2. Dry Eye Disease

Dry eye disease (DED) is defined as a “multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles” ^[6][16]. The ocular surface, epithelial sensory receptors, the innervation of the epithelial sensory receptors, secretory centers in the brain, and efferent nerves supplying the meibomian glands, goblet cells, and the main lacrimal gland form a functional unit. Any or all of these structures may be affected in DED ^[8][18]. The cornea is the most densely innervated part of the body. The cornea nerves serve the protective blink reflexes, help in tear secretion, and release neurotransmitters necessary for epithelial and stromal support as well as ocular homeostasis. They also serve the nociceptors associated with mechanical stimuli, pain, and cold sensations. Corneal nerves and their morphological changes can be seen under IVCM. With the help of analytic software, the corneal nerve plexuses can be evaluated quantitatively, typically by measuring the nerve fiber density, length, nerve branch density, and tortuosity ^[9][10][19,20]. Corneal dendritic cells (DC) have been shown to be increased in dry eye patients compared to controls ^[11][21]. In the pathogenesis of dry eye, DCs play an important role in inducing the activation of T cells ^[12][22], thus triggering an inflammatory cascade reaction. Reduced corneal nerve density and length indicate a greater degree of neural damage induced by ocular pathology ^[13][23]. It has been shown that reduced density of corneal nerves results in impairment of protective functions such as tear secretion and blink reflexes. This results in a reduction in the tear quality and even aqueous tear deficiency. A study has suggested that patients with DED (diagnosed using TBUT and Schirmer’s, as well as the presence of symptoms) had decreased corneal nerve density ^[14][24]. Nerve tortuosity, defined by the frequency and the amplitude of the variations in the nerve fiber orientation, suggests active regeneration of nerve fibers in damaged nerves ^[15][25]. Studies by Liu et al. ^[16][26], Tepelus et al. ^[14][24], and Baikai et al. ^[17][27] have shown that nerve tortuosity is positively correlated with the diagnosis of DED. A greater nerve tortuosity is linked to ocular discomfort, visual function disturbance, and tear film instability ^[17][27].

3. Sjogren’s-Related Dry Eye (SSDE)

Sjogren syndrome (SS) is a systemic autoimmune disease that initially targets the lacrimal and salivary glands primarily, resulting in keratoconjunctivitis sicca (SSDE) and stomatitis sicca (dry mouth). The prevalence of primary SS in the USA approaches 1.3 million, with a range of 0.4–3.1 million ^[18][28]. The IVCM studies related to Sjogren’s-related dry eye are summarized in Table 2 as well. Certain IVCM parameters in sub-basal nerves have been reported to be altered in SS. Nerve fiber density is significantly decreased in SS ^[19][20][29,30], and SSDE is associated with greater nerve tortuosity than non-Sjogren’s Syndrome Dry Eye (NSSDE) ^[19][29]. Light backscattering (LB) measured in light reflectivity unit (LRU) at the Bowman’s membrane (BM) at 50 μm, 100 μm, and 200 μm deep to the BM has been evaluated in SS using IVCM—this is a measure of corneal inflammation ^[21][31]. Higher levels of LB in each corneal layer compared with healthy controls could indicate increased levels of corneal inflammation in SSDE ^[22][32]. The corneal epithelium of DED patients shows morphological changes, such as areas of enlarged and irregularly-shaped cells, which can be quantified by IVCM. Compared to controls, the density of superficial epithelial cells was decreased in both the NSDE and SSDE groups ^[23][33]. In summary, IVCM represents a reliable technique for examining nerve tortuosity in DED, as well as documenting corneal epithelial changes and immune cell densities in SSDE.

4. The Use of IVCM to Evaluate the Treatment for Dry Eye

While disease outcomes have typically been measured using symptomatic questionnaires and clinical tools such as Schirmer Test, corneal and conjunctival staining, tear break up time (TBUT), and tear osmolarity, there has been increasing interest to document treatment outcomes with IVCM ^[24][25][26][34,35,36]. The most common anti-inflammatory treatment for DED is cyclosporin A (CsA), an immunosuppressant and a calcineurin inhibitor. It has been used in several trials since 1986 and continues to be the major anti-inflammatory drug in the treatment of DED. Six months following treatment with topical CsA in SSDE patients ^[27][37], symptoms of dry eye documented by the Ocular Surface Disease Index (OSDI) score improved together with a decrease in corneal nerve tortuosity. Ther same is study reported an increase in sub-basal nerve plexus (SNP) density and a decrease in DC density after treatment. Though increased nerve reflectivity was found, the association was not significant. The decrease in DC density was attributed to the decrease in antigen-presenting cells and local inflammation, and the increase in SNP density was due to the normalization of innervation by controlling the inflammatory reaction ^[27][37].

