[India]
INTRODUCTION
The exposed part of the ocular globe—the cornea and the bulbar conjunctiva is covered by a thin fluid film known as preocular tear film. Tear film is that surface of the eye, which remains most directly in contact with the environment. It is critically important for protecting the eye from external influences and for maintaining the health of the underlying cornea and conjunctiva. The optical stability and normal function of the eye depend on an adequate supply of fluid covering its surface.
The tear film is a highly specialized and well-organized moist film which covers the bulbar and palpebral conjunctiva and cornea. It is formed and maintained by an elaborate system' the lacrimal apparatus consisting of secretory, distributive and excretory parts. The secretory part includes the lacrimal gland, accessory lacrimal gland tissue, sebaceous glands of the eyelids, goblet cells and other mucin-secreting elements of the conjunctiva (Figure 1.1). The elimination of the lacrimal secretions is based on the movement of tears across the eye aided by the act of blinking and a drainage system consisting of lacrimal puncta, canaliculi, sac and nasolacrimal duct (Figure 1.2).
FIGURE 1.1: Cross-section of eye showing tear film (blue) in its natural distribution along with tear producing glands(Courtesy Allergan India Limited)
By definition, a film is a thin layer that can stand vertically without appreciable gravitational flow and the tear film meets this criteria very well.3
The presence of continuous tear film over the exposed ocular surface is imperative for good visual acuity and wellbeing of the epithelium and facilitates blinking. Tear film serves:
- An optical function by maintaining an optically uniform corneal surface
- A mechanical function by flushing cellular debris, foreign matter from the cornea and conjunctival sac and by lubricating the surface
- A corneal nutritional function
- An antibacterial function.
The composition of the tear film must be kept within rather narrow quantitative and qualitative limits in order to maintain the wellbeing and proper functioning of the visual system. Abnormalities of the tear film affecting its constituents or volume lead to serious dysfunction of the eyelids and the conjunctiva with the concomitant loss of corneal transparency. A thin tear film is uniformally spread over the cornea by blinking and ocular movements. The tear film can be arbitrarily divided into four main parts:
- The marginal tear film along the moist portions of the eyelid which lie posterior to the lipid strip secreted by the tarsal glands
- Portion covering the palpebral conjunctiva
- Portion covering the bulbar conjunctiva
- Precorneal tear film which covers the cornea.
The marginal, palpebral and conjunctival portions are regarded as making the preocular tear film.
Tears refers to the fluid present as the precorneal film and in the conjunctival sac. The volume of tear fluid is about 5 to 10 µl with normal rate of secretion about 1 to 2 µl/minute. About 95 percent of it is produced by the lacrimal gland and lesser amounts are produced by goblet cells and the accessory lacrimal glands of the conjunctiva. The total mass of the latter is about one-tenth of the mass of the main lacrimal gland.
The secretory part of the lacrimal apparatus provides the aqueous tear, lipids and mucus all the important components of the tear film and its boundary.
The tear film is composed of three layers (Figure 1.3).
1. Superficial Lipid Layer
The superficial layer at the air-tear interface is formed over the aqueous part of the tear film from the oily secretions of meibomian glands and the accessory sebaceous glands of Zeis and Moll. The meibomian gland openings are distributed along the eyelid margin immediately behind the lash follicles.
The chemical nature of the lipid layer is essentially waxy and consists of cholesterol esters and some polar lipids. The thickness of this layer varies with the width of the palpebral fissure and is between 0.1 and 0.2 µm. Being oily in nature it forms a barrier along the lid margins that retains the lid 5margin tear strip and prevents its overflow on to skin. This layer is so thin that there are no interference color patterns such as one normally sees on an oily surface. However, if one squints, the oily layer thickness and distinct interference colors may be seen.
While the bulk of tarsal gland secretions are nonpolar lipid compounds which do not spread over an aqueous surface alone, many surface active components are also present. It appears that the tarsal gland secretions which are transported to the cornea in the tear film are massaged into the outermost layer of corneal epithelial cells by eyelid action and then possibly are changed by local metabolic processes in the epithelium combining with conjunctival mucus to form a stable hydrophilic base for the precorneal tear film.
This outer lipid layer has the following main functions:
- It reduces the rate of evaporations of the underlying aqueous tear layer.
- It increases surface tension and assists in the vertical stability of the tear film so that tears do not overflow the lower lid margin.
- It lubricates the eyelids as they pass over the surface of the globe.
2. Middle Aqueous Layer
The intermediate layer of tear film is the aqueous phase which is secreted by the main lacrimal gland and the accessory glands of Krause and Wolfring.
This layer constitutes almost the total thickness of the tear film 6.5 to 10 µm, many times thicker than the fine superficial oily layer. This layer contains two phases—a more concentrated and a highly dilute one. The interfacial tension at the adsorbed mucin-aqueous layer is apt to be rather small due to the intensive hydrogen bond formation across the interface. This layer contains inorganic salts, water proteins, enzymes, glucose, urea, metabolites, electrolytes, glycoproteins and surface active biopolymers. Uptake of oxygen through the tear film is essential to normal corneal metabolism. This layer has four main functions:
- Most importantly it supplies atmospheric oxygen to the corneal epithelium.
- It has antibacterial substances like lactoferrin and lysozyme. Therefore, dry eye patients are more susceptible to infection than a normal eye.
- It provides smooth optical surface by removing any minute irregularities of the cornea.
