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Permeability of fluorescein-labelled dextrans in fundus fluorescein angiography of rats and birds
Exp. Eye Res. (1977) 24, 595-605
Permeability ~~ARGARET
of Fluorescein-labelled Dextrans in Fundus Fluorescein Angiography of Rats and Birds* B.
BELLHORN,
ROY
w.
BELLHORS
AND
DAVID
8. POLL
of Ophthaln~ology,MosteJiore Hospital a,& Medical Ceder/Albert Einst& College of Medicine, Bronx: X.Y. 10467, U.S.A.
Dq~~rtmeret
(Receiued16 Jurle 1976 and in revisedform 22 September1976, LomGm) Intravenously injected sodium fluorescein (mol. wt. 37G) does not leak from normal mammalian retinal vessels. However, sodium fluorescein (KaFl) leaks profusely from the avian pecten, a pigmented, vascularized organ locat.ed atop the optic nerve head and protruding into the vitreous cavity. Since molecular size is one property of molecules which ,governs t,heir permeability in a given blood vessel system, the permeability of these two intraocular blood vessel systems to fluorescein labelled compounds of selected molecular sizes was compared in vivo by standard fluorescein angiographic techniques. The compounds !lsed were fluorescein isothiocyanate (FITC) labelled dextmns of the following average dimensions: FITC-Dex 3 (mol. wt. 3000, radius, 12 if); FITC-Dex 20 (mol. wt. 20 000, ex 70 (mol. wt. ‘70 000, 58 $). Normal 32 A) ; FITC-Dex 40 (mol. wt. 40 000, 45 A) ; FITC-D rat retinal vessels were impermeable to any of these dextrans. In contrast to the known response to KaFI, the pigeon pecten effectively excluded each of the FITC Dextrans from ent.ering the vitreous. It is thought. that dissimilar mechanisms are responsible for t,he individual permeability responses of these mammalian and avian intraocular vessel systems. Key uor&: fluorescein; dextran; capillaries; permeability; angiography; retinal vessels; pecten: mierova,sculature; bird; rat; blood-retina barrier.
1. Introduction
Vascular permeability is an important functional parameter of all tissue systems, including those of the eye (Bill, 1955). The blood vesselsof each individual tissue system exhibit various anatomical and biochemical specificities which, in concert., control the transit of substancesfrom the blood vesse1lumen to the tissue substance. (Landis and Pappenheimer: 1963; Bruns and Palade, 1968; Studer and Potchen, 1971; Shakib and Cunha-Vaz, 1966). In the mammalian retina, for example: the capillaries have eont’inuous (nonfenestrated) endothelial cells joined by zonular tight junctions which are nonreactive to histamine. (Ashton and Cunha-Vaz, 1965; Shakib and Cunha-Vaz, 1966). Thesevesselsare not normally permeable to intravenously injected sodium fluorescein (NaFl), dyes covalently bound to serum albumin or to the electron microscopic tracer, horseradish peroxidase (Grayson end Laties, 1971; Machemer. 1970; Shioseand Oguri, 1969). These observations constitute examples of the bloodretina barrier. One function of such tight junctions is to prevent the passage of substancesfrom the capillary lumen to the neural retina based upon molecular size, and thus be the morphological equivalent of a physiologica~l pore (Landis and. Pappenheimer, 1963). Exclusion on the basis of molecular size is known to be an important discriminant in other blood vessel systems (Grotte, 1956). Our interest is in developing nondestructive in vivo methodologies with which permeability changescan be monitored, for example, on the basis of molecular size. inasmuch as such techniques would be useful in following the temporal course of alterations in retinal capillary permeability (Bellhorn, Bellhorn, Friedman and Henkind, 1973). *Reprint 210th Street,
requests to Sam Gartner Library, Bronx, N.Y. 10467, U.S.A.
Montefiore
Hospital
and
Medical
C!entor,
111 East
596
M.
B. BELLHORN,
R. W.
BELLHORN
AND
D. S. POLL
In this paper we introduce fluorescein isothiocyanate labelled dextrans of molecular sizes as an in vivo fundus fluorescein angiographic marker in models. As a comparative study we investigated the permeability of the avian an intraocular organ which normally is permeable to intravenously injected fluorescein (Bellhorn and Bellhorn, 1975), to these molecular weight markers.
selected animal pecten, sodium
2. Materials and Methods Fluorescein isothiocyanate (FITC) dextrans (Pharmacia Fine Chemicals, Inc.) are a homologous series of glucose polymers differing in molecular weight. The glycosidic linkages are primarily 1,4-a although some 1,6-a bonds are present. They are covalently labelled with fluorescein by condensation with fluoresceinisothiocyanate to form fluorescein thiocarbamoyl dextran derivatives (deBelder and Granath, 1973). Free fluorescein is removed by ethanol precipitation and molecular sieve chromatography. We have verified the absence of free fluorescein by chromatography on Sephadex G-25 in O-1 x-sodium phosphate buffer, pH 7.4. On the average there is one fluorescein residue per 100-1000 glucose residues. TABLE
I
Properties of tracersfey permeability studies Effective
Colllpouncl FITC-Dex 3 FITC-Dex 20 FITC-Dex 40 FITC-Dex 70 FITC-Des 150 Glucose Medium Fluorescein Myoglobin Bovine Serum Albumin Horseradish Peroxidase Ferritin Colloidal Carbon
&rolecular weight
3000* 20 000" 40000* 70000" 150000" 180 376 17800 69 000 40000 600000
diffusion radius
(A)
Diameter
121.
240
321 45t 5st
W
w
(A)
90s
1164 170s
3.6:
19.q 351 30**
614” -
30-i 508 200
* Average molecular weight, see text for explanation. f Averqe molecular size, see text for explanation. $ Grotte, 1956. $ Estimated as twice the effective diffusion radius. l[ Williams and Wissig, 1975. ** Caulfield and Farquhar, 1974.
The FITC-dextrans used in this study were of the average size and molecular weight shown in Table I; for comparison, properties of other compounds used in permeability studies are given. The FITC-dextrans are heterodisperse with respect to molecular weight (and therefore size) and encompass a range of molecular dimensions. For example, the FITC-Dex 3 used in this study ranged from mol. wt. 1200 to 5000. The average molecular weight by weight (L@~) is 3000 and the average molecular weight by number of molecules (B,) is 2400, indicating a greater number of molecules of mol. wt. less than 3000. Similar considerations are true for the other FITC-dextran fractions although the ranges are proportionally less widespread (Pharmacia Fine Chemicals, technical data bulletin).
PERMEABILITY
OF
FLUORESCEIN
DEXTRAKS
597
The FITC-dextrans were used as solutions of 1 g in 3.0 ml of 0.1 M-sodium phosphate buffer, pH 7,4, a, concentration empirically possible for all FITC-dextrans used in this study. The buffer is at physiological pH for both rats and birds. As a control, solutions of Dextran 40, unlabelled with fluorescein, were mixed with free KaFl to give a final concentration of 16% D eyi t ran 40 and 0.50/b sodium fluorescein in 0.1 &I-sodium phosphate buffer, pH 7.4.
