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Tear Film
Tear Film J P Craig, University of Auckland, Auckland, New Zealand A Tomlinson and L McCann, Glasgow Caledonian University, Glasgow, UK ã 2010 Elsevier Ltd. All rights reserved.
Glossary Fluorescein sodium – A topical agent used extensively as a diagnostic tool in ophthalmology to enhance tear film visibility or to highlight epithelial cell loss. The molecule is highly fluorescent, with excitation and emission occurring at 494 and 521 nm, respectively. Interference bandpass filters are commonly combined with the observation systems used in ophthalmology to optimize visualization of the fluorescence alone. Lacrimal gland – The lacrimal gland is a compound tubuloalveolar gland, similar to the salivary gland, situated superotemporally in the orbit, which secretes aqueous tear fluid. Lacrimal sac – The lacrimal sac forms part of the tear drainage system, collecting tear fluid from the ocular surface via the puncta and canaliculi. Blinking controls the pumping action of the lacrimal sac into the nasolacrimal duct for drainage into the nasal cavity. Meibomian gland – Vertically oriented tubulo-acinar glands, embedded in the upper and lower tarsal plates, which release meibum (lipid). Videokeratoscopy – A computerized, dynamic technique, based on the principle of keratoscopy, used traditionally to assess the shape of the anterior surface of the cornea (corneal topography) from the reflection of a series of projected concentric rings.
The tear film is a thin film of fluid, which covers the exposed ocular surface. Essential for the health and normal function of the eye and visual system, any abnormality in quantity or quality of the tear film can lead to signs and symptoms of dry eye disease and ultimately to a loss of vision.
The Role of the Tear Film The tear film has a number of important functions, the first of which, as the most anterior element of the visual system, is maintenance of high-quality vision. Alterations in the stability of the tear film due to abnormal tear evaporation, production, and/or drainage can cause optical aberrations and adversely affect retinal image quality.
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Secondly, the tear film plays an important role in ocular surface defence. Environmental challenges such as extremes of temperature or humidity, and exposure to irritants such as pollutants and allergens, can have a detrimental effect on the tear film. The tear film must be sufficiently robust to be able to withstand these challenges and be capable of responding rapidly with reflex tearing to help flush out irritants when required. External and adnexal infectious agents pose an additional risk to the exposed ocular surface. Antimicrobial components of the tear film, which include lysozyme, lactoferrin, and immunoglobulin A, help to protect the ocular surface from microbial infection. Lubrication is another important tear film function. The non-Newtonian rheological properties of the tear film mucins enable the tear film to lubricate the corneal and mucosal surfaces. The normal blinking mechanism draws the tear film across the ocular surface, enhancing comfort and cushioning the ocular surfaces from the shearing forces present during the blink, while the mucins that trap and coat foreign particles in the tear film for removal at the caruncle, confer further epithelial surface protection. Finally, the tear film plays a vital nutritive role in the transport of substances necessary for corneal metabolism and regeneration. Uniquely avascular for transparency, the cornea requires a nonvascular route for the supply of oxygen, electrolytes, growth factors, and nutrients to, and for the removal of metabolic by-products such as carbon dioxide from the ocular surface. While glucose diffuses primarily from, the aqueous humor, oxygen must be transported to the tissue through the tear film, either from the air in the open eye state or via the palpebral conjunctival vessels in the closed eye state.
Structure and Thickness of the Tear Film Initial reports described the tear film as trilaminar in structure, consisting of a thin superficial lipid layer, an intermediate aqueous layer, and an underlying mucous layer. Each of these layers has the potential to be affected by different conditions resulting in qualitative and quantitative changes. Almost half a century later, it was proposed that interfaces existed between the layers, giving rise to a six-layer model, with an oily layer, a polar lipid monolayer, an absorbed mucoid layer, an aqueous layer, and a mucoid layer on a glycocalyx base. The carbohydrate-rich
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Lipid component
Aqueous component
Mucous component Corneal epithelium
MUC5AC
SP-A
MUC5B
MUC7
MUC1
SP-D
MUC4
SP-B
MUC16
SP-C
Figure 1 Diagrammatic representation of our current understanding of tear film structure. The tear film comprises a thin superficial lipid layer, and an aqueous-mucin continuum increasing in mucin concentration toward the glycocalyx, adjacent to the ocular surface epithelium. Adapted from Bra¨uer, L. and Paulsen, F. P. (2008). Tear film and ocular surface surfactants. Journal of Epithelial Biology and Pharmacology 1: 62–67, with permission.
glycocalyx, produced by the surface cells of the corneal epithelium and subsurface vesicles of the conjunctival epithelium, is believed to attach the tear film to the surface of the epithelial cells. The most recent studies do not differentiate this number of distinct layers, but instead suggest the existence of an aqueous-mucin continuum that contains a decreasing concentration of dissolved mucus toward the superficial lipid layer, and is anchored to the epithelium by glycocalyx (Figure 1). The thickness of the precorneal tear film has proven to be a subject of great debate. Early estimates placed the thickness of the tear film in the region of between 4 and 8 mm. Later, on the basis of noninvasive techniques such as interferometry, it was proposed that due to a previously underestimated contribution from the mucous layer, the tear film thickness was closer to 40 mm in thickness. However, the most recent findings using techniques such as tomography and reflectance spectra propose values closer to the original measurements, suggesting that the tear film thickness is approximately 3 mm.