- It washes away debris from the cornea and conjunctiva.
3. Posterior Mucin Layer
The innermost layer of tear film is a thin mucoid layer elaborated by goblet cells of the conjunctiva and also by the crypts of Henle and glands of Manz. It is the deepest stratum of the precorneal tear film. This layer is even thinner than the lipid layer and is 0.02 to 0.04 µm thick. This adsorbs on the epithelial surface of the cornea and conjunctiva rendering them hydrophilic. It assumes the ridged appearance of the microvilli of superficial epithelial cells which it covers. The preocular tear film is dependent upon a constant supply of mucus which must be of proper chemical and physical nature to maintain corneal and conjunctival surfaces in the proper state of hydration. The mucous threads present in the tear film provides lubrication allowing the eyelid margin and palpebral conjunctiva to slide smoothly over one another with minimal energy lost as friction during blinking and ocular rotation movements. They also cover foreign bodies with a slippery coating thereby protecting the cornea and conjunctiva against the abrasive effects of such particles as they are moved about by the constant blinking movements of eyelids. The mucus contributes stability to the preocular tear film as well as furnishing an attachment for the tear film to the conjunctiva but not to the corneal surface. The corneal surface is covered with a myriad of fine microvilli which provides some support for the tear film. The mucus dissolved in the aqueous phase facilitates spreading of the tear film by smoothening the film over the corneal surface to form a perfect, regular refracting surface.
So the mucin layer which is a glycoprotein converts a hydrophobic surface into a hydrophilic surface and enables the corneal epithelium to be adequately wetted.
In addition to sufficient amounts of aqueous tears and mucin three other important factors are necessary for effective resurfacing of the cornea by the precorneal tear film.
- A normal blink reflex is essential to ensure that the mucin is brought from the inferior conjunctiva and rubbed into the corneal epithelium. Patients suffering from facial palsy and lagophthalmos therefore develop corneal drying.
- Congruity between external ocular surface and the eyelids ensures that the precorneal tear film shall spread evenly over the entire cornea. Patients suffering from limbal lesions like dermoids face the problem of apposition of the eyelids to the globe leading to local selective areas of drying.
- Normal epithelium is necessary for the adsorption of mucin on to its surface cells. Patients suffering from corneal scars and keratinizations have problem of interference with the corneal wetting.
The tear film is not visible apparently on the surface of the eye but at the upper and lower lid margins a 1 mm strip of tear fluid with concave outer surface can be seen. It is here that the oily surface prevents spillage of the tear fluid over the lid margin. Tears forming the upper tear strip are conducted nasally from the upper temporal fornix. At the lateral canthus the tears fall by gravity to form the lower strip, spreading medially the upper and lower strips reach the plica and caruncle where they join together. The tear fluid does not flow over the eye by gravity but a thin film is spread over the cornea by blinking and eye movements.
TEAR FILM FORMATION DYNAMICS
It is interesting to know the tear film formation. Generally during the closure of the eyelids the superficial lipid layer of the tear film is compressed by the eyelid edges because it is energetically unfavorable for the lipid to penetrate under the lids into the fornix. The thickness of lipid layer therefore increases by a factor of 1000 resulting in thickness of 0.1 mm which is easily contained between the adjacent eyelid edges. The aqueous tear layer remains uniform under the lids and acts as a lubricant between the eyelids and the globe. In a complete blink phenomenon, the two tear minisci join and most of their bulk is held at their junction to fill the slight bridge formed by the meeting eyelids and at the canthus.
When the eyelids open, first they form an aqueous tear surface on which the compressed lipid rapidly spread. Monomolecular lipid layer is the first to spread at speeds limited only by the moving eyelid. Following the spread of lipid monolayer, the excess lipid and associated macromolecules shall distribute themselves over the tear film surface at a lower speed, usually the lipid layer ceases within 1 second after the opening of the eye.
Under normal conditions a person blinks on an average 15 times per minute. Some of these blinks may not be complete (the upper eyelid descends only half way towards the lower eyelid). Normally the tear film break up time (BUT) is longer than the interval between blinks and no corneal drying occurs.
A deficiency in the conjunctival secretions can lead to dry eye symptoms even in the presence of an adequate aqueous tear component (Figure 1.4).8
BUT (Break up Time) is generally determined after the instillation of a drop of fluorescein solution in the eye or after staining the tear miniscus and the tear film by a wetted paper strip containing fluorescein. Normal BUT value ranges from 10 to 40 seconds for normal eyes (Figure 1.5) when the BUT is determined by a non-invasive method (e.g. by the toposcope). BUT values of as long as 3 to 5 minutes can be recorded.
If the BUT is shorter than the average time interval between two consecutive blinks, tear film rupture can cause pathological changes in the underlying epithelium. The tear film breaks up prematurely over the damaged epithelial surface thereby exacerbating the injury.
Generally there is balance between the secretion and excretion of tears and the rate of tear drainage increases with increased tear volume.
NORMAL TEAR DRAINAGE
In the normal tear film between 10 and 25% of the total tears secreted are lost by evaporation. Evaporation rate is low because of the protective oily surface.
