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   Table of Contents      
Year : 2001  |  Volume : 49  |  Issue : 3  |  Page : 153-168

New perspectives in ocular surface disorders. An integrated approach for diagnosis and management

1 Cornea Service, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad - 500 034, India
2 Cornea service, L.V. Prasad Eye Institute, Hyderabad, India (VSS), and Ocular Surface & Tear Center, Dept. of Ophthalmology, Bascom Palmer Eye Institute, and Dept. of Cell Biology & Anatomy, University of Miami School of Medicine, Miami, Florida, USA

Correspondence Address:
Virender S Sangwan
Cornea Service, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad - 500 034
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Source of Support: None, Conflict of Interest: None

PMID: 15887723

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The cornea, conjuctiva and the limbus comprise the tissues at the ocular surface. All of them are covered by stratified, squamous, non-keratinizing epithelium and a stable tear film. The ocular surface health is ensured by intimate relationship between ocular surface epithelia and the preocular team film. There are two types of ocular surface failure. The first one is characterized by squamous metaplasia and loss of goblet cells and mucin expression. This is consistent with unstable tear film which is the hallmark of various dry-eye disorders. The second type of ocular surface failure is characterized by the replacement of the normal corneal epithelium in a process called limbal stem cell deficiency. It is essential to establish accurate diagnosis for appropriate management of complex ocular surface disorders. There has been considerable advancement in the understanding of the pathophysiology of ocular surface disease. Management has improved with introduction of the limbal stem cell concept and use of amniotic membrane transplantation.

Keywords: Ocular surface, limbal stem cell, ocular surface disease, dry eye, ocular surface reconstruction, amniotic membrane grafting, limbal stem cell transplantation.

How to cite this article:
Sangwan VS, C.G.Tseng S. New perspectives in ocular surface disorders. An integrated approach for diagnosis and management. Indian J Ophthalmol 2001;49:153-68

How to cite this URL:
Sangwan VS, C.G.Tseng S. New perspectives in ocular surface disorders. An integrated approach for diagnosis and management. Indian J Ophthalmol [serial online] 2001 [cited 2023 Dec 11];49:153-68. Available from: https://journals.lww.com/ijo/pages/default.aspx/text.asp?2001/49/3/153/22629

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The cornea, conjunctiva, and the limbus comprise the tissues at the ocular surface. All three are covered by a stratified, squamous, nonkeratinizing epithelium. These epithelia sit on a basement membrane and are connected through an identical adhesion complex to an underlying connective tissue stroma. Functionally, all three regions of the epithelium support the tear film and protect against fluid loss and pathogen entrance. The connective tissue of all the three regions serves not only as a structural support but also act as the conduit for fluids and nutrients. It also houses support cells that provide for maintenance.

The primary function of the ocular surface is to provide clear vision when the eye is open as the anterior surface of the cornea contributes more than two-thirds of the total refractive power of the eye. To achieve this while maintaining comfort and preventing microbial invasion, the ocular surface must be covered by a stable tear film. The mechanism by which ocular surface health is ensured is built into the intimate relationship between ocular surface epithelia and the preocular tear film. Based on the five important concepts that have recently been reviewed and summarized,[1] several key elements have been identified within the realm of "ocular surface defense" that govern the function of ocular surface epithelia and the preocular tear film as one unit. Primary deficiency or alteration of any of these elements can lead to an unstable tear film, a hallmark of various forms of dry eye[2] and secondary changes of the epithelial phenotype. Conversely, primary changes of the ocular surface epithelial phenotype, a sign of ocular surface failure, can also lead to a secondary dry eye. Because all elements of ocular surface defense are integrated and work in concert with ocular surface epithelia, these diseases are better treated as a group than as isolated disease entities, even though they range from ocular irritation to defective wound healing and surface destruction. Furthermore, because dysfunction of one element can dynamically affect others, this review describes the rationale of how such an integrated view can direct us towards new diagnostic and therapeutic strategies for these ocular surface and tear film disorders.

  Relationship between ocular surface epithelia and preocular tear film Top

One way of demonstrating the intimate relationship between ocular surface epithelia and the preocular tear film is to take a closer look at the epithelial cell-mucin interphase. On the one hand a stable tear film protects ocular surface epithelia, and the other hand the epithelia also actively participate in forming a stable tear film. This notion is supported by the fact that the conjunctival goblet cells excrete gel-forming mucins, which form one of the important components of the tear film.[3] In addition, both corneal and conjunctival non-goblet cells express different types of transmembrane mucins on their superficial epithelial cells so that they can be wettable. [4,5] The latter mucins together with as yet poorly characterized "mucin-like glycoproteins"[6-8] in the vicinity of cell membranes may constitute the glycocalyx visualized by transmission electron microscopy.[9]

The normal phenotype of both corneal and conjunctival epithelia is non-keratinized and expresses keratins different from the normally keratinized epidermis[10-12]. Although the exact mechanism remains to be elucidated, such a pattern of keratin expression is closely linked with their mucin expression and together provides the salient feature of a wettable epithelial phenotype. The loss of these two features is the first step to ocular surface failure. To understand the mechanism of ocular surface health and failure, one has to consider the protective mechanism (ocular surface defense) leading to a stable tear film and how epithelial differentiation (or phenotype) is maintained.