The Lipid Layer The superficial lipid layer of the tear film forms the initial barrier between the ocular surface and the environment. This thin, oily layer approximates 100 nm in thickness, although values ranging between 10 and 600 nm have
been reported. It is derived primarily from the meibomian glands, with additional lipid secreted by the eyelid glands of Moll and Zeiss. The lipids are excreted as meibum onto the ocular surface through the gland orifices located at the mucocutaneous junction of the lid margins. Between blinks, the lipid layer forms in two distinct phases. An inner, thin, polar layer spreads as a monolayer across the aqueous in the initial phase after the blink, then a thicker, outer, nonpolar layer follows, creating a final lipid structure with multiple layers. The lipid layer must be spread evenly by the blink to form a continuous layer without excessively thin or thick patches in order to inhibit evaporation and to prevent accelerated tear breakup from mucin contamination, respectively. Table 1 describes the proportions of the major lipid components of meibum. The polar layer consists of phospholipids, free fatty acids, and cerebrosides, while the less surface-active, nonpolar layer comprises mainly wax esters and sterol esters. The lipid layer confers a number of important protective functions including the formation of a hydrophobic barrier to prevent tear overflow onto the lids and to provide a water-tight seal during overnight lid closure, and the prevention of tear film contamination by skin lipids. However, arguably one of the most critical roles of the superficial lipid layer is to retard evaporation from the ocular surface. The polar lipids of the ocular tear film in the normal eye are capable of reducing its rate of evaporation by about 80–90%.
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Tear Evaporation Numerous investigators have measured evaporation of fluid from the tear film, since it was established that the lipid layer retarded evaporation in a rabbit model, in 1961. Later work, also in a rabbit model, passed dry air over a cornea enclosed within a chamber. From the weight of water collected, the evaporative rate was measured as 10.1 10–7 g cm–2 s–1, and a fourfold increase in evaporation was found to occur with the removal of the rabbit tear film lipid layer. A similar increase in human tear film Table 1
Major lipid components of meibum
Component Synthesised lipids Wax esters Sterol esters Triglycerides Diglycerides Monoglycerides Fatty alchohol Hydrocarbons Membrane-derived lipids Cerebrosides Ceramides Phospholipids Degeneration products Free fatty acids
Percentage (%) 44 33 5 2 Trace Trace 2 4 Trace 8 2
Adapted from McCulley, J. P. and Shine, W. E. (2003). Meibomian gland function and the tear lipid layer. Ocular Surface 1(3): 97–106, with permission.
evaporation has since been confirmed in patients with incomplete or absent lipid layers (Figure 2). The use of different techniques for measurement of tear film evaporation makes comparison of evaporation rates in different studies difficult because the absolute values recorded are technique-dependent. However, a pattern to the observations reported in the literature does exist, making evaporation rate a useful measurement in the differential diagnosis of dry eye. In most cases, significant increases from normal tear film evaporation are seen in patients with aqueous deficient dry eye (ADDE), evaporative dry eye (EDE), and meibomian gland dysfunction (MGD). The evaporation in normal eyes averages 13.57 6.52 10–7 g cm–2 s–1, while in ADDE the values average 17.91 10.49 10–7 g cm–2 s–1, and in EDE, 25.34 13.8 10–7 g cm–2 s–1.
The Aqueous Layer The aqueous component of the tear film is a watery phase, bordering the lipid layer and comprising most of the tear film thickness. It is produced principally by the main lacrimal gland and accessory lacrimal glands of Krause and Wolfring although additional water and electrolytes are secreted by the epithelial cells of the ocular surface. The typical or basal level of tear flow present is believed to originate mainly from the accessory glands while the reflex tears, produced in response to mechanical, noxious, or emotional stimuli, arise from the main lacrimal gland.
