In vitro methods

Adhesive force falls rapidly after enucleation or death,^3,4 as is discussed later, and this puts a constraint of time on techniques for measuring adhesiveness in vitro.^5,6 One approach is to peel the retina within a fluid bath while recording the required force with a transducer.³ A faster method is to measure the amount of RPE pigment that remains attached to the retina after separation as the index of adhesiveness.⁷ An eye can be enucleated and small strips of the eyecup prepared within 30 seconds, after which the retina is gently peeled by hand from the RPE. The stronger the adhesive force, the more pigment adheres to the peeled retina.

In vivo methods

Kita et al.⁸ developed the most direct technique for quantifying adhesiveness within the living eye. A small detachment is made in the eye by injecting fluid into the subretinal space through a micropipette, and a second micropipette is inserted simultaneously into the detachment cavity to measure fluid pressure. The pressure that is required to expand the detachment can be converted mathematically, by Laplace’s law, into a value for adhesive force at the margin of the detachment (where the separation is taking place). This technique allows the evaluation in living animals of the effects of drugs and other agents that modify retinal adhesion.^9,10

Magnitude of adhesive force

In vitro measurements, 5–20 minutes after enucleation, have shown that about 25 mg of force is required to peel a 5-mm strip of rabbit retina from the RPE.^3,11,12 Pressure measurements in living eyes have shown an adhesive force in the rabbit of 100–180 dyn/cm.^8,9 Adhesion is stronger in cats and monkeys, which show mean values for adhesive force that are 180% (for cats) and 140% (for monkeys) of that in the rabbits.⁹

Sensitivity to temperature and ionic environment

Retinal adhesiveness drops rapidly postmortem at 37 °C, but remains near control levels for hours at 4 °C^7,13–15 (Fig. 19.1A). These effects of temperature are reversible. Figure 19.1B shows that changing the temperature from 37 °C to 4 °C or vice versa causes retinal adherence in the rabbit¹⁶ to rise or fall, respectively, and repeatedly. This reversibility has also been documented in primate and human tissue.^14,15,17 The mechanism of these reversible temperature effects is still obscure. Temperature could act directly on physicochemical components of adhesion or modulate metabolic systems. Some of the adhesion-enhancing effect of cold temperature may result from sodium pump inhibition and secondary tissue swelling, which would make the interdigitated outer segments and RPE microvilli harder to separate.^3,13 Because cellular swelling is a pathologic event, the actions of cold temperature may not be entirely relevant to normal adhesive processes.

Fig. 19.1 Relationship between retinal adhesiveness and temperature in the rabbit. (A) Cold temperatures slow down the postmortem failure in adhesiveness to the point that firm adhesion is maintained at 4°C for many hours. (Modified with permission from Endo EG, Yao XY, Marmor MF. Pigment adherence as a measure of retinal adhesion: dependence on temperature. Invest Ophthalmol Vis Sci 1988;29:1390–6.) (B) The effects of temperature on retinal adhesion are reversible within minutes, and adhesiveness can be repeatedly strengthened or weakened by cooling or warming the same piece of tissue.

(Modified with permission from Yao XY, Endo EG, Marmor MF. Reversibility of retinal adhesion in the rabbit. Invest Ophthalmol Vis Sci 1989;30:220–4.)

The ionic environment appears to be critical to adhesive strength. In in vitro experiments using tissue from rabbits, humans, and other primates, lowering the pH from 7.4 to 5.5, or removing calcium and magnesium ions from the bathing solution, weakened adhesive force and accelerated the rate at which adhesion falls postmortem^11,13,15,17 (Fig. 19.2A). These changes can be rapidly reversible¹⁶ (Fig. 19.2B), although cold temperature will block or mask the effects of pH and calcium ions. In vivo, the removal of calcium ions from the subretinal space weakens retinal adhesion in rabbits to about 30% of normal.¹⁸ In other words, calcium appears to be a necessary element for the maintenance of normal adhesiveness in the living eye.

