The Conjunctiva—Structure and Function
DARLENE A. DARTT
Table Of Contents
THE TEAR FILM
ANATOMY OF THE CONJUNCTIVA
INNERVATION OF THE CONJUNCTIVA
CONJUNCTIVAL EPITHELIAL CELL ELECTOLYTE AND WATER TRANSPORT
MODULATION OF CONJUNCTIVAL EPITHELIAL CELL ELECTROLYTE AND WATER TRANSPORT
SIGNALING PATHWAYS THAT STIMULATE CONJUNCTIVAL ELECTROLYTE AND WATER TRANSPORT
REGULATION OF MUCIN SECRETION
STRATIFIED SQUAMOUS CELLS OF THE CONJUNCTIVA AND CORNEA
STRUCTURE OF THE CONJUNCTIVAL STROMA
VASCULAR SUPPLY OF THE CONJUNCTIVA
LYMPH SUPPLY AND DRAINAGE
PLICA SEMILUNARIS AND CARUNCLE
|The conjunctiva is a thin, semitransparent mucous membrane that covers the posterior surface of the eyelids and is then reflected onto the eyeball, where it extends to the limbus of the cornea. The conjunctiva takes its name from the fact that it conjoins the eyelids with the eyeball. This junction is indirect, with the conjunctiva forming a fornix on three sides of the globe and an extendible plica on the fourth side. Such an arrangement allows the globe and the eyelids to move independently of each other. When viewed by light microscopy, the conjunctiva is revealed as a nonkeratinizing squamous epithelium containing mucin-secreting goblet cells. This epithelium overlies a loose connective tissue, the substantia propria, which is highly vascularized, contains afferent, sensory, and efferent (sympathetic and parasympathetic) innervation, and is well endowed with lymphoid tissue. Not only does the conjunctiva function to enable independent motion of the globe and eyelids, it also protects the cornea and hence the interior of the eye from the external environment by secreting mucins, antibacterial proteins, electrolytes, and water to form the inner mucous layer of the tear film and perhaps a portion of the aqueous layer. Without these normal quantitative and qualitative conjunctival secretions, a variety of mucous-deficiency diseases develop that in the worse possible case lead to extensive damage to the cornea and a sight-threatening condition.|
|THE TEAR FILM|
|The tear film is composed of three layers: The innermost mucous layer is
secreted by the conjunctiva; the middle aqueous layer is secreted by
the main lacrimal gland and accessory lacrimal glands; and the outer
lipid layer is secreted by the meibomian glands (Fig. 1). Between the mucous layer and the corneal and conjunctival epithelia
there is a mucin-containing glycocalyx that extends from the apical
membrane of the superficial cells of these epithelia, which acts as
an interface between the ocular surface cells and the tears (Fig. 2). The lipid, aqueous, and mucous layers have been measured to be 0.1 μm, 7 to 10 μm, and 0.2 to 1.0 μm thick, respectively.1 Measurements by Prydal et al2 indicated a far thicker mucous layer of about 30 μm and an aqueous
layer of 10 to 11 μm; however, it is thought that these measurements
included a portion of the epithelium, and they have not been reproduced. In
fact, recent studies again found a thinner tear film and a pre-lens
tear film layer that averaged 2.7 μm.3,4 One hypothesis about the structure of the tear film is that the mucous
and aqueous layers are not distinct, but rather are a gradient of decreasing
mucous and increasing aqueous content from the ocular surface
to the lipid layer. Another suggests that the lipid layer is a monolayer
consisting of two phases: a polar phase adjacent to the aqueous layer, and
a nonpolar phase at the tear film–air interface.5 Additional experiments are required to resolve this controversy.|
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.”