In the absence of the protective oily layer the rate of evaporation is increased 10 to 20 times. Normally tear flows along the upper and lower marginal strips and enters the upper and lower canaliculi by capillarity and possibly by suction also. About 70% of tear drainage is via the lower canaliculus and the remaining through the upper canaliculus. With each blink the superficial and deep heads of pretarsal orbicularis muscle compress the ampullae, shorten the horizontal canaliculi and move the puncta medially. Simultaneously the deep heads of preseptal orbicularis muscle which are attached to the fascia of the lacrimal sac contract and expand the sac. This creates a negative pressure which sucks the tears from the canaliculi into the sac. When the eyes are opened the muscles relax, the sac collapses and a positive pressure is created which forces the tear down the duct into the nose. Gravity also plays an important role in the sac emptying. The puncta move laterally, the canaliculi lengthen and become filled with tears.
TEAR COMPOSITION
Tears contain 98.2% water and 1.8% solids. The high percentage of water in tears is a natural consequence of the need for lubrication of the conjunctiva and corneal surface (Tables 1.1 and 1.2). The evaporation of water between blinks may influence the concentration of the tear film. The evaporation rate of water from the intact precorneal tear film through the superficial lipid layer has been shown to be 8 × 10−7 cm−2.sec−1. In a time interval of 10 seconds (between two consecutive blinks) the thickness of the tear film decreases about 0.1 mm resulting in nearly 1 to 2% decrease in water concentration. The solute concentration, however, increases about 20%.
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PHYSICAL PROPERTIES OF TEARS
Tear pH
The pH of unstimulated tears is about 7.4 and it approximates that of blood plasma. Although wide variations are found in normal individuals (between 5.0–8.35) the usual range is from 7.3 to 7.7. A more acidic pH of about 7.25 is found following prolonged lid closure possibly due to carbon dioxide produced by the cornea and trapped in the tear pool under the eyelids. Tear pH is characteristic for each individual and the normal buffering mechanism maintain the pH at a relatively constant level during waking hours. The permeability of the corneal epithelium does not seem to be affected by wide variations in the pH of tear fluid.
Osmotic Pressure
The osmotic pressure in tears mainly caused by the presence of electrolytes is about 305 mOsm/kg equivalent to 0.95% sodium chloride. Individual values over the waking day may range from 0.90 to 1.02% NaCl equivalents. A decrease to an average of 285 mOsm/kg equivalent to 0.89% NaCl has been reported following prolonged lid closure which accounts for the reduced evaporation. When the aqueous component of tears decreases, the tears become markedly hypertonic (0.97% NaCl solution or more) and corneal dehydration results. When the eyes are closed, there is no evaporation of tears and the precorneal tear film is in osmotic equilibrium with the cornea. When the eyes are open evaporation takes place, increasing the tonicity of the tear film and producing an osmotic gradient from the aqueous through the cornea to the tear film. This direction of flow will continue as long as evaporation maintains the hypertonicity of the tear film. Osmotic pressure is sensitive to changes in tear flow. Reflex stimulation of tears in early adaptation to contact lenses results in a decrease in electrolytes and in total protein leading to hypotonicity. This relative hypotonicity may account for the corneal edema often seen in early stages of contact lens wearing.
Other Physical Properties of Tear (Table 1.2)
- Refractive index—1.357
- Tear volume—0.50–0.67 g/16 hr (waking).
CHEMICAL COMPOSITION OF TEAR FLUID
The chemical composition Table 1.2 of tear fluid is quite complex. The first chemical analysis of tears was studied in 1791 by Fourcroy and Van Que Lin Fleming (1922) and Ridley (1934) demonstrated the detailed chemical composition of normal tears.
Immunoelectrophoretic studies have shown that tears contain lipids, proteins, enzymes, metabolites, electrolytes and hydrogen ions, etc.
Lipids
Lipids are present in small amount in tears as they are contained only in the very thin superficial lipid layer of the tear film. Chromatographic studies of meibomian lipids reveal the presence of all possible lipid classes mainly waxy esters, hydrocarbons, triglycerides, cholesterol esters and in lesser amount diglycerides, monoglycerides, free fatty acids, free cholesterol and phospholipid. However, great individual variations occur in lipid composition.
Cholesterol
Cholesterol has been reported to be present in tear fluid in concentrations of about 200 mg% which is same as in the blood. Like all lipids in biological fluids cholesterol has to be transported by α and β lipoproteins. In normal tears the very low protein content and the absence of lipoproteins is incompatible with a cholesterol concentration of 20 mg%.
Proteins
About 60 components to tear protein fraction have been reported which form the first line of defense against an external infection and seen to be more effective than systemically produced antibodies. The protein content of tears differ from that of blood plasma in several respects. Proteins can be divided in two groups.
Group A: Proteins which are similar to serum proteins with a low concentration representing less than 15% of all tear proteins. Some of them are always present in tears. Table 1.3 namely albumin, IgG, α-Lantitrypsin, transferrin, α-L antichymotrypsin and β-2 microglobulin others which appears sporadically are ceruloplasmin, haptoglobin and Zinc α-2 glycoprotein.
Group B: Specific proteins synthesized by tear gland are RMP (rapid migration protein) and some other proteins (Tables 1.4 and 1.5) which are also present in other external secretions (lysozyme, lactoferrin and IgA).13
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Tear Albumin
Albumin represents about 60% of the total protein in tears as it does in plasma. Tear albumin is a unique protein fraction. It is electrophoretically a prealbumin and migrates to a position similar to serum prealbumin. Genetic polymorphism has been reported of the tear albumin.
Electrophoresis of tears shows several peaks of migration. These peaks are main which correspond to proteins synthesized by the lacrimal gland—rapid migrant proteins and lactoferrin migrating to the anode and lysozyme migrating to the cathode.