5. Systemic Disease

Diabetic neuropathy, including diabetic corneal neuropathy, is one of the most common microvascular complications in diabetes ^[28][43]. There is increasing evidence to show that impairment of microvascular components is preceded by early neurodegenerative alterations primarily involving small nerve fibers, which can be demonstrated by IVCM ^[29][30][44,45]. Moreover, as small-fiber neuropathic changes can be picked up by IVCM, corneal nerve metrics have been used as surrogate markers for diabetic peripheral neuropathy ^[31][46]. Studies have shown that IVCM parameters such as CNFL ^[32][33][47,48], CNBD, CNFD, and CNFrD are reduced in patients with diabetes compared to controls, especially at the inferior whorl site ^[34][35][49,50]. A significant reduction in nerve beading frequency was also reported, which may be due to reduced metabolomic activity in diabetic patients ^[36][51]. IVCM is useful in analyzing the cornea and MG structures as well as the skin epidermis and dermis in ocular rosacea. It can quantify MG alterations based on meibum reflectivity, inflammation, and fibrosis, which correlated with the number of Demodex mites in both MG and cheek. However, no correlation was found between IVCM scores and both subjective and objective tests of dry eye ^[37][52]. Graves’ ophthalmopathy (GO) is often associated with DED, the most frequent cause of ocular discomfort in such patients ^[38][53]. GO is an autoimmune disease in which autoantibodies to the thyroid-stimulating hormone receptor lead to an inflammatory response in the orbital tissues ^[39][54]. Recent studies with IVCM have found changes in corneal nerves and MGs. Abnormal corneal SNP has been reported in active and inactive GO, suggesting nerve degeneration in GO. These central corneal SNP parameters of GO patients were significantly decreased compared with those of controls: corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), corneal nerve fiber total branch density (CTBD), corneal nerve fiber area (CNFA), corneal nerve fiber width (CNFW) and corneal nerve fiber fractal dimension (CNFrD). In addition, CNFD and ACNFrD values were significantly lower in the active GO compared with inactive GO patients. However, this study did not adjust for potential differences in DED between GO states did not adjust ^[40][55]. Hence, further studies could further stratify the active TAO further into mild, moderate, and severe states before comparing the difference in nerve parameters. IVCM also effectively revealed microstructural changes of MGs in eyes with GO and provided strong in vivo evidence for the roles of obstruction and inflammation in the disease process ^[41][56]. However, the patients in both groups had differing OSDI scores. Hence, it is unclear if the MG changes are related to concomitant DED in the GO patients or related to an extension of GO orbitopathy.

6. Glaucoma Treatment-Related Dry Eyes

Glaucoma is the leading cause of global irreversible blindness. The number of people with glaucoma worldwide will increase to 111.8 million in 2040, disproportionally affecting people residing in Asia and Africa ^[42][62]. Glaucoma is the leading cause of global irreversible blindness. The most common initial treatment for glaucoma is topical medical therapy and about half of glaucoma patients on topical anti-glaucomatous medications have the ocular surface disease ^[43][63]. Previous studies have demonstrated that toxic and proinflammatory effects of antiglaucoma ophthalmic solutions are mainly due to preservatives, though prostaglandins by themselves can cause periorbitopathy ^[44][45][64,65]. IVCM is useful in evaluating proinflammatory ocular surface changes induced by anti-glaucoma eye drops. These parameters may be affected: basal epithelial cell density, stromal reflectivity, number of sub-basal nerves, sub-basal nerve tortuosity, sub-basal nerve reflectivity, and endothelial cell density. One study found increased basal epithelial cells density, stromal reflectivity, sub-basal nerve tortuosity, and reduced sub-basal nerves in patients using glaucoma drops compared to healthy controls ^[46][66]. IVCM can also document changes in the cornea after glaucoma filtration surgery to evaluate for surgical success. For instance, preoperative DC density and goblet cell density (GCD) are correlated with filtration surgery outcomes ^[47][67]. These parameters were measured at the upper bulbar conjunctiva corresponding to the bleb site pre-operatively and at the bleb site postoperatively. Images were acquired from the epithelium and subepithelium (10–50 microns of depth). GCs may transport aqueous humor through the bleb wall ^[48][68] and DCs are the source of immune-regulatory cytokines ^[49][69], so increased GC and decreased DC are predictors of good outcomes. Hence, IVCM of the conjunctiva may represent an imaging tool to predict surgical success in glaucoma ^[47][67]. However, the study did not assess objective markers of dry eye, such as Schirmer’s test or TBUT. In addition, IVCM can be used to describe and compare the conjunctival filtering bleb features after XEN gel implantation and trabeculectomy, providing objective evaluation at a cellular level. For instance, IVCM was used to evaluate parameters like stromal meshwork reflectivity (SMR). As SMR represents an indirect indicator of the collagen content within the conjunctival stroma, a hyper-reflective pattern was a sign of collagen deposition, scarring, and potentially poorer clinical outcomes. After trabeculectomy, blebs showing a low degree of reflectivity and a thick wall are more likely to have a good filtering function ^[50][70].

