Experimental Models, Induction Protocols, and Measured Parameters in Dry Eye Disease: Focusing on Practical Implications for Experimental Research

Dry eye disease (DED) is one of the major ophthalmological healthcare challenges worldwide. DED is a multifactorial disease characterized by a loss of homeostasis of the tear film, and its main pathogenesis is chronic ocular surface inflammation related with various cellular and molecular signaling cascades. The animal model is a reliable and effective tool for understanding the various pathological mechanisms and molecular cascades in DED. Considerable experimental research has focused on developing new strategies for the prevention and treatment of DED. Several experimental models of DED have been developed, and different animal species such as rats, mice, rabbits, dogs, and primates have been used for these models. Although the basic mechanisms of DED in animals are nearly identical to those in humans, proper knowledge about the induction of animal models is necessary to obtain better and more reliable results. Various experimental models (in vitro and in vivo DED models) were briefly discussed in this review, along with pathologic features, analytical approaches, and common measurements, which will help investigators to use the appropriate cell lines, animal, methods, and evaluation parameters depending on their study design.


Introduction
Dry eye disease (DED) is a multifactorial disease characterized by inefficient tear secretion or increased evaporation of tear film, which hamper tear osmolarity and ocular surface lubrication, increase shear forces under the eyelids, and finally trigger inflammation and pathologic complexity. This can cause an imbalance of electrolytes, mucins, and proteins and damage the conjunctival and corneal epithelial cells and nerve fibers. It is also known as dry eye syndrome, keratoconjunctivitis sicca, and chronic inflammatory ocular surface disease. The primary clinical signs of DED are eye irritation, increased tear osmolarity, impairment of ocular surface epithelia, tear film instability, and blurred and fluctuating vision. Chronic inflammation, irritation, and pain induced by DED have a marked negative influence on the quality of life, hampering visual performance and the ability to perform daily tasks (e.g., reading and driving). These detrimental impacts on functioning lead to anxiety and depression [1]. Aged populations (approximately 10-20%) are more prevalent in DED [2]. It was surveyed that 1.68 million men over the age of 50 suffer DED in the United States, and this number is likely to grow to 2.79 million by 2030 as life expectancy elevates [3]. However, females of all age groups have a greater tendency to have DED than males, with DED prevalence increasing with age according to Drew et al. [4]. It was also reported that over 3.23 million women suffer from DED [5]. Therefore, considering several survey reports [5][6][7], DED is a significant health concern and has global socio-economic impacts.
The animal model in experimental biomedical research refers to a simplified representation of disease condition, hoping that obtaining information can be transferred to actual human or animal diseases. Nowadays, experimental basic animal research for application of medicinal practice and drug discovery has gained reliability. It became popular after its introduction in the National Institutes of Health Roadmap initiative [8,9]. Experimental animal research has also been widely applied in ocular medicine. The DED model is one of them. Most reports, including reviews [1,4,[10][11][12][13], have been focused on the discovery of drugs that would be relevant to study the human DED pathogenesis and evaluate the therapeutic outcomes of future pharmacological treatments. However, induction methods are important when evaluating the function of some mechanisms discovered in animal models in the pathophysiology of human disorders and when applying to discover new treatments for these conditions. Therefore, this review study focused on the design of experimental models-especially on the techniques, types of induction methods, types of cell lines for in vitro models, types of animals for in vivo models, dosages of drugs, frequency of drugs, pathological mechanisms-and cellular cascades of DED, and animal modeling. Evaluation methods and parameters in experimental DED such as specific for clinical signs tear deficiency, ocular surface structural damage, and molecular alterations such as inflammatory cytokines, apoptosis, fibrosis, or angiogenesis-related markers were also described briefly. In addition, this review also briefly described the anatomical location physiology function and pathological condition of DED related glands tissue or cells (lacrimal glands, meibomian glands (MBGs), conjunctival and corneal epithelium, goblet cells, tear film, Harderian gland, and nictitating membrane (NM)) for a better understanding of the pathophysiological mechanisms of experimental DED. We hope this study will provide clear information on experimental DED.

