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Localization of autologous antiperoxidase antibodies in the anterior uvea of the rabbit eye
Exp. Eye Res. (1980) 30, 253-260
Localization of Autologous Antiperoxidase Antibodies in the Anterior Uvea of the Rabbit Eye 31. HIRSCH".
C. H. G. HARTMANN*:
B. BELLON?,
K. KELLER*
AND P. DRCET~
* ILNXERM 17. 86 (PT. Dr Y. Pouliquen), Chnique Ophtalnrologiquede I’Hdtel-Die-u and t IA’SERM
17.28/CNRX ERA -28>Hdpital Broussais, Pa&, France (Received2 August 1979, Lo&o)!)
Using an immunoperoxidase technique, we have studied the distribution of autologous antiperoxidase antibodies in the anterior uvea of the albino rabbit eye. Experimental animals were first immunized with horseradish peroxidase and the antibodies were then revealed in vitro 4 weeks later. It was found that the immunological tracer was unevenly distributed within the anterior uvea. The antiperoxidase antibodies extravasated from the fenestrated capillaries, diffused into surrounding stromal tissue and were then stopped by occluding or tight junctions of the outer epithelial layer of the pars plicata. From the iridial and ciliary processes to the iris and ciliary body base, the labelling of antiperoxidase antibodies progressively decreased. They were detected in the lumen of the iris vessels. This method is considered to be more physiological than those using exogenous substances as tracers and may be used to study ultrastructurally the permeability properties of the ocular barriers to macromolecules in normal, abnormal or experimental conditions. Key words : iris ; ciliary body; blood-aqueous barrier; immunoperoxidase ; antiperoxidase antibodies ; light and electron microscopy.
1. Introduction In the anterior uvea of the eye, a physiological barrier exists which, in norma. conditions, restricts the passage of blood-born macromolecules into the aqueous humor (Davson, 1972). Morphologically, this blood-aqueous barrier is constituted by occluding or tight junctions located in the iridial and ciliary body epithelia and in the endothelium of the iris vessels. In contrast, no morphological barrier has been observed between the cells lining the anterior surface of the iris where large openings have been described (Gregersen, 1958; Tousimis and Fine, 1959; Hogan, Alvarado and Weddell, 1971; Raviola, 1977). Therefore, the permeability properties of the blood-aqueous barrier to compounds of various molecular size have been extensively investigated, both with the light and electron microscopy to identify the distribution of plasma proteins in tissues, and biochemically by studying the protein composition of the blood, uveal tissues and aqueous humor. It has been shown that the permeability of the anterior uvea to substanceswasdependent upon their molecular radius and size (Dernouchamps and Heremans, 1975; Burns-Bellhorn, Bellhorn and Benjamin, 1978). In the present study, we have used the recently described immunoperoxidase technique (Druet et al., 1978) to investigate the in situ distribution of the autologous antiperoxidase antibodies (APAB) in the iris and ciliary body of the albino rabbit Reprint requests to: Mr. Michel Hirsch, Laboratoire de Cytologie et Marie Curie 16, rue de 1’Ecole de MBdecine, 75270-Paris cedex 06. 00144835/80/030253+08 c
Ultrastructurale
0 1980 Academic
$01.00/O 253
Press Inc.
Universite (London)
Pierre Limited
‘54
31. HIRSC’H
ET
XL.
eye and to re-examine the morphological sites of the blood-aqueous barrier hy 1.1sing the APAB as a physiological protein tracer.
