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The use of fluorescein-labelled dextrans in investigation of aqueous humour outflow in the rabbit
Ezp. Eye Res. (1976) 23, 571-585
The Use of fluorescein-Labelled Dextrans in Investigation of Aqueous Humour Oufflow in the Rabbit* D. F.
COLE
AND
P.
A. G.
&fONRO
Institute of Ophthalmology, University of London, .Judd Street. London TT’Cl, P.K. and Department of A,natomy, University qf Camb~riclgr, U.K. (Received 10 December 1975 and in revisedform 19 February 1976, London) The anterior chamber of t*he rabbit eye was perfused with solutions containing fluoresceinlabelled dextrans (FDs) of various molecular weights from 3000 to 150,000. Using an adapted Zeiss slit-lamp, specially modified for rapid sequence flash photography, the outflow channels for the aqueous humour in the limbal region could be demonstrated in living, anaesthetized animals. Semi-permanent preparations showing the postmortem distribution of FD in t.he tissues were made by fixation in a mixture of neutral formaldehyde and 70:/, ethanol. The fixed material was dehydrated in the normal way and either cleared in bulk in metshy salicylate or embedded in paraffin for sectioning. Cleared whole-mount preparations showed FD precipitated in the angular aqueous plexus and the efferent veins together with some perivascular distribution. Paraffin sections showed that FD had penet,rated into the tissues of the iridocorneal angle and the suprachoroidal space. Further experiments were carried out using closed cycle perfusion of the anterior chamber with a mixture of FD (mol. wt. of 3000,40 000 or 150 000) and blue dextran (mol. wt. = 2000 000). Rat,e constants calculated for the loss of these substances from the anterior chamber showed that the rates for the FDs were 20-25Oh greater than the rate for blue dextran. On the basis of these findings it is suggested that an appreciable fraction of aqueous humour in the rabbit eye may leave via a uveoscleral route.
1. Introduction Tn order to demonstrate the outflow pathways for the aqueous humour it is an established procedure to inject a dye or coloured suspensioninto the anterior chamber and then to examine its subsequentdistribution (cf. Leber, 1903; Siedel, 1922). Thus, following Ascher’s demonstration of aqueous veins in man (Ascher, 1942), Weekers and Prijot (1950) and Greaves and Perkins (1951) employed respectively india ink and Evans blue to demonstrate outflow vesselsfor the aqueoushumour in viva in the rabbit. With the more recent availability of fluorescein-labelled dextrans of defined molecular sizes it seemedworthwhile to make use of these substancesfor a similar purpose and, at the sametime, to take advantage of their other properties in order to study aqueous outflow dynamics and post mortem tissue distribution. The present paper is concerned primarily to report the techniques which have been developed for this purpose. The fluorescein-labelleddextrans (FDs) are obtained by high resolution gel filtration and are labelled with fluorescein isothiocyanate. They are available commercially in a variety of different, well-defined molecular weights. They are readily identifiable by their fluorescenceand show no appreciable binding to plasma protein (Rutili and Arfors, 1972). This is a considerable advantage over dyes such as Evans blue, which bind extensively, since the tissue and plasma concentrations of FD measured by fluorimetry indicate the actual amount of the free material present. As will bc shown * Reprint d
requests
to: Dr D. P. Cole, Institute
of Ophthalmology, 571
Judd
St., London
WClH
9&Y.
,572
I).
I’.
(‘OLE
ANL)
I’. A. t:.
MOKRO
below, the FDs can bc precipitated ad fixed in situ for the post mortcxnl study of the tissues. The present investigation was carried out using three different E’D fractions having average molecular weights of 3000, 40 000 and 150 000 respectively. These. togrthcr with blue dextran (BD) (mol. wt. = 2000 000) were obtained from Pharmacia Lttl, Uppsala, Sweden.
2. Methods of t,he The apparatus designed by Monro (1971) f or rapid sequence photomicrography human conjunctival vessels was adapted to enable fluorescein angiogrnphy to be carried out in the limbal region of the rabbit eye after the anterior chamber had been perfused with PD. To enable an observer to make continuous photographic records while keeping the experimental eye under observation, a Zeiss slit lamp was modified as follows : the illumination system was replaced with a lamp-house containing (1) a xenon in quartz flash lamp working at 3 kV, with its own condenser lens and aperture stop; and (2) an alternative quartz iodine lamp with another condenser and a variable aperture field stop. Light from the quartz iodine lamp could be brought into the optical pathway by means of a pivoting F,O3QT CAMERA
[
FIG. 1. Diagram showing modifications to the Zeiss slit lamp microscope for use in rapid sequence photomicrography. The illuminating system (apart from the lamp house condenser, C) is not shown, but light from the illuminating system is indicated as broken lines. C, illuminating condenser; E,, inclined eyepieces; E,, second viewing eyepiece, F, barrier filter (Ilford 110); L,, objective lens; L,, anastigmat forming secondary image at lens forming primary image at V, ; L,, x 10 Complen eyepiece; L,, achromat V,; M, mirror on pivot (in position for photography); T, position of turret lens; V,, position of primary image; Vz, film plane (secondary image).
FLUORESCEIK-LABELLED
DEXTRANS
573
mirror actuated by a rotating solenoid (Monro, 1971), and afforded illumination for continuous observation. The xenon flash was triggered by the camera shutter contacts and was used for serial photography. For the present experiments the limiting aperture stop in the flash tube condenser was removed so that an area of 5-6 mm diam. could be illuminated at the object plane by means of a third aplanatic condenser (C, Fig. 1) with light from either the flash tube or the quartz iodine lamp. A Balzer FITC-3 filter was placed in the light path of the xenon flash and a heat filter in the condenser from the quartz iodine lamp, so that full colour was available for observation of the limbal region. The lamp house and its condenser could be rotated in a horizontal arc of radius 25 mm concentric with the animal’s eye into a position about 40” to the optical axis of t’he object,ive lens, L,. The Zeiss slit lamp is fitted with an achromatic lens of 100 mm focal length, placed at its focus from the object so that the rays to the image are esseutially parallel. In this pathway there is a turret lens system with powers from x 6 to x40 but the nominal position used in the present experiments contains no lenses. The modifications introduced into the microscope system are shown in Fig. 1. A mirror housing was built between the t,urret (T) and the ordinary binocular head, which produces virtual images for direct, observation in both inclined eyepieces (E,) when the mirror (M) is not in position for photography. The front-surfaced mirror (M) is pivoted about its lower edge by means of a second rotating solenoid into an angle of 45” to the rays to one eyepiece (the other being fitted with a graticule for checking the focus). The image rays are thus reflected into an anastigmat lens (L,) of 125 mm focal length at the top surface of the mirror housing so that a primary image (V,) is formed at its focus at a magnification of x 1.25. A positive “Cornplan” 10 eyepiece (Ls), with an achromatic lens (LJ of 85 mm focal length placed above it’, forms an image (V,) on the film plane. Immediately above the lens (LJ a rightangled prims reflects 10°,& of the light into a second viewing eyepiece (E,) used for checking the focus and flash illumination. The Robot Star camera can be made to operate at up t,o 8 frames jsec by means of its clockwork drive, but the shutter lies about half-way between the normal lens position and the film plane so that it was essential to use a lens system somewhere near the original design position in order to prevent vignetting of the image. A similar system operating at 2 frameslsec was used to study the localized leakage of fluorescein (Cole and Monro, 1976) and low molecular weight FDs from the conjunctival vessels. An Ilford 110 filter (F) is placed above the secondary objective lens as a barrier to ensure that only fluorescence from FD is recorded on the film. This filter combination (i.e. Balzer FITC-3 and Ilford 110) has previously been found to give good contrast during fluorescein angiography of the iris vessels (cf. Unger. Perkins and Bass, 1974). In the present experiments, photographs were taken on Kodak TriX film at 15-30 set intervals at a magnification of x 4 and using a flash of 20 J with a duration of less than 1 msec. The film was developed in Patterson “Acuspeed” to give a nominal ASA rating of 1200. After anaesthetizing the rabbit with intravenous urethane, 1.75 g /kg body weight, the right eye was transfixed with a perfusing needle and the animal mounted on a supporting cradle in front of the slit lamp. The eye was gently proptosed, covered with silicone oil and a suitable field identified in the inferior quadrant of the limbus. The eye was perfused with buffered Krebs Ringer solution at 10 $/min against an outflow pressure of 15.-20 mm/Hg and, once the limbal vessels had been focused, approximately 0.2 ml of 5% FD was injected slowly from a side syringe so as to fill the lower part of the anterior chamber. As soon as this was completed, photographic exposures were commenced at 2 frames/set every 15 or 30 set for a period of lo-12 min. Demonstration
of FD in$xed
tissue
The anterior chamber was perfused with FD-40 as described below except that it was allowed to fill completely with the FD solution. At the end of 10 or 20 min the eye was
574
1). F. (‘OLE
AND
P. -4. (l. MOSl
rapidly enucleated and placed in a fixative consisting of 30 ml neutralized formaldehyde and 70ml ethanol (since the dextrans are readily precipitated from alcoholic solutious when the ethanol concentration exceeds 509{,). The globe was opened at the equator to wash out the excess PD and fixation of the anterior segmeut continued overnight. The lens W;W removed and the tissue dehydrated in successive grades of ethanol, cleared in xylene and embedded in paraffin wax in the conventional manner. Sections of 5 -10 ~111 were cut. floated out on a warm bath of 70% isopropanol (floating out on water would have removed the FD) and taken up on microscope slides. The sect’ions were then de-waxed and mounted in DPX (B.D.H). In some cases large pieces of anterior segment were pinned out before the final fixation in order to prevent distortion. After dehydration these were prepared as whole mounts cleared in methyl salicylate and were studied using a low-power, wide-field objective iI1 order to obtain an overall picture of the FD-containing vessels. Microscopy was carried out with the Leitz Orthoplan microscope with fluorescein attachment using a UG5 filter on the illuminating side and a K530 filter as a barrier filter. Photomicrographs were taken using Kodak TriX film and developed in Promicrol (May & Baker, England). Recycling perfusion
experiments
After anaesthetizing with urethane as above, one eye was cannulated needle so that the anterior chamber could be continuously perfused containing 0.5% FD and 2% BD [see Fig. 2(a, b)].
with a transfixing with Krebs saline
D
C
(b)
(a) FIG. 2. (a) Diagram to show the arrangement of needle is represented by A and B [see also Fig. 2(b)],
the recycling perfusion system. The transfixing flow is maintained by the rotary pump, C, with B connected to its inflow and A to its outflow side so that there is continual recycling of the fluid contained in the pump-tube system (BCDA) and the anterior chamber of the eye. At given intervals the tubing is clipped at a point D and small samples arc removed from the outflow side of the pump at C. (b) Flow of perfusate through the transfixing needle in the anterior chamber. The animal was placed on its side and a drop of 1% amethocaine applied to the cornea. With the perfusing needle attached at one end (B) via a polythene tube to the inflow side of the pump system (C), which had already been filled with the perfusion fluid, the eye was transfixed (cf. Nagasubramanian, 1974) and the emergent tip of the needle connected to the outflow side of the pump at A, thus forming a closed system. Recycling perfusion was commenced at a measured 54 $/min and continued for 90-120 min. At approximately 20 min intervals the tubing was clipped at a point D samples of 5-10 ~1 fluid removed at the outflow side of the pump (C). These samples were diluted and used for the estimation of FD and BD. At the end of the experimental period the animal was killed, the eye was
FLUORESCEIN-LABELLED
575
DEXTRANS
enucleated and the attached conjunctival and subconjunctival tissue (1) snipped off with curved scissors. The globe was then opened about 2 mm in front of the equator and the lens (a), vitreous (3) and iris+ciliary body (4) removed, leaving the cornea and sclera. The outer rim of sclera (5) was removed to within about 1 mm of the limbus and an inner disc of cornea (6) was punched out with a cork-borer to within 1-2 mm of the lintbus. The remaining annulus of tissue was categorized as limbus (7). After weighing, each of these tissues. (1) Tao(7) \vas homogenizetl in 2.5 ml neutral ziuc hydroxide, centrifuged for 10 min to remove the precipitated protein and the supernatant usetl for the estimation of PD. In the present experiments it, was poysible to estituat,e the c.orlc.elltratioll of HD in the perfusiate Ijut, not in the tissues. (‘nlr:~lnfion.s. At zero time there is a closed system in which there is a known quantit> of FD (Jr,,). contained in a known volume of fluid (b;,) in the pump-tube system [WI>,\ i11 Fig. 2(a)]. In the remainder of the system, the anterior chamber has a volume I’, ;IILC~a zero concentratJion of FD. The pump recycles fluid through the anterior chamber :~II~ thr amount, of FD lost by normal drainage of a,queous humour will depend on its concentriltioll in the anterior c.hamber which will rise rapidly during the mixiug phase and thereafter will declirle exponentiallv. In order to calculate the total c:umulat,ive loss during t,he I\-hole course of the experiment it is necrssarv t’o include an estimate of the loss whklr ocmrs whilst the concentration is rising during the i&al mixing phase. Taking the pump volume, I’,,. :IS 270 ~1 and the flow ratca as .5-l-pi/ruin it WAS as~lrlerl t Ililt : (a)
100; of the solute present in the anterior chamber during the time interval the end of minute (~1) to t)he end of minute W, leaves during the following interval (from minute I/ to minute (H i -1)).
