*4.2. Aqueous Humor*

AH is primarily composed of water (99.9%) and small amounts of carbohydrates, vitamins, proteins, and other nutrients, as well as growth factors and cytokines. It is responsible for maintaining eye pressure among other things for the support of ocular health. It is important to know that AH is drain from the eye through one of two passive pathways: the traditional trabecular meshwork (TM) pathway and the unconventional one, which is the uveoscleral pathway [79,80].

The samples of AH are collected by aqueous route through a paracenthesis in patients undergoing cataract surgery, trabeculectomy, phacoemulsification, or post-mortem eyes. Collection volumes are small and range from 100 to 150 μL [81,82]. After aqueous humor samples are obtained they are usually stored undiluted directly at −80 ◦C; they can be aliquoted according to the volume obtained and no more than one freeze–thaw cycle is recommended prior to analysis [83]. Potentially valuable information can be obtained from the analysis of these samples, however, there are potential risks associated with their collection. The methods are highly invasive and can create a risk of additional damage to the cornea and lens [73]. Post-mortem eye samples will have di fferent profiles than those collected from living patients due to the accumulation of metabolic waste and other uncontrolled post-mortem processes. This is why samples collected from living patients are considered more useful.

## *4.3. Vitreous Humor*

The vitreous is located in the posterior segmen<sup>t</sup> between the lens and the retina. As stated earlier, it is mainly composed of water and a mesh of fine collagen fibrils embedded with dissolved hyaluronan molecules, inorganic salts and lipids [84]. In addition, the vitreous contains other proteins such as albumin, globulins, clotting proteins, and complement factors that have accumulated from blood filtration or the spread of surrounding tissue and vasculature [85]. The anatomical position of this matrix in contact and close with the retina makes it an ideal compartment for taking samples that reflect biochemical and pathophysiological changes when there are states of retinal or vitreoretinal disease, including PDR [73,86].

Vitreous samples in vivo are generally collected through vitreous taps from vitrectomy patients [87]. Samples obtained from vitreous humor (900–920 uL) are generally stored undiluted at −80 ◦C as well as aqueous humor, it is recommended to separate them in aliquots to avoid freeze–thaw cycles. For its analysis, the samples can be centrifuged to eliminate cellular components and debris [88]. Given the invasiveness of vitreous sampling, recently has been evaluated the possibility of collecting vitreous reflux after intravitreal injections, being the appropriate time and without the need to enter the operating room, however, the possible risks should be better assessed [89]. In a study by Srividya and colleagues, they used Schirmer's tear strips to collect vitreous reflux from patients with diabetic macular edema (DME) and PDR, where they compared their total protein concentration with undiluted vitrectomy specimens. The results showed similar total protein concentrations between the vitreous reflux and the vitrectomy samples ( *P* < 0.05) [89]. The most common method of analysis for biomarkers in this samples are enzyme-linked immunosorbent assays (ELISAs) and enzyme-colorimetric assays. Recently, multiplex bead array assays are now commonly used to maximize the usefulness of the vitreous sample, like in AH because they have the advantage that they can measure multiple analytes simultaneously in small volumes [90]. Proteomic and genomic analysis techniques are also used to analyze biomarkers of di fferent nature in vitreous samples.

#### **5. Measurement of Biomarkers**

A biomarker provide a powerful and dynamic approach to improve our understanding of the mechanisms underlying eye disease, supporting information for diagnosis, disease control, or to predict clinical response to treatment [73]. Biomarkers can help to understand DR and contribute to the development of new treatments or new clinical strategies to prevent vision loss [91]. The FDA-NIH Joint Leadership Council updated the BEST Resource (Biomarkers, Endpoints, and Other Tools), modify the original definition of biomarker to "a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions" [92]. New biomarkers are generally considered of interest, as they are associated to predict the disease or its response to treatments, however they are not proven, nor are they widely accepted or used in clinical practice as a decision-making tool [91]. The development of new biomarkers is a complex process that requires high refinement, and depends on the etiological disclosure that is presented. On the other hand, in clinical practice individual biomarkers but also a set or panel of biomarkers can be used, these can include biomarkers of di fferent types, such as exposure, effect, and susceptibility biomarkers. Multiplex immunoassays are ideal for analyzing small volumes of samples, such as tears and now also in AH where they have been shown to be e ffective in measuring cytokine levels. With Next Generation Sequencing Techniques (NGS), small-sample miRNome analysis can be performed that avoids some of the limitations of hybridization-based detection methods [93]. Genetic matrix analysis is an additional powerful technique for comparing gene expression profiles in AH [94].