  Neuroanatomic integration of ocular surface defense elements controlling a stable preocular tear film Top

Apart from the mucins and glycocalyx layer, the tear film consists of two major components. They are superficial meibom lipids produced by meibomian glands and the aqueous tear fluid produced primarily by lacrimal glands. Traditionally we have adopted the view popularised by Wolff (1946)[13] that these three major components exist as separate layers in the tear film. This view was challenged by Holly (1973).[14] He proposed that mucins might consist of a bottom coacervate layer together with an upper dilute solution. This notion was based on the observation that the surface tension of an aqueous solution lowered by lipids can be further lowered by mucins added in the subphase. Recently, using laser interferometry it has been shown that the mucin layer might be present throughout the tear film.[15] Another study using ultrastructural analysis following in vivo cryofixation suggests that a pure aqueous layer might not be present.[16] These new data lead to the view that mucins may form a gradient in the tear film to allow interactions among these three components as a unit. Taken together, we have to include two external adenexal glands in the system of ocular surface defense.

Even if there are ample amounts of tear with normal components, it cannot form a film without the hydrodynamic factor, which includes periodic and complete eyelid blinking to facilitate tear spread into a film to cover the entire ocular surface and tear clearance into the nasolacrimal drainage system for adequate turnover and refreshment. Therefore, another key element essential to achieve a stable tear film is the eyelid. All elements of both compositional and hydrodynamic factors are summarized in [Figure - 1]. It is important to point out that both compositional and hydrodynamic factors of the ocular surface defense are integrated with the status of ocular surface epithelia via two neuronal reflex arcs. Both reflexes are subserved by the first branch of trigeminal nerve (V1) controlling ocular sensitivity as the afferent sensory input, and by the parasympathetic branch, the motor branch of the facial nerve (VII) as the efferent output, respectively [Figure - 2]. Such neuro-anatomic integration explains how different tissue parts of the ocular surface including external adnexal glands and eyelids are integrated with ocular surface epithelia to maintain a stable tear film.

  Basic versus reflex tearing Top

Besides providing the aqueous fluid in the tears film, tear also contain essential components such as vitamin A,[17] Essential Growth Factor (EGF),[18-20] and Tissue Growth Factor β (TGF-β),[21] which support proliferation and differentiation of ocular surface epithelia. In addition, a stable tear film allows adequate oxygenation of the avascular corneal tissue when the eye is open. Decreased tearing may thus accelerate surface desiccation, deprive the ocular surface of these growth and differentiation-promoting factors, and facilitate microtrauma induced by eyelid blinking (vide infra) The concept illustrated in [Figure - 2] indicates that the amount of tearing is primarily controlled by reflex tearing driven by ocular sensitivity subserved by V1This concept contradicts our traditional view of basic and reflex tearing, of which the former is thought to be produced by accessory lacrimal glands and the latter by the lacrimal gland. It should be noted that innervation to accessory lacrimal glands has not been detected and that tear production is diminished during sleep, under general and local anesthesia, and neutrophic keratitis, when sensory drive is diminished.[22] For these reasons we believe that all tear production is controlled by sensory stimuli from different sources subserved by V1 and amounts of tearing vary according to the intensity of the summated sensory drive.[23] Norn (1973)[24] reported that ocular sensitivity varies across sites; the cornea is the most sensitive tissue followed by the lid margin and the conjunctiva, and that such a pattern of differential sensitivities decreases with age.

[Figure - 3] shows the source of sensory stimulation at the clinical setting to challenge the integrity of this reflex arc. For example, the so-called "basic tearing" measured by the Schirmer test with topical anaesthetics, reflects tear production under "minimal" stimulation because only the sensitivity of the cornea and the conjunctiva, but not that of lid margins is numbed. This test is not as sensitive and specific to detect the dry eye state as not all ocular sensitivity is taken into account. The function of the lacrimal gland to respond to sensory stimulation can be examined by detecting the presence of "reflex tearing" under maximal stimulation of the reflex arc. This is performed by the Schirmer test with or without anaesthesia followed by nasal stimulation. Based on the presence or absence of reflex tearing, aqueous tear deficiency (ATD) can be differentiated into non-Sjogren syndrome (non-SS) type and Sjogren syndrome (SS) type. As shown in [Figure - 4] the ocular surface damage is more severe in the SS type than the non-SS type.[25-27] This is evidenced by more severe squamous metaplasia revealed by rose bengal and fluorescein staining and impression cytology. The Schirmer test result with topical anaesthetics decreases in both types, indicating that both SS and non-SS dry eyes lose the ability to produce tears with little or no stimulation. However, reflex tearing remains intact in non-SS ATD but is lost in SS ATD, and such a loss in SS correlates with the presence of infiltrated lymphocytes in the lacrimal gland.[28] Further studies are needed to determine why and how sensory drive is insufficient to stimulate the presumably "normal" lacrimal gland in non-SS type ATD.