Pathophysiology of Dry Eye Disease (DED)
First, understanding the ocular pathophysiology is crucial for understanding the mechanisms of induction methods. The ocular surface is an exposed part of the eye. Therefore, the fluidity of this part evaporates owing to multiple environmental factors, including variable airflow, temperature, and humidity. Maintaining continuous lubrication in this part is challenging for maintaining normal health and performing normal functions. Lubrication depends on efficient tear production and turnover. The main and accessory lacrimal glands, MBGs, tear film, goblet cells, all ocular surface secretory cells, smooth pathways of lacrimal outflow, and conjunctival and corneal epithelia work simultaneously as a lacrimal functional unit (LFU) to maintain the tear film and protect the transparency of the cornea and the integrity of the ocular surface [12]. LFU is affected by many factors, such as nerve connections and hormones. Disturbances to any part, such as the afferent sensory nerves, efferent autonomic and motor nerves, tear-secreting glands, and lipid meibum-secreting glands, may lead to DED [1].

Lacrimal Glands
The lacrimal glands are exocrine glands. There are two types of glands: main and accessory lacrimal glands. The human main lacrimal gland is paired with each eye; it is situated in the upper lateral region of each orbit, in the lacrimal fossa of the orbit formed by the frontal bone. In rodents, including rats, mice have two pairs of lacrimal glands: the smaller intraorbital (infraorbital) gland is located slightly below the eye socket, and the larger exorbital lacrimal gland is located outside the eye socket, ventrally in front of the acoustic duct [14] (Figure 1). Lacrimal glands secrete aqueous tears composed of water, electrolytes, mucus, and proteins (lysozyme, immunoglobulins, lacritin, and cytokines) and form the tear film's aqueous layer [13]. There are two types of accessory lacrimal glands: Wolfring's/Ciaccio's glands and Krause's glands, as shown by Conrady et al. [15]. The glands of Wolfring and Ciaccio are small tubular-sized glands in the lacrimal caruncle of the eyelid and upper border of the tarsus, approximately in the middle between the extremities of the tarsal glands [16,17]. Approximately two to five glands are present in the upper eyelid. In contrast, Krause's glands are comparatively smaller in shape and more numerous than Wolfring's glands and are situated along the superior and inferior fornices of the conjunctival sac [18]. There are approximately 40 Krause glands in the upper eyelid and around six to eight in the lower lid [19]. Their function is to produce tears secreted onto the surface of the conjunctiva. However, the lacrimal gland epithelia also secrete small soluble mucin MUC7 [20].

Meibomian Glands (MBGs)
These are a special type of sebaceous gland, a holocrine-type exocrine gland arranged vertically within the tarsal plates of the upper and lower eyelids. MBGs are also known as the tarsal glands. Approximately, the upper eye lid tarsus contains about 30-40 glands, and the lower eye lid tarsus contains 20-40 MBG glands [21,22]. MBG primarily secretes lipid meibum, which is an oily substance that forms the lipid layer of the tear film and plays a pivotal physiological role in maintaining tear film stability, preventing evaporation of the ocular surface, preventing tear film contamination, and reducing surface tension [23,24]. In addition, lubricating molecules such as mucinous glycoprotein and lubricin synthesized by the MBG have been reported [24]. These molecules play an important role in the tribology of the eye and decrease the friction between the eyelid and the ocular surface [24]. The disturbances of MBG trigger abnormalities in the lipid layer of the tear film, leading to evaporative dry eye. This situation is defined as MBG dysfunction (MGD), which is one of the major causes of DED [22,23,25].

Conjunctival and Corneal Epithelium, Goblet Cells, and Mucins
The conjunctiva occupies two-thirds of the ocular surface, from the corneal rim to the lid margin. Both corneal and conjunctival epithelial cells participate in producing different mucins on the ocular surface, but the conjunctival goblet cells are the main mucin-producing cells [26]. Goblet cells are specialized epithelial cells that are present in the mucosal tissues along the body. The major function of these cells is to produce and secrete mucins that hydrate and lubricate mucosal surfaces [20]. In addition, the highly stratified corneal epithelium also produces mucins [13]. Mucins are highly glycosylated glycoproteins composed of a protein core and multiple side chains [27]. Mucins contain two types of transmembrane and secretory proteins. According to the polymer-forming ability, secretory mucin is further subdivided into gel-forming and soluble mucins. Mucins that have been identified in the eye are MUC1, MUC2, MUC4, MUC5AC, MUC7, MUC13, MUC15, MUC16, and MUC17 [2,28]. One of the most common large gel-forming secretory mucins in the conjunctiva is MUC5AC [26]. Mucin maintains tears' rheological properties and viscosity, providing a lubricative function during rapid and high share movements of the lid on the globe and vice versa, thereby protecting the eye from frictional damage. Therefore, a lack of mucin or goblet cells reduces tear breakup time. It also helps spread oil from MBG secretion, thus maintaining the lipid layer of tear film integrity, hence checking evaporation [29].

