**2. The layers of tear film**

Traditionally, the tear film has been described as a three-layered structure composed of the deep mucinous, middle aqueous, and superficial lipid layers (e.g. **Figure 1**). All three layers overlay the corneal and conjunctival epithelium, forming a full thickness of 2–5.5 μm [1]. Generally, the functions of the tear film are to lubricate the corneal and eyelid interface, form a protective covering and a smooth optical surface at the air-eye interface, and provide an antibacterial medium for the cornea and conjunctiva. The tear film also acts as the main oxygen supply to the corneal epithelium and functions as a temporary depository for instilling topical therapeutic drugs. (e.g. **Figure 1**).

#### *Dry Eye Syndrome - Modern Diagnostic Techniques and Advanced Treatments*


#### **Table 1.**

*Overview of the origin, composition, and function of the tear film layers.*

#### **Figure 1.**

*Traditional 3-layered composition of tear film. Available via license: Creative commons attribution 4.0 international.*

#### **2.1 The mucinous layer**

Most posteriorly sits the mucinous layer which has a thickness of 0.5 μm. It is composed of a mucin dominant gel formed by 2 layers: the glycocalyx and mucous layers. Posteriorly, the glycocalyx layer sits on the microvilli of the superficial corneal epithelium and is produced by the corneal and conjunctival epithelial cells [1, 2].

Overlying the glycocalyx layer is the mucous "blanket," a thick layer produced by the conjunctival goblet cells and the glands of Manz, lying in the crypts of Henle and in the bulbar conjunctiva, respectively [3]. This mucous layer is made of many gel-forming mucins and most significant of the mucin is MUC5-AC. Several studies

**Figure 2.** *TBUT with fluorescein dye tear break up (red arrow). Taken with permission from Kenny Chan [5].*

have linked MUC5-AC decrease to DES. The function of the mucin is to transform the corneal surface into a hydrophilic surface [1, 2]. This transformation results in a reduction in corneal surface tension and provides the cornea with tear film stability, allowing the adhesion of the overlying aqueous layer, preventing the formation of dry spots. This decrease in surface tension also serves to lubricate and cushion the eye during all movements [3].

In order to test the integrity of the mucinous layer, the Tear Break-up Time (TBUT) test can be done. This test is used to assess for evaporative dry eye disease from the deficiency of mucin [4]. It is carried out by first instilling fluorescein into the patient's tear film. Afterwards, a cobalt blue illumination is shown onto the effected eye to observe the time elapsed between the last blink and the appearance of the first dry spot in the tear film (e.g. **Figure 2**). A TBUT of under 10 seconds is abnormal, indicating a problem with the mucinous layer's ability to form a hydrophilic layer [4].

Other abnormalities can occur which affect this layer include Vitamin A deficiency, Ocular Cicatricial Pemphigoid, Stevens-Johnsons Syndrome, and Alkali burns [6]. All mentioned conditions lead to the destruction of goblet cells with consequent loss of mucin production. As a result, a rapid breakdown of tear film occurs, even with adequate volume of aqueous layer.

## **2.2 The aqueous layer**

The middle aqueous layer forms the largest part of the tear film thickness, at 2–6 μm [1]. Its main functions are to supply oxygen to the corneal epithelium, provide a protective layer against bacteria, and provide a healing media through VEGF. The aqueous layer is produced by the secretions of the lacriminal gland apparatus and its accessory glands. The aqueous layer can be secreted via reflex secretions or via its basal source. The reflex secretions are secreted by the main lacriminal gland whereas the basal source of the aqueous is secreted from the accessory lacriminal glands of Krause and Wolfring [7].

Unlike the mucinous layer, the release of aqueous is mediated by various methods: the autonomic nervous system, hormones, and psychological factors. The autonomic nervous system activates the lacriminal reflex through the sensory innervations at the corneal and conjunctival unmyelinated C-type fibers which form the subepithelial plexus at the superficial cornea [8]. The stimulation of the sensory nerves causes the parasympathetic system to increase the aqueous secretion and vasodilate the blood vessels supplying the lacriminal gland. Although the sympathetic nervous system plays a role in tear lacriminal aqueous secretion, the parasympathetic system predominates this reflex [8].

Androgens also play a role in the mediation of aqueous secretion from the lacriminal gland. Reduced serum androgen levels in women with altered endocrine states, such as women after menopause, ovariectomy, and during oral contraceptive use have been observed to have primary lacriminal deficiency, despite their variable estrogen levels. However, men who take anti-androgen therapy do not show signs of any change to tear secretion, suggesting that the androgen effect of the lacriminal gland may be sex specific [8]. Moreover, emotional expression also controls the secretion of aqueous from the lacriminal gland. The exact mechanism is currently unknown, and further research is needed to understand the neurobiology of human emotional crying [2, 8].

The aqueous layer is composed of 98% water, with the remaining 2% made up of Sodium, Potassium (6x that of plasma), Chloride, Bicarbonate, Calcium, Amino Acids, Oxygen, and VEGF (**Table 2**) [7]. The proteins found in the aqueous layer plays a significant role, as it supplies the cornea with a rich source of bactericidal enzymes. High in number of lysozymes, lactoferrin, betalysin, and immunoglobins, the aqueous layer provides a barrier to infection for the eye. The Immunoglobins, mostly IgA, are derived from the lymphoid tissue in the lacriminal gland stroma. Furthermore, the VEGF found in the aqueous provides the cornea with a source for healing [7, 9].

Due to the cornea's requirements to achieve transparency, there is no blood supply within its structure. Nevertheless, oxygen is needed for the corneal epithelium's aerobic metabolism. This oxygen is derived mainly from the aqueous layer of the tear film when the eyes are open and minorly from the conjunctival blood vessels when the eyes are closed. When the eyes are open, the tear film possesses a saturation of 155 mmHg of Oxygen which makes up 70% of ATP production at the corneal epithelium. The last 30% occurs when the eyes are closed, with the saturation of oxygen from the conjunctival blood vessels being 55 mmHg. If the individual is a contact lens wearer, the pO2 drops to around 15 mmHg when eyes are closed [9, 10]. Moreover, the aqueous layer smooths irregularities in the corneal surface providing an optical function.

To test the caliber of the aqueous layer, the Schirmer test can be performed. The Schirmer test is done using a special filter paper which is 5 mm wide and 35 mm long with the bent end placed between the palpebral conjunctiva of the lower eyelid and the bulbar conjunctiva of the eye. The eye is then closed for 5 minutes and the absorption of the fluid into the filter paper is measured in millimeters. The test can be done with or without the use of anesthetics (e.g. **Figure 3**) [11]. To evaluate the baseline secretions of the lacriminal gland, the test is done using anaesthestics, whereas the evaluation of reflex secretions along with baseline secretions is done without the use of anaesthestics. An individual with normal aqueous tear


#### **Table 2.** *Electrolytes and proteins making up 2% of aqueous [7].*

**Figure 3.** *Schirmer test. Available via license: Creative commons attribution-share alike 3.0 Unported.*

production will have a reading of >15 mm after 5 minutes. Mild–moderate reduction of aqueous production is a reading from 5 to 14 mm after 5 minutes, and severe dryness is a reading of less than 5 mm [11, 12].

A deficiency of the aqueous layer is responsible for about 20% of cases of DES [11]. Such deficiency can be a result of advanced age, Sjogrens syndrome, Keratoconjunctivitis Sicca, familial dysautonomia, and side-effects of common ocular surgeries such as LASIK, PRK, and phacoemulsification [13, 14].