The total tear proteins content strongly depends upon the method of collection of tears. Small unstimulated tears show levels of about 20 mg/ml while stimulated tears show much lower values in the range of 3 to 7 mg/ml reflecting the level of lacrimal gland fluid.
Lysozyme
Fleming first discovered an antibacterial substance and showed that this substance is an enzyme which he named lysozyme because of its capacity to lyze bacteria. In normal tears concentration of lysozyme is much higher than in any other body fluid. The normal level for human tear lysozyme (HTL) is 1 to 2 mg/ml. The enzymic activity of lysozyme is optimal at pH 5.2 and decreases above and below this pH value.
Lysozyme is a long chain, high molecular weight proteolytic enzyme produced by lysosomes—a known cellular ultra structure. Lysozyme acts upon certain bacteria and dissolves them by cleaning the polysaccharide component of their cell walls. As the function of cell wall in bacteria is to confer mechanical support a bacterium devoid of its cell wall usually bursts because of the high osmotic pressure inside the cell.
Lysozyme level in tears can be measured with a diffusion method or with a spectrophotometric assay.
In addition to lysozyme, presence of other antibacterial factors in human tears have been shown. The nonlysozymal bactericidal protein beta lysin has been reported to be derived chiefly from platelets but it exists in higher concentration in tears than in blood plasma. The lysozyme and beta lysin protein fractions can be separated by filtering the tears. The antibacterial activity of the filtrate results from lysozyme but in whole tears beta lysin is responsible for three-fourth of the bactericidal effect. Beta lysin acts primarily on cellular membrane while lysozyme dissolves bacterial cell walls.15
The action of lysozyme depends on the pH. The optimum pH for lysis varies with the solubility of the bacterial proteins but in general it ranges between 6.0 and 7.4. Low salt concentrations favor lysis by increasing solubility.
Human tear lysozyme (HTL) levels have been shown to be greatly decreased in tears of patients suffering from Sjögren's syndrome and ocular toxicity from long-term use of practolol therapy thus making it a useful diagnostic aid. Other disease states where HTL level is lowered include herpes simplex virus infection and malnutrition in children.
Lactoferrin
It is an iron carrying protein and appears to be a major tear protein in the intermediate fraction. Its property of iron binding (Fe III) is 300 times stronger than the other iron binding protein (transferrin). This is probably significant for its bacteriostatic activity in tears making essential metal ions unavailable for microbial metabolism.
Transferrin
Transferrin has been shown to be present in tears. Transferrin along with serum albumin and IgG can be detected only after mild trauma to the mucosal surface of the conjunctiva or in tears.
Ceruloplasmin
Ceruloplasmin, a copper carrying protein is regularly found in tears. In electrophoresis the migration rate of tear ceruloplasmin varies from its serum counter part.
Immunoglobulins
Tiselius (1939) for the first time separated the plasma proteins by electrophoresis and isolated three types of globulins—alpha, beta and gamma. Antibody property of the immune serum resides in the gamma globulin fraction. Immunoglobulins are elaborated by plasma cells following transformation of antigen stimulated B-lymphocytes. This elaboration constitutes the humoral immune system.
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Immunoglobulin A (IgA)
Immunoglobulin G (IgG)
Immunoglobulin G (IgG)
Immunoglobulin E (IgE)
Immunoglobulin D (IgD)
Immunoglobulin A (IgA): It is the major immunoglobulin present in tears, saliva and colostrum. Almost all of the IgA have a secretory component attached to them when they occur in external secretions. It participates in the functioning of IgA as antibody in the external environment. The possible functions of secretory IgA include prevention of viral and bacterial infections that may have an access to the external secretions, e.g. tears and participate as opsonins in the phagocytosis process.
The average levels of IgA—the predominant immunoglobulin in normal human tear is 14 mg/dl.
In the human lacrimal gland, IgA appears to be synthesized by interstitial plasma cells and after entry into the intercellular spaces it is coupled to SC and secreted as secretory IgA (IgA-SC) through the blood-tear barrier involving intracellular transport by acinar epithelial cells into the lumens. In the conjunctiva IgA and plasma cells are located in the substantia propria. Only in the acinar epithelium of the accessory lacrimal glands can SC material be present indicating that these are the sites of synthesis of secretory IgA of the conjunctival secretions. Depending upon the method of tear collection IgA values can vary from 10 to 100 mg%.
Immunoglobulin G (IgG): It is present in very low concentrations in normal tears. However, after mild trauma to the mucosal surface of the conjunctiva it can be easily detected.
IgG is the most prominent circulating (serum) immunoglobulin present in concentrations five times that of IgA. The average level of IgG in normal human tears range from 17 to 20 mg/100 ml.17
The serum level of IgG is about 1000 mg/dl. IgG molecule has a molecular weight of about 150,000. Each molecule of IgG consists of 2 L chains and 2 H chains linked by 20-25-S-S bonds. The antigenic analysis of IgG myelomas show four subclasses now termed as IgG1, IgG2, IgG3, and IgG4,. IgG1 is the predominant variant and together with IgG3 possesses the ability to combine with complement to bind to macrophages and to cross the placenta. IgG synthesis in humans is about 35 mg/kg/d and its half-life is about 23 days. IgG molecules are Y-shaped with a hinge region near the middle of the heavy chain connecting the 2 Fab segments to the Fc segment.