Since the degree of fluorescein substitution of the dextrans is low (0.01 to O.OOl), even a concentra,ted FITC-dextran solution is much less fluorescent than the 5-10% n’aF1 solutions commonly used. Therefore, as a standard reference, 0+0/6 KaFl in 0.1 x-sodium phospha.te buffer, pH 7.4, was used in all studies reported here. It was found by absorption spectrophotometry (Beckman DU spectrophotometer) that this concentration was comparable to that of the 33% solutions of FITC dextrans at their absorption maxima. The FITC-dextrans exhibit an absorption maximum at 493 nm, as eompa,red to 495 nm for NaFl in the same buffer. This shift could not influence the results as it is within the transmission range of the excitation filter used. The technical data from the manufacturer shows an emission spectrum maximum of 520 nm; the same as unbound sodium fluorescein.
Reca,use concentrated solutions of small molecular weight components will be appreciably hyperosmotic, we were concerned that a,rtifactual results could be obtained by, for exanlple, opening of the blood-retina barrier due to the hypertonicity of the dextran solutions (Rapoport and Thompson, 1973). The osmolality of the solutions used was mea,sured in an Adva,nced Osmometer, Advanced Instruments, Inc., courtesy of Dr Richard Lent, and were as follows : 0.1 M-sodium phosphate buffer, pH 7.4, 290 &osm/kg ; FITC-Des 3 in buffer, 416 mosm/kg ; FITC-Dex 20 in buffer, 309 mosm/kg ; FITC-Dex 40 in buffer, 356 mosm/kg; FITC-Dex 150 in buffer, 295 mosm/kg.
The dextrans are long chains rather than globular molecules and have an appreciable viscosity. This parameter was measured at 38°C in a Wells-Brookfield Micro Visc,ometer, Model LVT (courtesy of Dr Jack Novodoff): 0.1 al-sodium phosphate buffer, pH 7.4. 0.89 centipoise; FITC-Dex 3; 4.13 cP; FITC-Des 20, 19.6 cP; FITC-Dex 40, 36.1 cP; FITC-Dex 70, 75.3 cP; and FITC-Dex 150, 181.44 cP.
Kormal adult pigmented (Long-Evans strain) rats were anesthetized i.p. with sodium pentobarbital, 40 “g/kg, after pupillary dilation with 1% atropine sulfate and lo?; phenylephrine HCl. A femoral vein was surgically exposed and catheterized, and 0.I cc of a test compound was injected for fluorescein angiography. The rats used weighed approximately 250 g a.nd therefore had a pla.sma volume of 10 ml, or 100 times greater than the injection volume (Biology Da&a Book, 1974). Although dextran injections have caused anaphylactoid reactions in rats, the dosage of dextran in these exper&nents is less than that required to produce this effect (Norrison, Bloom and Richardson, 1951) and 110 severe reactions were observed in the current study. Twenty-four normal rats were used and each generally received two injections. Adult pigeons, after pupillary dilation with D-tubocurarine HCl in O.O25o/o benzalkonium chloride (3 mg/cc)> were anesthetized with a tiletamine HCl-zolazepam HCl combination (CL-744, Parke Davis & Co.) at 20 mg/kg i.m. A brachial vein was surgically exposed and catheterized and 0.25 cc of one of the test compounds injected. The pigeons used weighed
598
M.
B. BELLHORK,
R. IV.
BELLHORN
AND
D. S. POLL
250-350 g and had a plasma volume of approximately 13 ml (Biology Data Book, 1974), or approximately 50 times the injected volume of test compound. Twelve pigeons were used and each received multiple injections of FITC-dextran followed by sodium fluorescein. Fundus fluorescein photography was accomplished using a fundus camera (Fundus Flash II, Carl Zeiss, West Germany) equipped for fluorescein angiography with a Kikon F motorized camera a,nd Kodak Wratten 47A excitation and Wratten 15 barrier filters. it A 400 ASA black and white film (Kodak Tri-X) wras used to record the angiograms; wa.s routinely processed. The flash intensity of the fundus camera was set at maximum in order to elicit good photographic detail of the eye transit. The first frame was exposed simultaneously with the start of the injection in order to approximate the time of dye passage from the injection site. The minimum time between frames was of the order of Ot5 sec.
FIG. I. Standard retinal fluorescein angiogram utilizing 0.1 cc of 0.5% sodium fluorescein in a norma! rat. (a) 2.9 sec. Earliest indication of arterial phase. (Arrow). (b) 3.4 sec. Laminar flow (arrow) indicative of early venous phase only 0.5 set aft.er early arterial phase. Focal hyperfluorescent spots are end-on views of precapillary arterioles traversing from nerve fiber layer to superficial and deep capillary beds. appearance is not (c) 5.2 sec. First indication of full vascular phase. Elapsed time from first arterial more than 3.3 seo. The full capillary bed is apparent. (d) 446.7 sec. Late phase showing hazy and uncontrasty fundus appearance. The disc margin exhibits residual fluorescence.
PERMEABILITY
OF
FLUORESCEIN
DE;XTR’AP;S
599
3. Results In the normal rat, good quality fluorescein angiograms were obtained using 0.1 ml as compared to previous of 0.5% NaFl (Fig. 1). F emoral vein catheterization, techniques, provided more control over factors such as slight animal movement and assured compact bolus injection and control of the exact time of injection.
Flo. 2. Angiograms obtained utilizing fluorescein labelled dextrans in normal rats. (a) 4.8 sec. FITC-Des $ showing early venous laminar flow. (h) 454.1 sec. A late phase angiogram of FITC-Dex 3 in this same animal. (c) ‘i.1 sec. FITC-Dex 70 angiogram illustrating early venous phase. Note that all veins may not fill simultaneously. (d) 495.5 sec. FITC-Dex 70 angiogramillustrating late phase persistent vessel fluorescence. Compare vascular detail with that observed at same stage using 0.5% sodium Huorescein [Fig. l(d)].
It is evident in some’ normal rats that arterial and subsequent venous filling [Fig. 2(c)] may be sectorial in that certain vessels fill more quickly than others. This preferential filling is consistent in those rats each time angiography is performed. It may be that one or more arteries arise from the central retinal artery at a point slightly proximal to others. The FITC-Dex 3 angiogram was similar to that of NaFl in that no evidence of leakage or altered permeability of the retinal vessels was evidenced (Fig. 2). The
600
MI. B. BELLHORN,
R.
1%‘. BELLHORN
AND
D.