Tear Film
The tear film (TF) is a special form of the extracellular matrix part of the ocular surface approximately 3-8 µm thick [2,30] and 3 µL in volume in humans [30]. The thickness of TF may vary depending on the animal and the measuring methods (Table 1). It is essential for lubrication between the inner surface of the eyelids and cornea to rationalize the frictional forces during blinking, provide an optically smooth medium, and protect the ocular surface from potentially pathogenic microbes [24,30]. It comprises three layers: the outermost lipid layer, the middle aqueous layer, and the innermost mucous layer [13,15]. The middle aqueous layer is a major part of the TF [31].
The lipid layer of TF is produced by the MBG secretion. It is a bilayer and is the thinnest part of TF, approximately 50-100 nm in humans [30]. The upper part of the bilayer served as the air-TF interface, which is comprised of non-polar lipids (e.g., wax esters, cholesterol, and steroid esters) and lesser amounts of polar lipids such as O-acyl-ω-hydroxy fatty acids and phospholipids, which interact adjacent to the aqueous layer [24,30]. The vital role of this layer is to retard the evaporation of the underlying aqueous layer in the open eye, but it also maintains surface tension, elasticity, viscosity, and systematic pecking order, which aids the tear film in preserving the ocular surface integrity [13,30]. The middle aqueous layer of TF is produced by the main and accessory lacrimal gland secretions. Aqueous fluids contain water, electrolytes, and various proteins, including immunoglobulins, cytokines, and growth factors [13,30]. The conjunctival epithelium also contributes to electrolytes and water in tears [30]. The aqueous phase supplies oxygen and nutrients to the underlying adjacent avascular conjunctival and corneal tissue and rinses toxins, epithelial debris, and foreign bodies. It plays an important role in cell signaling and rehabilitation of the ocular surface during disease conditions. The aqueous phase contains electrolytes that maintain tear osmolarity [13,30]. The innermost mucous layer of TF is mainly formed by mucins, immunoglobulins, salts, urea, enzymes, glucose, and leukocytes [13]. Mucins are produced by the goblet cells in the conjunctival and corneal epithelium, and some transmembrane mucins are projected into the aqueous phase of TF from corneal and conjunctival epithelial cells [13,30], thereby stabilizing the TF. Soluble mucins connect with the transmembrane mucins and form a stable glycocalyx layer over the epithelium. The stratified corneal epithelium also produces O-glycosylated transmembrane mucins, contributing to the glycocalyx region. The microvilli of the superficial epithelium of the cornea also participate in the anchorage of the tear film [13].

Harderian Gland and Nictitating Membrane (NM)
The Harderian gland also plays an important role in the ocular surface homeostasis in some animals besides the lacrimal and MBG. Rodents (rats, mice, hamsters, gerbils, and guinea pigs) have the most developed Harderian gland compared to other animals. It is situated around the posterior half of the eyeball and occupies a significant part of the orbit in rodents [36]. The secretion component varies according to the different animal species; for example, mucolipidic in amphibians, mucoserous in reptiles, serous in snakes, and lipidic in bird secretions. In mammals, including rats, their secretions contain three components: lipids, indolic products, and porphyrins [37]. Its secretion also plays a role in lubricating the eye, especially by easing the NM in mammals and amphibians [36,38].
The NM, also known as the third eyelid, is present in most animals around the medial canthus, while humans and anthropoids maintain a vestigial remnant of this organ called plica semilunaris [39,40]. The physiological function of the plica semilunaris in humans is less in human, but the NM in animals contributes to a healthy animal eye by producing and distributing tears, secreting immune proteins, removing ocular debris, and acting as a mechanical barrier [40,41]. Therefore, removing the NM and the Harderian gland has been used to induce dry eye models [42][43][44][45].