2. Materials Immunization
and Methods
of animals
Six New Zealand albino rabbits weighing 2.5-3 kg were used for this experimentt Seven milligrams of horseradish peroxidase (Boehringer, grade 1, Jlannheim) emulsified with complete Freund’s adjuvant were injected into the hind foot pads of each rabbit. Four weeks later, the sera of the rabbits were tested by gel precipitation immunodiffusion technique for the presence of APAB. When tested by immunoelectrophoretic analysis, using peroxida,se as antigen, only IgG antibodies were detected. Preparation
of tissues
One hour before enucleation, two rabbits were treated with intravascular injections of 300 mg aspirin solution to minimize the supposed endogenous prostaglandins synthesis. These rabbits and two others non-treated with aspirin were anaesthetized with pentobarbital sodium injected in the marginal vein of the ear. Immediately after enucleation, the anterior segments were removed and pre-fixed for 1 hr at 4°C in 2.5% glutaraldehyde in 0.1 M-Sorensen phosphate buffer, pH 7.3-7.4. The anterior uvea rings were then separated from the corneas, cut into small pieces, post-fixed for 3 hr in the same fresh fixative and then washed in the buffer. A perfusion fixation procedure of the anterior chamber was also used in this study. A double needle was introduced into the anterior chamber of two living rabbits and the fixation solution slowly injected for 15 min. After this in situ pre-fixation, the eyes were treated as described above. IrrLrnunocytochemical
reaction
40 pm sections of the specimen were obtained with a Smith and Farquhar tissue-sectioner (Sorvall TC 2) and then incubated in buffered solution of horseradish peroxidase (0.5 “g/ml), at 0°C (Leduc, Avrameas and Bouteille, 1968; Druet et al., 1978). Tissue sections were rinsed in the buffer and incubated afterwards in 3,3’-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in O-2 M-Tris-HCl buffer (Graham and Karnovsky, 1966). They were rinsed in the Tris-HCl buffer and post-fixed in 1% osmium tetroxide in phosphate buffer, pH 7.4 at 4°C for 1 hr. Sections were dehydrated in a series of alcohol baths, placed in propylene oxide and embedded in Epon 812 epoxy-resin. Semi-thin and ultra-thin sections were cut on a Reichert OMU5 ultra-microtome and then examined with a Philips EM 300 electron microscope under 80 kV. Controls Some specimens was omitted.
were treated
as above,
but incubation
with
horseradish
peroxidase
3. Results In the rabbit, the pars plicata is divided into ciliary processessensustricto and iridial processes (Kozart, 1968). These processes are closely interconnected. In this paper, the inner epithelial cells correspond to the cells closest to the connective or stromal tissue and the outer epithelial cells correspond to the cells lining the posterior
chamber.
ANTIPEROXIDASE
AKTIBODIES
IN
ASTERIOR
UVEA
255
The immersion fixation of the anterior uvea pieces and the perfusion fixation in the anterior chamber of the eyes gave similar results. No difference was observed between the aspirin treated and non-treated animals. We noted that the lumen of the vessels in the iridial and ciliary processes was homogeneously filled with APAB as shown by the very denseperoxidase activity of the plasma. In the neighbouring connective tissues there was a gradient of this
FIG. 1. Semi-thin unstained section of an area of the anterior uvea. The stroma of the iridial procens (P) and the plasma in the lumen of the vessels (V) are strongly labelled. Note the progressive decrease of the labelling from the process of the iris (I). PC! = posterior chamber. (Perfusion fixation. x 150). PIG. 2. Semi-thin unstained section of a ciliary process. The labelling is seen in the stroma of t.he process (S) and in the intercellular spaces of the inner epithelial layer (IL). No labelling is observed beyond the tight junctions (TJ) sealing the intercellular spaces of the outer epithelial cell layer (OL). I’6 = posterior chamber. (Immersion fixation. x 360). FIG. 3. Control semi-thin unstained section of a ciliary stroma process (without horseradish peroxidase in the incubation medium). The labelling is not. seen either in the lumen of vessel (L) or in thr stromal tissue (S). (Perfusion fixation. x 150).
FIG. 4. Semi-thin unstained section of the posterior area of the iris. The labelling is localized only iu the lumen (L) of the “non-leaky” iris vessels. Iris stroma (8) is free of peroxidase activity. (T’erfuaion fixation. x 375). FIG. 5. Control semi-thin unstained section of the iris (without horseradish peroxidasc in the incuhation medium). The plasma of the lumen (L) of the vessels is not labelled. Sote the well-known endogenous peroxidase activity of the red blood cells (rbc). (Perfusion fixation. >, 700).