S. POLL
FIG. 3. Fluorescein angiogram of normal pigeon pecten after injection of a rn~sture of plain Dextl photograph. (b) Presence of fluorescence within pecten appro 413 and 0.5% NaFl. (a) Pre-injection n Lately 4 set after injection. (c) Virtual disappear rance of fluorescence from pecten about 8 set af (d) Beginning evidence of fluorescence adjacent to pecten at 28 set post injection. (e), in Ijection. evidence of fluorescence in vitreous at Ei6 and 220 set post injection. II xreasing
PERMEABILITY
OF
FL~ORESCEIW
DEXTKAN8
601
&+. 4. Fluorescein angiogram of normal pigcon pecten demonstrating sequence of evwts iLfttc[ !njection of FITC Dcstran 3 and a subsequent injection of O,5o/O NaFi. (a) l’re-injection photograph ,&owing pseudoflum-c,scenee of optic disc. Choroidai x~essek against light ha&ground on sclera ax (h) Presence of FITC Dextran 3 is evident within pccten 4.5 SW \-isible because t.he fundus is albinotic. post inject.ion. (c) Virtual absence of FITC De&ran 3 fluorescence evident at 87 set post injection. within pecten of same bird 4.1 WC after injection Compare with Fig. 3(e). (d) P resence of fluorescence of 0.5% NaPI. (e), (f) Presence of fluorescence adjacent to pecten 66 set and 257 see after injection of @50/o XaFl. Compare relative lack of parapectinal fluorescence of this bird to that depicted in Fig. 3. ‘~:he albinotic fundus allows scleral and ohoroidal fluorescence to partially mask that present, in the vitreous.
602
iM.B.RjEL~,$OR~,jR.W.BEL.LKOR~ANDD.S.POLT,
higher molecular weight FITC-dextrans similarly caused no signs of leakage across the blood-retina barrier under the given conditions (Fig. 2). An effect of increased molecular weight was a longer persistence of fluorescence in the vessels and an enhanced contrast with the background [compare Figs l(d) and 2(d)]. A mixed solution of Dextran 40 with free NaFl provided angiograms with characteristics similar to NaFI.
In the normal pigeon, 0.25 ml of a 0.5% solution of NaFl permits photographic visualization of the diffuse leakage into the vitreous, as previously observed at higher NaFl concentration (Bellhorn and Bellhorn, 1975). A mixture of unlabelled Dextran 40 and free NaFl given intravenously leaked profusely into the vitreous, as though the NaFl had been administered alone (Fig. 3). In contrast; no fluorescence within the vitreous was observed when any of the FITC-dextrans were administered [Fig. 4(a); (lo), (c)l. As an assurance that the FITC-dextran results were not due to, e.g. faulty positioning of the catheter, a subsequent injection of 0.5% NaFl was administered a few minutes later through the same catheter and in all instances the typical leakage phenomenon was observed [Fig. 4(d), (e), (f)]. Whenever NaFl was administered the initial appearance of dye in the pecten was followed by a brief interlude of darkness prior to observance of fluorescence in the vitreous (Fig. 3). Whenever FITC-dextrans were administered, they were observed to pass into the pecten and then to just fade away [Fig. 4(a), (b), (c)l. 4. Discussion The feasibility of using FITC-dextrans as in vivo molecular weight markers for fundus angiography has been demonstrated in rats and birds in these studies. Because the dextrans are not innocuous and may affect several aspects of the microvaseulature, the following factors were considered in evaluating this technique : (1) It is assumed that FITC-dextrans behave similarly to unlabelled dextrans of identical molecular weight and size. This seems reasonable in that the degree of labelling with fluorescein is low and unlikely to result in severe alterations of the dextran molecule configuration or chemistry. (2) An important effect of dextrans is that they may cause, in some strains of rats, histamine release and concomitant changes in vascular permeability. Although the effects of histamine are marked in peripheral vascular systems (Carter, Joyner and Renkin, 1974), it has been shown that the retinal vascular system does not exhibit permeability changes even to exogenously applied histamine (Ashton and Cunha-Vaz, 1965). The only observed effect which may have been due to dextrans was some respiratory difficulty in two rats. WTe are unaware of information concerning use of dextrans in birds but we did not observe untoward effects. (3) The osmolality of the solutions was of concern since local hypertonicity could cause alterations in capillary permeability (Rapoport and Thompson, 1973). The low molecular weight solutions were only slightly hyperosmotic to mammalian plasma, and in fact, no leakage from normal ma’mmalian vessels was noted with any of t,he dextrans used. Undoubtedly the fast heart beat in rats (approximately 350 beats/min) and birds (approximately 250 beats/min) helps to quickly dilute the small injection volume throughout the plasma volume (Riology Data Book, 1974).
PERMEABILITY
OF
FLUORESCEIN
DEXTRA,\TS
603
(4) Although the viscosity of the FITC-de&an solutions is appreciable and dextrans can increase blood viscosity (Singh and Coulter, 1973) or cause erythrocyte aggregation (Stalker, 1967) at high molecular weights, these properties did not appear to play a role in the results obtained. Once more, the rapid circulation in rats and birds and the small injection volume will help to diminish the import,ance of high viscosity solutions. (5) Increased persistence of dye in the retinal vasculature when compared to NaFl is due possibly to decreased kidney clearance of the high molecular weight compounds (Caulfield and Parquhar, 1974). An additional factor which may be involved in increased dye persistence is that the FITC-dextrans probably extravasate from even highly permeable blood vessel systems, such as skin or choroid, less rapidly than sodium fluorescein, thus effectively maintaining a higher blood concentration of dye. In turn, a less marked extravasation of FITC-dextrans from the choroidal circulation would enhance the photographic contrast of a late stage angiogram by reduction of background fluorescence [compare Figs l(d), 2(b), (d)]. Although dextrans are known to produce alterations in blood flow properties (e.g. Singh and Coulter, 1973) it is unlikely that those effects play a role in the late appearance of FITCdextrans in this study because the amount of dextran injected is small compared to the plasma volume. Such effects would become obvious if large quantities of dextrans were infused. This property of persistent vessel visualization could be useful in the study of abnormal retinal vessels prior, for example, to laser photocoagulation. (FITC-dextrans are not currently approved for use in humans). Dextrans have long been used to study peripheral microvasculature permeability (Grotte, 1956). Several properties enable the dextrans to be meaningful molecular size markers : (1) molecular size is both smaller and larger than serum albumin, considered to be at the limit of macromolecular transport by the pore theory (Landis and Pappenheimer, 1963) ; (2) no apparent in vivo aggregation to larger molecular sizes (Caulfield and Farquhar, 1974; Schrrijder, Arfors and Tangen, 1976) ; (3) binding to plasma proteins probably does not occur with the lower molec&r weight dextrans (Schroder et al., 1976). The fact that low molecular weight FITC-dextrans do not pass across a normal mammalian retinal vessel makes them invaluable for studies of abnormal retinal vesse1.s. Por example, does an abnormality which permits leakage of NaFl, a small molecule, simultaneously allow passage of larger fluorescein-labelled molecules and to what extent? Since NaFl is normally actively excluded from viable cells and retinal vessels, it is probable that the perm.eability mechanism which excludes NaFl is due to some property other than molecular size, e.g. anionic charge (Cunha-Vaz and Maurice, 1967). In order to obtain this kind of information, it is important that the fluorescein-dextran bond is stable under in vivo conditions for more than 24 hr (Arfors and Hint, 1971). The avian pecten is unusual in that it permits diffuse passage of NaPI across the capillary walls which have a unique elaborated surface unlike other known capillaries (Bellhorn and Bellhorn, 1975; Seaman and Storm, 1963). In contrast to the permeability to NaFI, the pecten d oes not permit passage into the vitreous of any of the tested PITC-dextrans. This suggests that two different permeability mechanisms exist in the pecten, which has some properties reminiscent of the ciliary body (Seaman and Storm: I963). The eiliary body is Iikewise permeable to NaPI, but excludes protein
6UI
11. 13. BELLHORN,
R.
