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The hydraulic conductivity of rabbit ciliary epithelium in vitro
Eiiy. E~P Res. (1982) 34, 121-129
The Hydraulic
Conductivity of Rabbit Epithelium in vitro
JON BRODWALL*AND Departments
JORGE
Ciliary
FISCHBARG
of Physiology and Ophthalmology, College of Physicians and Surgeons. Columbia University, New York, N.Y. 10032. U.S.A. (Received 2 March 1981 and accepted 12 April
1981, London)
The rate of fluid movement across rabbit ciliary body in vitro has been measured as a function of applied hydrostatic pressure. Previous studies from other laboratories carried out with the ciliary body clamped between two half chambers have yielded a value of 4600 k 600 pm/see. We have presently reproduced those results by usingasimilar mounting procedure. On the other hand. however, we report the development of a technique for edge-damage-free mounting of the ciliary body with which the tissue is not clamped and tissue glue is employed in order to achieve a proper seal. With this last procedure, the values thus obtained for the hydraulic conductivity were much lower than the previous ones, namely, 210 ,am/sec for pressure applied on the stromal side of the ciliary epithelium (pressure range: t&10 cm H,O, n = 13; T = 37’C), or 109 Pm/set (pressure difference range: Ck31 cm H,O) for pressure applied from the aqueous side. We conclude that the notion of aqueous production by ultrafiltration cannot be supported by the present results. Key words: ciliary epithelium; secretion; ultrafiltration; hydraulic permeability; edge damage.
1. Introduction
It is generally accepted (cf. Cole, 1977) that the aqueous humor is produced by the ciliary epithelium. This tissue indeed has the characteristics of a fluid-secreting structure; for instance, in rabbits, the total ciliary blood flow is about 80 pl/min or approximately 14 $/min/cm2 of epithelial area. For comparison, this is about one-fifth of the renal blood flow per cm2 of glomerular capillary area. Since the in vivo rate of rabbit aqueous humor formation is some 3 pl/min, it follows that some 3 Y! of the blood supply is utilized for fluid production. The blood capillaries that traverse the ciliary processes are embedded in the ciliary stroma, which consists of loose connective tissue, poor in cells. These capillaries are fenestrated and free diffusion into the stroma of water and plasma proteins (even with the size of myoglobin; Bill, 1968a, b) has been reported to occur. In spite of these facts, on the other hand, the aqueous humor differs somewhat from blood plasma (cf. Davson, 1969; Bito, 1977). The reasons for this may involve transport mechanisms and a physiological barrier, located both at the level of the ciliary epithelial cells. The ultrastructure of the ciliary epithelium is certainly that of a membrane with a well-defined barrier function, in which tight junctions [impermeable to La(OH), and horseradish peroxidase] are found at the apex of the unpigmented cell layer (Vegge. 1971; Uusitalo, Palkama and Stjernschantz, 1973). As for its cytochemistry, ouabainsensitive ATP-ase activity has been found in the ciliary epithelium (Cole, 1964), which is consistent with its postulated secretory function; likewise, the content of succinic and lactic dehydrogenases in the non-pigmented epithelial cells is high and its localization coincides with the more high vascularized zone of the ciliary processes (Cameron and Cole, 1963; Cole, 1963). Two principal mechanisms have been invoked in order to explain aqueous humor * Permanent address: Department 0014.4835/82/010121+09
of Ophthalmology,
$01.00/O
University
of Oslo, Oslo, h’orway.
@,! 1982 Academic Press Inc. (London) 121
Limited
, .,I 99
J. BRODWALL
AND
,J. FIS(‘HBARC:
which would take place due to hydrostatic pressure production : ultrafiltration, gradients across the ciliary epithelium and active transport of fluid, which would require direct energy expenditure by the ciliary epithelium. Some authors have argued that ultrafiltration contributes more than 70% to aqueous humor formation; this reasoning is based partly on studies of the hydraulic conductivity of t,he isolated ciliary epithelium (Green and Pederson, 1972. 1973). However, the validity of such evidence has been questioned (Bill, 1975). Given the central importance that measurements of the hydraulic permeability have for these arguments, the purpose of the present work was to develop a technical procedure with which such conductivity could be measured while inflicting as little damage as possible to the tissue. The present results validate this approach and show that techniques which are of common usage for other epithelia and that were previously applied to the ciliary body result in a sizeable overestimate of its hydraulic permeability.
2. Methods
and Results
Dissection of the ciliary body Male albino rabbits weighing 25-3.5 kg were killed with an iv. overdose of sodiumpentobarbital (300 mg/animal). Both eyes were enucleated and dissected. Subsequently, a given eye was trangentially cut some 4 mm behind the limbus and its anterior part was placed with its inside facing up on a Petri dish containing a solution with salts and glucose (BSG) or rabbit plasma (RP). The solution was preheated to 37’C and freshly made prior to each experiment; the composition of BSG has been previously detailed (Fischbarg, Lim and Bourguet, 1977). Posterior capsulectomy and removal of the lens nucleus was performed subsequently. During the dissection procedure, the eye was floating freely in BSG to avoid mechanical pressures on any part of the ciliary body. At this point, with the help of a dissection microscope, vitrectomy was performed down to about 1 mm from the surface of the ciliary processes. The anterior lens capsule suspended between the zonula fibers was carefully removed with forceps and cataract scissors, leaving merely minor parts of the lens capsule still hanging on the zonula fibers. A complete capsulectomy was impossible due to the high transparency and low contrast of the capsule. For the ensuing dissection, cutting the ciliary body free from the sclera with the help of a Gill knife was considered. However, we judged that procedure to be potentially traumatic for the ciliary body and therefore developed an alternative procedure. While still kept afloat in BSG or RP, the anterior part of the globe was turned so that the epithelium of the cornea was now facing up. A perforating incision was executed centrally in the cornea and, using cataract scissors, the cornea was completely removed merely leaving a scleral circle of approximately 1 mm in width. This scleral circle was then gently removed with Hodgkin’s forceps and cataract suture scissors, leaving the isolated ciliary body still floating in the solution. With this procedure, the ciliary epithelium was never mechanically irritated. The dissection of the tissue was always carried out by the same person and quickly became a standardized routine. Tissue clamped between chambers (previous procedure) A series of exploratory experiments were performed with the ciliary body clamped between two half chambers. The electrical resistance was measured as a function of increasing applied clamping pressure. The resistance dropped sharply at the clamping pressure required to avoid a gross fluid leak between the two half chambers. This was taken to mean that a leak had been created through the tissue. However, for control purposes, the hydraulic conductance of the tissue thus clamped was measured anyway with an automatic volumetric measuring device previously described (Fischbarg et al., 1977). For the present experiments its resolution was usually 10 nl. In Fig. 4 (below), the curve which represents the hydraulic conductivity of the ciiiary body thus clamped between our two half chambers (open circles) hm a remarkable resemblance to the curves which represent results from another laboratory. Given the