#### *5.1. Oxidative Stress: ROS and Relevance in DR*

The ocular surface is always subjected to intense exposure to light and solar ultraviolet radiation, also high metabolic activity increases the production of ROS and OS. This is a common adaptation secondary to inflammation and diabetes that produce more mediators to ROS in ocular surface as xanthine oxidase. It is hypothesized that the increase in antioxidant markers may reflect a local, compensatory response in the eye against OS [73]. Therefore, adequate levels of antioxidant enzymes responsible for free radical scavenging are essential for oxidation and reduction (redox) homeostasis [95]. Nezzar et al. analyzed the expression of glutathione peroxidase (GPx1–GPx8), catalase and SOD1 in human tissues of meibomian glands and conjunctiva and they found that both tissues express GPx2, GPx4, and GPx7 to control peroxide concentration in the lipid layer to protects the ocular surface from OS damage. Moreover, SOD1 and catalase were found expressed in conjunctiva and meibomian gland this might sugges<sup>t</sup> their role in recycling ROS and their interaction with inflammation caused by OS [96]. In addition, products such as GPx, SOD and malondialdehyde (MDA) have been observed at irregular levels in the AH of patients with PDR. SOD is a key antioxidant enzyme involved in the metabolism of oxygen free radicals and prevents the formation of other ROS [97].

GPXs are a family of selenium-dependent isoenzymes that catalyze the reduction of H2O2 or organic hydroperoxides in water and alcohols through oxidation of GSH to GSSG. Its activity can be measured using cumene hydroperoxide and GSH as substrates in a coupled reaction with glutathione reductase (GR) [98,99]. GPx messenger RNA express mainly in the ocular ciliary epithelium suggests that in AH glutathione peroxidase originates from these cells [100], its activity in the AH of glaucoma patients has been reported to be three times higher than in cataract patients, the mean value of GPx in AH of glaucoma patients was 18.4 ± 2.5 U/mL, and the mean value of the cataract group was 6.1 ± 0.6 U/mL (*p* < 0.001) [101] (see Table 1). Some antioxidant mechanisms, such as glutathione-related enzymes, are likely to work in normal and vitreous lenses to keep proteins in a reduced state. Unstable pro-oxidant molecules in the vitreous can play a role in cataract pathogenesis along with retinal disease. They can spread damage to lens membranes and vitreous proteins [102,103]. The study by Altomare et al. said specific activity of the GPx could not be detected in the vitreous humor; it is probably due to the high dilution of the vitreous humor used for the extraction procedure [103].

**Table 1.** Biomarker levels in tear, aqueous, and vitreous humor. Levels of the markers of oxidative stress, inflammation, and apoptosis measured in tear, aqueous, and vitreous humor in different ocular conditions, in addition to PDR, differentiating the different methodologies used. PDR: proliferative diabetic retinopathy, DED: dry eye disease, TAC: total antioxidant capacity, LPO: lipoperoxides, MDA: malondialdehyde, GSH: glutathione, GPx: glutathione peroxidase, SOD: superoxide dismutase, 8-OHdG: 8-hidroxi-2-deoxiguanosine, 8-IPGF: 8-isoprostaglandins, NO: nitric oxide, IL-6: interleukin-6, IL-8: interleukin-8, IL-10: interleukin-10, TNF-α: Tumour necrosis factor alpha, VEGF: vascular endothelial growth factor, EGF: epidermal grow factor, MMP-9: metalloproteinase-9, FN-kβ: Nuclear factor kβ, T1DM: type 1 diabetes mellitus, T2DM: type 2 diabetes mellitus.


\* Data are expressed as the mean ± SD. \*\* Median values and 5th and 95th percentile values in pg/mL.

SODs are a family of enzymes that catalyze the dismutation of superoxide into oxygen and H2O2 [99,131]. The fluid in the anterior chamber has very low SOD activity, so there are no apparent differences between diabetic patients and controls. However, it should be noted that the humor of the anterior chamber has a high content of ascorbate, which contributes significantly to protection against superoxide [132]. Significantly lower SOD levels in the vitreous have been found in diabetic patients with PDR than in non-diabetic controls examined post mortem [132]. There is a flow of fluid from the vitreous to the choriocapillary that holds the retina together, this is where SOD can contribute to protection against superoxide radicals in the extracellular space of the retina [132].

