Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

BCC is an actinic lesion and is more common in fairskinned individuals on sun-exposed parts of the body. It is most common on the lower lid, followed by the medial canthus, upper lid, and lateral canthus. The tumor is classically a firm, nodular lesion with a central ulcer and pearly, vascular border (Figure 9). It may also present as an irritating, erythematous patch or rarely be entirely subcutaneous. Lesions of the medial canthus have a propensity for deep invasion into the eye socket. BCC only rarely metastasizes and generally grows very slowly. The preferred treatment is complete excision with frozen section control or by Mohs micrographic surgery usually performed by a specially trained dermatologist. It provides the best cure rate (98–99%) while saving as much normal tissue as possible. Excision is followed by reconstruction of the eyelid defect or occasionally by spontaneous healing if the defect is small or does not involve the lid margin. Radiation therapy is an alternative to surgery but not as effective due to the relatively high recurrence rate (5–20%). Recurrence is also difficult to detect after radiation due to tissue alteration from the treatment. Cryotherapy is sometimes used for small lesions but has similar problems with a relatively high recurrence rate (20–30%) and depigmentation and atrophy of treated tissue.

Squamous cell carcinoma (SCC) accounts for about 5% of periocular malignancies. It too is an actinic lesion, being more common on sun-exposed areas of fair-skinned individuals. Again, the lower lid is the most common location. It most frequently presents as an erythematous, thickened patch with erosion of the involved tissue (Figure 10). A nodular subtype is occasionally observed. It may arise from preexisting actinic keratoses, which undergoes malignant transformation 20% of the time. SCC can metastasize through the regional lymphatics or spread along involved sensory nerves. Mohs removal and reconstruction are the preferred therapy. Radiation

Figure 10 Squamous cell carcinoma of the left medial canthus.

therapy is much less effective in SCC and thus is used only for palliation of surgically unresectable tumors. Topical therapy with 5-fluorouracil or imiquimod is a frequent treatment of the premalignant actinic keratosis.

Sebaceous cell carcinoma accounts for about 5% of periocular malignancies but is increasing in frequency, which may be due in part to better pathological diagnosis. It arises from the meibomian glands, the glands of Zeiss, and sebaceous glands of the caruncle. It presents as a nodular lesion, which is often mistaken for a chalazion or a diffuse intraepithelial pattern that looks like a chronic conjunctivitis (pagetoid spread) (Figure 11). Because of this, the diagnosis is often delayed until biopsied. It is capable of both regional lymphatic and vascular spread. It is considered deadlier than SCC. Mohs microsurgery may be effective in tumor removal if the tumor is nodular, but pagetoid spread and skip lesions often necessitate wide sampling of the bulbar and palpebral conjunctiva (map biopsy). If diffusely spread, orbital exenteration is

usually required. Sebaceous cell carcinoma is relatively radio resistant and responds poorly to chemotherapy.

Malignant melanoma accounts for less than 5% of periocular malignancies, but it too is increasing in frequency (Figure 12). It too is an actinic lesion but other less well-defined causes play a role. It arises spontaneously usually as a nodule or in an existing nevus or area of lentigo maligna (intraepithelial tumor). It too is capable of spread through the regional lymphatics and the blood stream. Surgical excision is the treatment of choice, but requires permanent section histology to adequately assess tumor margins. Because of this, the resection of the tumor is often spread over several days (Slow Mohs) followed by reconstruction when margins are clear.

Benign Eyelid Tumors

A variety of benign lesions are found on the lids and periocular skin. The vast majority are of minimal functional significance, but patients frequently request removal for cosmetic reasons. The most common are nevi, inclusion and glandular cysts, seborrheic keratoses, verruca, skin tags, and benign glandular tumors. Simple excision can usually be carried out in the office under local anesthesia.

Inflammatory and Infectious Disorders

of the Lids

Chalazion is a lipogranuloma of one of the meibomian glands of the tarsal plate. They arise relatively rapidly over a period of a few days, often with inflammation and discomfort. They can progress to form a chronic peasized, firm nodule in the lid (Figure 13). The initial treatment is to hot compress the involved lid frequently in the first few days in hopes of opening the obstructed gland. If not effective many will resolve spontaneously over the next several weeks to few months. Patients often

Figure 13 Chalazion of the right upper lid.

request removal for cosmetic reasons. This can usually be done in the office with local anesthesia. An alternative is to inject the chalazion with steroid, which is effective about 50% of the time.