Classification of Dry Eye
DED is mainly classified into two types: (a) aqueous tear-deficient DED and (b) evaporative DED. Aqueous tear-deficient DED is further classified into two types: Sjogren syndrome and non-Sjogren syndrome. Sjogren syndrome is mainly associated with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Wegener's granulomatosis, and other diseases (such as systemic sclerosis and primary biliary cirrhosis). Non-Sjögren syndrome is associated with lacrimal diseases (such as congenital alacrima, acquired primary lacrimal gland disease, and sarcoidosis), lacrimal gland obstruction such as trachoma, cicatrical pemphigoid erythema, and multiforme burns. On the other hand, evaporative DED may be caused by oil deficiency (absent glands MGD, distichiasis, blepharitis, obstructive MBGs), lid-related diseases (blink abnormalities, aperture abnormalities, lid surface incongruity), allergic conjunctivitis, or xerophthalmia (vitamin A deficiency) [46]. However, in an experimental study, the DED model can be broadly induced in two ways: in vitro and in vivo DED models. Furthermore, in vivo disease models can be classified into five ways: chemically induced model, surgically induced model, environmental factor induced model, genetic model, and combined methods induced model ( Figure 2).

In Vitro Dry Eye Model
In preclinical studies, an in vitro experiment provided tight control of the microphysiopathological environment of the animal body, which has the most obvious benefit of reducing the requirement of animal testing and is ethically more appropriate. It is also beneficial for obtaining quick, efficient, reliable, and robust results in the early stages of candidate drug screening. In DED, some established in vitro models have also been established: the Wong Kilbourne derivative of Chang conjunctival epithelial cell line (WKD; clone 1-5c-4) [47][48][49][50][51]; immortalized normal human conjunctival cell line (IOBA-NHC) [47,52,53]; human corneal epithelial cells (HCECs), a human transformed SV40 immortalized corneal epithelial cell line [54,55], and human conjunctival cell line (HCC) [56]. In vitro models with primary cell culture are also available: human corneal epithelial cells (HCECs) [57], rabbit corneal epithelial cells (CECs), or rabbit LG acinar cells (LGACs) [58]. The induction of DED-like symptoms in in vitro culture can be performed by the induction of hyperosmotic (500 mOsM) media in the culture media by the addition of 90 mM sodium chloride in the culture medium, which triggers inflammatory-like symptoms [47,48,55,57,59]. As in the in vivo experiment, benzalkonium chloride (BAC) (0.0001% (1 µg/mL) was also used to induce inflammatory cascades [47][48][49][50][51][52]. In addition, recombinant pro-inflammatory cytokine interleukin (IL)-1β (9 or 10 ng/mL) or tumor necrosis factor alpha (TNF α) (10 ng/mL) can be used to induce inflammatory cascades in the culture medium for 24 h or 48 h [53,58]. Cell culture methods and experimental schedules are described briefly in Table 2.