W.
BELLHORN
AKY
1). S. POLL
molecules on the basis of their molecular size, consistent with a pore theory of permeability (Dernouchamps and Heremans, 19’75) and similar to t’he peotan. In conclusion, FITC-dextrans can be profitably used for the study of permeability processes in retinal vessels which, unlike brain capillaries, are accessible to in vivo photography. Moreover, the FITC-dextrans will be meaningful in studies of ciliary body permeabilky to molecules of various sizes: an important aspect of aqueous humor formation. The permeability of the avian peeten, an organ more accessible to iu vivo photography than the ciliary body, may provide a model for mechanisms of fluid formation similar to those of the cjliary body, Because the FITC-dextrans arc not currently approved for human use and may cause a hypersensitivity reaction, the potent’ial clinical value of these compounds will have to be carefully evaluated. The LISA of these agents in studies of animal ocular physiology and pathology2 however, will have relevance to human pat~hophysiology.
We wish to thailk Dr Harold Kern for bringing these co~~~pounds to our attenbion. We are grateful to Noel Ron for excellent as&ance in aninlal surgery a,nd photography. We have profited from st~imulnting discussions with Drs Paul Henkind, Joseph Walsh, Daniel Gold and Barry Beckerman. Supported in part by USPHS Grants EY 01103 01 and EY 00613 05 (X.E.I.) and a gra’nt, from Research to Prevent Blindness, Inc., New York, N.Y. REFERE;NCES Arfors,
K-E. and Hint, H. (1971). Studies of the microcirculation using fluorescent destran. Abstr. Xicro~~ccsc. Res. 3, 437. d&on, I\ and Cunha-Vaz. J. G. (1965). Effect of hi&amine on the permeability of the ocular vessels.
Bellhorn,
Arch.
R’. W.,
OphthaZm.oZ.
Bellhorn,
73,
M.
211-23.
B..
Friedman, A. .H. and Honkind, Irvwst. Ophthnlmol. 12, 65-76. X B. (1975). The avian pecten.
I’.
(1973).
Urethan-induced
retinopathy in pigmented rats.
I. Fluorescein permeability. and Bellhorn, Res. 7, 1-7. Bill, A. (1975). Blood circulation and fluid dynamics in the eye. Physiol. Reu. 55, 383-417. Biology Data Book, Volume IIT, 2nd ed. (1974). (Eds Altman, P. L. and Dittmer, D. S.), Federation of American Societies for Experimental Biolo,gy, Bethesda, ?vId. Bruns, R. R. and Pnlade, G. E. (1968). Studies on blood capillaries. I. General organization of blood capillaries in muscle. J. Cell Biol. 37, 244-76. Cart’er, R. D., Joyner, W. L. and Renkin, E. M. (1974). Effect,s of histamine and some other substances on molecular selectivity of the capillary wall t’o plasma proteins and dextran. Nicrovasc. Res. 7, 31-48. Caulfield, J. P. and Farquhar, M. G. (1974). The permeability of glomerular capillaries to greded dextrans. J. Cell Biol. 63, 883-903. Cunha-Vaz, J. G. and Maurice, D. 31. (1967). The active transport of fluorescein by the retinal vessels and the retina. J. Physiol., Land. 191, 467-86. de Belder, A. N. and Granath, IX. (1973). Preparation and properties of fluorescein-labclled dextrans. Ccwbohyd. Res. 30, 375-8. Dernouchamps, J. P. and Heremans, J. F. (1975). Molecular sieve effect of the blood-aqueous harrier. &p. Eye Res. 21, 159-97. Grayson, M. C. and Laties, A. M. (1971). Ocular localizat,ion of sodium fluorescein. Arch. Ophth~lmol. $5, 600-609. Grotte, G. (1956). Passage of dextran molecules across the bloodMymph barrier. Beta C&r. &and. iYupp1. 211, I-84. Landis, E. 31. and Pappenheimer, J. R. (1963). Exchange of substance through the capillary walls. In Handbook of Physiology (Eds Hamilton, W. I?. and Dow, P.). Z(2), pp. 961-1034. American Physiology Society, Washington, D.C. Bellhorn,
R. W.
Ophthnlmol.
PERIVIEABILITY
OF
FLU
ORESCEIN
DEXTRANS
605
Machemer, R. (1970). Angiographic-histologic correlation of eye vessel permeability with protein bound fluorescent dye. Am. J. Ophthalmol. 69, 17-38. ?IIorrison, J. L., Bloom, W. L. and Richardson, A. P. (1951). Effect of dextran on the rat. J. Ph~armncol. Exp. Therap. 101, 27-8. Rspoport, S. I. and Thompson, H. K. (1973). Osmotic opening of the blood-brain barrier in the monkey without associated neurological deficits. science 180, 971. Schriider, U., Arfors, K.-E. and Tangen, 0. (1975). Stability of fluorescein labeled dextrans in vivo and in vitro. Microvascular Res. 11, 33-8. Seaman, A. R. and Storm, H. (1963). A correlated light and electron microscope study on the pecten oculi of the domestic fowl (Gallus domesticus). Exp. Eye Res. 2, 163-72. Shakib, M. and Cunha-Vaz, J. G. (1966). Studies on the permeability of the blood-retinal barrier. IV. Junctional complexes of the retinal vessels and their role in the permeability of the blood-retinal barrier. Exp. Eye Res. 5, 229-34. Shiose, Y. and Oguri, M. (1969). Electron microscopic studies on the blood-ret.inal barrier and the blood-aqueous barrier. Acta Sac. Ophthalmol. Jap. 73, 1606-12. Singh. M. and Coulter, N. A. (1973). Rheology of blood: effect of dilution with various dextrans. Microwasc. Rea. 5, 123-30. Stalker, A. L. (1967). The microcirculatory effects of dextran. J. Path. Bact. 93, 191-201. Studer, R. and Pot&en, J. (1971). The radioisotopic assessment of regional microvascular permeability to macromolecules. illicroz:asc. Res. 3, 3548. Williams, 31. C. and Wissig, S. L. (1975). The permeability of muscle capillaries to horseradish peroxidase. J. Cell Biol. 66, 531-55.