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suggestion of edge damage arising from the electricalresfstance measurements, we therefore set upon the design of a different type of chamber to mount the ciliary epithelium. Edge damage free mounting (our present procedure) (a) The chamber. A novel type of chamber was designed for this purpose which is shown schematically in Fig. 1. Two half chambers clamp the lucite insert (8) together, allowing fluid communication through the three banana shaped apertures (Fig. 1, bottom) which make up a total area of @32 cm2. The half-chamber A (Fig. 1) is open to the atmosphere; the half-chamber B is connected to the automatic volumetric measuring unit through the tubing had water jackets for temperature control. system (4). Both half-chambers
Fm. 1. Schematic diagram of the chamber built for edge-damage-free mounting. Top: (1) top and bottom chamber sections; (2) jackets for temperature control; (3) guide screw, there were six of them. and they served to clamp the lucite parts together: (4) stainless steel tubing connecting to fluid measurement apparatus; (5) cover for the top chamber, to avoid evaporation; (6) tissue; (7) t&on net; (8) lucite insert; (9) rubber gasket; (10) rubber stopper. Bottom: enlarged view of the lucite insert end tissue mounting arrangement; (11) outer and inner rubber ‘ 0 ’ rings; (12) grooves for the adhesive; (13) recess. Openings connecting the chambers are shaded.
The half-chamber B was tested for fluid leaks by using a lucite insert without apertures. The leak thus recorded was negligible. For added safety, this test was repeated often between experiments. Furthermore, the chamber was kept immersed in distilled water when not in use, so as to saturate the plastic matrix with fluid. The glass capillary in the detector of the automatic volumetric measuring unit was regularly resiliconized when necessary. In order to seal the edges of the tissue, high vacuum grease was tested; it proved acceptable at zero pressure but failed to prevent leaks when the pressure gradient pointed from the stroma towards the posterior chamber. Consequently, we chose to use instead tissue glue
124
,J. BROT)W.l\I,J,
i\ST) .J. FIS(‘HUAK(;
(Permabond 102) as a sealing agent. following previous examples in other rpithrlia (Walxrr. 1970; Helman and Miller. 1971). The seal of the chamber and the efficiency of the tissue glue were ascertained by using an isolated frog skin preparation cut into annular shape much like that of the ciliary body-iris preparation. The hydraulic conductance, total electrical resistance and electrical potrnt,ial difference were measured across this preparation and previous data typical of frog skins (cf. Fischbarg and Whittembury, 1977) were readily reproduced with our present technique. (b) The mountingprocedure. The isolated rabbit ciliary body floating in BSG or RP was eased onto a disc-shaped woven Teflon net 15.5 mm in diameter. The ciliary body now resting on the Teflon net was lifted with two forceps and the bottom (anterior) side was carefully dried with ‘tissue paper‘. Liquid glue had been placed shortly beforehand into the circular grooves of the lucite insert. The Teflon net was now lowered onto the center depression in the insert (Fig. 1) and upon contact both tissue and net were glued instantaneously to the insert in the proper position. Since the amount of glue employed was very small, its spread was limited and left free the portion of the net overlying the banana shaped apertures. At this point, two rubber rings were positioned as seen in Fig. 1 and glue was applied between the wall of the lucite insert and the outer ring, and also in the center of the smaller ring. In this way the periphery of the tissue and the pupilar opening were additionally sealed. Bathing solution (either BSG or RP) was added to the epithelial and stromal side of the ciliary body. and the half-chamber B (see Fig. 1) was connected to the fluid sensor system. The procedure of killing the rabbit, dissecting the tissue and mounting it usually lasted about 25-30 min. Experimental protocol For each pressure gradient value tested, flows were usually recorded for periods of about 50 min to allow for stabilization before the rate of fluid movement values were read. The temperature of the bathing solutions was controlled throughout the experiments wit’h a thermistor immersed into the solution on the epithelial side of the tissue. Except for the changes in hydrostatic pressure, the ciliary body was otherwise undisturbed for the usual duration of the experiments (4-7 hr). Rate of $uid movement and hydraulic conductance Experiments were conducted in which fluid movements were recorded as a function of applied hydrostatic pressure. The type of data obtained are exemplified in a typical chart recording shown in Fig. 2. The transient mechanical disturbances originating from pressure changes either on the stromal or on the aqueous sides of the ciliary body result in large initial movement of fluid. These disturbances, which are probably related to tissue displacement, subside later and the rates of fluid movement in good part seem to stabilize 15-30 min after the pressure change. The data from seven experiments in which the tissue was clamped are shown in Fig. 4 (open circles). From the slope of that curve, on the basis of a total epithelial area of 6 cm* (if the infoldings are taken into account), the hydraulic conductance found was 0.131 ,ul/min/mmHg (Lp. A) or 39OOpm/sec (Lp), which is comparatively an extremely large value. The procedure was subsequently changed, as explained above. Thirteen experiments were performed in which fluid movements as a function of hydrostatic pressure were investigated using the ‘edge-damage-free’ technique. The results for each one of these experiments are detailed in Fig. 3. In addition, for comparison, the averages of those data are shown in Fig. 4 (filled circles). On the basis of the slopes of the curve joining the average data, the hydraulic conductivities were as follows: (1) 37 x 10e3 pl/min/mmHg (Lp. A) or 109 km/see (Lp) in the third quadrant, for fluid moving from aqueous to stroma; (2) 7.1 x low3 pl/min/mmHg (Lp. A) or 210 pm/set (Lp) in the first quadrant, for fluid movements from stroma to aqueous and a pressure difference of less than 9 cm H,O. For pressure differences above 9 cm H,O, the hydraulic conductivity increased markedly in one experiment, but this issue was not pursued further. The implications of these results will be dealt with in the Discussion. The Lp units utilized were chosen to facilitate comparison with osmotic permeabilities. The dimensions for the driving force for the flow eliminated by dividing by the molar volume of water. A typical conversion is done as follows : Jv, flow per unit area; Anor AP, unit driving force; 1 mosm/cm3 = 2.6 x 10’ dynes/cm2 Lp (cm/set) = [Jv (cm3/sec/cma)] [(l/U)
(cm3/mosm)] [(l/vu)
(mosm/cm3)].
