Cellular and Molecular Mechanism of Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most common complications of diabetes affecting millions of working adults worldwide, in which the retina, a part of the eye becomes progressively damaged, leading to vision loss and blindness. Tremendous efforts have been made to identify biochemical mechanisms which led to the recognition of hyperglycemia, hypertension and dyslipidemia as major risk factors in DR. Consequently, tight glycemic control, blood pressure control and lipid-lowering therapy have all shown proven benefits in reducing the incidence and progression of DR. However, despite tight glycemic control, blood pressure control and lipid-lowering therapy, the number of DR patients keeps growing and therapeutic approaches are limited [Ismail-Beigi F, 2010; Patel A, 2008]. For last several decades, laser photocoagulation and vitrectomy remain as the two conventional approaches for treating sight-threatening conditions such as macular edema and proliferative DR (PDR).


Introduction
Diabetic retinopathy (DR) is one of the most common complications of diabetes affecting millions of working adults worldwide, in which the retina, a part of the eye becomes progressively damaged, leading to vision loss and blindness. Tremendous efforts have been made to identify biochemical mechanisms which led to the recognition of hyperglycemia, hypertension and dyslipidemia as major risk factors in DR. Consequently, tight glycemic control, blood pressure control and lipid-lowering therapy have all shown proven benefits in reducing the incidence and progression of DR. However, despite tight glycemic control, blood pressure control and lipid-lowering therapy, the number of DR patients keeps growing and therapeutic approaches are limited [Ismail-Beigi F, 2010; Patel A, 2008]. For last several decades, laser photocoagulation and vitrectomy remain as the two conventional approaches for treating sight-threatening conditions such as macular edema and proliferative DR (PDR).
The increased levels of metabolites in diabetic patients and in various animal models of the disease have been shown to induce several unrelated and interrelated biochemical pathways implicated in the progression of the DR. Disturbed level of several metabolites in addition to hyperglycemia and hormonal factors systemically and within diabetic retina change the production pattern of a number of mediators including growth factors, neurotrophic factors, cytokines/chemokines, vasoactive agents, inflammatory molecules, and adhesion molecules resulting in increased blood flow, increased capillary permeability, altered cell turnover (apoptosis) and finally in angiogenesis. In this chapter a major emphasis is given on diabetic induced metabolic changes in the retina which induces a range of molecules and pathways involved early in the pathophysiology of DR which are briefly discussed and those major cascades of events are shown in the schematic diagram as depicted in Fig.1.

Advanced Glycation end products (AGEs)
AGE's are formed via non-enzymatic condensation reaction between reducing glucoses and amine residues of proteins, lipids or nucleic acids that undergo a series of complex reaction to give irreversible cross linked complex group of compounds termed as AGEs. Some of the 6 In the diabetic retina, hyperglycemia not only activates protein kinase C but also mitogenactivated protein kinase (MAPK) to increase the expression of a unknown targets of PKC signaling, like SHP-1 (Src homology-2 domain-containing phosphatase-1), a protein tyrosine phosphatase. This signaling cascade leads to platelet-derived growth factor (PDGF) receptor-dephosphorylation and a reduction in downstream signaling from this receptor, resulting in pericyte apoptosis. [ Geraldes P, 2009].
PKC isoform selective inhibitors are likely new therapeutics, which can delay the onset or stop the progression of diabetic vascular disease. The highly selective PKCβ activation and its inhibition by ruboxistaurin mesylate have been most extensively studied [Davis MD, 2009]. Clinical studies have shown that ruboxistaurin prevented loss of visual acuity in diabetic patients [Gálvez MI, 2009]. Thus, PKC activation involving several isoforms is likely to be responsible for some of the pathologies in diabetic retinopathy.

