Deficient Autophagy Contributes to the Development of Diabetic Retinopathy

*Jacqueline M. Lopes de Faria and Marcella Neves Dátilo*

## **Abstract**

Autophagy is a self-degradation process essential to maintain intracellular homeostasis and cell survival, controlling elimination of pathogens, damage to organelles, and nutrient recycling to generate energy. Alterations in autophagic flux have been reported in the mechanisms of several diseases such as neurodegenerative diseases, cancer, diabetes mellitus, and its associated complications. Diabetic retinopathy (DR) is a microvascular complication of diabetes, affecting nearly 30% of diabetic patients. Several pathways are triggered and repressed in the development of DR, and autophagy showed to be relevant in the pathogenesis of this devastating complication. In this chapter, autophagy's involvement in the development and progression of DR will be discussed, mainly in retinal pigmented epithelial cells and retinal microvascular endothelial cells, as well as in Müller cells—the more prominent retinal glial cell.

**Keywords:** retina, diabetic retinopathy, autophagy, ARPE-19, endothelial cell, Müller cell

## **1. Introduction**

Autophagy (from Greek, meaning "self-eating") refers to a highly conserved process in eukaryotic cells, which coordinates the degradation of intracellular components and nutrient recycling. This process is essential for cellular homeostasis, survival, and differentiation. In basal conditions, the autophagic process happens in low levels to maintain cellular homeostasis. However, in such conditions as low levels of adenosine triphosphate (ATP) or depletion of essential amino acids and glucose, autophagic flux can increase to generate energy and raise basal levels. More recently, the understanding of this process has gained attention due to its pivotal role in cellular physiology and a variety of diseases from cancer, chronic degenerative diseases, and immune diseases (**Table 1**).

Autophagy is a primary cell response to stress and can be induced by starvation, endoplasmic reticulum (ER) stress, hypoxia, cytotoxicity, and infection (**Figure 1**). Sensation, initiation, and regulation of the autophagy–lysosomal pathway is controlled by the heterotrimeric serine/threonine kinase AMP (AMPK) and rapamycin complex 1 (mTORC1), either triggering or repressing autophagy and mitophagy. Unc-51-like kinase 1 (ULK1) is a primary initiating protein, as is mTORC1 supressed transcription factor EB (TFEB), which coordinates the synthesis of

#### **Table 1.**

*In this table, some examples of genetic diseases associated with autophagic impairment [1–8].*

#### **Figure 1.**

*Several cellular sensors regulate autophagic flux to maintain homeostasis.*

lysosomes and other essential proteins maintaining the autophagic flux [9–12]. In addition, sirtuin-1—a class III deacetylase dependent on nicotinamide adenine dinucleotide (NAD+)—becomes a positive autophagy regulator, since it may also be considered a cellular sensor [13].

This process is mainly regulated at a post-translational level, increasing mRNA expression of autophagy genes [14]. Under stress conditions, TFEB is translocated from cytosol to the nucleus, activating transcription of ATG genes and coordinating upregulation of the entire autophagy–lysosomal pathway [15].

**15**

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy*

or 5 genes leads to postnatal neurodegeneration [16, 17].

pathways that similarly modulate AMPK and mTOR [20–22].

autophagy-related genes (ATG). For example, deletion of specific neurons of ATG7

leads to an increase in the AMP/ADP ratio, activating the AMPK α subunit [10].

This discussion includes a short overview of the more common types of autophagy and will highlight the role of autophagy in retinal diseases, with special

There are three forms of autophagy previously described in the literature: macroautophagy, chaperone-mediated autophagy, and microautophagy (**Figure 2**).

Usually known as autophagy, this intracellular pathway includes cytosolic components such as proteins, lipids, organelles, and parts of the nucleus [23, 24]. Autophagy was first described by Christian du Duve 50 years ago and has been highly preserved across the species. From beginning to end, the whole process is controlled by the ATG protein family, and more than 35 genes have been identified

Autophagosome formation is the hallmark of this process. The well-coordinated process begins with an initiation phase, when ULK1 kinase forms a complex with ATG13, ATG10, and FIP200 (known as RB1CC1) at a specific cell site located in the perivacuolar region known as the phagophore assembly site (PAS). ULK1 kinase activity triggers the formation of the phosphoinositide 3-kinase (PI3K) complex, which favors the formation of phosphatidylinositol 3-phosphate, initiating the nucleation phase [26]. Ubiquitin-like conjugation systems are then activated, catalyzed by ATG7. ATG12 is conjugated to ATG5, then phosphatidylethanolamine to microtubule-associated protein 1A/1B-light chain 3 (LC3) through ATG7 kinase, forming an autophagosome bound to LC3 (also called LC3-II) [27, 28]. The late stage of autophagy is controlled by molecules that regulate maturation of the autophagosome, fusion with lysosomes, acidification of the inside compartment of the autophagosome components, and recycling of metabolites from the lysosomal compartment. This coordinated process—including a sequence of protein–protein and protein–lipid interaction—is a dynamic process, where the autophagosome formation, fusion to the lysosome, and digestion of the inside components occur in less than 10 minutes. Therefore, any sort of autophagy dysfunction (such as blockage of lysosomal fusion or lysosomal function impairment) may lead to accumulation of harmful damaged organelles and protein aggregates inside the cell [29] (**Figure 2**).

In chaperone-mediated autophagy, there is no reorganization of the lysosomal membrane. This selective autophagy is only described in mammals [30], which

Intrinsically, cellular sensors detect changes in levels of glucose, cytosolic Ca++, reactive oxygen species (ROS), and metabolic intermediates. Therefore, a decrease in glucose availability or impairment of mitochondrial respiration-compromising ATP production

An example of extrinsic sensing occurs via drug-targetable mechanisms at the plasma membrane level. Tyrosine kinase receptors converge on mTOR, AMPK, or Beclin-1-Vps complex by modulating autophagy following growth factors [18, 19]. Even G-protein-coupled receptors (GPCRs) control autophagy via intracellular

*DOI: http://dx.doi.org/10.5772/intechopen.89339*

attention to diabetic retinopathy.

**2. Types of autophagy**

**2.1 Macroautophagy**

to orchestrate the process [25].

**2.2 Chaperone-mediated autophagy**

Autophagy can be constitutive or inducible, rapidly adjusting to alterations within the internal and external environment of the cells. Autophagy serves as a housekeeping system, demonstrated by animal models deficient in

#### *Deficient Autophagy Contributes to the Development of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.89339*

autophagy-related genes (ATG). For example, deletion of specific neurons of ATG7 or 5 genes leads to postnatal neurodegeneration [16, 17].

Intrinsically, cellular sensors detect changes in levels of glucose, cytosolic Ca++, reactive oxygen species (ROS), and metabolic intermediates. Therefore, a decrease in glucose availability or impairment of mitochondrial respiration-compromising ATP production leads to an increase in the AMP/ADP ratio, activating the AMPK α subunit [10].

An example of extrinsic sensing occurs via drug-targetable mechanisms at the plasma membrane level. Tyrosine kinase receptors converge on mTOR, AMPK, or Beclin-1-Vps complex by modulating autophagy following growth factors [18, 19]. Even G-protein-coupled receptors (GPCRs) control autophagy via intracellular pathways that similarly modulate AMPK and mTOR [20–22].

This discussion includes a short overview of the more common types of autophagy and will highlight the role of autophagy in retinal diseases, with special attention to diabetic retinopathy.

## **2. Types of autophagy**

*The Eye and Foot in Diabetes*

**14**

**Figure 1.**

**Table 1.**

*Several cellular sensors regulate autophagic flux to maintain homeostasis.*

upregulation of the entire autophagy–lysosomal pathway [15].

considered a cellular sensor [13].

lysosomes and other essential proteins maintaining the autophagic flux [9–12]. In addition, sirtuin-1—a class III deacetylase dependent on nicotinamide adenine dinucleotide (NAD+)—becomes a positive autophagy regulator, since it may also be

*In this table, some examples of genetic diseases associated with autophagic impairment [1–8].*

Autophagy can be constitutive or inducible, rapidly adjusting to alterations within the internal and external environment of the cells. Autophagy serves as a housekeeping system, demonstrated by animal models deficient in

This process is mainly regulated at a post-translational level, increasing mRNA expression of autophagy genes [14]. Under stress conditions, TFEB is translocated from cytosol to the nucleus, activating transcription of ATG genes and coordinating

There are three forms of autophagy previously described in the literature: macroautophagy, chaperone-mediated autophagy, and microautophagy (**Figure 2**).

#### **2.1 Macroautophagy**

Usually known as autophagy, this intracellular pathway includes cytosolic components such as proteins, lipids, organelles, and parts of the nucleus [23, 24]. Autophagy was first described by Christian du Duve 50 years ago and has been highly preserved across the species. From beginning to end, the whole process is controlled by the ATG protein family, and more than 35 genes have been identified to orchestrate the process [25].

Autophagosome formation is the hallmark of this process. The well-coordinated process begins with an initiation phase, when ULK1 kinase forms a complex with ATG13, ATG10, and FIP200 (known as RB1CC1) at a specific cell site located in the perivacuolar region known as the phagophore assembly site (PAS). ULK1 kinase activity triggers the formation of the phosphoinositide 3-kinase (PI3K) complex, which favors the formation of phosphatidylinositol 3-phosphate, initiating the nucleation phase [26]. Ubiquitin-like conjugation systems are then activated, catalyzed by ATG7. ATG12 is conjugated to ATG5, then phosphatidylethanolamine to microtubule-associated protein 1A/1B-light chain 3 (LC3) through ATG7 kinase, forming an autophagosome bound to LC3 (also called LC3-II) [27, 28]. The late stage of autophagy is controlled by molecules that regulate maturation of the autophagosome, fusion with lysosomes, acidification of the inside compartment of the autophagosome components, and recycling of metabolites from the lysosomal compartment. This coordinated process—including a sequence of protein–protein and protein–lipid interaction—is a dynamic process, where the autophagosome formation, fusion to the lysosome, and digestion of the inside components occur in less than 10 minutes. Therefore, any sort of autophagy dysfunction (such as blockage of lysosomal fusion or lysosomal function impairment) may lead to accumulation of harmful damaged organelles and protein aggregates inside the cell [29] (**Figure 2**).

#### **2.2 Chaperone-mediated autophagy**

In chaperone-mediated autophagy, there is no reorganization of the lysosomal membrane. This selective autophagy is only described in mammals [30], which

#### **Figure 2.**

*Types of autophagy. (1) Macroautophagy: initiation of autophagy through isolation membrane, extension of membrane, and closure forming the autophagosome. Finally, the autophagosome merges with lysosome. Lysosomal hydrolases digest the contents to recycling nutrients. (2) Chaperone-mediated autophagy: identification of KFERQ-motif by Hsc70. Transportation of damage protein to lysosome. Recognition and multimerization of LAMP-2. Damage proteins are translocated to inside of the lysosome to suffer the action of lysosomal hydrolases. (3) Microautophagy: recognition and internalization of cytoplasmatic component.*

mediates delivery of specific proteins to the lysosome. The distinction occurs because the cytosolic proteins need to be degraded by the presence of a pentapeptide amino acid sequence, KFERQ. This sequence permits recognition of the target protein by a family of chaperones and co-chaperones: the heat shock cognate, 70-kDa (Hsc70)—the most abundant in the family. After recognition of the KFERQ sequence, Hsc70 presents the unfolded proteins to the lysosome, one by one, where they are recognized by the transmembrane domain of lysosome-associated membrane protein type 2A (LAMP2-A). After this, the multimerization of LAMP2-A occurs, allowing transportation of the substrate into the lysosome for degradation. At the end of this process, the LAMP2-A complex is disassembled, and the chaperone Hsp70 is released to start a new cycle [31].

## **2.3 Microautophagy**

Microautophagy is not well described in mammalian cells. However, recent evidence has shown that there is recognition and internalization of small cytoplasmatic components in late endosomes. This type of autophagy requires the chaperone Hsc70. However, the microautophagy process is independent of the unfolding of KFERQ and the multimerization of LAMP2-A [32, 33].

## **2.4 Role of autophagy in disease development**

Since the primary function of autophagy is to eliminate harmful components from cells (aggregated proteins, damaged organelles, and pathogens), malfunctioning of this mechanism implicit in diseases—such as Huntington's and Parkinson's diseases [34, 35]—results in protein accumulation.

**17**

as DR [71].

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy*

In physiological conditions, autophagy is involved in cellular homeostasis, as demonstrated in heart diseases, as seen in heart failure and ischemia–reperfusion injuries [36]. In the pancreas, autophagy is required to maintain function of β cells, revealing significance in the pathogenesis of diabetes. Alterations in autophagy have also been described in a more complex model in cancer research: it can suppress tumors but also helps the tumor adapt to metabolic stress in its late

Diabetes mellitus is a public health issue, estimated to affect about 500 million people by 2035 [38]. Nearly 30% of patients are likely to suffer from retinal microvascular complications and 10% may experience visual threatening due to macular

Multiple mechanisms are triggered under hyperglycemic conditions (hexosamine and polyol pathways [41], synthesis de novo of diacylglycerol-PKC [42, 43], low grade oxidative stress [44–46], inflammation [47–51], and advanced glycation end products [52, 53]). Although vascular changes are presumed to be the hallmarks of DR, abnormalities in retinal function are detected in patients with diabetes who

The characteristics of retinal neurodegeneration are apoptosis of neuro cells and dysfunction of glial cells—mainly Müller cells [29, 50, 60]. In microvascular disease of diabetic retinopathy, both inner and outer blood retinal barrier break down [61].

Since their pioneering studies, Remé et al. —describing the presence of active autophagy in photoreceptors during hibernation with a decreased number of mitochondria and organelles compared to animals in non-hibernating conditions observed an increased number of autophagosomes [62]. These data show the pivotal role of autophagy in the retina, degrading cellular components (such as mitochon-

Implications of autophagy in retinal ganglion cells (RGCs) attracted interest as a potential tool for neuroprotection in glaucoma. The first evidence of the cytoprotective role of autophagy in RGCs was shown by Rodríguez-Muela et al. using autophagy-deficient mice, which displayed increased axonal damage following

The main function of the blood-retina barrier (BRB) is maintenance of retinal homeostasis, regulating the transport of blood stream molecules to provide an appropriate supply for the neuroretina and to protect neural tissue against harmful agents present in the blood. The BRB is formed by two types of barriers: the inner blood-retina barrier (iBRB) and the outer blood-retina barrier (oBRB) [66].

Both outer and inner retinal barriers are affected by the toxic metabolic effects of hyperglycemia [67]. Alterations in the iBRB are more studied than the oBRB among the mechanisms of development and progression of DR [68–70]. The appropriated function of autophagy flux is important for maintenance of cellular viability and confers stress tolerance in retinal cells under adverse conditions such

optic nerve transection (ONT) models of optic neuropathy [63–65].

**3.2 Autophagy in blood retinal barriers and implications on diabetic**

*DOI: http://dx.doi.org/10.5772/intechopen.89339*

edema or proliferative diabetic retinopathy [39, 40].

stages [37].

**3. Diabetic retinopathy**

have good visual acuity [54–59].

dria) during hibernation.

