Since the many exocrine glands surrounding the orbit (the ocular adnexa) and the ocular surface epithelia secrete the tear film, it is a necessarily a complicated fluid. The functions of the tear film are varied and vital to vision. Tears are the first refractive surface encountered by light rays, and therefore they must remain clear to form a refractive surface that will allow light to reach the retina. The transparency of the cornea, the second refractive surface that light encounters, is maintained by the tears. Another crucial role of tears is to protect the ocular surface and to maintain its health and normal function. The tear film protects the ocular surface from the external environment by responding to wide-ranging exterior conditions and potential threats to the eye. The environmental stresses placed on the eye are a result of desiccation, bright light, extreme thermal conditions, physical injury, and noxious chemicals, and infection by bacteria, viruses, and parasites. Tears also lubricate the surface of the eye to avoid mechanical damage from the high pressures generated by the blink. Tears transport oxygen and provide a limited number of other nutrients to the avascular cornea. They also contain proteins and other molecules that are involved in the defense of the ocular surface and regulate cellular functions of the conjunctiva and cornea. To meet the needs of the cornea and conjunctiva, and to respond to changes in the external environment, the volume, composition (including the electrolyte composition, osmolality, protein composition, and pH), and structure of the tears are extraordinarily well regulated.

Three factors control the volume and composition of the tear film: control of secretion by the lacrimal and accessory glands, the cornea, and the conjunctiva; changes in the evaporation of tears; and regulation of tear drainage. The control of secretion and absorption of tears by the conjunctiva and meibomian glands is covered in the present chapter, and the control of lacrimal and accessory lacrimal gland secretion and tear drainage is discussed in another chapter,”The Lacrimal Gland.”

For descriptive purposes the conjunctiva may be divided into three major subdivisions (as shown in Fig. 3):

1. Tarsal, or palpebral, lining the eyelids
   
2. Forniceal, lining the upper and lower the fornices
   
3. Bulbar, overlaying the sclera on the anterior portion of the globe
   

Approximately 2 mm from the eyelid on the tarsal conjunctiva lies a shallow (less than 1 mm deep) groove called the subtarsal groove. This groove runs parallel to the eyelid margin for most of the length of the tarsal conjunctiva. The groove marks the area of transition from the nonkeratinized, stratified squamous epithelium of the eyelid margin to the cuboidal epithelium of the tarsal conjunctiva (Fig. 3). The groove provides an ideal anatomical trap for small foreign objects before they can reach the cornea and bulbar conjunctiva. The objects trapped in the subtarsal groove are almost immediately covered with a thick coating of mucus and are moved medially by the blink to be carried out on, and trapped by, the hairs of the caruncle. There are multiple smaller ridges and grooves between the eyelid margin and the tarsal groove that contain a series of small saccular mucus glands. Each of these glands has a tubular lumen 15 to 30 μm in diameter that opens onto the conjunctival surface. These glands are formed by an invagination of the conjunctival epithelium and are lined with mucus-secreting epithelial cells.⁶

The nasal tarsal conjunctiva contains numerous structures known as Henle's mucus crypts. These crypts change in the midtarsal conjunctiva to form a network of subepithelial grooves. The grooves become tunnels with multiple interconnections that have small openings onto the ocular surface. The grooves and tunnels divide the surface of the conjunctiva into irregular papillae averaging approximately 100 μm in diameter (Fig. 5). At the orbital margin of the tarsus, the tunnel system opens onto the surface to form a network of grooves that divide the surface into bulges averaging 300 μm in diameter. In the fornix, these grooves and clefts are called Stieda's cleft and plateau system. These structures are generally larger and more prominent on the temporal side,⁷ and are usually more prominent in the upper eyelid than in the lower fornix. The patterns made by these clefts and grooves differ greatly between the conjunctivas of different individuals, as well as between the eyes of one individual. Local or systemic disease processes may result in hypertrophy of the underlying lymphoid layer, with follicle formation and profound alterations in the surface features of the conjunctiva. The conjunctival papillae that are normally present may also hypertrophy in response to infectious or toxic agents, and thus further modify the surface topography.⁷

This complex groove, crypt, and gland system covering the tarsal and forniceal conjunctivas provides an efficient trap system whereby small foreign bodies, bacteria, and cellular debris are sequestered, coated with mucus, and neutralized. These conjunctival surface folds greatly increase the total surface area and at the same time decrease the area of contact between the bulbar and palpebral conjunctiva, thereby reducing friction force as the two surfaces glide over one another during blinking and rotation of the globe.

The bulbar conjunctiva is smoother than the palpebral conjunctiva and its surface features are less consistent. There are often four to six low broad papillae about the corneoscleral limbus that range in size from barely visible irregularities to the hypertrophic follicles that are occasionally seen in vernal conjunctivitis. The palisades of Vogt are structures around the limbus that radiate outward from the cornea. These structures consist of a series of 1.5- to 2-mm ridges and are formed by the epithelial rete ridges and the underlying stromal condensations. One of their functions is to trap small foreign objects that were on the cornea and were subsequently dislocated by blinking. Often the palisades are outlined by a fine brown line that radiates from the cornea. This is caused by the production of pigment from an increase in the local melanocyte population, which makes these structures easy to identify.

The basal conjunctival epithelial cells are attached by desmosomes to a basement membrane. The tarsal conjunctiva is firmly attached to the tarsi and requires careful dissection. The upper tarsus is usually more tightly adhered than the lower tarsus. Meibomian glands are visible in both the upper and lower eyelids as yellow streaks running perpendicular to the eyelid margin. The conjunctiva of the fornix forms an almost continuous sac that is interrupted medially by the plica semilunaris. From the fornices the conjunctiva sweeps posteriorly to attach to and cover the eyeball, approximately 10 mm posterior to the limbus superiorly, 12 to 14 mm posterior to the limbus laterally, and 8 to 10 mm from the limbus inferiorly. Medially it variably covers the globe for approximately 7 mm posterior to the limbus.

Four fornices are formed by the conjunctiva where it is reflected from the eyelids to the globe. The superior fornix is the largest in both size and volume. It is formed and maintained by fine smooth muscle slips that pass from the levator palpebral superioris muscle and insert into the conjunctiva. This attachment prevents the conjunctiva from folding downward over the cornea. It also prevents the fornix from developing sags as the globe moves upward, which might obstruct vision. The temporal fornix is attached by fine fibrous slips to the tendon of the lateral rectus muscle, again maintaining the relative position of the fornix during horizontal movements of the globe. The inferior fornix is attached to the tendon of the inferior rectus, which prevents its movement. The plica semilunaris performs the same function as a fornix medially, and is a reversed fornix with the fold of conjunctiva lying externally (Fig. 3). During a medial gaze, the fornix has a variable depth. This occurs because the fibrous slips that link the conjunctiva to the tendon of the medial rectus muscle insert onto the deep surface of the plica and caruncle. Contraction of the medial rectus tightens these slips, forming a cul-de-sac medially as the globe adducts. On maximal medial rotation, the plica partially unfolds to form a true fornix similar to that present in other areas.

The conjunctival epithelium changes from cuboidal shaped at the anterior surface to nonkeratinized-stratified squamous epithelium 2 to 3 mm from the corneal limbus. It is in this area, where the conjunctival stratified squamous epithelium becomes continuous with the corneal epithelium, that the corneal epithelial stem cells reside. It is well known that if the corneal epithelial cells are removed but the limbus remains intact, the cornea will re-epithelize and remain clear. If the limbus and its stem cells are lost, the cornea will vascularize.⁸ In addition, goblet cells can be observed migrating onto the corneal surface with the new epithelial cells after the corneal epithelium, including the limbus, is completely removed. As the new epithelium matures and becomes isotopic with normal corneal epithelium, the goblet cells sometimes disappear. It is not known what processes the goblet cells use to migrate, or what effects they have on the corneal epithelium.

