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

generally considered to correspond to borderline dry eye, although it should be noted that this reported range was originally established for Caucasian eyes, and Asian eyes may exhibit significantly shorter tear film stabilities. In noninvasive techniques, without instillation of fluorescein, the reported cut-off values are longer with mean values around double those of the traditional fluorescein breakup test.

The distribution of the tear film can further be observed in vivo using thin film interferometry. Interference fringes are produced by light reflected at the air-lipid and at the lipid-aqueous boundaries of the tear film due to the changes in refractive index. Specular reflection from the lipid layer precludes a clear view of the aqueous layer of the precorneal tear film although where the lipid layer is very thin or absent, aqueous fringes may be observed.

Based on this optical principle, a number of clinical instruments, together with qualitative grading systems have been developed. These are useful for observing the structure of the tear film and offer some insight into its stability. Significant differences in appearance (and grade)

have been observed in dry eye conditions, with the partial or complete absence of the lipid layer being a feature. Recent work in this field has concentrated on developing quantitative analyses of interferometric images from the tear film of normal and dry eye patients (Figures 4 and 5, respectively). With the use of kinetic analysis of sequential interference images, it has been possible to quantify the lipid-spread time of tears in normal and dry eye patients. This spread time, defined as the time taken for the lipid film to reach a stable interference image, is significantly slower in ADDE, at 2.17 1.09 s, than it is in normal eyes (0.36 0.22 s). Because of this slower spread time, the resultant lipid film has been found to be thicker on the inferior cornea than the superior cornea, with the thickness being measured from a color reference chart created from the reflectance images of thin film interference generated by a white light source. Almost 90% of the patients with aqueous tear deficiency exhibit an interferometric pattern with vertical streaking, rather than the horizontal propagation typically observed in the superior corneal region.

Evaluation of tear film particle movement can also provide an indication of the time necessary to obtain stability of the tear film after the blink. The observed particles are thought to be accumulations of newly secreted lipid from the meibomian glands. Measuring the displacement of these tear film particles immediately after a blink has shown that the time necessary to reach zero velocity (tear stabilization time) is 1.05 0.3 s.

A commercial thin film interferometer has been developed, which enables the specular reflection from the tear surface to be monitored digitally and the tear film interference patterns classified. Research with this apparatus has shown that thicker lipid layers are associated with greater tear film stability. A number of grading systems have been developed mostly assessing the uniformity of the interference fringe pattern. A change in color and loss of uniformity in distribution indicates tear film instability. Such patterns are found more commonly in dry eyes in association with thin lipid layers and reduced stability.

Assessment of the reflected images from the cornea and tear film has been used to evaluate tear film quality and stabilization following the blink. High-speed videokeratoscopy assesses the regularity indices, such as surface regularity index (SRI) and surface asymmetry index (SAI), in the time interval following a blink. These indices

have been found to correlate significantly with the results of standard diagnostic tests for dry eye, such as symptoms, tear breakup time, Schirmer test, fluorescein staining score, and best corrected visual acuity.

Tear Film Osmolarity

Adequate production, retention, distribution, and balanced elimination of tears are necessary for ocular surface health and normal function. Any imbalance of these components can lead to the condition of dry eye. A single biophysical measurement that captures the balance of inputs and outputs from the tear film dynamics is tear osmolarity, the end-product of variations in tear dynamics. Normal homeostasis requires regulated tear flow, the primary driver of which is osmolarity. Hyperosmolarity is thus an important biomarker for dry eye disease.

Tear hyperosmolarity has been found to be the primary cause of discomfort, ocular surface damage, and inflammation in dry eye. In studies of rabbit eyes, tear osmolarity has been found to be a function of tear flow rate and evaporation. In rabbit conjunctival cell cultures, hyperosmolarity has been demonstrated to decrease the density of goblet cells and, in humans, a 17% decrease in

goblet cells density for subjects with dry eye has been reported. Granulocyte survival is significantly decreased with increases in solute concentration. Rabbit cells cultured in hyperosmolar states, above 330 mOs ml–1, show significant morphological changes, similar to those seen in subjects with dry eye. Hyperosmolarity-induced changes in surface cells in dry eye can be correlated with the degree and distribution of rose bengal staining.