activity which increased towards the posterior part of the iridial and ciliary processes and decreased towards the iris and ciliary body base (Fig. 1). The APAB escaped freely from the fenestrated “leaky” capillaries, completely invaded the surrounding stromal tissue (Figs 1 and 8) and reached the intercellular spaces between the inner epithelial cells and between the apices of the inner and the outer epithelial cell layers (Figs 2 and 6). They were stopped by occluding or tight junctions sealing the intercellular spaces of the outer epithelial cells lining the posterior chamber. No APAB could be detected beyond this epithelial barrier (Figs 6 and 7). Iris vessels contained large amounts of APAB in contrast to the absence of peroxidase activity in the iris stroma (Fig. 4). The APAB were readily stopped at the level of intercellular tight junctions of the endothelial cells as shown in a moderately magnified electron micrograph (Fig. 9). In the control experiments performed with the in viva peroxidase-free incubation medium, no peroxidase activity could be seen, except for the endogenous peroxidase activity of red blood cells. Some mitochondrial cristae are also revealed (Figs 3 and 5).
ANTIPEROXIDASE
AXTIBODIES
IN
AXTERIOR
CVEA
“57
FIG. 6. Ultra-thin non-contrasted section of a ciliary process epithelium. Note the labelling in the intercellular spaces between cells of the inner epithelial layer (IL) and between the apices of the ir lner epithelial layer and the outer epithelial layer (OL). No l&belling in the intercellular spaces beyond the tight junctions (TJ) and no significant vesicular transport near the posterior chamber (PC!) could L he seen. (Immersion fixation. x 14 000).
PIG. 7. Ultra-thin non-contrasted section of the ciliary epithelium. The end-product of the pcroxitlase reaction is not observed in the intercellular spaces (curved arrow) between cells forming the oats epithelial layer (OL). Tight junctions are impermeable to AI’AK. IL = inner cpithelial layer: I\1 mitochondria. (Immersion fixation. x 22 000). Ik. 8. Ultra-thin non-contrasted sect,ion showing the intense labelling in the lumen (L) of t,ho ciliary capillary and in the surrounding stromal tissue (S). Note t,he presence of APAB in the endothelial fenestrae (arrows). (Immersion fixation. .,; 40 000). FIG. 9. Ultra-thin non-contrasted section of an iris vessel. The endothelial tight junctions (TJ) do not, permit the leakage of APAR from the lumen (L) to the perivascular space. EN> = cndothelial cells; M = mitochondria. (Immersion fixation. x 30 000).
AXTIPEROXIDASE
ANTIBODIES
I?;
AXTERIOR
UVEA
259
4. Discussion The major advantage of the immunoperoxidase technique used for this study is that it provides a physiological model for the in situ detection of plasma proteins in the anterior uvea of the eye. It differs from methods involving exogenous protein substances as tracers in that foreign substances introduced into the blood stream are considered as non-physiologic, being chemically different from plasma proteins. They probably interact with tissue components such as collagen fibers, or stimulate entlocytic activities, vesicular transports and changes in the permeability properties of the vessels. We have shown in this study, that APAB revealed in vitro by the end-product of t’he peroxidase reaction, escape from the fenestrated capillaries of the iridial and ciliary processes, invade massively the surrounding stromal tissue and are stopped by the tight junctions of the outer epithelial cells lining the posterior chamber. In the iris vessels, no leakage of APAB could be seen. No significant vesicular transport of these antibodies was detected through the outer epithelial cells, nor through the endothelial cells of the iris. We have also shown a progressive decrease of the immunostaining from the iridial and ciliary processes towardsthe iris st’roma and ciliary body base. This confirms the observations of Allanxmitht Newman and Whitney (1971) who employed the light microscopic technique of indirect’ immunofluorescence to identify the IgG in the rabbit eye. However, the present immunoperoxidase technique permits a better understanding of phenomena since tissues can be observed at, the ultrastructural level. It is a well-known fact that no morphological barrier exist’s on the anterior surfa,ce of the iris (Gregersen, 1958; Tousimis and Fine, 1959), so in theory, there is no reason why diffusible substances could not reach the aqueous humor (Grayson, Tsukahara a.nd Laties, 1974; Vegge, Neufeld and Sears, 1976). The absence in practice of APAB in the anterior area of the anterior uvea can be partially errplained by the constant, removal and dilution of proteins by the aqueous humor flow. The two methods of fixation used gave similar results, both in the distribution of the APAB in the anterior uvea and in the structure of junctional barriers. Since no leakage of APAB was observed, there can have been no rupture of the tight junctions, and so it seems that these fixation procedures do not, affect their permeability properties. In conclusion, this method will permit, in previously immunized animals: (I) the detection and location of blood-born APAB in both normal a,nd abnormal tissues and (2) the correlation of immunological phenomena and their corresponding morphological aspects ACKNOWLEDGMESTS
This investigation was supported by a grant from the Institut de la Recherche Mkdicale (A.T.P. 78/100 INSERM).