|ANATOMY OF THE CONJUNCTIVA|
|The conjunctiva starts at the posterior third of the meibomian (also
known as the tarsal) gland openings and along the palpebral margin
of each eyelid (Fig. 3). In this area, the cells change from the keratinized stratified
squamous epithelium of the eyelid to the nonkeratinized stratified squamous
epithelium of the marginal conjunctiva. This forms a mucocutaneous
junction that is covered with lipids secreted by the meibomian glands. The
lipids restrict the tears behind the openings of the glands, resulting
in a division of the eyelid margin into an anterior, dry, keratinized
part and a posterior, wet, nonkeratinized conjunctival portion. The
tarsal conjunctiva is continually covered with tears, and it is
this area that the lacrimal puncta open to connect the conjunctival sac
with the nose (Fig. 4). The conjunctiva then passes over the eyelid margin, forming a right
angle to the tarsi forming the superior and inferior fornix, which
allows for a sweeping mechanism to clean the bulbar conjunctiva and
the cornea. From the fornix, the conjunctiva proceeds to the eyeball, forming
the anterior portion. The conjunctiva ends at the corneal limbus. This
arrangement forms a mucous membrane-lined sac, with the eye positioned
in the posterior part of the sac. The palpebral fissure is the
only opening to the external environment. |
For descriptive purposes the conjunctiva may be divided into three major subdivisions (as shown in Fig. 3):
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 nonkeratinized conjunctival epithelium consists of several layers of
stratified squamous cells that do not substantially vary over the different
regions of the conjunctiva.11 The often-reported variation in the number of cell layers is thought to
be the result of variable stretching of the elastic conjunctiva.11 The basal layer of the epithelium undulates along with the rippling of
the underlying substantia propria. Scattered within the epithelium of
the conjunctival surface are numerous mucin-secreting goblet cells and
mucous glands (Fig. 6). Human conjunctiva contain intraepithelial clusters of cells known
as the glands of Manz and the crypts of Henle.12 Goblet cells are not evenly distributed throughout the conjunctiva, and
range from 1,000 to 56,000 per square millimeter of conjunctival surface
depending on the presence or absence of inflammation and the area
studied.13 The fornices contain more goblet cells per unit area than the tarsal and
bulbar conjunctivas. The inferior conjunctiva contains more goblet cells
compared to the superior conjunctiva, and the nasal conjunctiva contains
more than the temporal conjunctiva.14 |
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
|INNERVATION OF THE CONJUNCTIVA|
|The external portion of the visual system–the eyebrows, eyelids, conjunctiva, and
cornea–is richly endowed with sensory nerve endings. This
abundant and highly integrated sensory system is necessary
for the protection, coordination, and general well-being of the eyeball
and the aforementioned periorbital structures.|
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:
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
|CONJUNCTIVAL EPITHELIAL CELL ELECTOLYTE AND WATER TRANSPORT|
|Under basal conditions, fluid secretion from conjunctival epithelial cells
into tears prevails over absorption to account for the volume of the
tear film.37,38 Studies on fluid secretion from the conjunctiva have employed a mixed
population of both stratified squamous cells and goblet cells. Ion transporters, which
are necessary for electrolyte and water secretion, have
been localized to both of these cell types. Thus, both must be considered
as secreting electrolytes and water. The movement of two ions (Cl−and Na+) drives conjunctival epithelial fluid transport (Fig. 8). Under short-circuit conditions, Cl−secretion (in a basolateral to apical movement) accounts for
about 80% of the ion transport, while Na+ absorption (apical to basolateral) accounts for about 20% of
the ion transport measured.39,40 Because the Na+/glucose transporter SGLUT1, NKCC, and Na+, K+-ATPase are all located on conjunctival stratified squamous cells and goblet
cells, it appears that the same cell type is both secretory and
absorptive. There are no conjunctival cells that have only SGLUT1 and
not NKCC or Na+, K+-ATPase. This is unique to the conjunctiva.41,42|
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
|MODULATION OF CONJUNCTIVAL EPITHELIAL CELL ELECTROLYTE AND WATER TRANSPORT|
|Conjunctival fluid secretion can be stimulated by numerous t ypes of neurotransmitters
and agonists. The activation of β2-adrenergic receptors by the release of norepinephrine from sympathetic
nerves stimulates Cl−secretion (Fig. 8).40,42,48 Although receptors for β-adrenergic agonists have been localized
in the mouse, rat, and human conjunctiva, it appears that humans have β1- but not β2-adrenergic receptors.49 In all three species these receptors were found on the basolateral membranes
of conjunctival epithelial cells. Neither β-adrenergic nor
cholinergic agonists stimulate conjunctival fluid secretion, despite
the presence of several subtypes of muscarinic receptors (activated
by cholinergic agonists) on stratified squamous and goblet cells.42 It is not clear whether β-adrenergic receptors occur in the conjunctival
epithelium, since these receptors have not been found consistently. Thus
far, only sympathetic nerves using β2-adrenergic receptors are known to stimulate conjunctival fluid secretion.