**retinopathy**

**3.1 Autophagy in diabetic retinopathy**

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.89339*

In physiological conditions, autophagy is involved in cellular homeostasis, as demonstrated in heart diseases, as seen in heart failure and ischemia–reperfusion injuries [36]. In the pancreas, autophagy is required to maintain function of β cells, revealing significance in the pathogenesis of diabetes. Alterations in autophagy have also been described in a more complex model in cancer research: it can suppress tumors but also helps the tumor adapt to metabolic stress in its late stages [37].

## **3. Diabetic retinopathy**

*The Eye and Foot in Diabetes*

mediates delivery of specific proteins to the lysosome. The distinction occurs because the cytosolic proteins need to be degraded by the presence of a pentapeptide amino acid sequence, KFERQ. This sequence permits recognition of the target protein by a family of chaperones and co-chaperones: the heat shock cognate, 70-kDa (Hsc70)—the most abundant in the family. After recognition of the KFERQ sequence, Hsc70 presents the unfolded proteins to the lysosome, one by one, where they are recognized by the transmembrane domain of lysosome-associated membrane protein type 2A (LAMP2-A). After this, the multimerization of LAMP2-A occurs, allowing transportation of the substrate into the lysosome for degradation. At the end of this process, the LAMP2-A complex is disassembled, and the chaperone Hsp70 is released to start a new cycle [31].

*Types of autophagy. (1) Macroautophagy: initiation of autophagy through isolation membrane, extension of membrane, and closure forming the autophagosome. Finally, the autophagosome merges with lysosome. Lysosomal hydrolases digest the contents to recycling nutrients. (2) Chaperone-mediated autophagy: identification of KFERQ-motif by Hsc70. Transportation of damage protein to lysosome. Recognition and multimerization of LAMP-2. Damage proteins are translocated to inside of the lysosome to suffer the action of lysosomal hydrolases. (3) Microautophagy: recognition and internalization of cytoplasmatic component.*

Microautophagy is not well described in mammalian cells. However, recent evidence has shown that there is recognition and internalization of small cytoplasmatic components in late endosomes. This type of autophagy requires the chaperone Hsc70. However, the microautophagy process is independent of the unfolding

Since the primary function of autophagy is to eliminate harmful components from cells (aggregated proteins, damaged organelles, and pathogens), malfunctioning of this mechanism implicit in diseases—such as Huntington's and Parkinson's

of KFERQ and the multimerization of LAMP2-A [32, 33].

**2.4 Role of autophagy in disease development**

diseases [34, 35]—results in protein accumulation.

**16**

**2.3 Microautophagy**

**Figure 2.**

Diabetes mellitus is a public health issue, estimated to affect about 500 million people by 2035 [38]. Nearly 30% of patients are likely to suffer from retinal microvascular complications and 10% may experience visual threatening due to macular edema or proliferative diabetic retinopathy [39, 40].

Multiple mechanisms are triggered under hyperglycemic conditions (hexosamine and polyol pathways [41], synthesis de novo of diacylglycerol-PKC [42, 43], low grade oxidative stress [44–46], inflammation [47–51], and advanced glycation end products [52, 53]). Although vascular changes are presumed to be the hallmarks of DR, abnormalities in retinal function are detected in patients with diabetes who have good visual acuity [54–59].

The characteristics of retinal neurodegeneration are apoptosis of neuro cells and dysfunction of glial cells—mainly Müller cells [29, 50, 60]. In microvascular disease of diabetic retinopathy, both inner and outer blood retinal barrier break down [61].

#### **3.1 Autophagy in diabetic retinopathy**

Since their pioneering studies, Remé et al. —describing the presence of active autophagy in photoreceptors during hibernation with a decreased number of mitochondria and organelles compared to animals in non-hibernating conditions observed an increased number of autophagosomes [62]. These data show the pivotal role of autophagy in the retina, degrading cellular components (such as mitochondria) during hibernation.

Implications of autophagy in retinal ganglion cells (RGCs) attracted interest as a potential tool for neuroprotection in glaucoma. The first evidence of the cytoprotective role of autophagy in RGCs was shown by Rodríguez-Muela et al. using autophagy-deficient mice, which displayed increased axonal damage following optic nerve transection (ONT) models of optic neuropathy [63–65].

### **3.2 Autophagy in blood retinal barriers and implications on diabetic retinopathy**

The main function of the blood-retina barrier (BRB) is maintenance of retinal homeostasis, regulating the transport of blood stream molecules to provide an appropriate supply for the neuroretina and to protect neural tissue against harmful agents present in the blood. The BRB is formed by two types of barriers: the inner blood-retina barrier (iBRB) and the outer blood-retina barrier (oBRB) [66].

Both outer and inner retinal barriers are affected by the toxic metabolic effects of hyperglycemia [67]. Alterations in the iBRB are more studied than the oBRB among the mechanisms of development and progression of DR [68–70]. The appropriated function of autophagy flux is important for maintenance of cellular viability and confers stress tolerance in retinal cells under adverse conditions such as DR [71].

Retinal endothelial cells of microcirculation of the retina form the iBRB. This barrier selectively allows passage of molecules from systemic circulation to retinal tissue. As a constituent of this barrier, there are tight junctions and adherens junctions such as zonula occludens-1 (ZO-1), occludin, VE-cadherin, and N-cadherin [72]. Endothelial cells are warped by pericytes, which are highly specialized. Pericytes play an essential role in the structure and stability of the iBRB, coordinating angiogenesis and vascular remodeling [73, 74].

Few articles have highlighted the autophagic process in retinal endothelial cells under diabetic conditions [75, 76]. Exposure to high glucose leads to an increase in retinal endothelial cell apoptosis, and this mechanism is mediated by the enhancement of ROS production. This phenomenon is correlated with a reduction in the AMPK pathway [76], which is well described as a direct activator of ULK-1 in the autophagy process [77]. Reestablishing the level of AMPK using specific activators—such as AICAR or antioxidant treatment—is effective in the protection of endothelial retinal cells from damage caused by diabetic conditions [75, 76]. A recent study from Niu et al. described the importance of the protective properties of metformin on retinal endothelial cells and human umbilical vascular endothelial cells (HUVECs) via autophagy in diabetic conditions. In this work, the authors showed that there was an increased LC3 puncta formation, which is an indicative of autophagy, in retinal vascular endothelium from db/db (diabetic) mice compared with control (non-diabetic) mice. This is indicative that metformin protects the retinal microvascular cells by diminishing LC3 formation. To further understand this mechanism, HUVECs were exposed to high levels of glucose and treated with metformin, resulting in a clear increase of LC3 formation. In HUVECs transfected with sh-PRKAA1/2 (AMP catalytic subunit), the protective effect of metformin was abrogated, indicating that metformin acts via AMPK activation [78] and improving autophagy in these cells.

The oBRB is a monolayer formed by retinal pigment epithelial cell layer that separates the neuro retina from choriocapillaris. Impairment of this barrier is implicated in diabetic retinopathy development [79–81]. The major functions of the oBRB are to provide glucose, fatty acids, and retinol to photoreceptors from choriocapillaris and reisomerise all-trans-retinal in 11-cis-retinal after photon absorption of the photoreceptor [66, 82, 83]. Therefore, any disturbance in this structure may have detrimental effects on the retina. A number of sight-threatening diseases display RPR dysfunction, such as age-related macular degeneration, proliferative vitreoretinopathy, and diabetic retinopathy [84].

It is well described in the literature that human retinal pigmented epithelial (RPE) immortalized cells (ARPE-19) exposed to high concentrations of glucose present molecular changes, including a decrease of proliferation, an increase in oxidative stress mediated by ROS production, and augmented lipid droplets and inflammation [85–88]. These alterations can activate or repress the autophagic flux in RPE cells. Studies have shown that, until 48 hours of exposure to high glucose levels, ARPE-19 cells present an increase in lipid droplets, which can contribute to ROS production [71, 85, 89]. This increase in ROS production can initiate autophagy, enhancing the numbers of autophagosomes, increasing conversion of LC3-I to LC3-II, and decreasing levels of p62/SQSTM1 as a defense mechanism against damage caused by high glucose. However, Chen et al. found that an increase in autophagic flux promoted by high glucose cannot be maintained long-term. After 7 days in high glucose, ARPE-19 presented impairment in the degradation of p62/ SQSTM1 and an increase in apoptotic cells. These findings indicated that autophagy was the first defense against oxidative stress in high-glucose conditions. In the longterm, this protective pathway became saturated and inefficient, thus contributing to RPE degeneration in DR [87].

**19**

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy*

because the biogenesis of mitochondria becomes compromised [90].

Zhang et al. have shown that high glucose concentrations can attenuate the PINK1 and parkin pathways involved in controlling cellular mitophagy. Downregulation of mitophagy can lead to an increase in cellular stress levels

**3.3 Autophagy in Müller glial cells and implications in diabetic retinopathy**

Müller cells are the predominant glial cell in the retina. Its unique morphology allows the Müller cell to directly interact with neighboring neural and vascular cells, expanding through the entire retina from the inner limiting membrane to the photoreceptor layer. Müller cells are closely related with vitreous, blood vessels, and sub retinal space. Each Müller cell interacts with one cone and 10 rods [91]. This configuration of Müller cells inside the retina explains the diversity of its function, responsible for the metabolic, functional, and structural support of the retina [92]. There are several functions attributed to Müller cells, such as the release of trophic factors [93, 94], neurotransmitter recycling [95], and phagocytosis of external photoreceptor segments [96, 97]. Müller cells, depending upon the stimulus (trauma, vascular, or metabolic), may react with phenotype changes called gliosis, which consist in adaptive morphological, biochemical, and physiological alterations. Among the more interesting biochemical changes in Müller cells are increased vascular endothelial growth factor (VEGF) [98] and glial fibrillary acidic protein (GFAP) production, both with pro-angiogenic and pro-inflammatory effects. Massive VEGF release is present in the proliferative stages of DR and diabetic macular edema, representing a major therapeutic target for pharmacological treatment of

There are few studies showing the effects of high glucose on autophagy in retinal

In the previously published work addressing the mechanism by which Müller cells exposed to high glucose release high amounts of VEFG and trigger increased apoptosis, it was shown that the autophagic process was defective in Müller cells among diabetic conditions. In cells exposed to high glucose, autophagy markers both early Beclin and late LC3-I and LC3-II—were increased, but p62/SQSTM1 accumulated in the cytosol compartment of Müller cells, accompanied by an increased apoptotic rate. To further understand how p62/SQSTM1 could modulate the autophagy and apoptosis in Müller cells exposed to high glucose, p62/SQSTM1 was suppressed. In this condition, there was less endoplasmic reticulum stress, lowering the interaction with caspase-8 and, by extension, less apoptosis. The presence of rapamycin, an mTOR blocker, triggered the formation of autophagosome

Müller cells. Devi et al. described the implications of autophagy dysfunction in the mechanisms of DR [99]. In their study, Müller cells exposed to high glucose conditions for 5 days displayed an increase of autophagosome and mitophagosome in the cytosol, suggesting high glucose conditions activated the autophagy process. Despite activation of the protective process (autophagy), they observed an association with an increased proapoptotic caspase-3, leading to programmed cell death. This scenario elucidates that diabetic conditions induce activation of autophagy

The role of autophagy in retinal diabetic complications is not simply a matter of inhibiting its initiation or progression. Inhibition of autophagy in ARPE-19 during its initial phase with 3-methyladenine (3-MA) or during the fusion of autophagosome and lysosome using bafilomycin aggravates oxidative stress and exacerbates secretion of the pro-inflammatory interleukin-1β promoted by high glucose [88]. The appropriated autophagic process is important as a mechanism of cell homeosta-

*DOI: http://dx.doi.org/10.5772/intechopen.89339*

sis in diabetic conditions.

these devastating complications.

followed by dysfunction, leading to cellular death.

**pathogenesis**

#### *Deficient Autophagy Contributes to the Development of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.89339*

Zhang et al. have shown that high glucose concentrations can attenuate the PINK1 and parkin pathways involved in controlling cellular mitophagy. Downregulation of mitophagy can lead to an increase in cellular stress levels because the biogenesis of mitochondria becomes compromised [90].

The role of autophagy in retinal diabetic complications is not simply a matter of inhibiting its initiation or progression. Inhibition of autophagy in ARPE-19 during its initial phase with 3-methyladenine (3-MA) or during the fusion of autophagosome and lysosome using bafilomycin aggravates oxidative stress and exacerbates secretion of the pro-inflammatory interleukin-1β promoted by high glucose [88]. The appropriated autophagic process is important as a mechanism of cell homeostasis in diabetic conditions.

## **3.3 Autophagy in Müller glial cells and implications in diabetic retinopathy pathogenesis**

Müller cells are the predominant glial cell in the retina. Its unique morphology allows the Müller cell to directly interact with neighboring neural and vascular cells, expanding through the entire retina from the inner limiting membrane to the photoreceptor layer. Müller cells are closely related with vitreous, blood vessels, and sub retinal space. Each Müller cell interacts with one cone and 10 rods [91]. This configuration of Müller cells inside the retina explains the diversity of its function, responsible for the metabolic, functional, and structural support of the retina [92].

There are several functions attributed to Müller cells, such as the release of trophic factors [93, 94], neurotransmitter recycling [95], and phagocytosis of external photoreceptor segments [96, 97]. Müller cells, depending upon the stimulus (trauma, vascular, or metabolic), may react with phenotype changes called gliosis, which consist in adaptive morphological, biochemical, and physiological alterations. Among the more interesting biochemical changes in Müller cells are increased vascular endothelial growth factor (VEGF) [98] and glial fibrillary acidic protein (GFAP) production, both with pro-angiogenic and pro-inflammatory effects. Massive VEGF release is present in the proliferative stages of DR and diabetic macular edema, representing a major therapeutic target for pharmacological treatment of these devastating complications.

There are few studies showing the effects of high glucose on autophagy in retinal Müller cells. Devi et al. described the implications of autophagy dysfunction in the mechanisms of DR [99]. In their study, Müller cells exposed to high glucose conditions for 5 days displayed an increase of autophagosome and mitophagosome in the cytosol, suggesting high glucose conditions activated the autophagy process. Despite activation of the protective process (autophagy), they observed an association with an increased proapoptotic caspase-3, leading to programmed cell death. This scenario elucidates that diabetic conditions induce activation of autophagy followed by dysfunction, leading to cellular death.

In the previously published work addressing the mechanism by which Müller cells exposed to high glucose release high amounts of VEFG and trigger increased apoptosis, it was shown that the autophagic process was defective in Müller cells among diabetic conditions. In cells exposed to high glucose, autophagy markers both early Beclin and late LC3-I and LC3-II—were increased, but p62/SQSTM1 accumulated in the cytosol compartment of Müller cells, accompanied by an increased apoptotic rate. To further understand how p62/SQSTM1 could modulate the autophagy and apoptosis in Müller cells exposed to high glucose, p62/SQSTM1 was suppressed. In this condition, there was less endoplasmic reticulum stress, lowering the interaction with caspase-8 and, by extension, less apoptosis. The presence of rapamycin, an mTOR blocker, triggered the formation of autophagosome

*The Eye and Foot in Diabetes*

autophagy in these cells.

ing angiogenesis and vascular remodeling [73, 74].

vitreoretinopathy, and diabetic retinopathy [84].