National de la SantP et
REFERENCES Allansmith, M. R., Newman, L. P. and Whitney, C. R. (1971). I mmunoglobulins in the rabbit eye. Arch. Ophthalmd. 86,6M. Burns-Bellhorn, M. S., Bellhorn, R. W. and Benjamin, J. V. (1978). Anterior segment permeability to fluorescein-labeled dextrans in the rat. Invest. Ophthulrnol. Fis. Sci. 17,857-62. Davson, H. (1972). The Physiology of the Eye. 3rd ed. London, Churchill.
960
Il.
HIRSCH
ET
AL.
Drrnouchamps, J. P. and Heremans, J. F. (1975). ;\lolerular sieve effeect, of t’hr l~lootl~nr~~~c~r~c~~ barrier. Eq). I$ye Res. 21, 289-97. B., Belair, 11. F. and Paing, M. (1978). Distriblltioll Druet, P., Bar&y, J.. LalibertB, F., Bellon, of heterologous antiperoxidase antibodies and their fragments in the superficial renal cwrtrl of normal Wistar Munich rat. An ultrastructural study. Lab. Invest. 39,623-31. Graham, R,. c’. and Karnovsky, X J. (1966). Th e early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. J. Histochem. Cytochem. 14, 391-302. Grsyson, M., Tsukahara, S. and Laties. A. M. (1974). Tn E’luoreseein. A+ogrnphy Bhimizu. Ii. (Ed.). P. 236. Igaku Shoin Ltd., Tokyo. Gregersen, E. (1958). The tissue spaces in t’he human iris and their communications wit,11 the anterior chamber by way of the iridic crypts. Bctn Ophthnlmol. 36,819-28. Hogan, M. J., Alvarado, J. A. and Weddell, J. E. (1971). Histoloyy ofthe Hwnun Eye. An L4tZrrs trnrl Textbook. 11’. B. Saunders clo. Philadelphia, London, Toronto. Mozart, D. M. (1968). Light and electron microsropic study of regional morphological differences in the processes of the ciliary body in the rabbit. Invest. Ophthulmol. 7, 15-33. Leduc, E. H., Avrameas, S. and Bouteille, M. (1968). Ultrastructural localization of ant,ibody in differentiating plasma cells. J. E.rp. Med. 127, 109-18. Raviola, G. (1977). The structural basis of t’he blood-ocular barriers. In The Ocular rend Cerebrospinal Fluids. F0gart.y International Centre Symposium (Eds Bito, L. Z., Davson. H. and Fenstermacher, J. D.). Exp. Eye Res. 24 Suppl. Pp. 27-63. Tousimis, A. J. and Fine, B. S. (1959). Ultrast’ructure of the iris: the intercellular components. A,rn. J. Ophthalmol. 48, 397417. Vegge, T., Neufeld, -4. H. and Sears, M. L. (1976). Movement of a protein tracer (horseradish peroxidase) in the anterior uvea. In The Structure of the Eye III. (Eds Yamada, E. and Mishima, S.). Pp. 103-110. JUP. J. Ophthalmol., Tokyo.
Localization of Autologous Antiperoxidase Antibodies in the Anterior Uvea of the Rabbit Eye 31. HIRSCH".
C. H. G. HARTMANN*:
B. BELLON?,
K. KELLER*
AND P. DRCET~
* ILNXERM 17. 86 (PT. Dr Y. Pouliquen), Chnique Ophtalnrologiquede I’Hdtel-Die-u and t IA’SERM
17.28/CNRX ERA -28>Hdpital Broussais, Pa&, France (Received2 August 1979, Lo&o)!)
