**1. Introduction**

The retina is a transparent tissue of the eye which has an intricate arrangemen<sup>t</sup> of neurons and also requires a highly complex circulation to meet metabolic requirements and the proper functioning of neurotransmission, phototransduction, and complex interaction of metabolites, vasoactive agents, and growth factors [1,2]. The central retinal artery passes through the optic nerve to ensure blood flow,

and gas and nutrient exchange, while the central retinal vein is involved in the elimination of waste products [3]. The retinal vasculature is of grea<sup>t</sup> importance and within its physiological functions; the most important is to maintain the internal blood–retinal barrier (iBRB), which prevents nonspecific penetration of macromolecules into the retinal neuropile. The outer blood–retinal barrier (oBRB), formed between the tight junctions of retinal pigment cells, maintains ionic concentrations in the avascular region of the retina and the interstitial space for neurotransmission [3]. Retinal vascular dysfunction occurs shortly after the onset of diabetes and is characterized by impaired microvasculature and transport across the blood–retinal barrier playing an important role in the onset and progression of vascular lesions in diabetic retinopathy (DR) [2,4]. There is currently a wide range of treatments available for diabetes mellitus (DM) which has dramatically increased the lifespan of diabetic patients, but in turn gives time for clinically significant microvascular complications to develop [5]. Currently, there is a wide variety of effective treatments for DR, diagnosing the disease in its early stages helps prevent progression to blindness [5,6].

In this narrative review, we aim to approach various biomarkers associated with the development of diabetic retinopathy. In particular, our objective is to focus on the importance of molecules that are promising as detection tools and their measurement in different biological matrices obtained from the eye, in order to achieve an early disease detection or, ideally, even before the actual start of the DR. Articles in English were included that showed relevance both in preclinical and clinical stages of the DR. We take into account the articles that contribute to the discussion of the use of biomarkers of different nature to identify and estimate the stage of the disease in which patients with DR are, in addition to the use of different biological matrices obtained directly from the eyeball such as the tear, aqueous humor, and vitreous humor.

## **2. Diabetic Retinopathy**

Among the 468 million people with diabetes mellitus worldwide, approximately 90 million suffer from some form of DR [7]. It is the number one cause of blindness in people between the ages of 27 and 75. The prevalence of DR is approximately 25% and 90% at 5 and 20 years, respectively, from its diagnosis. Furthermore, it is estimated that ~191 million people will be diagnosed with this microvascular complication by 2030 [8,9], and the number of DR patients whose vision is threatened will increase from 37.3 to 56.3 million. This disturbing prospect makes the DR a significant global public health and economic problem [10].

Chronic hyperglycemia is the main risk factor affecting DR, as part of its pathophysiology it has been shown to induce vascular endothelial dysfunction in the retina [11]. When this state persist, activation of other pathways occurs in addition to glycolysis (such as polyol, hexosamine, and advanced glycation), which are known to induce apoptosis and pericyte degeneration, eventually damaging the retina [12].

DR is classified into nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) stages according to the presence of visible ophthalmological changes and the manifestation of retinal neovascularization [13,14]. NPDR is usually asymptomatic except when associated with macular edema; however, cases with uncontrolled DM or where retinopathy's progression is not monitored tend to progress to PDR, which is generally linked to complications that could lead to impaired visual acuity [15]. The first clinical sign of DR is the presence of microaneurysms in the retina during the mild version of the disease. In moderate diabetic retinopathy, exudates, hemorrhages, and minimal intraretinal microvascular abnormalities appear and may increase their proportion in severe stages [8]. Retinal detachment, neovascularization, along with fibrovascular tissue proliferation are features of PDR where newly formed vessels are leaky, fragile, misdirected, and the contraction of the aging vitreous cause them to rupture or if a greater force is created, it can lead to tractional retinal detachment resulting in acute or gradual loss of vision [2,9].