Relative humidity sensor
Temperature sensor
Water vapour from ocular surface
Figure 2 Tear film evaporation rate measured by a modified ServoMed EP-3 Evaporimeter (Kinna, Sweden). This technique involves the measurement of the vapor pressure gradient from recordings of relative humidity and temperature at two points a known distance above the ocular surface. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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During sleep, tear production is minimal but in the normal eye, in the open eye state, sensory stimulation of the exposed ocular surface induces tear production at a rate that varies according to the demands of the external environment. The secretion of electrolytes, protein, and water onto the ocular surface serves to nourish and protect the epithelia and convey messages between the structures bathed in aqueous. Corneal innervation is denser than that of any other part of the body, resulting in extreme pain if the corneal epithelium is damaged. Sensory nerve supply to the ocular surface arises from the trigeminal nerve. Stimulation of these nerve endings causes the release of neuropeptides such as substance P and calcitronin gene-related peptide (CGRP), which, through initiation of the inflammatory cascade, is believed to be an important step in the pathogenesis of many cases of dry eye. The lacrimal and meibomian glands are innervated by parasympathetic efferent nerve fibers (muscarinic and vaso-intestinal peptide (VIP)-ergic fibers) and to some extent they, and the blood vessels supplying them, are sympathetically innervated through tyrosine hydroxylase (TH) and neuropeptide Y fibers. Parasympathetic efferent nerve terminals surrounding the goblet cells suggest that conjunctival secretions are also under neurogenic control. The aqueous phase has a number of important responsibilities. These include creating a nurturing environment for the epithelial cells of the ocular surface, carrying essential nutrients and oxygen to the cornea, allowing cell movement over the ocular surface, and washing away epithelial debris, toxic elements, and foreign bodies. The major electrolytes present in the tear film are sodium, potassium, bicarbonate, and chloride, with magnesium, calcium, nitrate phosphate, and sulfate present in smaller quantities. The electrolytes dictate the osmolarity of tears, besides acting as a buffer to maintain pH and playing a role in maintaining epithelial integrity. An increase in the electrolyte concentration, described as hyperosmolarity, can cause damage to the ocular surface. The tear film protein concentration is approximately 10% that of plasma. The proportion of lacrimal gland versus serum-derived proteins and enzymes varies with tear flow rate, epithelial surface stimulation, blinking, and ocular surface disease. The tear proteins are involved in defense of the ocular surface and the maintenance of tear film stability. Electrophoresis has confirmed the presence of approximately 80 different components of human tear proteins. Around 30 proteins have been identified, half of which are enzymes. The principal tear proteins are lysozyme, lactoferrin, albumin, tear-specific pre-albumin, and globulins. Table 2 shows typical concentrations of the most significant tear proteins. The tear film also contains antioxidants such as vitamin C and tyrosine, which scavenge free radicals from within the tear film, while the
Table 2
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Average concentration of the principal tear proteins
Protein component Total protein Lysozyme Albumin Tear specific prealbumin Lactoferrin Immunoglobulins (IgA, IgG, IgM, and IgE)
Average concentration (mg ml 1) 7.51 2.36 1.30 1.23 1.84 0.43
Adapted from Sariri R. and Ghafoori, H. (2008). Tear proteins in health, disease, and contact lens wear. Biochemistry (Moscow) 73(4): 381–392, with permission.
abundance of growth factors facilitates constant epithelial regeneration and promotes wound healing. Alterations in tear composition or inflammatory changes within the conjunctival vascular endothelia can act as the stimulus to ocular surface inflammation in which both cellular and soluble mediators play a significant role. The numbers of T lymphocytes and the relative proportions of activated T cells are increased in dry eye. The ocular surface epithelial cells are directly involved in such ocular surface inflammation with the release of a number of pro-inflammatory cytokines such as interleukin (IL)-1a, IL-1b, IL6, IL8, transforming growth factor beta 1 (TGF-b1) and tumor necrosis factor alpha (TNFa), and increased expression of immune activation molecules such as CD54 and HLA-DR. Increased proteolytic enzyme levels and activity have been observed in dry eye with, in particular, high levels of matrix metalloproteinase 9 (MMP9), which are not present on the normal ocular surface. The inflammatory markers described as precipitating dry eye are also recognized to perpetuate ocular surface inflammation, triggering an escalating cycle of ocular irritation, inflammation, apoptosis, and tear film dysfunction and instability, epithelial cell disease, and disruption of corneal epithelial barrier function.
Tear Production Traditional methods of measuring tear production rates are based on absorption of tears by Schirmer strips or cotton threads; however, both tests have been found to be poor quantifiers of tear production; the Schirmer test is marred by low specificity and sensitivity and the exact parameter measured with the cotton thread test has been questioned. As a result, a number of tests have been devised to measure the rate of disappearance of a dye marker placed in the tear film, as new tears are produced and the waste eliminated. In most studies in recent years, the rate of disappearance of instilled sodium fluorescein dye has been used to determine tear turnover (TTR) by the technique of fluorophotometry (Figure 3).