7. Corneal Graft Versus Host Disease (GVHD)

Patients with ocular GVHD adjusted for ODSI and corneal staining displayed significantly decreased corneal epithelial cell density, SNP fiber density, and reflectivity compared to DED from other causes and healthy controls, while nerve tortuosity and epithelial DC density were increased in both oGVHD and DED groups ^[51][72]. This is in agreement with previous cross-sectional studies done ^[52][53][54][73,74,75]. As patients with DED unrelated to GVHD and ocular GVHD typically present with similar symptoms, IVCM could be used to evaluate and monitor patients with dry eyes due to GVHD and non-GVHD. Patients with chronic GVHD had worse meibography scores, reduced corneal sub-basal nerve plexus densities, lower TBUT scores, lower Schirmer I values and higher corneal staining scores. There was extensive loss of meibomian glands in both superior and inferior eyelids. In patients with chronic GVHD, the ensuing long-term inflammation often results in fibrosis of the ocular surface and cicatrizing conjunctivitis ^[55][76]. Hence, patients with chronic GVHD are at high risk for developing DED and MG dysfunction ^[56][77]. It is unclear if the IVCM signs of GVHD are linked to the more severe MG dysfunction compared to the DED group.

8. Contact Lens-Related Conditions

Estimates of total contact lens (CL) wearers worldwide in 2005 were as high as 140 million and hence even complications with a low incidence may affect a large number of individuals ^[57][78]. While the majority of complications are minor such as conjunctival hyperemia and corneal edema from overwear, there are serious sight-threatening complications such as infectious keratitis ^[58][79]. IVCM of the central cornea observed a higher density of DCs in contact lens wearers compared with non–contact lens wearers. CL lens has been known to activate and increase DC, contributing to ocular surface inflammation and a decrease in SNP. This decrease in SNP has been hypothesized to be due to increased DC and activated inflammation ^[59][80]. This finding has also been confirmed in soft lens wearers ^[60][81]. The precise etiology of “corneal infiltrative events” (CIE) which arise during CL wear, including both corneal infections and noninfectious inflammatory events ^[61][82], is not well understood. The incidence of symptomatic CIEs during extended soft lens wear ranges from 2.5 to 6%; when asymptomatic CIEs are included, the incidence can be as high as 20–25% ^[62][83]. IVCM can thus be potentially used to assess the subclinical response of the ocular surface in CL wearer. The risk of developing CIEs is 12.5 times higher in reusable lenses (those stored overnight in disinfecting solution throughout their usage period, which is typically 2 weeks or 1 month) compared with daily disposable lenses ^[59][80]. Interestingly, DC density was higher in reusable lens wearers than in daily disposable CL wearers ^[61][82]. IVCM can also study changes in corneal nerves associated with contact lenses. Orthokeratology (OK) involves using specially designed and fitted GP contact lenses to reshape the corneal surface for the temporary correction of refractive error. Lenses are only worn at night during sleep and removed on waking to provide clear, unaided vision throughout the day. IVCM has found that nerve fiber density (NFD) is decreased in OK wear ^[63][64][84,85]. This reduced NFD is associated with reduced corneal sensitivity and increased nerve tortuosity as well ^[64][85].