Retinal endothelial cells of microcirculation of the retina form the iBRB. This barrier selectively allows passage of molecules from systemic circulation to retinal tissue. As a constituent of this barrier, there are tight junctions and adherens junctions such as zonula occludens-1 (ZO-1), occludin, VE-cadherin, and N-cadherin [72]. Endothelial cells are warped by pericytes, which are highly specialized. Pericytes play an essential role in the structure and stability of the iBRB, coordinat-

Few articles have highlighted the autophagic process in retinal endothelial cells under diabetic conditions [75, 76]. Exposure to high glucose leads to an increase in retinal endothelial cell apoptosis, and this mechanism is mediated by the enhancement of ROS production. This phenomenon is correlated with a reduction in the AMPK pathway [76], which is well described as a direct activator of ULK-1 in the autophagy process [77]. Reestablishing the level of AMPK using specific activators—such as AICAR or antioxidant treatment—is effective in the protection of endothelial retinal cells from damage caused by diabetic conditions [75, 76]. A recent study from Niu et al. described the importance of the protective properties of metformin on retinal endothelial cells and human umbilical vascular endothelial cells (HUVECs) via autophagy in diabetic conditions. In this work, the authors showed that there was an increased LC3 puncta formation, which is an indicative of autophagy, in retinal vascular endothelium from db/db (diabetic) mice compared with control (non-diabetic) mice. This is indicative that metformin protects the retinal microvascular cells by diminishing LC3 formation. To further understand this mechanism, HUVECs were exposed to high levels of glucose and treated with metformin, resulting in a clear increase of LC3 formation. In HUVECs transfected with sh-PRKAA1/2 (AMP catalytic subunit), the protective effect of metformin was abrogated, indicating that metformin acts via AMPK activation [78] and improving

The oBRB is a monolayer formed by retinal pigment epithelial cell layer that separates the neuro retina from choriocapillaris. Impairment of this barrier is implicated in diabetic retinopathy development [79–81]. The major functions of the oBRB are to provide glucose, fatty acids, and retinol to photoreceptors from choriocapillaris and reisomerise all-trans-retinal in 11-cis-retinal after photon absorption of the photoreceptor [66, 82, 83]. Therefore, any disturbance in this structure may have detrimental effects on the retina. A number of sight-threatening diseases display RPR dysfunction, such as age-related macular degeneration, proliferative

It is well described in the literature that human retinal pigmented epithelial (RPE) immortalized cells (ARPE-19) exposed to high concentrations of glucose present molecular changes, including a decrease of proliferation, an increase in oxidative stress mediated by ROS production, and augmented lipid droplets and inflammation [85–88]. These alterations can activate or repress the autophagic flux in RPE cells. Studies have shown that, until 48 hours of exposure to high glucose levels, ARPE-19 cells present an increase in lipid droplets, which can contribute to ROS production [71, 85, 89]. This increase in ROS production can initiate autophagy, enhancing the numbers of autophagosomes, increasing conversion of LC3-I to LC3-II, and decreasing levels of p62/SQSTM1 as a defense mechanism against damage caused by high glucose. However, Chen et al. found that an increase in autophagic flux promoted by high glucose cannot be maintained long-term. After 7 days in high glucose, ARPE-19 presented impairment in the degradation of p62/ SQSTM1 and an increase in apoptotic cells. These findings indicated that autophagy was the first defense against oxidative stress in high-glucose conditions. In the longterm, this protective pathway became saturated and inefficient, thus contributing

**18**

to RPE degeneration in DR [87].

and ameliorated the degradation of p62/SQSTM1. Rapamycin showed to improve proteolytic activity of the lysosome, reducing the release of VEGF. Corresponding findings were also demonstrated in models using diabetic animals. In the retinas of diabetic rats, there was a significant increase in p62/SQSTM1 accumulation, particularly in cells located in the inner nuclear layer [29]. Lysosomal impairment and autophagic flux dysfunction are early indicators of the pathogenesis of DR.

## **4. Conclusion**

Diabetic retinopathy is a neurodegenerative disease presenting vascular changes in its late stages. Multiple factors are associated with the development and progression of DR. Recently, better understanding at cellular and molecular levels of its process has been identified through the pathways and intracellular signaling involved in cells exposed to diabetic conditions. This has allowed identification of new therapeutic approaches. Recent concepts of this disease have been analyzed here, with special focus on the process of autophagy using experimental models in different retinal cells targeted by hyperglycemia in the developmental stages of the disease.

## **Author details**

Jacqueline M. Lopes de Faria\* and Marcella Neves Dátilo Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**21**

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy*

[7] Sumpter R, Sirasanagandla S,

cell.2016.04.006

immuni.2016.05.007

10.1038/s41586-018-0162-7

DOI: 10.4161/auto.3636

[10] Melendez A. Autophagy genes are essential for Dauer development and life-span extension in C. elegans. Science. 2003;**301**(5638):1387-1391. DOI: 10.1038/s41586-018-0162-7

[11] Hars ES, Qi H, Jin SV, Cai L, Hu C, Liu LF. Autophagy Regulates Ageing in C. elegans. Autophagy. 2007;**3**(2):93-95.

[12] Hansen M, Chandra A, Mitic LL, Onken B, Driscoll M, Kenyon CA. Role for autophagy in the extension of lifespan by dietary restriction in C. elegans. PLoS Genetics. 2008;**4**(2):e24. DOI: 10.1371/journal.pgen.0040024.

Neufeld TP. Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in drosophila. Genes & Development. 2007;**21**(23):3061-3066. DOI: 10.1101/

[14] Feng Y, Yao Z, Klionsky DJ. How to control self-digestion: Transcriptional,

[13] Juhasz G, Erdi B, Sass M,

gad.1600707

Fernández ÁF, Wei Y, Dong X, Franco L, et al. Fanconi anemia proteins function in Mitophagy and immunity. Cell. 2016;**165**(4):867-881. DOI: 10.1016/j.

[8] Lassen KG, McKenzie CI, Mari M, Murano T, Begun J, Baxt LA, et al. Genetic coding variant in GPR65 alters Lysosomal pH and links Lysosomal dysfunction with colitis risk. Immunity. 2016;**44**(6):1392-1405. DOI: 10.1016/j.

[9] Fernández ÁF, Sebti S, Wei Y, Zou Z, Shi M, McMillan KL, et al. Disruption of the beclin 1–BCL2 autophagy regulatory complex promotes longevity in mice. Nature. 2018;**558**(7708):136-140. DOI:

*DOI: http://dx.doi.org/10.5772/intechopen.89339*

[1] Schapira AHV. Glucocerebrosidase and parkinson disease: Recent advances. Molecular and Cellular Neurosciences.

2015;**66**:37-42. DOI: 10.1016/j.

[2] Kim MJ, Deng H-X, Wong YC, Siddique T, Krainc D. The Parkinson's disease-linked protein TMEM230 is required for Rab8a-mediated secretory vesicle trafficking and retromer

[3] Lee J-H, Yu WH, Kumar A,

trafficking. Human Molecular Genetics. 2017;**26**(4):729-741. DOI: 10.1093/hmg/

Lee S, Mohan PS, Peterhoff CM, et al. Lysosomal proteolysis and autophagy require Presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010;**141**(7):1146-1158. DOI: 10.1016/j.

[4] Reddy PH, Yin X, Manczak M, Kumar S, Pradeepkiran JA, Vijayan M, et al. Mutant APP and amyloid betainduced defective autophagy,

mitophagy, mitochondrial structural and functional changes and synaptic damage in hippocampal neurons from Alzheimer's disease. Human Molecular Genetics. 2018;**27**(14):2502-2516. DOI:

**References**

mcn.2015.03.013

ddw413

cell.2010.05.008

10.1093/hmg/ddy154

[5] Scharl M, Wojtal KA,

DOI: 10.1002/ibd.21891

Becker HM, Fischbeck A, Frei P, Arikkat J, et al. Protein tyrosine phosphatase nonreceptor type 2 regulates Autophagosome formation in human intestinal cells. Inflammatory Bowel Diseases. 2012;**18**(7):1287-1302.

[6] Vega-Rubín-de-Celis S, Zou Z, Fernández ÁF, Ci B, Kim M, Xiao G, et al. Increased autophagy blocks HER2-

mediated breast tumorigenesis.

10.1073/pnas.1717800115

Proceedings of the National Academy of Sciences. 2018;**115**(16):4176-4181. DOI:

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.89339*

## **References**

*The Eye and Foot in Diabetes*

**4. Conclusion**

stages of the disease.

**Author details**

Jacqueline M. Lopes de Faria\* and Marcella Neves Dátilo

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and ameliorated the degradation of p62/SQSTM1. Rapamycin showed to improve proteolytic activity of the lysosome, reducing the release of VEGF. Corresponding findings were also demonstrated in models using diabetic animals. In the retinas of diabetic rats, there was a significant increase in p62/SQSTM1 accumulation, particularly in cells located in the inner nuclear layer [29]. Lysosomal impairment and autophagic flux dysfunction are early indicators of the pathogenesis of DR.

Diabetic retinopathy is a neurodegenerative disease presenting vascular changes in its late stages. Multiple factors are associated with the development and progression of DR. Recently, better understanding at cellular and molecular levels of its process has been identified through the pathways and intracellular signaling involved in cells exposed to diabetic conditions. This has allowed identification of new therapeutic approaches. Recent concepts of this disease have been analyzed here, with special focus on the process of autophagy using experimental models in different retinal cells targeted by hyperglycemia in the developmental

**20**

[1] Schapira AHV. Glucocerebrosidase and parkinson disease: Recent advances. Molecular and Cellular Neurosciences. 2015;**66**:37-42. DOI: 10.1016/j. mcn.2015.03.013

[2] Kim MJ, Deng H-X, Wong YC, Siddique T, Krainc D. The Parkinson's disease-linked protein TMEM230 is required for Rab8a-mediated secretory vesicle trafficking and retromer trafficking. Human Molecular Genetics. 2017;**26**(4):729-741. DOI: 10.1093/hmg/ ddw413

[3] Lee J-H, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, et al. Lysosomal proteolysis and autophagy require Presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010;**141**(7):1146-1158. DOI: 10.1016/j. cell.2010.05.008

[4] Reddy PH, Yin X, Manczak M, Kumar S, Pradeepkiran JA, Vijayan M, et al. Mutant APP and amyloid betainduced defective autophagy, mitophagy, mitochondrial structural and functional changes and synaptic damage in hippocampal neurons from Alzheimer's disease. Human Molecular Genetics. 2018;**27**(14):2502-2516. DOI: 10.1093/hmg/ddy154

[5] Scharl M, Wojtal KA, Becker HM, Fischbeck A, Frei P, Arikkat J, et al. Protein tyrosine phosphatase nonreceptor type 2 regulates Autophagosome formation in human intestinal cells. Inflammatory Bowel Diseases. 2012;**18**(7):1287-1302. DOI: 10.1002/ibd.21891

[6] Vega-Rubín-de-Celis S, Zou Z, Fernández ÁF, Ci B, Kim M, Xiao G, et al. Increased autophagy blocks HER2 mediated breast tumorigenesis. Proceedings of the National Academy of Sciences. 2018;**115**(16):4176-4181. DOI: 10.1073/pnas.1717800115

[7] Sumpter R, Sirasanagandla S, Fernández ÁF, Wei Y, Dong X, Franco L, et al. Fanconi anemia proteins function in Mitophagy and immunity. Cell. 2016;**165**(4):867-881. DOI: 10.1016/j. cell.2016.04.006

[8] Lassen KG, McKenzie CI, Mari M, Murano T, Begun J, Baxt LA, et al. Genetic coding variant in GPR65 alters Lysosomal pH and links Lysosomal dysfunction with colitis risk. Immunity. 2016;**44**(6):1392-1405. DOI: 10.1016/j. immuni.2016.05.007

[9] Fernández ÁF, Sebti S, Wei Y, Zou Z, Shi M, McMillan KL, et al. Disruption of the beclin 1–BCL2 autophagy regulatory complex promotes longevity in mice. Nature. 2018;**558**(7708):136-140. DOI: 10.1038/s41586-018-0162-7

[10] Melendez A. Autophagy genes are essential for Dauer development and life-span extension in C. elegans. Science. 2003;**301**(5638):1387-1391. DOI: 10.1038/s41586-018-0162-7

[11] Hars ES, Qi H, Jin SV, Cai L, Hu C, Liu LF. Autophagy Regulates Ageing in C. elegans. Autophagy. 2007;**3**(2):93-95. DOI: 10.4161/auto.3636

[12] Hansen M, Chandra A, Mitic LL, Onken B, Driscoll M, Kenyon CA. Role for autophagy in the extension of lifespan by dietary restriction in C. elegans. PLoS Genetics. 2008;**4**(2):e24. DOI: 10.1371/journal.pgen.0040024.