Using an immunoperoxidase technique, we have studied the distribution of autologous antiperoxidase antibodies in the anterior uvea of the albino rabbit eye. Experimental animals were first immunized with horseradish peroxidase and the antibodies were then revealed in vitro 4 weeks later. It was found that the immunological tracer was unevenly distributed within the anterior uvea. The antiperoxidase antibodies extravasated from the fenestrated capillaries, diffused into surrounding stromal tissue and were then stopped by occluding or tight junctions of the outer epithelial layer of the pars plicata. From the iridial and ciliary processes to the iris and ciliary body base, the labelling of antiperoxidase antibodies progressively decreased. They were detected in the lumen of the iris vessels. This method is considered to be more physiological than those using exogenous substances as tracers and may be used to study ultrastructurally the permeability properties of the ocular barriers to macromolecules in normal, abnormal or experimental conditions. Key words : iris ; ciliary body; blood-aqueous barrier; immunoperoxidase ; antiperoxidase antibodies ; light and electron microscopy.
1. Introduction In the anterior uvea of the eye, a physiological barrier exists which, in norma. conditions, restricts the passage of blood-born macromolecules into the aqueous humor (Davson, 1972). Morphologically, this blood-aqueous barrier is constituted by occluding or tight junctions located in the iridial and ciliary body epithelia and in the endothelium of the iris vessels. In contrast, no morphological barrier has been observed between the cells lining the anterior surface of the iris where large openings have been described (Gregersen, 1958; Tousimis and Fine, 1959; Hogan, Alvarado and Weddell, 1971; Raviola, 1977). Therefore, the permeability properties of the blood-aqueous barrier to compounds of various molecular size have been extensively investigated, both with the light and electron microscopy to identify the distribution of plasma proteins in tissues, and biochemically by studying the protein composition of the blood, uveal tissues and aqueous humor. It has been shown that the permeability of the anterior uvea to substanceswasdependent upon their molecular radius and size (Dernouchamps and Heremans, 1975; Burns-Bellhorn, Bellhorn and Benjamin, 1978). In the present study, we have used the recently described immunoperoxidase technique (Druet et al., 1978) to investigate the in situ distribution of the autologous antiperoxidase antibodies (APAB) in the iris and ciliary body of the albino rabbit Reprint requests to: Mr. Michel Hirsch, Laboratoire de Cytologie et Marie Curie 16, rue de 1’Ecole de MBdecine, 75270-Paris cedex 06. 00144835/80/030253+08 c
Ultrastructurale
0 1980 Academic
$01.00/O 253
Press Inc.
Universite (London)
Pierre Limited
‘54
31. HIRSC’H
ET
XL.
eye and to re-examine the morphological sites of the blood-aqueous barrier hy 1.1sing the APAB as a physiological protein tracer.
2. Materials Immunization
and Methods
of animals
Six New Zealand albino rabbits weighing 2.5-3 kg were used for this experimentt Seven milligrams of horseradish peroxidase (Boehringer, grade 1, Jlannheim) emulsified with complete Freund’s adjuvant were injected into the hind foot pads of each rabbit. Four weeks later, the sera of the rabbits were tested by gel precipitation immunodiffusion technique for the presence of APAB. When tested by immunoelectrophoretic analysis, using peroxida,se as antigen, only IgG antibodies were detected. Preparation
of tissues
One hour before enucleation, two rabbits were treated with intravascular injections of 300 mg aspirin solution to minimize the supposed endogenous prostaglandins synthesis. These rabbits and two others non-treated with aspirin were anaesthetized with pentobarbital sodium injected in the marginal vein of the ear. Immediately after enucleation, the anterior segments were removed and pre-fixed for 1 hr at 4°C in 2.5% glutaraldehyde in 0.1 M-Sorensen phosphate buffer, pH 7.3-7.4. The anterior uvea rings were then separated from the corneas, cut into small pieces, post-fixed for 3 hr in the same fresh fixative and then washed in the buffer. A perfusion fixation procedure of the anterior chamber was also used in this study. A double needle was introduced into the anterior chamber of two living rabbits and the fixation solution slowly injected for 15 min. After this in situ pre-fixation, the eyes were treated as described above. IrrLrnunocytochemical
reaction
40 pm sections of the specimen were obtained with a Smith and Farquhar tissue-sectioner (Sorvall TC 2) and then incubated in buffered solution of horseradish peroxidase (0.5 “g/ml), at 0°C (Leduc, Avrameas and Bouteille, 1968; Druet et al., 1978). Tissue sections were rinsed in the buffer and incubated afterwards in 3,3’-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in O-2 M-Tris-HCl buffer (Graham and Karnovsky, 1966). They were rinsed in the Tris-HCl buffer and post-fixed in 1% osmium tetroxide in phosphate buffer, pH 7.4 at 4°C for 1 hr. Sections were dehydrated in a series of alcohol baths, placed in propylene oxide and embedded in Epon 812 epoxy-resin. Semi-thin and ultra-thin sections were cut on a Reichert OMU5 ultra-microtome and then examined with a Philips EM 300 electron microscope under 80 kV. Controls Some specimens was omitted.