(1)) the pump tube recycling time is 5 min so that, the efflux during equsl t,o t’he influx during the (/[+5)th minme. The total amount of dextran in the anterior chamber (Qc) cessive 1 min intervals (i.e. Q;1, Q;n+l,, etc) starting from various values for the fractional outflow loss (k) from the a,nterior veins and surrounding tissues. At the end of t,he & ntinute the ant,erior chamber is:
where the values, Q,: incorporat.e chamber:
correction
for fract,ional
the lath minute
zero time aud assuming chamber into the aqueous the amount of dextran iI1
loss due to outflow
from the (3
and Q, represents the amount which would have been present in the chamber been no outflow (i.e. with /I: = 0). With the values indicated above, knowing volume flow in the system and when ~004 ( 1; < 0.006 the time course for concentration (C,) was calculated and was found to approximate to a simple tleaa,v :tfter the first 25 min (Fig. 3). Furthermore, the time course of the total
1
is
inay be caloulat.etlat suc-
Q: = &,(I-4
toss. d ( : I; f Qc) f or Il’ff 1 erent values of &. within
from 1 min
this range,
approximated
had there the rate of the effluent, exponential cumulative to a set
of straight lines passing through zero (Fig. 4) so that, knowing the value for A at an assumed value for k (say 0.005 min-l) and knowing also the true value, Ic’ (from the decay rate after 25 min), the actual cumulative loss, A’ could be obtained from: ,4’ = (k’/k).A. (3) In order to validate the procedure a series of experiments were performed in which the concentration of FD was estimated in small (5 ~1) samples of effluent in the first 20 min of
I 20
0
I 40
I 00
I 60
I 100
120
Time (min 1
Flo. 3. Semilogarithmic plot of preliminary experiments showin, v the effluent concentration of ED (Cl,). The broken line (. . . ). during the first 25 min shows the predicted time course calculated from equations (1) and (7) in the text). The solid line shows the loss during the period of exponent,ial dccag (k’ z 0.00512 min-l). Thtr symbols represent cxpcrimcntal values obtained in each of four animals. 40
0
20
40
60
Perfusion
time
80 (min)
FIG. 4. Time course of the total cumulative loss, A, calculated for different ralues of lz (see text). In the sample calculation shown in the text, this graph is used to determine the total cumulativr loss at the end of 91 min. Assuming L = 0,005, this yields a value of 306 pg. Volume of pump+tubing (VP) = 272 ~1 Concentration of FD at start = 5000 pg/ml Iuitial amount of FD iu system (M,) = 1360 pg Concentration in system at end of experiment (after 91 min) = 1,975 pg/m Experimental value for k’ (from Fig. 3) = O-00512 min-l (a) From the graph (Fig. 4) the cumulative loss in 91 min at a k: value of 0.005 mine’ would be 305 pg and so the actual cumulative loss, i4’, must have been : 0.00512
3o5x isiG-
= 313 pg (from (3) above)
FLUORESCEIN-LABELLED
DEXTRANS
577
Amount of FD present in sclera+suprachoroidal
tissue at the end of the experiment 7.80 x 100 = 74 pg . .. Tissue level as “/o of act,& cumulative loss = ~313
Final amount of FD remaining in the system = Ala--A’ = M, = 1047 pg The total volume of the pump system plus the anterior chamber is (B,+ VP) so that :
va = .v,/c,--B*, Substituting
the experimental values: 1047 J’, zzz ~ -0.272 = 0258 ml 1975
The value for k, is then calculated with reference to this figure for V%: k, = k’ ( va+Vp) _ (,.(,(,512 ‘< , ((‘.258+o*272) 0.258 = 0.0106 min-l Similar corrections were applied to estimate the k, values for BD. . _^_ Preliminary studies had shown that the osmolarit,y of the aqueous humour was 283& 2.5 @/ml H,O and solutions of BD and FD were made up as 2.0:/: and 0*5’h respectively in buffered Krebs Ringer and adjusted to this osmolarity. In the case of FD three different molecular sizes were used: FITC-Dextran 3 (average mol. wt. = 3000, Stokes’s radius = 1.2 nm), FITC-Dextran 40 (mol. wt. = 40 000, Stokes’s radius = 4.4 nm), and FITCDextran 150 (mol. wt. = 150 000, Stokes’s radius = 8.7 nm). The BD was Dextran 2000 (mol. at. = 2000 000, Stokes’s radius = 30-O nm). The information on the molecular radii was obtained from the manufacturers. FD was estimated using a 2.5 mm-quare cell in the Aminco-Bowman Spectrophotofluorimeter with an excitation wavelength of 493 nm and an emission wavelength of 513 am and using solutions of the appropriate FD in buffered (pH 74) isotonic saline as standards. BD was estimated using a Unicam SP 500 spectrophot,oruet,er at a wavelength setting of 625 nm with a specially constructed micro-cell holtliil~g 0.15 ml. 3. Results
Figure 5 shows a typical set of photographs of the inferior quadrant seenat intervals from 3 to 10 min after the introduction of 50/bFD 40 into the anterior chamber. The FD soIut,ion is present in the lower part of the anterior chamber and after 3 min has commenced filling the outflow channels [Fig. 5(a)] and by 49 min is contained in a network of vesselsmost of which underlie the limbal and conjunctival vessels[Fig. 5 (b, c)~]although somebranches appear to lie closer to the surface [Fig. 5(d)]. At the end of 10 niin someleakage has commenced into the surrounding tissues. When the lower molecular weight substance (FD 3) was used this leakage was more rapid [Fig. 6(a, b)].
Figure 7(ac) shows the pattern seen by low-power fluorescence microscopy in whole mounts of tissue from the limbal region after dehydration and clearing in methyl salicylate. FD may be identified in the anterior aqueous plexus and in the vesselsleading away from it. especially when the specimenis viewed from the vitreal
!
FLUORESCEIN-LABELLED
579
DESTRANS
side [Fig. 7(a)]. Deposits of FD were also observed in close proximity to the larger blood vessels and from Fig. 7(b, c) it is clear that this material was not actually in the lumen of the vessels, which remained filled with blood, but was located in the perivascular space. Sections through the corneoscIera1 junction show the intrascleral and limbal vessels in cross section filled with fluorescent material (presumably aqueous veins) whilst others appear empty (Fig. 8). There is considerable penetration of FD into the sugrachoroidal space running posteriorly from the iridocorneal angle (Fig. 8, SCS) ant1 there are deposits in the iris stroma (Fig. 9).
FIG. 6. (a, b) In viva. fluorescent photographs of aqueous outflow channels after intracameral infusion of FL) 3. (a) 2 min, (b) 4 min. As with FD 40, the outflow channels AV appear deep to the conjunctival vc~sscls, but in this case considerable extravascular diffusion has occurred after 4 min (h, r).
TABLE
I
Values for anterior chamber,volume nrzd efllux rates (Va and k,) c&d&l
for
XntcGr chamber rolume (J-8.) W) Meann&s.E.iv. EfHus (k,,t) Mzans+s.E.M.
millrl
BD, FD 150, FD 40 and FD 3
38 * 1o.i (20) 0~0081 +0.0006
“61 2 8.3 (6) 0.0105 f 0~0009
(6)
(19)
Individual differences .h(FD)-kmt(BD) Means$s.a.w.
“75
“94 79.8
7 9.8 (7
(7)
0.01 I + 0.0010 (7)
I~~0116 k 0.0011
(6)
+0.0024**
-/ 0.0029**
&0.0034**
*0.0004
$0~0006
~0@005
(6)
(6)
(7)
The significance of the individual differences (line 3) is indicated by asterisks. Figures in parentheses represent thr number of ryes in each grump.
Thus
** = 0.01
I’.
I). F. (‘OI,E
FIG.
7. (a),
AK
(11).
1.) I’. A. c:. n1os
See
caption
I(0
opposite.
FLUORESCEIN-LABELLED
DEXTRANS
FIG.
581
7. (c).
FIG. 7. Fluorescent photomicrographs of whole mount preparations (a) Regions near the angular aqueous plexus, (AAP), showing efferent PD. Viewed from the vitreal side. (b) The terminal branches of the diverging to join the major iris circle, MIC. Perivascular deposits Viewed from the scleral side. (c) Intrascleral vessel containing blood of FD (1). Viewed from the scleral side.
after clearing in methyl salicylate. vessels (1) containing precipitated long ciliary artery, LCA, are seen of FD are shown by arrows (I). ( 4 ) with perivascular deposits
FIG. 8. Fluorescent photomicrographs of section through corncoscleral junction showing the intrascleral channels (aqueous outflow channels) filled with FD (1). There is considerable penetration into the iris root and the supra-choroidal space, SCS. C. cornea: AC’: anterior chamber; I, iris; LC, limbal conjunctiva; S, sclera; ev, empty vessel.
Figures
in parentheses
represent
the number
of eyes m each group
FLUORESCEIN-LABELLED
DEXTRANS
583
Recycling perfusion Figure 10 shows typical results from three eyes in which the exponential decay phase of the three fluorescein dextrans (FD 3, FD 40 and FD 150) are compared with the corresponding decay rates for BD. In all cases the rate for FD was greater than the corresponding rate for BD and the results from the whole series are summarized in Table I which gives the calculated values for anterior chamber volume (7,) and outflow constant (k,), calculated as described above. The last line of the table shows the means of individual differences (i.e. 7c, for FD less the corresponding k, for BD) and in each case the outflow constant for the FD is significantly higher than the corresponding value for BD. Table II shows the amomrts of FD remaining in the tissues at the end of the perfusion period expressed as percentages of the total amount of FD lost from the perfusate during this period. Only insignificant amounts were present in the lens and the vitreous humour ; values for these tissues are not included in t,he table. 4. Discussion The illustrations above show that the FDs can be used to demonstrate aqueous humour outflow channels both in vivo, and in fixed material post mortem. The invivo, and post mortem appearances are consistent with one another and the outflow channels seen in the living eye may be readily identified in whole mount flat preparations of fixed and cleared material, or in sections through the limbus [Figs 7(a-c), 8(a-c)]. The appearance of the sectioned material shows FD located mainly in the suprachoroid and sclera, limbus and iris, and this pattern agrees with the quantitative distribution in Table II. The relatively small amounts present in the cornea are confined to the endothelium. The fact that FD 3 penetrates the limbus and sclera in a significantly greater amount than the larger molecules of FD 40 or FD 150 (Table II) is consistent with the more rapid spread of FD 3 seen in vivo (cf. Figs 5 and 6). $ 4-o1 E 2-o2 z .E I.0 c z 0’ :, 0
20
-5
:
z .5
- 2.5 ii
*50
I 20
I 40
I 60 Time
I 00
I 100
I 120
(min)
FIG. IO. Typical results from three experiments showing decay rates of FD and BD in recycling perfusion experiments (semilogarithmic plot). . . . values for BD k, = 0.0081; l , FD 150, k, = 0.0106; 0, FD 40, 7cf = 0.0108; 0, FD 3, k, = 0.0110.