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4.2
C0
FCE (ng ml−1)
4.0 3.8 3.6 3.4 3.2 3.0
0
2
4
6
8
10
12
14
16
Time (min) Figure 3 Commercial fluorophotometer (Fluorotron Master, Coherent Radiation Inc, CA, USA) shown with a typical trace of ocular surface fluorescence decay following instillation of fluorescein sodium into the eye. A biphasic curve of fluorescence is observed with initial rapid decay (due to reflex tearing) followed by a more gradual decay (due to basal tear turnover). Adapted from The Ocular Surface (www.theocularsurface.com), with permission.
The values reported for tear turnover (%min–1) and tear flow (ml min–1) in the major studies in the literature for normal and dry eye subjects of studies using the commercial fluorophotometer have recently been collated. The data reported for normals in the majority of studies ranges from 10% to 20% min–1, which equates to an average basal tear flow rate of 1.03 0.39 ml min–1 (16.19 5.10% min–1). For dry eye, in all its forms, it averages 0.58 0.28 ml min–1 (9.36 5.68% min–1) and, within the dry eye subtypes, averages 0.40 0.10 ml min–1 (7.71 1.02% min–1) and 0.71 0.25 ml min–1 (11.95 4.25% min–1) for ADDE and for EDE, respectively. These are the rates of tear production under nonstimulated conditions in normal and dry eyes. However, the eye is capable of producing copious reflex tears under provocative conditions, providing the lacrimal gland has the ability to function at the required capacity. Reflex rates have been quoted as approximately 100-fold those under basal conditions.
The Mucin Layer The innermost, mucin layer of the tear film lies adjacent to the hydrophobic epithelial cells of the ocular surface. The layer consists of soluble, gel-forming mucins, which are capable of retaining large quantities of water, and corneal and conjunctival epithelial mucins (principally MUC1, 2, 4, and 16), which form the glycocalyx. The glycocalyx functions, through the membrane-spanning domain of MUC1, to anchor the soluble mucin layer to the plasma membrane of the corneal and conjunctival epithelial cells, while the soluble mucins interact with these transmembrane mucins and with the overlying aqueous layer, to form a water-retaining gel. The most significant soluble mucin for the ocular surface is MUC5AC, secreted by the goblet cells of the conjunctiva. The high-molecular-weight glycoproteins, with additional proteins, electrolytes, and cellular material that
contribute to the mucous layer, enable fulfilment of several important functions in the maintenance of a healthy ocular surface. In addition to providing a hydrophilic surface upon which to support a stable aqueous layer, the mucous layer offers protection against the shear force of blinking and environmental insult, and facilitates maintenance of a smooth ocular surface for optical clarity. The constituents are also believed to protect the ocular surface by inhibiting inflammatory cell adhesion.
Tear Distribution and Stability The distribution of tear fluid on the ocular surface is highly dependent on the blink. Lid closure during a blink progresses from the temporal to the nasal side of the eye spreading tears across the ocular surface and facilitating tear drainage through the lacrimal puncta. The inter-blink period in normal individuals averages 4.0 2.0 s and is significantly decreased in patients with dry eye (to 1.5 0.9 s); a high blink rate in dry eye patients maximizes the tear supply to the ocular surface. In detailed reading tasks, requiring concentration, the blink rate drops to about a half (from 22.4 8.9 to 10.5 6.5 min–1). In the clinical setting, tear film stability has traditionally been measured following the instillation of fluorescein sodium solution into the tear film, to improve visualization of the film. Tear breakup time has been defined as the time taken for the tear film to form a dark spot or streak, following a blink. However, subsequent awareness of the disruptive effect of fluorescein instillation on the tear film has encouraged use of noninvasive techniques where tear film stability is determined by observing mires reflected from the tear film surface, for signs of disruption or distortion following a blink. A tear breakup time of greater than 10 s is considered normal while values less than 5 s are suggestive of dry eye. Values between 5 and 10 s are
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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)
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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.
Figure 4 Series of images obtained by dynamic thin film interferometry in a normal, asymptomatic subject. The images are obtained at 1 s intervals, following a blink. The lipid layer of the normal tear film reaches a relatively stable pattern within the first second after the blink. This pattern is then stable for about 6 s. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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Figure 5 Series of thin film interferometry images obtained from a patient with severe dry eye. The patient had primary Sjo¨gren’s syndrome with a tear turnover rate of 4% min–1, evaporation of 25.5 g cm 2 s 1, volume of 3.9 ml and osmolarity of 337.6 mOsm ml–1. The images are obtained at 1 s intervals following a blink. The lipid layer of the tear film is incomplete and variable in thickness, exhibiting color fringe patterns. A stable pattern is reached in 2–3 s after the blink, but this pattern begins to be disrupted within the next 3 s. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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
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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
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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
(a)
(b)
(c)
(d)
(e)
Figure 6 Gamma camera (a–c) used in the recording of intensity of a radioactive dye at various stages as it passes through the lacrimal system (d). In many cases of normal systems, the tracer does not proceed beyond the lacrimal sac (e). Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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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.