Determination of total antioxidant capacity (TAC) is a method for rapid quantification of antioxidant efficacy in disease prevention [133,134], defined as the "cumulative action of all antioxidants present in plasma and body fluids, providing an integrated parameter rather than the simple sum of measurable antioxidant values" [135]. Reduced capacity has been observed in the AH of patients with PDR [104] and there is a close correlation between OS and morphological changes in the TM [136], in patients with PDR suggests that the involvement of the anterior chamber may be caused in part by redox state imbalances [104]. Actually, the antioxidants of each system can exert their activity with different mechanisms and different efficiency; therefore, the assessment of TAC could be much more important than the concentration of antioxidants individually [104]. Izuta et al. reported that the TAC scan of the vitreous actually increases in patients with PDR [137].

Oxidation of DNA components by ROS and reactive nitrogen species (RNS) is the main source of DNA damage leading to various types of DNA modification, including chain breakage, nucleotide oxidation, base loss, and adduct formation [138]. The radicals can react with all the purine and pyrimidine bases, as well as with the deoxyribose skeleton, generating several products, one of them 8-hidroxi-2-deoxiguanosine (8-OHdG) [139]. It has been estimated that several thousand 8-OHdG lesions can form daily in a mammalian cell, this represents 5% of all oxidative lesions, which is why it is one of the most widely used DNA oxidation biomarkers to measure OS [140]. Their levels in aqueous humor increases and the total antioxidant status decreases in the serum and aqueous humor of glaucoma patients [141]. High myopia is a degenerative disease [142]. Therefore, 8-OHdG was found at lower levels in it compared to the control group; their levels are positively correlated with central corneal thickness (CCT) and negatively correlated with long axial length (AXL). Myopic patients presented 212.5 ± 103.2 μg/mL versus 311.6 ± 127.7 μg/mL in the control group with cataract patients [109] (see Table 1).

In diabetic patients with PDR, lipid peroxidation is a very pronounced process in humoral parts of the body, which is why it is responsible for the OS induced in diabetic patients [143]. Polyunsaturated fatty acid (PUFA) molecules are present in cell membranes and are prone to oxidation due to the presence of susceptible carbon double bonds [144,145]. The eye, in general, but particularly the macula, is susceptible to OS due to its high metabolic activity and the large amount of PUFA in the photoreceptor membranes [145]. The determination of the final product of lipid oxidation is widely used as a marker of OS. MDA together with 4-hydroxy-2-nonenal (4-HNE) represent the most investigated final product of lipid oxidation. 4-HNE is a stable and biologically very active molecule whose presence is not restricted to the site of its origin, since it can diffuse through the membrane barrier [146]. The elevated blood levels of MDA in patients with diabetes reflect similar increases in the level of AH, which points to the involvement of OS and lipid peroxidation in the progression of DR to the proliferative form [95]. Cell-free vitreous is the target of 4-HNE, although aging has been reported to increase specific conjugates in retinal proteins [147]. After laser treatment, this biomarker accumulates in this relatively separate compartment giving rise to conjugated humor proteins and deterioration of the antioxidant activity by the lipoperoxidation product will make the vitreous more vulnerable to pro-oxidative effect [148]. Besides, it could interfere with the expression of different collagen subtypes effecting a change in the expression profile of the collagen subclasses, having consequences for the functional integrity of the vitreous as seen in the aged eye [149]. On the other hand, the results on the changes of MDA in the vitreous of diabetic patients with PDR obtained by Brzovi´c-Šari´c et al. agree with what has been reported, which showed an increase in vitreous MDA in patients with PDR. So in general, direct lipid peroxidation (LPO) method is more sensitive and provides a better picture of the

status than MDA [105]. A significant correlation has been shown between the increase in LPO in the vitreous and an increase in the expression of VEGF; therefore, it seems that the determination of LPO in serum could be a good predictor of the onset of OS in the vitreous [105,150]. Because the retina is rich in PUFAs and there is an increase in glucose oxidation and oxygen absorption, it is susceptible to an increase in OS. The structural and functional changes observed in it may be due to lipid peroxidation of the vascular endothelium [151]. Isoprostanes (F2-IsoPs) are stable products, whose formation increases with exposure to OS, they have gained acceptance as a reliable marker of oxidative damage in both in vivo and in vitro animal models and their use is becoming more frequent [32], as a chemically stable prostaglandin-like isomers generated by the reaction of PUFAs in membrane phospholipids. They are formed in lipid membranes and then released freely by the action of phospholipase. An important fact is that they are not a ffected by lipid content in the diet and, therefore, its measurement in biological fluids can provide an estimate of endogenous production [152].