Measuring tear osmolarity is of benefit in the diagnosis of conditions such as dry eye. In a meta-analysis of human tear osmolarity values recorded in studies between 1978 and 2004 with freezing point depression (FPD) and vapor pressure (VP) osmolarity tests, normal values averaged 302.0 9.7 compared with 326.9 22.1 mOs ml–1 for patients with dry eye disease.

Drainage of Tears

A principal means of elimination of tears from the eye is by drainage through the puncta of the eye. Tears then pass through the canaliculi, the lacrimal sac, and finally the nasolacrimal duct before reaching the nose. A technique for measuring tear turnover, which allows direct observation of tear drainage, involves instilling a radioactive dye into the tear film. In the technique of lacrimal scintigraphy a small quantity (0.013 mls) radioactive tracer such as technetium 99 (99M Tc), is introduced into the lower marginal tear strip. The distribution of the tracer is imaged serially by a gamma camera as it passes down the lacrimal drainage system (Figure 6 a–c). Images are typically taken at 10-s intervals for 1 min and then at less frequent intervals until all of the tracer has drained into the nasal cavity. The technique has been used to quantify tear turnover from the eye and drainage through

the lacrimal system. The drainage through this system is not linear, as a significant number of naso-lacrimal folds and ducts offer physiological obstruction to normal tear flow, and variable tear flow has been shown to be a typical feature of the drainage facility in asymptomatic individuals (Figure 6(d) and (e)). Therefore, most models of lacrimal drainage favor compartmental analysis to evaluate tear flow through the system, with separate components for the conjunctival sac, lacrimal sac, the nasolacrimal duct, and the nasal cavity. Although most quantitative lacrimal scintigraphy measurements describe the transit time of the radioactive tracer through the system, the compartmental model has be used to estimate tear flow rates. Depending on the number of compartments considered, basal flow rates have been estimated to fall between 0.45 and 8 ml min–1. Using a single compartment model for decay of the radioactive tracer on the conjunctival surface, mean values of reflex and basal turnover of 3.33 1.95 ml min–1 and 0.56 0.32 ml min–1, respectively, have been recorded by gamma scintigraphy.

The mechanism of lacrimal drainage and the influence of blinking on the mechanics of the system have been observed by high-speed photography and by intracanalicular pressure measurements. Taking an anatomical approach and observing the lacrimal systems of human cadavers has shown that the surrounding vascular plexus of the lacrimal sac and the nasolacrimal duct is comparable to a cavernous body. While regulating the blood flow, the specialized blood vessels of this body permit opening and closing of the lumen of the lacrimal passage, which is effected by the bulging and subsiding of the cavernous body, thereby regulating tear outflow from the eye. Attempts have been made to quantify the regulation of tear outflow by measurement of the transit time of a fluorescein drop from the conjunctival sac into the inferior meatus

of the nose. Application of a decongestant drug or placement of a foreign body on the ocular surface have both been found to significantly prolong the dye transit time, indicated restricted drainage through the lacrimal system in these conditions. It has therefore been concluded that the cavernous body of the lacrimal sac and naso-lacrimal duct plays an important role in the physiology of tear outflow regulation; it is subject to autonomic control and is integrated into a complex neural reflex feedback mechanism between the blood vessels, the cavernous body, and the ocular surface.

Absorption of Tears by the Ocular Surface

Another method by which tears can be eliminated from the eye is by absorption into the tissues of the ocular surface and the drainage system. The possibility has been suggested that the epithelial lining of the drainage system absorbs tear fluid before it reaches the nose. It has been shown in an animal model that lipophilic substances are absorbed from the tear fluid by the epithelium of the naso-lacrimal duct and that the cavernous body surrounding this duct may play a role in drainage of absorbed fluid. No quantification of fluid volume eliminated by this route has been reported. However, tears absorbed in the blood vessels of the cavernous body may, because these vessels connect to the blood vessels of the outer eye, have a role in a biofeedback mechanism for tear production.

Observations of the absorption of tear film onto the anterior ocular surface have been made in studies of corneal permeability. The proportion absorbed, in the absence of compromised corneal function, appears to be small at 0.24 0.13% of the dye instilled in the eye.