  Aqueous tear deficiency (ATD ) versus Lipid tear deficiency (LTD) Top

Compared to the ocular surface epithelial changes described above for ATD, those caused by LTD are not characterized well. In the context of blepharitis, various forms of meibomian gland dysfunction (MGD) have been classified (reviewed in[29-30]). It has been recognised that meibom lipids constitute the superficial tear layer, stabilize the tear film, and prevent aqueous tear evaporation.[31] Consistent with this premise, patients with MGD reveal short tear break-up time,[32] rapid evaporation rate,[33-34] high osmolarity,[35] and positive dye staining.[36] A recent study using impression cytology has shown a unique "lytic" change on the non-exposure zone of the ocular surface of these patients[37] (see [Figure - 5] for different patterns of exposure zone and non-exposure zone).

Interestingly, this cytologic change also correlates well with rose bengal staining in that region [Figure - 5]. The lytic change of epithelial cells, different from squamous metaplasia, a cytologic finding frequently seen in ATD, suggests that the underlying pathogenic process is either inflammatory or toxic, but not dry. Thus one may wonder whether the lytic change is a result of inflammation derived from associated bacterial infection[38-39] or from a toxic effect of qualitative/quantitative changes of meibom lipids.[40-42] In this context two polar lipids, phosphoethanolamine and sphigomyelin, have been found deficient in the "evaporative" form of LTD.[43] The epithelial cells on the non-exposed ocular surface covered by the lid margins are altered. Future studies are needed to determine whether this change is sufficient to alter mucin expression and thereby destabilize the tear film. The lipid film is the most superficial layer. Based on the above differences, patients with pure LTD frequently show minimal signs and can be differentiated from ATD [Figure - 6].

  Compensatory effects of eyelid blinking on unstable tear film Top

In the normal open-eye situation, the tear film needs to be stable only during the time period between blinks (inter-blink interval) and such a demand increases when the exposure zone enlarges. Thus, when the tear film stability is compromised by compositional deficiency, it can be compensated by an increase in the rate of blinking. This has an inverse relationship with the inter-blink interval. This can also be compensated by narrowing of the exposure zone, for example, via squinting or closing eyelids, or a combination of both. By the same token, even for a normal individual with the normal compositional factor, the demand for a stable tear film increases when the blink rate slows or the exposure zone enlarges, or a combination of both. For example, as shown in [Figure7], the tear evaporation rate increases from downgaze to normal gaze to upgaze as a result of an increasing exposure zone. This is normally compensated for by an increase in the blink rate. However, it should be noted that control of lid blinking, exerted by a motor branch of the facial nerve, is a complex task mediated by local neuronal reflexes driven by corneal and supraorbital touch (pain), and light stimuli (see [Figure - 2]. This is also modified by supranuclear pathways (reviewed by Ongerboer de Visser[44]). The blink rate slows down when one is engaged in concentrated visual tasks such as reading, knitting, watching TV or driving, or in darkness; this explains how the defense is decompensated even for normal individuals working on video display terminals.[45] This also explains why symptoms of dry eye patients often increase during the aforementioned activities.

One can thus conclude that the compositional factor works in concert with the hydrodynamic factor, and that deficiency in one increases the demand for the other. Failure to meet such a demand by the other can lead to an unstable tear film. For this reason, clinical work-ups should always aim to explore the status of all elements in these two factors for a complete understanding of each patient's ocular surface defense. Failure to do so will provide incomplete information to arrive at the pathogenesis of the patient's dry eye problems.

  Blink-related ocular surface disorders Top

Although eyelid blinking is essential to create tear spread and to minimise the interblink interval, frequent and uncontrolled blinking, such as in blepharospasm, can be hazardous because it causes mechanical microtrauma to the ocular surface [Figure - 8]. A similar blink-related microtrauma can also occur in patients with ATD. Recently, a regional (i.e., superior more than the inferior bulbar conjunctiva) tear deficiency has been implicated as a cause for superior limbic keratoconjunctivitis (SLK), and hence punctal occlusion directed to the upper punctum has been found effective.[46] The blink-related microtrauma is also pathogenic in patients with LTD because meibom lipids play an important role in lubricating the movement of eyelid blinking to ameliorate friction-induced trauma, and concurrently lowers surface tension, prevents evaporation, and helps aqueous tear spread. The friction increases when the spatial relationship between the lids and the globe is tight, for example in thyroid eye disease. Corneal microtrauma is also increased in patients with abnormal lid margins with keratinization, meibomian gland orifice metaplasia, and misdirected lashes. That is why patients with cicatricial keratoconjunctivitis should also be worked up in terms of their ocular surface defense with respect to compositional and hydrodynamic factors.