FD is scattered diffusely throughout the iris stroma (Fig. 9) and, while it does not enter the iridial portion of the ciliary processes,it appears to be more concentrated at the posterior iris epithelium. Fowlks and Havener (1964) described such distribution in the case of nitro-blue tetrazolium and also envisaged the possibility of perivascular drainage of aqueous
5x1
1). F. (~or,b: ASI) I’. A. t:. iuo~ttco
humour. Some further evidence for the latter is af?orllecI here by the presence of PD deposits outside the lumen of blootl-filled vesselS~. in and immediately posterior to. the limbal region [pig. 7(c)]. In the recycling perfusion experiments there If-as an obvious and consistent, difference between t.he A, values for PD and those for BL) (Table 1 and Pig. 10). Were this difference solely clue to diffusional loss of FD it would be reasonable to expect a greater difference between the L, values for the larger 171) molecules (i.e. FD 150 ant1 FD 40) and the smaller FD 3 than was actually observed. Thus, the observetl differences (Table 1, line 3) for k, (FD) ~- L, (BD) arc in the proportion of 1 : 1.2 : 1.4 for FD 150, FD 40and PI) 3 respectively, but on the basis of diffusion being approximately proportional to 1 .L/ mol. wt.. these ratios should have been of the order 1 : 1.9: 6.9. If diffusion is ruled out as the main cause of the difference between the Jc~values the alternative hypothesis seems to he that some part of the outflow pathwa.> is available to FDs hut not to BD. Here there are two main possibilities: ( i) that some of the loss of FDs occurs via uveoscleral routes similar to those described in monkeys (Bill, 1965; 1966a; 1967) and that k, for BD represents only the loss which takes place via the angular aqueous plexus and its efflus channels; (ii) that the kf values for FD represent loss via the angular aqueous plexus but that there is some restriction on the entry of BD into this system. The obvious objection to (i) is that Bill was unable to demonstrate any appreciable uveoscleral flow in cats and rabbits, either using red dextran (mol. wt. = 40 000) or lalI-labelled albumin (Bill. 1966b,c). In the present experiments it may be seen from Fig. 8 that FD 40 certainly penetrates into the suprachoroidal tissues and Tripathii (1975) has reported the penetration of an electron dense tracer (Thorotrast, particle size approximately 10 nm) into the suprachoroidal space of the rabbit. FD is much easier to identify in the tissues than is the red dextran used by Bill (1966c) and it is possible that this fact alone may account for his failure to detect any penetration into the suprachoroidal tissue in normal rabbit eyes. The amounts of FD 150 and FD 40 in the sclera and suprachoroid at the end of the experiment are only about 2.57; of the total amount lost from the perfusion system (Table II) but this may represent only the material “in transit” at that particular time, and does not necessarily give a quantitative estimate of uveoscleral flow unless it is assumed that all the tracer substance, in this case FD, is retained in the tissues. The second possibility (ii) implies that both FD and BD leave entirely via the angular aqueous plexus but that BD molecules, presumably on account of their size (30 nm) do not have free accessto the system. While this cannot be entirely discounted, it seemsunlikely if only becauseit is known that much larger particles (up to 800 nm) can penetrate into the plexus (Huggert, 1955). In addition, it has been shown that when the anterior chamber is perfused with a mixture of carbohydrates of differing molecular sizes, such as xylose and starch, and the effluent collected from the aqueous drainage channels, it is the smaller, rather than the larger molecules which are impeded (Berggren, 1963). This observation has tentatively been ascribed to a gel-filtration effect of hyaluronic acid in the anterior chamber angle but whatever the explanation, it renders it unlikely that the exit of the high molecular weight BD would be restricted in comparison with the smaller FD molecules. It is hoped that further studies on the tissue distribution of BD may throw more light on this question.
FLUORESCEIN-LABELLED
DEXTRAXS
585
REFERESCES Ascher, K. W. (1942). The aqueous veins. Am. J. O$thnZmoZ. 25,, 1174209. Berggren, L. (1963). Passage out of the eye of substances of low and high molecular weights. Invest. Ophthalmol. 2, 305-15. Bill, A. (1965) The aqueous humor drainage mechanism in the C’ynomolgus monkey (Macaca irusj with evidence for unconventional routes. Incest. Ophthtrlmol. 2, 911-19. Bill, A. (1966a). The routes for bulk drainage of aqueous humour in the rervet monkey (C’ercopithecus ethiopsj. Exp. Eye Res. 5, 56-7. Bill, A. (1966b). Formation and drainage of aqueous humour in cars. Exp. Eye Res. 5, 185-90. Bill, A. (1966c) The routes for bulk drainage of aqueous humorrr in rabbits with and w-ithout cyclodialysis. Documenta. Ophthnl. 20, 157-69. Bill, A. (1967) Further studies on the influence of the intra-ocular pressure on aqueous humor dynamics in Cynomolgus monkeys. Incest. Ophthalmol. 6, 364-?2. Cole, D. F. and Monro, P. A. G. (1976) Demonstration of outflow pathways for aqueous humour in the rabbit eye using fluorescein angiography. Biorheology. 13, 266. Fowlks, W. L. and Havener, V. R. (1964). Bqueous flow into the perivascular space of the rabbit ciliary body. Invest. Ophthalmol. 3, 374-83. Greaves, D. P. and Perkins. E. S. (1951). Aqueous veins in rabbi&. Br. J. Ophthnlmot. 35,119-23. Huggert, A. (1955). Pore size in the filtration angle of the eye. Acta Ophthnlmol. (Kbh) 33, 272-84. Leber, Th. (1903). In Graefe-Snemisch Hnndbuch der gesamten Augenheilkunde Bd. 2. Abt. p. 279. W. Englemann, Leipzig. Monro, P. A. G. (1971). Rapid sequence photomicrography of the human bulbar conjunctival vessels. 6th European Conference on Microcirculation. Aalborg, 1970. (Ed. Ditzel, J., Lewis, D. H.), pp. 90-94. S. Karger, Basel. Kagasubramanian, S. (1974). The effect of vasopressin on the facility of aqueous humour outflow in the rabbit. Ophthal. Res. 6, 301-7. Rutili. G. and Arfors, K. E. (1972). The usefulness of fluorescein labelled dextrans in the study of macromolecular passage across the capillary and blood lymph barrier. Abstracfs VII Microcirculation Coqference, Aberdeen, 1972, p. 101. Siedel, E. (1922). Weitere experimentelle Untersuchungen iiber die Quelle und den Verlauf der intraokularen Saftstriimung-(XVIII). r,o12 Graefes Archic. 111, 167-95. Tripathi, R. C. (1975). Comparative aspects of aqueous outflow. In The Eye (Ed. Davson, H. and Graham, L. T.), vol. V. pp. 193-356. Academic Press. London. Unger, W. G., Perkins, E. 8. and Bass, hr. S. (1974). The response of t,he rabbit eye to laser irradiation of the iris. Exp. Eye Res. 19, 367-77. Weekers, R. and Prijot, E. (1950). Recherches experimentales sur les fonrtions desveines aqueuses. Ophthulmologica, Basel. 119, 321-35.