#### **3. Role of Oxidants and Antioxidants in the Eye with Diabetic Retinopathy**

The eye is an organ exposed to multiple exogenous factors, which are potentially precipitants of injury, including visible light, ultraviolet light, environmental toxins, and ionizing radiation, as well as the endogenous stress induced by the mitochondria within the eye tissues during the physiological functions of the eye [16]. This endogenous and exogenous stress produces an imbalance between oxidants and antioxidants, generating unstable reactive oxygen species (ROS) characterized by one or two unpaired electrons within their outer orbit [17]. ROS generation is normally correlated with cellular metabolic rate. The ocular surface produces lipids, aqueous, and mucin, all together form the tear film, which serves to protect and maintain the health of the ocular surface when it spreads over the eyelids [18]. The aqueous layer is produced by the tear and accessory glands. Mucins are secreted by corneal epithelial cells and conjunctival goblet cells. The lipids are secreted by the Meibomian glands located in the eyelids [18]. The ocular surface is further compromised in those patients with more severe and longer duration of diabetes disease, including those with higher A1c values, or retinopathy [19,20]. That is why the eyeball, as well as being exposed to attacks by ROS, is also provided with different antioxidants in its different segments, especially in the tear film, aqueous humor and vitreous humor.

#### *3.1. Oxidative Stress and the Damage It Causes to the Eye*

Oxidative stress (OS) is known as the interruption in free radical production homeostasis during various vital processes, such as the electron transport chain reaction and the sweeping of the oxidant products, or defense mechanisms designed to neutralize these harmful molecules. This imbalance is closely related to the pathophysiology of DR [21]. The addition of an electron to the dioxygen molecule creates the superoxide anion radical, which is generated mainly during the mitochondrial respiration process. Subsequently, the dismutation of this free radical by superoxide dismutase enzymes forms hydrogen peroxide (H2O2) [22]; decomposition of this molecule by various transition metals through the Fenton reaction can generate a high reactive hydroxyl radical [23,24]. Moreover, the reaction of the superoxide or hydroxyl radical with polyunsaturated fatty acids can generate the peroxyl radical (Figure 1). The human eye is constantly subject to OS, due to frequent exposure to light, in addition to high metabolic activity and oxygen tension. Solar ultraviolet radiation (UVR) turns out to be the main inducer in the external environment for ROS formation in the eye [24]. Other mechanisms like increased vascular endothelial growth factor (VEGF) production, alteration of the extracellular matrix architecture, genetic factors, and redox signaling are also present along with angiogenesis, collateral vessel formation, and increased permeability in PDR [25–27]. Stress induced by oxygen-derived free radicals such as hydroxyl radical, superoxide anion, and hydrogen peroxide can be harmful to cells [28], due to its ability to diffuse across hydrophobic membranes and their participation in the production of more reactive species, being H2O2 the most extensively studied oxygen metabolite [29].

In the anterior segmen<sup>t</sup> of the eye, H2O2 is present in the uvea and in the aqueous humor of mammals at concentrations between 30 and 70 μM [30]. H2O2 is a product of many antioxidant reactions of ascorbate such as those with oxygen and superoxide. High concentrations of this oxidant have been shown to be toxic to the lens [29,31]. Elevated levels up to seven times the normal range of H2O2 have been demonstrated in the aqueous humor and the lens of some human patients with cataracts [32]. H2O2 injected into the anterior chamber of the eye caused significant morphological changes in the iris and ciliary body and decreased intraocular pressure (IOP) [29].

In the posterior segment, H2O2 has been associated with tissue damage in the retina due to light and oxygen [33]. One of the main causes of DR is the development of glycosylated proteins, which generate free radicals, resulting in oxidative tissue damage and subsequent glutathione (GSH) depletion [34]. Glycosylated proteins can even combine with lipids and be further damaged by free radicals, forming advanced glycated end products (AGE), which can then deposit in blood vessels of the retina and promote neovascularization [35]. Diabetics with retinopathy have higher levels of oxidative damage markers in subretinal fluid when compared to diabetics without retinopathy and healthy controls [36]. The retina responds to OS with reactive gliosis: the activation of astrocytes, microglial, and macroglial cells. Microglial cells are resident retinal macrophages that confer neuroprotection against ROS damage and other injuries. OS promotes the degradation of sialic acid residues in membrane proteins, leaving photoreceptors and other cells with a damaged glycocalyx, this leads to greater phagocytosis by microglial cells, and increases neuronal cells death, worsening the pathology [37,38].