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EPITHELIUM
050 f
i E E
iii 0 2LL
O-25
0
1
I
I
I
I
I
L
I
2
3
4
5
6
Ttme i hrj
FIG. 2. At each arrow, the hydrostatic pressure difference applied to the tissue was changed for the value given in the graph (in cm H,O). Positive pressures are applied on the stromal side; positive fluid movement goes from stroma to aqueous, in the same direction as aqueous production.
3. Discussion Tissue cyanoacrylate glue has been used previously (cf. Walser, 1970 ; Helman and Miller, 1971) in other epithelial studies without apparent adverse effects but since the use of such adhesive was of great importance to our mounting procedure, we examined the total electrical resistance and the electrical potential difference across our tissue using both high-vacuum grease (Dow Corning, 970V) and tissue glue as sealing agents. The results, which will be detailed elsewhere, were the same with either procedure. In addition, current studies (Brodwall, unpublished) show normal morphology for the ciliary epithelium (i.e. normal cell membrane structures and intracellular organelles) when the tissue is fixed after treatment with tissue glue as described above. Data taken in separate experiments showed that the resistance was about 300 ohm. cm2 and stayed almost constant as a function of applied pressure in the range between - 12 and + 16 cm H,O, indicating that the permeability of the ciliary epithelium is relatively unaffected and that the tissue glue provides an efficient seal. From the present results, at zero pressure difference, the rate of fluid movement is about 62 ,ul/min. This movement could be due to active secretion but this point cannot be ascertained from the present results, since a demonstration of pumping against a pressure gradient (i.e. positive flow in the first quadrant in Fig. 3) would be needed for that purpose. The value above is some three times larger than the figure
-0-5
-
I
- 32
\ .-0’
.’
1
-24
I
I
1
-16 Hydrostatic
-
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I
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-8 pressure
I
8 dlfference
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L
16
(cm ti20)
FIQ. 3. Points represents data obtained as shown in Fig. 2 from each of the 13 experiments in which the tissue was mounted in the chamber shown in Fig. 1. The lines link the data coming from each given experiment.
of 0067 pl/min reported by Green and Griffin (1978) for flow at zero pressure but is still only 6-10 o/oof the in vivo value for aqueous humor formation. One has to conclude that the active transport mechanism which has been accepted to be responsible for the bulk of ciliary secretion (Cole, 1966, 1977; Ill, 1975) is somehow inhibited, totally or partially, when the tissue is mounted in vitro or that, as noted above, in spite of our precautions, a leak is present across the preparation. It is possible that the transport mechanisms may be impaired as an unavoidable consequence of mechanical handling of the tissue. Other possible adverse effects may arise from the present use of pentobarbital to kill the rabbits. Thus, Eakins (1969) reported that the intraocular pressure in cats is reduced by some 30% due to pentobarbital, which was confirmed by Cevario and Macri (1974) for the rhesus monkey. As for yet another important factor, Cole (1977) argued that the in vitro preparations lack the extensive blood supply which might be needed for normal fluid production. We will now focus our attention on the mechanical factors involved. The geometry of this preparation is a complicating factor in that the ciliary epithelium is continued without interruption by the posterior epithelium of the iris and by the epithelial layer of the pars plana, both of which are tight epithelia. Clearly, any damage to the cells that make up those epithelial layers may well introduce a leak through the preparation. In many previous in vitro experimental studies, as has been customary in studies in other epithelia, the ciliary body has been clamped between two half-chambers (e.g. Cole, 1962; Green and Pederson, 1972). Yet, since the chambers are made of hard materials, such a procedure would be expected to produce a leak to some degree. With their pioneering technique, Green and Pederson (1972, 1973) found an Lp value of 4600 + 600 pm/set room temperature, and Green and Griffin (1978) nearly reproduced
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EPITHELIUM
-I
-30
-20
-10 Hydrostatic
IO pressure difference
20
30
(cm H20)
Fm. 4. The different data given are aa follows : (0) average of the data for individual experiments shown in Fig. 3 in which the tissue w&8 mounted with an edge-damage-free technique; (0) average data for fluid movement obtained in experiments in which the tissue was clamped between two lucite chambers : (A) average data reported by Green and Pederson, 1972 (room Temperature).