[13] Juhasz G, Erdi B, Sass M, Neufeld TP. Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in drosophila. Genes & Development. 2007;**21**(23):3061-3066. DOI: 10.1101/ gad.1600707

[14] Feng Y, Yao Z, Klionsky DJ. How to control self-digestion: Transcriptional, post-transcriptional, and posttranslational regulation of autophagy. Trends in Cell Biology. 2015;**25**(6):354- 363. DOI: 10.1016/j.tcb.2015.02.002

[15] Sardiello M, Palmieri M, di Ronza A, Medina DL, Valenza M, Gennarino VA, et al. A gene network regulating Lysosomal biogenesis and function. Science. 2009;**325**(5939):473- 477. DOI: 10.1126/science.1174447

[16] Lipinski MM, Zheng B, Lu T, Yan Z, Py BF, Ng A, et al. Genomewide analysis reveals mechanisms modulating autophagy in normal brain aging and in Alzheimer's disease. Proceedings of the National Academy of Sciences. 2010;**107**(32):14164-14169. DOI: 10.1016/j.cellsig.2013.06.013

[17] Chen Y, Sawada O, Kohno H, Le YZ, Subauste C, Maeda T, et al. Autophagy protects the retina from light-induced degeneration. The Journal of Biological Chemistry. 2013;**288**(11):7506-7518. DOI: 10.1074/jbc.M112.439935

[18] Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, et al. FoxO3 coordinately activates protein degradation by the Autophagic/ Lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metabolism. 2007;**6**(6):472-483. DOI: 10.1016/j.cmet.2007.11.004

[19] Sarkis GJ, Ashcom JD, Hawdon JM, Jacobson LA. Decline in protease activities with age in the nematode caenorhabditis elegans. Mechanisms of Ageing and Development. 1988;**45**(3):191-201. DOI: 10.1016/0047-6374(88)90001-2

[20] Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;**432**(7020):1032-1036. DOI: 10.1038/nature03029

[21] Kapahi P, Kaeberlein M, Hansen M. Dietary restriction and lifespan: Lessons from invertebrate models. Ageing Research Reviews. 2017;**39**:3-14. DOI: 10.1016/j.arr.2016.12.005

[22] Mattison JA, Colman RJ, Beasley TM, Allison DB, Kemnitz JW, Roth GS, et al. Caloric restriction improves health and survival of rhesus monkeys. Nature Communications. 2017;**8**(1):14063. DOI: 10.1038/ ncomms14063

[23] Boya P, Reggiori F, Codogno P. Emerging regulation and functions of autophagy. Nature Cell Biology. 2013;**15**(7):713-720. DOI: 10.1038/ ncb2788

[24] Dou Z, Xu C, Donahue G, Shimi T, Pan JA, Zhu J, et al. Autophagy mediates degradation of nuclear lamina. Nature. 2015;**527**(7576):105-109. DOI: 10.1038/ nature15548

[25] Ktistakis NT, Tooze SA. Digesting the expanding mechanisms of autophagy. Trends in Cell Biology. 2016;**26**(8):624-635. DOI: 10.1016/j. tcb.2016.03.006

[26] Bento CF, Renna M, Ghislat G, Puri C, Ashkenazi A, Vicinanza M, et al. Mammalian autophagy: How does it work? Annual Review of Biochemistry. 2016;**85**(1):685-713. DOI: 10.1146/ annurev-biochem-060815-014556

[27] Choi AMK, Ryter SW, Levine B. Mechanisms of disease: Autophagy in human health and disease. The New England Journal of Medicine. 2013;**368**(7):651-662. DOI: 10.1056/ NEJMra1205406

[28] Suzuki H, Osawa T, Fujioka Y, Noda NN. Structural biology of the core autophagy machinery. Current Opinion in Structural Biology. 2017;**43**:10-17. DOI: 10.1016/j.sbi.2016.09.010

**23**

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy*

[36] Lavandero S, Chiong M,

64. DOI: 10.1172/JCI73943

DOI: 10.1016/j.tcb.2015.11.001

diacare.27.5.1047

dc11-1909

Rothermel BA, Hill JA. Autophagy in cardiovascular biology. The Journal of Clinical Investigation. 2015;**125**(1):55-

[37] Galluzzi L, Bravo-San Pedro JM, Kroemer G. Autophagy mediates tumor suppression via cellular senescence. Trends in Cell Biology. 2016;**26**(1):1-3.

[38] Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: Estimates for the year 2000 and projections for 2030. Diabetes Care. 2004;**27**(5):1047-1053. DOI: 10.2337/

[39] Yau JWY, Rogers SL, Kawasaki R, Lamoureux EL, Kowalski JW, Bek T, et al. Global prevalence and major risk factors of diabetic retinopathy. Diabetes Care. 2012;**35**(3):556-564. DOI: 10.2337/

[40] Kempen JH, O'Colmain BJ, Leske MC, Haffner SM, Klein R, Moss SE, et al. The prevalence of diabetic retinopathy among adults in the United States. Archives of Ophthalmology. 2004;**122**(4):552-563. DOI: 10.1001/archopht.122.4.552

[41] Berrone E, Beltramo E, Solimine C, Ape AU, Porta M. Regulation of intracellular glucose and polyol pathway by thiamine and benfotiamine in vascular cells cultured in high glucose. The Journal of Biological Chemistry. 2006;**281**(14):9307-9313. DOI: 10.1074/

[42] Geraldes P, King GL. Activation of protein kinase C isoforms and its impact on diabetic complications. Circulation Research. 2010;**106**(8):1319-1331. DOI: 10.1161/CIRCRESAHA.110.217117

[43] Lee TS, Saltsman KA, Ohashi H, King GL. Activation of protein kinase C

jbc.M600418200

*DOI: http://dx.doi.org/10.5772/intechopen.89339*

[29] Lopes de Faria JM, Duarte DA, Montemurro C, Papadimitriou A, Consonni SR, Lopes de Faria JB. Defective autophagy in diabetic retinopathy. Investigative

Ophthalmology & Visual Science. 2016;**57**(10):4356. DOI: 10.1167/

[30] Arias E, Cuervo AM. Chaperonemediated autophagy in protein quality control. Current Opinion in Cell Biology. 2011;**23**(2):184-189. DOI:

10.1016/j.ceb.2010.10.009

[31] Orenstein SJ, Cuervo AM. Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance. Seminars in Cell & Developmental Biology. 2010;**21**(7):719-726. DOI: 10.1074/jbc.

[32] Sahu R, Kaushik S, Clement CC, Cannizzo ES, Scharf B, Follenzi A, et al. Microautophagy of cytosolic proteins by late endosomes. Developmental Cell. 2011;**20**(1):131-139. DOI: 10.1016/j.

Chaperone-mediated autophagy and endosomal microautophagy: Jointed by a chaperone. The Journal of Biological Chemistry. 2018;**293**(15):5414-5424.

[34] Moloudizargari M, Asghari MH, Ghobadi E, Fallah M, Rasouli S, Abdollahi M. Autophagy, its mechanisms and regulation: Implications in neurodegenerative diseases. Ageing Research Reviews. 2017;**40**:64-74. DOI: 10.1016/j.

iovs.16-19197

R117.818237

devcel.2010.12.003

arr.2017.09.005

[33] Tekirdag K, Cuervo AM.

DOI: 10.1074/jbc.R117.818237

[35] Menzies FM, Fleming A,

Caricasole A, Bento CF, Andrews SP, Ashkenazi A, et al. Autophagy and Neurodegeneration: Pathogenic mechanisms and therapeutic

opportunities. Neuron. 2017;**93**(5):1015- 1034. DOI: 10.1016/j.neuron.2017.01.022

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.89339*

[29] Lopes de Faria JM, Duarte DA, Montemurro C, Papadimitriou A, Consonni SR, Lopes de Faria JB. Defective autophagy in diabetic retinopathy. Investigative Ophthalmology & Visual Science. 2016;**57**(10):4356. DOI: 10.1167/ iovs.16-19197

*The Eye and Foot in Diabetes*

post-transcriptional, and posttranslational regulation of autophagy. Trends in Cell Biology. 2015;**25**(6):354- 363. DOI: 10.1016/j.tcb.2015.02.002

[21] Kapahi P, Kaeberlein M, Hansen M. Dietary restriction and lifespan: Lessons from invertebrate models. Ageing Research Reviews. 2017;**39**:3-14. DOI:

Beasley TM, Allison DB, Kemnitz JW, Roth GS, et al. Caloric restriction improves health and survival of rhesus monkeys. Nature Communications. 2017;**8**(1):14063. DOI: 10.1038/

[23] Boya P, Reggiori F, Codogno P. Emerging regulation and functions of autophagy. Nature Cell Biology. 2013;**15**(7):713-720. DOI: 10.1038/

[24] Dou Z, Xu C, Donahue G, Shimi T, Pan JA, Zhu J, et al. Autophagy mediates degradation of nuclear lamina. Nature. 2015;**527**(7576):105-109. DOI: 10.1038/

[25] Ktistakis NT, Tooze SA. Digesting

[26] Bento CF, Renna M, Ghislat G, Puri C, Ashkenazi A, Vicinanza M, et al. Mammalian autophagy: How does it work? Annual Review of Biochemistry. 2016;**85**(1):685-713. DOI: 10.1146/ annurev-biochem-060815-014556

[27] Choi AMK, Ryter SW, Levine B. Mechanisms of disease: Autophagy in human health and disease. The New England Journal of Medicine. 2013;**368**(7):651-662. DOI: 10.1056/

[28] Suzuki H, Osawa T, Fujioka Y, Noda NN. Structural biology of the core autophagy machinery. Current Opinion in Structural Biology. 2017;**43**:10-17. DOI: 10.1016/j.sbi.2016.09.010

the expanding mechanisms of autophagy. Trends in Cell Biology. 2016;**26**(8):624-635. DOI: 10.1016/j.

10.1016/j.arr.2016.12.005

ncomms14063

ncb2788

nature15548

tcb.2016.03.006

NEJMra1205406

[22] Mattison JA, Colman RJ,

[15] Sardiello M, Palmieri M, di Ronza A, Medina DL, Valenza M, Gennarino VA, et al. A gene network regulating Lysosomal biogenesis and function. Science. 2009;**325**(5939):473- 477. DOI: 10.1126/science.1174447

[16] Lipinski MM, Zheng B, Lu T, Yan Z, Py BF, Ng A, et al. Genomewide analysis reveals mechanisms modulating autophagy in normal brain aging and in Alzheimer's disease. Proceedings of the National Academy of Sciences. 2010;**107**(32):14164-14169. DOI: 10.1016/j.cellsig.2013.06.013

[17] Chen Y, Sawada O, Kohno H, Le YZ, Subauste C, Maeda T, et al. Autophagy protects the retina from light-induced degeneration. The Journal of Biological Chemistry. 2013;**288**(11):7506-7518. DOI: 10.1074/jbc.M112.439935

[18] Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, et al. FoxO3 coordinately activates protein degradation by the Autophagic/ Lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metabolism. 2007;**6**(6):472-483. DOI:

10.1016/j.cmet.2007.11.004

of Ageing and Development. 1988;**45**(3):191-201. DOI: 10.1016/0047-6374(88)90001-2

DOI: 10.1038/nature03029

[20] Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;**432**(7020):1032-1036.

[19] Sarkis GJ, Ashcom JD, Hawdon JM, Jacobson LA. Decline in protease activities with age in the nematode caenorhabditis elegans. Mechanisms

**22**

[30] Arias E, Cuervo AM. Chaperonemediated autophagy in protein quality control. Current Opinion in Cell Biology. 2011;**23**(2):184-189. DOI: 10.1016/j.ceb.2010.10.009

[31] Orenstein SJ, Cuervo AM. Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance. Seminars in Cell & Developmental Biology. 2010;**21**(7):719-726. DOI: 10.1074/jbc. R117.818237

[32] Sahu R, Kaushik S, Clement CC, Cannizzo ES, Scharf B, Follenzi A, et al. Microautophagy of cytosolic proteins by late endosomes. Developmental Cell. 2011;**20**(1):131-139. DOI: 10.1016/j. devcel.2010.12.003

[33] Tekirdag K, Cuervo AM. Chaperone-mediated autophagy and endosomal microautophagy: Jointed by a chaperone. The Journal of Biological Chemistry. 2018;**293**(15):5414-5424. DOI: 10.1074/jbc.R117.818237

[34] Moloudizargari M, Asghari MH, Ghobadi E, Fallah M, Rasouli S, Abdollahi M. Autophagy, its mechanisms and regulation: Implications in neurodegenerative diseases. Ageing Research Reviews. 2017;**40**:64-74. DOI: 10.1016/j. arr.2017.09.005

[35] Menzies FM, Fleming A, Caricasole A, Bento CF, Andrews SP, Ashkenazi A, et al. Autophagy and Neurodegeneration: Pathogenic mechanisms and therapeutic opportunities. Neuron. 2017;**93**(5):1015- 1034. DOI: 10.1016/j.neuron.2017.01.022

[36] Lavandero S, Chiong M, Rothermel BA, Hill JA. Autophagy in cardiovascular biology. The Journal of Clinical Investigation. 2015;**125**(1):55- 64. DOI: 10.1172/JCI73943

[37] Galluzzi L, Bravo-San Pedro JM, Kroemer G. Autophagy mediates tumor suppression via cellular senescence. Trends in Cell Biology. 2016;**26**(1):1-3. DOI: 10.1016/j.tcb.2015.11.001

[38] Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: Estimates for the year 2000 and projections for 2030. Diabetes Care. 2004;**27**(5):1047-1053. DOI: 10.2337/ diacare.27.5.1047

[39] Yau JWY, Rogers SL, Kawasaki R, Lamoureux EL, Kowalski JW, Bek T, et al. Global prevalence and major risk factors of diabetic retinopathy. Diabetes Care. 2012;**35**(3):556-564. DOI: 10.2337/ dc11-1909

[40] Kempen JH, O'Colmain BJ, Leske MC, Haffner SM, Klein R, Moss SE, et al. The prevalence of diabetic retinopathy among adults in the United States. Archives of Ophthalmology. 2004;**122**(4):552-563. DOI: 10.1001/archopht.122.4.552

[41] Berrone E, Beltramo E, Solimine C, Ape AU, Porta M. Regulation of intracellular glucose and polyol pathway by thiamine and benfotiamine in vascular cells cultured in high glucose. The Journal of Biological Chemistry. 2006;**281**(14):9307-9313. DOI: 10.1074/ jbc.M600418200

[42] Geraldes P, King GL. Activation of protein kinase C isoforms and its impact on diabetic complications. Circulation Research. 2010;**106**(8):1319-1331. DOI: 10.1161/CIRCRESAHA.110.217117

[43] Lee TS, Saltsman KA, Ohashi H, King GL. Activation of protein kinase C by elevation of glucose concentration: Proposal for a mechanism in the development of diabetic vascular complications. Proceedings of the National Academy of Sciences. 2006;**86**(13):5141-5145. DOI: 10.1073/ pnas.86.13.5141

[44] Wu M, Yiang G, Lai T, Li C. The oxidative stress and mitochondrial dysfunction during the pathogenesis of diabetic retinopathy. Oxidative Medicine and Cellular Longevity. 2018;**2018**:1-12. DOI: 10.1155/2018/3420187

[45] Rosales MAB, Silva KC, Duarte DA, Rossato FA, Lopes de Faria JB, Lopes de Faria JM. Endocytosis of tight junctions Caveolin Nitrosylation dependent is improved by cocoa via opioid receptor on RPE cells in diabetic conditions. Investigative Ophthalmology & Visual Science. 2014;**55**(9):6090. DOI: 10.1167/ iovs.14-14234

[46] Brownlee M. The pathobiology of diabetic complications: A unifying mechanism. Diabetes. 2005;**54**(6):1615- 1625. DOI: 10.3390/nu8090585

[47] Li J, Schmidt AM. Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products. The Journal of Biological Chemistry. 1997;**272**(26):16498-16506. DOI: 10.1074/jbc.272.26.16498

[48] Rajagopal R, Bligard GW, Zhang S, Yin L, Lukasiewicz P, Semenkovich CF. Functional deficits precede structural lesions in mice with high-fat diet-induced diabetic retinopathy. Diabetes. 2016;**65**:1-35. DOI: 10.2337/db15-1255

[49] Shelton MD, Kern TS, Mieyal JJ. Glutaredoxin regulates nuclear factor κ-B and intercellular adhesion molecule in Müller cells: Model of diabetic retinopathy. The Journal of Biological

Chemistry. 2007;**282**(17):12467-12474. DOI: 10.1074/jbc.M610863200

[50] Duarte DA, Papadimitriou A, Gilbert RE, Thai K, Zhang Y, Rosales MAB, et al. Conditioned medium from early-outgrowth bone marrow cells is retinal protective in experimental model of diabetes. PLoS One. 2016;**11**(2):e0147978. DOI: 10.1371/ journal.pone.0147978

[51] Dátilo MN, Sant'Ana MR, Formigari GP, Rodrigues PB, de Moura LP, da Silva ASR, et al. Omega-3 from flaxseed oil protects obese mice against diabetic retinopathy through GPR120 receptor. Scientific Reports. 2018;**8**(1):14318. DOI: 10.1038/ s41598-018-32553-5

[52] Genuth S, Sun W, Cleary P, Gao X, Sell DR, Lachin J, et al. Skin advanced glycation end products glucosepane and methylglyoxal hydroimidazolone are independently associated with long-term microvascular complication progression of type 1 diabetes. Diabetes. 2015;**64**(1):266-278. DOI: 10.1007/ s00726-010-0773-2

[53] Treins C, Giorgetti-Peraldi S, Murdaca J, Van Obberghen E. Regulation of vascular endothelial growth factor expression by advanced Glycation end products. The Journal of Biological Chemistry. 2001;**276**(47):43836-43841. DOI: 10.1074/jbc.M106534200

[54] Hyvarärinen L, Laurinen P, Rovamo J. Contrast sensitivity in evaluation of visual impairment due to diabetes. Acta Ophthalmologica. 2009;**61**(1):94-101. DOI: 10.1111/j.1755- 3768.1983.tb01399.x

[55] Sokol S, Moskowitz A, Skarf B, Evans R, Molitch M, Senior B. Contrast sensitivity in diabetics with and without background retinopathy. Archives of Ophthalmology. 1985;**103**(1):51-54. DOI: 10.1001/archopht.1985.01050010055018

**25**

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy*

[63] Rodríguez-Muela N, Germain F, Mariño G, Fitze PS, Boya P. Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice. Cell Death and Differentiation. 2012;**19**(1):162-169. DOI: 10.1038/

[64] Rodríguez-Muela N, Boya P. Axonal damage, autophagy and neuronal survival. Autophagy. 2012;**8**(2):286-288.