While it is well established that the corneal stem cells are located in the corneal limbus, the site of the stem cells in the conjunctival epithelium is controversial. Clonal cultures of human conjunctival epithelial cells suggest that stem cells are dispersed throughout the epithelium.⁹ This has been substantiated by studies in mice containing green fluorescent protein in the conjunctival epithelial cells.¹⁰ Because the amount of this protein varies between cells, these studies were able to track specific cells and monitor their mitotic activity. The results indicated that the epithelial cells of the bulbar conjunctiva are mitotically active and are generally immobile in the lateral direction, which suggests that these cells are self-sufficient. Other investigators observed that label-retaining cells had a uniform distribution, which implies that stem cells are uniformly distributed in the bulbar conjunctiva.⁹ Still other studies have suggested that there are stem cells in the conjunctival fornix, as well as at the mucocutaneous junction.

The apical cells of the conjunctival epithelium are not flattened like those of the cornea. However, like the apical squamous cells of the cornea, the stratified squamous cells have a complicated arrangement of microvilli and microplicae on the apical-most membranes.^15,16 The microvilli are 0.5 μm in diameter and 0.5 to 1 μm high. The microplicae are of a similar size, measuring 0.5 μm in width, 1 to 3 μm in length, and 0.5 μm in height. This system of outfoldings is present over the entire conjunctival surface, and is continued over the apical membrane of the goblet cells. In these cells the apical cell membrane disintegrates during secretion and is released into the tear film along with the secretory granule membranes and their components. After the secretory process is completed, the goblet cell resynthesizes mucin by packaging it into secretory granules that are separated from the tear film by the apical membrane. This complex system of micro-outfoldings of the surface cell membranes may aid in supporting, stabilizing, and anchoring the tear film against the effects of gravity and eyelid movement, thereby preventing irregular streams and flow patterns from developing that would interfere with clear vision. In addition, membrane-spanning mucins are inserted into the microplicae of the apical membrane to form the glycocalyx. Notable in the stratified squamous cells of the conjunctiva are numerous clear vesicles in the cytoplasm, which give the cells a lacy appearance. These vesicles contain mucin that is perhaps being transported to the apical membranes.

The total surface area of an adult conjunctival sac in one eye, including the cornea, averages 16 cm², or roughly 17 times more surface area than the cornea. Because of its large surface area, the conjunctiva may be a significant source of electrolytes and water in the tear film.¹⁷ The blood vessels and the epithelial cells of the conjunctiva may transport fluid across the conjunctiva. An increase in vascular permeability of the blood vessels may contribute fluid to tears by leaking plasma. This may occur in pathologic conditions, such as inflammation or topical application of drugs, that increase vascular permeability. Epithelial cells can also be a source of fluid for tears. Under normal conditions, the goblet cells and the stratified squamous cells are the primary sources of fluid transport in the conjunctiva. The conjunctiva can also absorb electrolytes and water.^18,19 In addition, the conjunctiva absorbs drugs that are applied to the ocular surface.²⁰ The conjunctiva is permeable to hydrophobic molecules up to 40 kDa²¹ and actively transports drugs using nucleoside, monocarboxylate, dipeptide, and sodium-dependent transporters.^22–24 Conjunctival epithelial cells can also absorb compounds by endocytosis.²⁵ For a comprehensive review of conjunctival fluid secretion, see Dartt.²⁶

The conjunctival epithelium is a low-resistance, permeable epithelium, particularly in contrast to the high-resistance, impermeable corneal epithelium. The permeability of the conjunctival epithelium arises from the attachment of the stratified squamous and goblet epithelial cells to each other by tight junctions and desmosomes. Furthermore, the plasma membranes of adjoining cells do not totally interdigitate, and thus leave large intercellular spaces. These intercellular spaces provide antibodies, other plasma constituents, and inflammatory cells from the abundant underlying conjunctival capillaries ready access to the surface of the conjunctiva, where irritative and infectious processes arise. This pathway may operate in the reverse direction, such that infectious agents on the conjunctival surface may gain access to the circulatory system. Proteins applied topically to the conjunctiva percolate easily down to and into the lumen of the conjunctival capillaries, and appear shortly thereafter in the systemic circulation.²⁷ Certain topically applied ophthalmic medications may accumulate intra- or extracellularly within the conjunctival epithelium, resulting in discoloration or an accumulation of deposits that may be mistaken for various disease processes. An example of this process is the accumulation of brownish black deposits that are often encountered clinically after topically applied epinephrine is used as an antiglaucomatous medication.²⁸

The conjunctiva has a rich sensory innervation, but because it lacks muscular structures and innervated glands, no motor nerves are present. Autonomic fibers are present surrounding the blood vessels, and may on occasion supply twigs to scattered and inconsistently present smooth muscle fibers in the plica semilunaris. The sensory nerve supply to the conjunctiva is derived from the first or ophthalmic branch of the trigeminal nerve. This sensory system is closely associated with that of the eyelids, eyebrows, and cornea. The same main functional classes of sensory afferents that have been identified and well-studied in the cornea also innervate the bulbar conjunctiva.^29,30 Because the corneal and conjunctival nerves are similar, and because both arise from the trigeminal ganglion, only the characteristics of the corneal nerves are described here.

Ocular surface nerves can be classified as thin myelinated (A-δ type) or unmyelinated (C type). These two types of nerves differ in that the former conduct nerve impulses faster than the latter. They also differ in their passive and active electrophysiological properties. All of these peripheral axons lose their myelin sheath when they enter the stroma. They then branch extensively, forming a subepithelial plexus.²⁹ Thin branches derived from this plexus ascend up into the basal layer of the epithelium, where they run parallel to the surface and form leashes that terminate in the apical layers of the epithelium. It is of functional importance that the sensory nerves do not surround the goblet cells but are restricted to the stratified squamous cells. Morphologically, the sensory nerves appear homogeneous: approximately 58% of the sensory nerves contain the neuropeptide CGRP in their nerve endings and axonal fibers, and about 20% contain Substance P.³¹ Electrophysiologically, these nerves are very different and can be divided into four categories:

1. Mechanoreceptor (low threshold)
   
2. Mechano-nociceptor (high threshold)
   
3. Polymodal nociceptor
   
4. Cold receptor
   

About 20% of the sensory nerves are mechanoreceptor and mechano-nociceptor, and respond to mechanical forces that are similar to those required to damage the cornea. These receptors are likely responsible for the acute, sharp sensation of pain that occurs with mechanical contact with the cornea. The majority of sensory nerves (about 70%) are polymodal nociceptors that are activated by mechanical energy, heat, exogenous chemical irritants, and endogenous chemical mediators released by damaged tissue, resident inflammatory cells, or plasma leaking from blood vessels.²⁹ Most of these nerves are of the C type, and they signal both the presence of the stimulus and its intensity and duration. These nerves contribute to the sharp pain that occurs when the cornea is mechanically stimulated, as well as to the pain caused by chemical irritation, heat, cold, or inflammation. The final category of nerves, which comprises 10% to 15% of the sensory nerves, consists of cold-sensitive receptors that contain both A-δ and C fibers. The cold-sensitive nerves fire continuously, but increase their rate of firing as the temperature of the surface decreases because of evaporation, application of cold solutions, or blowing of cold air.