Hordeolum is a staphylococcal infection of one of the sebaceous glands of the lid. It presents as an acutely swollen, erythematous, painful nodule on the lid margin (external hordeolum) or on the palpebral conjunctiva (internal hordeolum). Hot compress, topical antibiotics, and surgical drainage are effective. Oral antibiotics may be needed if cellulitis occurs.

Blepharitis is the most common inflammatory disorder of the eyelids characterized by redness, swelling, and irritation with visible crusting. It is a chronic condition characterized by intermittent exacerbations. There are three subtypes, but many patients show combinations of all three. In staphylococcal blepharitis, chronic colonization of the lid margin leads to inflammation from the bacterial toxins and antigens. Slit-lamp examination

reveals characteristic white collarettes around the base of the lashes. In seborrheic blepharitis, there are greasy scales (scurf) found on the lashes and often associated seborrheic dermatitis of the face and scalp. The third form, posterior blepharits (posterior lid margin disease, meibomian gland dysfunction, and meibomianits) is characterized by a change in the normal clear meibomian gland secretion to a thick, cloudy to yellow, oily discharge. Posterior blepharitis is often associated with acne rosacea and chalazia. All forms of blepharitis lead to chronic redness of the conjunctiva, sometimes with papillary hypertrophy. The tear film is often unstable as manifested by a rapid tear break-up time. Dry eye is a frequent association. In addition to the above findings, slit-lamp examination may reveal punctate epithelial erosions (PEEs) of the cornea and marginal corneal erosions due to staphylococcal hypersensitivity. There may be small, rounded domes on the meibomian orifices, manifestations of the thick, inspissated secretions. Digital pressure on the lid may cause the meibomian glands to express material that can, in severe cases, have a cheesy consistency.

Treatment consists of warm compresses to thin the meibomian secretions and eyelid scrubs with baby

shampoo or commercially available products to clean the lid margin and express the meibomian glands. Topical antibiotic drops or ointment reduces the bacterial colonization of the lid margin and topical steroids help to control the inflammation. Oral, low-dose tetracycline (50 mg doxycycline per day) is helpful in reducing the meibomian gland discharge and normalizing the pH of the tear film. Oral erythromycin can be used if the patient is intolerant to tetracyclines, pregnant, nursing, or under the age of 12.

See also: Eyelid Anatomy and the Pathophysiology of Blinking; Lacrimal Gland Overview; Tear Drainage.

Further Reading

Jordan, D. R. and Anderson, R. A. (2000). Surgical Anatomy of the Ocular Adnexa: A Clinical Approach. San Francisco, CA: American Academy of Ophthalmology.

Stewart, W. B. (2000). Surgery of the Eyelid, Orbit, and Lacrimal System.

San Francisco, CA: American Academy of Ophthalmology.

Glossary

Short-circuit current – The short-circuit current (Isc) across tissues isolated within an Ussing chamber is defined as the charge flow per time, per the crosssectional area of the epithelium that is exposed to the bathing solutions, when the tissue is short-circuited by clamping the transepithelial voltage (PDt) to zero with an external circuit. The Isc is a current that circulates through the tissue and the external circuit thereby completing a closed loop. Because of this, the Isc enters the tissue across one surface and leaves across the other. Monitoring the Isc provides a continuous measure of the net charged flow of current across the transcellular pathways of the tissue. There is no net flow across the paracellular pathways in the short-circuited condition if the tissue is bathed bilaterally with solutions containing identical ionic concentrations. Adding drugs that affect the ionic channels or electrogenic elements within the membranes of the epithelium will affect the magnitude of the Isc.

Transepithelial resistance – An epithelium can be considered to be comprised of an arrangement of resistance elements or resistors. This arrangement is most simply modeled in a multilayered epithelium as two resistors in series, namely Ra (the resistance of the apical membrane) and Rb (the resistance of the basolateral membrane). These resistances are shunted by a parallel resistor, Rshunt, which is the cumulative resistance of the paracellular pathways. As such, transepithelial resistance (Rt) is defined as follows:

Rt ¼ ðRa þ Rb Þ Rshunt

Ra þ Rb þ Rshunt

In the conjunctiva, Rshunt tends to be lower than the transcellular pathway. This leads to small measured changes in Rt when drugs that selectively affect either Ra or Rb are added. For example, potassium channel blockers should selectively increase Rb, but the effect on the measured Rt parameter is relatively smaller in the conjunctiva than in the corneal epithelium, which is a tight epithelium, that is, a tissue with a high Rshunt.