  Corneal sensitivity and neurotrophic keratopathy Top

As mentioned above, the integration of the two reflex arcs controlling compositional and hydrodynamic factors, is through V1, and the corneal sensitivity plays a dominant, but not exclusive role. Pain is thought to be the sensory input for triggering the corneal reflex controlling both eyelid blinking and tearing. Using infrared radiation thermometry,[47] new evidence has been gathered to suggest that a change in corneal temperature, when the tear film becomes unstable, might also be involved in arousing corneal sensitivity, thus triggering lid blinking. Further investigation is needed to determine if different types of sensation can be used to control compositional and hydrodynamic factors.

Based on the scheme shown in [Figure - 2], one can appreciate that ocular sensitivity mediated by V1is the single most important driving force controlling the integrity of both compositional and hydrodynamic factors in ocular surface defence. Deficiency in ocular sensitivity can lead to a deficiency of the compositional factor resulting in ATD. This in turn leads to inadequate blinking, resulting in increased exposure, and finally leading to an unstable tear film. This explains why decreased corneal sensitivity (hypoaesthesia) noted in a number of disorders (reviewed by Martin and Safran[48]) is frequently associated with dryness-induced keratopathy. Such a pathologic effect mediated via disturbance of ocular surface defence is termed the "secondary" neurotrophic effect on the ocular surface, and is clinically indistinguishable from that of keratitis sicca due to ATD. Nevertheless, denervation of the corneal nerve can cause a "primary" neurotrophic effect to the corneal epithelium. Recently Insulin Growth Factor - I (IGF-I), substance P,[49] and Nerve Growth Factor (NGF)[50] have been regarded as putative factors involved in neurotrophic keratitis. A recent study[51] has reported the beneficial effects of topical NGF in healing moderate to severe neurotrophic keratitis. Because corneal sensitivity decreases with age,[52] it is no surprise that there is a high prevalence of dry eye among the elderly.

  Tear clearance: final integration of both reflex arcs] Top

The final neuro-anatomic integration of these two reflex arcs is manifested in tear clearance (turnover). The reflex arc giving rise to lid blinking generates a pump function to remove tear fluid from the tear meniscus into the nasolacrimal drainage system.[53] Increased aqueous tear secretion and thus large tear volume also facilitates tear clearance. The latter also explains the reasons for decreased clearance rate, measured by fluorophotometry, in ATD.[54-55] Using fluorescein as a tracer, the tear clearance rate, called fluorescein clearance test (FCT) can be measured by a modified method using serial Schirmer paper strips.[56-57] Such a measurement made with this modified method is consistent with that measured by fluorophotometry.[58] This test is useful in the following clinical situations:

  1. 1. To accurately diagnose the dry eye state[56]

  2. 2. To distinguish SS ATD dry eye from non-SS ATD in conjunction with nasal stimulation[59]

  3. 3. To distinguish ATD from LTD[37] (also see [Figure - 5].

Adequate tear clearance is important to allow constant refreshing of tear components and effective elimination of debris, which can irritate the ocular surface. Measurement of tear clearance is thus clinically important to determine the healthy status of ocular surface defence. The dysfunction in tear clearance, such as delayed tear clearance (DTC) is quite common. It is often overlooked, and contributes to pathogenesis of several ocular surface disorders[Lee and Rose;[37] [Figure:9]. Besides decreased tear secretion as a cause, DTC can be induced by ineffective or decreased blinking (functional block) or by mucosal inflammation and oedema in the nasolacrimal drainage system (partial anatomic block). The former is contributed by such risk factors as old age, female gender, decreased corneal sensitivity, and lid laxity including floppy lids. Relevant to the issue is the fact that this finding of decrease corneal sensitivity (e.g., neurotrophic keratopathy), mentioned above, induces an unstable tear film and causes DTC. Allergy, atopy, rosacea, and meibomian gland disease (MGD) can intrinsically generate mucosal oedema and inflammation. Once set in, a vicious cycle can form between DTC, the mucosal inflammation and oedema leading to ocular irritation, medicamentosa, drug-induced pseudopemphigoid, and steroid-induced ocular hypertension. Several inflammatory cytokines are elevated in DTC explaining in part how DTC becomes pathogenic[60]. Therefore, we advise that tear clearance together with secretion, distribution, and evaporation should become a routine part of the examination to fully understand all aspects of the tear function.

  Interaction Top

Unstable tear film and delayed tear clearance (DTC) leading to accumulation of inflammatory debris, are the two major pathogenic processes thus identified. Therefore it is important to identify and differentiate them. Based on our recent report,[57] this can be achieved clinically using the fluorescein clearance test. (The method and interpretation are illustrated in [Figure:10] and [Figure:11] respectively). The patients with DTC carry the features listed in [Figure:12], that differentiates from unstable tear film caused by compositional tear deficiency, such as ATD and LTD.