The parent axons of trigeminal ganglion nerves branch extensively when they enter the stroma, and cover an area of tissue surface called a “receptive field.”²⁹ The functional receptive fields of the mechano-nociceptors and polymodal nociceptors are large. In contrast, the cold receptors have small receptive fields.

Tissue injury and inflammation modify the activity of sensory nerves. An important feature of ocular-surface polymodal nociceptor nerves is that when a noxious stimulus is repeated within a short time, the impulse-firing threshold decreases and the mean firing frequency in response to a given stimulus increases. Often a low spontaneous firing activity develops. These characteristics are called “sensitization.” Sensitization develops because tissue injury from the noxious stimulus releases endogenous inflammatory mediators from damaged cells and neighboring resident inflammatory cells activated by the injury. These inflammatory mediators cause changes in the ion channels that are responsible for the electrical activity of the nerve. Prostaglandin and bradykinin are two inflammatory compounds know to cause sensitization of corneal nerves. Another consequence of an injurious stimulus is that the stimulus causes the nerve endings of the polymodal nociceptors to release their neuropeptide content as a consequence of membrane depolarization and antidromic (opposite direction) conduction of the nerve impulse. The released sensory neuropeptides, CRRP and Substance P, produce vasodilation and plasma extravasation, and stimulate cytokine release, leading to a local inflammatory response known as neurogenic inflammation. Although the noxious stimulus may be limited to a restricted area, the excited endings produce impulses that propagate centripetally and antidromically stimulate other, noninjured branches of the parent axon. Neuropeptides are subsequently released from nerve endings that were not originally injured. This antidromic stimulation appears to be the origin of the extension to noninjured conjunctiva of the neurogenic inflammation that follows a limited corneal or conjunctiva injury.

The development of the gas esthesiometer by Belmonte et al³² has enabled researchers to study the application of controlled mechanical impulses, irritant chemical stimuli, and hot or cold air pulses separately in a limited area of the conjunctiva, and to measure the psychophysical characteristics for each type of stimulus. In one study the same responses were elicited from human conjunctiva and cornea, except that the overall sensitivity was comparatively lower in the conjunctiva and the light stimuli were not felt as irritating.³⁰ The sensations produced by heat and suprathreshold mechanical or chemical stimulation always included a component of irritation, although each sensation was identified as being distinct from the others.

Other types of nerves that innervate the conjunctiva include the efferent sympathetic and parasympathetic nerves.^12,33,34 These unmyelinated nerves send free nerve endings into blood vessels and epithelium around goblet cells and between stratified squamous cells.¹² External stimuli activate the different types of sensory nerves in both the cornea and the conjunctiva. However, one study found that the polymodal nociceptors in the cornea are the primary nerves that stimulate reflex tear production.³⁵ In that study, activation of the corneal mechanoreceptors and cold receptors was less effective, and activation of the conjunctival sensory receptors did not induce tear secretion. When corneal sensory receptors are activated, they use a local neural reflex conducted in the dromic direction to stimulate the parasympathetic and sympathetic nerves in the conjunctiva to release their neurotransmitters (Fig. 7). These neurotransmitters may then activate conjunctival stratified squamous cells to increase electrolyte and water transport, and goblet cells to secrete mucins.³⁶

For Cl⁻secretion, Na⁺, K⁺, and Cl⁻enter the cell by means of the NKCC transporter located on the basolateral membrane (Fig. 8)^39–41and exit the cell through a Cl⁻channel in the apical membrane. Na⁺ is pumped out of the cell via the Na⁺, K⁺-ATPase located in the basolateral membrane, while K⁺ diffuses out of the cell via a K⁺ channel that is also located in the basolateral membrane. To maintain electroneutrality, the Na⁺ that is pumped out of the cell diffuses back into tears through the paracellular pathway (Fig. 8). This ion movement drives water to enter the tears using both the paracellular and transcellular pathways. The transcellular pathway, which probably involves aquaporin type 3, is the predominant mechanism by which water enters the tears.^37,38,43,44

Absorption of Na⁺ is mediated by the Na⁺ coupled-glucose and -amino acid transport (Fig. 8),^21,45–47 which is located on the apical membrane of conjunctival cells. The Na⁺, K⁺-ATPase pumps the Na⁺ that enters the cell on the apical side, using the coupled transporters, out of the cell on the basolateral side. Interestingly, unlike many other tissues, the Na⁺/H⁺ exchanger does not play a role in conjunctival Na⁺ absorption.⁴⁰

The activation of purinergic receptors also stimulates conjunctival fluid secretion (Fig. 8).^37,50,51 A comparison of a variety of nucleotides indicated that the purinergic receptors P2Y₂ or P2Y₄ were activated. In contrast to β₂-adrenergic agonists, the P2Y₂ or P2Y₄ agonist, serotonin (also known as 5-hydroxytryptamine (5-HT)), inhibits Cl⁻secretion by downregulating apical Cl⁻and basolateral K⁺ channels, which inhibits Cl⁻secretion.⁵² This is unusual because in most other cell types, 5-HT stimulates secretion by either increasing Cl⁻secretion or inhibiting Na⁺ absorption.

The Ca²⁺- and protein kinase C (PKC)-dependent signaling pathway also alters conjunctival electrolyte and water secretion. Activation of these pathways results in a transient increase in Cl⁻secretion followed by a sustained decrease in secretion.^53,54 This decrease is mediated by an inhibition of Na⁺, K⁺-ATPase.

In summary, sympathetic nerves release norepinephrine, which binds to and activates β₂-adrenergic receptors to increase cellular cAMP levels. This is an important pathway for stimulating conjunctival fluid secretion (Fig. 8). Ca²⁺ is another possible stimulus of conjunctival fluid secretion, although it is less effective than cAMP.⁵³ It is possible that P2Y₂ agonists stimulate secretion via the Ca²⁺ signaling pathway. Finally, activation of PKC inhibits secretion; however, the agonists that use this pathway are as yet unidentified.

Several types of mucins have been localized to either the goblet cells or conjunctival stratified squamous epithelial cells. MUC5AC, a gel-forming mucin, is synthesized and secreted by the goblet cells.⁵⁶ The conjunctival epithelium also produces MUC2 and MUC7, two membrane-bound mucins.^57,58 MUC1, -4, and -16 are membrane-bound mucins that are produced in the stratified squamous cells of the conjunctiva.^59–61 Since mucins secreted from stratified squamous cells are secreted by different mechanisms than mucins secreted from goblet cells, protein secretion from these two cell types will be discussed separately.

REGULATION OF STRATIFIED SQUAMOUS CELL MUCIN SECRETION

The stratified squamous cells of the conjunctiva synthesize the membrane-bound mucins MUC1, -4, and -16. Two of these mucins, MUC1 and -4, are known to be inserted into the plasma membrane and released into the tear film by ectodomain shedding. While little is known about how the release of these mucins is regulated, it has been shown that stimulation with the eicosanoid 15(S)HETE causes secretion of MUC1 (but not MUC4 or MUC5AC) into the tear film.^62,63

No evidence is available regarding the regulation of soluble mucin (MUC4) secretion. It is known that MUC4 is cleaved intracellularly to form a soluble mucin, which is probably stored in the secretory vesicles in stratified squamous cells, but this has not been demonstrated. It is also not known which stimuli release the soluble portion of MUC4 into the tear film.