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.
|SIGNALING PATHWAYS THAT STIMULATE CONJUNCTIVAL ELECTROLYTE AND WATER TRANSPORT|
|As described above, β-adrenergic agonists stimulate conjunctival fluid
secretion40,42,48 (Fig. 8). They do so by activating the cAMP-dependent signally
pathway, which results in activation of adenylyl cyclase. This enzyme
increases the cellular cAMP level, which results in stimulation of
Cl−secretion. The addition of permeable cAMP analogs, or the inhibition of
cAMP phosphodiesterase activity, also stimulates Cl−secretion. Furthermore, the inhibition of adenylyl cyclase or protein kinase
A blocks secretion stimulated by epinephrine.
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.
|REGULATION OF MUCIN SECRETION|
|The innermost layer of the tear film is the mucous layer. This layer consists
of electrolytes, water, and mucins. Mucins are highly glycosylated
glycoproteins that consist of a protein core with multiple heterogeneous
oligosaccharide side chains. At least 20 different mucins, known
as MUCs, have been identified and classified according to the structure
of their protein cores.11 MUCs can be classified as either soluble or membrane-bound. Some soluble
mucins can be large-molecular-weight molecules that form gels to provide
the scaffold for the mucous layer. These gel-forming MUCs are stored
in secretory granules in condensed form and are secreted upon stimulation. Other
soluble mucins have smaller molecular weights than the
gel-forming mucins. These mucins, like the gel-forming MUCs, are also
stored in secretory vesicles and secreted upon stimulation. Membrane-bound
mucins have a cytoplasmic tail, transmembrane domain, and extracellular
domain, and have a smaller molecular weight than the gel-forming
mucins. These mucins are found in many cellular membranes, particularly
the plasma membrane, where they form the glycocalyx of the apical
surface of the ocular surface epithelia. Membrane-bound mucins can also
become incorporated into the mucous layer in a process known as ectodomain
shedding. This occurs when the extracellular domain is cleaved
by a specific metalloprotease. In addition, the membrane-bound mucin, MUC4 (also
known as asialoglycoprotein (ASPG)), has
also been found in the mucous layer. This molecule is cleaved intracellularly
shortly after synthesis to form a membrane-bound dimer and
a soluble portion.55 The soluble portion is stored in secretory granules and released into
the tear film upon stimulation.|
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.
|STRATIFIED SQUAMOUS CELLS OF THE CONJUNCTIVA AND CORNEA|
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.
|STRUCTURE OF THE CONJUNCTIVAL STROMA|
|The conjunctival stroma lies beneath the epithelial basement membrane. The
stroma is composed of two layers. The most superficial layer, the
reticulum, is a complex of connective tissue. This is intertwined with
a homogeneous lamina of mature lymphocytes. This lymphocyte or “adenoid” layer
is 50 to 70 μm thick, and does not appear until 8 to 12 weeks
of age. No follicles are present in the noninflamed conjunctiva; however, nodules are common and may raise the epithelium. This
can result in irregularities on the ocular surface. If infectious
agents are present or irritation of the conjunctiva occurs, true follicles
can form and may be visible. These areas of lymphocytic infiltration
can raise the conjunctival epithelium, such that a system of crests, valleys, and
troughs is formed on the surface. A central vascular
core can occur. This changes the avascular follicle into a papilla, which
is an indication of a persistent and severe inflammatory response
or a toxic reaction.|
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.