DOI: 10.4161/auto.8.2.18982

[65] Kurz T, Karlsson M, Brunk UT, Nilsson SE, Frennesson C. ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron. Autophagy. 2009;**5**(4):494-501. DOI: 10.4161/

[66] Cunha-Vaz J, Bernardes R,

Journal of Ophthalmology.

Lobo C. Blood-retinal barrier. European

[67] Di Rosa M, Distefano G, Gagliano C, Rusciano D, Malaguarnera L. Autophagy

[68] Rübsam A, Parikh S, Fort P. Role of inflammation in diabetic retinopathy. International Journal of Molecular Sciences. 2018;**19**(4):942. DOI: 10.3390/

[69] Díaz-Coránguez M, Ramos C, Antonetti DA. The inner blood-retinal barrier: Cellular basis and development. Vision Research. 2017;**139**:123-137. DOI:

10.1016/j.visres.2017.05.009

S0039-6257(02)00387-9

[70] Gardner TW, Antonetti DA, Barber AJ, LaNoue KF, Levison SW. Diabetic retinopathy: More than meets the eye. Survey of Ophthalmology. 2002;**47**:S253-S262. DOI: 10.1016/

2011;**21**(Suppl 6):3-9. DOI: 10.5301/

in diabetic retinopathy. Current Neuropharmacology. 2016;**14**(8):810- 825. DOI: 10.2174/1570159X1466616032

cdd.2011.88

auto.5.4.7961

EJO.2010.6049

1122900

ijms19040942

*DOI: http://dx.doi.org/10.5772/intechopen.89339*

[57] Bresnick GH. Electroretinographic

oscillatory potentials predict progression of diabetic retinopathy. Archives of Ophthalmology. 1984;**102**(9):1307. DOI: 10.1167/

[58] Lopes de Faria J, Katsumi O, Cagliero E, Nathan D, Hirose T. Neurovisual abnormalities preceding the retinopathy in patients with longterm type 1 diabetes mellitus. Graefe's Archive for Clinical and Experimental Ophthalmology. 2001;**239**(9):643-648.

DOI: 10.1007/s004170100268

Neurodegeneration in the diabetic eye: New insights and therapeutic perspectives. Trends in Endocrinology and Metabolism. 2014;**25**(1):23-33. DOI:

[59] Simó R, Hernández C.

10.1016/j.tem.2013.09.005

jnutbio.2014.09.003

1977;**16**(9):815-840

DOI: 10.1038/nrdp.2016.12

[60] Duarte DA, Rosales MAB, Papadimitriou A, Silva KC,

Amancio VHO, Mendonça JN, et al. Polyphenol-enriched cocoa protects the diabetic retina from glial reaction through the sirtuin pathway. The Journal of Nutritional Biochemistry. 2015;**26**(1):64-74. DOI: 10.1016/j.

[61] Li PSH, Wong TH, Tang WWT, Lai JSM. Diabetic retinopathy. Hong Kong Practice. 2004;**26**(8):346-353.

[62] Remé CE, Young RW. The effects of hibernation on cone visual cells in the ground squirrel. Investigative Ophthalmology and Visual Science.

[56] Hardy KJ, Lipton J, Scase MO, Foster DH, Scarpello JHB. Detection of colour vision abnormalities in uncomplicated type 1 diabetic patients with angiographically normal retinas. The British Journal of Ophthalmology. 1992;**76**(8):461-464. DOI: 10.1136/

bjo.76.8.461

iovs.04-1409

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.89339*

[56] Hardy KJ, Lipton J, Scase MO, Foster DH, Scarpello JHB. Detection of colour vision abnormalities in uncomplicated type 1 diabetic patients with angiographically normal retinas. The British Journal of Ophthalmology. 1992;**76**(8):461-464. DOI: 10.1136/ bjo.76.8.461

*The Eye and Foot in Diabetes*

pnas.86.13.5141

of diabetic retinopathy.

10.1155/2018/3420187

iovs.14-14234

by elevation of glucose concentration: Proposal for a mechanism in the development of diabetic vascular complications. Proceedings of the National Academy of Sciences. 2006;**86**(13):5141-5145. DOI: 10.1073/

Chemistry. 2007;**282**(17):12467-12474.

Rosales MAB, et al. Conditioned medium from early-outgrowth bone marrow cells is retinal protective in experimental

Moura LP, da Silva ASR, et al. Omega-3 from flaxseed oil protects obese mice against diabetic retinopathy through GPR120 receptor. Scientific Reports. 2018;**8**(1):14318. DOI: 10.1038/

[52] Genuth S, Sun W, Cleary P, Gao X, Sell DR, Lachin J, et al. Skin advanced glycation end products glucosepane and methylglyoxal hydroimidazolone are independently associated with long-term microvascular complication progression of type 1 diabetes. Diabetes. 2015;**64**(1):266-278. DOI: 10.1007/

DOI: 10.1074/jbc.M610863200

model of diabetes. PLoS One. 2016;**11**(2):e0147978. DOI: 10.1371/

[51] Dátilo MN, Sant'Ana MR, Formigari GP, Rodrigues PB, de

journal.pone.0147978

s41598-018-32553-5

s00726-010-0773-2

3768.1983.tb01399.x

[53] Treins C, Giorgetti-Peraldi S,

DOI: 10.1074/jbc.M106534200

[54] Hyvarärinen L, Laurinen P, Rovamo J. Contrast sensitivity in evaluation of visual impairment due to diabetes. Acta Ophthalmologica. 2009;**61**(1):94-101. DOI: 10.1111/j.1755-

[55] Sokol S, Moskowitz A, Skarf B, Evans R, Molitch M, Senior B. Contrast sensitivity in diabetics with and without background retinopathy. Archives of Ophthalmology. 1985;**103**(1):51-54. DOI: 10.1001/archopht.1985.01050010055018

Murdaca J, Van Obberghen E. Regulation of vascular endothelial growth factor expression by advanced Glycation end products. The Journal of Biological Chemistry. 2001;**276**(47):43836-43841.

[50] Duarte DA, Papadimitriou A, Gilbert RE, Thai K, Zhang Y,

[44] Wu M, Yiang G, Lai T, Li C. The oxidative stress and mitochondrial dysfunction during the pathogenesis

Oxidative Medicine and Cellular Longevity. 2018;**2018**:1-12. DOI:

[45] Rosales MAB, Silva KC, Duarte DA, Rossato FA, Lopes de Faria JB, Lopes de Faria JM. Endocytosis of tight junctions Caveolin Nitrosylation dependent is improved by cocoa via opioid receptor on RPE cells in diabetic conditions. Investigative Ophthalmology & Visual Science. 2014;**55**(9):6090. DOI: 10.1167/

[46] Brownlee M. The pathobiology of diabetic complications: A unifying mechanism. Diabetes. 2005;**54**(6):1615-

[47] Li J, Schmidt AM. Characterization

1625. DOI: 10.3390/nu8090585

and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products. The Journal of Biological Chemistry. 1997;**272**(26):16498-16506. DOI:

10.1074/jbc.272.26.16498

DOI: 10.2337/db15-1255

[49] Shelton MD, Kern TS, Mieyal JJ. Glutaredoxin regulates nuclear factor κ-B and intercellular adhesion molecule in Müller cells: Model of diabetic retinopathy. The Journal of Biological

[48] Rajagopal R, Bligard GW, Zhang S, Yin L, Lukasiewicz P, Semenkovich CF. Functional deficits precede structural lesions in mice with high-fat diet-induced diabetic retinopathy. Diabetes. 2016;**65**:1-35.

**24**

[57] Bresnick GH. Electroretinographic oscillatory potentials predict progression of diabetic retinopathy. Archives of Ophthalmology. 1984;**102**(9):1307. DOI: 10.1167/ iovs.04-1409

[58] Lopes de Faria J, Katsumi O, Cagliero E, Nathan D, Hirose T. Neurovisual abnormalities preceding the retinopathy in patients with longterm type 1 diabetes mellitus. Graefe's Archive for Clinical and Experimental Ophthalmology. 2001;**239**(9):643-648. DOI: 10.1007/s004170100268

[59] Simó R, Hernández C. Neurodegeneration in the diabetic eye: New insights and therapeutic perspectives. Trends in Endocrinology and Metabolism. 2014;**25**(1):23-33. DOI: 10.1016/j.tem.2013.09.005

[60] Duarte DA, Rosales MAB, Papadimitriou A, Silva KC, Amancio VHO, Mendonça JN, et al. Polyphenol-enriched cocoa protects the diabetic retina from glial reaction through the sirtuin pathway. The Journal of Nutritional Biochemistry. 2015;**26**(1):64-74. DOI: 10.1016/j. jnutbio.2014.09.003

[61] Li PSH, Wong TH, Tang WWT, Lai JSM. Diabetic retinopathy. Hong Kong Practice. 2004;**26**(8):346-353. DOI: 10.1038/nrdp.2016.12

[62] Remé CE, Young RW. The effects of hibernation on cone visual cells in the ground squirrel. Investigative Ophthalmology and Visual Science. 1977;**16**(9):815-840

[63] Rodríguez-Muela N, Germain F, Mariño G, Fitze PS, Boya P. Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice. Cell Death and Differentiation. 2012;**19**(1):162-169. DOI: 10.1038/ cdd.2011.88

[64] Rodríguez-Muela N, Boya P. Axonal damage, autophagy and neuronal survival. Autophagy. 2012;**8**(2):286-288. DOI: 10.4161/auto.8.2.18982

[65] Kurz T, Karlsson M, Brunk UT, Nilsson SE, Frennesson C. ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron. Autophagy. 2009;**5**(4):494-501. DOI: 10.4161/ auto.5.4.7961

[66] Cunha-Vaz J, Bernardes R, Lobo C. Blood-retinal barrier. European Journal of Ophthalmology. 2011;**21**(Suppl 6):3-9. DOI: 10.5301/ EJO.2010.6049

[67] Di Rosa M, Distefano G, Gagliano C, Rusciano D, Malaguarnera L. Autophagy in diabetic retinopathy. Current Neuropharmacology. 2016;**14**(8):810- 825. DOI: 10.2174/1570159X1466616032 1122900

[68] Rübsam A, Parikh S, Fort P. Role of inflammation in diabetic retinopathy. International Journal of Molecular Sciences. 2018;**19**(4):942. DOI: 10.3390/ ijms19040942

[69] Díaz-Coránguez M, Ramos C, Antonetti DA. The inner blood-retinal barrier: Cellular basis and development. Vision Research. 2017;**139**:123-137. DOI: 10.1016/j.visres.2017.05.009

[70] Gardner TW, Antonetti DA, Barber AJ, LaNoue KF, Levison SW. Diabetic retinopathy: More than meets the eye. Survey of Ophthalmology. 2002;**47**:S253-S262. DOI: 10.1016/ S0039-6257(02)00387-9

[71] Huang C, Lu H, Xu J, Yu H, Wang X, Zhang X. Protective roles of autophagy in retinal pigment epithelium under high glucose condition via regulating PINK1/ Parkin pathway and BNIP3L. Biological Research. 2018;**51**(1):22. DOI: 10.1186/ s40659-018-0169-4

[72] Daruich A, Matet A, Moulin A, Kowalczuk L, Nicolas M, Sellam A, et al. Mechanisms of macular edema: Beyond the surface. Progress in Retinal and Eye Research. 2018;**63**:20-68. DOI: 10.1016/j. preteyeres.2017.10.006

[73] Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell and Tissue Research. 2003;**314**(1):15-23. Available from: http://link.springer.com/10.1007/ s00441-003-0745-x

[74] Park DY, Lee J, Kim J, Kim K, Hong S, Han S, et al. Plastic roles of pericytes in the blood–retinal barrier. Nature Communications. 2017;**8**(1):15296. DOI: 10.1007/ s00441-003-0745-x

[75] Mao XB, You ZP, Wu C, Huang J. Potential suppression of the high glucose and insulin-induced retinal neovascularization by Sirtuin 3 in the human retinal endothelial cells. Biochemical and Biophysical Research Communications. 2017;**482**(2):341-345. DOI: 10.1016/j.bbrc.2016.11.065

[76] Li J, Yu S, Ying J, Shi T, Wang P. Resveratrol prevents ROS-induced apoptosis in high glucose-treated retinal capillary endothelial cells via the activation of AMPK/Sirt1/PGC-1 α pathway. Oxidative Medicine and Cellular Longevity. 2017;**2017**:1-10. DOI: 10.1155/2017/7584691

[77] Kim J, Kundu M, Viollet B, Guan K-L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature Cell Biology. 2011;**13**(2):132-141. DOI: 10.1038/ncb2152

[78] Niu C, Chen Z, Kim KT, Sun J, Xue M, Chen G, et al. Metformin alleviates hyperglycemia-induced endothelial impairment by downregulating autophagy via the hedgehog pathway. Autophagy. 2019;**15**(5):843-870. DOI: 10.1080/15548627.2019.1569913

[79] Simó R, Villarroel M, Corraliza L, Hernández C, Garcia-Ramírez M. The retinal pigment epithelium: Something more than a constituent of the blood-retinal barrier-implications for the pathogenesis of diabetic retinopathy. Journal of Biomedicine and Biotechnology. 2010;**2010**:190724. DOI: 10.1155/2010/190724

[80] Xu H-Z, Le Y-Z. Significance of outer blood–retina barrier breakdown in diabetes and ischemia. Investigative Ophthalmology & Visual Science. 2011;**52**(5):2160. DOI: 10.1167/ iovs.10-6518

[81] Xia T, Rizzolo LJ. Effects of diabetic retinopathy on the barrier functions of the retinal pigment epithelium. Vision Research. 2017;**139**:72-81. DOI: 10.1016/j.visres.2017.02.006

[82] Strauss O. The retinal pigment epithelium in visual function. Physiological Reviews. 2005;**85**(3):845- 881. DOI: 10.1152/physrev.00021.2004

[83] Amram B, Cohen-Tayar Y, David A, Ashery-Padan R. The retinal pigmented epithelium – From basic developmental biology research to translational approaches. The International Journal of Developmental Biology. 2017;**61**(3-4- 5):225-234. DOI: 10.1387/ijdb.160393ra

[84] Vinores SA, Gadegbeku C, Campochiaro PA, Green WR. Immunohistochemical localization of blood-retinal barrier breakdown in

**27**

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy*

2019;**111**:1315-1325. DOI: 10.1016/j.