that value working at 37V. However, a hydraulic conductivity in this order of magnitude is relatively high when compared with those seen in other epithelia, so that the possibility ofepithelial damage has to be considered. The fact that clamping does seem to inflict damage is suggested by the different results presently obtained with the two different techniques. Thus, when the ciliary body is clamped between two half chambers, the Lp values found is of the same order of magnitude as that given by Green and Pederson (cf. Fig. 4). On the other hand, with our chamber for edgedamage-free mounting of the tissue, the Lp value is at least 18 times less than previously reported, or 210 ,um/sec (maximal value) in the pressure difference range from 0 to about + 10 cm H,O (filled circles, first quadrant, Fig. 4). Even this low value is probably an overestimate, since, as can be noted from the same Fig. 4, in the pressure difference range from - 2 to - 31 cm H,O (with the pressure applied on the epithelial side of the tissue) the curve was nearly parallel to the abscissa, which implies an even lower Lp value. It may be speculated that, in this range, the ciliary processes may be flattened and the epithelial cells may be squeezed together, but still the very low slope of the curve is suggestive. UltraJiltration
as a mechanism for aqueous production
Macri and Cevario (1973), when perfusing the ophthalmic artery in enucleated cat eyes, found that vaso-active drugs which changed the arterial pressure also caused changes in aqueous humor production. Hence, in their interpretation, ultrafiltration
128
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as a function of ciliary capillary pressure was not excluded as an important factor ill ciliary secretion. Similar results had been reported by Best, Blumenthal, Futterman and Galin (1969) in experiments in vivo. On the other hand, Bill (1970) found only small changes in ciliary secretion due to blood pressure variations in monkeys. Given the state of that argument’, the data of Green and Pederson (1972,1973) gained added relevance. Since their results from in vitro experiments showed a near-linear increase in flow and a value of flow similar to that of in vivo secretion was recorded at a hydrostatic pressure difference of 10-15 mmHg (13620.4 cm H,O), they argued that a ciliary capillary pressure of some 53 mmHg (4957 mmHg) could account for secretion. They went on further to propose that ultrafiltration is the major mechanism behind ciliary secretion. However, as the present study shows, the ciliary hydraulic conductance is much lower than previously reported, so that such an argument does not appear tenable. Furthermore, the ciliary capillary pressure in monkeys has been estimated to be about 25 mmHg (Bill, 1973), which, as that author argues, would exclude an important role for ultrafiltration. Landis (1930) estimated the capillary pressure at ocular level at some 17.5 mmHg, so an estimated capillary pressure of 53 mmHg (average) again appears excessive. The present results support Bill’s arguments against ultrafiltration as a major mechanism, and highlight the conclusions in support of the fluid transport hypothesis arising from Cole’s (1960) in vivo experiments, in which it was shown that the secretion of aqueous humor is reduced some 75 “b by the use of metabolic inhibitors. ACKNOWLEDGMENTS This work was supported by U.S.P.H.S. Research Grant EY 01080 to J. F. and in part by Grants to J.B. from The Norwegian Department of Social Affairs and Skipreder T. Wilhelmsens Stiftelse. REFERENCES Best, M., Blumenthal, M., Futterman, M. A. and Galin, H. A. (1969). Critical closure of intraocular blood vessels. Arch. Ophthalmol. 82, 385-92. Bill, A. (1968a). Capillary permeability to and extravascular dynamics of myoglobin, albumin and gammaglobulin the uvea. Acta Physiol. Scud 73, 204-19. Bill, A. (1968b). A method to determine osmotically effective albumin and gammaglobulin concentrations in tissue fluids, its application to the uvea and a note on the effects of capillary ‘leaks’ on tissue fluid dynamics. Acta Physiol. Scand 73, 51 l-22. Bill, A. (1970). The effect of changes in arterial blood pressure on the rate of aqueous humor formation in a primate (Cercopithecus ethiops) Ophthdmol. Res. 1, 193-200. Bill, A. (1973). The role of ciliary blood flow and ultrafiltration in aqueous humor formation. Exp. Eye Res. 16, 281-98. Bill, A. (1975). Blood circulation and fluid dynamics in the eye. Physiol. Rev. 55, 383417. Bito, L. (1977). The physiology and pathophysiology of intraocular fluids. Exp. Eye Res. 25, 273-89. Cameron, E. and Cole, D. F. (1963). Succinic dehydrogenase in the rabbit ciliary epithelium. Exp. Eye Rex 2, 25-7. Ckvario, S. J. and Macri, F. J. (1974). The inhibitory effect of pentobarbital Na on aqueous humor formation. Invest. Ophthalmol. 13, 348-86. Cole, D. F. (1960). Effects of some metabolic inhibitors on the formation of the aqueous humor in rabbits. Br. J. Opht?udmol 44, 739-50. Cole, D. F. (1962). Transport across the isolated ciliary body of ox and rabbit. Br. J. 0phtha1mo1. 46, 577-91. Cole, D. F. (1963). Utilization of carbohydrate metabolites in the rabbit ciliary epithelium. Exp. Eye Res. 2, 284-95.
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Cole, D. F. (1964). Location of ouabain-sensitive adenosine triphosphatase in ciliary epithelium. Exp. Eye Res. 3, 72-5. Cole, D. F. (1966). Aqueous humor formation. Dot. OphthuZmoE. 21, 116-38. Cole, D. F. (1977). Secretion of aqueous humor. Exp. Eye Res. Suppl. 161-76. Davson, H. (1969). The intraocular fluids. In The Eye (Ed. Davson, H.), Academic Press, New York, London. Eakins, K. E. (1969). A comparative study of intraocular pressure and gross outflow facility in the cat eye during anaesthesia. Exp. Eye Res. 8, 10615. Fischbarg, J., Lim, J. J. and Bourguet, J. (1977). Adenosine stimulation of fluid transport across cornea1 endothelium. J. Memb. Biol. 35, 95-l 12. Fischbarg, J. and Whittemburg, G. (1978). The effect of external pH on osmotic permeability, ion and fluid transport across isolated frog skin. J. Physiol. (London) 275, 403-17. Green, K. and Griffin, C. (1978). Adrenergic effects on the isolated rabbit ciliary epithelium. Exp. Eye Res. 27, 143-9. Green, K. and Pederson, J. E. (1972). Contribution of secretion and filtration to aqueous humor formation. Am. J. Physiol. 222, 1218-26. Green, K. and Pederson, J. E. (1973). Aqueous humor formation. Exp. Eye Res. 16, 273-86. Helman, S. I. and Miller, D. A. (1971). In vitro techniques for avoiding edge damage in studies of frog skin. Science 173, 146-8. Landis. C. (1930). Blood pressure changes in deception. J. Comp. Psych&. 10, 437-9. Macri, F. J. and Cevario, S. J. (1973). The induction of aqueous humor formation by the use of Ach +eserine. Invest. OphthaZmoZ. 12, 91&6. Pederson, 0. 0. and Tonjum, A. M. (1975). In vitro studies on peroxidase movement in the epithelium of prostaglandin-treated rabbit ciliary bodies. Acta OptihaEmoZ. 53, 673-84. Uusitalo. R., Palkama, A. and Stjernschantz, J. (1973). An electron microscopical study of the blood-aqueous barrier in the ciliary body and iris of the rabbit. Exp. Eye Res. 17. 49-63. Vegge, T. (1971). An epithelial blood-aqueous barrier to horseradish peroxidase in the ciliary processes of the vervet monkey (Cercopithecus ethiops). 2. Zellforsch 114, 309-20. Walser, M. (1970). Role of edge damage in sodium permeability of toad bladder and a means of avoiding it. Am. J. PhysioZ. 219, 252-5.