During the secondary response, IgG is the major immunoglobulin to be synthesized probably because of its small size, IgG diffuses more readily than other immunoglobulins into the tears, therefore as the predominating immunoglobulin it carries the major burden of neutralizing bacterial toxins and of binding to microorganisms (specially streptococci, pneumococci and staphylococci) to enhance their phagocytosis. IgG is most efficient in killing and stopping the progress of microorganism's invasion.
Immunoglobulin M (IgM): It is present in very low concentrations in normal tears. The average level of IgM in normal tears range from 5 to 7 mg%. Barnett (1968) reported first the presence of IgM in normal tears.
The serum level of IgM is about 100 mg/dl. The IgM molecule with a molecular weight 900,000 is the largest of the immunoglobulins. Often referred to as macroglobulin because of its size, the IgM molecule are pentamers with a high valency or anticombining capacity. Due to its high valency IgM is extremely efficient agglutinating and cytolytic agent and is the first type of antibody which is formed after the initial encounter with antigen. It appears early in response to infection and is confined mainly to the bloodstream.
Even minimum trauma to conjunctiva would cause serum proteins to leak into the tears. There is increased concentrations of IgA, IgG and IgE in tears. Either these immunoglobulins are selectively excreted into the tears or they are locally synthesized. Increased concentrations of IgA, IgG and IgM are reported in cases of blepharoconjunctivitis, herpes keratitis, vernal conjunctivitis, acute follicular conjunctivitis, phlyctenular conjunctivitis, keratomalacia, corneal ulcer and acute endogenous uveitis.
Immunoglobulin E (IgE): It is mostly extravascular in distribution. IgE values ranges from 26 to 144 µ g/ml in normal tears. Normal serum contains only traces of IgE but greatly elevated levels are seen in atopic conditions.18
Immunoglobulin D (IgD): IgD levels are quite low in tears as well as in serum. It is mostly intravascular.
Complement
Complement in tears has been shown in hemolytic assays up to dilution of 1.4 whereas serum is active in this system up to 1:32.
Glycoproteins
Glycoproteins are present in the mucoid layer as well as in the tear fluid since they are highly soluble in water. Glycoproteins contribute significantly to the stickiness of the material forming the mucoid layer. N-acetyeneuraminic acid (a sialic acid) has been indentified in normal tears. Glycoproteins may play a critical role in the lubrication of the corneal surface by rendering its hydrophobic surface more hydrophilic permitting spreading and stabilization of the tear film. The mucus is secreted by the conjunctival goblet cells as a solution of glycoproteins (mucoids) and this sticky mixture adheres to the surface of the epithelium even though the glycoproteins are water soluble.
The glycoproteins are carbohydrate-protein complexes characterized by the presence of hexosamines, hexoses and sialic acid. In normal tears relative hexosamine content of the protein which is used as indicator for glycoproteins varies from 0.5 to 17%, the hexosamine concentration from 0.05 to 3 g/l. Sialic acid concentration of human tears has been reported to be 114 µmol/100 ml.
Antiproteinases
Antiproteinases, inhibitors of proteinases are present in tears at levels much lower than in plasma (Table 1.7).
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These includes α1-antitrypsin, α1-antichymotrypsin, inter-α-trypsin inhibitor and α2-macroglobulin. The source of-α1 antitrypsin is the lacrimal gland while other antiproteinases originate from corneal and conjunctival surfaces. In various inflammatory conditions of the eye the levels of α1-at and α2-m in tear fluid are increased.
In bacterial and viral infections of the eye (Table 1.8) and in corneal ulceration the levels of α1-at and α2-m in tear fluids are increased. Using albumin as a marker protein there is evidence suggesting that these two collagenase inhibitors are derived either from plasma by a general increase in vascular permeability to proteins or they are produced locally.
Metabolites
A number of metabolites have been reported to be present in normal human tears. These include organic constituents of low molecular weight like glucose, urea, amino acids and other metabolites like lactate, histamine, prostaglandins and catecholamines.
Glucose
Glucose is present in minimal amounts of about 0.2 mmol/liter in tear fluids of normal glycemic persons. This low concentration of glucose appear to be insufficient for corneal nutrition. There is no definitive evidence that cornea metabolizes glucose emanating from the tears.20
It has been shown that some glucose in tears originates from the goblet cells of the conjunctiva. There is corresponding rise in tear glucose level with elevation of plasma glucose level above 100 mg%. However, there is no significant rise in tear glucose levels in diabetics with blood glucose level of more than 20 mmol/liter which demonstrates the barrier function of the corneal and conjunctival epithelium against loss of glucose from the tissues into the tear fluid. It is the tissue fluid which contributes to the tear glucose after mechanically stimulated methods of tear collection.
Urea
Urea concentration in tear fluid and plasma have been found to be equivalent suggesting an unrestricted passage through the blood-tear barrier in the lacrimal gland. Urea concentration in tears decreases with increasing secretion rate.
Amino Acids
Free amino acid concentration in tears is reported to be 7.58 mg/100 ml. This value is 3 to 4 times higher than the free amino acid concentration in serum.
Lactate
Lactate levels of 1 to 5 mmol/l in tears are far higher than the normal blood levels of 0.5 to 0.8 mmol/l. Pyruvate from 0.05 to 0.35 mmol/l is about the same as is normal for blood (0.1–0.2 mmol/l). These levels do not show significant alterations after mechanical irritation. The epithelium does not possess a barrier function for lactate and pyruvate.