were treated
as above,
but incubation
with
horseradish
peroxidase
3. Results In the rabbit, the pars plicata is divided into ciliary processessensustricto and iridial processes (Kozart, 1968). These processes are closely interconnected. In this paper, the inner epithelial cells correspond to the cells closest to the connective or stromal tissue and the outer epithelial cells correspond to the cells lining the posterior
chamber.
ANTIPEROXIDASE
AKTIBODIES
IN
ASTERIOR
UVEA
255
The immersion fixation of the anterior uvea pieces and the perfusion fixation in the anterior chamber of the eyes gave similar results. No difference was observed between the aspirin treated and non-treated animals. We noted that the lumen of the vessels in the iridial and ciliary processes was homogeneously filled with APAB as shown by the very denseperoxidase activity of the plasma. In the neighbouring connective tissues there was a gradient of this
FIG. 1. Semi-thin unstained section of an area of the anterior uvea. The stroma of the iridial procens (P) and the plasma in the lumen of the vessels (V) are strongly labelled. Note the progressive decrease of the labelling from the process of the iris (I). PC! = posterior chamber. (Perfusion fixation. x 150). PIG. 2. Semi-thin unstained section of a ciliary process. The labelling is seen in the stroma of t.he process (S) and in the intercellular spaces of the inner epithelial layer (IL). No labelling is observed beyond the tight junctions (TJ) sealing the intercellular spaces of the outer epithelial cell layer (OL). I’6 = posterior chamber. (Immersion fixation. x 360). FIG. 3. Control semi-thin unstained section of a ciliary stroma process (without horseradish peroxidase in the incubation medium). The labelling is not. seen either in the lumen of vessel (L) or in thr stromal tissue (S). (Perfusion fixation. x 150).
FIG. 4. Semi-thin unstained section of the posterior area of the iris. The labelling is localized only iu the lumen (L) of the “non-leaky” iris vessels. Iris stroma (8) is free of peroxidase activity. (T’erfuaion fixation. x 375). FIG. 5. Control semi-thin unstained section of the iris (without horseradish peroxidasc in the incuhation medium). The plasma of the lumen (L) of the vessels is not labelled. Sote the well-known endogenous peroxidase activity of the red blood cells (rbc). (Perfusion fixation. >, 700).
activity which increased towards the posterior part of the iridial and ciliary processes and decreased towards the iris and ciliary body base (Fig. 1). The APAB escaped freely from the fenestrated “leaky” capillaries, completely invaded the surrounding stromal tissue (Figs 1 and 8) and reached the intercellular spaces between the inner epithelial cells and between the apices of the inner and the outer epithelial cell layers (Figs 2 and 6). They were stopped by occluding or tight junctions sealing the intercellular spaces of the outer epithelial cells lining the posterior chamber. No APAB could be detected beyond this epithelial barrier (Figs 6 and 7). Iris vessels contained large amounts of APAB in contrast to the absence of peroxidase activity in the iris stroma (Fig. 4). The APAB were readily stopped at the level of intercellular tight junctions of the endothelial cells as shown in a moderately magnified electron micrograph (Fig. 9). In the control experiments performed with the in viva peroxidase-free incubation medium, no peroxidase activity could be seen, except for the endogenous peroxidase activity of red blood cells. Some mitochondrial cristae are also revealed (Figs 3 and 5).
ANTIPEROXIDASE
AXTIBODIES
IN
AXTERIOR
CVEA
“57
FIG. 6. Ultra-thin non-contrasted section of a ciliary process epithelium. Note the labelling in the intercellular spaces between cells of the inner epithelial layer (IL) and between the apices of the ir lner epithelial layer and the outer epithelial layer (OL). No l&belling in the intercellular spaces beyond the tight junctions (TJ) and no significant vesicular transport near the posterior chamber (PC!) could L he seen. (Immersion fixation. x 14 000).