Polyol pathway
In diabetes, hyperglycemia activates polyol pathway, where a part of excess glucose are metabolized to sorbitol which is then converted to fructose [Lorenzi M, 2007]. Aldose reductase (AR) is the key and rate limiting enzyme in polyol pathway, and both galactose and glucose are substrates to this enzyme and compete with each other while being reduced to galactitol and sorbitol, respectively. Under physiological conditions glucose is poorly reduced by AR to sorbitol. By contrast, under diabetic condition the intracellular glucose levels are elevated, the polyol pathway of glucose metabolism becomes active and sorbitol is produced [Lorenzi M, 2007;Gabbay KH, 1973;Barba I, 2010]. AR, reduces glucose to sorbitol using NADPH as a cofactor, thereby reducing the NADPH level [B. Lass` egue, 2003] which results in less glutathione and increase in oxidative stress, a major factor in retinal damage [Chung SS, 2003;Brownlee M, 2002]. Retinas from diabetic patients with retinopathy showed high expression of AR protein in nerve fibers, ganglion cells and Müller cells than from nondiabetic individuals [Dagher Z, 2004]. Similarly excess accumulation of sorbitol has been found in various tissues including retina of diabetic animals and also in human retinas from nondiabetic eye donors exposed to high glucose similar to the level in nondiabetic rats retina incubated under identical conditions [Lorenzi M, 2007;Chung SS, 2005]. We also measured rate of polyols formation in ex vivo rat retinas that gave evidence of increased flux through the polyol pathway with increase in the duration of diabetes and with hyperglycemia [Ola MS, 2006]. The use of inhibitor of aldose reductase in many animal models has prevented the early activation of complement in the wall of retinal vessels, apoptosis of vascular pericytes and endothelial cells and the development of acellular capillaries [Dagher Z, 2004].
Accumulated sorbitol within retina may cause osmotic stress and also the byproducts of polyol pathway, fructose-3-phosphtae and 3-deoxyglucosone are powerful glycosylating agents that enter in the formation of AGEs, which are an important factor for the pathogenecity of diabetic retinopathy. Biochemical consequences of polyol pathway activation as studied in the retina of experimentally diabetic rats show an increased nitrotyrosine [Obrosova IG, 2005], lipid peroxidation products and depletion of antioxidant enzymes [Obrosova IG, 2003].Thus, activation of the polyol pathway initiate and multiply several mechanisms of cellular damage by activation and interaction of aldose reductase and other pathogenetic factors such as formation of AGE, activation of oxidative-nitrosative 7 stress, PKC pathway and poly(ADP-ribose) polymerase that may further lead to initiation of inflammation and growth factor imbalances [Obrosova IG, 2011]. The use of fidarestat, an inhibitor of aldose reductase counteracts diabetes-associated retinal oxidative-nitrosative stress and poly (ADP-ribose) polymerase formation [Obrosova IG, 2005] supporting an important role for aldose reductase in diabetes and rationale for the development of aldose reductase inhibitors for counteraction of polyol pathway [Drel VR, 2008].

Hexosamine pathway
The hexosamine biosynthesis pathway is another hyperglycemic induced pathway which has been implicated in diabetic pathogenesis [Giacco F, 2010]. Increased expression of an enzyme called GFAT (glutamine: fructose-6 phosphate amidotransferase) causes the diversion of some of glycolytic metabolites such as fructose-6 phosphate to the hexosamine pathway producing UDP (uridine diphosphate)-N-acetylglucosamine which is a substrate used for the post-translational modification of intracellular factors including transcription factors [Nerlich AG, 1998;Brownlee M, 2005]. Du and coworkers have shown the role of hyperglycemia in activation of hexosamine pathway that increases the expression of plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-1 (TGF-1), which are deleterious for diabetic blood vessels and may contribute to the pathogenesis of diabetic complications [Du XL, 2000]. Hyperglycaemia results in increased glucosamines may cause insulin resistance in skeletal muscle and adipocytes and heamoglobin-A1c (HbA1c) which significantly correlates with basal GFAT activity in Type 2 diabetes [Yki-Järvinen H, 1996; Buse MG, 2006]. Few studies suggest that hexosamine biosynthetic pathway may cause retinal neurodegeneration via either affecting the neuroprotective effect of insulin or through the induction of apoptosis possibly by altered glycosylation of proteins [Nakamura M, 2001].
The ability of benfotiamine, a lipid soluble thiamine, to inhibit simultaneously the hexosamine pathway along with AGE formation and PKC pathways might be clinically useful in preventing the development and progression of diabetic pathogenesis arising due to hyperglycemia induced vascular damage [Hammes HP, 2003].