FUNCTIONAL ANATOMY OF GOBLET CELLS

Goblet cells are highly polarized cells that provide for the unidirectional synthesis and secretion of mucins. These cells occur throughout the conjunctiva either singly or in clusters, depending on the species. Mucins are synthesized in the endoplasmic reticulum, modified in the Golgi apparatus, and stored in secretory granules in the apical portion of the cell. Upon the appropriate stimulus, the secretory granule membrane fuses with other secretory granule membranes and the apical membrane of the cell to release the stored mucin (Fig. 9). Entire populations of granules in the stimulated cell are released at once by a mechanism known as apocrine secretion. The goblet cell body then resynthesizes the mucins to secrete again. Several techniques have been used to identify goblet cells by staining the secretory proteins in the granules. However, this means that once a goblet cell has secreted, the stains can no longer identify it. Thus, these types of techniques tend to underestimate the number of goblet cells in a given sample.

REGULATION OF CONJUNCTIVAL GOBLET CELL SECRETION

Early studies suggested that the goblet cells are not innervated,²⁶ despite the presence of sensory, sympathetic, and parasympathetic nerves in the conjunctiva. However, when the activation of nerves was prevented so that goblet cell secretion would not occur, nerves were identified surrounding conjunctival goblet cells in rats, mice, and humans.^49,64,65 Parasympathetic and sympathetic (but not sensory) nerves were detected surrounding conjunctival goblet cells. Activation of these nerves can stimulate conjunctival goblet cell secretion, because a strong sensory stimulus to the cornea caused goblet cell secretion in the conjunctiva that was blocked by local anesthetic.⁶⁶ This implies that activation of afferent sensory nerves stimulates efferent parasympathetic or sympathetic nerves to stimulate secretion of the goblet cells. This hypothesis is supported by the fact that three different types of neurotransmitter receptors have been localized on goblet cells. These receptors include three subtypes of muscarinic receptors (m₁, m₂, and m₃) that are activated by the parasympathetic neurotransmitter acetylcholine, the vasoactive intestinal polypeptide (VIP)2 receptor that is activated by the parasympathetic neurotransmitter VIP, one subtype of the α-adrenergic receptor on human goblet cells that is activated by the sympathetic neurotransmitter norepinephrine, and three subtypes of β-adrenergic receptors (β₁ and β₂-adrenergic receptors in mice and rats, and β₃-adrenergic receptor in humans) that are also activated by norepinephrine.^49,65 Thus, nerves surround the goblet cells and stimulate them to secrete, and neurotransmitter receptors are present on goblet cells.

To date, two neurotransmitters have been shown to stimulate conjunctival goblet cell secretion. These neurotransmitters are the parasympathetic neurotransmitters ACh and VIP.^67–69 In contrast, activators of the α- and β-adrenergic receptors and Substance P do not stimulate goblet cell secretion. Thus, the parasympathetic nerves appear to be a major neural stimulus of conjunctival goblet cell secretion.

The purinergic agonists UTP and ATP have been shown to stimulate goblet cell secretion.⁷⁰ By determining the potency of a series of nucleotides, Jumblatt and Jumblatt⁷⁰ implicated the P2Y₂ receptors in the stimulation of goblet cell secretion. The source of these agonists in the conjunctiva is unknown, but they may be released from nerve endings, platelets, or damaged cells.

SIGNALING PATHWAYS THAT REGULATE CONJUNCTIVAL GOBLET CELL SECRETION

The signaling pathway that is activated by the cholinergic agonists (which are released from parasympathetic nerves) to stimulate goblet cell secretion has been characterized (Fig. 10). However, the pathway that is activated by another parasympathetic neurotransmitter, VIP, has not. Increasing the intracellular Ca+ with the use of a Ca²⁺ ionophore (ionomycin) or the cholinergic agonist carbachol stimulates goblet cell secretion.^69,71 Activation of PKC by phorbol esters also produces goblet cell secretion; however, a direct role for PKC in cholinergic agonist stimulation has not yet been determined, because the PKC inhibitors themselves stimulate goblet cell secretion.⁷¹

In addition to the Ca²⁺/PKC pathway, cholinergic agonists use an additional signaling pathway to stimulate conjunctival goblet cell secretion. As demonstrated in several other tissues, cholinergic agonists in the conjunctiva transactivate the EGF receptor.⁷² The EGF receptor is actually a family of four different receptors, termed erbB1–4. The EGF receptors erbB1–3 have been found in conjunctival goblet cells.^73,74 Cholinergic agonists transactivate the EGF receptor by first activating the non-receptor tyrosine kinases Pyk2 and p60Src (Fig. 8). This leads to the activation of a cascade of kinases, culminating in the activation of p42/p44 mitogen-activated protein kinase (MAPK). Inhibitors of both the EGF receptor and p42/p44 MAPK block cholinergic agonist-stimulated mucin secretion. In addition, stimulation of p42/p44 MAPK is dependent on Ca²⁺ and the activation of PKC. Interestingly, cultured human and rat goblet cells react similarly to cholinergic agonist stimulation in terms of p42/p44 MAPK activation.⁷⁵

Stratified squamous cells secrete the membrane-spanning mucins MUC1 and -4, while goblet cells secrete the gel-forming mucin MUC5AC. It is hypothesized, then, that the three mucins play different roles in the tear film. Thus it is possible that their secretion is regulated differently, and the secretion of either mucin might depend upon the stimulus. However, the relative contribution of the stratified squamous cells and goblet cells to the mucin layer is not known.

The lymphocytes present in the stroma are predominantly T cells, and the remaining lymphocytes have been identified as B cells. B cells are found only in the substantia propria. Langerhans' cells are found in the epithelium. The immunology of the surface of the conjunctiva is similar to that of other mucus membranes and the skin.⁷⁶ Mucosa-associated lymphoid tissue (MALT) represents an important portion of the immune response of mucosal surfaces, including the conjunctiva, where it is termed conjunctival associated-lymphoid tissue (CALT).⁷⁷ The MALT system consists of an arrangement of lymphatic cells situated in and closely underneath the epithelium. It detects antigens and induces an immune response by the direct action of lymphatic cells or the secretion of soluble antibodies. It also induces tolerance of ubiquitous nonpathogenic antigens. Specialized blood vessels known as high endothelial venules allow the recirculation of lymphocytes and interaction with the central immune system. All of these components of MALT have been detected in the human conjunctiva. There are two main types of CALT: diffuse CALT and dense follicular spots.⁷⁷ In the diffuse form, plasma cells and lymphocytes form a diffuse layer that extends like a carpet in the stroma underneath the epithelium. Embedded in the diffuse layer are occasional dark areas that resemble solitary lymphoid follicles. Lymphatic cells are present in the conjunctival crypts. Within the epithelium are intraepithelial lymphocytes. Associated with the diffuse lymphoid layer is a network of vessels with high endothelial venules. Expressed within the conjunctiva are immunoglobulins of the IgA type and a secretory component consistent with the conjunctiva belonging to the secretory immune system.

The blood vessels beneath the bulbar conjunctiva are visible and can be easily examined. In contrast, the blood vessels below the palpebral conjunctiva are much less visible. The conjunctival vascular supply appears as a random distribution of branching, overlapping, and crossing vessels that do not appear to differentiate into specific functional units.⁷⁸ The capillaries run parallel and below the basement membrane. Larger vessels and lymphatics lie deeper in the conjunctiva. It is thought that this arrangement may improve the exchange of nutrients and gases when the eyelids are closed.⁷⁹ Interestingly, the pattern of capillaries in the bulbar conjunctiva is as individual as a fingerprint.