Transepithelial voltage – Epithelial cells transport ions and thereby generate a transepithelial voltage (PDt). The generation of PDt requires (i) an asymmetric distribution of ion channels and

electrogenic transporters on the apical and basolateral sides of the tissue, and (ii) the presence of tight junction proteins between adjacent cells in the superficial epithelium to impede the flow of ions along the paracellular pathways.

Unidirectional fluxes – Across epithelia isolated in Ussing-type chambers, a unidirectional flux of a substance (e.g., a radiolabeled electrolyte or water molecule) is its rate of translocation across the tissue from the bathing solution within one hemichamber to the contralateral bath, disregarding any counterbalancing flux in the opposite direction.

In practice, unidirectional fluxes are measured in the apical-to-basolateral direction, and again in the basolateral-to-apical direction. A difference in the magnitude of these two unidirectional fluxes provides a measure of the net flux, for example, there is a net chloride flux across the conjunctiva in the basolateral-to-apical direction.

Ussing chamber – A device designed by Hans Ussing in 1951 to originally study vectorial ion transport across the frog skin. It has since been modified (i.e., an Ussing-type arrangement) by many investigators to characterize electrolyte transport across various epithelial tissues including epithelia of the eye. This approach has significantly contributed to our understanding of how electrolytes are transported. The Ussing-type methodology entails two aspects. One is the chamber itself, which is constructed to hold the dimensions of a particular tissue, and enable it to be bathed bilaterally. The second aspect is the external electrical circuitry, which can be designed to measure transepithelial voltage, resistance, and current. The effects of pharmacological agents on the electrical parameters generated by the epithelium can be studied by applying test compounds unilaterally to either the apical-side or basolateral-side baths.

Introduction

The conjunctiva and the corneal epithelium together form the ocular surface. The conjunctiva (from late Latin, feminine of conjunctivus, or conjoining) is in essence a connection (conjunction) between the eyelids, the sclera of the eyeball, and the cornea. It lines the posterior surface of the

eyelids (the palpebral conjunctiva) and the exposed, anterior aspect of the globe (the bulbar conjunctiva). The latter is loosely attached to the sclera of the eyeball, and translucent, thereby exposing the so-called ‘white of the eye’; it merges with the corneal epithelium at the limbus, which constitutes the edge of the cornea. The palpebral conjunctiva is tightly adherent to the eyelid.

The space, lined by the conjunctiva, between the lids and the globe is known as the conjunctival sac. The bottom of the sac, which is unattached to the eyelids or to the eyeball, is known as the fornix, forniceal region, or conjunctival fold. The untethered nature of the fornix allows the eyeball to move freely. The conjunctival sac varies in size depending upon the degree to which the lids are open. The depths of the unextended sac in humans are about 14–16 mm superiorly and 9–11 mm inferiorly. The total surface area of the conjunctiva is about 9 and 17 times larger than that of the cornea in rabbits and humans, respectively. The lacrimal glands, which secrete tears, open into the superior fornix.

The palpebral conjunctiva contains the openings of the lacrimal canaliculi, which allow tears within the conjunctival sac to drain into the nasal cavity. The vasculature of the palpebral conjunctiva is clearly visible within the tissue upon examining the posterior surface of the eyelid. In contrast, the bulbar conjunctiva is normally colorless, unless its vessels are dilated as a result of inflammation (conjunctivitis). The conjunctival vessels arise from a peripheral palpebral arcade and from the anterior ciliary arteries. Blood comes mostly from the orbit, but anastomoses with the facial system.

Conjunctival innervation is mediated by the ophthalmic division of the trigeminal nerve. The conjunctiva is extensively innervated with adrenergic, cholinergic, and peptidergic fibers identified in various species. In general, the largest numbers of nerves present are sympathetic, with fewer parasympathetic and sensory nerves. The parasympathetic nerves contain the neurotransmitters acetylcholine and vasoactive intestinal peptide; the sympathetic nerves, norepinephrine and neuropeptide Y; and the sensory nerves, substance P and calcitonin-gene-related peptide.

The major roles of the conjunctiva are:

(1)to contribute to tear production by secreting electrolytes and fluid;

(2)to modify the composition of the tear film by secreting mucins and lipids, and absorbing various organic compounds found in tears; and

(3)to contribute to the resistance of the eye to infection by providing protection against microorganisms.