Poly (ADP-ribose) Polymerase (PARP)
Poly (ADP-ribose) Polymerase (PARP) is a nuclear enzyme residing as an inactive form which gets activated after the cell receives the DNA damaging signals. Increased intracellular glucose generates increased ROS in the mitochondria, which induces DNA strand breaks, thereby activating PARP. Once activated, PARP depletes its substrate, NAD + molecule, by breaking into nicotinic acid and ADP-ribose, slowing the rate of glycolysis and mitochondrial function. By inhibiting mitochondrial superoxide or ROS production with either MnSOD or UCP-1, prevented both modification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by ADP-ribose and reduction of its activity by hyperglycemia [Du X, 2003]. PARP was found to decrease the GAPDH activity, activate the polyol and PKC pathways, increases intracellular AGE formation and activates hexosamine pathway flux which trigger the production of reactive oxygen and nitrogen species, playing a role in the pathogenesis of endothelial dysfunction and diabetic complications. PARP also potentiates NF-κB activation resulting in increase of the expression of NF-κB dependent genes such as ICAM-1, MCP-1 and TNF-with increase in leukostasis and producing greater oxidative www.intechopen.com Diabetic Retinopathy 8 stress. PARP inhibition suppresses NF-κB activation and expression of adhesion molecule in cultured endothelial cells under high glucose [Zheng L, 2004]. More recently, Drel et al., demonstrated an increase in PARP activity in streptozotocin induced diabetic rats and PARP inhibitors reduced retinal oxidative-nitrosative stress, glial activation, and cell death in palmitate exposed pericytes and endothelial cells [Drel VR, 2009].

Peroxisome Proliferator Activator Receptor-γ (PPAR-γ)
PPAR-is a member of ligand-activated nuclear receptor superfamily, which plays an important role in carbohydrate metabolism, angiogenesis and inflammation [Malchiodi-Albedi F, 2008; Yanagi Y, 2008]. PPAR-is highly expressed in retinal cells, macropahges and other cell types that influence inflammation such as microglial cells, a resident macrophage present both in brain and retina, indicating that PPAR-might modulate diabetes induced activation of these cells involved in inflammation and neurodegeneration [Bernardo A, 2006]. The recent work by Tawfik and group has shown the down regulation of PPAR-expression in oxygen induced retinopathy in an experimental model of diabetes [Tawfik A, 2009]. In streptozotocin induced diabetic mice deficient in PPAR-expression had increased leukostasis and leakage compared to wild type control mice, indicating that endogeneous PPAR-and its activation by specific ligands is critical for inhibiting leukostasis and leakage in diabetic mice [Muranaka K, 2006]. PPAR-also acts as agonist by inhibiting the VEGF-stimulated proliferation, migration and tube formation in PPARexpressing retinal endothelial cells [Murata T, 2000]. In diabetic patients, PPAR-agonists have been shown to reduce several markers of inflammation such as serum levels of creactive protein, interleukin-6 (IL-6), monocyte chemoattractant protein (MCP-1) and matrix metallo ptoteinase 9 (MMP-9) [Agarwal R, 2006]. In-vitro studies showed that PPARagonists suppress activated NF-κB and decrease ROS generation in blood mononuclear cells [Aljada A, 2001]. Many such studies suggest the use of PPAR-agonists in the treatment of diabetic retinopathy.

Oxidative stress
The retina is highly metabolic active tissue, making it susceptible to increased oxidative stress. Diabetes disturbs the cellular homeostasis in the normal retina by metabolic dysregulation of glucose, lipids, amino acids and other metabolites which causes oxidative stress, implicating in the in the pathogenesis of diabetic retinopathy.
Oxidative stress is believed to play a pivotal role in the development of diabetic retinopathy by damaging retinal cells [Sato H, 2005]. However, the potential sources of ROS, is still unclear although a number of studies showed that high glucose and the diabetic state stimulate flux through the glycolytic pathway, increases cytosolic NADH, tissue lactate-topyruvate ratios, and tricarboxylic acid cycle flux thereby producing excess level of ROS [Madsen-Bouterse SA, 2008; Ido Y, 1997; Obrosova IG, 2001]. ROS can be produced by activation of AGE, aldose reductase, hexosamine and PKC pathways induced by hyperglycemia, altered lipoprotein metabolism, excess level of excitatory amino acids and altered growth factor or cytokines/chemokines activities [Ola MS, 2006;Kanwar M, 2009]. Oxidative stress creates a vicious cycle of damage to macromolecules by amplifying the production of more ROS and activates other metabolic pathways that are detrimental to the 9 development of diabetic retinopathy. However, it is still unclear whether oxidative stress has a primary role in the pathogenesis of diabetic complication, occurs at an early stage in diabetes or it is a consequence of the tissue damage. Other sources of oxidative stress are the activation of NADPH oxidase which may increase superoxide, induction of xanthine oxidase, decreased tissue concentration of endogenous antioxidants such as glutathione and impaired activities of antioxidant defense enzymes such as superoxide dismutase (SOD) and catalase [Sonta T, 2004 To develop novel therapeutic strategies that specifically target ROS is actually desired for patients with PDR. The use of PEDF as a therapeutic option which has a anti-oxidative, antiangiogenic, neuroprotective and anti-inflammatory properties could be used to block pathways that leads the production of ROS [Yamagishi S, 2011]. Vitamin E has a protective role against lipid peroxidation, whereas its effects on protein and DNA oxidation are less pronounced [Pazdro R, 2010]. The major sources of fatty acids/lipids are from the modern diets (Western in particular) that have a high fat content [Hu FB, 2001]. Not only these diets have high caloric content, but also have high levels of saturated and trans-fatty acids (SFA), rather than the generally beneficial cis-monounsaturated or polyunsaturated fatty acids. Thus understanding the details of metabolic response of diabetic mice to Western diets may aid in understanding, how dietary lipid/fatty acids contribute to the complication of diabetes. The sensitivity of retina to fatty acid is well documented and thus understanding how diet affects the levels of these key metabolites will provide important new information about their role in DR [Giovanni JP, 2005; Adibhatla RM, 2007]. Very long chain unsaturated fatty acids such as docosahexaenoic acids (DHA) are essential for retinal development and function, and free fatty acids in this class have been shown to be protective against age related macular degeneration in a mouse model [Connor KM, 2007]. Diet high in SFA and deficient in the precursors of important retinal fatty acids may adversely affect retinal function or increase the pathology. In the context of type I diabetes, a high fat diet may also increase oxidative stress [Kowluru RA, 2007] and contributes to the inflammatory response [Fox TE, 2006