The lacrimal system of the human eye is, in the vast majority of individuals, a robust system, which allows the ocular surface to maintain its health and normal function throughout life, and under modest provocation. It is only in a relatively small proportion ( 15%) that the imbalance between evaporative loss and tear production results in dry eye. Recent research has confirmed that an increase in this ratio of approximately 2–3 times, as most often occurs in older individuals, appears to lead the condition of dry eye.

The tears covering the anterior ocular surface, form a dynamic structure with a complex nature and a number of important functions. The tear film components are

interdependent and have a close relationship with those of the adjacent ocular tissues such that failure of any one of aspect of the tear film or lacrimal system can cause imbalance and result in dry eye.

See also: Conjunctival Goblet Cells; Contact Lenses; Defense Mechanisms of Tears and Ocular Surface; Dry Eye: An Immune-Based Inflammation; Eyelid Anatomy and the Pathophysiology of Blinking; Inflammation of the Conjunctiva; Lacrimal Gland Overview; Lids: Anatomy, Pathophysiology, Mucocutaneous Junction; Meibomian Glands and Lipid Layer; Tear Drainage.

Further Reading

Bron, A. J., Yokoi, N., Gaffney, E., and Tiffany, J. M. (2009). Predicted phenotypes of dry eye: Proposed consequences of its natural history. Ocular Surface 7(2): 78–92.

Craig, J. P. (2002). Structure and function of the preocular tear film. In: Korb, D. R. (ed.) The Tear Film: Structure, Function and Clinical Examination, pp. 18–50. London: Elsevier Health Sciences.

Dartt, D. A. (2004). Dysfunctional neural regulation of lacrimal gland secretion and its role in the pathogenesis of dry eye syndromes.

Ocular Surface 2(2): 76–91.

Doane, M. G. (1994). Abnormalities of the structure of the superficial lipid layer on the in vivo dry-eye tear film. Advances in Experimental Medicine and Biology 350: 489–493.

Gilbard, J. P. (1985). Tear film osmolarity and keratoconjunctivitis sicca.

Contact Lens Association of Ophthalmologists Journal 11(3): 243–250.

Gipson, I. K., Hori, Y., and Argu¨eso, P. (2004). Character of ocular surface mucins and their alteration in dry eye disease. Ocular Surface 2(2): 131–148.

King-Smith, P. E., Fink, B. A., Fogt, N., et al. (2000). The thickness of the human precorneal tear film: Evidence from reflection spectra.

Investigative Ophthalmology and Visual Science 41(11): 3348–3359. Mathers, W. D. and Choi, D. (2004). Cluster analysis of patients with

ocular surface disease, blepharitis, and dry eye. Archives of Ophthalmology 122(11): 1700–1704.

McCulley, J. P. and Shine, W. E. (2003). Meibomian gland function and the tear lipid layer. Ocular Surface 1(3): 97–106.

Sariri, R. and Ghafoori, H. (2008). Tear proteins in health, disease, and contact lens wear. Biochemistry (Moscow) 73(4): 381–392.

Stern, M. E., Beuerman, R. W., and Pflugfelder, S. (2004). Dry Eye and Ocular Surface Disorders; the Normal Tear Film and Ocular Surface. New York: Marcel Dekker.

Tiffany, J. M. (2008). The normal tear film. Developments in Ophthalmology 41: 1–20.

Tomlinson, A. and Khanal, S. (2005). Assessment of tear film dynamics: Quantification approach. Ocular Surface 3(2): 81–95.

van Best, J. A., Benitez del Castillo, J. M., and Coulangeon, L. M. (1995). Measurement of basal tear turnover using a standardized protocol. European concerted action on ocular fluorometry. Graefes Archive for Clinical and Experimental Ophthalmology 233(1): 1–7.

Glossary

Acinus – A gland that is shaped like a hollow sphere with the gland cells lining the sphere and secreting into the center of the sphere. The secretions are removed from the center of the sphere by a duct. HMG-CoA – The 3-hydroxy-3-methyl-glutaryl- coenzyme A is a precursor molecule for lipid synthesis with the small precursor molecule attached to a carrier molecule, coenzyme A.

Holocrine – A mechanism of secretion by a gland wherein the whole gland cell is secreted. Hydrophilic – A substance that dissolves readily in water (water loving).