The Hydraulic
Conductivity of Rabbit Epithelium in vitro
JON BRODWALL*AND Departments
JORGE
Ciliary
FISCHBARG
of Physiology and Ophthalmology, College of Physicians and Surgeons. Columbia University, New York, N.Y. 10032. U.S.A. (Received 2 March 1981 and accepted 12 April
1981, London)
The rate of fluid movement across rabbit ciliary body in vitro has been measured as a function of applied hydrostatic pressure. Previous studies from other laboratories carried out with the ciliary body clamped between two half chambers have yielded a value of 4600 k 600 pm/see. We have presently reproduced those results by usingasimilar mounting procedure. On the other hand. however, we report the development of a technique for edge-damage-free mounting of the ciliary body with which the tissue is not clamped and tissue glue is employed in order to achieve a proper seal. With this last procedure, the values thus obtained for the hydraulic conductivity were much lower than the previous ones, namely, 210 ,am/sec for pressure applied on the stromal side of the ciliary epithelium (pressure range: t&10 cm H,O, n = 13; T = 37’C), or 109 Pm/set (pressure difference range: Ck31 cm H,O) for pressure applied from the aqueous side. We conclude that the notion of aqueous production by ultrafiltration cannot be supported by the present results. Key words: ciliary epithelium; secretion; ultrafiltration; hydraulic permeability; edge damage.
1. Introduction
It is generally accepted (cf. Cole, 1977) that the aqueous humor is produced by the ciliary epithelium. This tissue indeed has the characteristics of a fluid-secreting structure; for instance, in rabbits, the total ciliary blood flow is about 80 pl/min or approximately 14 $/min/cm2 of epithelial area. For comparison, this is about one-fifth of the renal blood flow per cm2 of glomerular capillary area. Since the in vivo rate of rabbit aqueous humor formation is some 3 pl/min, it follows that some 3 Y! of the blood supply is utilized for fluid production. The blood capillaries that traverse the ciliary processes are embedded in the ciliary stroma, which consists of loose connective tissue, poor in cells. These capillaries are fenestrated and free diffusion into the stroma of water and plasma proteins (even with the size of myoglobin; Bill, 1968a, b) has been reported to occur. In spite of these facts, on the other hand, the aqueous humor differs somewhat from blood plasma (cf. Davson, 1969; Bito, 1977). The reasons for this may involve transport mechanisms and a physiological barrier, located both at the level of the ciliary epithelial cells. The ultrastructure of the ciliary epithelium is certainly that of a membrane with a well-defined barrier function, in which tight junctions [impermeable to La(OH), and horseradish peroxidase] are found at the apex of the unpigmented cell layer (Vegge. 1971; Uusitalo, Palkama and Stjernschantz, 1973). As for its cytochemistry, ouabainsensitive ATP-ase activity has been found in the ciliary epithelium (Cole, 1964), which is consistent with its postulated secretory function; likewise, the content of succinic and lactic dehydrogenases in the non-pigmented epithelial cells is high and its localization coincides with the more high vascularized zone of the ciliary processes (Cameron and Cole, 1963; Cole, 1963). Two principal mechanisms have been invoked in order to explain aqueous humor * Permanent address: Department 0014.4835/82/010121+09
of Ophthalmology,
$01.00/O
University
of Oslo, Oslo, h’orway.
@,! 1982 Academic Press Inc. (London) 121
Limited
, .,I 99
J. BRODWALL
AND
,J. FIS(‘HBARC:
which would take place due to hydrostatic pressure production : ultrafiltration, gradients across the ciliary epithelium and active transport of fluid, which would require direct energy expenditure by the ciliary epithelium. Some authors have argued that ultrafiltration contributes more than 70% to aqueous humor formation; this reasoning is based partly on studies of the hydraulic conductivity of t,he isolated ciliary epithelium (Green and Pederson, 1972. 1973). However, the validity of such evidence has been questioned (Bill, 1975). Given the central importance that measurements of the hydraulic permeability have for these arguments, the purpose of the present work was to develop a technical procedure with which such conductivity could be measured while inflicting as little damage as possible to the tissue. The present results validate this approach and show that techniques which are of common usage for other epithelia and that were previously applied to the ciliary body result in a sizeable overestimate of its hydraulic permeability.
2. Methods
and Results
Dissection of the ciliary body Male albino rabbits weighing 25-3.5 kg were killed with an iv. overdose of sodiumpentobarbital (300 mg/animal). Both eyes were enucleated and dissected. Subsequently, a given eye was trangentially cut some 4 mm behind the limbus and its anterior part was placed with its inside facing up on a Petri dish containing a solution with salts and glucose (BSG) or rabbit plasma (RP). The solution was preheated to 37’C and freshly made prior to each experiment; the composition of BSG has been previously detailed (Fischbarg, Lim and Bourguet, 1977). Posterior capsulectomy and removal of the lens nucleus was performed subsequently. During the dissection procedure, the eye was floating freely in BSG to avoid mechanical pressures on any part of the ciliary body. At this point, with the help of a dissection microscope, vitrectomy was performed down to about 1 mm from the surface of the ciliary processes. The anterior lens capsule suspended between the zonula fibers was carefully removed with forceps and cataract scissors, leaving merely minor parts of the lens capsule still hanging on the zonula fibers. A complete capsulectomy was impossible due to the high transparency and low contrast of the capsule. For the ensuing dissection, cutting the ciliary body free from the sclera with the help of a Gill knife was considered. However, we judged that procedure to be potentially traumatic for the ciliary body and therefore developed an alternative procedure. While still kept afloat in BSG or RP, the anterior part of the globe was turned so that the epithelium of the cornea was now facing up. A perforating incision was executed centrally in the cornea and, using cataract scissors, the cornea was completely removed merely leaving a scleral circle of approximately 1 mm in width. This scleral circle was then gently removed with Hodgkin’s forceps and cataract suture scissors, leaving the isolated ciliary body still floating in the solution. With this procedure, the ciliary epithelium was never mechanically irritated. The dissection of the tissue was always carried out by the same person and quickly became a standardized routine. Tissue clamped between chambers (previous procedure) A series of exploratory experiments were performed with the ciliary body clamped between two half chambers. The electrical resistance was measured as a function of increasing applied clamping pressure. The resistance dropped sharply at the clamping pressure required to avoid a gross fluid leak between the two half chambers. This was taken to mean that a leak had been created through the tissue. However, for control purposes, the hydraulic conductance of the tissue thus clamped was measured anyway with an automatic volumetric measuring device previously described (Fischbarg et al., 1977). For the present experiments its resolution was usually 10 nl. In Fig. 4 (below), the curve which represents the hydraulic conductivity of the ciiiary body thus clamped between our two half chambers (open circles) hm a remarkable resemblance to the curves which represent results from another laboratory. Given the