NO is one of the most abundant free radicals in the human body, which can also react with other ROS, this causes cellular dysfunction and apoptosis [153]. It is synthesized by endothelial cells and is an important vasoactive agen<sup>t</sup> that a ffects blood flow along with other vascular functions [154]. In the eye, neuronal nitric oxide synthase is believed to be responsible for producing NO in photoreceptors and bipolar cells, and as far as endothelial nitric oxide synthase is concerned, it is present in vascular endothelial cells [155]. However, inducible nitric oxide synthase found in Muller's cells and in the retinal pigment epithelium could be involved in phagocytosis of the external segmen<sup>t</sup> of the photoreceptor, in addition to infectious, inflammatory, and ischemic processes, and in the pathogenesis of DR [156]. In a Kulaksızoglu's study, the NO levels found by the measurement of nitrite and nitrate in the aqueous humor were significantly higher in patients with PDR than in controls without diabetes [95]. Another study noted that improved NO production in the eyes of people with diabetes is consistent with reports of it as a mediator of physiological and pathological processes in the retina [157]. NO is a highly important intercellular signaling molecule that plays a role in vasodilatory responses. In addition, it is involved in basal conditions and after retinal ischemia in the control of retinal blood flow [158]. The use of aqueous humor as a biological sample is proposed to assess the course of di fferent eye disorders showing an increase in pro-oxidative molecules and reduction in antioxidants [159,160]. In the study by Yilmaz et al. nitrite levels in vitreous humor of patients with PDR were higher than those of the control group (patients undergoing vitrectomy for idiopathic macular hole). However, among patients with PDR, there were no significant di fferences between the levels corresponding to type I and type II diabetes ( *P* = 0.56). On the other hand, patients with type I diabetes had a mean concentration of 0.524 ± 0.27 μM in vitreous, while those with type II diabetes had a mean of 0.383 ± 0.17 μM [112] (see Table 1). Other possibility is that overproduction of NO can cause damage to the retina by disrupting the rod outer segmen<sup>t</sup> membrane phagocytosis by retinal pigment epithelium (RPE) cells. Then, an accumulation of ROS occurs between the photoreceptors and the RPE cells, resulting in the degeneration of the photoreceptors [161].

#### *5.2. Inflammation: Cytokines and Relevance in DR*

DM patients with advanced diabetic retinopathy have more dry eye issues associated with neurotrophic keratopathy which could cause severe vision loss due to cornea ulcer or neurotrophic ulcer, this could lead an increased expression of proinflammatory cytokines, like inducible protein-10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), and decreased levels of antiangiogenic cytokines which demonstrate that an inflammatory reaction occurs in the ocular surface of diabetic patients [162]. The alterations in the ocular surface have been associated to inflammatory processes and microvascular damage that involves mediators like Th1 and Th17 and IL-1ß, IL-17A, TNFα, and mainly Epidermal Growth Factor (EGF) found elevated in patients with diabetes mellitus [126].

TNFα a cytokine strongly correlated with insulin resistance by changing phosphorylation of insulin receptor substrate-1 and interfering with insulin signaling cascade and leading chronic inflammation due to leukostasis induce by VEGF, IL-1 α and platelet-activating factor in the retinal