Permeability ~~ARGARET
of Fluorescein-labelled Dextrans in Fundus Fluorescein Angiography of Rats and Birds* B.
BELLHORN,
ROY
w.
BELLHORS
AND
DAVID
8. POLL
of Ophthaln~ology,MosteJiore Hospital a,& Medical Ceder/Albert Einst& College of Medicine, Bronx: X.Y. 10467, U.S.A.
Dq~~rtmeret
(Receiued16 Jurle 1976 and in revisedform 22 September1976, LomGm) Intravenously injected sodium fluorescein (mol. wt. 37G) does not leak from normal mammalian retinal vessels. However, sodium fluorescein (KaFl) leaks profusely from the avian pecten, a pigmented, vascularized organ locat.ed atop the optic nerve head and protruding into the vitreous cavity. Since molecular size is one property of molecules which ,governs t,heir permeability in a given blood vessel system, the permeability of these two intraocular blood vessel systems to fluorescein labelled compounds of selected molecular sizes was compared in vivo by standard fluorescein angiographic techniques. The compounds !lsed were fluorescein isothiocyanate (FITC) labelled dextmns of the following average dimensions: FITC-Dex 3 (mol. wt. 3000, radius, 12 if); FITC-Dex 20 (mol. wt. 20 000, ex 70 (mol. wt. ‘70 000, 58 $). Normal 32 A) ; FITC-Dex 40 (mol. wt. 40 000, 45 A) ; FITC-D rat retinal vessels were impermeable to any of these dextrans. In contrast to the known response to KaFI, the pigeon pecten effectively excluded each of the FITC Dextrans from ent.ering the vitreous. It is thought. that dissimilar mechanisms are responsible for t,he individual permeability responses of these mammalian and avian intraocular vessel systems. Key uor&: fluorescein; dextran; capillaries; permeability; angiography; retinal vessels; pecten: mierova,sculature; bird; rat; blood-retina barrier.
1. Introduction
Vascular permeability is an important functional parameter of all tissue systems, including those of the eye (Bill, 1955). The blood vesselsof each individual tissue system exhibit various anatomical and biochemical specificities which, in concert., control the transit of substancesfrom the blood vesse1lumen to the tissue substance. (Landis and Pappenheimer: 1963; Bruns and Palade, 1968; Studer and Potchen, 1971; Shakib and Cunha-Vaz, 1966). In the mammalian retina, for example: the capillaries have eont’inuous (nonfenestrated) endothelial cells joined by zonular tight junctions which are nonreactive to histamine. (Ashton and Cunha-Vaz, 1965; Shakib and Cunha-Vaz, 1966). Thesevesselsare not normally permeable to intravenously injected sodium fluorescein (NaFl), dyes covalently bound to serum albumin or to the electron microscopic tracer, horseradish peroxidase (Grayson end Laties, 1971; Machemer. 1970; Shioseand Oguri, 1969). These observations constitute examples of the bloodretina barrier. One function of such tight junctions is to prevent the passage of substancesfrom the capillary lumen to the neural retina based upon molecular size, and thus be the morphological equivalent of a physiologica~l pore (Landis and. Pappenheimer, 1963). Exclusion on the basis of molecular size is known to be an important discriminant in other blood vessel systems (Grotte, 1956). Our interest is in developing nondestructive in vivo methodologies with which permeability changescan be monitored, for example, on the basis of molecular size. inasmuch as such techniques would be useful in following the temporal course of alterations in retinal capillary permeability (Bellhorn, Bellhorn, Friedman and Henkind, 1973). *Reprint 210th Street,
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In this paper we introduce fluorescein isothiocyanate labelled dextrans of molecular sizes as an in vivo fundus fluorescein angiographic marker in models. As a comparative study we investigated the permeability of the avian an intraocular organ which normally is permeable to intravenously injected fluorescein (Bellhorn and Bellhorn, 1975), to these molecular weight markers.
selected animal pecten, sodium
2. Materials and Methods Fluorescein isothiocyanate (FITC) dextrans (Pharmacia Fine Chemicals, Inc.) are a homologous series of glucose polymers differing in molecular weight. The glycosidic linkages are primarily 1,4-a although some 1,6-a bonds are present. They are covalently labelled with fluorescein by condensation with fluoresceinisothiocyanate to form fluorescein thiocarbamoyl dextran derivatives (deBelder and Granath, 1973). Free fluorescein is removed by ethanol precipitation and molecular sieve chromatography. We have verified the absence of free fluorescein by chromatography on Sephadex G-25 in O-1 x-sodium phosphate buffer, pH 7.4. On the average there is one fluorescein residue per 100-1000 glucose residues. TABLE
I
Properties of tracersfey permeability studies Effective
Colllpouncl FITC-Dex 3 FITC-Dex 20 FITC-Dex 40 FITC-Dex 70 FITC-Des 150 Glucose Medium Fluorescein Myoglobin Bovine Serum Albumin Horseradish Peroxidase Ferritin Colloidal Carbon
&rolecular weight
3000* 20 000" 40000* 70000" 150000" 180 376 17800 69 000 40000 600000
diffusion radius
(A)
Diameter
121.
240
321 45t 5st
W
w
(A)
90s
1164 170s
3.6:
19.q 351 30**
614” -
30-i 508 200
* Average molecular weight, see text for explanation. f Averqe molecular size, see text for explanation. $ Grotte, 1956. $ Estimated as twice the effective diffusion radius. l[ Williams and Wissig, 1975. ** Caulfield and Farquhar, 1974.
The FITC-dextrans used in this study were of the average size and molecular weight shown in Table I; for comparison, properties of other compounds used in permeability studies are given. The FITC-dextrans are heterodisperse with respect to molecular weight (and therefore size) and encompass a range of molecular dimensions. For example, the FITC-Dex 3 used in this study ranged from mol. wt. 1200 to 5000. The average molecular weight by weight (L@~) is 3000 and the average molecular weight by number of molecules (B,) is 2400, indicating a greater number of molecules of mol. wt. less than 3000. Similar considerations are true for the other FITC-dextran fractions although the ranges are proportionally less widespread (Pharmacia Fine Chemicals, technical data bulletin).
PERMEABILITY
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FLUORESCEIN
DEXTRAKS
597
The FITC-dextrans were used as solutions of 1 g in 3.0 ml of 0.1 M-sodium phosphate buffer, pH 7,4, a, concentration empirically possible for all FITC-dextrans used in this study. The buffer is at physiological pH for both rats and birds. As a control, solutions of Dextran 40, unlabelled with fluorescein, were mixed with free KaFl to give a final concentration of 16% D eyi t ran 40 and 0.50/b sodium fluorescein in 0.1 &I-sodium phosphate buffer, pH 7.4.