A continual cycle of capillary filling is seen in the vessels and consists of variations in flow and filling and emptying of adjacent capillary beds. This results in an average oxygen tension in the upper palpebral conjunctiva of 61 mmHg.⁸⁰ Cyclic arteriovenous shunts can be used to bypass inactive capillary beds.^81,82 Conjunctival capillaries are similar to the choroids in that they are fenestrated, and each fenestration is bridged with a fine diaphragm of endothelial cell plasma membrane.⁸³ Some capillaries in the stroma appear to be without fenestrations.⁸⁴ In addition, under normal conditions the conjunctival blood vessels are leaky, because intravenous injection of fluorescein dye results in a leakage of fluorescein from these capillaries. Under conditions of infection or irritation of the conjunctiva, or in severe intraorbital inflammatory reactions, the conjunctival capillaries leak plasma faster than the fluid can escape to the surface or be absorbed by the lymphatics. Thus the conjunctiva becomes thick with the superficial blood vessels that arise, resulting in conjunctival edema or chemosis.^85,86

When epinephrine is topically applied, the precapillary sphincter constricts to close and empty the capillary bed.⁸⁷ The velocity of blood flow in conjunctival arteries has been reported to be about 100 μm/second. This slows to average values of 26 and 56 μm/second in the capillary bed and the collecting veins, respectively.⁸⁷ Examination of the conjunctival blood vessels often shows localized areas of nonspecific venous dilatation, and formation of microaneurysms. Such findings have little clinical significance because these changes can be found in normal conjunctivas, as well as in the conjunctivas of patients with an assortment of systemic or localized disease processes.^89,90 The conjunctival capillaries may be affected by systemic diseases, such as in vitamin C deficiency, in which the capillaries leak and subconjunctival hemorrhage can occur.⁹¹ In addition, the number of conjunctival arterioles has been reported to decrease in essential hypertension.⁹² Finally, external conditions such as wind, heat, and cold, and the hormonal changes associated with menstruation and early pregnancy dilate the venous side of the capillary bed.⁹³

The conjunctival blood vessels around the corneoscleral limbus maintain their superficial position in the stroma, while the blood supply to the peripheral corneal arcades, the iris, and the ciliary body lies below. When the conjunctiva is inflamed or infected, the conjunctival superficial blood vessels dilate, resulting in a pattern that increases as the distance to the limbus increases. When the cornea or anterior segment is inflamed, the deeper blood vessels dilate. This results in a pattern that increases as the distance to the limbus decreases. Superficial inflammatory processes can be distinguished from deep inflammatory processes because of the violet hue and straight course of the deep blood vessels.

The caruncle is a raised soft body that is 4 to 5 mm long and 3 to 4 mm wide. It is located in the medial portion of the palpebral fissure, and has numerous functions. Stratified squamous epithelium (similar to that seen in the marginal conjunctiva) is present on the caruncle. Present on the surface are small hairs located on the interpalpebral surface of the caruncle. These hairs are about 1 mm long and are directed nasally. They form an efficient trap in which debris and foreign bodies are caught and retained in the inferior and superior fornix mucous threads. Several large sebaceous glands are present below the epithelium of the caruncle. These glands are similar to the tarsal glands and open directly onto the ocular surface. Surrounding each hair are smaller sebaceous glands that empty into the ciliary canal. The lipid secretion from these sebaceous glands coats, lubricates, and softens foreign objects that are deposited on the caruncle by the blinking action of the eyelids. Accessory lacrimal glands may also be present and their secretory product is released onto the ocular surface via a duct that opens near the base of the plica. The caruncle is also attached to the medial rectus by fibrous bands. Therefore, the movement of the caruncle is the same as that of the plica.

The relationship between the plica semilunaris and caruncle and the bulbar conjunctiva, eyelids, and lacrimal puncta is important in several ways. Any change in these structures due to scarring or other fibrous changes could mechanically limit rotation of the globe. In addition, keratinization, hypertrophy, or retraction of the caruncle may interfere with mucus and foreign body excretion, resulting in dysfunction of the lacrimal drainage system.

ANATOMY OF THE MEIBOMIAN GLANDS

In the superior and inferior eyelids lies a row of meibomian glands (Fig. 3). Each gland has one straight duct that opens directly onto the margin of the eyelids next to the mucocutaneous junction. Each duct consists of four cell layers of epithelial cells before it branches into smaller ducts, each of which terminates in an acinus.¹⁰¹ The acinus is made up of several layers of epithelial cells. The outer layer of cells consists of germinal basal cells that do not synthesize lipids (Fig. 3). The cells migrate toward the center of the acinus as they mature. This movement has been measured at a rate of 0.62 μ per day.^102,103 As the cells mature and migrate, the endoplasmic reticulum of each acinar cell also matures. As this occurs, the endoplasmic reticulum starts to synthesize lipids. The lipids are stored in secretory granules. As the cell gets closer to the center of the acinus, the number of lipid secretory granules increases.¹⁰³ When the mature cells reach the center of the acinus, the cells disintegrate in a process known as holocrine secretion. Not only is the lipid content of the secretory granules released, but the secretory granule membranes and the contents of the cell are also released into the duct. Since meibomian gland fluid contains lipid secretory products and the cellular contents, the meibomian gland fluid is necessarily complex. This fluid is stored in the ducts until it is released by the action of the blink.

The meibomian gland is innervated with parasympathetic, sympathetic, and sensory nerves.^{102,104–106} The parasympathetic nerves are the predominant nerve type and contain acetylcholine and VIP as neurotransmitters.¹⁰⁵ The sympathetic nerves contain the neurotransmitter norepinephrine. Sensory nerves contain the neuropeptides Substance P and calcitonin gene-related peptide (CGRP). Neuropeptide Y (NPY) is also found in the meibomian glands. Normally NPY is found in sympathetic nerves, but in the meibomian glands its distribution is similar to that in parasympathetic nerves.¹⁰⁶ Although the meibomian gland is known to be innervated, the functional role of these nerves has not yet been determined.

It is well established that meibomian gland secretion is regulated by sex hormones. The receptors for androgens, progesterone, and estrogen are located in the nucleus of the acinar cell (Fig 5).^107–109 Androgens increase the size and activity of lipid production in the meibomian glands, whereas estrogens and progestins decrease them.¹¹⁰ This accounts for the substantial sex-based differences between the meibomian glands of males and females.

Lipids Secreted by the Meibomian Glands

Meibomian glands secrete a complex fluid containing hydrocarbons, wax esters, triglycerides, diesters, free sterols, sterol esters, free fatty acids, and polar lipids.¹¹¹ As stated above, the complexity of meibomian gland fluid reflects the products of the disintegrating cells and the synthesized lipids. The lipids secreted by the meibomian gland are mainly wax monoesters and sterol esters, which account for about 60% to 70% of the meibomian gland fluid.¹¹¹ Diester compounds, such as those that form ester linkages with fatty acids, fatty alcohols, or sterols, make up about 8% of the fluid.

