonstrated a homogeneous distribution. Minimal amounts of radioactivity were found in the choroid. Hematoxylin-eosin stained sections revealed that the blood vessels of the choroid and iris were free from erythrocytes after perfusion. The sclera was diffusely stained with ureaC-14 (fig. 5 ) . The superficial layers of the conjunctiva contained more radioactivity than did the deeper portions. The concentration of C-14-labelled urea in the blood plasma remained fairly constant throughout the course of the experiment. After two hours, the anterior chamber aqueous humor contained more radioactivity than did the vitreous humor and the amounts of radioactivity in these two chambers appeared to be unaffected by the perfusion. DISCUSSION
F i g . 2 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a m demonstrating that the anterior surface of the iris ( A ) concentrates more urea-C-14 than does the posterior surface ( B ) , while the stroma contains lesser amounts.
The finding that the inner and outer surfaces of the cornea contained more radioactivity than did the stroma is probably the result of both surfaces being bathed with ureaC-14 and diffusion taking place in both directions. Urea-C-14 is present in tear fluid ; this would also explain why the superficial layers of the conjunctiva contained more radioactivity than did the deeper layers. The ciliary processes and anterior surface of the
AUTORADIOGRAPHIC LOCALIZATION OF
UREA
65
F i g . 3 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a p h of an oblique section through the angle demonstrating the relatively high concentration of C-14-labelled urea in the ciliary processes and anterior surface of the iris as compared with surrounding structures.
iris contained large amounts of radioactivity, which is in agreement with the assumption that diffusional exchange takes place between the blood and posterior chamber across the ciliary epithelium and between the blood and anterior chamber across the anterior surface
F i g . 4 ( G o r e n and N e w e l l ) . A u toradiogram of the equatorial region o f the lens. T h e lens capsule, cortex, and nucleus contain u r e a - C 14 in a gradient decreasing f r o m the periphery to the center o f the lens. Distribution is similar in the a n terior, equatorial, and posterior p o r tions.
of the iris. The lens capsule, cortex, and nucleus contained urea in a gradient decreasing from the periphery to the center of the lens. Distribution was similar in the anterior, equatorial and posterior portions. It appears that urea diffuses into the lens
66
S E Y M O U R Β. G O R E N A N D F R A N K W .
NEWELL
F i g . 5 ( G o r e n and N e w e l l ) . A u t o r a d i o g r a m s h o w i n g h o m o g e n e o u s distribution in the retina ( R ) , minimal infiltration of the choroid ( C ) and diffuse staining of the sclera ( S ) .
from both the posterior chamber aqueous humor and the vitreous humor in equal amounts two hours after its intravenous administration. After two hours, the retina was homogeneously infiltrated with radioactivity while minimal amounts were found in the choroid. The level in the choroid was difficult to determine because of the irregular shrinkage of the posterior uveal tract during freezing and dehydration. The concentration of the C-14-labelled urea in the plasma and anterior chamber aqueous humor was similar to that reported by Kinsey, et al. After two hours, the concentration in relative units to the plasma was 61 percent in the anterior chamber and 36 percent in the vitreous cavity. 2
SUMMARY
The autoradiographic localization of ureaC-14 in the rabbit eye has been investigated. Urea-C-14 was found in all ocular tissues two hours after the intravenous administration of the compound. High concentrations were present in the ciliary processes and anterior surface of the iris which is in agreement with the generally accepted theory of the sites of diffusional exchange. Urea entered the cornea via the anterior chamber aqueous humor and tear fluid. Equal amounts diffused into the lens from the posterior chamber aqueous humor and vitreous humor. The relationships between blood, anterior chamber aqueous humor and vitreous humor concentrations have also been studied. 950 East 59th Street
(37).
REFERENCES
1. Moore, E . , Scheie, H . G., and Adler, F. H . : Chemical equilibrium b e t w e e n blood and a q u e o u s humor: Further studies. A r c h . Ophth., 2 3 : 1 7 0 , 1939. 2. Kinsey, V . E., Reddy, D . V . N . , and Skrentny, Β . Α . : Intraocular transport of C-14-labelled urea and the influence of D i a m o x on its rate of accumulation in aqueous humors. A m . J. Ophth., 5 0 : 1 1 3 0 , 1960. 3. Goren, S. Β., N e w e l l , F . W . , and O T o o l e , J. J.: T h e localization of D i a m o x - S - 3 5 in the rabbit eye. A m . J. Ophth., 5 1 : 8 7 , 1961.