**Figure 1.** Reactive oxygen species and antioxidants in the eye. In the eye, chronic hyperglycemic state, atmospheric O2, and inflammation processes alter different metabolic pathways which stimulates the formation of reactive oxygen species (ROS) in the anterior and posterior segment, starting with oxygen (O2) to which the addition of one electron forms the superoxide anion radical (O2-•), the dismutation of this molecule by superoxide dismutases (SOD) forms hydrogen peroxide (H2O2), and the breakdown of this molecule can generate hydroxyl radical (OH•) which is highly reactive. In addition the reaction of O2- • or OH• radical with polyunsaturated fatty acids generates the peroxyl radical (LOO•). The formation of this radicals can be countered by enzymatic and non-enzymatic antioxidants like vitamin C, vitamin E, glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), among others. (Modified from ref. [24]).

Free radicals have also been reported to cause lipid peroxidation and a decrease in potassium-evoked dopamine release in vitro. OS induced by H2O2 has shown to enhance basal release of [3H] d-aspartate but decreased potassium (K+)—evoked release of this amino acid [39]. In one study, H2O2 caused a decrease in concentrations of glutamate and glycine in the retina. While low concentrations of H2O2 also produced decrease in glycine concentration in the vitreous humor, but had no significant action on glutamate levels [40]. The catalase 3-AT inhibitor caused reduction in both the retina and vitreous humor of the glutamate and glycine concentrations, indicating an important role of endogenously produced peroxides in the regulation of retinal amino acid neurotransmission. The observed inhibitory action of H2O2 on glutamate concentrations in ex vivo experiments emulate the effects observed in in vitro assays [40]. Nitric oxide synthase—the enzyme that catalyzes the formation of nitric oxide (NO)—is located in retinal neurons and the pigment epithelium. NO can participate in reactions with superoxide radicals to form the more potent and long-lived oxidant, peroxynitrite, from which there is evidence that can inhibit the absorption of glutamate in the rat's brain [41]. H2O2 interacts with the COX pathway that leads to the formation of prostanoids both in vitro and in vivo as well as simulating the biosynthesis of PGE*2* and PGI2 and the production of thromboxane B2. Isoprostanes are compounds derived from the free radical catalyzed peroxidation of arachidonic acid independent of COX. Then products such as PGE2 and 8-*iso*-PGF2<sup>α</sup>, regulate H2O2 and its inhibitory action on glutaminergic transmission in the isolated bovine retina [42]. It can be

concluded that in the posterior segmen<sup>t</sup> H2O2 has the ability to alter the availability of amino acids in bovine eyes [29].

#### *3.2. Antioxidants Present in the Tear Film, Aqueous Humor, and Vitreous Humor*

The eye is packed with a variety of antioxidants, which mitigates the damaging effects of ROS. An antioxidant is frequently defined as the substance that, when present in low concentration compared to that of an oxidizable substrate, significantly delays or inhibits the oxidation of the substrate [43]. Overproduction or inadequate elimination of ROS beyond the ability to counteract the antioxidant system can cause OS and overload the eye tissues [44]. Tear film and aqueous humor are important components of defense mechanisms on the ocular surface.

The tear film covers the anterior surface of the cornea and is the first line of defense against external aggressions [24]; it contains both non-enzymatic and enzymatic antioxidants. In human tears, ascorbic acid (665 μM) and uric acid (328 μM) represent ~50% of the total antioxidant activity, with ascorbic acid being the most abundant followed by uric acid; some other small molecules found are GSH (107 μM), L-cysteine (48 μM), and L-tyrosine (45 μM). The only antioxidant enzyme reported in the tear film is superoxide dismutase (SOD), which has an activity at 1–32 U/mg protein [24,45].