|VASCULAR SUPPLY OF THE CONJUNCTIVA|
|The blood vessels and nerves of the conjunctiva are located below the lymphoid
layer in a layer of fibrous tissue of variable thickness (Fig. 11). The muscular, medial palpebral, and lacrimal branches of the ophthalmic
artery supply the conjunctiva with blood and are derived from
the internal carotid artery. The tarsal arcades are comprised of the
medial palpebral and lacrimal arteries, and lie between the tarsus and
the orbicularis. Arteries penetrate the tarsi near the subtarsal groove, and
arterial branches supply the marginal, pretarsal fornices and
most of the bulbar conjunctiva. The areas of the bulbar conjunctiva that
lie within 3 to 4 mm of the corneal limbus do not contain any arteries. The
perilimbal conjunctiva receives its blood supply from the anterior
ciliary arteries, which are derived from the muscular branches that
serve the rectus muscles.|
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.
|LYMPH SUPPLY AND DRAINAGE|
|The conjunctiva contains a rich network of lymphatic tissue with extensive subepithelial lymphatic tissue. Many small, irregular lymphatic channels are present approximately 1 mm around the corneoscleral limbus. These join with one another to form large collecting channels in the stroma. Drainage from this collecting system joins the lymphatic system of the eyelids.94 Dilation of one of these large lymph-collecting channels may become obvious and is known clinically as a lymphangiectasis.|
|PLICA SEMILUNARIS AND CARUNCLE|
|The plica semilunaris is a crescent-shaped fold of conjunctiva that is
situated medially and conjoins the bulbar conjunctiva with the caruncle
and lacrimal portion of the eyelids (Fig. 3). It is located
in the superior fornix at the junction of extending downward, surrounding
the limbus to end in the inferior fornix. It extends 3 to 6 mm laterally
from the caruncle. The nictitating membrane present in some animals
is the counterpart to the plica, and is a partial or complete third
eyelid. Although humans do not have a nictitating membrane, occasionally
smooth muscle fibers may be present that are innervated with sympathetic
nerves. Goblet cells are present in the plica either singly
or in clusters. In humans, the plica functions as the opposite of a fornix; that
is, if the conjunctiva were to directly join the eyelids to
the globe, the globe and eyelids would both be restricted in movement. The
fornix provides for a fold of conjunctiva that may be extended
or retracted as the globe moves. Extension occurs because of fibrous slips
that connect the fornix to its extraocular rectus muscle. As the
muscle contracts, the globe rotates and the adjacent conjunctiva is retracted. This
occurs above, laterally, and below the globe, but not medially, which
would not allow the lacrimal puncta to drain the lacrimal
lake. On abduction the plica tends to unfold and flatten, whereas on
adduction it is drawn posteriorly and is unfolded by the fibrous slips
that extend to the plica and caruncle from the medial rectus. While
it never completely unfolds, extreme adduction of the plica causes it
to form a true fornix. As this occurs, a small movement of the globe occurs
as a result of the retraction of the medial canthal tendon. This
keeps the lacrimal puncta properly positioned with the lacrimal lake. The
puncta now dips into the lacrimal strip to allow continuous drainage
despite the position of the globe. In addition, the plica helps to
maintain the lacrimal lake in its proper position and location in the
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.
|As humans age, the conjunctival epithelial cells change from nearly square (in childhood) to vertically elongated, and decrease in height as the aging process progresses. In addition, the number of epithelial layers is reduced during aging.95 A decrease in collagen within the conjunctival stroma, and a more tenuous attachment to the Tenon's fascia result in much easier movement of the conjunctival flap.96 Blood vessels in the bulbar conjunctiva become more random, with an increase in capillary fragility.97,98 Some medications, such as glaucoma medication, can worsen the shortening of the inferior fornix that occurs naturally with age.99|
|The meibomian glands are found in the upper and lower eyelids, and secrete
the lipid layer of the tear film. The lipid secreted by the meibomian
glands is a complex fluid that contains a large number of lipids, all
of which are fluid in the eye. The lipid layer moves independently
of the aqueous layer underneath. The lipid layer extends from the orifices
of the meibomian glands at the top and bottom. It is not involved
in the flow of tears from the lateral canthus to the lacrimal puncta.1 When the eyelid closes, such as during a blink, the lipid layer thickens
as it is compressed over the aqueous layer.100 When the eyelid opens, the lipid layer begins to thin to again spread
over the aqueous layer. The aqueous layer is never exposed, because the
lipid layer spreads faster than the lid can open.100|
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
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