The conjunctiva is comprised of an epithelium and an underlying stroma. The epithelium is embryologically related to, and anatomically continuous with, the epithelium of the upper airway. Within the conjunctival epithelium are goblet cells, which are specialized epithelial cells that function as unicellular mucus glands. The goblet cells secrete the

mucin component of the tear film, which consists of three layers, each of which is secreted by different cells. Secreted mucins constitute the inner layer of the tear film and serve as wetting agents that keep the apical, hydrophobic aspects of the corneal and conjunctival epithelia hydrated. The middle, aqueous layer of the tear film contains water, electrolytes, immunoglobulin A (IgA), glucose, and proteins (including antibacterial enzymes). This layer is primarily secreted by the main and accessory lacrimal glands; the latter glands of Krause and Wolfring flank the main lacrimal duct near the superior fornix. It is possible that the conjunctiva also contributes to this layer under basal conditions when the lacrimal glands are not stimulated. The outer, lipid layer of the tear film contains a fat mixture that is secreted by the meibomian glands that line the eyelids. This layer functions to reduce evaporation of the aqueous layer.

Underlying the conjunctival epithelium, the connective tissue contains blood vessels, nerves, conjunctival glands, mast cells, and leukocytes including macrophages. The latter, defensive cells can be recruited in large numbers to an injury site on the ocular surface due to disruption of the barrier properties of the epithelium. They may also release paracrine-signaling agents that affect the transport properties of the epithelium, and certain leukocyte populations can also serve as antigen-presenting cells.

Recent work has characterized the active transport properties of the conjunctival epithelium. The epithelium is capable of transporting fluid as a consequence of a sufficiently high water permeability bestowed by endogenous water channels (aquaporins) and transepithelial solute movement due to active transport mechanisms. This article includes a synopsis of the current understanding of the electrolyte and fluid transport across the conjunctiva.

Conjunctival Epithelium

A primary role of all epithelial tissues, including those in the conjunctiva, is the absorption and/or secretion of fluid. In brief, two main elements are necessary for fluid movement across a membrane or a set of membranes: (1) the driving force represented by an osmotic gradient (or hydrostatic pressure), and (2) a water pathway represented by water channels (aquaporins) and the lipid bilayer. Thus, all fluid secretion or reabsorption is a consequence of the osmotic gradient created by active electrolyte transport, with the direction of fluid movement identical to that of the net transepithelial solute transport. To date, extensive, functional characterizations of the electrolyte transport properties of the conjunctival epithelium have been done only on the isolated rabbit conjunctiva. The epithelium of this species exhibits mechanisms that simultaneously mediate Na+ absorption and Cl secretion. The relative proportions of these oppositely directed functions varies considerably from one individual rabbit conjunctival

specimen to another, for reasons that are unknown, but in general Cl transport is predominant. On average, the ratio of Cl secretion to Na+ absorption is about 1.5 to 1, suggesting that the rabbit epithelium can function primarily as a chloride-secreting epithelium potentially capable of moving fluid into the conjunctival sac. However, it must also be noted that a small proportion of conjunctival specimens exhibited a Na+ absorptive rate larger than the rate of Cl secretion.

Morphologically, the epithelia of the rabbit bulbar, forniceal, and palpebral regions have distinct appearances (Figure 1). The bulbar epithelium appears columnar and thinner than the other sections with goblet cells present. It is as thick as two to three cell layers and packed irregularly. In the forniceal area, the number of cell layers increases to three or four with a greater abundance of goblet cells. From this region to the lid margin, a transition within the palpebral epithelium is evident with the number of goblet cells diminishing, and the epithelial

cells becoming more stratified. Species differences in morphology among mammalian conjunctivae have been described.

Excluding variations in the number of goblet cells, the epithelial cells within each conjunctival region appear homogeneous, which suggests that both absorptive and secretory activities coexist within an individual epithelial cell. If so, the conjunctival epithelium exhibits a rare property among epithelia in that the transport functions for Na+ absorption and Cl secretion are not segregated in distinct cell types, and the transport rates for these oppositely directed functions are nearly equivalent in isolated conjunctivae under in vitro conditions.

Regardless of the normal, physiologic direction of fluid movement across the human conjunctiva, it is clear that inhibiting reabsorption and/or stimulating secretion may have a beneficial effect by increasing the aqueous layer of the tear film in individuals with a tear-fluid deficit due to various lacrimal gland deficiencies.