Since the degree of fluorescein substitution of the dextrans is low (0.01 to O.OOl), even a concentra,ted FITC-dextran solution is much less fluorescent than the 5-10% n’aF1 solutions commonly used. Therefore, as a standard reference, 0+0/6 KaFl in 0.1 x-sodium phospha.te buffer, pH 7.4, was used in all studies reported here. It was found by absorption spectrophotometry (Beckman DU spectrophotometer) that this concentration was comparable to that of the 33% solutions of FITC dextrans at their absorption maxima. The FITC-dextrans exhibit an absorption maximum at 493 nm, as eompa,red to 495 nm for NaFl in the same buffer. This shift could not influence the results as it is within the transmission range of the excitation filter used. The technical data from the manufacturer shows an emission spectrum maximum of 520 nm; the same as unbound sodium fluorescein.
Reca,use concentrated solutions of small molecular weight components will be appreciably hyperosmotic, we were concerned that a,rtifactual results could be obtained by, for exanlple, opening of the blood-retina barrier due to the hypertonicity of the dextran solutions (Rapoport and Thompson, 1973). The osmolality of the solutions used was mea,sured in an Adva,nced Osmometer, Advanced Instruments, Inc., courtesy of Dr Richard Lent, and were as follows : 0.1 M-sodium phosphate buffer, pH 7.4, 290 &osm/kg ; FITC-Des 3 in buffer, 416 mosm/kg ; FITC-Dex 20 in buffer, 309 mosm/kg ; FITC-Dex 40 in buffer, 356 mosm/kg; FITC-Dex 150 in buffer, 295 mosm/kg.
The dextrans are long chains rather than globular molecules and have an appreciable viscosity. This parameter was measured at 38°C in a Wells-Brookfield Micro Visc,ometer, Model LVT (courtesy of Dr Jack Novodoff): 0.1 al-sodium phosphate buffer, pH 7.4. 0.89 centipoise; FITC-Dex 3; 4.13 cP; FITC-Des 20, 19.6 cP; FITC-Dex 40, 36.1 cP; FITC-Dex 70, 75.3 cP; and FITC-Dex 150, 181.44 cP.
Kormal adult pigmented (Long-Evans strain) rats were anesthetized i.p. with sodium pentobarbital, 40 “g/kg, after pupillary dilation with 1% atropine sulfate and lo?; phenylephrine HCl. A femoral vein was surgically exposed and catheterized, and 0.I cc of a test compound was injected for fluorescein angiography. The rats used weighed approximately 250 g a.nd therefore had a pla.sma volume of 10 ml, or 100 times greater than the injection volume (Biology Da&a Book, 1974). Although dextran injections have caused anaphylactoid reactions in rats, the dosage of dextran in these exper&nents is less than that required to produce this effect (Norrison, Bloom and Richardson, 1951) and 110 severe reactions were observed in the current study. Twenty-four normal rats were used and each generally received two injections. Adult pigeons, after pupillary dilation with D-tubocurarine HCl in O.O25o/o benzalkonium chloride (3 mg/cc)> were anesthetized with a tiletamine HCl-zolazepam HCl combination (CL-744, Parke Davis & Co.) at 20 mg/kg i.m. A brachial vein was surgically exposed and catheterized and 0.25 cc of one of the test compounds injected. The pigeons used weighed
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250-350 g and had a plasma volume of approximately 13 ml (Biology Data Book, 1974), or approximately 50 times the injected volume of test compound. Twelve pigeons were used and each received multiple injections of FITC-dextran followed by sodium fluorescein. Fundus fluorescein photography was accomplished using a fundus camera (Fundus Flash II, Carl Zeiss, West Germany) equipped for fluorescein angiography with a Kikon F motorized camera a,nd Kodak Wratten 47A excitation and Wratten 15 barrier filters. it A 400 ASA black and white film (Kodak Tri-X) wras used to record the angiograms; wa.s routinely processed. The flash intensity of the fundus camera was set at maximum in order to elicit good photographic detail of the eye transit. The first frame was exposed simultaneously with the start of the injection in order to approximate the time of dye passage from the injection site. The minimum time between frames was of the order of Ot5 sec.
FIG. I. Standard retinal fluorescein angiogram utilizing 0.1 cc of 0.5% sodium fluorescein in a norma! rat. (a) 2.9 sec. Earliest indication of arterial phase. (Arrow). (b) 3.4 sec. Laminar flow (arrow) indicative of early venous phase only 0.5 set aft.er early arterial phase. Focal hyperfluorescent spots are end-on views of precapillary arterioles traversing from nerve fiber layer to superficial and deep capillary beds. appearance is not (c) 5.2 sec. First indication of full vascular phase. Elapsed time from first arterial more than 3.3 seo. The full capillary bed is apparent. (d) 446.7 sec. Late phase showing hazy and uncontrasty fundus appearance. The disc margin exhibits residual fluorescence.
PERMEABILITY
OF
FLUORESCEIN
DE;XTR’AP;S
599
3. Results In the normal rat, good quality fluorescein angiograms were obtained using 0.1 ml as compared to previous of 0.5% NaFl (Fig. 1). F emoral vein catheterization, techniques, provided more control over factors such as slight animal movement and assured compact bolus injection and control of the exact time of injection.
Flo. 2. Angiograms obtained utilizing fluorescein labelled dextrans in normal rats. (a) 4.8 sec. FITC-Des $ showing early venous laminar flow. (h) 454.1 sec. A late phase angiogram of FITC-Dex 3 in this same animal. (c) ‘i.1 sec. FITC-Dex 70 angiogram illustrating early venous phase. Note that all veins may not fill simultaneously. (d) 495.5 sec. FITC-Dex 70 angiogramillustrating late phase persistent vessel fluorescence. Compare vascular detail with that observed at same stage using 0.5% sodium Huorescein [Fig. l(d)].