Identification of the presence of DTC is particularly important in the work-up of patients complaining of ocular irritation. Theoretically [Figure:13], the source of ocular irritation can come from unstable tear film, DTC, or intrinsic irritative stimuli caused by inflammation, infection, toxicity, and allergy, or a combination of the three. Failure to identify DTC will present a difficulty in unsuccessful treatment even if the other two sources are identified. This is because ocular irritation caused by intrinsic irritative stimuli is worsened by DTC. Therefore, under such a situation, preexisting DTC or artificially induced by punctal occlusion, will worsen the ocular irritation. However, in the absence of intrinsic irritative stimuli, preexisting DTC is desirable for patients with an unstable tear film because it functions like punctal occlusion, and thus should be preserved and not eliminated. Inadvertent removal of such DTC will make dry eye worse.

Based on the dynamic interactions between unstable tear film and DTC a logical therapeutic strategy is proposed to include several principles outlined above. First, the work up should be complete to include history taking, external and slitlamp examinations and special tests, directed at identifying intrinsic irritative stimuli, unstable tear film, and DTC. After identifying and differentiating these, the therapeutic regimen should begin with eliminating intrinsic irritative stimuli particularly when associated with DTC. In this regard, we have recently noted that an effective therapy is to use topical application of a non-preserved corticosteroid, e.g., Methyl Prednisolone.[57] After intrinsic irritative stimuli are eliminated or controlled, DTC should be left alone especially if it is accompanied by unstable tear film. Finally, unstable tear film can then best be treated in the absence of intrinsic irritative stimuli. For unstable tear film, therapies should first be directed to ATD before LTD can be better managed.

  Complexity of ocular surface disorders Top

It is worth noting that many diseases, when classified in the traditional manner, frequently manifest dysfunction in more than one element of ocular surface defense. One good example is so-called "blepharitis" in the context of MGD. Depending on the stage of MGD, it can manifest several different dysfunctional elements leading to ocular irritation. During the acute stage, inflammation of MGD can spill over to the mucosa and the tear fluid. When the ocular mucosa suffers from chronic inflammation, DTC can set in and create a vicious cycle to aggravate underlying irritation. During the chronic stage when inflammation is controlled or subsides, orifice metaplasia and acinar drop-out frequently lead to LTD, which can contribute to the aforementioned unstable tear film together with ATD, if present. Occasionally, due to the lack of lipid layer or chronic inflammation at the lid margin, irregular lid margins can lead to irritation from microtrauma during blinking. For each patient with MGD, it is thus imperative to determine the dysfunctional elements in order to formulate effective therapies. Failure to do so and attempts to use the same regimen, i.e., lid hygiene and hot compress, will not be successful.

Another good example is conjunctivochalasis of which different extents of severity manifest different dysfunctional elements. Conjunctivochalasis is defined as a redundant, loose, non-oedematous inferior bulbar conjunctiva interposed between the globe and lower eyelid, which tends to be bilateral, is more prevalent in older populations, and often has been overlooked. The conjunctivochalasis contributes to an unstable tear film at the mild stage, causes an interference in tear clearance (DTC) leading to epiphora at the moderate stage, and exposure-derived problems at the severe stage.[61-64] Again, it is conceivable that difficulty in treating patients with this disorder may emerge when each pathogenic process is not identified and their interactions are not considered.

  Types of ocular surface failure Top

Two major types of ocular surface failure have been identified by impression cytology based upon resultant epithelial phenotype.[65] The first type of surface failure shows pathologic transition of normal non-keratinized ocular surface epithelia into keratinized epithelia in a process termed squamous metaplasia.[66] For conjunctiva, squamous metaplasia is preceded with loss of goblet cells. The loss of goblet cells and mucin expression are accompanied by a switch to epidermal keratins[12]. Such phenotypic changes represent altered epithelial differentiation rendering ocular surface epithelia non-wettable. Squamous metaplasia therefore is considered a common phenotype of unstable tear film, the hallmark of various dry eyes disorders.[2] The diagnosis and management have been described in the preceding section.

The second type of ocular surface failure is characterised by the replacement of the normal corneal epithelial phenotype with an invaded conjunctival epithelium in a process termed limbal (stem cell) deficiency.[67] In the next section we discuss the pathophysiology and mangement of stem cell related diseases affecting the ocular surface and the role of the stromal factors in stem cell biology.

  Ocular surface epithelia and stem cells Top

In the adult many tissues undergo rapid continuous cell turnover. These tissues, that include simple and stratified epithelium as well as the hematopoietic system, must repopulate and continuously maintain the integrity of the tissue. Similarly the corneal epithelium exists in a state of dynamic equilibrium, with the superficial cells being constantly shed into the tear pool.[68] Terminal differentiation of cells', coupled with cell death by apoptosis, prompts the cell loss via desquamation.[68] The cells ultimately responsible for repopulation are termed 'stem cells'. The stem cell can be defined as any cell with high capacity for self-renewal extending through adult life of the organism.[69] These cells form a small sub-population of the total tissue and have been estimated to make up 0.5% to 10% of the total cell population[70]. Stem cells are thought to share a common set of characteristics including high proliferative potential and enjoy a long cell cycle.