Bioelectric Studies on the Isolated Rabbit Conjunctiva

The ionic transport systems potentially mediating the absorptive and/or secretory functions of an epithelium can be efficiently characterized by isolating the epithelium under an Ussing-type arrangement. With this method, a flat piece of an epithelium is dissected with some of its underlying connective tissue or stroma maintained in place to provide structural support. The thickness of the entire dissected preparation is usually about 1 mm, of which about 0.05 mm represents the epithelial cellular compartment. The isolated tissue is then positioned between two hemichambers, which when closed together result in the tissue serving as a partition separating the cavities of the two hemichambers. In this situation, the apical surface of the epithelium interfaces with the cavity of one hemichamber, while the basal surface attached to the underlying stroma interfaces with the contralateral chamber. Each hemichamber is then filled with a physiological solution to simultaneously bathe the two surfaces of the in vitro preparation.

The rabbit conjunctiva serves as a fairly good specimen for this approach given its relatively large surface area, as well as the fact that the conjunctival sac can be removed nearly intact as a cylinder and then cut longitudinally to convert it to a flat epithelium that is easily mounted between Ussing-type hemichambers. Typically, about 0.5 cm2 of cross-sectional area of tissue is bilaterally exposed to the bathing solutions within the chambers. A negative aspect of this approach with the conjunctiva is that the epithelium seems to be relatively frail (when compared to the corneal epithelium) as its integrity deteriorates following several hours of isolation within the chambers. Nevertheless, useful electrophysiological experiments can be designed and informative data are obtainable.

Upon isolation of an epithelial preparation, such as the conjunctiva, within the divided chambers, a potential

difference (PD) develops across the tissue. The PD is a consequence of the active transport of electrolytes by the epithelium, which spends metabolic energy to maintain ionic gradients between the cellular compartment and the extracellular bath. An epithelium will exhibit a negative intracellular voltage with respect to both the apical-side bathing solution and the stromal-side (basolateral) solution. Typically, the positive voltage of the stromal-side bath (PDs) with respect to the cellular compartment occurs because of the electrogenic Na+–K+ ATPase, which extrudes three Na+ ions for two imported K+ ions, and the fact that the cellular K+ ion concentration is above equilibrium, so that K+ will constantly efflux through K+ channels toward the stromal (basolateral) bath (Figure 2 shows an overview of the major transport elements found in the conjunctival epithelium). The Na+–K+ ATPase functions incessantly to maintain cellular K+ above equilibrium. The positive voltage of the apical-side bath (PDa) with respect to the cellular compartment is less positive than PDs. As such, a transepithelial PD (PDt) exists, which equals the difference between the PDs across the respective contralateral surfaces of the epithelium (PDt = PDa – PDs; and has a negative sign with PDs taken as reference). PDa is less positive with respect to the cellular compartment than is PDs because of the efflux of Cl via channels in the apical domain toward the apical-side bath, and in the case of the conjunctiva, an influx of Na+ also occurs via electrogenic transporters that are in the apical membrane (Figure 2).

PDt can only exist if tight junctions are present in the epithelium, which is the case in the conjunctiva. These elements are located between the lateral membranes of the most superficial epithelial cells and form a resistance barrier that impedes the diffusion of ions from the contralateral bathing solutions through the paracellular pathways between the epithelial cells. Without the presence of tight junctions, ionic flows in the paracellular pathway would

short-circuit PDt, because PDa and PDs would still exist and result in a net movement of anions from the apical-side bath to the stromal-side bath through the paracellular pathways, and a net movement of cations in the opposite direction through the paracellular pathways.

The Ussing-type chambers used to isolate the conjunctiva have ports for inserting electrodes into the bathing solutions to directly measure PDt. In addition, there are ports for current-sending electrodes. These are used to short-circuit PDt with an external circuit connected to an automatic voltage clamp that constantly maintains PDt ¼ 0 mV. The amount of current needed for this is continuously recorded and known as the short-circuit current (Isc). The Isc represents a real-time measure of the net transepithelial movement of electrolytes across the transcellular pathways of the tissue due to metabolically dependent active transport. As PDt is maintained at 0 mV, there is no net movement of electrolytes through the paracellular pathways, even if tight junctions were, in principle, not present.

Transepithelial electrical resistance (Rt) can be determined by applying Ohm’s law to the measured PDt (under open-circuit conditions) and the measured Isc (under short-circuited conditions), or by measuring the amount of current necessary to offset the short-circuited condition by a few mV for a few seconds. Either approach gives identical measures of Rt with the conjunctiva.