PIG. 7. Ultra-thin non-contrasted section of the ciliary epithelium. The end-product of the pcroxitlase reaction is not observed in the intercellular spaces (curved arrow) between cells forming the oats epithelial layer (OL). Tight junctions are impermeable to AI’AK. IL = inner cpithelial layer: I\1 mitochondria. (Immersion fixation. x 22 000). Ik. 8. Ultra-thin non-contrasted sect,ion showing the intense labelling in the lumen (L) of t,ho ciliary capillary and in the surrounding stromal tissue (S). Note t,he presence of APAB in the endothelial fenestrae (arrows). (Immersion fixation. .,; 40 000). FIG. 9. Ultra-thin non-contrasted section of an iris vessel. The endothelial tight junctions (TJ) do not, permit the leakage of APAR from the lumen (L) to the perivascular space. EN> = cndothelial cells; M = mitochondria. (Immersion fixation. x 30 000).
AXTIPEROXIDASE
ANTIBODIES
I?;
AXTERIOR
UVEA
259
4. Discussion The major advantage of the immunoperoxidase technique used for this study is that it provides a physiological model for the in situ detection of plasma proteins in the anterior uvea of the eye. It differs from methods involving exogenous protein substances as tracers in that foreign substances introduced into the blood stream are considered as non-physiologic, being chemically different from plasma proteins. They probably interact with tissue components such as collagen fibers, or stimulate entlocytic activities, vesicular transports and changes in the permeability properties of the vessels. We have shown in this study, that APAB revealed in vitro by the end-product of t’he peroxidase reaction, escape from the fenestrated capillaries of the iridial and ciliary processes, invade massively the surrounding stromal tissue and are stopped by the tight junctions of the outer epithelial cells lining the posterior chamber. In the iris vessels, no leakage of APAB could be seen. No significant vesicular transport of these antibodies was detected through the outer epithelial cells, nor through the endothelial cells of the iris. We have also shown a progressive decrease of the immunostaining from the iridial and ciliary processes towardsthe iris st’roma and ciliary body base. This confirms the observations of Allanxmitht Newman and Whitney (1971) who employed the light microscopic technique of indirect’ immunofluorescence to identify the IgG in the rabbit eye. However, the present immunoperoxidase technique permits a better understanding of phenomena since tissues can be observed at, the ultrastructural level. It is a well-known fact that no morphological barrier exist’s on the anterior surfa,ce of the iris (Gregersen, 1958; Tousimis and Fine, 1959), so in theory, there is no reason why diffusible substances could not reach the aqueous humor (Grayson, Tsukahara a.nd Laties, 1974; Vegge, Neufeld and Sears, 1976). The absence in practice of APAB in the anterior area of the anterior uvea can be partially errplained by the constant, removal and dilution of proteins by the aqueous humor flow. The two methods of fixation used gave similar results, both in the distribution of the APAB in the anterior uvea and in the structure of junctional barriers. Since no leakage of APAB was observed, there can have been no rupture of the tight junctions, and so it seems that these fixation procedures do not, affect their permeability properties. In conclusion, this method will permit, in previously immunized animals: (I) the detection and location of blood-born APAB in both normal a,nd abnormal tissues and (2) the correlation of immunological phenomena and their corresponding morphological aspects ACKNOWLEDGMESTS
This investigation was supported by a grant from the Institut de la Recherche Mkdicale (A.T.P. 78/100 INSERM).
National de la SantP et
REFERENCES Allansmith, M. R., Newman, L. P. and Whitney, C. R. (1971). I mmunoglobulins in the rabbit eye. Arch. Ophthalmd. 86,6M. Burns-Bellhorn, M. S., Bellhorn, R. W. and Benjamin, J. V. (1978). Anterior segment permeability to fluorescein-labeled dextrans in the rat. Invest. Ophthulrnol. Fis. Sci. 17,857-62. Davson, H. (1972). The Physiology of the Eye. 3rd ed. London, Churchill.
960
Il.
HIRSCH
ET
AL.
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