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suggestion of edge damage arising from the electricalresfstance measurements, we therefore set upon the design of a different type of chamber to mount the ciliary epithelium. Edge damage free mounting (our present procedure) (a) The chamber. A novel type of chamber was designed for this purpose which is shown schematically in Fig. 1. Two half chambers clamp the lucite insert (8) together, allowing fluid communication through the three banana shaped apertures (Fig. 1, bottom) which make up a total area of @32 cm2. The half-chamber A (Fig. 1) is open to the atmosphere; the half-chamber B is connected to the automatic volumetric measuring unit through the tubing had water jackets for temperature control. system (4). Both half-chambers
Fm. 1. Schematic diagram of the chamber built for edge-damage-free mounting. Top: (1) top and bottom chamber sections; (2) jackets for temperature control; (3) guide screw, there were six of them. and they served to clamp the lucite parts together: (4) stainless steel tubing connecting to fluid measurement apparatus; (5) cover for the top chamber, to avoid evaporation; (6) tissue; (7) t&on net; (8) lucite insert; (9) rubber gasket; (10) rubber stopper. Bottom: enlarged view of the lucite insert end tissue mounting arrangement; (11) outer and inner rubber ‘ 0 ’ rings; (12) grooves for the adhesive; (13) recess. Openings connecting the chambers are shaded.
The half-chamber B was tested for fluid leaks by using a lucite insert without apertures. The leak thus recorded was negligible. For added safety, this test was repeated often between experiments. Furthermore, the chamber was kept immersed in distilled water when not in use, so as to saturate the plastic matrix with fluid. The glass capillary in the detector of the automatic volumetric measuring unit was regularly resiliconized when necessary. In order to seal the edges of the tissue, high vacuum grease was tested; it proved acceptable at zero pressure but failed to prevent leaks when the pressure gradient pointed from the stroma towards the posterior chamber. Consequently, we chose to use instead tissue glue
124
,J. BROT)W.l\I,J,
i\ST) .J. FIS(‘HUAK(;
(Permabond 102) as a sealing agent. following previous examples in other rpithrlia (Walxrr. 1970; Helman and Miller. 1971). The seal of the chamber and the efficiency of the tissue glue were ascertained by using an isolated frog skin preparation cut into annular shape much like that of the ciliary body-iris preparation. The hydraulic conductance, total electrical resistance and electrical potrnt,ial difference were measured across this preparation and previous data typical of frog skins (cf. Fischbarg and Whittembury, 1977) were readily reproduced with our present technique. (b) The mountingprocedure. The isolated rabbit ciliary body floating in BSG or RP was eased onto a disc-shaped woven Teflon net 15.5 mm in diameter. The ciliary body now resting on the Teflon net was lifted with two forceps and the bottom (anterior) side was carefully dried with ‘tissue paper‘. Liquid glue had been placed shortly beforehand into the circular grooves of the lucite insert. The Teflon net was now lowered onto the center depression in the insert (Fig. 1) and upon contact both tissue and net were glued instantaneously to the insert in the proper position. Since the amount of glue employed was very small, its spread was limited and left free the portion of the net overlying the banana shaped apertures. At this point, two rubber rings were positioned as seen in Fig. 1 and glue was applied between the wall of the lucite insert and the outer ring, and also in the center of the smaller ring. In this way the periphery of the tissue and the pupilar opening were additionally sealed. Bathing solution (either BSG or RP) was added to the epithelial and stromal side of the ciliary body. and the half-chamber B (see Fig. 1) was connected to the fluid sensor system. The procedure of killing the rabbit, dissecting the tissue and mounting it usually lasted about 25-30 min. Experimental protocol For each pressure gradient value tested, flows were usually recorded for periods of about 50 min to allow for stabilization before the rate of fluid movement values were read. The temperature of the bathing solutions was controlled throughout the experiments wit’h a thermistor immersed into the solution on the epithelial side of the tissue. Except for the changes in hydrostatic pressure, the ciliary body was otherwise undisturbed for the usual duration of the experiments (4-7 hr). Rate of $uid movement and hydraulic conductance Experiments were conducted in which fluid movements were recorded as a function of applied hydrostatic pressure. The type of data obtained are exemplified in a typical chart recording shown in Fig. 2. The transient mechanical disturbances originating from pressure changes either on the stromal or on the aqueous sides of the ciliary body result in large initial movement of fluid. These disturbances, which are probably related to tissue displacement, subside later and the rates of fluid movement in good part seem to stabilize 15-30 min after the pressure change. The data from seven experiments in which the tissue was clamped are shown in Fig. 4 (open circles). From the slope of that curve, on the basis of a total epithelial area of 6 cm* (if the infoldings are taken into account), the hydraulic conductance found was 0.131 ,ul/min/mmHg (Lp. A) or 39OOpm/sec (Lp), which is comparatively an extremely large value. The procedure was subsequently changed, as explained above. Thirteen experiments were performed in which fluid movements as a function of hydrostatic pressure were investigated using the ‘edge-damage-free’ technique. The results for each one of these experiments are detailed in Fig. 3. In addition, for comparison, the averages of those data are shown in Fig. 4 (filled circles). On the basis of the slopes of the curve joining the average data, the hydraulic conductivities were as follows: (1) 37 x 10e3 pl/min/mmHg (Lp. A) or 109 km/see (Lp) in the third quadrant, for fluid moving from aqueous to stroma; (2) 7.1 x low3 pl/min/mmHg (Lp. A) or 210 pm/set (Lp) in the first quadrant, for fluid movements from stroma to aqueous and a pressure difference of less than 9 cm H,O. For pressure differences above 9 cm H,O, the hydraulic conductivity increased markedly in one experiment, but this issue was not pursued further. The implications of these results will be dealt with in the Discussion. The Lp units utilized were chosen to facilitate comparison with osmotic permeabilities. The dimensions for the driving force for the flow eliminated by dividing by the molar volume of water. A typical conversion is done as follows : Jv, flow per unit area; Anor AP, unit driving force; 1 mosm/cm3 = 2.6 x 10’ dynes/cm2 Lp (cm/set) = [Jv (cm3/sec/cma)] [(l/U)
(cm3/mosm)] [(l/vu)
(mosm/cm3)].