Hydrophobic – A substance that does not like water. Fats, lipids, and oils are common hydrophobic substances.

mN m 1(millinewtons per meter) – A unit used for measuring surface pressure relative to that of water which is regarded as 0 mN m 1.

Osmolarity – A measure of the number of individual molecules dissolved in water. It is important to cells because water can pass readily through a cell membrane, but the dissolved chemicals in the cytoplasm cannot. Hence, a cell will either take on water or release water depending upon whether its osmolarity is more (hyperosmolar) or less (hypoosmolar) than its environment, respectively. Refractive index – When light travels from one medium to another, for example, from air into water, it is bent. The refractive index is a measure of the extent to which the light is bent and is a constant for a particular substance.

Tarsal plate – A sheet of fibrous cartilage in the eyelids of mammals that gives the eyelids their stiffness and shape.

Overview

Meibomian glands are a series of fat (lipid)-producing glands found in the upper and lower eyelids of mammals, named after a German anatomist, Heinrich Meibom (1638–1700), who recorded their presence in De Vasis Palpebrarum Novis Epistola (1666). In humans, there are 30–40 evenly spread glands in the upper lid and 20–30 in the lower lid (Figure 1). Each gland is aligned vertically in

the eyelid and located within the tarsal plate (a sheet of fibrous cartilage that gives the eyelids their stiffness and shape) which lies closer to the ocular surface than the dermal surface. Structurally, meibomian glands have a central tubular duct surrounded by grape-like acini (glands). The duct is blind at one end and the other end opens onto the eyelid margin. By everting the lower eyelid, the openings can be readily seen as a row of small dots behind the eyelashes (Figure 2).

Phylogenetically, meibomian glands are present in marsupials, but are absent in the two monotremes (echidna,

Tachyglossus aculeatus; platypus, Ornithorhynchus anatinus) that we have studied. They are not present in reptiles or birds. It is believed that many of these animals use a different gland, the harderian gland, to secrete lipids onto the ocular surface. While the distribution and appearance of meibomian glands in other mammals are generally similar to those of humans, this is not always the case. Some species of voles and musk rats have few glands, for example, Microtus pinetorum, which has only two large glands in the upper eyelid – one at the medial canthus and the other at the lateral canthus. Whales have neither meibomian glands nor a tarsal plate in the eyelid. Dolphins and sea lions have a very oily secretion in their tears, but this is thought to originate from the harderian gland. Currently, the literature is not clear about the presence or absence of meibomian glands in sea-dwelling mammals. However, it is of interest to note that dolphins, sea lions, and sea otters have no eyelashes (or eyebrows), which means that they also lack the other major sebaceous glands in the eyelid, for example, the glands of Zeis which are the sebaceous glands of the eyelashes.

The major function of the meibomian gland is to supply the main components of the outer layer of the tear film. The tear film is a thin (7–10 mm thick), watery fluid that covers the exposed surface of our eyes (Figure 3). Lipids from the meibomian glands are secreted onto the inner margin of the eyelids where they contact and then spread over the aqueous part of the tear film to form a covering layer ( 90 nm thick) in contact with the air. This layer is referred to as the lipid layer of the tear film. It is believed to decrease evaporation from the tear film and hence prevent dry eyes. However, this role for the meibomian lipids is by no means certain, and it is likely that it has other roles such as preventing tears from flowing onto the skin and skin lipids from flowing onto the ocular surface, assisting the spread of the tear film over the eye by lowering the surface tension, and forming a watertight seal when the lids are closed. The Meibomian lipids provide a smooth and

Figure 1 Arrangement of the meibomian glands in the upper and lower eyelids.

Figure 2 Meibomian gland orifices (arrows) in the lower lid margin.

highly refractive (1.4766 at 589 nm and 35 C) surface. Clinically, a mechanism for measuring tear breakup time (TBUT) is to observe changes to interference colors of the surface layer of the tear film. This can be used as one measure of the tear film performance.