Regulation of Meibomian Gland Secretion

The secretion of meibomian gland fluid includes the synthesis and release of lipids from the cells, and the release of secretory fluid from the ducts. Differentiation of the acinar cells controls the synthesis of meibomian lipids. The more differentiated the cell, the more lipid secretory product is synthesized. The cellular differentiation and lipid synthesis are controlled by androgens.¹⁰⁷ Androgens increase the gene expression of the proteins necessary for the synthesis and secretion of lipids. Androgens also alter genes that are responsible for fatty acid and cholesterol synthesis, the degree of fatty acid saturation and branching, incorporation of fatty acids into phospholipids and neutral lipids, the total amount of lipids, the secretion of wax esters and other lipids, and the metabolism of lipoproteins.¹⁰⁷ The regulatory processes by which the acinar cells disintegrate and release their contents are unknown. It is believed that the blinking action regulates the final step in the secretion of meibomian gland fluid (i.e., the release of preformed meibomian gland fluid from the ducts in which it has been stored).

Nerves may also be involved in regulating the meibomian gland secretions, since nerves innervate the gland and surround the acini. Nerves may regulate the release of lipids stored in the secretory granules by stimulating fusion of the secretory granule membranes with the apical membrane or by inducing the disruption of the entire cell. However, no role for nerves in regulating meibomian gland function has been demonstrated.

Functions of the Lipid Layer

The major functions of the lipid layer are to prevent the spillover of tears and contain the tears within the palpebral opening, prevent damage of the lid margin skin by tears, and prevent evaporation from the exposed portion of the eye during sleep.¹¹¹ The lipid layer may also decrease evaporation during eyelid opening, but there are conflicting experimental data regarding this issue. Another possible function of the lipid layer is that it may protect the eye from microorganisms, pollen, and other organic matter by trapping such particles.⁵ However, there is no evidence that it serves this function.¹¹¹

1. Lamberts DW: Physiology of the tear film. In: Smolin G, Thoft RA (eds). The Cornea. 3rd ed. Boston: Little, Brown and Company, 1994

2. Prydal JI, Artal P, Woon H, Campbell FW: Study of human precorneal tear film thickness and structure using laser interferometry. Invest Ophthalmol Vis Sci 33:2006–2011, 1992

3. Nichols JJ, King-Smith PE: Thickness of the pre- and post-contact lens tear film measured in vivo by interferometry. Invest Ophthalmol Vis Sci 44:68–77, 2003

4. King-Smith PE, Fink BA, Fogt N, et al: The thickness of the human precorneal tear film: Evidence from reflection spectra. Invest Ophthalmol Vis Sci 41:3348–3359, 2000

5. McCulley JP, Shine WE: Meibomian gland and tear film lipids: structure, function and control. Adv Exp Med Biol 506(Pt A):373–378, 2002

6. Greiner JV, Allansmith MR: Effect of contact lens wear on the conjunctival mucous system. Ophthalmology 88:821–832, 1981

7. Kessing SV: Mucous gland system of the conjunctiva. A quantitative normal anatomical study. Acta Ophthalmol (Copenh) Suppl 95:91+, 1968

8. Kruse FE, Chen JJ, Tsai RJ, Tseng SC: Conjunctival transdifferentiation is due to the incomplete removal of limbal basal epithelium. Invest Ophthalmol Vis Sci 31:1903–1913, 1990

9. Wei ZG, Cotsarelis G, Sun TT, Lavker RM: Label-retaining cells are preferentially located in fornical epithelium: Implications on conjunctival epithelial homeostasis. Invest Ophthalmol Vis Sci 36:236–246, 1995

10. Nagasaki T, Zhao J: Uniform distribution of epithelial stem cells in the bulbar conjunctiva. Invest Ophthalmol Vis Sci 46:126–132, 2005

11. Gipson IK, Joyce N, Zieske J: The anatomy and cell biology of the human cornea, limbus, conjunctiva, and adnexa. In: Foster C, Azar D, Dohlman C (eds). The Cornea. Philadelphia: Lippincott Williams & Wilkens, 2005:1–35

12. Pepperl JE, Ghuman T, Gill KS, et al: Conjunctiva. In: Jeager E (ed). Duane's Foundations of Clinical Ophthalmology. Philadelphia: Lippincott, Williams & Wilkins, 1996:1–30

13. Allansmith MR, Baird RS, Greiner JV: Density of goblet cells in vernal conjunctivitis and contact lens-associated giant papillary conjunctivitis. Arch Ophthalmol 99:884–885, 1981

14. Lemp MA: The Dry Eye: A Comprehensive Guide. Heidelberg, Germany: Springer Verlag, 1992

15. Carroll JM, Kuwabara T: Ocular pemphigus. An electron microscopic study of the conjunctival and corneal epithelium. Arch Ophthalmol 80:683–695, 1968

16. de Toledo C, Brunner A Jr: Superficial digitiform structures of the human conjunctiva studied on the electron microscope. Rev Bras Oftalmol 26:283–287, 1967

17. Watsky MA, Jablonski MM, Edelhauser HF: Comparison of conjunctival and corneal surface areas in rabbit and human. Curr Eye Res 7:483–486, 1988

18. Maurice DM: Electrical potential and ion transport across the conjunctiva. Exp Eye Res 15:527–532, 1973

19. Hosoya K, Horibe Y, Kim KJ, Lee VH: Na⁺-dependent L-arginine transport in the pigmented rabbit conjunctiva. Exp Eye Res 65:547–553, 1997

20. Yang JJ, Ueda H, Kim K, Lee VH: Meeting future challenges in topical ocular drug delivery: Development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drug transport studies. J Control Release 65:1–11, 2000

21. Horibe Y, Hosoya K, Kim KJ, Lee VH: Kinetic evidence for Na⁺-glucose co-transport in the pigmented rabbit conjunctiva. Curr Eye Res 16:1050–1055, 1997

22. Basu SK, Haworth IS, Bolger MB, Lee VH: Proton-driven dipeptide uptake in primary cultured rabbit conjunctival epithelial cells. Invest Ophthalmol Vis Sci 39:2365–2373, 1998

23. Horibe Y, Hosoya K, Kim KJ, Lee VH: Carrier-mediated transport of monocarboxylate drugs in the pigmented rabbit conjunctiva. Invest Ophthalmol Vis Sci 39:1436–1443, 1998

24. Gukasyan HJ, Kannan R, Lee VH, Kim KJ: Regulation of L-cystine transport and intracellular GSH level by a nitric oxide donor in primary cultured rabbit conjunctival epithelial cell layers. Invest Ophthalmol Vis Sci 44:1202–1210, 2003

25. Steuhl KP: Ultrastructure of the conjunctival epithelium. Dev Ophthalmol 191–104, 1989

26. Dartt DA: Regulation of mucin and fluid secretion by conjunctival epithelial cells. Prog Retin Eye Res 21:555–576, 2002

27. Chait R: Absorption of protein through the conjunctival mucus membrane. Arch Ophthalmol 43526, 1950

28. Hansen E: Conjunctivale auleiringer red bruk av adrenalin oyedraper. Tidsskr Nor Laegeforen 84:678, 1964

29. Belmonte C, Acosta MC, Gallar J: Neural basis of sensation in intact and injured corneas. Exp Eye Res 78:513–525, 2004

30. Acosta MC, Tan ME, Belmonte C, Gallar J: Sensations evoked by selective mechanical, chemical, and thermal stimulation of the conjunctiva and cornea. Invest Ophthalmol Vis Sci 42:2063–2067, 2001

31. Muller LJ, Marfurt CF, Kruse F, Tervo TM: Corneal nerves: Structure, contents and function. Exp Eye Res 76:521–542, 2003

32. Belmonte C, Garcia-Hirschfeld J, Gallar J: Neurobiology of ocular pain. Prog Retin Eye Res 16:117–156, 1997

33. Macintosh SR: The innervation of the conjunctiva in monkeys. An electron microscopic and nerve degeneration study. Albrecht Graefes Arch Klin Exp Ophthalmol 192:105–116, 1974