vasculature, but also it is a mediator of apoptosis in retinal neurons and endothelial cells. TNFα in tears was significantly higher in patients with PDR (13.5 pg/mL) compared with NPDR (2.8 pg/mL) [122]. In a recent cross-sectional study in Asian patients with DR, there was evaluated the di fferences in Total Protein Concentrations (TPC) and TNFα in tears correlated with the three stages of NPDR (mild, moderate and severe), they found a decreased in TPC in moderate and severe NPDR compared with higher concentrations of TNFα in the same stages, but in mild NPDR and patients without retinopathy the concentrations were similar. In this study, there was also higher levels of TNFα correlated with higher levels of HbA1c which are explained by AGEs that activate proinflammatory pathways and promotes angiogenesis and microvascular changes in the retina [163]. TNFα is one of the cytokines that induces disruption of the BRB by loosening the tight junctions between individual retinal endothelial cells and also between cells of the RPE, causing BRB breakdown. It promotes the irreversible adhesion of leukocytes to the endothelium and increases the production of ROS [164]. It also plays an important role in neovascularization and vasomotor reactions [165]. Actually, increased levels of TNFα have been found in the vitreous body of patients with T2DM and PDR compared with a control group [166]. There is a study where patients with PDR were divided into subgroups based on disease progression and regression, the vitreous levels of VEGF and IL-6 were significantly higher in the eyes of patients in the progression group with 1789.2 pg/mL (198.5–3436.8) and 347.2 pg/mL (26.2–758.6), respectively (see Table 2), than they were in eyes with PDR regression [116].

**Table 2.** miRNA's found in human eye fluids of patients. A comparison of some of the most commonly found miRNAs in vitreous or aqueous humor from patients with and without proliferative diabetic retinopathy. PDR: proliferative diabetic retinopathy, NDM: non-diabetic, PVD: proliferative vitreoretinal disease, MH: macular hole without any other condition, DME: diabetic macular edema.


+Biomarker found but not compared to another matrix or group.

In the inflammatory pathway, IL-6 has a role with induction of multiple process like synthesis and release of acute phase reactants and matrix metalloproteinase 9 (MMP-9), decreased tear production and apoptosis, induced di fferentiation of Th17, and the release of other proinflammatory cytokines like IL-1ß, the increased levels in this cytokine are particularly associated with increased metaplasia and keratinization of the ocular surface. Additionally, the secretion of interferon gamma (IFN-y) plays a multiple role with adaptive and innate response on the ocular surfaces inducing cell loss in the conjunctival goblet cells that reduces mucin production and apoptosis on lacrimal acini, associating high levels of this cytokine with severity of the tear film dysfunction [73,113,185]. Furthermore, decrease in the concentrations of proteins lysozyme, lactoferrin and albumin is more frequent in patients with PDR than NPDR [186], that could be associated with changes on ocular surface like tear film dysfunction, progressive loss of corneal epithelia and degeneration of nerve fibers [187], increasing the risk of corneal ulcerations and deteriorate their visual quality. In other studies have been analyze tear samples in di fferent stages of diabetic retinopathy and found a decreased in the content of tears proteins in the onset of DR as well, tear film dysfunction due to malfunctioning in the tear formation or more diluted tears. Lactotransferrin (LTF) and Immunoglobulin λ are increased in PDR together with another tear proteins expressed with frequency in PDR like lipocalin 1 (LCN1), lysozyme C (LYZ), lipophilin A, lacritin (LACRT) lipidic carriers for retinoids required for tear production, their increase are specifically associated as predictors for DR progression, some are linked to inflammation secondary to neovascularization, bleeding or macular edema [188]. Interestingly, IL-6 levels were positively correlated with the DR stage (PDR: 47.68 vs. NPDR: 29.68 pg/mL; *p* < 0.001) [115]. Elevated levels of IL-6 have been reported to participate as a proinflammatory and angiogenic factor in PDR and DM, is also involved in crossing both the blood–brain barrier and the blood–retinal barrier [189,190], and their levels in DR patients increased significantly compared to those in a non-diabetic control group. IL-6 levels were significantly correlated with PDR and it can act as one of the main drivers to generate a change in the cytokine profile in the aqueous humor of DR patients [115,191].

AH analysis provides useful information to understand the pathogenesis and responses to treatment of various eye conditions. The analysis of AH of DR patients has allow to identify some of the mediators (cytokines, chemokines, among other factors) involved in the pathogenesis of DR. Like IL-8, the concentration of VEGF is found to be increased in AH of diabetic patients [192]. Most studies have worked on aqueous humor cytokines in patients with PDR. However, the dynamic changes between the levels of cytokines in the di fferent states of severity of DR must be analyzed in greater detail. Chen et al. found that IL-6 had positive correlations with IL-8, IP-10, leukemia inhibitory factor (LIF) and hepatocyte growth factor (HGF) in the DR group, while it presented negative correlations with IL-9, IL-21, IL-23, IL-27, and IL-31.