Histamine
Histamine is present in normal tears collected from the conjunctival sac at a level of about 10 mg/ml. In vernal conjunctivitis specifically a variable increase up to 125 mg/ml has been observed.
Prostaglandins
Prostaglandins are present in normal tears at the level of 75 pg prostaglandin F/ml and it is little lower than in serum. In inflammatory conditions of the eye significant higher values are found up to 300 pg/ml of tears.21
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Catecholamines, Dopamine, Noradrenaline and Dopa
Catecholamines, dopamine, noradrenaline and dopa have been found in the tear fluid. The levels vary from 0.5 to 1.5 mg/ml. Dopamine has values as high as 280 mg/ml.
In glaucoma patients lower values have been reported for these compounds which reflect the diminished activity of the sympathetic innervation of the eye. The determination of catecholamines in tears has been advocated as a test in glaucoma diagnosis.
Electrolytes and Hydrogen Ions
The predominant positively charged electrolytes (cation) in tears are mainly sodium and potassium while the negative ions (anions) are chloride and bicarbonate (Table 1.9).
Sodium
Sodium concentration in tears 120 to 170 mmol/liter is about equal to that in plasma suggesting a passive secretion into the tears. While potassium with an average value of about 20 mmol/l is much higher than the corresponding plasma concentration of about 5 mmol/l. This indicates an active secretion of potassium into the tears. It is interesting to observe that while the main cationic constituent of the aqueous and vitreous humor is sodium while cornea (mainly corneal epithelium) contains a much higher concentration of potassium than sodium. These two cations play an essential role in the osmotic regulation of the extracellular and intracellular spaces and in general changes in sodium level are the reverse of changes in potassium level.
Calcium
Calcium is independent of the tear production and is lower than the free fraction of plasma. In cystic fibrosis patients have much higher calcium values. 22An average of 2.5 mmol/l have been shown only at slow rates concomitant with lower tear sodium values.
Magnesium
Magnesium in tears is little lower than corresponding serum value possibly reflecting the free fraction of magnesium. Both calcium and magnesium play a role in controlling membrane permeability.
Chloride
Chloride, an anion essential to all tissues also plays an important role in osmotic regulation much like sodium and potassium. The chloride concentration is slightly higher in tears than in serum.
Bicarbonate
The bicarbonate together with the carbonate ions in tears may be involved in the regulation of pH. This buffer system maintains the near neutral pH of the tear film, the surface of which is exposed to atmospheric changes.
Enzymes
Enzymes of Energy Producing Metabolisms
Glycolytic enzymes and enzymes of tricarboxylic acid cycle can be detected in high values only in human tear samples. These enzymes form a blood-tear barrier against penetration from the blood. The source of these enzymes is in the conjunctiva where they are secreted in small amounts. The lacrimal gland apparently does not secrete these enzymes. These enzymes can be obtained during mechanical irritation.
Lactate Dehydrogenase
Lactate dehydrogenase (LDH) is the enzyme in the highest concentration in tears. It can be separated electrophoretically into its five isoenzymes showing a pattern with more of the slower migrating muscle type isoenzymes. This is closely related to the distribution pattern of corneal tissue in contrast to serum LDH where the faster migrating heart type isoenzymes prevail.
These findings indicate that tear LDH originates from the corneal epithelium. Therefore, in patients suffering from corneal disease, the distribution of LDH isoenzymes in tears differs from those found in healthy 23individuals. LDH isoenzymes bound to immunoglobulin have been found in blood and it is probable that here an analogous binding takes place in tears.
Lysosomal Enzymes
Lysosomal enzymes include a number of lysosomal acid hydrolases which are present in tears in concentration of 2 to 10 times than those in serum. The lacrimal gland is the main source of the lysosomal enzymes but conjunctiva may act as a second source for lysosomal enzymes after mild trauma. The relative high values are found in tear fluid collection where the epithelial cells of conjunctiva remain intact and contain very low levels of lactate dehydrogenase or other cytoplasmic enzymes. Lysosomal enzyme activities in tears are used for diagnosis and identification of carriers of several inborn errors of metabolism.
The concentration of β-hexosaminidase in tears collected on filter paper strips is an index for the development and prognosis of diabetic retinopathy. The tears would reflect the decreased enzyme activity of β-hexosaminidase and of other lysosomal glycosidases in the retina showing a negative correlation with the increased plasma levels of these enzymes.
Amylase
Amylase is the enzyme present in tear fluid in relatively moderate levels. The origin of this enzyme is in lacrimal gland. The reported presence of amylase in the cornea might be due to contamination by tear fluid.
Peroxidase
Peroxidase (POD) is present in human tears originating from the lacrimal gland and not from the conjunctiva. The level of tear POD in human tears is 103 µ/l. POD activity found in the conjunctiva is probably derived from the tears.
Plasminogen Activator
Plasminogen activator has been demonstrated in tear fluid and corneal epithelium is suggested to be the source of this urokinase-like fibrinolytic activity.
Collagenase
Collagenase has been shown to be present in tear fluid in the presence of corneal ulceration, due to infection, chemical burn, trauma and desiccation. 24Corneal collagenase is present as an inactive precursor “latent collagenase” which can be activated with trypsin and in vivo possibly by plasmin resulting from plasminogen activator activity in tears.