In Vivo Dry Eye Models
In preclinical studies, in vivo testing is a major part of safety assessment and is a regulatory requirement prior to clinical trials as it provides a similar biological environment and properties to humans. Therefore, it is more realistic and reliable than in vitro studies and provides important data for function validation, clinical trials, and approval by the FDA and regulatory authorities. In vivo experimental DED is generally induced in laboratory animals by several methods, including chemical, surgical, controlled environmental system, genetic (immunological) manipulations, and combined methods. Most of the experiments in DED are carried out in rabbits and rodents, although some studies are still performed on other animals, such as monkeys and pigs. Among these models, the chemically induced model is the most commonly used technique.  BAC is an organic salt, a quaternary ammonium compound, and is a type of cationic surfactant. It is also known as the alkyl dimethyl benzyl ammonium chloride. It is mainly used in three ways: an antimicrobial, a cationic surfactant, and a phase transfer agent [60]. In ocular medicine, it is widely used as a preservative in eye drops, with typical safe concentrations of 0.004-0.01%, which are most commonly used at a concentration of 0.01% in ophthalmic preparations [61]. However, experimental and clinical studies have found that the long-term use of topical drugs with BAC may alter ocular surface integrity, tear film instability, inflammation, and loss of goblet cells, conjunctival squamous metaplasia, epithelial apoptosis, subconjunctival fibrosis, and the potential risk of failure for further glaucoma surgery [62]. Liang et al. [63] showed that short and repeated exposure to 0.02% BAC induced a large number of inflammatory cells in the rabbit corneal epithelial basal layer and stroma [64]. They experimentally applied 0.01%, 0.05%, or 0.1% twice daily to one eye each of rabbits for 4 days and showed that BAC at 0.1% resulted in significant increases in central corneal thickness, endothelial CF permeability, endothelial cell damage with dislocation of ZO-1, and disruption of peri-junctional actomyosin ring (PAMR) compared with those in control eyes with other doses. In addition, Pauly et al. [65] (2005) applied 0.01%, 0.1%, 0.25%, and 0.5% to rat corneas for 11 days and found that 0.25% and 0.5% induced increased corneal thickness, loss of goblet cells, reversible corneal inflammation, and persistent neovascularization. Since then, experimental evidence has shown that the toxic effect of BAC on the ocular surface is mainly related to its concentration. Nonetheless, 0.1% BAC is commonly used to induce the DE model. Li et al. [66] applied 0.1% BAC twice daily for 2 weeks, 3 weeks, 4 weeks, or 5 weeks and concluded stable, sustainable DE induced by a 5-week BAC treatment group. Furthermore, the effects of different concentrations of BAC (0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, and 1.0%), different frequencies of topical application (two to four times daily), and different lengths of treatment (1-4 weeks) were evaluated for their effects on the rabbit ocular surface using the Schirmer test, ocular surface staining, CIC, and microscopy in the study by Xiong et al. [67]. It was found that twice-daily topical administration of 0.1% BAC drops for 2 weeks was the optimal procedure to induce dry eye syndrome in white New Zealand rabbits. However, using lower BAC concentrations was insufficient to induce tear deficiency over 2 weeks, while higher concentrations and multiple applications induced serious ocular surface damage and corneal ulcer, vascularization, and scarring, similar to chemical burns. Nonetheless, using 0.2% BAC for 2 weeks was also found to induce dry eye in mice [68].
Finasteride, atropine sulfate, scopalamine hydrobromide, and N-acetyl cysteine were also used to induce DED. The doses and experimental protocol are shown in Table 3. Androgen deficiency is one of the main causes of dry eye, as androgen plays a vital role in the regulation of lacrimal gland secretory functions [69], consequently inducing decreased tear secretion, ocular surface damage, and lacrimal gland inflammation, which are the characteristics of dry eye [70]. Finasteride is a 5α-reductase inhibitor that works by decreasing the production of dihydrotestosterone; therefore, it is called antiandrogen. In animal studies, it was also shown that finasteride administration decreased testosterone levels and DED [71].
Atropine is an anticholinergic antimuscarinic drug that inhibits the parasympathetic nervous system. Therefore, the topical administration of atropine sulfate has been used to induce dry eye models because of its ability to decrease tear production [72][73][74]. Moreover, as a muscarinic receptor blocker [75] and parasympathetic nerve inhibitor [76], scopolamine hydrobromide is also used to induce dry eye models. Scopolamine reduces the action of the central nervous system, such as atropine, by inducing nerve paralysis, mydriasis, and inhibition of secretion [75]. N-acetylcysteine (NAC) is a derivative of the amino acid L-cysteine that acts as a mucolytic agent that breaks mucoprotein disulfide bonds, thereby converting low molecular weight mucin molecules. Multiple or long-time topical exposure 0.1 M or 10% NAC induces the loss of the conjunctival mucus layer or mucin-like substances in rabbit eyes [77,78]. In addition, corrosive 50% trichloroacetic acid is also used to induce the dry eye model [45].  Another way to induce experimental DE is via excision of the ocular secretory glands. The lacrimal glands, MBGs, Harderian gland, and NM work together to maintain the tear film's moisture and lubrication of the ocular surface. Therefore, these parts are the surgical targets for inducing the dry eye model. After removing the lacrimal gland, dry eye occurred due to fluid deficiency [14], and the Harderian gland was removed together with severe fluid deficiency. Blocking the MBG induces oil deficiency in the lacrimal fluid of the ocular surface; consequently, the fluid evaporates easily and induces dry eye. Surgically, it can be induced by removing the lacrimal gland [14,95]; removal of the lacrimal gland (LG), Harderian gland (HG), and NM [42][43][44][45] or by thermal obstruction of MBG along with removal of the lacrimal and Harderian glands [96].

DED can also be induced by another surgical technique by excision of the left inferior
LGs for the isolation of purified epithelial cells and peripheral blood from each rabbit to collect peripheral blood lymphocytes. Activated lymphocytes are injected into the right LGs to induce autoimmune dacryoadenitis (AID), which results in tear deficiency. Consequently, DED is induced within 2 weeks of disease [97,98].
Concanavalin A (Con A) jack bean plant-derived lectins when injected into the body bind to various glycoproteins and activated lymphocytes, trigger inflammatory cascades, and cause tissue injury [99,100]. Therefore, Con A injection into the lacrimal gland allowed the study of the pathophysiology of immunologically mediated lacrimal gland-related DED [100][101][102][103][104]. Similarly, injection of botulin toxin B into the lacrimal gland also induced DED [105]. The experimental methods are listed in Table 4.