The Lipid Layer of the Tear Film

The lipid layer of the tear film provides an optically smooth surface at the interface between the air and the aqueous part of the tear film. Although the structure of this layer has not been determined, the meibomian lipids form a major component (Figure 3). A useful model of the lipid layer, based on the idea that only lipids are present, was developed by McCulley and Shine. The model is presented as a crystalline array, and, while very useful

for developing understanding, the lipid layer is unlikely to be crystalline in practice. This model proposed that the wax esters, steryl esters, triglycerides, and hydrocarbons (hydrophobic lipids) from the meibomian glands reside in the outermost layers and are linked to the aqueous subphase by polar lipids (phospholipids, free cholesterol, and free fatty acids). Some of these polar lipids may arise from the aqueous layer of the tear film and others from meibomian lipids and hydrolysis (cleavage) of the wax esters, steryl esters, and triglycerides. These polar lipids have a hydrophobic part which interacts with the hydrophobic lipids and a hydrophilic part that is able to interact with water (the aqueous layer). Assuming that the outer layer of the tear film is entirely made from lipids implies that the interfacial molecules (surfactants) must be polar lipids.

An alternate model proposing that proteins and mucins also contribute to this layer is more realistic. Strong evidence comes from surface tension measurements. It has been found that the surface tension of tears is 42–46 mN m 1 and this can only be achieved by a mixture of lipids and proteins and not with meibomian lipids alone. The interaction of meibomian lipids with tear proteins is often cautiously presented as lipids occupying the outer surface of the lipid layer, and the inner surface interacting with proteins from the aqueous layer. A more recent model includes proteins and mucins as integrated parts of this layer (Figure 3). Although there has been a focus on lipocalin, an abundant lipid-binding protein in tears, being the main tear protein interacting with the meibomian lipids, lysozyme and lactoferrin may be more involved. Lipocalin is thought to scavenge lipids that have adhered to the epithelial cells of the ocular surface and lipids that are in the aqueous layer. Although it has been claimed that these are then transported to the outer lipid layer, this may not be the case. Once a lipid is bound into the central pocket of lipocalin, lipocalin is in a low-energy state and unlikely to interact with the meibomian lipids at the outer surface of the tear film.

The presence of proteins in the lipid layer has important conceptual implications because they are large molecules with complex mixtures of hydrophobic, hydrophilic, and distinctly charged components. These properties mean that they can unfold and form a range of shapes according to their local molecular environment. Due to this unfolding, it is possible for them to extend across the lipid layer and interact with the hydrophobic lipids and other proteins. As the model suggests, this means that the layer comprises a complex mixture of islands of proteins, islands of lipids, islands of mucins, and various mixtures of these (Figure 3). This model is more akin to models for cell membranes and for lung surfactant. Some advantages of this model are that: the outer layer would be a noncollapsible viscoelastic gel; it would allow for the lowest free energy states of the proteins in contact with lipids; and the changes in salt concentrations in the tears

(e.g., hyperosmolarity) would affect the ability of the proteins to unfold and interact with lipids in the outer layer. Taking these one at a time, the lipid-only model needs some mechanism to spread the lipids over the aqueous surface after a blink. Both phospholipids and mucins have been proposed as enabling this by spreading slightly ahead of the hydrophobic lipids, and this concept also means that a new lipid layer is formed at each blink. Lack of change over a number of blinks to the interference patterns formed by the outer layer of the tear film does not support this concept. However, a noncollapsible viscoelastic gel that would be formed with proteins and mucins in the layer obviates the need for spreading and would account for the consistent interference pattern over a number of blinks. Furthermore, in other fields of study, mixtures of molecules in a liquid environment autoassemble into states of lowest free energy, which means that when lipids and proteins are placed together, they will mix at the molecular level rather than remain separate as the lipid-only model suggests. Some of the proteins can act as the surfactants, meaning that while phospholipids may co-jointly serve as surfactants, they are not absolutely necessary. It has also been found that while lipids alone are not capable of lowering the surface tension to the levels found in tears, mixtures of tear proteins and tear lipids possess that ability. Hyperosmolarity of the tears has the strongest correlation with a dry eye. Salt concentrations have strong effects on how proteins fold; therefore, it is possible that high salt concentrations would affect the folding of proteins and hence their ability to interact with the lipid layer to form a stable outer layer.

In turn, this could affect the surface tension and spreading of the outer layer and, consequently, its protective function.