Fig. 19.2 Effects of pH and calcium/magnesium ion concentration on retinal adhesiveness in the rabbit. (A) The force required to peel retina from retinal pigment epithelium (RPE) drops within seconds after lowering pH (by injecting 1 mL of 1 mol/L HCl near the tissue) or lowering external Ca/Mg-free solution. The tracings represent a continuous measurement of the peeling force; chemical changes were made at time 0. (Modified with permission from Marmor MF, Maack T. Local environmental factors and retinal adhesion in the rabbit. Exp Eye Res 1982;34:727–33.) (B) The effects of changing pH or Ca/Mg concentration are rapidly reversible. Pigment adherence was used as an index of adhesiveness. Dotted line, tissue maintained in Hanks balanced salt solution. Dashed line, pH changed from 7.4 to 6.0 and back again. Solid line, Hanks solution changed to Ca/Mg-free solution and back again.

(Modified with permission from Yao XY, Endo EG, Marmor MF. Reversibility of retinal adhesion in the rabbit. Invest Ophthalmol Vis Sci 1989;30:220–4.)

Mechanical forces outside the subretinal space

A number of forces play upon the retina from outside the subretinal space, strengthening or weakening retinal adhesion. Most important are fluid and vitreous pressure, not only because they contribute to adhesion, but also because they may cause detachment when altered by pathologic conditions.

Fluid pressure: hydrostatic and osmotic

Fluid is driven passively from vitreous to choroid by both intraocular pressure and the osmotic pressure of the extracellular fluid in the choroid (estimated to be about 12 mmHg in the rabbit,¹⁹ but possibly lower in humans²⁰). However, under normal conditions there appears to be relatively little posterior flow, and most of the fluid that enters the eye as aqueous leaves the eye through anterior drainage channels.^19,21 The posterior route is limited because the retina and RPE^22,23 provide a substantial resistance to water movement, and little fluid can cross these layers under the available heads of pressure. A side-effect of this retinal flow resistance is that the outward movement of fluid acts to push the retina against the RPE (Fig. 19.3). This is one mechanism contributing to normal retinal attachment. The actual pressure difference across retina or RPE is probably small because intraocular pressure is ultimately contained by the sclera, and tissue pressures will therefore equalize to a large degree between the vitreous and choroid. However, Fatt and Shantinath²² have calculated that even a very small pressure difference (only 0.52 × 10⁻³ mmHg) across the retina would generate a force sufficient to keep the retina firmly fixed against the wall of the eye. This low value may explain the fact that no one^14,19 has been able to measure a pressure difference between the vitreous cavity and subretinal space.

Fig. 19.3 Intraocular pressure and retinal flow resistance as a factor in retinal adhesion. (A) Intraocular pressure is evenly distributed within the vitreous cavity so that fluid is continually being pushed through the coats of the eye. (B) Magnified view of the tissue layers in the posterior of the eye, including cortical vitreous. The flow resistance of each layer is indicated on the right; note that the retinal pigment epithelium (RPE) and choroid can also modify fluid movement by active transport and osmotic pressure, respectively.

These physical forces can also work in the other direction and cause retinal detachment. For example, if hyperosmotic fluid is introduced into the vitreous cavity, fluid moving from the choroid into the vitreous will elevate the retina^24,25 (Fig. 19.4). This is an important concern in the evaluation or use of intravitreal drugs that may damage the retina osmotically independently of pharmacologic effects.

Fig. 19.4 Effects of injecting hyperosmolar solution into the vitreous. (A) Monkey eye enucleated after a mid-vitreal hyperosmolar injection. Bullous detachment was present in the posterior pole and peripapillary region. (B) Degree of serous retinal detachment in rabbit eyes 15 minutes after injecting 0.05 mL of different solutions and osmolarities into the vitreous. Incipient detachment, the vitreoretinal interface glistened but did not visibly separate; low detachment, separation occurred without bullous elevation; moderate detachment, the bullae were confined to the posterior pole; large detachment, there was extension near or beyond the equator.

(Reproduced with permission from Marmor MF. Retinal detachment from hyperosmotic intravitreal injection. Invest Ophthalmol Vis Sci 1979;18:1237–44.)