Environmental Factors-Induced DED Model
Another way to induce experimental DE is by applying an ICES [109,110]. An ICES is an artificial environment in which temperature, humidity, and airflow are regulated and recorded. This environment causes a deficiency of ocular surface fluidity by quick evaporation, in which dry eye can be induced due to critical environmental factors. However, the environmental factors and time of induction vary from article to article and are displayed in Table 5, which would be very helpful in ICES-induced experimental evaporative DED. Furthermore, the prevalence of DED in fine dust particulate matter-exposed persons is very high [111,112]. Recently, researchers found that the exposure of fine dust particulate matter induced DED in rodents [112][113][114].

Combining Methods
To increase the induction time of dry eye, two or three methods are combined to aggravate ocular surface dysfunction. For example, when C57BL/6 mice were used to induce DED, 21 days [115][116][117] and 42 days [110] were required (Table 5). However, when scopolamine was used along with ICES, DED was confirmed on day 9-14 [75,[128][129][130][131][132]. Likewise, the topical application of atropine and ICES was used, and DED was induced after 48 h [130]. Surgically dissected NM rabbit were housed in low humidity and higher air flow-controlled environment to induce DED [133]. Interestingly, three factors, i.e., genetic mouse, chemical administration, and ICES, were also applied [122,134,135] (Table 7). In brief, 0.5 mg/0.2 mL scopolamine hydrobromide was injected subcutaneously in the dorsal skin of mice three times daily.
The mice placed in the CEC were continuously exposed to a relative humidity < 30%, a constant temperature of 21-23 • C, and airflow of 15 L/min, 24 h a day From day 3 to day 9, 2 µL of the topical agent was applied to both eyes of each mouse, twice a day (9:00 a.m. and 5:00 p.m.).
On day 9 Lee et al. [129] 2011 C57BL/6 mice Topical application of atropine sulfate, 1%, twice daily for the first 48 h. In addition, the mice also received subcutaneous 0.1-mL injections of 5 mg/mL scopolamine hydrobromide three times a day (9:00 a.m., 1:00 p.m., and 5:00 p.m.) on their dorsal surface for the duration of the experiment.

Evaluation of DED Severity and Therapeutic Efficacy of Candidate Drugs in an Experimental Model
Several parameters are used in experimental studies to evaluate the severity of DED and therapeutic efficacy of candidate drugs. However, not all of these are used in one experiment, which are based on the ocular abnormalities that occur following DED and therapeutic targets of the candidate drugs. Some tests are specific for clinical signs, some are specific for tear deficiency, and some are specific for ocular surface structural damage, along with molecular alterations such as inflammatory cytokines, apoptosis, fibrosis, or angiogenesis-related markers (Table 7).

Inflammatory Index and Clinical Scoring
Both inflammatory index and clinical scoring can be used in rabbits or large animals, but only the inflammatory index is used in rodents. The inflammatory index can be assessed as described previously [83,136] by considering the following criteria: ciliary hyperemia (0-3), central corneal edema (0-3), and peripheral corneal edema (0-3). The total inflammatory index results were measured for each criterion from each individual animal and divided by a factor of 9 (Table 8).  In disease models, clinical signs are important criteria for accepting experimental research as a viable means of declaring scientific validity and practical reliability similar to an actual disease. Moreover, the experimental clinical criteria should be met by measurement scores for easy differentiation among the experimental groups. However, there is no gold standard for clinical scores in the dry eye model. The severity score was modified from that of Eaton et al. [137] and Silva et al. [74]. The score varies from 0 to 16 and is defined as the sum of the individual scores graded from conjunctival hyperemia (0-3), conjunctival swelling (chemosis) (0-4), conjunctival discharge (0-3), corneal opacity (area) (0-4), and corneal vascularization (0-2). Ophthalmologic examinations can be performed using a portable slit lamp and visual examination. Clinical signs should be scored before induction of DE, prior to treatment, and then several times at time intervals until the end of the experiment to evaluate the therapeutic efficacy of drugs (Table 8).