34. Elsas T, Edvinsson L, Sundler F, Uddman R: Neuronal pathways to the rat conjunctiva revealed by retrograde tracing and immunocytochemistry. Exp Eye Res 58:117–126, 1994

35. Acosta MC, Peral A, Luna C, et al: Tear secretion induced by selective stimulation of corneal and conjunctival sensory nerve fibers. Invest Ophthalmol Vis Sci 45:2333–2336, 2004

36. Diebold Y, Rios JD, Hodges RR, et al: Presence of nerves and their receptors in mouse and human conjunctival goblet cells. Invest Ophthalmol Vis Sci 42:2270–2282, 2001

37. Li Y, Kuang K, Yerxa B, et al: Rabbit conjunctival epithelium transports fluid, and P2Y2(2) receptor agonists stimulate Cl^– and fluid secretion. Am J Physiol Cell Physiol 281:C595–C602, 2001

38. Shiue MH, Kulkarni AA, Gukasyan HJ, et al: Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva. Life Sci 66:L105–111, 2000

39. Kompella UB, Kim KJ, Lee VH: Active chloride transport in the pigmented rabbit conjunctiva. Curr Eye Res 12:1041–1048, 1993

40. Shi XP, Candia OA: Active sodium and chloride transport across the isolated rabbit conjunctiva. Curr Eye Res 14:927–935, 1995

41. Turner HC, Alvarez LJ, Bildin VN, Candia OA: Immunolocalization of Na-K-ATPase, Na-K-Cl and Na-glucose cotransporters in the conjunctival epithelium. Curr Eye Res 21:843–850, 2000

42. Turner HC, Alvarez LJ, Candia OA: Cyclic AMP-dependent stimulation of basolateral K⁺ conductance in the rabbit conjunctival epithelium. Exp Eye Res 70:295–305, 2000

43. Zwick E, Daub H, Aoki N, et al: Critical role of calcium-dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J Biol Chem 272:24767–24770, 1997

44. Hamann S, Zeuthen T, La Cour M, et al: Aquaporins in complex tissues: distribution of aquaporins 1–5 in human and rat eye. Am J Physiol 274(5 Pt 1):C1332–1345, 1998

45. Candia OA, Shi XP, Alvarez LJ: Reduction in water permeability of the rabbit conjunctival epithelium by hypotonicity. Exp Eye Res 66:615–624, 1998

46. Hosoya K, Kompella UB, Kim KJ, Lee VH: Contribution of Na+-glucose cotransport to the short-circuit current in the pigmented rabbit conjunctiva. Curr Eye Res 15:447–451, 1996

47. Kompella UB, Kim KJ, Shiue MH, Lee VH: Possible existence of Na⁺-coupled amino acid transport in the pigmented rabbit conjunctiva. Life Sci 57:1427–1431, 1995

48. Kompella UB, Kim KJ, Shiue MH, Lee VH: Cyclic AMP modulation of active ion transport in the pigmented rabbit conjunctiva. J Ocul Pharmacol Ther 12:281–287, 1996

49. Diebold Y, Rios JD, Hodges RR, et al: Presence of nerves and their receptors in mouse and human conjunctival goblet cells. Invest Ophthalmol Vis Sci 42:2270–2282, 2001

50. Hosoya K, Ueda H, Kim KJ, Lee VH: Nucleotide stimulation of Cl^– secretion in the pigmented rabbit conjunctiva. J Pharmacol Exp Ther 291:53–59, 1999

51. Murakami T, Fujihara T, Nakamura M, Nakata K: P2Y₂ receptor stimulation increases tear fluid secretion in rabbits. Curr Eye Res 21:782–787, 2000

52. Alvarez LJ, Turner HC, Zamudio AC, Candia OA: Serotonin-elicited inhibition of Cl^– secretion in the rabbit conjunctival epithelium. Am J Physiol Cell Physiol 280:C581–592, 2001

53. Shiue MH, Kim KJ, Lee VH: Modulation of chloride secretion across the pigmented rabbit conjunctiva. Exp Eye Res 66:275–282, 1998

54. Alvarez LJ, Candia OA, Turner HC, Zamudio AC: Phorbol ester modulation of active ion transport across the rabbit conjunctival epithelium. Exp Eye Res 69:33–44, 1999

55. Komatsu M, Arango ME, Carraway KL: Synthesis and secretion of Muc4/sialomucin complex: Implication of intracellular proteolysis. Biochem J 368(Pt 1):41–48, 2002

56. Argueso P, Gipson IK: Epithelial mucins of the ocular surface: structure, biosynthesis and function. Exp Eye Res 73:281–289, 2001

57. Jumblatt MM, McKenzie RW, Steele PS, et al: MUC7 expression in the human lacrimal gland and conjunctiva. Cornea 22:41–45, 2003

58. McKenzie RW, Jumblatt JE, Jumblatt MM: Quantification of MUC2 and MUC5AC transcripts in human conjunctiva. Invest Ophthalmol Vis Sci 41:703–708, 2000

59. Argueso P, Spurr-Michaud S, Russo CL, et al: MUC16 mucin is expressed by the human ocular surface epithelia and carries the H185 carbohydrate epitope. Invest Ophthalmol Vis Sci 44:2487–2495, 2003

60. Inatomi T, Spurr-Michaud S, Tisdale AS, Gipson IK: Human corneal and conjunctival epithelia express MUC1 mucin. Invest Ophthalmol Vis Sci 36:1818–1827, 1995

61. Inatomi T, Spurr-Michaud S, Tisdale AS, et al: Expression of secretory mucin genes by human conjunctival epithelia. Invest Ophthalmol Vis Sci 37:1684–1692, 1996

62. Gamache DA, Wei ZY, Weimer LK, et al: Corneal protection by the ocular mucin secretagogue 15(S)-HETE in a rabbit model of desiccation-induced corneal defect. J Ocul Pharmacol Ther 18:349–361, 2002

63. Jumblatt JE, Cunningham LT, Li Y, Jumblatt MM: Characterization of human ocular mucin secretion mediated by 15(S)-HETE. Cornea 21:818–824, 2002

64. Dartt DA, McCarthy DM, Mercer HJ, et al: Localization of nerves adjacent to goblet cells in rat conjunctiva. Curr Eye Res 14:993–1000, 1995

65. Rios JD, Forde K, Diebold Y, et al: Development of conjunctival goblet cells and their neuroreceptor subtype expression. Invest Ophthalmol Vis Sci 41:2127–2137, 2000

66. Kessler TL, Mercer HJ, Zieske JD, et al: Stimulation of goblet cell mucous secretion by activation of nerves in rat conjunctiva. Curr Eye Res 14:985–992, 1995

67. Rios JD, Zoukhri D, Rawe IM, et al: Immunolocalization of muscarinic and VIP receptor subtypes and their role in stimulating goblet cell secretion. Invest Ophthalmol Vis Sci 40:1102–1111, 1999

68. Dartt DA, Kessler TL, Chung EH, Zieske JD: Vasoactive intestinal peptide-stimulated glycoconjugate secretion from conjunctival goblet cells. Exp Eye Res 63:27–34, 1996

69. Jumblatt JE, Jumblatt MM: Detection and quantification of conjunctival mucins. Adv Exp Med Biol 438:239–246, 1998

70. Jumblatt JE, Jumblatt MM: Regulation of ocular mucin secretion by P2Y2 nucleotide receptors in rabbit and human conjunctiva. Exp Eye Res 67:341–346, 1998