As alluded to above, the integrity of the conjunctival epithelium appears to degenerate following a prolonged period in the chamber. This is observed as a spontaneous, gradual decline in Rt. It appears that there is a loss in paracellular resistance (i.e., tight-junction structure may change with time in vitro) since the Rt decline occurs in the presence of a steady Isc. As noted, under the shortcircuited conditions, increases in paracellular ion movement do not result in a net flow across this pathway given the absence of a PD across the epithelium and identical electrolyte concentrations on each side of the preparation. As such, the Isc continues to measure net transcellular flow of electrolytes in experiments with symmetrical solutions. However, changes in conjunctival Rt in response to the additions of various drugs frequently underestimate changes in membrane resistance. This is because transcellular resistance is proportionally larger than paracellular resistance, which means that large changes in transcellular resistance are recorded as smaller changes when measuring conjunctival Rt.

Nonetheless, the initial measurements of conjunctival Rt upon the isolation of fresh preparations within the divided chambers provide a good indication of the barrier properties of the epithelium. This is because the electrical resistances of both the cell membranes as well as the paracellular pathways contribute to the Rt measurement. The paracellular pathways of the in situ conjunctiva at the ocular surface allow for the passive diffusion of

hydrophilic solutes across the conjunctiva. Passive paracellular transport of electrolytes across the conjunctiva in vivo, which is analogous to the open-circuit situation in vitro, occurs because of gradients created by the transcellular mechanisms. In addition, cell-impermeable, hydrophilic solutes applied to the conjunctival sac may diffuse across the epithelium through the paracellular route. The transepithelial permeability of such solutes decreases with increased solute size. Tight junctions located at the apical-most aspect of an epithelium create the major barrier for the movement of solutes across all epithelia including the conjunctiva. However, the paracellular route varies considerably among epithelia in terms of permeability to solutes and electrical resistance. Rt measurements can range at least 1000-fold between highly resistant and so-called leaky epithelia. In some tissues, electron microscopy studies have correlated the ultrastructure of the tight junctions with the measured Rt values obtained in vitro. In general, the number of tight-junction strands along the apical-basal axis is proportional to the junctional resistance. Rt values from freshly isolated rabbit conjunctival epithelia are in the range of 1–2 kO cm2, with many studies reporting an average value of 1 kO cm2. This suggests that the conjunctival epithelium is a moderately tight epithelium. For comparison, Rt measurements of the rabbit corneal epithelium and rabbit corneal endothelium are 7–9 kO cm2 and 0.01–0.07 kO cm2, respectively. As such, the electrical resistances of the corneal epithelium and endothelium vary over a range of about 100-fold, and the conjunctiva exhibits an intermediary value. The fact that the measured Rt of the freshly isolated conjunctiva is so high also indicates that tight junctions must exist between the surface goblet cells and the most superficial stratified epithelial cells. An explanation as to why Rt declines with prolonged time in vitro remains to be evinced, but barrier resistance is physiologically regulated in other systems. The stable Isc recorded during the spontaneous Rt decline indicates that the epithelial cells have remained metabolically viable.

Electrolyte Transport Systems of the

Rabbit Conjunctiva

The bioelectrical approach discussed above has been used to determine the major electrolyte transport systems present in the rabbit conjunctival epithelium. In work done to date, the short-circuiting methodology was used to characterize transcellular transport. Such transport is energy dependent, and controlled by the tissue-specific profile of transporters and channels along the apical and basolateral membranes of the epithelium. The conjunctival apical membranes interface with the tears and the basolateral membranes interface with the paracellular pathways from the tight junctions to the basement membranes at the

stroma. Transport mechanisms in the tissue are generally uncovered by electrolyte substitution experiments, and the application of relatively specific drugs (i.e., channel blockers, channel openers, transporter inhibitors, etc.) to the bathing solutions of conjunctiva isolated in the divided chambers. In some cases, the identity of channels and transporters that were identified in electrophysiological experiments were corroborated with immunoblotting and immunohistochemcial observations.

Identical to other Cl -secreting epithelia, the rabbit conjunctival epithelium has a basolateral bumetanidesensitive Cl uptake process (mediated by the Na+–K+–2Cl cotransporter, NKCC1) positioned in series with apical Cl channels, including cystic fibrosis transmembrane conductance regulator (CFTR). In addition, Na+/H+ and Cl /HCO3 exchangers exist in parallel in the basolateral membrane and can also mediate Cl uptake (Figure 2).