  Basic concept of stem cells Top

Stem cells (SC) are by definition present in all self-renewing tissue.[71] These cells are long-lived, have great potential for clonogenic cell division, and are ultimately responsible for cell replacement and tissue regeneration. Most of our knowledge about stem cells are derived from studies on blood cells and some epithelial tissue e.g. intestinal epithelia, semineferous epithelia, and skin epidermis.[71] Based on cell kinetic studies[72-73] all cells in a tissue consisting of a clonogenic cell lineage can be placed into either one of the following two tissue compartments: proliferative or non-proliferative[71]. Cells in the proliferative compartment are capable of preceding cell mitosis with DNA synthesis. This compartment includes limbal stem cells (SC) and transient amplifying cells (TAC) that are derived from each limbal SC mitosis and amplify their number by undergoing a few rounds of cell division. Cells in the non-proliferative differentiative compartment are in theory all post-mitotic cells (PMC) that are committed to cellular differentiation. In the latter compartment, cells at different stages of differentiation can be identified during the process of tissue maturation. The terminally differentiated cells (TDC) achieve the ultimate expression of functional tissue.

All cells except limbal SC have a limited life span and are destined to die. These serial steps of clonogenic cell lineage in a defined tissue are summarised in [Figure:14] and [Figure:15] In these illustrations one can see an organised cellular hierarchy containing heterogeneous populations of cells that are arranged in the order of limbal SC-TAC-PMC-TDC. From this schematic diagram [Figure:14], we can appreciate the fact that the loss of TDC is compensated for by the gradual terminal differentiation of the preceding higher hierarchy, the PMC and eventually by the source of cellular proliferation the limbal SC, at the highest rank. Furthermore, to ensure the normal health of the tissue, cellular proliferation and differentiation in a coordinated manner at different levels of this hierarchy is important, even indispensable. Several laboratory and clinical studies reported limbal location of corneal stem cells.[67-71] The fact that the limbus contains a self-renewing stem cell population of the corneal epithelium explains why ocular surface dysplasia and neoplasia are known to have a limbal predilection[74]. The conjunctival stem cells are reported to be either in the fornix or at the mucocutaneous junction[75].

  Clinical application of the concept of limbal stem cell Top

There is a well-defined group of human corneal diseases with limbal stem cell deficiency.[76] The stem cell deficiency could be focal or diffuse [Figure:17] depending upon the extent of limbal involvement with the underlying disease process. Patients with this group of diseases often suffer from severe photophobia, decreased vision, and other complications. The patients with total stem cell deficiency are poor candidates for conventional corneal transplantation (where limbal stem cells are not the part of the graft) for several following reasons:

  1. (1) Only corneal transient amplifying cells are transplanted.

  2. (2) Pre-existing corneal vascularisation and inflammation increase the risk of allograft rejection.

  3. (3) They tend to undergo recurrent conjunctivalization owing to the loss of stem cell function.[77]

Ocular surface diseases such as Stevens-Johnson syndrome, chemical and thermal burns, ocular surface tumors, immunologic conditions, radiation injury, inherited syndromes (such as aniridia), and ocular pemphigoid can severely compromise the ocular surface and cause catastrophic visual loss in otherwise healthy eyes. The common pathogenic feature of this seemingly diverse group of diseases is the depletion of the stem cell population from corneal limbus.[67] Damage or depletion of the corneal stem cells results in "conjunctivalisation" or ingrowth of conjunctival elements on the surface of the cornea with associated profound visual loss [Figure:16].

Based on the pathogenic nature of limbal involvement, corneal surface diseases can be divided in two categories [Table - 1]. Category I diseases destroy the epithelial stem cell population. They are characterised by a clear pathogenic cause and are identified from history. The destruction can be caused by chemical/ thermal injuries, Stevens-Johnson syndrome, and multiple surgeries or cryotherapies applied on the limbal regions. In the rare situation, contact lens-induced keratopathy, lens-wearing injuries or toxic effects from lens-cleansing solutions can also cause limbal stem cell (SC) damage. Category II diseases include diverse causes such as aniridia, keratitis associated with multiple endocrinal deficiencies, neurotrophic keratopathy, and pterygium/ pseudopterygium. The category II disease represents a milder form of corneal disease where limbal SC dysfunction is not due to the total loss of limbal SC, but rather is associated with either a gradual loss of limbal SC population or poor TAC generation and amplification. Because it has not resulted from traumatic loss, the underlying pathogenesis might arise from poor microenvironment support of limbal SC or TAC, or a poor regulatory mechanism. In the case of aniridia, a hereditary example of limbal SC dysfunction, such poor regulation is probably associated with microenvironment alteration due to the anomalous development of the adjacent angle-iris structures. Poor nutritional supply of endocrine factors and of neuronal trophic cytokines might be the basis for the development of limbal SC deficiency in keratitis associated with multiple endocrine deficiency and neurotrophic keratopathy derived from a primary neuronal or ischaemic component respectively. The introduction of adverse undesirable cytokines secreted by chronic inflammation of various natures might inhibit or antagonize normal regulators and create a state of limbal SC dysfunction. These mechanisms might explain the poor support of SC function in clinical examples of chronic limbitis and pterygium or pseudopterygium.