Aqueous humor is a clear, slightly alkaline liquid that occupies the space between the cornea and the lens, formed and secreted by the ciliary bodies. It plays a crucial role in the nutrition and protection of the corneal endothelium and in the anterior epithelial lining of the lens. Another of its functions is to eliminate metabolic waste and biochemical products generated by the cornea and the lens. ROS can be continuously generated in the aqueous humor in the form of hydrogen peroxide, superoxide anion, singlet oxygen, and peroxyl radicals [46]. The antioxidants found in the aqueous humor are almost the same as in the tear film, among the non-enzymatic antioxidants there is ascorbic acid (530 μM), L-tyrosine (78 μM), uric acid (43 μM), L-cysteine (14.3 μM), and glutathione (5.5 μM) [45]. Ascorbic acid has three different protective mechanisms in the aqueous humor: quenching or blocking the fluorescence of biomolecules, control of the biotransformation generated by the same fluorescence, and the direct absorption of UVR. The amino acid L-tyrosine is electrochemically active and removes hydroxyl radicals and singlet oxygen species. Uric acid (UA), a water-soluble molecule with high reactivity towards singlet oxygen and hydroxyl radicals, serves as a powerful scavenger of ROS [24,47]. In summary, the tear film and aqueous humor are packed with low-molecular weight, water-soluble antioxidants, which support the cornea's defense mechanisms against OS [24].

The vitreous humor is the structure that fills the space within the posterior segmen<sup>t</sup> of the eye; it is surrounded by the surface of the posterior lens and by the internal limiting membrane (ILM) of the retina. The vitreous body has a total volume of approximately 4 mL, mainly composed of water (98–99%), collagen fibers, glycosaminoglycans, non-collagen proteins, and small amounts of trace elements [48]. The nature of the vitreous gel is attributed to the interaction between its two main components: collagen and hyaluronan (HA) [44]. The concentration of HA within the vitreous gel varies between 0.02 and 1 mg/cm3 and plays a synergistic role with collagen and other proteoglycans for the regulation of vitreous stiffness [44,49]. The vitreous cortex is a lamellar structure attached to the ILM of the retina posterior to the peripheral vitreous base by an extracellular matrix "adhesive" consisting of laminin, opticin, fibronectin, chondroitin sulfate, and heparan sulfate. It can be said that the vitreous is acellular since it only presents a monolayer of mononuclear phagocytes, hyalocytes, located within the posterior vitreous cortex [50]. Among its functions, the vitreous contributes to the clarity of the intraocular media, the maintenance of IOP, and the regulation of intraocular oxygen tension [51]. In addition, the vitreous body provides protection by acting as a shock absorber, due to the collagen fibers that reduce the compressive forces of HA when the globe is exposed to external pressure [52].

The vitreous accumulates a high amount of water-soluble antioxidants, which could protect the eye from OS. These antioxidants can also be classified into enzymatic and non-enzymatic antioxidants [29]. Non-enzymatic antioxidants have the ability to quickly inactivate radicals and oxidants. Considering the

source of non-enzymatic vitreous antioxidants, these can be classified into nutrient non-enzymatic and metabolic antioxidants [53]. Nutrient non-enzymatic antioxidants include those obtained exogenously through food and supplements, such as vitamin C, vitamin B2, and trace metals like zinc and selenium. Metabolic antioxidants are endogenous antioxidants produced by the body itself, such as GSH, metal-chelating proteins like transferrin, and uric acid [44,54].

Vitamin C, also known as ascorbic acid, is a water-soluble molecule present in most tissues in its anionic state but cannot be synthesized by humans: they can only obtain it exogenously. The vitreous gel receives its vitamin C supply from the plasma through active transport from the ciliary body. The ascorbic acid found inside the vitreous body reaches concentrations of approximately 2 mmol/L; this is 33 times more than the plasma concentration. As an antioxidant, ascorbic acid is oxidized to convert superoxide anions and lipid hydroperoxidases into stable forms, thereby preventing lipid peroxidation [55]. Vitamin B2 (riboflavin) has been detected in the human vitreous (0.8 μg/100 mL) and animal (8.0 μg/L). Riboflavin protects against lipid peroxidation and plays an essential role in the glutathione redox cycle [56]. Zinc is the most abundant trace element within the eye that exerts antioxidant effects by protecting sulfhydryl groups from oxidation; its concentration is close to 1.95 μMol/L. Zinc works as a scavenger of free oxygen radicals like hydroxyl as it acts as a stimulus for metallothionein synthesis, it also protects tissues from various forms of oxidative damage, including lipid peroxidation and glycoxidation [57,58]. Selenium is an essential trace element, it has an average concentration of 0.1035 μMol/L. Selenium works indirectly as an antioxidant being incorporated into antioxidant enzymes, such as selenoenzymes [59].