The Use of fluorescein-Labelled Dextrans in Investigation of Aqueous Humour Oufflow in the Rabbit* D. F.
COLE
AND
P.
A. G.
&fONRO
Institute of Ophthalmology, University of London, .Judd Street. London TT’Cl, P.K. and Department of A,natomy, University qf Camb~riclgr, U.K. (Received 10 December 1975 and in revisedform 19 February 1976, London) The anterior chamber of t*he rabbit eye was perfused with solutions containing fluoresceinlabelled dextrans (FDs) of various molecular weights from 3000 to 150,000. Using an adapted Zeiss slit-lamp, specially modified for rapid sequence flash photography, the outflow channels for the aqueous humour in the limbal region could be demonstrated in living, anaesthetized animals. Semi-permanent preparations showing the postmortem distribution of FD in t.he tissues were made by fixation in a mixture of neutral formaldehyde and 70:/, ethanol. The fixed material was dehydrated in the normal way and either cleared in bulk in metshy salicylate or embedded in paraffin for sectioning. Cleared whole-mount preparations showed FD precipitated in the angular aqueous plexus and the efferent veins together with some perivascular distribution. Paraffin sections showed that FD had penet,rated into the tissues of the iridocorneal angle and the suprachoroidal space. Further experiments were carried out using closed cycle perfusion of the anterior chamber with a mixture of FD (mol. wt. of 3000,40 000 or 150 000) and blue dextran (mol. wt. = 2000 000). Rat,e constants calculated for the loss of these substances from the anterior chamber showed that the rates for the FDs were 20-25Oh greater than the rate for blue dextran. On the basis of these findings it is suggested that an appreciable fraction of aqueous humour in the rabbit eye may leave via a uveoscleral route.
1. Introduction Tn order to demonstrate the outflow pathways for the aqueous humour it is an established procedure to inject a dye or coloured suspensioninto the anterior chamber and then to examine its subsequentdistribution (cf. Leber, 1903; Siedel, 1922). Thus, following Ascher’s demonstration of aqueous veins in man (Ascher, 1942), Weekers and Prijot (1950) and Greaves and Perkins (1951) employed respectively india ink and Evans blue to demonstrate outflow vesselsfor the aqueoushumour in viva in the rabbit. With the more recent availability of fluorescein-labelled dextrans of defined molecular sizes it seemedworthwhile to make use of these substancesfor a similar purpose and, at the sametime, to take advantage of their other properties in order to study aqueous outflow dynamics and post mortem tissue distribution. The present paper is concerned primarily to report the techniques which have been developed for this purpose. The fluorescein-labelleddextrans (FDs) are obtained by high resolution gel filtration and are labelled with fluorescein isothiocyanate. They are available commercially in a variety of different, well-defined molecular weights. They are readily identifiable by their fluorescenceand show no appreciable binding to plasma protein (Rutili and Arfors, 1972). This is a considerable advantage over dyes such as Evans blue, which bind extensively, since the tissue and plasma concentrations of FD measured by fluorimetry indicate the actual amount of the free material present. As will bc shown * Reprint d
requests
to: Dr D. P. Cole, Institute
of Ophthalmology, 571
Judd
St., London
WClH
9&Y.
,572
I).
I’.
(‘OLE
ANL)
I’. A. t:.
MOKRO
below, the FDs can bc precipitated ad fixed in situ for the post mortcxnl study of the tissues. The present investigation was carried out using three different E’D fractions having average molecular weights of 3000, 40 000 and 150 000 respectively. These. togrthcr with blue dextran (BD) (mol. wt. = 2000 000) were obtained from Pharmacia Lttl, Uppsala, Sweden.
2. Methods of t,he The apparatus designed by Monro (1971) f or rapid sequence photomicrography human conjunctival vessels was adapted to enable fluorescein angiogrnphy to be carried out in the limbal region of the rabbit eye after the anterior chamber had been perfused with PD. To enable an observer to make continuous photographic records while keeping the experimental eye under observation, a Zeiss slit lamp was modified as follows : the illumination system was replaced with a lamp-house containing (1) a xenon in quartz flash lamp working at 3 kV, with its own condenser lens and aperture stop; and (2) an alternative quartz iodine lamp with another condenser and a variable aperture field stop. Light from the quartz iodine lamp could be brought into the optical pathway by means of a pivoting F,O3QT CAMERA
[
FIG. 1. Diagram showing modifications to the Zeiss slit lamp microscope for use in rapid sequence photomicrography. The illuminating system (apart from the lamp house condenser, C) is not shown, but light from the illuminating system is indicated as broken lines. C, illuminating condenser; E,, inclined eyepieces; E,, second viewing eyepiece, F, barrier filter (Ilford 110); L,, objective lens; L,, anastigmat forming secondary image at lens forming primary image at V, ; L,, x 10 Complen eyepiece; L,, achromat V,; M, mirror on pivot (in position for photography); T, position of turret lens; V,, position of primary image; Vz, film plane (secondary image).
FLUORESCEIK-LABELLED
DEXTRANS
573
mirror actuated by a rotating solenoid (Monro, 1971), and afforded illumination for continuous observation. The xenon flash was triggered by the camera shutter contacts and was used for serial photography. For the present experiments the limiting aperture stop in the flash tube condenser was removed so that an area of 5-6 mm diam. could be illuminated at the object plane by means of a third aplanatic condenser (C, Fig. 1) with light from either the flash tube or the quartz iodine lamp. A Balzer FITC-3 filter was placed in the light path of the xenon flash and a heat filter in the condenser from the quartz iodine lamp, so that full colour was available for observation of the limbal region. The lamp house and its condenser could be rotated in a horizontal arc of radius 25 mm concentric with the animal’s eye into a position about 40” to the optical axis of t’he object,ive lens, L,. The Zeiss slit lamp is fitted with an achromatic lens of 100 mm focal length, placed at its focus from the object so that the rays to the image are esseutially parallel. In this pathway there is a turret lens system with powers from x 6 to x40 but the nominal position used in the present experiments contains no lenses. The modifications introduced into the microscope system are shown in Fig. 1. A mirror housing was built between the t,urret (T) and the ordinary binocular head, which produces virtual images for direct, observation in both inclined eyepieces (E,) when the mirror (M) is not in position for photography. The front-surfaced mirror (M) is pivoted about its lower edge by means of a second rotating solenoid into an angle of 45” to the rays to one eyepiece (the other being fitted with a graticule for checking the focus). The image rays are thus reflected into an anastigmat lens (L,) of 125 mm focal length at the top surface of the mirror housing so that a primary image (V,) is formed at its focus at a magnification of x 1.25. A positive “Cornplan” 10 eyepiece (Ls), with an achromatic lens (LJ of 85 mm focal length placed above it’, forms an image (V,) on the film plane. Immediately above the lens (LJ a rightangled prims reflects 10°,& of the light into a second viewing eyepiece (E,) used for checking the focus and flash illumination. The Robot Star camera can be made to operate at up t,o 8 frames jsec by means of its clockwork drive, but the shutter lies about half-way between the normal lens position and the film plane so that it was essential to use a lens system somewhere near the original design position in order to prevent vignetting of the image. A similar system operating at 2 frameslsec was used to study the localized leakage of fluorescein (Cole and Monro, 1976) and low molecular weight FDs from the conjunctival vessels. An Ilford 110 filter (F) is placed above the secondary objective lens as a barrier to ensure that only fluorescence from FD is recorded on the film. This filter combination (i.e. Balzer FITC-3 and Ilford 110) has previously been found to give good contrast during fluorescein angiography of the iris vessels (cf. Unger. Perkins and Bass, 1974). In the present experiments, photographs were taken on Kodak TriX film at 15-30 set intervals at a magnification of x 4 and using a flash of 20 J with a duration of less than 1 msec. The film was developed in Patterson “Acuspeed” to give a nominal ASA rating of 1200. After anaesthetizing the rabbit with intravenous urethane, 1.75 g /kg body weight, the right eye was transfixed with a perfusing needle and the animal mounted on a supporting cradle in front of the slit lamp. The eye was gently proptosed, covered with silicone oil and a suitable field identified in the inferior quadrant of the limbus. The eye was perfused with buffered Krebs Ringer solution at 10 $/min against an outflow pressure of 15.-20 mm/Hg and, once the limbal vessels had been focused, approximately 0.2 ml of 5% FD was injected slowly from a side syringe so as to fill the lower part of the anterior chamber. As soon as this was completed, photographic exposures were commenced at 2 frames/set every 15 or 30 set for a period of lo-12 min. Demonstration
of FD in$xed
tissue
The anterior chamber was perfused with FD-40 as described below except that it was allowed to fill completely with the FD solution. At the end of 10 or 20 min the eye was
574
1). F. (‘OLE
AND
P. -4. (l. MOSl
rapidly enucleated and placed in a fixative consisting of 30 ml neutralized formaldehyde and 70ml ethanol (since the dextrans are readily precipitated from alcoholic solutious when the ethanol concentration exceeds 509{,). The globe was opened at the equator to wash out the excess PD and fixation of the anterior segmeut continued overnight. The lens W;W removed and the tissue dehydrated in successive grades of ethanol, cleared in xylene and embedded in paraffin wax in the conventional manner. Sections of 5 -10 ~111 were cut. floated out on a warm bath of 70% isopropanol (floating out on water would have removed the FD) and taken up on microscope slides. The sect’ions were then de-waxed and mounted in DPX (B.D.H). In some cases large pieces of anterior segment were pinned out before the final fixation in order to prevent distortion. After dehydration these were prepared as whole mounts cleared in methyl salicylate and were studied using a low-power, wide-field objective iI1 order to obtain an overall picture of the FD-containing vessels. Microscopy was carried out with the Leitz Orthoplan microscope with fluorescein attachment using a UG5 filter on the illuminating side and a K530 filter as a barrier filter. Photomicrographs were taken using Kodak TriX film and developed in Promicrol (May & Baker, England). Recycling perfusion
experiments
After anaesthetizing with urethane as above, one eye was cannulated needle so that the anterior chamber could be continuously perfused containing 0.5% FD and 2% BD [see Fig. 2(a, b)].