Oppositely directed, electrogenic Na+ reabsorption is amiloride-insensitive, indicating the absence of the major epithelial Na+ channel (ENaC) at the apical surface, and occurs through a Na+-dependent cotransporter for glucose. Nonselective cation channels (NSCCs) were identified in whole-cell patch clamping of freshly isolated conjunctival epithelial cells and such channels may also reside at the apical surface. A transcellular Na+ movement occurs because the apical uptake mechanisms for Na+ exist in series with the basolaterally located Na+–K+ pump (Figure 2).

Other electrogenic Na+-dependent uptake mechanisms at the apical surface were demonstrated by adding the transported substrates to the apical bath. Transport systems for amino acids, nucleosides, L-lactate, and diand tri-pep- tides were evidenced in this manner because such compounds are normally not included in the physiological solutions used to bathe the in vitro preparations. The addition of such compounds to the apical bath results in a shortcircuit current stimulation. In the cases of the amino acids and nucleosides, the largest Isc stimulations occurred with L-arginine and uridine, respectively. The roles of these carriers are not firmly established. It is thought that these transport systems may clear the tear fluid of these compounds in either nonphysiologic or pathophysiological states of the ocular surface when excess amounts of such solutes might have leaked into tears.

Results from protocols for immunoblotting and the immunofluorescent labeling of frozen sections from separately isolated bulbar and palpebral regions of the conjunctival epithelium indicated that the proteins for the Na+–glucose cotransporter, Na+–K+ ATPase, and Na+–K+–2Cl cotransporter are uniformly distributed throughout the conjunctiva. These observations suggest that despite stark differences in the regional morphology of the bulbar and palpebral regions, the entire conjunctival epithelium exhibits the elements for transepithelial Na+ and Cl transport.

Regulation of Epithelial Ion Transport

in Rabbit Conjunctiva

Currently, information on the regulation of electrolyte transport by the conjunctival epithelium is somewhat limited. This is because the characterization of the macroscopic electrolyte transport properties of this tissue, as measured in bicameral Ussing-type chambers, was begun relatively recently. Hence, many fundamental aspects of the tissue have not been elucidated. One underlying rationale for studying conjunctival transport is to define the secretory functions of the epithelium. This effort could prove to have utility in ameliorating complications from dry-eye diseases, and some progress has been made in this regard. Because of the large surface area of the conjunctival epithelium, active transport by conjunctiva with accompanying fluid secretion could, hypothetically, contribute a significant fraction of tear production, which is normally provided in healthy individuals by the lacrimal gland. Upon stimulation, the transepithelial conjunctival contribution could be greater.

As commonly found in Cl -secreting epithelia, the exposure of the conjunctiva to secretogogues that increase either cell calcium or cyclic adenosine monophosphate (cAMP) stimulates transepithelial Cl fluxes and the Isc. The latter intracellular messenger can be increased in the conjunctival epithelium with forskolin (a direct stimulator of adenylyl cyclase), dibutyryl-cAMP (a cell permeable form of cAMP), 3-isobutyl-1-methyl-xanthine (IBMX, a nonselective phosphodiesterase inhibitor), rolipram (an inhibitor specific for cAMP-phosphodiesterase type IV), or epinephrine (a nonselective adrenergic agonist). In addition, these agents also increase the transconjunctival Isc under Cl -free conditions indicating that the increased cAMP levels also stimulate the Na+ absorptive activity of the epithelium. The increase in Na+ absorption has been attributed to a protein kinase A (PKA)-regulated, bariuminhibitable, basolateral K+ conductance in the rabbit conjunctival epithelial cells. One or more different types of K+ channels that have not yet been identified may mediate this K+ conductance. The stimulation of the PKA-gated K+ channels hyperpolarizes the negative cell potential relative to the bathing solutions and favors both the uptake of Na+ across the apical face and the efflux of Cl into the tears. There is evidence that apical Cl channels are also gated by PKA, particularly in the case of CFTR. In the shortcircuited conjunctiva, cAMP has a central role in coordinating simultaneous changes in apical Cl and basolateral K+ conductances to enable stimulations in the transcellular transport of Cl in the stromal-to-apical direction and of Na+ in the opposite direction.