It may seem puzzling that, although most intraocular fluid leaves the eye anteriorly rather than posteriorly, ample animal data^26–30 indicated that the RPE can pump fluid from the subretinal space to choroid at a very high rate (about 0.3 µL/h/mm² of RPE) comparable with that of aqueous secretion. How can the RPE have such enormous power for fluid removal, yet very little fluid leaves the normal eye by a posterior route? The answer probably lies in the flow resistance of the retina. Under normal conditions, given the flow resistance of the tissue and the tiny pressure differential across it,^22,23 water will cross the retina only at a very slow rate. In other words, the tissue resistance is rate-limiting, and most aqueous fluid must leave by the anterior route. Under pathologic conditions of retinal detachment (whether rhegmatogenous or nonrhegmatogenous), fluid is present within the subretinal space and is thus available for transport at a maximal rate. Measurements in humans for the resorption of detachments showed a rate of 0.11 µL/h/mm² of RPE, and the rate may well be higher in eyes without pathology.³¹ This translates into roughly 3.5 mL of fluid per day, which explains why a rhegmatogenous detachment can settle within 24 hours, and emphasizes the power of RPE fluid transport.

The clinical importance of fluid pressure as a factor in preventing retinal detachment is uncertain. It undoubtedly helps to keep the retina in place when the retina is intact, but raising intraocular pressure in rabbits to 38 mmHg or lowering it to 0 mmHg had only a modest effect on the rate of subretinal fluid absorption.³² Furthermore, clinical retinal (as opposed to choroidal) detachment is uncommon in hypotonus eyes, and even a small hole in the retina would theoretically allow fluid to reach the subretinal space and circumvent fluid pressure mechanisms of adhesion. Retinal holes are, in fact, found in about 10% of autopsy eyes without detachment,³³ but many of these holes may not be functionally open because of surrounding pigmentation or because of “tamponade” by the cortical vitreous gel.³⁴

Once the retina has detached and adhesion mechanisms that depend on retina–RPE contact can no longer work, fluid dynamics become much more important, even in the presence of a hole. Hammer³⁵ has calculated that fluid entering a retinal hole over a scleral buckle will, by its flow pattern, create a suction force that pulls the retina backward against the buckle.

Osmotic pressure can be manipulated more easily and may turn out to have therapeutic applications. Both in vitro and in vivo experiments, using rabbits and primates,^9,36,37 have shown that an intravenous injection of mannitol will increase retinal adhesiveness by roughly 50% within 1–2 hours of injection (Fig. 19.5). The magnitude of the effect correlates rather closely with blood osmolality. Mannitol is known to enhance the absorption of fluid out of the subretinal space.³⁸ Some of the adhesive effect may come from fluid absorption “pulling” the retina against the RPE; however, most of the effect probably comes from dehydration of the subretinal space (which enhances the binding properties of the interphotoreceptor matrix (IPM)), because the mannitol effect is still evident in excised tissue where there is no flow toward the choriocapillaris.

Fig. 19.5 Time course of change in retinal adhesive force after intravenous injection of mannitol in rabbits (broken line, 2.5 g/kg) and monkeys (solid lines, 2.0 g/kg).

(Reproduced with permission from Kita M, Marmor MF. Retinal adhesive force in living rabbit, cat, and monkey eyes: normative data and enhancement by mannitol and acetazolamide. Invest Ophthalmol Vis Sci 1992;33:1879–82.)

Vitreous support and other physical aspects of adhesion

The role of the vitreous in creating retinal detachments through traction and contraction is well known. The role of the vitreous in maintaining retinal attachment is less clear. Vitreous gel has a physical structure that may help to keep the retina in place,^34,39 although retinas do not just come off when vitreous detachment or syneresis occurs. A thin cortical layer of vitreous might remain after vitreous detachment or syneresis and serve as a seal or tamponade for retinal holes,³⁴ thus aiding the action of fluid pressure in keeping the retina apposed (Fig. 19.3).