It is evident in some’ normal rats that arterial and subsequent venous filling [Fig. 2(c)] may be sectorial in that certain vessels fill more quickly than others. This preferential filling is consistent in those rats each time angiography is performed. It may be that one or more arteries arise from the central retinal artery at a point slightly proximal to others. The FITC-Dex 3 angiogram was similar to that of NaFl in that no evidence of leakage or altered permeability of the retinal vessels was evidenced (Fig. 2). The
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FIG. 3. Fluorescein angiogram of normal pigeon pecten after injection of a rn~sture of plain Dextl photograph. (b) Presence of fluorescence within pecten appro 413 and 0.5% NaFl. (a) Pre-injection n Lately 4 set after injection. (c) Virtual disappear rance of fluorescence from pecten about 8 set af (d) Beginning evidence of fluorescence adjacent to pecten at 28 set post injection. (e), in Ijection. evidence of fluorescence in vitreous at Ei6 and 220 set post injection. II xreasing
PERMEABILITY
OF
FL~ORESCEIW
DEXTKAN8
601
&+. 4. Fluorescein angiogram of normal pigcon pecten demonstrating sequence of evwts iLfttc[ !njection of FITC Dcstran 3 and a subsequent injection of O,5o/O NaFi. (a) l’re-injection photograph ,&owing pseudoflum-c,scenee of optic disc. Choroidai x~essek against light ha&ground on sclera ax (h) Presence of FITC Dextran 3 is evident within pccten 4.5 SW \-isible because t.he fundus is albinotic. post inject.ion. (c) Virtual absence of FITC De&ran 3 fluorescence evident at 87 set post injection. within pecten of same bird 4.1 WC after injection Compare with Fig. 3(e). (d) P resence of fluorescence of 0.5% NaPI. (e), (f) Presence of fluorescence adjacent to pecten 66 set and 257 see after injection of @50/o XaFl. Compare relative lack of parapectinal fluorescence of this bird to that depicted in Fig. 3. ‘~:he albinotic fundus allows scleral and ohoroidal fluorescence to partially mask that present, in the vitreous.
602
iM.B.RjEL~,$OR~,jR.W.BEL.LKOR~ANDD.S.POLT,
higher molecular weight FITC-dextrans similarly caused no signs of leakage across the blood-retina barrier under the given conditions (Fig. 2). An effect of increased molecular weight was a longer persistence of fluorescence in the vessels and an enhanced contrast with the background [compare Figs l(d) and 2(d)]. A mixed solution of Dextran 40 with free NaFl provided angiograms with characteristics similar to NaFI.
In the normal pigeon, 0.25 ml of a 0.5% solution of NaFl permits photographic visualization of the diffuse leakage into the vitreous, as previously observed at higher NaFl concentration (Bellhorn and Bellhorn, 1975). A mixture of unlabelled Dextran 40 and free NaFl given intravenously leaked profusely into the vitreous, as though the NaFl had been administered alone (Fig. 3). In contrast; no fluorescence within the vitreous was observed when any of the FITC-dextrans were administered [Fig. 4(a); (lo), (c)l. As an assurance that the FITC-dextran results were not due to, e.g. faulty positioning of the catheter, a subsequent injection of 0.5% NaFl was administered a few minutes later through the same catheter and in all instances the typical leakage phenomenon was observed [Fig. 4(d), (e), (f)]. Whenever NaFl was administered the initial appearance of dye in the pecten was followed by a brief interlude of darkness prior to observance of fluorescence in the vitreous (Fig. 3). Whenever FITC-dextrans were administered, they were observed to pass into the pecten and then to just fade away [Fig. 4(a), (b), (c)l. 4. Discussion The feasibility of using FITC-dextrans as in vivo molecular weight markers for fundus angiography has been demonstrated in rats and birds in these studies. Because the dextrans are not innocuous and may affect several aspects of the microvaseulature, the following factors were considered in evaluating this technique : (1) It is assumed that FITC-dextrans behave similarly to unlabelled dextrans of identical molecular weight and size. This seems reasonable in that the degree of labelling with fluorescein is low and unlikely to result in severe alterations of the dextran molecule configuration or chemistry. (2) An important effect of dextrans is that they may cause, in some strains of rats, histamine release and concomitant changes in vascular permeability. Although the effects of histamine are marked in peripheral vascular systems (Carter, Joyner and Renkin, 1974), it has been shown that the retinal vascular system does not exhibit permeability changes even to exogenously applied histamine (Ashton and Cunha-Vaz, 1965). The only observed effect which may have been due to dextrans was some respiratory difficulty in two rats. WTe are unaware of information concerning use of dextrans in birds but we did not observe untoward effects. (3) The osmolality of the solutions was of concern since local hypertonicity could cause alterations in capillary permeability (Rapoport and Thompson, 1973). The low molecular weight solutions were only slightly hyperosmotic to mammalian plasma, and in fact, no leakage from normal ma’mmalian vessels was noted with any of t,he dextrans used. Undoubtedly the fast heart beat in rats (approximately 350 beats/min) and birds (approximately 250 beats/min) helps to quickly dilute the small injection volume throughout the plasma volume (Riology Data Book, 1974).
PERMEABILITY
OF
FLUORESCEIN
DEXTRA,\TS
603
(4) Although the viscosity of the FITC-de&an solutions is appreciable and dextrans can increase blood viscosity (Singh and Coulter, 1973) or cause erythrocyte aggregation (Stalker, 1967) at high molecular weights, these properties did not appear to play a role in the results obtained. Once more, the rapid circulation in rats and birds and the small injection volume will help to diminish the import,ance of high viscosity solutions. (5) Increased persistence of dye in the retinal vasculature when compared to NaFl is due possibly to decreased kidney clearance of the high molecular weight compounds (Caulfield and Parquhar, 1974). An additional factor which may be involved in increased dye persistence is that the FITC-dextrans probably extravasate from even highly permeable blood vessel systems, such as skin or choroid, less rapidly than sodium fluorescein, thus effectively maintaining a higher blood concentration of dye. In turn, a less marked extravasation of FITC-dextrans from the choroidal circulation would enhance the photographic contrast of a late stage angiogram by reduction of background fluorescence [compare Figs l(d), 2(b), (d)]. Although dextrans are known to produce alterations in blood flow properties (e.g. Singh and Coulter, 1973) it is unlikely that those effects play a role in the late appearance of FITCdextrans in this study because the amount of dextran injected is small compared to the plasma volume. Such effects would become obvious if large quantities of dextrans were infused. This property of persistent vessel visualization could be useful in the study of abnormal retinal vessels prior, for example, to laser photocoagulation. (FITC-dextrans are not currently approved for use in humans). Dextrans have long been used to study peripheral microvasculature permeability (Grotte, 1956). Several properties enable the dextrans to be meaningful molecular size markers : (1) molecular size is both smaller and larger than serum albumin, considered to be at the limit of macromolecular transport by the pore theory (Landis and Pappenheimer, 1963) ; (2) no apparent in vivo aggregation to larger molecular sizes (Caulfield and Farquhar, 1974; Schrrijder, Arfors and Tangen, 1976) ; (3) binding to plasma proteins probably does not occur with the lower molec&r weight dextrans (Schroder et al., 1976). The fact that low molecular weight FITC-dextrans do not pass across a normal mammalian retinal vessel makes them invaluable for studies of abnormal retinal vesse1.s. Por example, does an abnormality which permits leakage of NaFl, a small molecule, simultaneously allow passage of larger fluorescein-labelled molecules and to what extent? Since NaFl is normally actively excluded from viable cells and retinal vessels, it is probable that the perm.eability mechanism which excludes NaFl is due to some property other than molecular size, e.g. anionic charge (Cunha-Vaz and Maurice, 1967). In order to obtain this kind of information, it is important that the fluorescein-dextran bond is stable under in vivo conditions for more than 24 hr (Arfors and Hint, 1971). The avian pecten is unusual in that it permits diffuse passage of NaPI across the capillary walls which have a unique elaborated surface unlike other known capillaries (Bellhorn and Bellhorn, 1975; Seaman and Storm, 1963). In contrast to the permeability to NaFI, the pecten d oes not permit passage into the vitreous of any of the tested PITC-dextrans. This suggests that two different permeability mechanisms exist in the pecten, which has some properties reminiscent of the ciliary body (Seaman and Storm: I963). The eiliary body is Iikewise permeable to NaPI, but excludes protein
6UI
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molecules on the basis of their molecular size, consistent with a pore theory of permeability (Dernouchamps and Heremans, 19’75) and similar to t’he peotan. In conclusion, FITC-dextrans can be profitably used for the study of permeability processes in retinal vessels which, unlike brain capillaries, are accessible to in vivo photography. Moreover, the FITC-dextrans will be meaningful in studies of ciliary body permeabilky to molecules of various sizes: an important aspect of aqueous humor formation. The permeability of the avian peeten, an organ more accessible to iu vivo photography than the ciliary body, may provide a model for mechanisms of fluid formation similar to those of the cjliary body, Because the FITC-dextrans arc not currently approved for human use and may cause a hypersensitivity reaction, the potent’ial clinical value of these compounds will have to be carefully evaluated. The LISA of these agents in studies of animal ocular physiology and pathology2 however, will have relevance to human pat~hophysiology.