71. Dartt DA, Rios JR, Kanno H, et al: Regulation of conjunctival goblet cell secretion by Ca2+ and protein kinase C. Exp Eye Res 71:619–628, 2000

72. Kanno H, Horikawa Y, Hodges RR, et al: Cholinergic agonists transactivate the EGFR and stimulate MAPK to induce goblet cell secretion. Am J Physiol Cell Physiol 284:C988, 2003

73. Shatos MA, Rios JD, Horikawa Y, et al: Isolation and characterization of cultured human conjunctival goblet cells. Invest Ophthalmol Vis Sci 44:2477–2486, 2003

74. Narawane MA, Lee VH: IGF-I and EGF receptors in the pigmented rabbit bulbar conjunctiva. Curr Eye Res 14:905–910, 1995

75. Horikawa Y, Shatos MA, Hodges RR, et al: Activation of mitogen-activated protein kinase by cholinergic agonists and EGF in human compared with rat cultured conjunctival goblet cells. Invest Ophthalmol Vis Sci 44:2535–2544, 2003

76. Sacks EH, Wieczovek R, Jakobiec FA, Knowles DM: Lymphocytic subpopulations in the normal human conjunctiva: A monoclonal antibody study. Ophthalmology 93:1276, 1986

77. Knop N, Knop E: Conjunctiva-associated lymphoid tissue in the human eye. Invest Ophthalmol Vis Sci 41:1270–1279, 2000

78. Spyratos S: Étude de la vascularisation superficielle de l'oeil. Ann Ocul 199:754, 1966

79. Oduntan AO: Organization of capillaries in the primate conjunctiva. Ophthalmic Res 24:40–44, 1992

80. Holden BA, Sweeney DF: The oxygen tension and temperature of the superior palpebral conjunctiva. Acta Ophthalmol (Copenh) 63:100–103, 1985

81. Graffin A, Coddry E: A note on peripheral blood vascular beds in the bulbar conjunctiva of man. Bull Johns Hopkins Hosp 92:423, 1953

82. Graffin A, Coddry E: Studies of peripheral blood vascular beds in the bulbar conjunctiva man. Bull Johns Hopkins Hosp 93:275, 1953

83. Scarpelli P, Pellegrini M, Brancato R: L'ultrastruttura die capillari della conjiuntiva umana. Ann Ottal 92:977, 1966

84. Tamura T: Ultrastructure of human conjunctival capillaries. Acta Soc Ophthalmol Jpn 71:109, 1967

85. Deodati F: L'angiographie fluoresceinique du segment anterieu: son interet ses possibilities. Bull Soc Ophthalmol 70:33–52, 1970

86. Lockard I, Debacker H: Conjunctival circulation in relation to circulatory disorders. J South Carolina Med Assoc 63:201, 1967

87. Lee R: Anatomical and physiologic aspects of the capillary bed in the bulbar conjunctiva of man in health and disease. Angiology 63:69, 1955

88. Romani J: Manifestations angiopathiques chez les diabetiques et chez les obeses. Presse Med 77:1969, 1969

89. Francois J, Neetens A: Importance clinique de l'angioscopie conjonctivale. Acta Cardiol Angiol 16:109, 1967

90. Agarwal L, Chabra H, Batta R: Conjunctival vessels in diabetes mellitus. Orient Arch Ophthalmol 41:41, 1966

91. Hood J, Hodges RE: Ocular lesions in scurvy. Am J Clin Nutr 22:559–567, 1969

92. Wolf S, Arend O, Schulte K, et al: Quantification of retinal capillary density and flow velocity in patients with essential hypertension. Hypertension 23:464–467, 1994

93. Landesman R, Douglas RG, Dreishpoon G, Holze E: The vascular bed of the bulbar conjunctiva in the normal menstrual cycle. Am J Obstet Gynecol 66:988–998, 1953

94. Sugar HS, Riazi A, Schaffner R: The bulbar conjunctival lymphatics and their clinical significance. Trans Am Acad Ophthalmol Otolaryngol 61:212–223, 1957

95. Takayama T: Studies in histological changes of bulbar conjunctiva with special regard to the arterioles. Acta Soc Ophthalmol Jpn 64:1962, 1960

96. Kojo T: Changes on bulbar conjunctiva with age with special regard to the nature to be split and extensibility. Acta Soc Ophthalmol Jpn 64:2895, 1960

97. Frayser R, Knisely WH, Barnes R, Satterwhite WM Jr: In vivo observations on the conjunctival circulation in elderly subjects. J Gerontol 19:494–500, 1964

98. Ivanor V: On the resistance of the bulbar conjunctival vessels. Vestn Oftalmol 55:6, 1970

99. Schwab IR, Linberg JV, Gioia VM, et al: Foreshortening of the inferior conjunctival fornix associated with chronic glaucoma medications. Ophthalmology 99:197–202, 1992

100. Holly FJ: Formation and rupture of the tear film. Exp Eye Res 15:515–525, 1973

101. Jester JV, Nicolaides N, Smith RE: Meibomian gland studies: histologic and ultrastructural investigations. Invest Ophthalmol Vis Sci 20:537–547, 1981

102. Seifert P, Spitznas M: Immunocytochemical and ultrastructural evaluation of the distribution of nervous tissue and neuropeptides in the meibomian gland. Graefes Arch Clin Exp Ophthalmol 234:648–656, 1996

103. Olami Y, Zajicek G, Cogan M, et al: Turnover and migration of meibomian gland cells in rats' eyelids. Ophthalmic Res 33:170–175, 2001

104. LeDoux MS, Zhou Q, Murphy RB, et al: Parasympathetic innervation of the meibomian glands in rats. Invest Ophthalmol Vis Sci 42:2434–2441, 2001

105. Seifert P, Spitznas M: Vasoactive intestinal polypeptide (VIP) innervation of the human eyelid glands. Exp Eye Res 68:685–692, 1999

106. Chung CW, Tigges M, Stone RA: Peptidergic innervation of the primate meibomian gland. Invest Ophthalmol Vis Sci 37:238–245, 1996

107. Sullivan DA, Yamagami H, Liu M, et al: Sex steroids, the meibomian gland and evaporative dry eye. Adv Exp Med Biol 506(Pt A):389–399, 2002

108. Wickham LA, Gao J, Toda I, et al: Identification of androgen, estrogen and progesterone receptor mRNAs in the eye. Acta Ophthalmol Scand 78:146–153, 2000

109. Esmaeli B, Harvey JT, Hewlett B: Immunohistochemical evidence for estrogen receptors in meibomian glands. Ophthalmology 107:180–184, 2000

110. Suzuki T, Sullivan BD, Liu M, et al: Estrogen and progesterone effects on the morphology of the mouse meibomian gland. Adv Exp Med Biol 506(Pt A):483–488, 2002

111. Tiffany JM: Physiological properties of the meibomian glands. Prog Retin Eye Res 14:47–74, 1995

112. Hodges RR, Dartt DA: Regulatory pathways in lacrimal gland epithelium. Int Rev Cytol 231:129–196, 2003

113. Greiner JV, Covington HI, Allansmith MR: Surface morphology of the human upper tarsal conjunctiva. Am J Ophthalmol 83:892–905, 1977

114. Shatos MA, Rios JD, Tepavcevic V, et al: Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro. Invest Ophthalmol Vis Sci 42:1455–1464, 2001