[91] Reichenbach A, Bringmann A. Müller Cells in the Healthy and Diseased Retina. Müller Cells in the Healthy and Diseased Retina. New York, NY: Springer New York; 2010. DOI:

[92] Vecino E, Rodriguez FD, Ruzafa N, Pereiro X, Sharma SC. Glia–neuron interactions in the mammalian retina. Progress in Retinal and Eye Research. 2016;**51**:1-40. DOI: 10.1016/j.

10.1007/978-1-4419-1672-3

preteyeres.2015.06.003

[93] Pow DV, Crook DK. Direct

of glutamate and glutamine.

10.1016/0306-4522(95)00363-N

Goldman D. Analysis of DNA methylation reveals a partial reprogramming of the Muller glia genome during retina regeneration. Proceedings of the National Academy of Sciences. 2013;**110**(49):19814-19819.

DOI: 10.1073/pnas.1312009110

of glutathione in the ischemic rat retina. Neuroscience Letters. 1998;**246**(1):53-56. DOI: 10.1016/

[96] Long KO, Fisher SK, Fariss RN, Anderson DH. Disc shedding and autophagy in the cone-dominant ground squirrel retina. Experimental Eye Research. 1986;**43**(2):193-205. DOI:

10.1016/S0014-4835(86)80087-2

[97] Wang JS, Kefalov VJ. The conespecific visual cycle. Progress in Retinal and Eye Research. 2011;**30**(2):115-128. DOI: 10.1016/j.preteyeres.2010.11.001

S0304-3940(98)00229-8

[95] Schütte M, Werner P. Redistribution

immunocytochemical evidence for the transfer of glutamine from glial cells to neurons: Use of specific antibodies directed against thed-stereoisomers

Neuroscience. 1996;**70**(1):295-302. DOI:

[94] Powell C, Grant AR, Cornblath E,

biopha.2019.01.034

*DOI: http://dx.doi.org/10.5772/intechopen.89339*

human diabetics. The American Journal of Pathology. 1989;**134**(2):231-235

[85] Zhang W, Song J, Zhang Y, Ma Y, Yang J, He G, et al. Intermittent high glucose-induced oxidative stress modulates retinal pigmented epithelial cell autophagy and promotes cell survival via increased HMGB1. BMC Ophthalmology. 2018;**18**(1):192. DOI:

10.1016/j.biopha.2019.01.034

preteyeres.2016.08.001

[88] Shi H, Zhang Z, Wang X, Li R, Hou W, Bi W, et al. Inhibition of autophagy induces IL-1β release from ARPE-19 cells via ROS mediated NLRP3 inflammasome activation under high glucose stress. Biochemical and Biophysical Research Communications. 2015;**463**(4):1071-1076. DOI: 10.1016/j.

[89] Filomeni G, De Zio D, Cecconi F. Oxidative stress and autophagy: The clash between damage and metabolic needs. Cell Death and Differentiation. 2015;**22**(3):377-388. DOI: 10.1038/

[90] Zhang Y, Xi X, Mei Y, Zhao X, Zhou L, Ma M, et al. High-glucose induces retinal pigment epithelium mitochondrial pathways of apoptosis and inhibits mitophagy by regulating ROS/PINK1/Parkin signal pathway. Biomedicine & Pharmacotherapy.

bbrc.2015.06.060

cdd.2014.150

[86] Boya P, Esteban-Martínez L, Serrano-Puebla A, Gómez-Sintes R, Villarejo-Zori B. Autophagy in the eye: Development, degeneration, and aging. Progress in Retinal and Eye Research. 2016;**55**:206-245. DOI: 10.1016/j.

[87] Chen Q, Tang L, Xin G, Li S, Ma L, Xu Y, et al. Oxidative stress mediated by lipid metabolism contributes to high glucose-induced senescence in retinal pigment epithelium. Free Radical Biology & Medicine. 2019;**130**:48-58. DOI: 10.1016/j. freeradbiomed.2018.10.419

*Deficient Autophagy Contributes to the Development of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.89339*

human diabetics. The American Journal of Pathology. 1989;**134**(2):231-235

*The Eye and Foot in Diabetes*

s40659-018-0169-4

preteyeres.2017.10.006

s00441-003-0745-x

s00441-003-0745-x

[73] Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell and Tissue Research. 2003;**314**(1):15-23. Available from: http://link.springer.com/10.1007/

[74] Park DY, Lee J, Kim J, Kim K, Hong S, Han S, et al. Plastic roles of pericytes in the blood–retinal barrier. Nature Communications. 2017;**8**(1):15296. DOI: 10.1007/

[75] Mao XB, You ZP, Wu C, Huang J. Potential suppression of the high glucose and insulin-induced retinal neovascularization by Sirtuin 3 in the human retinal endothelial cells. Biochemical and Biophysical Research Communications. 2017;**482**(2):341-345.

DOI: 10.1016/j.bbrc.2016.11.065

10.1155/2017/7584691

[76] Li J, Yu S, Ying J, Shi T, Wang P. Resveratrol prevents ROS-induced apoptosis in high glucose-treated retinal capillary endothelial cells via the activation of AMPK/Sirt1/PGC-1 α pathway. Oxidative Medicine and Cellular Longevity. 2017;**2017**:1-10. DOI:

[77] Kim J, Kundu M, Viollet B, Guan K-L. AMPK and mTOR regulate autophagy through direct

phosphorylation of Ulk1. Nature Cell

[71] Huang C, Lu H, Xu J, Yu H, Wang X, Zhang X. Protective roles of autophagy in retinal pigment epithelium under high glucose condition via regulating PINK1/

Parkin pathway and BNIP3L. Biological Research. 2018;**51**(1):22. DOI: 10.1186/

Biology. 2011;**13**(2):132-141. DOI:

[78] Niu C, Chen Z, Kim KT, Sun J, Xue M, Chen G, et al. Metformin alleviates hyperglycemia-induced endothelial impairment by

downregulating autophagy via the hedgehog pathway. Autophagy. 2019;**15**(5):843-870. DOI: 10.1080/15548627.2019.1569913

[79] Simó R, Villarroel M, Corraliza L, Hernández C, Garcia-Ramírez M. The retinal pigment epithelium: Something

retinopathy. Journal of Biomedicine and Biotechnology. 2010;**2010**:190724. DOI:

[80] Xu H-Z, Le Y-Z. Significance of outer blood–retina barrier breakdown in diabetes and ischemia. Investigative Ophthalmology & Visual Science. 2011;**52**(5):2160. DOI: 10.1167/

[81] Xia T, Rizzolo LJ. Effects of diabetic retinopathy on the barrier functions of the retinal pigment epithelium. Vision Research. 2017;**139**:72-81. DOI:

10.1016/j.visres.2017.02.006

[82] Strauss O. The retinal pigment epithelium in visual function.

Physiological Reviews. 2005;**85**(3):845- 881. DOI: 10.1152/physrev.00021.2004

[83] Amram B, Cohen-Tayar Y, David A, Ashery-Padan R. The retinal pigmented epithelium – From basic developmental biology research to translational approaches. The International Journal of Developmental Biology. 2017;**61**(3-4- 5):225-234. DOI: 10.1387/ijdb.160393ra

[84] Vinores SA, Gadegbeku C, Campochiaro PA, Green WR.

Immunohistochemical localization of blood-retinal barrier breakdown in

more than a constituent of the blood-retinal barrier-implications for the pathogenesis of diabetic

10.1155/2010/190724

iovs.10-6518

10.1038/ncb2152

[72] Daruich A, Matet A, Moulin A, Kowalczuk L, Nicolas M, Sellam A, et al. Mechanisms of macular edema: Beyond the surface. Progress in Retinal and Eye Research. 2018;**63**:20-68. DOI: 10.1016/j.

**26**

[85] Zhang W, Song J, Zhang Y, Ma Y, Yang J, He G, et al. Intermittent high glucose-induced oxidative stress modulates retinal pigmented epithelial cell autophagy and promotes cell survival via increased HMGB1. BMC Ophthalmology. 2018;**18**(1):192. DOI: 10.1016/j.biopha.2019.01.034

[86] Boya P, Esteban-Martínez L, Serrano-Puebla A, Gómez-Sintes R, Villarejo-Zori B. Autophagy in the eye: Development, degeneration, and aging. Progress in Retinal and Eye Research. 2016;**55**:206-245. DOI: 10.1016/j. preteyeres.2016.08.001

[87] Chen Q, Tang L, Xin G, Li S, Ma L, Xu Y, et al. Oxidative stress mediated by lipid metabolism contributes to high glucose-induced senescence in retinal pigment epithelium. Free Radical Biology & Medicine. 2019;**130**:48-58. DOI: 10.1016/j. freeradbiomed.2018.10.419

[88] Shi H, Zhang Z, Wang X, Li R, Hou W, Bi W, et al. Inhibition of autophagy induces IL-1β release from ARPE-19 cells via ROS mediated NLRP3 inflammasome activation under high glucose stress. Biochemical and Biophysical Research Communications. 2015;**463**(4):1071-1076. DOI: 10.1016/j. bbrc.2015.06.060

[89] Filomeni G, De Zio D, Cecconi F. Oxidative stress and autophagy: The clash between damage and metabolic needs. Cell Death and Differentiation. 2015;**22**(3):377-388. DOI: 10.1038/ cdd.2014.150

[90] Zhang Y, Xi X, Mei Y, Zhao X, Zhou L, Ma M, et al. High-glucose induces retinal pigment epithelium mitochondrial pathways of apoptosis and inhibits mitophagy by regulating ROS/PINK1/Parkin signal pathway. Biomedicine & Pharmacotherapy.

2019;**111**:1315-1325. DOI: 10.1016/j. biopha.2019.01.034

[91] Reichenbach A, Bringmann A. Müller Cells in the Healthy and Diseased Retina. Müller Cells in the Healthy and Diseased Retina. New York, NY: Springer New York; 2010. DOI: 10.1007/978-1-4419-1672-3

[92] Vecino E, Rodriguez FD, Ruzafa N, Pereiro X, Sharma SC. Glia–neuron interactions in the mammalian retina. Progress in Retinal and Eye Research. 2016;**51**:1-40. DOI: 10.1016/j. preteyeres.2015.06.003

[93] Pow DV, Crook DK. Direct immunocytochemical evidence for the transfer of glutamine from glial cells to neurons: Use of specific antibodies directed against thed-stereoisomers of glutamate and glutamine. Neuroscience. 1996;**70**(1):295-302. DOI: 10.1016/0306-4522(95)00363-N

[94] Powell C, Grant AR, Cornblath E, Goldman D. Analysis of DNA methylation reveals a partial reprogramming of the Muller glia genome during retina regeneration. Proceedings of the National Academy of Sciences. 2013;**110**(49):19814-19819. DOI: 10.1073/pnas.1312009110

[95] Schütte M, Werner P. Redistribution of glutathione in the ischemic rat retina. Neuroscience Letters. 1998;**246**(1):53-56. DOI: 10.1016/ S0304-3940(98)00229-8

[96] Long KO, Fisher SK, Fariss RN, Anderson DH. Disc shedding and autophagy in the cone-dominant ground squirrel retina. Experimental Eye Research. 1986;**43**(2):193-205. DOI: 10.1016/S0014-4835(86)80087-2

[97] Wang JS, Kefalov VJ. The conespecific visual cycle. Progress in Retinal and Eye Research. 2011;**30**(2):115-128. DOI: 10.1016/j.preteyeres.2010.11.001

#### *The Eye and Foot in Diabetes*

[98] Bai Y, Ma J, Guo J, Wang J, Zhu M, Chen Y, et al. Müller cell-derived VEGF is a significant contributor to retinal neovascularization. The Journal of Pathology. 2009;**219**(4):446-454. DOI: 10.1002/path.2611

[99] Devi TS, Lee I, Hüttemann M, Kumar A, Nantwi KD, Singh LP. TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: Implications for diabetic retinopathy. Experimental Diabetes Research. 2012;**2012**:1-19. DOI: 10.1155/2012/438238

**29**

**Chapter 3**

**Abstract**

*Ogugua N. Okonkwo*

offer even more benefit.

**1. Introduction**

treatment [5–8].

should be done on a case-by-case basis.

Diabetic Vitrectomy

Diabetic retinopathy (DR) in its advanced stage is a leading cause of blindness and visual impairment. Despite efforts at early detection of DR, disease monitoring, and medical therapy, significant proportions of people living with diabetes still progress to develop the advanced proliferative disease, which is characterized by neovascularization, actively proliferating fibrovascular membranes, and retinal traction. The surgical removal of this proliferating tissue and the treatment of the retinal ischemic drive can be very rewarding, providing significant stability of the retina and in several cases improved retinal anatomy and vision. Diabetic vitrectomy comprises a broad range of surgical techniques and maneuvers, which offer the surgeon and patient opportunity to reverse deranged vitreoretinal anatomy and improve or stabilizes vision. Advances in vitreoretinal technology have contributed greatly to more recent improved outcomes; it is expected that future advances will

**Keywords:** diabetic retinopathy, vitreous hemorrhage, proliferative diabetic retinopathy, tractional retinal detachment, macular edema, vitrectomy

Global estimates of diabetes have been on the rise [1]. Diabetic retinopathy (DR) is a leading cause of blindness among the working age group, with increasing numbers of persons being affected worldwide [2, 3]. It is a microvascular complication of diabetes which progresses to advanced disease in several cases. It is a global concern as indicated by a recent review published in Lancet [4]. The microvascular complications of diabetes result in macular leakage or exudation and vasoproliferative retinal disease, which are the hallmarks of advanced DR. Despite treatment of earlier stages of DR with medical therapy, which include intravitreal injection of anti-vascular endothelial growth factor (VEGF), intravitreal injection of steroids, and retinal laser photocoagulation, several eyes will progress to require surgical

Surgical treatment for the advanced complications of DR can range from more straightforward cases involving the removal of a non-clearing vitreous hemorrhage from an eye in which vitreous separation has already occurred, to more complicated surgical techniques such as in dealing with a combined tractional and rhegmatogenous retinal detachment (TRD/RRD) or tractional retinal detachment (TRD) involving the macula [9, 10]. The preoperative considerations, intraoperative techniques, and the postoperative outcome, including the complications of surgery, could vary considerably, depending on the risk factors and complexity of the vitreoretinal presentation associated with each case. Therefore surgical planning

## **Chapter 3** Diabetic Vitrectomy

*Ogugua N. Okonkwo*

## **Abstract**

*The Eye and Foot in Diabetes*

10.1002/path.2611

10.1155/2012/438238

[98] Bai Y, Ma J, Guo J, Wang J, Zhu M, Chen Y, et al. Müller cell-derived VEGF is a significant contributor to retinal neovascularization. The Journal of Pathology. 2009;**219**(4):446-454. DOI:

[99] Devi TS, Lee I, Hüttemann M, Kumar A, Nantwi KD, Singh LP. TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: Implications for diabetic retinopathy. Experimental Diabetes Research. 2012;**2012**:1-19. DOI:

**28**

Diabetic retinopathy (DR) in its advanced stage is a leading cause of blindness and visual impairment. Despite efforts at early detection of DR, disease monitoring, and medical therapy, significant proportions of people living with diabetes still progress to develop the advanced proliferative disease, which is characterized by neovascularization, actively proliferating fibrovascular membranes, and retinal traction. The surgical removal of this proliferating tissue and the treatment of the retinal ischemic drive can be very rewarding, providing significant stability of the retina and in several cases improved retinal anatomy and vision. Diabetic vitrectomy comprises a broad range of surgical techniques and maneuvers, which offer the surgeon and patient opportunity to reverse deranged vitreoretinal anatomy and improve or stabilizes vision. Advances in vitreoretinal technology have contributed greatly to more recent improved outcomes; it is expected that future advances will offer even more benefit.