IL-8 is an important chemoattractant that regulates chronic leukocytic inflammation in the vascular walls and ultimately leads to capillary occlusion and retinal ischemia [193], its levels have been reported in the AH of DR patients with 42.20 ± 33.03 pg/mL (mean ± SD) [115]. IL-8 expression can be hypoxia-induced and mediated by nuclear factor kappa B (NF-kB). IL-8 can induce angiogenesis in vitro and in vivo, and its elevated levels have been found in aqueous and vitreous of patients with ocular vascular disease, including DR [194]. Other studies have reported that elevated levels of vitreous IL-8 are associated with increasing levels of retinal ischemia and an increased degree of gliotic obliteration of large vessels in patients with PDR (see Table 2) [119,193]. Sun et al. found that the level of IL-8 in aqueous humor was significantly higher than that in the vitreous in PDR patients [119].

IL-10 is an anti-inflammatory cytokine and antiangiogenic mediator produced by monocytes and macrophages its antitumor e ffects have been associated with its ability to prevent angiogenesis associated with tumor growth [195]. In Mao C study, the median IL-10 concentration was higher in the vitreous of the PDR patients (224.789 pg/mL) than in the control group (160.143 pg/mL) and it was statistically significantly, which was di fferent from Hernández C study [121,196]. A predominance of macrophages (50%) has been determined in the vitreous samples of patients with PDR by cytological examination, this could explain the greater production of IL-10 resistant to other proinflammatory cytokines due to the increase in macrophages [125]. It suppresses the expression of the receptor for proinflammatory cytokines such as IL-1 and tumor necrosis factor α (TNFα) and inhibits the activation of its receptors by reducing the synthesis of these cytokines it limits inflammation. Its antiangiogenic effect has recently been shown to be associated with the downregulation of VEGF expression [197]. Dong et al. found that IL-10 levels in the AH decreased with increasing severity of DR.

TNFα is one of the main inflammatory response cytokines with chemotactic action on monocytes and neutrophils that activates them as macrophages, improving their cytotoxicity, while being one of the mediators of this cytotoxicity [198], with levels in aqueous humor of 4.04 ± 1.83 pg/mL (mean ± SD) in DR patients [115]. VEGF, also known as vascular permeability factor (VPF or vasculotropin), considered today the main factor controlling angiogenesis and vascular permeability and causes much of the pathogenesis of PDR and diabetic macular edema [199]. It is produced by endothelial cells, macrophages, CD4 lymphocytes, plasma cells, among others [200,201]. One study showed that the VEGF level was significantly elevated in diabetic subjects compared to the non-diabetic control group. However, there was no correlation between VEGF and DR severity (*p* = 0.357) [115].

The vitreous cavity contains different types of unique cells, such as retinal cells, RPE, choroid, and retinal vessels. Although the AH and the vitreous fluid do not flow with each other, some proteins can be exchanged between these two compartments. IL-6, IL-8, VEGF, and TNF-α have been observed to be elevated in serum and vitreous fluid from patients with DR [202]. Murugeswari et al. found that the levels of IL-6, IL-8, MCP-1, and VEGF present in the vitreous humor were significantly higher in PDR patients than in patients with macular hole [203]. In another study, a comprehensive analysis of mediators in the vitreous fluids of patients with PDR and in patients with other eye diseases was performed and elevated levels of VEGF, MCP-1, IL-8 and IL-6 were found compared to the control group [204]. IL-6 normally participates in acute phase reactions such as hematopoiesis, IL-2 induction, and differentiation of keratinocytes and B and T lymphocytes. In vitro studies sugges<sup>t</sup> that NO may be acting as the intermediate molecule in IL-6-induced VEGF synthesis. Precisely, the synthesis of IL-6 can be induced in vitro by hypoxia and tissue hyperglycemia [205].

Interleukin 17 (IL-17) plays an important role in a wide variety of immunological diseases, but on the ocular surface it promotes neutrophil infiltration in tissues that induce synthesis and secretion of matrix metalloproteinases and ROS that generate disruption in the corneal epithelium, loss of epithelial functionality and induction of apoptosis. This contributes to the induction of neovascularization with stimulation of proangiogenic factor and modulation of cytoskeleton, which was examined in the vitreous and plasma of patients with DR, with levels significantly increased in PDR [114,206,207].