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: Blinking Mechanisms; 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; Tear Film Overview.
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
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 Fluorescein sodium – A topical agent used extensively as a diagnostic tool in ophthalmology to enhance tear film visibility or to highlight epithelial cell loss. The molecule is highly fluorescent, with excitation and emission occurring at 494 and 521 nm, respectively. Interference bandpass filters are commonly combined with the observation systems used in ophthalmology to optimize visualization of the fluorescence alone. Lacrimal gland – The lacrimal gland is a compound tubuloalveolar gland, similar to the salivary gland, situated superotemporally in the orbit, which secretes aqueous tear fluid. Lacrimal sac – The lacrimal sac forms part of the tear drainage system, collecting tear fluid from the ocular surface via the puncta and canaliculi. Blinking controls the pumping action of the lacrimal sac into the nasolacrimal duct for drainage into the nasal cavity. Meibomian gland – Vertically oriented tubulo-acinar glands, embedded in the upper and lower tarsal plates, which release meibum (lipid). Videokeratoscopy – A computerized, dynamic technique, based on the principle of keratoscopy, used traditionally to assess the shape of the anterior surface of the cornea (corneal topography) from the reflection of a series of projected concentric rings.
The tear film is a thin film of fluid, which covers the exposed ocular surface. Essential for the health and normal function of the eye and visual system, any abnormality in quantity or quality of the tear film can lead to signs and symptoms of dry eye disease and ultimately to a loss of vision.
The Role of the Tear Film The tear film has a number of important functions, the first of which, as the most anterior element of the visual system, is maintenance of high-quality vision. Alterations in the stability of the tear film due to abnormal tear evaporation, production, and/or drainage can cause optical aberrations and adversely affect retinal image quality.
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Secondly, the tear film plays an important role in ocular surface defence. Environmental challenges such as extremes of temperature or humidity, and exposure to irritants such as pollutants and allergens, can have a detrimental effect on the tear film. The tear film must be sufficiently robust to be able to withstand these challenges and be capable of responding rapidly with reflex tearing to help flush out irritants when required. External and adnexal infectious agents pose an additional risk to the exposed ocular surface. Antimicrobial components of the tear film, which include lysozyme, lactoferrin, and immunoglobulin A, help to protect the ocular surface from microbial infection. Lubrication is another important tear film function. The non-Newtonian rheological properties of the tear film mucins enable the tear film to lubricate the corneal and mucosal surfaces. The normal blinking mechanism draws the tear film across the ocular surface, enhancing comfort and cushioning the ocular surfaces from the shearing forces present during the blink, while the mucins that trap and coat foreign particles in the tear film for removal at the caruncle, confer further epithelial surface protection. Finally, the tear film plays a vital nutritive role in the transport of substances necessary for corneal metabolism and regeneration. Uniquely avascular for transparency, the cornea requires a nonvascular route for the supply of oxygen, electrolytes, growth factors, and nutrients to, and for the removal of metabolic by-products such as carbon dioxide from the ocular surface. While glucose diffuses primarily from, the aqueous humor, oxygen must be transported to the tissue through the tear film, either from the air in the open eye state or via the palpebral conjunctival vessels in the closed eye state.
Structure and Thickness of the Tear Film Initial reports described the tear film as trilaminar in structure, consisting of a thin superficial lipid layer, an intermediate aqueous layer, and an underlying mucous layer. Each of these layers has the potential to be affected by different conditions resulting in qualitative and quantitative changes. Almost half a century later, it was proposed that interfaces existed between the layers, giving rise to a six-layer model, with an oily layer, a polar lipid monolayer, an absorbed mucoid layer, an aqueous layer, and a mucoid layer on a glycocalyx base. The carbohydrate-rich
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Lipid component
Aqueous component
Mucous component Corneal epithelium
MUC5AC
SP-A
MUC5B
MUC7
MUC1
SP-D
MUC4
SP-B
MUC16
SP-C
Figure 1 Diagrammatic representation of our current understanding of tear film structure. The tear film comprises a thin superficial lipid layer, and an aqueous-mucin continuum increasing in mucin concentration toward the glycocalyx, adjacent to the ocular surface epithelium. Adapted from Bra¨uer, L. and Paulsen, F. P. (2008). Tear film and ocular surface surfactants. Journal of Epithelial Biology and Pharmacology 1: 62–67, with permission.
glycocalyx, produced by the surface cells of the corneal epithelium and subsurface vesicles of the conjunctival epithelium, is believed to attach the tear film to the surface of the epithelial cells. The most recent studies do not differentiate this number of distinct layers, but instead suggest the existence of an aqueous-mucin continuum that contains a decreasing concentration of dissolved mucus toward the superficial lipid layer, and is anchored to the epithelium by glycocalyx (Figure 1). The thickness of the precorneal tear film has proven to be a subject of great debate. Early estimates placed the thickness of the tear film in the region of between 4 and 8 mm. Later, on the basis of noninvasive techniques such as interferometry, it was proposed that due to a previously underestimated contribution from the mucous layer, the tear film thickness was closer to 40 mm in thickness. However, the most recent findings using techniques such as tomography and reflectance spectra propose values closer to the original measurements, suggesting that the tear film thickness is approximately 3 mm.