with a transfixing with Krebs saline
D
C
(b)
(a) FIG. 2. (a) Diagram to show the arrangement of needle is represented by A and B [see also Fig. 2(b)],
the recycling perfusion system. The transfixing flow is maintained by the rotary pump, C, with B connected to its inflow and A to its outflow side so that there is continual recycling of the fluid contained in the pump-tube system (BCDA) and the anterior chamber of the eye. At given intervals the tubing is clipped at a point D and small samples arc removed from the outflow side of the pump at C. (b) Flow of perfusate through the transfixing needle in the anterior chamber. The animal was placed on its side and a drop of 1% amethocaine applied to the cornea. With the perfusing needle attached at one end (B) via a polythene tube to the inflow side of the pump system (C), which had already been filled with the perfusion fluid, the eye was transfixed (cf. Nagasubramanian, 1974) and the emergent tip of the needle connected to the outflow side of the pump at A, thus forming a closed system. Recycling perfusion was commenced at a measured 54 $/min and continued for 90-120 min. At approximately 20 min intervals the tubing was clipped at a point D samples of 5-10 ~1 fluid removed at the outflow side of the pump (C). These samples were diluted and used for the estimation of FD and BD. At the end of the experimental period the animal was killed, the eye was
FLUORESCEIN-LABELLED
575
DEXTRANS
enucleated and the attached conjunctival and subconjunctival tissue (1) snipped off with curved scissors. The globe was then opened about 2 mm in front of the equator and the lens (a), vitreous (3) and iris+ciliary body (4) removed, leaving the cornea and sclera. The outer rim of sclera (5) was removed to within about 1 mm of the limbus and an inner disc of cornea (6) was punched out with a cork-borer to within 1-2 mm of the lintbus. The remaining annulus of tissue was categorized as limbus (7). After weighing, each of these tissues. (1) Tao(7) \vas homogenizetl in 2.5 ml neutral ziuc hydroxide, centrifuged for 10 min to remove the precipitated protein and the supernatant usetl for the estimation of PD. In the present experiments it, was poysible to estituat,e the c.orlc.elltratioll of HD in the perfusiate Ijut, not in the tissues. (‘nlr:~lnfion.s. At zero time there is a closed system in which there is a known quantit> of FD (Jr,,). contained in a known volume of fluid (b;,) in the pump-tube system [WI>,\ i11 Fig. 2(a)]. In the remainder of the system, the anterior chamber has a volume I’, ;IILC~a zero concentratJion of FD. The pump recycles fluid through the anterior chamber :~II~ thr amount, of FD lost by normal drainage of a,queous humour will depend on its concentriltioll in the anterior c.hamber which will rise rapidly during the mixiug phase and thereafter will declirle exponentiallv. In order to calculate the total c:umulat,ive loss during t,he I\-hole course of the experiment it is necrssarv t’o include an estimate of the loss whklr ocmrs whilst the concentration is rising during the i&al mixing phase. Taking the pump volume, I’,,. :IS 270 ~1 and the flow ratca as .5-l-pi/ruin it WAS as~lrlerl t Ililt : (a)
100; of the solute present in the anterior chamber during the time interval the end of minute (~1) to t)he end of minute W, leaves during the following interval (from minute I/ to minute (H i -1)).