There is other, indirect, evidence for a vitreous role in adhesion. It is very difficult to produce and maintain an experimental rhegmatogenous detachment if the vitreous body remains intact.^24,40 Vitreous removal (either mechanical or with hyaluronidase) presumably weakens the tamponade and the structural qualities of the gel, but it also allows liquid vitreous to reach the subretinal space. The status of the gel may help account for the vastly different incidence of retinal detachment among young individuals, in whom the gel is largely intact, and among older individuals, in whom syneresis and vitreous detachment have occurred. A retinal hole in a young eye is blocked by gel pressure and can seal uneventfully; a hole in an old eye is more likely to allow fluid to enter the subretinal space and cause detachment. On the other hand, the integrity of the vitreous gel seems of little consequence to the formation or persistence of experimental nonrhegmatogenous detachments³² or to the retinal adhesive force measured by peeling,¹¹ so the role of the vitreous gel in attachment may be more one of preventing pathologic fluid access to the subretinal space than one of providing a direct adhesive support or force.

The weight of retina is potentially a factor in attachment and detachment, as influenced by gravity and ocular movement. Once a detachment has occurred, the influence of gravity is well known, because appropriate positioning of the patient can be enormously effective in inducing retina to settle. However, it is hard to envision retinal weight as a major factor in retinal attachment under normal conditions,^27,32 because our upright posture and eye movements would dictate that benefits and adverse effects be simultaneously present in different parts of the eye.

Mechanical interdigitation

RPE microvilli wrap closely around the tips of the outer segments (Fig. 19.6), and this connection is strong enough to allow for daily phagocytosis of outer-segment fragments as the photoreceptors renew their disc material.^1,41,42 However, as a factor in adhesion, interdigitation seems of variable importance. The RPE microvilli are subject to continuous cellular remodeling during the outer-segment renewal cycle. After surgical repair of a retinal detachment, reattachment takes place rather promptly, before the outer segments have regenerated and microvillous connections have been re-established.^43,44 Interdigitation begins to develop within 3 days of reattachment, but retinal adhesiveness does not return to normal until 5–6 weeks after reapposition of experimentally detached retina in the rabbit⁴⁵ (Fig. 19.7).

Fig. 19.6 Interdigitation between photoreceptor outer segments and the microvilli of the retinal pigment epithelium (RPE). Note that the microvilli actually indent the outer segments during phagocytosis of “outdated” discs. The cone outer segments are shorter, and the microvilli form long pedicles in order to reach them.

Fig. 19.7 Time course of the recovery of adhesive strength after separated retina becomes reattached. Experimental detachments were made in rabbit eyes by injecting a balanced salt solution into the subretinal space; the fluid absorbed within several hours, and at intervals after settling of the detachment, a comparison was made between the force required to peel the retina from normal and reattached areas of retinal pigment epithelium (RPE).

(Reproduced with permission from Yoon YH, Marmor MF. Rapid enhancement of retinal adhesion by laser photocoagulation. Ophthalmology 1988;95:1385–8. Copyright 1988, with permission from the American Academy of Ophthalmology.)

The mechanisms by which interdigitation could produce adhesion are not known. During outer-segment phagocytosis, the microvilli indent the outer segments and must physically impede attempts to withdraw them. Close ensheathment might also provide a frictional resistance to withdrawal, just as a finger is hard to pull from a narrow tube. There may be electrostatic forces that oppose separation of the membranes.⁴⁶ The magnitude of all of these effects will also depend on other factors, such as the composition of the intervening matrix and capability of the RPE microvilli for motility and remodeling.

Interphotoreceptor matrix properties

The IPM is a viscous material composed largely of proteins, glycoproteins, and proteoglycans^47,48 but containing a substantial concentration of glycosaminoglycans.⁴⁹ The idea that IPM might serve as a viscous glue was proposed many years ago by Berman,⁵⁰ but current evidence indicates the IPM is not just a layer of glue but has component structures that play a role in the adhesive process. For example, the cones are surrounded by a specialized sheath of matrix^51,52 that can be recognized histochemically by the binding of peanut agglutinin (PNA) (Fig. 19.8A). These cone matrix sheaths remain attached to both RPE and photoreceptor cells when the neural retina is peeled from the RPE.^48,51,53 If fresh primate or human retina is peeled from the RPE, the matrix material will stretch markedly before breaking (Fig. 19.8B), suggesting a rather strong bonding at both the outer-segment and RPE interfaces.^17,54 The evidence for a bonding function of the IPM is strongest with respect to the cone matrix sheaths, but the structural material that surrounds the rods may play a similar role. It can be recognized histochemically by staining with wheatgerm agglutinin (WGA) or other lectins,^55,56 and stretching of WGA-staining material may be observed on peeling freshly enucleated eyecup material.^17,54,57 The role of newly recognized matrix components such as galectins and IPM proteoglycans remains to be determined.^58,59