The Lipid Layer The superficial lipid layer of the tear film forms the initial barrier between the ocular surface and the environment. This thin, oily layer approximates 100 nm in thickness, although values ranging between 10 and 600 nm have
been reported. It is derived primarily from the meibomian glands, with additional lipid secreted by the eyelid glands of Moll and Zeiss. The lipids are excreted as meibum onto the ocular surface through the gland orifices located at the mucocutaneous junction of the lid margins. Between blinks, the lipid layer forms in two distinct phases. An inner, thin, polar layer spreads as a monolayer across the aqueous in the initial phase after the blink, then a thicker, outer, nonpolar layer follows, creating a final lipid structure with multiple layers. The lipid layer must be spread evenly by the blink to form a continuous layer without excessively thin or thick patches in order to inhibit evaporation and to prevent accelerated tear breakup from mucin contamination, respectively. Table 1 describes the proportions of the major lipid components of meibum. The polar layer consists of phospholipids, free fatty acids, and cerebrosides, while the less surface-active, nonpolar layer comprises mainly wax esters and sterol esters. The lipid layer confers a number of important protective functions including the formation of a hydrophobic barrier to prevent tear overflow onto the lids and to provide a water-tight seal during overnight lid closure, and the prevention of tear film contamination by skin lipids. However, arguably one of the most critical roles of the superficial lipid layer is to retard evaporation from the ocular surface. The polar lipids of the ocular tear film in the normal eye are capable of reducing its rate of evaporation by about 80–90%.
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Tear Evaporation Numerous investigators have measured evaporation of fluid from the tear film, since it was established that the lipid layer retarded evaporation in a rabbit model, in 1961. Later work, also in a rabbit model, passed dry air over a cornea enclosed within a chamber. From the weight of water collected, the evaporative rate was measured as 10.1 10–7 g cm–2 s–1, and a fourfold increase in evaporation was found to occur with the removal of the rabbit tear film lipid layer. A similar increase in human tear film Table 1
Major lipid components of meibum
Component Synthesised lipids Wax esters Sterol esters Triglycerides Diglycerides Monoglycerides Fatty alchohol Hydrocarbons Membrane-derived lipids Cerebrosides Ceramides Phospholipids Degeneration products Free fatty acids
Percentage (%) 44 33 5 2 Trace Trace 2 4 Trace 8 2
Adapted from McCulley, J. P. and Shine, W. E. (2003). Meibomian gland function and the tear lipid layer. Ocular Surface 1(3): 97–106, with permission.
evaporation has since been confirmed in patients with incomplete or absent lipid layers (Figure 2). The use of different techniques for measurement of tear film evaporation makes comparison of evaporation rates in different studies difficult because the absolute values recorded are technique-dependent. However, a pattern to the observations reported in the literature does exist, making evaporation rate a useful measurement in the differential diagnosis of dry eye. In most cases, significant increases from normal tear film evaporation are seen in patients with aqueous deficient dry eye (ADDE), evaporative dry eye (EDE), and meibomian gland dysfunction (MGD). The evaporation in normal eyes averages 13.57 6.52 10–7 g cm–2 s–1, while in ADDE the values average 17.91 10.49 10–7 g cm–2 s–1, and in EDE, 25.34 13.8 10–7 g cm–2 s–1.
The Aqueous Layer The aqueous component of the tear film is a watery phase, bordering the lipid layer and comprising most of the tear film thickness. It is produced principally by the main lacrimal gland and accessory lacrimal glands of Krause and Wolfring although additional water and electrolytes are secreted by the epithelial cells of the ocular surface. The typical or basal level of tear flow present is believed to originate mainly from the accessory glands while the reflex tears, produced in response to mechanical, noxious, or emotional stimuli, arise from the main lacrimal gland.