(1)) the pump tube recycling time is 5 min so that, the efflux during equsl t,o t’he influx during the (/[+5)th minme. The total amount of dextran in the anterior chamber (Qc) cessive 1 min intervals (i.e. Q;1, Q;n+l,, etc) starting from various values for the fractional outflow loss (k) from the a,nterior veins and surrounding tissues. At the end of t,he & ntinute the ant,erior chamber is:
where the values, Q,: incorporat.e chamber:
correction
for fract,ional
the lath minute
zero time aud assuming chamber into the aqueous the amount of dextran iI1
loss due to outflow
from the (3
and Q, represents the amount which would have been present in the chamber been no outflow (i.e. with /I: = 0). With the values indicated above, knowing volume flow in the system and when ~004 ( 1; < 0.006 the time course for concentration (C,) was calculated and was found to approximate to a simple tleaa,v :tfter the first 25 min (Fig. 3). Furthermore, the time course of the total
1
is
inay be caloulat.etlat suc-
Q: = &,(I-4
toss. d ( : I; f Qc) f or Il’ff 1 erent values of &. within
from 1 min
this range,
approximated
had there the rate of the effluent, exponential cumulative to a set
of straight lines passing through zero (Fig. 4) so that, knowing the value for A at an assumed value for k (say 0.005 min-l) and knowing also the true value, Ic’ (from the decay rate after 25 min), the actual cumulative loss, A’ could be obtained from: ,4’ = (k’/k).A. (3) In order to validate the procedure a series of experiments were performed in which the concentration of FD was estimated in small (5 ~1) samples of effluent in the first 20 min of
I 20
0
I 40
I 00
I 60
I 100
120
Time (min 1
Flo. 3. Semilogarithmic plot of preliminary experiments showin, v the effluent concentration of ED (Cl,). The broken line (. . . ). during the first 25 min shows the predicted time course calculated from equations (1) and (7) in the text). The solid line shows the loss during the period of exponent,ial dccag (k’ z 0.00512 min-l). Thtr symbols represent cxpcrimcntal values obtained in each of four animals. 40
0
20
40
60
Perfusion
time
80 (min)
FIG. 4. Time course of the total cumulative loss, A, calculated for different ralues of lz (see text). In the sample calculation shown in the text, this graph is used to determine the total cumulativr loss at the end of 91 min. Assuming L = 0,005, this yields a value of 306 pg. Volume of pump+tubing (VP) = 272 ~1 Concentration of FD at start = 5000 pg/ml Iuitial amount of FD iu system (M,) = 1360 pg Concentration in system at end of experiment (after 91 min) = 1,975 pg/m Experimental value for k’ (from Fig. 3) = O-00512 min-l (a) From the graph (Fig. 4) the cumulative loss in 91 min at a k: value of 0.005 mine’ would be 305 pg and so the actual cumulative loss, i4’, must have been : 0.00512
3o5x isiG-
= 313 pg (from (3) above)
FLUORESCEIN-LABELLED
DEXTRANS
577
Amount of FD present in sclera+suprachoroidal
tissue at the end of the experiment 7.80 x 100 = 74 pg . .. Tissue level as “/o of act,& cumulative loss = ~313
Final amount of FD remaining in the system = Ala--A’ = M, = 1047 pg The total volume of the pump system plus the anterior chamber is (B,+ VP) so that :
va = .v,/c,--B*, Substituting
the experimental values: 1047 J’, zzz ~ -0.272 = 0258 ml 1975
The value for k, is then calculated with reference to this figure for V%: k, = k’ ( va+Vp) _ (,.(,(,512 ‘< , ((‘.258+o*272) 0.258 = 0.0106 min-l Similar corrections were applied to estimate the k, values for BD. . _^_ Preliminary studies had shown that the osmolarit,y of the aqueous humour was 283& 2.5 @/ml H,O and solutions of BD and FD were made up as 2.0:/: and 0*5’h respectively in buffered Krebs Ringer and adjusted to this osmolarity. In the case of FD three different molecular sizes were used: FITC-Dextran 3 (average mol. wt. = 3000, Stokes’s radius = 1.2 nm), FITC-Dextran 40 (mol. wt. = 40 000, Stokes’s radius = 4.4 nm), and FITCDextran 150 (mol. wt. = 150 000, Stokes’s radius = 8.7 nm). The BD was Dextran 2000 (mol. at. = 2000 000, Stokes’s radius = 30-O nm). The information on the molecular radii was obtained from the manufacturers. FD was estimated using a 2.5 mm-quare cell in the Aminco-Bowman Spectrophotofluorimeter with an excitation wavelength of 493 nm and an emission wavelength of 513 am and using solutions of the appropriate FD in buffered (pH 74) isotonic saline as standards. BD was estimated using a Unicam SP 500 spectrophot,oruet,er at a wavelength setting of 625 nm with a specially constructed micro-cell holtliil~g 0.15 ml. 3. Results
Figure 5 shows a typical set of photographs of the inferior quadrant seenat intervals from 3 to 10 min after the introduction of 50/bFD 40 into the anterior chamber. The FD soIut,ion is present in the lower part of the anterior chamber and after 3 min has commenced filling the outflow channels [Fig. 5(a)] and by 49 min is contained in a network of vesselsmost of which underlie the limbal and conjunctival vessels[Fig. 5 (b, c)~]although somebranches appear to lie closer to the surface [Fig. 5(d)]. At the end of 10 niin someleakage has commenced into the surrounding tissues. When the lower molecular weight substance (FD 3) was used this leakage was more rapid [Fig. 6(a, b)].
Figure 7(ac) shows the pattern seen by low-power fluorescence microscopy in whole mounts of tissue from the limbal region after dehydration and clearing in methyl salicylate. FD may be identified in the anterior aqueous plexus and in the vesselsleading away from it. especially when the specimenis viewed from the vitreal
!
FLUORESCEIN-LABELLED
579
DESTRANS
side [Fig. 7(a)]. Deposits of FD were also observed in close proximity to the larger blood vessels and from Fig. 7(b, c) it is clear that this material was not actually in the lumen of the vessels, which remained filled with blood, but was located in the perivascular space. Sections through the corneoscIera1 junction show the intrascleral and limbal vessels in cross section filled with fluorescent material (presumably aqueous veins) whilst others appear empty (Fig. 8). There is considerable penetration of FD into the sugrachoroidal space running posteriorly from the iridocorneal angle (Fig. 8, SCS) ant1 there are deposits in the iris stroma (Fig. 9).
FIG. 6. (a, b) In viva. fluorescent photographs of aqueous outflow channels after intracameral infusion of FL) 3. (a) 2 min, (b) 4 min. As with FD 40, the outflow channels AV appear deep to the conjunctival vc~sscls, but in this case considerable extravascular diffusion has occurred after 4 min (h, r).
TABLE
I
Values for anterior chamber,volume nrzd efllux rates (Va and k,) c&d&l
for
XntcGr chamber rolume (J-8.) W) Meann&s.E.iv. EfHus (k,,t) Mzans+s.E.M.
millrl
BD, FD 150, FD 40 and FD 3
38 * 1o.i (20) 0~0081 +0.0006
“61 2 8.3 (6) 0.0105 f 0~0009
(6)
(19)
Individual differences .h(FD)-kmt(BD) Means$s.a.w.
“75
“94 79.8
7 9.8 (7
(7)
0.01 I + 0.0010 (7)
I~~0116 k 0.0011
(6)
+0.0024**
-/ 0.0029**
&0.0034**
*0.0004
$0~0006
~0@005
(6)
(6)
(7)
The significance of the individual differences (line 3) is indicated by asterisks. Figures in parentheses represent thr number of ryes in each grump.
Thus
** = 0.01
I’.
I). F. (‘OI,E
FIG.
7. (a),
AK
(11).
1.) I’. A. c:. n1os
See
caption
I(0
opposite.
FLUORESCEIN-LABELLED
DEXTRANS
FIG.
581
7. (c).
FIG. 7. Fluorescent photomicrographs of whole mount preparations (a) Regions near the angular aqueous plexus, (AAP), showing efferent PD. Viewed from the vitreal side. (b) The terminal branches of the diverging to join the major iris circle, MIC. Perivascular deposits Viewed from the scleral side. (c) Intrascleral vessel containing blood of FD (1). Viewed from the scleral side.
after clearing in methyl salicylate. vessels (1) containing precipitated long ciliary artery, LCA, are seen of FD are shown by arrows (I). ( 4 ) with perivascular deposits
FIG. 8. Fluorescent photomicrographs of section through corncoscleral junction showing the intrascleral channels (aqueous outflow channels) filled with FD (1). There is considerable penetration into the iris root and the supra-choroidal space, SCS. C. cornea: AC’: anterior chamber; I, iris; LC, limbal conjunctiva; S, sclera; ev, empty vessel.
Figures
in parentheses
represent
the number
of eyes m each group
FLUORESCEIN-LABELLED
DEXTRANS
583
Recycling perfusion Figure 10 shows typical results from three eyes in which the exponential decay phase of the three fluorescein dextrans (FD 3, FD 40 and FD 150) are compared with the corresponding decay rates for BD. In all cases the rate for FD was greater than the corresponding rate for BD and the results from the whole series are summarized in Table I which gives the calculated values for anterior chamber volume (7,) and outflow constant (k,), calculated as described above. The last line of the table shows the means of individual differences (i.e. 7c, for FD less the corresponding k, for BD) and in each case the outflow constant for the FD is significantly higher than the corresponding value for BD. Table II shows the amomrts of FD remaining in the tissues at the end of the perfusion period expressed as percentages of the total amount of FD lost from the perfusate during this period. Only insignificant amounts were present in the lens and the vitreous humour ; values for these tissues are not included in t,he table. 4. Discussion The illustrations above show that the FDs can be used to demonstrate aqueous humour outflow channels both in vivo, and in fixed material post mortem. The invivo, and post mortem appearances are consistent with one another and the outflow channels seen in the living eye may be readily identified in whole mount flat preparations of fixed and cleared material, or in sections through the limbus [Figs 7(a-c), 8(a-c)]. The appearance of the sectioned material shows FD located mainly in the suprachoroid and sclera, limbus and iris, and this pattern agrees with the quantitative distribution in Table II. The relatively small amounts present in the cornea are confined to the endothelium. The fact that FD 3 penetrates the limbus and sclera in a significantly greater amount than the larger molecules of FD 40 or FD 150 (Table II) is consistent with the more rapid spread of FD 3 seen in vivo (cf. Figs 5 and 6). $ 4-o1 E 2-o2 z .E I.0 c z 0’ :, 0
20
-5
:
z .5
- 2.5 ii
*50
I 20
I 40
I 60 Time
I 00
I 100
I 120
(min)
FIG. IO. Typical results from three experiments showing decay rates of FD and BD in recycling perfusion experiments (semilogarithmic plot). . . . values for BD k, = 0.0081; l , FD 150, k, = 0.0106; 0, FD 40, 7cf = 0.0108; 0, FD 3, k, = 0.0110.