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050 f
i E E
iii 0 2LL
O-25
0
1
I
I
I
I
I
L
I
2
3
4
5
6
Ttme i hrj
FIG. 2. At each arrow, the hydrostatic pressure difference applied to the tissue was changed for the value given in the graph (in cm H,O). Positive pressures are applied on the stromal side; positive fluid movement goes from stroma to aqueous, in the same direction as aqueous production.
3. Discussion Tissue cyanoacrylate glue has been used previously (cf. Walser, 1970 ; Helman and Miller, 1971) in other epithelial studies without apparent adverse effects but since the use of such adhesive was of great importance to our mounting procedure, we examined the total electrical resistance and the electrical potential difference across our tissue using both high-vacuum grease (Dow Corning, 970V) and tissue glue as sealing agents. The results, which will be detailed elsewhere, were the same with either procedure. In addition, current studies (Brodwall, unpublished) show normal morphology for the ciliary epithelium (i.e. normal cell membrane structures and intracellular organelles) when the tissue is fixed after treatment with tissue glue as described above. Data taken in separate experiments showed that the resistance was about 300 ohm. cm2 and stayed almost constant as a function of applied pressure in the range between - 12 and + 16 cm H,O, indicating that the permeability of the ciliary epithelium is relatively unaffected and that the tissue glue provides an efficient seal. From the present results, at zero pressure difference, the rate of fluid movement is about 62 ,ul/min. This movement could be due to active secretion but this point cannot be ascertained from the present results, since a demonstration of pumping against a pressure gradient (i.e. positive flow in the first quadrant in Fig. 3) would be needed for that purpose. The value above is some three times larger than the figure
-0-5
-
I
- 32
\ .-0’
.’
1
-24
I
I
1
-16 Hydrostatic
-
I
I
I
-8 pressure
I
8 dlfference
I
L
16
(cm ti20)
FIQ. 3. Points represents data obtained as shown in Fig. 2 from each of the 13 experiments in which the tissue was mounted in the chamber shown in Fig. 1. The lines link the data coming from each given experiment.
of 0067 pl/min reported by Green and Griffin (1978) for flow at zero pressure but is still only 6-10 o/oof the in vivo value for aqueous humor formation. One has to conclude that the active transport mechanism which has been accepted to be responsible for the bulk of ciliary secretion (Cole, 1966, 1977; Ill, 1975) is somehow inhibited, totally or partially, when the tissue is mounted in vitro or that, as noted above, in spite of our precautions, a leak is present across the preparation. It is possible that the transport mechanisms may be impaired as an unavoidable consequence of mechanical handling of the tissue. Other possible adverse effects may arise from the present use of pentobarbital to kill the rabbits. Thus, Eakins (1969) reported that the intraocular pressure in cats is reduced by some 30% due to pentobarbital, which was confirmed by Cevario and Macri (1974) for the rhesus monkey. As for yet another important factor, Cole (1977) argued that the in vitro preparations lack the extensive blood supply which might be needed for normal fluid production. We will now focus our attention on the mechanical factors involved. The geometry of this preparation is a complicating factor in that the ciliary epithelium is continued without interruption by the posterior epithelium of the iris and by the epithelial layer of the pars plana, both of which are tight epithelia. Clearly, any damage to the cells that make up those epithelial layers may well introduce a leak through the preparation. In many previous in vitro experimental studies, as has been customary in studies in other epithelia, the ciliary body has been clamped between two half-chambers (e.g. Cole, 1962; Green and Pederson, 1972). Yet, since the chambers are made of hard materials, such a procedure would be expected to produce a leak to some degree. With their pioneering technique, Green and Pederson (1972, 1973) found an Lp value of 4600 + 600 pm/set room temperature, and Green and Griffin (1978) nearly reproduced
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-30
-20
-10 Hydrostatic
IO pressure difference
20
30
(cm H20)
Fm. 4. The different data given are aa follows : (0) average of the data for individual experiments shown in Fig. 3 in which the tissue w&8 mounted with an edge-damage-free technique; (0) average data for fluid movement obtained in experiments in which the tissue was clamped between two lucite chambers : (A) average data reported by Green and Pederson, 1972 (room Temperature).