We wish to thailk Dr Harold Kern for bringing these co~~~pounds to our attenbion. We are grateful to Noel Ron for excellent as&ance in aninlal surgery a,nd photography. We have profited from st~imulnting discussions with Drs Paul Henkind, Joseph Walsh, Daniel Gold and Barry Beckerman. Supported in part by USPHS Grants EY 01103 01 and EY 00613 05 (X.E.I.) and a gra’nt, from Research to Prevent Blindness, Inc., New York, N.Y. REFERE;NCES Arfors,
K-E. and Hint, H. (1971). Studies of the microcirculation using fluorescent destran. Abstr. Xicro~~ccsc. Res. 3, 437. d&on, I\ and Cunha-Vaz. J. G. (1965). Effect of hi&amine on the permeability of the ocular vessels.
Bellhorn,
Arch.
R’. W.,
OphthaZm.oZ.
Bellhorn,
73,
M.
211-23.
B..
Friedman, A. .H. and Honkind, Irvwst. Ophthnlmol. 12, 65-76. X B. (1975). The avian pecten.
I’.
(1973).
Urethan-induced
retinopathy in pigmented rats.
I. Fluorescein permeability. and Bellhorn, Res. 7, 1-7. Bill, A. (1975). Blood circulation and fluid dynamics in the eye. Physiol. Reu. 55, 383-417. Biology Data Book, Volume IIT, 2nd ed. (1974). (Eds Altman, P. L. and Dittmer, D. S.), Federation of American Societies for Experimental Biolo,gy, Bethesda, ?vId. Bruns, R. R. and Pnlade, G. E. (1968). Studies on blood capillaries. I. General organization of blood capillaries in muscle. J. Cell Biol. 37, 244-76. Cart’er, R. D., Joyner, W. L. and Renkin, E. M. (1974). Effect,s of histamine and some other substances on molecular selectivity of the capillary wall t’o plasma proteins and dextran. Nicrovasc. Res. 7, 31-48. Caulfield, J. P. and Farquhar, M. G. (1974). The permeability of glomerular capillaries to greded dextrans. J. Cell Biol. 63, 883-903. Cunha-Vaz, J. G. and Maurice, D. 31. (1967). The active transport of fluorescein by the retinal vessels and the retina. J. Physiol., Land. 191, 467-86. de Belder, A. N. and Granath, IX. (1973). Preparation and properties of fluorescein-labclled dextrans. Ccwbohyd. Res. 30, 375-8. Dernouchamps, J. P. and Heremans, J. F. (1975). Molecular sieve effect of the blood-aqueous harrier. &p. Eye Res. 21, 159-97. Grayson, M. C. and Laties, A. M. (1971). Ocular localizat,ion of sodium fluorescein. Arch. Ophth~lmol. $5, 600-609. Grotte, G. (1956). Passage of dextran molecules across the bloodMymph barrier. Beta C&r. &and. iYupp1. 211, I-84. Landis, E. 31. and Pappenheimer, J. R. (1963). Exchange of substance through the capillary walls. In Handbook of Physiology (Eds Hamilton, W. I?. and Dow, P.). Z(2), pp. 961-1034. American Physiology Society, Washington, D.C. Bellhorn,
R. W.
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PERIVIEABILITY
OF
FLU
ORESCEIN
DEXTRANS
605
Machemer, R. (1970). Angiographic-histologic correlation of eye vessel permeability with protein bound fluorescent dye. Am. J. Ophthalmol. 69, 17-38. ?IIorrison, J. L., Bloom, W. L. and Richardson, A. P. (1951). Effect of dextran on the rat. J. Ph~armncol. Exp. Therap. 101, 27-8. Rspoport, S. I. and Thompson, H. K. (1973). Osmotic opening of the blood-brain barrier in the monkey without associated neurological deficits. science 180, 971. Schriider, U., Arfors, K.-E. and Tangen, 0. (1975). Stability of fluorescein labeled dextrans in vivo and in vitro. Microvascular Res. 11, 33-8. Seaman, A. R. and Storm, H. (1963). A correlated light and electron microscope study on the pecten oculi of the domestic fowl (Gallus domesticus). Exp. Eye Res. 2, 163-72. Shakib, M. and Cunha-Vaz, J. G. (1966). Studies on the permeability of the blood-retinal barrier. IV. Junctional complexes of the retinal vessels and their role in the permeability of the blood-retinal barrier. Exp. Eye Res. 5, 229-34. Shiose, Y. and Oguri, M. (1969). Electron microscopic studies on the blood-ret.inal barrier and the blood-aqueous barrier. Acta Sac. Ophthalmol. Jap. 73, 1606-12. Singh. M. and Coulter, N. A. (1973). Rheology of blood: effect of dilution with various dextrans. Microwasc. Rea. 5, 123-30. Stalker, A. L. (1967). The microcirculatory effects of dextran. J. Path. Bact. 93, 191-201. Studer, R. and Pot&en, J. (1971). The radioisotopic assessment of regional microvascular permeability to macromolecules. illicroz:asc. Res. 3, 3548. Williams, 31. C. and Wissig, S. L. (1975). The permeability of muscle capillaries to horseradish peroxidase. J. Cell Biol. 66, 531-55.