The GSH peptide has cysteine and a thiol antioxidant in its constitution, and is found in an average concentration of 0.26 mmol/L [60]. As an antioxidant, glutathione can directly remove selected oxygen radicals and indirectly aid in the recycling of vitamins C and E [61], it also functions as a cofactor for glutathione peroxidase (GPx) activity allowing the reduction of lipid hydroperoxides [62]. Transferrin (molecular weight ~80 kDa), is found in an average concentration in the vitreous of 0.0878 g/<sup>L</sup> [44]. As an antioxidant, transferrin is an iron chelator that keeps ionic iron sequestered at physiological pH, and thus minimizes the participation of iron in radical iron-dependent reactions; its activity helps reduce the toxicity of intravitreal iron during vitreous hemorrhage [63]. Uric acid is a breakdown product of purine nucleotides and works as an antioxidant at normal concentrations. However, in the presence of oxidative stress, there is an upregulation of UA concentrations and a change related to redox balance, causing UA to become oxidative [64].

The enzymatic antioxidants detected in the vitreous are GPx, SOD, and catalase. From the glutathione peroxidase family, extracellular GPx3 and phospholipid GPx4 are found within the vitreous body [65]. As a homotetrameric protein, GPx3 catalyzes the reduction of organic hydroperoxides and H2O2 until alcohol and water are obtained by using GSH as an electron donor. GPx4 is a monomeric protein capable of directly reducing phospholipid and cholesterol hydroperoxides [66]. SOD is a metalloprotein enzyme that is responsible for catalyzing superoxide radicals into hydrogen peroxide and molecular oxygen. SOD is made up of three isoforms: cytosolic SOD (SOD1), mitochondrial SOD (SOD2), and extracellular SOD (SOD3). SOD1 and SOD3 contain copper and zinc (Cu/Zn-SOD), while SOD2 contains manganese (Mn-SOD) [44,67]. In the vitreous base and cortex we find the concentrated SOD3 isoenzyme where it interacts with proteoglycans and regulates the response to OS in the vitreous preventing local oxidative damage [67]. Catalase is a tetrahedral hemoprotein that also protects tissues from the toxic effects of peroxide by converting peroxides to water and oxygen. The vitreous body in humans has an average concentration of 58 μL O2 per mg of soluble catalase protein. It has been detected in the vitreous of patients with PDR; this suggests that catalase may be a potential target for the treatment of acute ischemic diseases of the retina [68]. Besides, along with GPx are found in other ocular tissues, including the iris and the ciliary body [30]. Fluorometric and postmortem toxicological analysis studies have shown that the passage of molecules from the systemic circulation to the vitreous through the blood–aqueous and blood–retina barriers is mediated by diffusion, hydrostatic and osmotic pressure gradients, convection, and active transport [69,70]. Repeated long-term administration of these agents may be necessary to achieve su fficient therapeutic doses of exogenous nutrients within the vitreous [71].

#### **4. Ocular Matrices: Tears, Aqueous Humor, and Vitreous Humor**

There are many di fferent microenvironments in the body; each organ and tissue can have its own microenvironment, including blood and cells. For example, a given biomarker can be present at multiple sites, and its relationship to the state of retinopathy can vary according to the site where it is measured [72]. The eye is a complex sensory organ that has the ability to receive light and convert it into electrical impulses, which are transmitted to the brain through the optic nerve, resulting in visual perception. In the case of animal models with ocular disorders, the variety of ocular matrices that can be collected and analyzed for biomarker measurement is wide, but the implementation of this biomarker measurement in the clinic together with the type of ocular matrix to be sampled is a key consideration. In humans, the most easily obtained eye matrices are tears and tissues of the ocular surface, such as the cornea and conjunctiva, they provide exact information regarding disorders in the anterior segment. Aqueous humor (AH) and vitreous are the most suitable matrices for evaluating relevant biomarkers for posterior segmen<sup>t</sup> disorders, such as DR. They are di fficult to access matrices, requiring an invasive procedure performed in the clinic to facilitate specimen collection, leading to significant ocular complications [73]. Evaluation of ocular biomarkers provides valuable information regarding disease progression and this makes it a critical component of the discovery and development of ophthalmic drugs.