Schirmer's Test and Phenol Red Thread (PRT) Test
Schimer's test and the PRT test are similar types of experiments that are used to evaluate tear secretion in response to both basal non-reflex secretion and conjunctival stimulation in DED in conscious animals without anesthesia. Schimer's test was first introduced by German ophthalmologist Otto Schirmer over 100 years ago, and the PRT test was conducted by Japanese scientists in 1982 [151]. Comparatively, the PRT test is more sensitive and quicker than the Scimer strips. Only 15-60 s is required to obtain the result, while 5-10 min is needed for the Schirmer test per eye. In addition, the thread size is smaller than that of Schimer's strip. Therefore, it is suitable for experimental animals, including mice [152]. The strip/thread was inserted over the inferior lid margin toward the lateral canthus for five min. The eyes are allowed to blink normally. The wetting length was measured in millimeters after an equal selection time for all animals.

Tear Breakup Time
The TBUT test is a specific test to evaluate the quality of the tear film. In DED, tear film becomes unstable due to tear deficiency that may cause either a lack of tear production or increase in the evaporation of tears due to environmental factors, lack of MBG secretion, or mucin. Therefore, tear film breaks quicker in DED than in normal eyes. Usually, TBUT is ≤10 s for DED in mice [144], rats [141], and rabbits [145], and it may vary but is always lower than that in normal eyes. TBUT can be measured in two ways: by a slit lamp with a cobalt blue filter and by tear scope-plus (2413-P-2003, Keeler, UK) or Corneal Topographer (Carl Zeiss Meditec Ag, Oberkochen, Germany) [153]. For the former, one drop of 1% fluorescein eye drops was instilled into the lower conjunctival fornix. Then, the eyelids were blinked manually for one time, and then, the lids were held open. A slit lamp with a cobalt blue filter was used to evaluate the time required for the appearance of the first dark spot. This time is called the TBUT. On the other hand, noninvasive TBUT is measured by hand and held tear scope-plus. The eyelids were manually blinked to distribute the tear film; then, the eye was held open, and the time taken for distortion of the reflected image of the tear scope grid was recorded and considered as TBUT. In both methods, to obtain accurate results, the TBUT test was performed twice to obtain the average result.

Surface Structural Damage
Corneal fluorescein staining (CFS), Rose Bengal staining (RBS), and Lisamine green staining (LGS) are similar types of experiments used to evaluate the lesions of the ocular surface epithelium in DED (Table 8). Only damaged surface epithelial cells are stained by CFS and RBS, which is the principal mechanism of these methods. Therefore, as many cells are damaged, staining and darkness of staining are observed. Therefore, these techniques provide useful information about the severity of DED and the efficacy of therapeutic agents. Either CFS [66] or RBS [88,98] or LGS [112,146] can be selected for a particular experiment to avoid corneal tissue stress. However, two tests ((CFS and RBS) [67,79,83], (CFS and LGS) [133]) were also performed in one experiment. Three tests (CFS, RBS and LGS) were also performed in one experiment [114]. Even though two or three tests were performed on the same day, there was no discussion of the interval between the two experiments, and there was no information on washing after performing one test. Practically, this is time consuming, injurious to epithelial cells, and difficult to perform, as both tests are performed in the anesthetic state. Usually, 1% fluorescein sodium or 1% Rose Bengal stain or 1% Lisamine green stain is instilled (2-25 µL) into the conjunctival sac. Then, the eyelids were blinked manually for one time, and then, the lids were held open. After 3-10 min, cobalt blue light or red fluorescence staining or green staining, respectively, of the cornea was evaluated using a slit-lamp and scored.
There are various grading systems available for the evaluation of severity by staining, such as the van Bijsterveld scale (0-3, scoring from the whole cornea), National Eye Institute (NEI) scale (0-3, scoring from selected five zones), the Sjogren's International Collaborative Clinical Alliance ocular staining score/ICCA OSS Group Scale (0-3, scoring from the whole cornea), Baylor scale (0-4, scoring from selected five zones), and Oxford scale (0-5, scoring from whole corneas) [154]. Among them, Oxford scale scoring and NEI scales are the most popular in experimental studies. However, the Oxford scale scoring system could be convenient for statistical analysis and comparing and finding statistical significance among experimental groups (Table 9). Table 9. Ocular surface scoring methods by the Oxford scoring system (Bron et al. [155]).