**Keywords:** diabetic retinopathy, vitreous hemorrhage, proliferative diabetic retinopathy, tractional retinal detachment, macular edema, vitrectomy

## **1. Introduction**

Global estimates of diabetes have been on the rise [1]. Diabetic retinopathy (DR) is a leading cause of blindness among the working age group, with increasing numbers of persons being affected worldwide [2, 3]. It is a microvascular complication of diabetes which progresses to advanced disease in several cases. It is a global concern as indicated by a recent review published in Lancet [4]. The microvascular complications of diabetes result in macular leakage or exudation and vasoproliferative retinal disease, which are the hallmarks of advanced DR. Despite treatment of earlier stages of DR with medical therapy, which include intravitreal injection of anti-vascular endothelial growth factor (VEGF), intravitreal injection of steroids, and retinal laser photocoagulation, several eyes will progress to require surgical treatment [5–8].

Surgical treatment for the advanced complications of DR can range from more straightforward cases involving the removal of a non-clearing vitreous hemorrhage from an eye in which vitreous separation has already occurred, to more complicated surgical techniques such as in dealing with a combined tractional and rhegmatogenous retinal detachment (TRD/RRD) or tractional retinal detachment (TRD) involving the macula [9, 10]. The preoperative considerations, intraoperative techniques, and the postoperative outcome, including the complications of surgery, could vary considerably, depending on the risk factors and complexity of the vitreoretinal presentation associated with each case. Therefore surgical planning should be done on a case-by-case basis.

Furthermore, in recent times, there have been significant improvements in preoperative care and evaluation, and intraoperative surgical technique, including the administration of preoperative intravitreal pharmacotherapy, development of small-gauge transconjunctival instrumentation [11–13], and availability of multifunctional vitrectomy probes with high cut rates [14]. These advances have made surgical outcome more predictable and have resulted in an expansion of the indication for vitrectomy in the management of the tractional complications seen in DR. This review will focus on highlighting the indications, pathophysiology and principles of surgery, preoperative considerations, intraoperative surgical techniques, and outcome of diabetic vitrectomy in contemporary times.

## **2. Indications for surgery**

The indications for vitrectomy in advanced DR have increased over the years from the situation in the early years of diabetic vitrectomy, when surgery was used for removing non-clearing vitreous hemorrhage. The first vitrectomy performed by Machemer was for the removal of non-clearing vitreous hemorrhage in a patient living with diabetes, and suffering from proliferative diabetic retinopathy (PDR) [15]. The Diabetic Retinopathy Vitrectomy Study (DRVS) was the first randomized, large series study evaluating the outcome of early versus deferral of vitrectomy in eyes with vitreous hemorrhage secondary to advanced DR [16]. It highlighted the benefit of early vitrectomy especially in type 1 diabetics with more severe disease. The DRVS also demonstrated that the benefit of surgery was maintained over a 4-year study period. Since then, the indications for diabetic vitrectomy (DV) are now known to include non-clearing vitreous hemorrhage, TRD, combined RRD/TRD, vitreomacular traction, traction-induced diabetic macular edema (DME), rubeosis iridis, and macular distortion (including dragging of the macula), to mention a few [17–21].

**31**

glaucoma.

*Diabetic Vitrectomy*

*DOI: http://dx.doi.org/10.5772/intechopen.91360*

advanced tractional complications.

**3. Preoperative systemic considerations**

neovascular tuft.

Surgery is indicated when the traction occurs in the macula or if there is obvious progression of an extra macular TRD towards the macula. In some cases the retinal traction is significant and creates a retinal tear. This adds a rhegmatogenous component to the already existing TRD. In combined TRD/RRD, progression to

In these situations of significant traction involving the macula or threatening the macula, surgery is indicated to relieve the traction and reattach the retina or to prevent progression of TRD to the macula. However, there are several cases in which extra macular TRD remains stable after adequate panretinal laser photocoagulation (PRP) and good control of systemic parameters have been achieved. Such cases can be observed since there is no progression. In one review comparing African-Americans with Caucasians requiring diabetic vitrectomy, patients of African-American descent were found to be more likely to have TRD/RRD than Caucasians, and it was concluded that African-Americans might have a greater risk of developing this advanced complication [24]. In the light of this, African-Americans and others at increased risk could benefit from earlier vitrectomy, before the onset of vision damaging

3.Persistent retinal neovascularization despite adequate laser PRP may result in recurrent VH and requires surgical removal of the vitreous scaffolding on which such neovascularization would progress. In such cases, adequate retinal laser fails to cause a complete regression of neovascularization. The vascular tuft invades the vitreous scaffold and forms neovascular pegs. This vitreous attachment to the neovascular peg has to be removed to prevent the recurrent VH, which recurs whenever there is significant vitreous traction on the

4.Severe FVP, especially if associated with significant traction involving or threatening the macula, or if obscuring the macula, may require surgical removal. In some instances, FVP occurs in the retina periphery and may be associated with proliferation extending from sclerotomy sites. This was more common in the era of large sclerotomies using the 20-gauge vitrectomy systems. Residual postoperative peripheral vitreous and significant untreated ischemia in the peripheral retina using either endoretinal laser photocoagulation or cryotherapy predispose to the formation of this complication better known as anterior hyaloidal fibrovascular proliferation (AHFVP) [25]. This is a known complication of diabetic vitrectomy which has also been reported to occur after cataract surgery in poorly controlled diabetic patients [25, 26].

Other indications for diabetic vitrectomy include macular ectopia and rubeotic

Diabetes is a multisystem disease. The presence of DR suggests microvascular affectation, which may include an effect on the microvasculature in other organs, especially the kidney resulting in diabetic nephropathy. Advanced retinopathy requiring surgery has been found to be associated with reduced life expectancy [27]. Also patients with diabetic macular edema have been noted to have a higher

incidence of cerebrovascular accidents and myocardial infarcts [28].

involve the macula could be rapid, and early surgery is advised.

#### *Diabetic Vitrectomy DOI: http://dx.doi.org/10.5772/intechopen.91360*

*The Eye and Foot in Diabetes*

**2. Indications for surgery**

ultrasound, which will reveal the TRD.

Furthermore, in recent times, there have been significant improvements in preoperative care and evaluation, and intraoperative surgical technique, including the administration of preoperative intravitreal pharmacotherapy, development of small-gauge transconjunctival instrumentation [11–13], and availability of multifunctional vitrectomy probes with high cut rates [14]. These advances have made surgical outcome more predictable and have resulted in an expansion of the indication for vitrectomy in the management of the tractional complications seen in DR. This review will focus on highlighting the indications, pathophysiology and principles of surgery, preoperative considerations, intraoperative surgical tech-

The indications for vitrectomy in advanced DR have increased over the years from the situation in the early years of diabetic vitrectomy, when surgery was used for removing non-clearing vitreous hemorrhage. The first vitrectomy performed by Machemer was for the removal of non-clearing vitreous hemorrhage in a patient living with diabetes, and suffering from proliferative diabetic retinopathy (PDR) [15]. The Diabetic Retinopathy Vitrectomy Study (DRVS) was the first randomized, large series study evaluating the outcome of early versus deferral of vitrectomy in eyes with vitreous hemorrhage secondary to advanced DR [16]. It highlighted the benefit of early vitrectomy especially in type 1 diabetics with more severe disease. The DRVS also demonstrated that the benefit of surgery was maintained over a 4-year study period. Since then, the indications for diabetic vitrectomy (DV) are now known to include non-clearing vitreous hemorrhage, TRD, combined RRD/TRD, vitreomacular traction, traction-induced diabetic macular edema (DME), rubeosis iridis, and macular distortion (including dragging of the macula), to mention a few [17–21].

1.Vitreous hemorrhage (VH): vitrectomy for vitreous hemorrhage removal, in several studies, remains the most common indication for diabetic vitrectomy [20, 22, 23]. The scope of this will depend on the surgeon's personal experience, state of the fellow eye, previous retinal laser, recurrence of VH, and systemic control of glycemic levels. Non-clearing vitreous hemorrhage cases with complete separation of the posterior hyaloid are rather uncommon and the vitreous can be easily removed with expectation of improved vision in a majority of eyes. Vitrectomy for a case in which prior retinal laser photocoagulation has been applied also tends to progress quite well as the prior laser would have reduced the activity of the retinopathy, treated the retinal ischemia, and slowed the momentum of the disease. Moreover, in eyes with prior preoperative retinal laser, the occurrence of iatrogenic breaks within the areas of retinal laser scars prevents progression to retinal detachment. VH can at times be associated with more severe proliferative retinal disease such as a macula involving TRD. In this case both VH and TRD are indications for surgery and eventual visual outcome will be affected by the occurrence of TRD. This situation can be identified using a preoperative B scan

2.Retinal traction involving the macula can be an important indication for vitrectomy. This can occur as a result of an epiretinal membrane (ERM), TRD, TRD/ RRD and vitreomacular traction (VMT). Fibrovascular proliferation (FVP) in the sub-hyaloid space is responsible for the retinal traction, which could initially occur in an extra macular site, and then progress to involve the macular area. Also, TRD could develop primarily in the macula and have an early impact on vision.

niques, and outcome of diabetic vitrectomy in contemporary times.

**30**

Surgery is indicated when the traction occurs in the macula or if there is obvious progression of an extra macular TRD towards the macula. In some cases the retinal traction is significant and creates a retinal tear. This adds a rhegmatogenous component to the already existing TRD. In combined TRD/RRD, progression to involve the macula could be rapid, and early surgery is advised.

In these situations of significant traction involving the macula or threatening the macula, surgery is indicated to relieve the traction and reattach the retina or to prevent progression of TRD to the macula. However, there are several cases in which extra macular TRD remains stable after adequate panretinal laser photocoagulation (PRP) and good control of systemic parameters have been achieved. Such cases can be observed since there is no progression.

In one review comparing African-Americans with Caucasians requiring diabetic vitrectomy, patients of African-American descent were found to be more likely to have TRD/RRD than Caucasians, and it was concluded that African-Americans might have a greater risk of developing this advanced complication [24]. In the light of this, African-Americans and others at increased risk could benefit from earlier vitrectomy, before the onset of vision damaging advanced tractional complications.

Other indications for diabetic vitrectomy include macular ectopia and rubeotic glaucoma.

## **3. Preoperative systemic considerations**

Diabetes is a multisystem disease. The presence of DR suggests microvascular affectation, which may include an effect on the microvasculature in other organs, especially the kidney resulting in diabetic nephropathy. Advanced retinopathy requiring surgery has been found to be associated with reduced life expectancy [27]. Also patients with diabetic macular edema have been noted to have a higher incidence of cerebrovascular accidents and myocardial infarcts [28].

As the patient for diabetic vitrectomy could be ill before or after the surgery, careful review by the internist and anesthesia team is required before the decision to proceed to surgery is taken. If the patient is on routine dialysis, heparin-free dialysis may be beneficial in reducing the incidence of intraoperative and postoperative hemorrhage. Preoperative administration of intravitreal VEGF injection has become popular in recent times and has been shown to decrease the rate of intraoperative and postoperative hemorrhage; it also improves intraoperative visibility and reduces surgery time.

Importantly, an internist clearance is required before preoperative adjunctive anti-VEGF is administered to avoid a situation in which following the administration of anti-VEGF, surgery is postponed due to the patient's ill health. This may result in an overactivity of the anti-VEGF with severe contraction of the fibrous component of the fibrovascular membrane resulting in a worsening TRD (and perhaps more retinal ischemia); this is known as a "Crunch." The use of anti-VEGF will be discussed in more detail later on.

## **4. Preoperative ocular considerations**

**33**

*Diabetic Vitrectomy*

*DOI: http://dx.doi.org/10.5772/intechopen.91360*

5.Lens: the presence of significant cataract may require that a combined vitrectomy and cataract removal be performed during the same surgery. The cataract is first removed using a phacoemulsification technique, then the diabetic vitrectomy is performed. The intraocular lens (IOL) could be inserted before or at the conclusion of the vitrectomy. This combined procedure has become a popular technique in recent years. It provides a clearer view and lends itself to improved access to the retina periphery with the use of a wide-angle viewing lens. However, it can also be associated with significant complications of the anterior segment, since there could be enhanced diffusion of the growth factors including VEGF from the posterior segment to the anterior segment of the

eye, resulting in the formation of rubeosis iridis and its sequelae.

ing its presence is essential for good outcome.

**5. Relevant pathophysiology and surgical principles**

Advanced stages of DR are characterized by retina edema and ischemia, consequent to vascular hyperpermeability and vascular occlusion, respectively. The chronic hyperglycemia results in progressive damage to the retinal capillary network resulting in retinal hypoxia and release of hypoxia-inducible factor (HIF) from the affected areas of the retina. The resultant ischemic retina due to the action of HIF then releases pro angiogenesis growth factors which include basic fibroblast growth factor (FGF), insulinlike growth factor 1 (IGF 1), erythropoietin, and, the most known growth factor, VEGF [29]. Also, cytokines such as IL-6, IL-8, and MCP-1 are released. The interaction of these growth factors and cytokines stimulate angiogenesis. VEGF-mediated new blood vessels sprout out from the surrounding vessels, i.e., capillaries and venules, and invade the vitreous. Progressive vasoproliferation occurs in response to increasing levels of the growth factors; and this is associated with proliferation of fibrous tissue resulting in the characteristic fibrovascular membranes. The fibrovascular tissue proliferates and extends across the retina in the preretinal space (or sub hyaloid space). In eyes with PVD, fibrovascular membranes can only grow on the surface of the retina; therefore, retinal detachments do not tend to occur. However in eyes without a PVD (which is often the case), the posterior hyaloid acts as a scaffold that allows the fibrovascular tissue to grow, leading to traction on the retina and retinal detachments. Tractional forces within the vitreous exert effect on these rather brittle new blood vessels resulting in different degrees of hemorrhage. The resulting hemorrhage can range from a small leakage of blood on the surrounding retina to larger preretinal hemorrhage (**Figure 1a**) and to a more severe break through intragel vitreous hemorrhage.