Drugs Excreted in Tears
Tears represent a potentially more stable body fluid of low protein content and with modest variations of pH. Passage of drugs from the plasma to the tears apparently takes place by diffusion of the non-protein bound fraction. However, presence of tight junctions between the acinar epithelial cells in the lacrimal gland forming a blood-tear barrier, the lipid solubility is expected to play a major role. The blood-tear barrier shows the same characteristics as that of cell membrane. Phenobarbital and carbamazepine are excreted in tears in about 0.5% of corresponding plasma concentration.
Methotrexate, an antimetabolite reaches tear levels of 5% of the corresponding plasma concentrations and is in equilibrium with the unbound fraction in plasma. Ampicillin is present in tears in concentration of about 0.02 of the corresponding serum level.
APPLIED PHYSIOLOGY
Basic secretion of tear fluid is made up of the secretions of the lacrimal gland and accessory lacrimal gland tissue together with the secretions of meibomian glands and the mucous glands of the conjunctiva. Reflex secretions of tears is hundreds time greater than basal or resting secretion. The stimulus to reflex secretions appears to be derived from the superficial corneal and conjunctival sensory stimulation as a result of tear break up and dry spot formation. The secretory stimulus to the lacrimal glands is parasympathetic with reflex secretions occurring in both eyes following superficial stimulation of one eye. The whole mass of lacrimal tissue responds as one unit to reflex tearing. Reflex secretion is reduced by topical corneal and conjunctival anesthesia.
HYPOSECRETION OF TEARS
Hyposecretion means decreased formation of tears.
Lacrimal hyposecretion may be congenital although not very common. Acquired lacrimal hyposecretion may be due to:
- Atrophy and fibrosis of lacrimal tissue due to a destructive infiltration by mononuclear cells as in keratoconjunctivitis sicca and Sjögren' s syndrome.
- Local inflammatory diseases of the conjunctiva commonly conjunctival scarring secondary to bacterial or viral infection.
- Chronic inflammatory disease of the salivary and lacrimal glands (Mikulicz's syndrome).
- Damage or destruction of lacrimal tissue by granulomatous (sarcoidosis), pseudotumor or neoplastic lesions.
- Absence of lacrimal gland.
- Blockage of excretory ducts of the lacrimal gland.
- Neurogenic lesions.
- Meibomian gland dysfunction.
Diagnostic Tests for Tear Hyposecretions (Table 1.10)
Tear Film Break-up Time (BUT)
The tear film break-up time is a simple physiological test to assess the stability of the precorneal tear film. This test is performed by instilling fluorescein into the lower fornix, taking precaution not to touch cornea. The patient is asked to blink several times and then to refrain from blinking. The tear film is scanned with a broad beam and cobalt blue filter. After an interval of time black spots or line indicating dry spots appear in the tear film. :BUT is the interval between the last blink and appearance of the first randomly distributed dry spot. Ideally average of three measurements is taken. A normal BUT is more than 10 seconds and a BUT of less than 10 seconds is considered abnormal. This test may also be abnormal in eyes with mucin or lipid deficiency.
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Schirmer's Test
The rate of tear formation is estimated by measuring the amount of wetting on a special filter paper which is 5 mm wide and 35 mm long (Figure 1.6).
Previously Schirmer's test 1 and 2 were used in diagnostic practice but nowadays modified Schirmer-I test is employed. This test is performed as follows.
Schirmer strips are prepared by cutting out Whatman filter paper No. 41 into the strips of 5 mm × 35 mm dimensions. A 5 mm tab is folded over at one end. Before use, these strips are autoclaved.
The bent end is placed into lower conjunctival sac at the junction of lateral one-third and medial two-third of the lower eyelid so that a 5 mm bent end rests on the palpebral conjunctiva and the folding crease lies over the eyelid margin. This test is usually performed in sitting posture in dim light.
The patient is asked to keep the eyelid open and look slightly upwards at a fixation point. Blinking is allowed while the patient gazes at the fixation point.
After one minute, the strips are carefully removed and moistening of the exposed portion of the strip is measured in millimeters with the help of a millimeter ruler.
The measurements are made from the notch at the bend of the Schirmer strip to the distal end of the wetting on the strip (excluding the folded over tab). The amount of wetting of the Schirmer strip in one minute is multiplied by three to correspond roughly to the amount of wetting that would have 27occurred in five minutes (Jones, 1972). It is a measure of the rate of tear secretion in a five-minute period.
A normal eye will wet between 10 to 25 mm during that period. Measurements between 5 and 10 mm are considered borderline and values less than 5 mm is indicative of impaired secretion.
Vital Dye Staining
- Rose Bengal 1% has an affinity for devitalized epithelial cells and mucus in contrast to fluorescein which remains extracellular and is more useful in showing up epithelial defects. Rose Bengal is very useful in detecting even mild cases of keratoconjunctivitis sicca (KCS) by staining the interpalpebral conjunctiva in the form of two triangles with their base at the limbus.The only disadvantage with Rose Bengal staining is that it may cause ocular irritation specially in eyes with severe KCS. In order to reduce that amount of irritation only a small drop should be instilled into the eye. A topical anesthetic should not be used prior to the instillation of Rose Bengal as it may produce a false-positive result.
- Alcian blue has similar properties as Rose Bengal and is less irritant but it is not generally available.
Lysozyme Assay
Lysozyme assay is based on the fact that in hyposecretion of tears, there may be reduction in the concentration of lysozyme. This test is performed by placing the wetted filter strip into an agar plate containing specific bacteria. The plate is then incubated for 24 hours and the zone of the lysis is measured. The zone will be reduced if the concentration of lysozyme in the tears is decreased.