that value working at 37V. However, a hydraulic conductivity in this order of magnitude is relatively high when compared with those seen in other epithelia, so that the possibility ofepithelial damage has to be considered. The fact that clamping does seem to inflict damage is suggested by the different results presently obtained with the two different techniques. Thus, when the ciliary body is clamped between two half chambers, the Lp values found is of the same order of magnitude as that given by Green and Pederson (cf. Fig. 4). On the other hand, with our chamber for edgedamage-free mounting of the tissue, the Lp value is at least 18 times less than previously reported, or 210 ,um/sec (maximal value) in the pressure difference range from 0 to about + 10 cm H,O (filled circles, first quadrant, Fig. 4). Even this low value is probably an overestimate, since, as can be noted from the same Fig. 4, in the pressure difference range from - 2 to - 31 cm H,O (with the pressure applied on the epithelial side of the tissue) the curve was nearly parallel to the abscissa, which implies an even lower Lp value. It may be speculated that, in this range, the ciliary processes may be flattened and the epithelial cells may be squeezed together, but still the very low slope of the curve is suggestive. UltraJiltration
as a mechanism for aqueous production
Macri and Cevario (1973), when perfusing the ophthalmic artery in enucleated cat eyes, found that vaso-active drugs which changed the arterial pressure also caused changes in aqueous humor production. Hence, in their interpretation, ultrafiltration
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as a function of ciliary capillary pressure was not excluded as an important factor ill ciliary secretion. Similar results had been reported by Best, Blumenthal, Futterman and Galin (1969) in experiments in vivo. On the other hand, Bill (1970) found only small changes in ciliary secretion due to blood pressure variations in monkeys. Given the state of that argument’, the data of Green and Pederson (1972,1973) gained added relevance. Since their results from in vitro experiments showed a near-linear increase in flow and a value of flow similar to that of in vivo secretion was recorded at a hydrostatic pressure difference of 10-15 mmHg (13620.4 cm H,O), they argued that a ciliary capillary pressure of some 53 mmHg (4957 mmHg) could account for secretion. They went on further to propose that ultrafiltration is the major mechanism behind ciliary secretion. However, as the present study shows, the ciliary hydraulic conductance is much lower than previously reported, so that such an argument does not appear tenable. Furthermore, the ciliary capillary pressure in monkeys has been estimated to be about 25 mmHg (Bill, 1973), which, as that author argues, would exclude an important role for ultrafiltration. Landis (1930) estimated the capillary pressure at ocular level at some 17.5 mmHg, so an estimated capillary pressure of 53 mmHg (average) again appears excessive. The present results support Bill’s arguments against ultrafiltration as a major mechanism, and highlight the conclusions in support of the fluid transport hypothesis arising from Cole’s (1960) in vivo experiments, in which it was shown that the secretion of aqueous humor is reduced some 75 “b by the use of metabolic inhibitors. ACKNOWLEDGMENTS This work was supported by U.S.P.H.S. Research Grant EY 01080 to J. F. and in part by Grants to J.B. from The Norwegian Department of Social Affairs and Skipreder T. Wilhelmsens Stiftelse. REFERENCES Best, M., Blumenthal, M., Futterman, M. A. and Galin, H. A. (1969). Critical closure of intraocular blood vessels. Arch. Ophthalmol. 82, 385-92. Bill, A. (1968a). Capillary permeability to and extravascular dynamics of myoglobin, albumin and gammaglobulin the uvea. Acta Physiol. Scud 73, 204-19. Bill, A. (1968b). A method to determine osmotically effective albumin and gammaglobulin concentrations in tissue fluids, its application to the uvea and a note on the effects of capillary ‘leaks’ on tissue fluid dynamics. Acta Physiol. Scand 73, 51 l-22. Bill, A. (1970). The effect of changes in arterial blood pressure on the rate of aqueous humor formation in a primate (Cercopithecus ethiops) Ophthdmol. Res. 1, 193-200. Bill, A. (1973). The role of ciliary blood flow and ultrafiltration in aqueous humor formation. Exp. Eye Res. 16, 281-98. Bill, A. (1975). Blood circulation and fluid dynamics in the eye. Physiol. Rev. 55, 383417. Bito, L. (1977). The physiology and pathophysiology of intraocular fluids. Exp. Eye Res. 25, 273-89. Cameron, E. and Cole, D. F. (1963). Succinic dehydrogenase in the rabbit ciliary epithelium. Exp. Eye Rex 2, 25-7. Ckvario, S. J. and Macri, F. J. (1974). The inhibitory effect of pentobarbital Na on aqueous humor formation. Invest. Ophthalmol. 13, 348-86. Cole, D. F. (1960). Effects of some metabolic inhibitors on the formation of the aqueous humor in rabbits. Br. J. Opht?udmol 44, 739-50. Cole, D. F. (1962). Transport across the isolated ciliary body of ox and rabbit. Br. J. 0phtha1mo1. 46, 577-91. Cole, D. F. (1963). Utilization of carbohydrate metabolites in the rabbit ciliary epithelium. Exp. Eye Res. 2, 284-95.
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Cole, D. F. (1964). Location of ouabain-sensitive adenosine triphosphatase in ciliary epithelium. Exp. Eye Res. 3, 72-5. Cole, D. F. (1966). Aqueous humor formation. Dot. OphthuZmoE. 21, 116-38. Cole, D. F. (1977). Secretion of aqueous humor. Exp. Eye Res. Suppl. 161-76. Davson, H. (1969). The intraocular fluids. In The Eye (Ed. Davson, H.), Academic Press, New York, London. Eakins, K. E. (1969). A comparative study of intraocular pressure and gross outflow facility in the cat eye during anaesthesia. Exp. Eye Res. 8, 10615. Fischbarg, J., Lim, J. J. and Bourguet, J. (1977). Adenosine stimulation of fluid transport across cornea1 endothelium. J. Memb. Biol. 35, 95-l 12. Fischbarg, J. and Whittemburg, G. (1978). The effect of external pH on osmotic permeability, ion and fluid transport across isolated frog skin. J. Physiol. (London) 275, 403-17. Green, K. and Griffin, C. (1978). Adrenergic effects on the isolated rabbit ciliary epithelium. Exp. Eye Res. 27, 143-9. Green, K. and Pederson, J. E. (1972). Contribution of secretion and filtration to aqueous humor formation. Am. J. Physiol. 222, 1218-26. Green, K. and Pederson, J. E. (1973). Aqueous humor formation. Exp. Eye Res. 16, 273-86. Helman, S. I. and Miller, D. A. (1971). In vitro techniques for avoiding edge damage in studies of frog skin. Science 173, 146-8. Landis. C. (1930). Blood pressure changes in deception. J. Comp. Psych&. 10, 437-9. Macri, F. J. and Cevario, S. J. (1973). The induction of aqueous humor formation by the use of Ach +eserine. Invest. OphthaZmoZ. 12, 91&6. Pederson, 0. 0. and Tonjum, A. M. (1975). In vitro studies on peroxidase movement in the epithelium of prostaglandin-treated rabbit ciliary bodies. Acta OptihaEmoZ. 53, 673-84. Uusitalo. R., Palkama, A. and Stjernschantz, J. (1973). An electron microscopical study of the blood-aqueous barrier in the ciliary body and iris of the rabbit. Exp. Eye Res. 17. 49-63. Vegge, T. (1971). An epithelial blood-aqueous barrier to horseradish peroxidase in the ciliary processes of the vervet monkey (Cercopithecus ethiops). 2. Zellforsch 114, 309-20. Walser, M. (1970). Role of edge damage in sodium permeability of toad bladder and a means of avoiding it. Am. J. PhysioZ. 219, 252-5.