Fig. 19.8 Fluorescence micrographs of monkey retina showing cone sheaths of the interphotoreceptor matrix (IPM) stained with peanut agglutinin. Photoreceptors are above and retinal pigment epithelium (RPE) below. (A) Normal retina. The cone sheaths are short and thick. (B) Partially peeled retina. The cone sheaths have been stretched markedly between the photoreceptors and the RPE surface, showing the strength of IPM bonding to both sides (original magnification ×130).

(Reproduced with permission from Hageman GS, Marmor MF, Yao XY, et al. The interphotoreceptor matrix mediates primate retinal adhesion. Arch Ophthalmol 1995;113:655–60.

The ability of cone or rod matrix sheaths to serve as a structural bond between retina and RPE may well depend on the presence of specific receptors that bind IPM components to the cell membranes. (“Cell adhesion molecules” represent a group of substances that mediate adhesion between individual cells or between cells and substrates.⁶⁰) Experiments have shown that extracted IPM material does not automatically stick cells to one another,⁶¹ but adhesion is unlikely unless specific receptors are present to bind the macromolecular substances. Several receptor systems, including sites for fibronectin, integrins, and mannose, have been proposed and disputed as being involved with retinal adhesion.^48,62–65 There is functional evidence that inhibition of chondroitin sulfate proteoglycan synthesis with xylosides causes spontaneous detachments in primates.^48,66

Other types of evidence support the concept that IPM contributes significantly to retinal adhesion. Physical factors that affect adhesiveness, such as temperature, pH, and calcium concentration, are known to be modulators of the physicochemical properties of IPM macromolecules and may also affect the bonding of large-molecular species at receptoral sites.^67,68 There is also a marked loss of retinal adhesiveness when the subretinal space is exposed to enzymes that degrade matrix components,^15,69 such as chondroitinase ABC (which degrades chondroitin sulfate⁷⁰) or neuraminidase (which breaks sialic acid bonds⁷¹). These enzymes, given intravitreally or into the subretinal space itself, weaken adhesiveness markedly in correlation with cytochemical evidence of damage to the IPM (Figs 19.9 and 19.10). It is unknown whether the effects of these enzymes on adhesiveness are a result of changes in viscosity or damage to the structural elements of the IPM. However, adhesiveness returns to normal roughly 3 weeks after exposure of an eye to neuraminidase or chondroitinase ABC, and the recovery of adhesiveness correlates closely with the recovery of normal PNA-binding properties in the IPM.⁷²

Fig. 19.9 Fluorescence light micrographs depicting binding of peanut agglutinin (PNA) to rabbit retinas in the region between the retinal pigment epithelium (RPE) and outer plexiform layer (OPL). (A) In a normal eye, PNA bound intensely and specifically to cone matrix sheaths (arrows). (B) After injection of neuraminidase, PNA binds throughout the interphotoreceptor matrix, with binding in the region of cones being diminished (note gaps indicated by arrows). (C) After intravitreal injection of testicular hyaluronidase, cone matrix sheaths were disrupted, especially at their apices (asterisks), and shallow separations between the photoreceptors and RPE were commonly observed.

(Reproduced with permission from Yao XY, Hageman GS, Marmor MF. Retinal adhesiveness is weakened by enzymatic modification of the interphotoreceptor matrix in vivo. Invest Ophthalmol Vis Sci 1990;31:2051–8.)

Fig. 19.10 Peeled whole-mount retinas after subretinal injection of enzymes. The areas of strong adhesion show retinal pigment epithelium (RPE) pigment adherent to the retina after peeling; the areas of weak adhesion are clear of pigment. (A) Three days after injection of control (Hanks) solution, adhesion is normal (strong) except at the injection site. One (B), 2 (C), and 3 (D) days after testicular hyaluronidase, there was a progressive increase in the area of weakened adhesion beyond the injection site. A similar progression of adhesive loss was observed after neuraminidase.