Meibomian Glands

Anatomy and Histology

There are no anatomical differences in the meibomian glands of human males and females. The ducts in humans are approximately 1.6 mm long with the central ducts being slightly longer than the nasal and temporal ducts, and are surrounded by a dense banding of elastic fibers. The ducts are lined by keratinized epithelium and lie nearly 780 mm from the dermal surface of the eyelid. A horny cell layer overlies one or two layers of intermediate cells that rest on cuboidal basal cells connected to a basement membrane, and there is no difference in this appearance between the proximal and distal portions of the duct (Figure 4). Acini are arranged circularly around the central duct and are connected to it by short ductules. The acinar cells are distinct from the ductal cells with no keratohyaline granules or lamellar bodies in the acinar cells, and no lipid vesicles in the ductal cells. The acinar cells continually differentiate into holocrine-secret- ing cells from basal acinar cells. They contain an abundance of smooth endoplasmic reticulum that surrounds the lipid vesicles. These cells also develop from the division of basal cells and move toward the center of the acinus (migration rate of 0.62 mm d 1 in rats), and slowly increase in neutral lipid content and in the size of the lipid-containing vesicles. The acinar cells die and gradually breakdown, leaving a

Figure 4 An illustration of meibomian glands and duct. A: Ghosts of acini not filled in with detail. B: Acini showing nucleated basal cells around the periphery which gradually

lose their nuclei as they mature and move to the center of the acinus. C: Secondary duct surrounded by ductal cells (stratified epithelium) containing meibomian secretion and cell remnants. D: Main duct surrounded by ductal cells containing meibomian secretion and cell remnants. The arrow shows a bundle of nerve endings close to the acinus but separated by collagen fibers.

lipid mass that is secreted via the duct. In rats, it takes close to 9 days for a cell to migrate to the center of the acinus, and it probably divides twice during this time.

Surrounding the glands is a distinct extracellular matrix comprising collagen types I, II, and IV, aggrecan, dermatan sulfate, and chondroitin-6-sulfate. Close to the acini are numerous unmyelinated varicose nerve fibers with boutons in contact with collagen fibers within the basal lamina of the basal acinar cells. These are mainly parasympathetic fibers which contain the neurotransmitter, acetylcholine, and the neuropeptide vasoactive intestinal polypeptide. The cell bodies for these fibers lie in the pterygopalatine ganglion and their fibers reach the eyelid by the greater petrosal nerve. Preganglionic neurons are ipsilateral and cholinergic, and lie in the superior salivary nucleus located lateral, dorsal, and caudal to the superior olive and lateral, dorsal, and rostral to the facial nucleus. Sympathetic innervation is sparse and mainly associated with blood vessels. Sensory nerve fibers are also sparsely distributed close to the basal region of acini, and these are immunoreactive for the neuropeptides substance P and calcitonin gene-related peptide.

Development

Meibomian glands in humans develop as small, solid epithelial invaginations from the ocular face of the eyelid margin at approximately 9 weeks of development (crownrump length of 40 mm). By 12 weeks (60 mm), the epithelial growth has extended the depth of the tarsal plate and has a central tube that was formed from apoptosis of the

central epithelial cells. At 15 weeks (100 mm), cuboidal secretory cells line the ducts. The upper and lower eyelids are fused during this whole process. At 7.5 months (250 mm), after the eyelids have separated, acini are present, epithelial cells plugging the meibomian gland orifices disintegrate, and secretion begins just prior to birth. A similar process takes place in the mouse; however, in this case, the eyelids are still fused at birth, which is also the time when the first signs of meibomian gland development show, again from the eyelid margin. In contrast to meibomian gland development, eyelashes in humans begin development earlier at 8 weeks (35 mm) as solid epithelial invaginations from the external face of the eyelid margin.