FD is scattered diffusely throughout the iris stroma (Fig. 9) and, while it does not enter the iridial portion of the ciliary processes,it appears to be more concentrated at the posterior iris epithelium. Fowlks and Havener (1964) described such distribution in the case of nitro-blue tetrazolium and also envisaged the possibility of perivascular drainage of aqueous
5x1
1). F. (~or,b: ASI) I’. A. t:. iuo~ttco
humour. Some further evidence for the latter is af?orllecI here by the presence of PD deposits outside the lumen of blootl-filled vesselS~. in and immediately posterior to. the limbal region [pig. 7(c)]. In the recycling perfusion experiments there If-as an obvious and consistent, difference between t.he A, values for PD and those for BL) (Table 1 and Pig. 10). Were this difference solely clue to diffusional loss of FD it would be reasonable to expect a greater difference between the L, values for the larger 171) molecules (i.e. FD 150 ant1 FD 40) and the smaller FD 3 than was actually observed. Thus, the observetl differences (Table 1, line 3) for k, (FD) ~- L, (BD) arc in the proportion of 1 : 1.2 : 1.4 for FD 150, FD 40and PI) 3 respectively, but on the basis of diffusion being approximately proportional to 1 .L/ mol. wt.. these ratios should have been of the order 1 : 1.9: 6.9. If diffusion is ruled out as the main cause of the difference between the Jc~values the alternative hypothesis seems to he that some part of the outflow pathwa.> is available to FDs hut not to BD. Here there are two main possibilities: ( i) that some of the loss of FDs occurs via uveoscleral routes similar to those described in monkeys (Bill, 1965; 1966a; 1967) and that k, for BD represents only the loss which takes place via the angular aqueous plexus and its efflus channels; (ii) that the kf values for FD represent loss via the angular aqueous plexus but that there is some restriction on the entry of BD into this system. The obvious objection to (i) is that Bill was unable to demonstrate any appreciable uveoscleral flow in cats and rabbits, either using red dextran (mol. wt. = 40 000) or lalI-labelled albumin (Bill. 1966b,c). In the present experiments it may be seen from Fig. 8 that FD 40 certainly penetrates into the suprachoroidal tissues and Tripathii (1975) has reported the penetration of an electron dense tracer (Thorotrast, particle size approximately 10 nm) into the suprachoroidal space of the rabbit. FD is much easier to identify in the tissues than is the red dextran used by Bill (1966c) and it is possible that this fact alone may account for his failure to detect any penetration into the suprachoroidal tissue in normal rabbit eyes. The amounts of FD 150 and FD 40 in the sclera and suprachoroid at the end of the experiment are only about 2.57; of the total amount lost from the perfusion system (Table II) but this may represent only the material “in transit” at that particular time, and does not necessarily give a quantitative estimate of uveoscleral flow unless it is assumed that all the tracer substance, in this case FD, is retained in the tissues. The second possibility (ii) implies that both FD and BD leave entirely via the angular aqueous plexus but that BD molecules, presumably on account of their size (30 nm) do not have free accessto the system. While this cannot be entirely discounted, it seemsunlikely if only becauseit is known that much larger particles (up to 800 nm) can penetrate into the plexus (Huggert, 1955). In addition, it has been shown that when the anterior chamber is perfused with a mixture of carbohydrates of differing molecular sizes, such as xylose and starch, and the effluent collected from the aqueous drainage channels, it is the smaller, rather than the larger molecules which are impeded (Berggren, 1963). This observation has tentatively been ascribed to a gel-filtration effect of hyaluronic acid in the anterior chamber angle but whatever the explanation, it renders it unlikely that the exit of the high molecular weight BD would be restricted in comparison with the smaller FD molecules. It is hoped that further studies on the tissue distribution of BD may throw more light on this question.
FLUORESCEIN-LABELLED
DEXTRAXS
585
REFERESCES Ascher, K. W. (1942). The aqueous veins. Am. J. O$thnZmoZ. 25,, 1174209. Berggren, L. (1963). Passage out of the eye of substances of low and high molecular weights. Invest. Ophthalmol. 2, 305-15. Bill, A. (1965) The aqueous humor drainage mechanism in the C’ynomolgus monkey (Macaca irusj with evidence for unconventional routes. Incest. Ophthtrlmol. 2, 911-19. Bill, A. (1966a). The routes for bulk drainage of aqueous humour in the rervet monkey (C’ercopithecus ethiopsj. Exp. Eye Res. 5, 56-7. Bill, A. (1966b). Formation and drainage of aqueous humour in cars. Exp. Eye Res. 5, 185-90. Bill, A. (1966c) The routes for bulk drainage of aqueous humorrr in rabbits with and w-ithout cyclodialysis. Documenta. Ophthnl. 20, 157-69. Bill, A. (1967) Further studies on the influence of the intra-ocular pressure on aqueous humor dynamics in Cynomolgus monkeys. Incest. Ophthalmol. 6, 364-?2. Cole, D. F. and Monro, P. A. G. (1976) Demonstration of outflow pathways for aqueous humour in the rabbit eye using fluorescein angiography. Biorheology. 13, 266. Fowlks, W. L. and Havener, V. R. (1964). Bqueous flow into the perivascular space of the rabbit ciliary body. Invest. Ophthalmol. 3, 374-83. Greaves, D. P. and Perkins. E. S. (1951). Aqueous veins in rabbi&. Br. J. Ophthnlmot. 35,119-23. Huggert, A. (1955). Pore size in the filtration angle of the eye. Acta Ophthnlmol. (Kbh) 33, 272-84. Leber, Th. (1903). In Graefe-Snemisch Hnndbuch der gesamten Augenheilkunde Bd. 2. Abt. p. 279. W. Englemann, Leipzig. Monro, P. A. G. (1971). Rapid sequence photomicrography of the human bulbar conjunctival vessels. 6th European Conference on Microcirculation. Aalborg, 1970. (Ed. Ditzel, J., Lewis, D. H.), pp. 90-94. S. Karger, Basel. Kagasubramanian, S. (1974). The effect of vasopressin on the facility of aqueous humour outflow in the rabbit. Ophthal. Res. 6, 301-7. Rutili. G. and Arfors, K. E. (1972). The usefulness of fluorescein labelled dextrans in the study of macromolecular passage across the capillary and blood lymph barrier. Abstracfs VII Microcirculation Coqference, Aberdeen, 1972, p. 101. Siedel, E. (1922). Weitere experimentelle Untersuchungen iiber die Quelle und den Verlauf der intraokularen Saftstriimung-(XVIII). r,o12 Graefes Archic. 111, 167-95. Tripathi, R. C. (1975). Comparative aspects of aqueous outflow. In The Eye (Ed. Davson, H. and Graham, L. T.), vol. V. pp. 193-356. Academic Press. London. Unger, W. G., Perkins, E. 8. and Bass, hr. S. (1974). The response of t,he rabbit eye to laser irradiation of the iris. Exp. Eye Res. 19, 367-77. Weekers, R. and Prijot, E. (1950). Recherches experimentales sur les fonrtions desveines aqueuses. Ophthulmologica, Basel. 119, 321-35.