Relative humidity sensor
Temperature sensor
Water vapour from ocular surface
Figure 2 Tear film evaporation rate measured by a modified ServoMed EP-3 Evaporimeter (Kinna, Sweden). This technique involves the measurement of the vapor pressure gradient from recordings of relative humidity and temperature at two points a known distance above the ocular surface. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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During sleep, tear production is minimal but in the normal eye, in the open eye state, sensory stimulation of the exposed ocular surface induces tear production at a rate that varies according to the demands of the external environment. The secretion of electrolytes, protein, and water onto the ocular surface serves to nourish and protect the epithelia and convey messages between the structures bathed in aqueous. Corneal innervation is denser than that of any other part of the body, resulting in extreme pain if the corneal epithelium is damaged. Sensory nerve supply to the ocular surface arises from the trigeminal nerve. Stimulation of these nerve endings causes the release of neuropeptides such as substance P and calcitronin gene-related peptide (CGRP), which, through initiation of the inflammatory cascade, is believed to be an important step in the pathogenesis of many cases of dry eye. The lacrimal and meibomian glands are innervated by parasympathetic efferent nerve fibers (muscarinic and vaso-intestinal peptide (VIP)-ergic fibers) and to some extent they, and the blood vessels supplying them, are sympathetically innervated through tyrosine hydroxylase (TH) and neuropeptide Y fibers. Parasympathetic efferent nerve terminals surrounding the goblet cells suggest that conjunctival secretions are also under neurogenic control. The aqueous phase has a number of important responsibilities. These include creating a nurturing environment for the epithelial cells of the ocular surface, carrying essential nutrients and oxygen to the cornea, allowing cell movement over the ocular surface, and washing away epithelial debris, toxic elements, and foreign bodies. The major electrolytes present in the tear film are sodium, potassium, bicarbonate, and chloride, with magnesium, calcium, nitrate phosphate, and sulfate present in smaller quantities. The electrolytes dictate the osmolarity of tears, besides acting as a buffer to maintain pH and playing a role in maintaining epithelial integrity. An increase in the electrolyte concentration, described as hyperosmolarity, can cause damage to the ocular surface. The tear film protein concentration is approximately 10% that of plasma. The proportion of lacrimal gland versus serum-derived proteins and enzymes varies with tear flow rate, epithelial surface stimulation, blinking, and ocular surface disease. The tear proteins are involved in defense of the ocular surface and the maintenance of tear film stability. Electrophoresis has confirmed the presence of approximately 80 different components of human tear proteins. Around 30 proteins have been identified, half of which are enzymes. The principal tear proteins are lysozyme, lactoferrin, albumin, tear-specific pre-albumin, and globulins. Table 2 shows typical concentrations of the most significant tear proteins. The tear film also contains antioxidants such as vitamin C and tyrosine, which scavenge free radicals from within the tear film, while the
Table 2
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Average concentration of the principal tear proteins
Protein component Total protein Lysozyme Albumin Tear specific prealbumin Lactoferrin Immunoglobulins (IgA, IgG, IgM, and IgE)
Average concentration (mg ml 1) 7.51 2.36 1.30 1.23 1.84 0.43
Adapted from Sariri R. and Ghafoori, H. (2008). Tear proteins in health, disease, and contact lens wear. Biochemistry (Moscow) 73(4): 381–392, with permission.
abundance of growth factors facilitates constant epithelial regeneration and promotes wound healing. Alterations in tear composition or inflammatory changes within the conjunctival vascular endothelia can act as the stimulus to ocular surface inflammation in which both cellular and soluble mediators play a significant role. The numbers of T lymphocytes and the relative proportions of activated T cells are increased in dry eye. The ocular surface epithelial cells are directly involved in such ocular surface inflammation with the release of a number of pro-inflammatory cytokines such as interleukin (IL)-1a, IL-1b, IL6, IL8, transforming growth factor beta 1 (TGF-b1) and tumor necrosis factor alpha (TNFa), and increased expression of immune activation molecules such as CD54 and HLA-DR. Increased proteolytic enzyme levels and activity have been observed in dry eye with, in particular, high levels of matrix metalloproteinase 9 (MMP9), which are not present on the normal ocular surface. The inflammatory markers described as precipitating dry eye are also recognized to perpetuate ocular surface inflammation, triggering an escalating cycle of ocular irritation, inflammation, apoptosis, and tear film dysfunction and instability, epithelial cell disease, and disruption of corneal epithelial barrier function.
Tear Production Traditional methods of measuring tear production rates are based on absorption of tears by Schirmer strips or cotton threads; however, both tests have been found to be poor quantifiers of tear production; the Schirmer test is marred by low specificity and sensitivity and the exact parameter measured with the cotton thread test has been questioned. As a result, a number of tests have been devised to measure the rate of disappearance of a dye marker placed in the tear film, as new tears are produced and the waste eliminated. In most studies in recent years, the rate of disappearance of instilled sodium fluorescein dye has been used to determine tear turnover (TTR) by the technique of fluorophotometry (Figure 3).
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4.2
C0
FCE (ng ml−1)
4.0 3.8 3.6 3.4 3.2 3.0
0
2
4
6
8
10
12
14
16
Time (min) Figure 3 Commercial fluorophotometer (Fluorotron Master, Coherent Radiation Inc, CA, USA) shown with a typical trace of ocular surface fluorescence decay following instillation of fluorescein sodium into the eye. A biphasic curve of fluorescence is observed with initial rapid decay (due to reflex tearing) followed by a more gradual decay (due to basal tear turnover). Adapted from The Ocular Surface (www.theocularsurface.com), with permission.