Composition of Meibomian Lipids

The meibomian gland is often referred to as a modified sebaceous gland. In this case, the term sebaceous means lipid producing rather than sebum producing because the composition of the lipids differs from those produced by the sebaceous glands of hair follicles. The main lipid types produced by meibomian glands are wax and steryl esters (60–70%) which are very hydrophobic (dislike water). Wax esters are formed by linking a long-chain carboxylic acid (fatty acid) to a long-chain alcohol (fatty alcohol). Since different fatty acids and fatty alcohols are linked together, the wax esters are a complex family of lipids and their detailed structure varies between species. In humans, oleic acid is the most prevalent fatty acid found in these waxes. Similarly, the steryl esters are mainly cholesterol esters which are formed by linking cholesterol to a longchain fatty acid. These fatty acids are generally longer than those found in the wax esters. Small amounts of other lipids (mainly polar) – such as mono-, di-, and triglycerides; fatty acids; fatty alcohols; free cholesterol; and phospholipids – make up the remainder. While phospholipids are readily detected in meibomian lipids of rabbits, there is contention as to whether they are a component of human meibomian lipids. This is important because in models of the lipid layer of the tear film, phospholipids are crucial as a link between the hydrophobic molecules (wax and cholesterol esters) and the aqueous layer. If they are not present in the meibomian lipids, then they must be derived from elsewhere, such as the aqueous layer of the tear film, or alternative surfactants need to be present in the model. The nature and mixture of the lipids give them a melting range of 19–33 C, which means that they are fluid on the ocular surface.

Meibomian Lipid Turnover and Synthesis

Since the meibomian gland is a holocrine gland, lipid turnover is related to the cell turnover rate and the lipids are synthesized by the glands rather than being adsorbed from the bloodstream. For instance, the levels of

cholesterol found in meibomian lipids are independent of cholesterol levels in the blood. Synthesis of the straightchain fatty acids is typical of elsewhere in the body, occurring in the cytoplasm using acetyl-coenzyme A (CoA) and malonyl-CoA as the starting molecules and fatty acid synthase as the major enzyme. Some of the fatty acids have branching and, at least in rabbits, the branched carbon chains are derived mainly from the amino acids valine and isoleucine. The acyltransferases seem to be nonselective and will connect any fatty acid to a fatty alcohol. Similarly, cholesterol synthesis is typical of other tissues converting b-hydroxy-b-methylglutaryl- CoA to mevalonate, isoprene, squalene, and cholesterol. The acylcholesterol transferase, involved with cholesterol ester synthesis, appears to be selective for longer-chain fatty acids based on the predominance of long-chain fatty acids in cholesterol esters.

The meibomian acinar cells have nuclear androgen receptors and the level of androgens or, more likely, the ratio between androgens and estrogens is critical for controlling lipid synthesis in the meibomian glands. Stimulating androgen receptors increases gene transcription in enzymes associated with fatty acid and cholesterol synthetic pathways (adenosine triphosphate (ATP)-citrate lyase, acetyl-CoA synthase, acetoacetyl-CoA synthase, 3-hydroxy-3-methyl- glutary (HMG)-CoA synthase 1, HMG-CoA reductase, acetyl-CoA carboxylase, glyceraldehyde-3-phosphate dehydrogenase, and sterol regulatory element-binding protein 1 and 2) and hence stimulates lipid production. Androgen deficiency has been associated with meibomian gland dysfunction (MGD) and dry eye. P2Y2 receptor gene expression has also been detected in meibomian gland acini. This suggests that extracellular ATP or UTP might influence lipid synthesis or composition through activation of G proteins. Specific changes to lipid composition through this pathway have not been investigated.

The total amount of meibomian lipids on the lid margins has been estimated and children under 14 years of age showed the lowest levels (1.5 mg mm 2 lid margin). After puberty, there is a steady increment with age until the late 60s (3.26 mg mm 2) and throughout this period, males have nearly 10% greater levels than females. Given that one of the reasons for dry eye is insufficient meibomian lipid secretion and that there is an increase in the incidence of dry eye with age, it is surprising that the lowest levels of meibomian lipids have been found in children. Morning and afternoon basal levels are the same and there is no correlation between lid temperature (30–34 C) and basal levels of meibomian lipids. However, deliberately increasing the eyelid temperature from 33 to 37 C increases the lipid values on the lid margin by close to 25%. Despite the presence of parasympathetic nerves around the acini, lipid secretion is most likely due to the mechanical force of blinking which causes compression of the territorial fibrocartilaginous matrix that surrounds the

meibomian glands. This has been shown by measuring the reappearance of lipids on the eyelid margin after cleaning with an organic solvent. No lipids appear until a blink occurs (3 min was the longest). After approximately 10 blinks, the levels return to nearly a third of their basal levels. It is estimated that close to 10 mg of lipids are delivered per blink and that there is approximately 20–40 times excess basal amount of lipids available on the eyelid margin than what is required for forming a complete lipid layer on the tear film.