(Reproduced with permission from Yao XY, Hageman GS, Marmor MF. Retinal adhesiveness is weakened by enzymatic modification of the interphotoreceptor matrix in vivo. Invest Ophthalmol Vis Sci 1990;31:2051–8.)

Experiments have shown that the chemical nature of the IPM may change with light and dark adaptation.⁷³ To the extent that photic effects might modify the viscosity or bonding properties of the IPM, light could be a modulating factor of retinal adhesion. However, experimental data on adhesion in the light and dark have been contradictory and of uncertain significance.^11,74,75

Subcellular components and mobility

Subcellular components of the RPE, such as microtubules and microfilaments, will be relevant to retinal adhesion to the extent that they influence remodeling and movement of the RPE microvilli.⁷⁶ Actin filaments control melanin movement in the amphibian RPE¹¹ and are present in mammalian RPE,^77,78 but a movement of melanin granules has never been observed in mammals. A few attempts have been made to alter adhesion by inhibiting subcellular components of the RPE. Cytochalasin B, which blocks microfilaments, was found to have no effect on adhesion when the drug was presented to excised tissue briefly after enucleation.¹² However, injecting cytochalasin into the vitreous 4–72 hours before enucleation caused a 70–90% reduction in pigment adherence as a measure of adhesiveness.⁷⁹

When retina detaches, the surface morphology of the RPE changes literally within minutes.²³ The evolution of these changes can be altered by both colchicine (which dissembles microtubules) and cytochalasin,⁸⁰ suggesting that microtubules and microfilaments are important to apical RPE morphology. Actin filament contraction and subcellular motility are known to require calcium,⁸¹ the absence of which is severely detrimental to retinal adhesion. However, we do not know whether the action of calcium in weakening adhesiveness relates to this mechanism or to effects that calcium may have on transport systems and matrix-binding properties.

Metabolic factors

The question of whether or why metabolic activity is necessary or even contributory to retinal adhesion is extremely important. From a physiologic standpoint, we would like to know the interaction between RPE transport, IPM properties, and the passive physical forces that impinge on the subretinal space. From a clinical standpoint, if adhesion is purely passive (i.e., dependent on pressure, or “glue”), therapy for clinical detachments would be primarily mechanical, whereas if active metabolism is necessary for adhesion, physicians may have a very different range of therapeutic options.

Critical dependence on oxygen

It was noted previously that adhesiveness in living rabbit eyes falls severely within a few minutes of death⁵ (Fig. 19.1A). Furthermore, just 1 minute of ocular ischemia in living rabbits weakens adhesion dramatically, whereas restoration of circulation restores adhesiveness.^82,83 The postmortem adhesive loss appears to be slower in primates and humans, and under similar in vitro conditions, at least a moderate adhesive force was measurable for 30 minutes or more after enucleation of primate and human tissue^15,17,83 (Fig. 19.11A).

Fig. 19.11 Dependence of retinal adhesion on metabolic activity. (A) Scanning electron micrograph of the photoreceptor surface of human retina peeled from the retinal pigment epithelium (RPE) within 4 minutes of enucleation. The outer segments are largely covered by apical fragments of RPE cells, indicating that the strength of retinal adhesion in living or fresh tissue is greater than that of the RPE cell membrane. Retinas peeled 1 hour after enucleation showed little or no adherence of RPE fragments. Bar = 10 µm. (Reproduced with permission from Marmor MF, Yao XY. The metabolic dependency of retinal adhesion in rabbit and primate. Arch Ophthalmol 1995;113:232–8.) (B) Effect of oxygenation on the adhesiveness of rabbit retina in vitro. Left, Postmortem adhesive failure was delayed markedly by the incubation of tissue in an oxygenated rather than static bath. Right, Switching tissue from a static bath to an oxygenated one caused a prompt recovery of adhesive strength.

(Modified with permission from Marmor MF, Yao XY. The metabolic dependency of retinal adhesion in rabbit and primate. Arch Ophthalmol 1995;113:232–8.)

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