6.A B scan ultrasound is a useful ocular investigation to have, especially in situations in which there is limited or no view of the retina as a result of vitreous hemorrhage, opacities in the vitreous, and cataract. A B scan can detect the presence or absence of posterior vitreous detachment (PVD) and provide information useful for preparing the eye for diabetic vitrectomy. For instance, some surgeons would give preoperative intravitreal anti-VEGF in eyes without a PVD and refrain from doing so in eyes in which a PVD already exists. Also a B scan can detect the presence of vitreoschisis (aka second membrane). Vitreoschisis is common in diabetic retinopathy eyes, and for this reason re-staining using multiple intravitreal triamcinolone injections is important to detect the residual vitreous layer when vitreoschisis is present. Vitreoschisis is thought to be due to vitreous hemorrhage in the gel splitting the vitreous fibers. Recogniz-

#### *Diabetic Vitrectomy DOI: http://dx.doi.org/10.5772/intechopen.91360*

*The Eye and Foot in Diabetes*

reduces surgery time.

will be discussed in more detail later on.

**4. Preoperative ocular considerations**

obscure the view of the macula.

tation such as using iris hooks.

As the patient for diabetic vitrectomy could be ill before or after the surgery, careful review by the internist and anesthesia team is required before the decision to proceed to surgery is taken. If the patient is on routine dialysis, heparin-free dialysis may be beneficial in reducing the incidence of intraoperative and postoperative hemorrhage. Preoperative administration of intravitreal VEGF injection has become popular in recent times and has been shown to decrease the rate of intraoperative and postoperative hemorrhage; it also improves intraoperative visibility and

Importantly, an internist clearance is required before preoperative adjunctive anti-VEGF is administered to avoid a situation in which following the administration of anti-VEGF, surgery is postponed due to the patient's ill health. This may result in an overactivity of the anti-VEGF with severe contraction of the fibrous component of the fibrovascular membrane resulting in a worsening TRD (and perhaps more retinal ischemia); this is known as a "Crunch." The use of anti-VEGF

1.Visual acuity: preoperative acuity has been shown to be an important factor in determining the eventual postoperative visual outcome, with eyes having better preoperative vision tending to have improved postoperative vision. Also, TRD involving the macula will have poorer preoperative vision than a "macular sparing" TRD. Therefore surgery should be performed once the macula is perceived to be threatened. Macular ischemia remains an important reason for poor preoperative and postoperative vision; this can be determined by the use of fundus fluorescein angiography (FFA) to assess for macular non-perfusion. Optical coherence angiography (OCT angiography or OCTA) can also be used and has the advantage of repeatability of the test. However, in several cases, it is not possible to perform this FFA assessment of the macular vasculature before surgery because of opacities in the medium, including VH and FVP, which

2.Intraocular pressure (IOP): this may be normal or elevated. When IOP is elevated, it is important to assess the anterior chamber angles and anterior uvea carefully, in search of rubeosis. The finding of rubeosis suggests very significant retinal ischemia and further worsens the prognosis for recovery of vision. The rise in IOP may also have damaging effects on the cornea, including cornea

3.Cornea: the clarity of this structure is required for proper access and visibility required for diabetic vitrectomy. The use of contact lens viewing systems significantly increases the incidence of cornea opacity and may require the removal of cornea epithelium during the surgery. Such scrapping off of cornea epithelium could result in postoperative cornea defects, which could take some time to heal. The aforementioned situation has been greatly reduced with the

4.Pupil: it is vital to assess for adequate pupillary dilatation prior to surgery. A poorly dilating pupil may require more than pharmacological mydriasis. In some instances pupillary synechiae may exist and will require mechanical dila-

edema, and result in decreased visibility during surgery.

more frequent use of non-contact lens viewing systems.

**32**

## **5. Relevant pathophysiology and surgical principles**

Advanced stages of DR are characterized by retina edema and ischemia, consequent to vascular hyperpermeability and vascular occlusion, respectively. The chronic hyperglycemia results in progressive damage to the retinal capillary network resulting in retinal hypoxia and release of hypoxia-inducible factor (HIF) from the affected areas of the retina. The resultant ischemic retina due to the action of HIF then releases pro angiogenesis growth factors which include basic fibroblast growth factor (FGF), insulinlike growth factor 1 (IGF 1), erythropoietin, and, the most known growth factor, VEGF [29]. Also, cytokines such as IL-6, IL-8, and MCP-1 are released. The interaction of these growth factors and cytokines stimulate angiogenesis. VEGF-mediated new blood vessels sprout out from the surrounding vessels, i.e., capillaries and venules, and invade the vitreous. Progressive vasoproliferation occurs in response to increasing levels of the growth factors; and this is associated with proliferation of fibrous tissue resulting in the characteristic fibrovascular membranes. The fibrovascular tissue proliferates and extends across the retina in the preretinal space (or sub hyaloid space). In eyes with PVD, fibrovascular membranes can only grow on the surface of the retina; therefore, retinal detachments do not tend to occur. However in eyes without a PVD (which is often the case), the posterior hyaloid acts as a scaffold that allows the fibrovascular tissue to grow, leading to traction on the retina and retinal detachments. Tractional forces within the vitreous exert effect on these rather brittle new blood vessels resulting in different degrees of hemorrhage. The resulting hemorrhage can range from a small leakage of blood on the surrounding retina to larger preretinal hemorrhage (**Figure 1a**) and to a more severe break through intragel vitreous hemorrhage.

#### **Figure 1.**

*Fundus photograph of PDR. (a) Left eye showing diffuse vitreous hemorrhage and preretinal hemorrhage in an eye diagnosed to have PDR. Notice also the absence of retinal laser marks and presence of macula hard exudates, with some significant cataract. (b) Same eye as in 1a after vitrectomy; view of the fundus is clearer and some hard exudates persist in the macular area.*

This hemorrhage can often be removed using vitrectomy technique (**Figure 1b**). The fibrous component of the fibrovascular tissue contracts, due to a myofibroblastic effect [30], and causes traction on the inner retina, resulting in a TRD that may initially involve an extra macular site, and then subsequently progresses to the macula and damages vision. In some cases the traction results in a split in the layers of the retina (retinoschisis or foveoschisis) that can be appreciated on optical coherence tomography (OCT) scan. Similarly, ERM may be present in the macular area and result in considerable macular traction, worsening already existing macular edema. Since such diabetic macular edema (DME) has a traction-induced component as its causation, surgery will be required to remove the traction if resolution of the edema is to be achieved. VEGF suppression alone is unlikely to achieve complete resolution of this sort of DME, and this needs to be recognized.

The surgical principles of diabetic vitrectomy include performing a core vitrectomy. In some cases, a posterior vitreous separation already exists preoperatively and the goal of surgery is simply vitreous hemorrhage removal. This is a rather uncommon presentation. In cases of vitreous hemorrhage removal, in addition to the hemorrhage (since the vitreous cavity acts as a reservoir of several proinflammatory and pro-angiogenic factors that result in macular edema, neovascularization, and proliferation of fibrous tissue), diabetic vitrectomy also achieves immediate removal of these factors and cytokines. It facilitates access to the retina and permits release of the posterior hyaloid and further dissection of the tractional fibrotic membranes that create the TRD.

Separation of the anterior vitreous from the more posterior cortical vitreous can be easily accomplished using any standard vitreous cutter, allowing the release of the anteroposterior traction induced by the vitreous. Careful dissection of the posterior vitreous cortex from the underlying retina and the removal of proliferating fibrovascular membranes and fibrous bands from the retina surface (and at times from the subretinal space) are the highlights of the surgery. This should be done, avoiding the creation of iatrogenic breaks and creation of false passages. Identification of the right plane of vitreoretinal separation is the key to proper dissection and avoiding unnecessary iatrogenic breaks. One tip to achieve entry into the right vitreoretinal plane in difficult situations is to commence dissection from the optic disc and then move out towards the macula and retina periphery, the "inside out approach." In

**35**

*Diabetic Vitrectomy*

*DOI: http://dx.doi.org/10.5772/intechopen.91360*

complete TRD repair using only the vitreous cutter [33, 34].

to ensure maximum obliteration of the ischemic drive.

tive status over some weeks.

**6. Intraoperative considerations**

are as enumerated and discussed below:

practice, it is possible to detach the vitreous at the optic disc with gentle traction on the adjoining vitreous or fibrous tissue close to the optic disc using an intraocular forceps or the aspiration port of a vitreous cutter. This lifts the vitreous off the disc and ensures a safe entry into the vitreoretinal space, from where dissection can continue outward. In some cases a moderate to large amount of retrohyaloid blood already exists; this provides a useful entry point into the desired vitreoretinal space. Techniques for fibrovascular proliferation removal have been well described and include en bloc dissection in which vitreous and proliferating tissue is removed as one, segmentation of fibrovascular tissue into islands of tissue using straight scissors or small-gauge cutter, and delamination involving careful removal of the islands of tissue using a small-gauge cutter or a curved intraocular scissors [31, 32]. Various ancillary instrumentation including picks, vertical and horizontal scissors, blades, membrane peeler cutters, forceps, scrapers, and other instruments can be used for the removal of proliferating fibrovascular membranes. However the use of newer high cut rate multifunctional vitreous cutters enables surgeons to often

Upon completion of membrane dissection, ERM in the macular area may require

There may be a need for longer acting tamponade such as silicone oil in the more complex retina detachments such as in TRD cases with the occurrence of significant iatrogenic breaks, TRD/RRD situation, or in cases with existing traction [36]. Silicone oil is also used in monocular patients and patients who cannot position or who have to undertake air travel soon. Otherwise air, saline or shorter acting tamponade such as SF6 is sufficient in cases of low to medium complexity, especially if there are no iatrogenic breaks and release of traction is considered adequate. C3F8 can be used if longer duration of tamponade is required. It is important to ensure that sclerotomy ports are well closed, with no leakage. If required and judged to be necessary, sclerotomy sites should be sutured using, e.g., 8-0 vicryl suture, to prevent hypotony and reduce the risk of postoperative vitreous hemorrhage. A reported disadvantage of the sutured sclerotomy is postoperative patient discomfort and induction of cornea astigmatism, which tends to settle and return to preopera-

Diabetic vitrectomy has benefited from the overwhelming advances that have occurred in vitrectomy over the past decade. This includes advances in surgical technique, instrumentation, improved preoperative patient work-up, and case selection. All this has resulted in improved surgical outcome, which has further increased surgeon confidence in performing surgery, even in the more complex vitreoretinal cases. Some of these advances in diabetic vitrectomy and their impact

1.Improvements in vitrectomy machines and probes, which includes faster cutting rates and smaller gauges (27 G, 25 G, and 23 G) trans conjunctival vitrectomy

identification and removal. Subretinal fluid drainage may be required. Existing retinal ischemia is treated with the application of laser panretinal photocoagulation [35]. PRP should be done up to the extreme retinal periphery, i.e., ora serrata. Scleral indentation is performed in search of iatrogenic retina breaks (which if undetected and treated can result in postoperative RRD and need for re-vitrectomy). Indentation is also done to ensure PRP has been extended to all areas of peripheral ischemia. Some surgeon will apply cryotherapy to the peripheral retina,

#### *Diabetic Vitrectomy DOI: http://dx.doi.org/10.5772/intechopen.91360*

*The Eye and Foot in Diabetes*

**Figure 1.**

This hemorrhage can often be removed using vitrectomy technique (**Figure 1b**). The fibrous component of the fibrovascular tissue contracts, due to a myofibroblastic effect [30], and causes traction on the inner retina, resulting in a TRD that may initially involve an extra macular site, and then subsequently progresses to the macula and damages vision. In some cases the traction results in a split in the layers of the retina (retinoschisis or foveoschisis) that can be appreciated on optical coherence tomography (OCT) scan. Similarly, ERM may be present in the macular area and result in considerable macular traction, worsening already existing macular edema. Since such diabetic macular edema (DME) has a traction-induced component as its causation, surgery will be required to remove the traction if resolution of the edema is to be achieved. VEGF suppression alone is unlikely to achieve complete

*Fundus photograph of PDR. (a) Left eye showing diffuse vitreous hemorrhage and preretinal hemorrhage in an eye diagnosed to have PDR. Notice also the absence of retinal laser marks and presence of macula hard exudates, with some significant cataract. (b) Same eye as in 1a after vitrectomy; view of the fundus is clearer*

The surgical principles of diabetic vitrectomy include performing a core vitrectomy. In some cases, a posterior vitreous separation already exists preoperatively and the goal of surgery is simply vitreous hemorrhage removal. This is a rather uncommon presentation. In cases of vitreous hemorrhage removal, in addition to the hemorrhage (since the vitreous cavity acts as a reservoir of several proinflammatory and pro-angiogenic factors that result in macular edema, neovascularization, and proliferation of fibrous tissue), diabetic vitrectomy also achieves immediate removal of these factors and cytokines. It facilitates access to the retina and permits release of the posterior hyaloid and further dissection of the tractional

Separation of the anterior vitreous from the more posterior cortical vitreous can be easily accomplished using any standard vitreous cutter, allowing the release of the anteroposterior traction induced by the vitreous. Careful dissection of the posterior vitreous cortex from the underlying retina and the removal of proliferating fibrovascular membranes and fibrous bands from the retina surface (and at times from the subretinal space) are the highlights of the surgery. This should be done, avoiding the creation of iatrogenic breaks and creation of false passages. Identification of the right plane of vitreoretinal separation is the key to proper dissection and avoiding unnecessary iatrogenic breaks. One tip to achieve entry into the right vitreoretinal plane in difficult situations is to commence dissection from the optic disc and then move out towards the macula and retina periphery, the "inside out approach." In

resolution of this sort of DME, and this needs to be recognized.

fibrotic membranes that create the TRD.

*and some hard exudates persist in the macular area.*

**34**

practice, it is possible to detach the vitreous at the optic disc with gentle traction on the adjoining vitreous or fibrous tissue close to the optic disc using an intraocular forceps or the aspiration port of a vitreous cutter. This lifts the vitreous off the disc and ensures a safe entry into the vitreoretinal space, from where dissection can continue outward. In some cases a moderate to large amount of retrohyaloid blood already exists; this provides a useful entry point into the desired vitreoretinal space.

Techniques for fibrovascular proliferation removal have been well described and include en bloc dissection in which vitreous and proliferating tissue is removed as one, segmentation of fibrovascular tissue into islands of tissue using straight scissors or small-gauge cutter, and delamination involving careful removal of the islands of tissue using a small-gauge cutter or a curved intraocular scissors [31, 32]. Various ancillary instrumentation including picks, vertical and horizontal scissors, blades, membrane peeler cutters, forceps, scrapers, and other instruments can be used for the removal of proliferating fibrovascular membranes. However the use of newer high cut rate multifunctional vitreous cutters enables surgeons to often complete TRD repair using only the vitreous cutter [33, 34].