How the lipids are removed from the eye is uncertain. It is believed that most of them flow over the eyelid margin onto the skin and eyelashes. This constant flow prevents the skin lipids from contaminating the tear film. It has been shown that skin lipids disrupt the tear film. Some lipids are likely to bind to proteins of the aqueous layer, particularly lipocalin, and are removed with the aqueous layer through the lacrimal ducts. The crusty buildup that collects in the corner of the eye during sleep is primarily a mixture of lipids and mucins and thus another mechanism for removing lipids from the ocular surface.

Pathology of the Meibomian Gland

Disorders of the meibomian glands are manifest by the obstruction of the gland orifices, inflammation, or loss of the glands. This is often associated with one or more of the following: thickening of the lid margin, exaggerated vascularization around the gland orifices, and hyperkeratinization. Meibomian gland diseases are usually more uncomfortable rather than painful and, when chronic, are associated with dry eye which can be very painful. Absence or deficiency of meibomian glands is often congenital.

Clinically, the state of meibomian glands is determined by examining their morphology and function. In eye clinics, the orifices of the glands lining both the upper and lower eyelids are observed through a slit lamp biomicroscope. If a gland is blocked, the orifice appears swollen on the lid margin. Some practitioners also squeeze the lower eyelid gently to expel meibomian lipids. A clear fluid is considered to be normal, whereas a thick, yellowy secretion is an indication of meibomian gland disorder. If excessive pressure is applied, a thick pasty expression can be obtained from people with normal meibomian gland function (Figure 5). This technique is used for obtaining meibomian lipids for experimental purposes or for analysis. In research settings, more specific tests are performed to assess meibomian gland function. Transillumination of the lower eyelids is widely used to evaluate the morphology of the glands. In particular, shorter-than-normal meibomian glands and meibomian gland dropout are strong indications of MGD (Figure 6). Changes to the shape and form of the glands do not occur with aging,

although expression of secretion is commonly more difficult. A clear, noninvasive view of meibomian glands can be achieved by using infrared light, but the need for specialized equipment means that this technique is only used in a few research laboratories.

Rather than examine the meibomian glands themselves, observation of the lipid layer of the tear film is regarded as an indirect measure of both the quantity and the quality of meibomian gland secretions. Using specialized interferometry, the lipid layer stability, distribution, dynamics, and thickness can be assessed. Generally, a thick amorphous layer is an indication of high-quality meibomian oil secretions and, hence, excellent meibomian gland function. Such a layer is also commonly associated with longer TBUTs, presumably because of the lowered surface tension. The

Figure 5 Hard squeezing of meibomian glands. The secretions are indicated by arrows.

results obtained from tear breakup tests have been shown to be comparable to interferometry findings, although a cause–effect relationship between the two is yet to be established.

Another method for evaluating meibomian gland function indirectly is to measure evaporation from the ocular surface. This is a difficult technique and is normally confined to research settings. Evaporimetry is based on the theory that evaporation from the ocular surface is normally minimal due to the well-spread lipid layer acting as a blanket, and that an inadequate lipid layer or disruption to this lipid layer causes tear evaporation to increase. It is thought to be the cause of evaporative dry eye. However, this idea has been difficult to substantiate and a wide variability in tear evaporation rates has been reported irrespective of the presence or absence of dry eye and, more importantly, the appearance of the lipid layer. Further, a large quantity of lipid on the tear surface does not necessarily correlate with an adequate barrier to evaporation. Evaporative dry eye can occur with an excessively thick lipid layer. Current areas of research center on whether the biochemical composition of the meibomian lipids can influence their surface activity and ability to diminish evaporation, but clear outcomes are still in the future.

Chronic Blepharitis

The term blepharitis has different meanings depending upon the user. Acute blepharitis (normally just called blepharitis) is an infection of the anterior eyelid and

Staphylococcus epidermidis or Staphylococcus aureus are the most likely cause. Chronic blepharitis is caused by dysfunction of the meibomian glands and is synonymous with