With the advent of the limbal stem cell concept, Kenyon and Tseng[70] developed new surgical procedure termed as "limbal transplantation". In experimental rabbits Tsai and coworkers[78] demonstrated that limbal transplantation could restore effectively the corneal epithelial phenotype on severely damaged corneal surface; in contrast conjunctival transplantation results in conjunctival epithelial phenotype. These results further confirmed limbal location of corneal epithelial stem cells; they also established limbal autograft as an ideal method of corneal surface reconstruction for patients suffering from the diseases [Table - 1], especially for focal or unilateral limbal deficiency. [70,79-81] Patients with diffuse and bilateral limbal deficiency, corneal surface reconstruction requires an allograft from either HLA-matched living[80-82] or nonmatched cadaver[76] [Figure:17], [Figure:18]. Unlike autografts, allografts can be rejected, especially when they are transplanted to such vascular tissues as the limbus and conjunctiva. The clinical features and treatment of limbal allografts rejection has been recently reported.[83]

Schwab and co-workers[84] reported a successful outcome with cultured limbal stem cells (autologus procedures). The success was defined as restoration or improvement of vision, along with maintenance of corneal re-epithelialisation and absence or recurrence of surface disease, in 6 of 10 patients. Another study reported the use of "bioengineered cornea" for reconstruction of severely damaged corneal surface with stem cell deficiency.[85] [Figure:19] shows slitlamp photo before (a) and after (b) cultured autologus limbal stem cell by one of us (VSS).

  Role of stroma and amniotic membrane in ocular surface reconstruction Top

Tsai and Tseng[86] found that chronic inflammation in the perilimbal region precedes progressive failure of limbal autografts in rabbit. This raises the possibility of gradual dysfunction of the limbal stromal microenvironment as a result of inflammation. This explains possible pathogenesis of the diseases that have been classified as category II [Table - 1]. It also emphasizes that limbal stem cells can be regulated by the underlying stroma. The notion that limbal stem cell deficiency can be caused by a dysfunctional stromal microenvironment mimics murine hematopoeitic stem cell deficiency.[76]

Amniotic membrane has been reported to have anti-inflammatory properties due to down-regulation of key proinflammatory cytokines such as IL-1α and IL-1β and TGF-β.[87] Shimmura et al[88] reported the anti-inflammatory effects of amniotic membrane transplantation in ocular surface disorders. Tseng and coworkers reported that amniotic membrane transplantation is useful as the first-stage procedure to reduce inflammation and scarring before the limbal allografts in patients with total limbal deficiency.[76]

The amniotic membrane consists of a thick basement membrane rich in collagens type IV and V and a single layer of columnar epithelial cells that secrete amniotic fluid.[89] Amniotic membrane lines the innermost layer of the foetal membrane, that protects the fetus during pregnancy.[90] The use of amniotic membrane as a surgical material dates as far back as 1913, when Stern[91] reported the use of amniotic membrane in surgical procedures of the skin. Also, the use of amniotic membrane in ophthalmology was practiced in the 1940s, when it was used as a patch to cover defects in the conjunctival epithelium caused by trauma and burns.[92-93] Amniotic membrane transplantation subsequently did not appear in the literature for several decades, until Kim and Tseng[94] reinstated the technique for the reconstruction of the ocular surface in experimental corneal disease in the rabbit. Immediately thereafter, a surge was observed in the use of amniotic membrane in clinical practice[88] for the treatment of difficult disorders such as Stevens -Johnson syndrome, ocular cicatricial pemphigoid, recurrent pterygia, persistent epithelial defects (PEDs), acute chemical and thermal injuries,[95] sheild ulcer of VKC,[96] neurotrphic keratitis, and partial stem cell deficiency.