Analysis of Molecular Changes and Histopathological Changes
DED is a chronic inflammatory disease. Therefore, evaluation of inflammatory cytokines in the ocular surface epithelial (epithelium of cornea or conjunctiva) cells, such as IL-1β, IL-8, TNF-α, and matrix metalloproteinases (MMPs), is the most common feature in experimental models [81,84,[138][139][140]. Alongside the underlying stimulating factors of inflammation in the ocular surface, such as MAP kinase pathways (ERK, JNK, and P38) [140,148], NFkB has also been measured by many researchers. Since ocular surface stress and inflammation induce apoptosis, apoptosis-related factors BAX, BCl2, and their ratio were also measured [88]. As chronic DED fibrosis may induce apoptosis as well, fibrosis-related TGF-β1 [150] was also measured. Furthermore, angiogenesis in the ocular surface may be triggered by inflammation, so the angiogenesis-related factor VEGF-A was measured by Kwon J.W. et al. [139]. Endothelial cell damage with dislocation of ZO-1, and disruption of PAMR [63] (Table 8).
Hyperosmolarity stress and amplified inflammatory cascades in the ocular surface of DED significantly alter the thickness of the corneal epithelium to thinning. Therefore, the histologically most used parameter in experimental DED is the corneal epithelial thickness [79,81,84,88,147]. In DED, goblet cells were also reduced, which is responsible for mucin secretion. Conjunctival impression cytology (CIC) using hematoxylin and periodic acid-Schiff (PAS) reagent [79,148] or counting goblet cells in the conjunctiva using periodic acid-Schiff (PAS) staining [44,84]. Immunohistochemical slides were also stained with MUC5AC in the conjunctiva to count the goblet cells [79,83,138]. Furthermore, polymorphonuclear cells (PMNs) in the cornea and limbus by hematoxylin and eosin stains were evaluated to measure the infiltration of inflammatory cells [138].

Conclusions
Tear hyperosmolarity is a key factor responsible for DED. Tear hyperosmolarity induces loss of surface integrity, tear film instability, and surface epithelial stress, which triggers the inflammatory cascades via MAP kinase and NFkB signaling pathways. This inflammation is responsible for multifactorial clinicopathological outcomes, such as epithelial cell damage, apoptosis, pain, fibrosis, and neovascularization ( Figure 3, Table 8). Tear hyperosmolarity may be induced by a lack of lacrimal secretion called tear or higher tear evaporation due to abnormality of MBG secretion, goblet cell secretion, or rough environmental factors (low moisture, higher airflow). In an experimental study, the administration of scopolamine chloride, finasteride, atropine sulfate, and injection of Con-A or BTX-B activated lymphocytes to the LG or excision of LG and results in LGS deficiency, thereby inducing dry eye. BAC or NAC induces tear film instability, and the loss of the epithelium and mucus layer or mucin-like substances results in hyperosmolarity, which in turn leads to dry eye. Thermal obstruction of the MBG results in oil deficiency, such as susceptance, which results in tear evaporation and DED (Figure 3). Furthermore, the addition of 90 mM sodium chloride to the culture medium induced a hyperosmolarity environment and DED-like symptoms in the in vitro DED model, while the addition of benzalkonium chloride (0.0001% (1 µg/mL) was also used to induce inflammatory cascades, as in the in vivo experiment. The animal models described above share similar features of DED in humans, and each model is an essential tool for investigating morphologic changes and underlying etiopathogenic molecular mechanisms that could also be involved during the evolution of DED. To develop new therapeutic drugs, it is necessary to test many agents in the preclinical research environment, and the use of smaller mammal models, such as mice or rats, will reduce the expense of producing test screening. Some advanced efficacy studies or toxicological examinations that require invasive procedures and large tear samples and tissue samples may be facilitated using animals with large body sizes, such as rabbits or non-human primates. In addition, the selection of a specific animal model depends on the type of drug being screened, the aim of the scientific strategy, investigator's choice, institutional financial and facility resources in the DED research, and pharmaceutical drug discovery and development program. Importantly, the combination of two or three induction methods (genetic, chemical, or surgical methods with ICES) shortens the DED induction time, which is important for experimental research in saving time and money. In addition, the repetition of similar types of experiments, such as corneal surface staining tests, Schirmer's test, and PRT test can be avoided for experimental schedule traffic and to relieve stress to animals and tissue injury.