Should both absorptive and secretory mechanisms coexist within the same cell, such cAMP-evoked stimulations of the in vivo conjunctiva would, in principle, deplete the epithelium of KCl and reduce cellular volume, while Na+ and Cl move in opposite directions both transand

paracellularly. Experimental measurements of net water fluxes across the isolated conjunctiva (under open-circuit conditions) indicate an increased fluid movement in the stromal (basolateral)-to-apical direction in response to cAMP, likely due to the higher rate of Cl secretion relative to Na+ absorption. Fluid flow occurs under open-circuit conditions, which is the situation in vivo. The dominant transport system, apparently Cl , will be transported mainly transcellularly, while Na+ will reverse from its net tear-to-stroma direction found under shortcircuit conditions to move paracellularly as a companion ion to neutralize the Cl charge and create a possible isotonic fluid at the apical surface. In open circuit, there would still be a transcellular movement of Na+ toward the stroma, and a paracellular movement of Cl toward the stroma, but the magnitude of these flows will be less than the net of Na+ and Cl secreted into tears (Figure 3). cAMP stimulates all flows and increases the net.

Other effective secretogogues in the rabbit conjunctiva are: (1) 1-ethyl-2-benzimidazolinone (EBIO), a Cl and K+ channel opener that elicits electrophysiological effects similar to those of cAMP, although different subtypes of channels are likely involved; and (2) the nucleotide uridine 50-triphos- phate (UTP), which stimulates Cl secretion through P2Y2 receptors upon exogenous application to the apical-side bath. Of these, only the latter has been tested on net fluid movement across isolated conjunctivae and found to be a useful stimulant. Recently, synthetic P2Y2 agonists (e.g., diquafosol tetrasodium, which is also known as INS365) have been studied in clinical trials. Such agents are administered 4–5 times daily, and there is a time-dependent loss of efficacy that is observable in the data produced by such trials. This may be because P2Y2-receptor activation is often transitory due to the nature of the Ca2+ signal itself (through the phospholipase C-sensitive calcium signaling pathways) and the fact that purinergic agonists produce receptor desensitization from which recovery is slow. Yet currently, the use of purinergic agonists appears a suitable approach to palliate dry eye because of not only the stimulatory effects on conjunctival Cl secretion and fluid transport, but also the fact that purinergics serve as mucin secretogogues from conjunctival goblet cells. As such, purinergics appear to have utility in conserving the composition of the tear film.

The established receptors that stimulate electrolyte and fluid secretion in the stromal-to-tear direction under open-circuit conditions are schematically presented in Figure 4. The specific channel subtypes activated by calcium and cAMP remain to be conclusively identified.

Fluid Transport Studies across Isolated Rabbit Conjunctiva

Two commonly used methods to measure water fluxes across various epithelia have been applied to the excised,

HCO3

Cl1 + Cl2 > Cl3

Na3 > Na1 + Na2

(Cl1 + Cl2) − Cl3 = Clnet

Na3 − (Na1 + Na2) = Nanet

Clnet = Nanet

Figure 3 A simplified model of the sodium and chloride flows across the rabbit conjunctiva under open-circuit conditions, which are analogous to the in vivo situation. Some transporters present in the epithelium have been omitted for clarity. Cl1 is the transcellular efflux of chloride via chloride channels in the apical domain. Cl2 is the paracellular movement of chloride in the stromal-to-tear direction. Cl3 is the paracellular movement of chloride in the tear-to-stromal direction. Na1 is the sodium efflux mediated by the sodium-potassium ATPase pump. Na2 is the paracellular movement of sodium in the tear-to-stromal direction, while Na3 is the paracellular movement of sodium in the opposite direction. In open-circuit, the tear-side (apical) bath will have a negative potential relative to that of the basolateral-side bath. Cations will thus flow in the paracellular pathways toward the tear side, while anions will flow in the opposite direction. There is also the possibility that some potassium will move along the paracellular pathways toward the tears. The sodium–potassium–chloride cotransporter in the basolateral membranes drives the transcellular movement of chloride. The net flux of sodium and chloride into the tears results in a net fluid transport across the conjunctiva. Flux relationships are indicated at the bottom of the figure by equations.

isolated rabbit conjunctiva: (1) unidirectional/diffusional flow with tritiated water (3H2O); and (2) net water flow by volumetric procedures.

With method 1, the diffusion permeability coefficient, Pdw , is expressed in cm s 1 and given by:

Pdw ¼ Jdw=A Vw Cw

where, A is the area of the membrane (cm2), Vw is the partial volume of water (cm3 mol 1), Cw is the concentration of water (mol cm 3), and Jdw is the measured unidirectional H2O flux in cm3 s 1.

In this case, a two-compartment chamber is used. The tissue is mounted between compartments; 3H2O is added to one side and samples are taken periodically from both