The potential mechanism of action [Table - 2] could be that the basement side of the amniotic membrane provides an ideal substrate for supporting the growth of epithelial progenitor cells by prolonging their life span and maintaining their clonogenicity.[94] This action supports the amniotic membrane transplantation in so as to expand remaining limbal stem cells and corneal transient amplifying cells partial limbal deficiency[97] and to facilitate epithelialisation for persistent corneal epithelial defects with stromal ulceration.[98-100] In tissue cultures, amniotic membrane supports limbal epithelial cells grown from explant cultures and resultant epithelial cells -amniotic membrane can be transplanted back to reconstruct the damaged corneal surface.[101] The amniotic membrane can also be used to promote non-goblet cell differentiation of the conjunctival epithelium,[102] and conjunctival goblet cell differentiation is further promoted by coculturing with conjunctival fibroblasts on the same side of the basement membrane.[94] This data explains the promotion of conjunctival goblet cell density following amniotic membrane transplantationin vivo.[103] The stromal side of the membrane contains a unique matrix component that suppresses TGF-signaling, proliferation and myofibroblast differentiation of normal human corneal and limbal fibroblast.[104] This action explains why and how the amniotic membrane transplantation helps reduce scars during conjunctival surface reconstruction,[105] preventing recurrent scarring after pterygium removal,[106] and reducing corneal haze following PTK and PRK.[107-108] Although such an action is more potent when fibroblasts are in contact with the stromal matrix, a lesser effect is also noted when fibroblasts are separated from the membrane by a distance, suggesting that some diffusible factors might also be involved besides the insoluble matrix components in the membrane. In concurrence with this thinking, several growth factors have been identified in the amniotic membrane.[109]

Future studies are needed to understand the exact mechanism of action. The stromal matrix of the membrane can also exclude inflammatory cells by causing rapid apoptosis.[107-110] It also contains various forms of protease inhibitors.[111] This also explains why stromal inflammation is reduced after amniotic membrane transplantation[98-105] and corneal neovascularisation is mitigated[112] - actions important for preparing the stroma to support limbal stem cells transplantation either at the same time or later. [95,113-115] This action also explains why keratocyte apoptosis can be reduced and hence prevent the stromal haze is prevented in PRK or PTK by amniotic membrane. [107, 109, 116]

With any type of ocular surface failure manifesting either as squamous metaplasia or limbal deficiency, it should be aimed at restoring the ocular surface defence prior to or during the corneal surface reconstruction with amniotic membrane transplantation with or without limbal stem cell transplantation. These measures include punctal occlusion or application of autologus serum drops for severe aqueous tear deficiency,[113] plastic correction of lid margin and lash problems, and tarsorrhaphy or refractory exposure. Severe dry eyes, diffuse keratinization and stromal ischaemia remain to be difficult to overcome.

  Practical approach to diagnosis and management of ocular surface disorders Top

Patients with ocular surface disease could present with mild ocular irritation or with severe decrease in vision due to ocular surface keratinization or due to destruction of limbal or conjunctival epithelial stem cells. The history taking, examination, and diagnostic testing should be directed to identifying type of surface failure: the type I and II. The algorithm in [Figure:20] (modified from Pflugfelder, Solomon and Stern[117] outlines the diagnostic approach for patients with ocular irritation, which represents type one ocular surface failure. The management principles of type one surface failure are as outlined below:

  1. 1. For each patient, diagnostic work-up should determine any intrinsic irritative stimuli derived from allergy, atopy, inflammation, toxic or infection, unstable tear film caused byATD and /or LTD, or delayed tear clearance.

  2. 2. Treatment should be directed to the intrinsic irritative stimuli first especially if accompanied by DTC even in the presence of unstable tear film.

  3. 3. Treatment of unstable tear film begins after the intrinsic irritative stimuli are eliminated.

  4. 4. For unstable tear film, ATD should be treated before LTD.

Type II ocular surface (stem cell deficiency) failure is diagnosed by specific clinical features with cytologic evidence by impression cytology (diagnosis is usually clinical). For established limbal stem cell deficiency, the following steps are recommended.

  1. 1. Initial treatment is for non-specific as well as immune - mediated inflammation after taking care of confounders such as misdirected cilia or keratinisation.

  2. 2. Identification and treatment of compositional and neuroanatomic factors contributing to pathogenesis of stem cell deficiency

  3. 3. Use of artificial tears and autologus serum drops before and after surgery.

  4. 4. Rectification of associated ocular adnexal anatomical deformaties such as entropion / ectopion or neurotrophic keratitis.

  5. 5. Amniotic membrane transplantation with removal of pannus is recommended as initial procedure to reduce stromal inflammation, thus increasing chances of limbal graft survival.

  6. 6. We recommend staged (step-wise) surgical procedure for ocular surface reconstruction such as limbal grafts to be followed by PKP if required and not as combined procedure.

  7. 7. Adequate topical and systemic immuno-suppression is the key factor for survival of transplanted allogenic limbal tissue. Immunosuppression is not required for cultured autologus limbal stem cell transplantation and there is no risk of rejection.

  8. 8. Frequent clinical examinations to detect the earliest signs of rejection and laboratory test to identify drug toxicity are mandatory.

Detailed history, careful clinical examination, and clinical and laboratory diagnostic testing would help in diagnosis of type of ocular surface failure, to differentiate various types of dry eye syndromes, and distinguish delayed tear clearance from unstable tear film. The dry eye or ocular surface disease is not a diagnosis in itself but represents an end result of variety of aetiologic mechanisms.

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  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7], [Figure - 8]

  [Table - 1], [Table - 2]

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