Tear Globulin Assay
Tear IgA levels are measured in this test. This test is also based on the principle that decreased tear formation will lead to decreased IgA (immunoglobulin A) levels in tears. This test is performed on a specific tripartigan immunodiffusion plates containing specific agar gel in wells (Figures 1.7 and 1.8). 20 αl of tear samples is put into these wells and plates are incubated for 48 hours. The diffusion of rings around wells are measured to the nearest 0.1 mm with a partigen ruler. The ring will be reduced if the concentration of IgA in tears is decreased. This is a reliable test for measuring tear globulins.28
FIGURE 1.8: Tripartigen immunodiffusion plates (diffusion of rings around agar wells is measured up to 0.1 mm)
Tear Osmolarity
Tear osmolarity is increased in cases of hyposecretion.
Biopsy of the Conjunctiva
Biopsy of the conjunctiva and an estimation of the number of goblet cells are other tests which can be done. In mucin deficiency states the number of goblet cells shall be decreased.
HYPERSECRETION OF TEARS
In practice when patient complains of a wet eye there are two possibilities of excessive watering of the eye.
- Lacrimation from reflex hypersecretion due to irritation of cornea and conjunctiva.
- Obstructive epiphora as a result of failure of tear drainage or evacuation system. The main causes are lacrimal pump failure due to lower lid laxity or weakness of the orbicularis muscle and more commonly due to mechanical obstructions of the drainage system.If the wet eye is caused by hypersecretion the Schirmer's test values (technique already mentioned) will be increased and the Jones Fluorescein dye test will reveal normal outflow function.
Physiological Diagnostic Test for Hypersecretions
Jones I (Primary) Test
This is a physiological test which differentiates an excessive watering due to a partial obstruction of the lacrimal passages from primary hypersecretion of tears (Figure 1.9).
In this test 1 drop of 2% fluorescein solution is instilled into the conjunctival sac. After about 5 minutes a cotton-tipped bud or applicator (moistened in coccaine 4% or proparacaine 0.75%) is inserted under the inferior turbinate at the nasolacrimal duct opening. This is situated about 3 cm from the external nares.
The results are interpreted as follows.
- If the fluorescein is recovered from the nose on the applicator and aqueous solution passes from the conjunctival sac to the nose in 1 minute then the excretory system is patent and cause of watering is primary hypersecretion. No further tests are required then and the test is inferred as positive.30
- If no dye is recovered from the nose a partial obstruction is present or there is failure of the lacrimal pump mechanism. In this situation secondary dye test or Jones II test is required.
Jones II (Secondary Irrigation) Test
This test helps to identify the probable site of partial obstruction.
In this procedure topical anesthesia (4% Xylocaine or 0.5% proparacaine) is instilled into the conjunctival sac and any residual fluorescein is washed out. The nasolacrimal system is then irrigated with normal saline. The patient is positioned with his or her head down by about 45° so that the saline runs out of the nose into white paper tissues and not into the pharynx.
This test is interpreted as follows.
- Positive—if fluorescein-stained saline is recovered from the nose, the dye must have reached the lacrimal sac during the primary dye test but was stopped from entering the nose by a partial obstruction in the nasolacrimal duct. However, syringing of the lacrimal system had pushed the dye past the obstruction into the nose. A positive secondary dye test indicates a partial obstruction to the nasolacrimal duct which can be treated by a dacryocystorhinostomy (DCR) procedure.
- Negative—if unstained saline is recovered from the nose it means that no dye has entered the lacrimal sac during the primary dye test. This means a partial obstruction in the upper drainage system (punctum, canaliculi or common canaliculus) or a defective lacrimal pump mechanism. In such a situation DCR would fail and some other operative procedure will be required.
Fluorescein Dye Disappearance Test
An accurate status of the excretory capability of the lacrimal system can be obtained by observing the behavior of a single drop of 2% fluorescein solution instilled into the inferior conjunctival cul-de-sac. The color intensity after 5 minutes is measured and graded on a scale of 0 to 4+. The normal excretion of the retained fluorescein shall be 0–1+. Any greater residual then is indicative of impaired outflow. However, by this test one cannot distinguish between impairment of the upper and lower segments of the system, but it may complement the Jones tests.
- Nasal examination should be performed in order to determine the position of normal nasal structures specially the position of the anterior end of the middle turbinate when surgery is contemplated. It will also detect the presence of polyps or tumors, etc.
Special Tests
Intubation Dacryocystography
The conventional method of dacryocystography consists of injecting contrast medium into one of the canaliculi followed by the taking of posteroanterior (PA) and lateral views, radiographs. However, far superior status of the canalicular system can be obtained by using a technique that combines injection of lipoidol ultra fluid through a catheter with macrography. In common canalicular lesions, subtraction macrodacryocystography may provide more sophisticated details.
These specific investigations are not only extremely valuable in depicting the exact location of the obstruction but they are also of help in the diagnosis of diverticula, fistulae, filling defects due to tumors, stones and infections by streptothrix species.
Scintillography (Radionuclide Testing)
This test involves the labeling of tears with gamma-emitting substances such as technetium-99m and monitoring their progress through the drainage 32system. This is a sophisticated and reliable test for better understanding of excretory physiology.
Color Doppler Scanography
Color Doppler scanography is the latest technique for evaluating the status of the drainage system. It is a recently introduced test with accurate results.