The values reported for tear turnover (%min–1) and tear flow (ml min–1) in the major studies in the literature for normal and dry eye subjects of studies using the commercial fluorophotometer have recently been collated. The data reported for normals in the majority of studies ranges from 10% to 20% min–1, which equates to an average basal tear flow rate of 1.03 0.39 ml min–1 (16.19 5.10% min–1). For dry eye, in all its forms, it averages 0.58 0.28 ml min–1 (9.36 5.68% min–1) and, within the dry eye subtypes, averages 0.40 0.10 ml min–1 (7.71 1.02% min–1) and 0.71 0.25 ml min–1 (11.95 4.25% min–1) for ADDE and for EDE, respectively. These are the rates of tear production under nonstimulated conditions in normal and dry eyes. However, the eye is capable of producing copious reflex tears under provocative conditions, providing the lacrimal gland has the ability to function at the required capacity. Reflex rates have been quoted as approximately 100-fold those under basal conditions.
The Mucin Layer The innermost, mucin layer of the tear film lies adjacent to the hydrophobic epithelial cells of the ocular surface. The layer consists of soluble, gel-forming mucins, which are capable of retaining large quantities of water, and corneal and conjunctival epithelial mucins (principally MUC1, 2, 4, and 16), which form the glycocalyx. The glycocalyx functions, through the membrane-spanning domain of MUC1, to anchor the soluble mucin layer to the plasma membrane of the corneal and conjunctival epithelial cells, while the soluble mucins interact with these transmembrane mucins and with the overlying aqueous layer, to form a water-retaining gel. The most significant soluble mucin for the ocular surface is MUC5AC, secreted by the goblet cells of the conjunctiva. The high-molecular-weight glycoproteins, with additional proteins, electrolytes, and cellular material that
contribute to the mucous layer, enable fulfilment of several important functions in the maintenance of a healthy ocular surface. In addition to providing a hydrophilic surface upon which to support a stable aqueous layer, the mucous layer offers protection against the shear force of blinking and environmental insult, and facilitates maintenance of a smooth ocular surface for optical clarity. The constituents are also believed to protect the ocular surface by inhibiting inflammatory cell adhesion.
Tear Distribution and Stability The distribution of tear fluid on the ocular surface is highly dependent on the blink. Lid closure during a blink progresses from the temporal to the nasal side of the eye spreading tears across the ocular surface and facilitating tear drainage through the lacrimal puncta. The inter-blink period in normal individuals averages 4.0 2.0 s and is significantly decreased in patients with dry eye (to 1.5 0.9 s); a high blink rate in dry eye patients maximizes the tear supply to the ocular surface. In detailed reading tasks, requiring concentration, the blink rate drops to about a half (from 22.4 8.9 to 10.5 6.5 min–1). In the clinical setting, tear film stability has traditionally been measured following the instillation of fluorescein sodium solution into the tear film, to improve visualization of the film. Tear breakup time has been defined as the time taken for the tear film to form a dark spot or streak, following a blink. However, subsequent awareness of the disruptive effect of fluorescein instillation on the tear film has encouraged use of noninvasive techniques where tear film stability is determined by observing mires reflected from the tear film surface, for signs of disruption or distortion following a blink. A tear breakup time of greater than 10 s is considered normal while values less than 5 s are suggestive of dry eye. Values between 5 and 10 s are
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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)
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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.
Figure 4 Series of images obtained by dynamic thin film interferometry in a normal, asymptomatic subject. The images are obtained at 1 s intervals, following a blink. The lipid layer of the normal tear film reaches a relatively stable pattern within the first second after the blink. This pattern is then stable for about 6 s. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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Figure 5 Series of thin film interferometry images obtained from a patient with severe dry eye. The patient had primary Sjo¨gren’s syndrome with a tear turnover rate of 4% min–1, evaporation of 25.5 g cm 2 s 1, volume of 3.9 ml and osmolarity of 337.6 mOsm ml–1. The images are obtained at 1 s intervals following a blink. The lipid layer of the tear film is incomplete and variable in thickness, exhibiting color fringe patterns. A stable pattern is reached in 2–3 s after the blink, but this pattern begins to be disrupted within the next 3 s. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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
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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
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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
(a)
(b)
(c)
(d)
(e)
Figure 6 Gamma camera (a–c) used in the recording of intensity of a radioactive dye at various stages as it passes through the lacrimal system (d). In many cases of normal systems, the tracer does not proceed beyond the lacrimal sac (e). Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.
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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.
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: Blinking Mechanisms; 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; Tear Film Overview.
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
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.