Renin Angiotensin System (RAS)
Hypertension has been identified as a major risk factor of microvascular complications leading to small vessel dysfunction, manifesting the state of diabetic retinopathy. In patients with diabetic retinopathy, tight control of blood pressure delays the progression of the disease and growing evidence suggests that RAS plays an important role in the regulation of blood pressure. The RAS is an enzymatic cascade in which angiotensinogen is the precursor of the angiotensin peptides. The cascade begins with the conversion of the inactive form of renin, prorenin, to active renin [Satofuka S, 2009]. Renin converts angiotensinogen to angiotensin-1 (Ang I) which is further cleaved by angiotensin converting enzyme (ACE) to angiotensin-II (Ang II). Ang II is the main effector peptide of the RAS, acting primarily on two receptors, the angiotensin type I (AT-1) and angiotensin type 2 (AT22). Ang II is known to cause systemic and, local blood pressure via its constrictor effect by upregulation of angiotensin II type 1 receptor.
A number of investigators studied components of retinal RAS (Ang I, Ang II, renin, ACE, AT-1, AT-2) in the retina and increased levels of prorenin, rennin and angiotensin II have been reported in the vitreous of patients with PDR and diabetic macular edema (DME) suggesting the involvement of RAS in pathogenesis of diabetic retinopathy [Noma H, 2009;Nagai N, 2005]. Ang II is also a growth factor, promoting differentiation, apoptosis and the deposition of extracellular matrix [Otani A, 2001;Suzuki Y, 2003]. Ang II potentiates deleterious effect of AGEs by inducing RAGE expression in hypertensive eye and can be blocked by telmisartan, an inhibitor of ACE, indicating a link between AGE-RAGE and the RAS which may be involved in the pathogenesis of diabetic retinopathy.
Angiotensin induce cell growth, proliferation and the deposition of extracellular matrix proteins via stimulation of growth factors such as transforming growth factor (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) [Ruperez M, 2003]. There is evidence that the AT-2 receptor also influences pathological angiogenesis in rats with oxygen induced retinopathy and blockade of the AT-2 receptor was shown to reduce retinal angiogenesis and expression of VEGF, VEGFR-2 and angiopoietin-2. In diabetic rats both AT-1 and AT-2 receptor blockade attenuate the rise in retinal VEGF expression [Zhang X, 2004]. Blockade of the RAS at the level of ACE inhibition or angiotensin reduces the rise in retinal VEGF and VEGFR-2 that occurs in diabetic rats and transgenic rats with OIR and attenuates vascular pathology including vascular leakage, proliferation of endothelial cells, angiogenesis [Kim JH, 2009], leukostasis [Chen P, 2006] and inflammation [Egami K, 2003]. Recently, Nagai et al. studied the involvement of RAS and NF-kB pathway in diabetic induced retinal inflammation by upregulation of ICAM-1, MCP-1 and VEGF which are attenuated by AT-1 receptor blocker [Nagai N, 2007]. Therefore, RAS plays an important role in the pathogenesis of diabetic retinopathy and this has led a major interest in RAS inhibitors to prevent retinopathy.

Hormones
Several hormones such as insulin, aldosterone, adrenomesdulin, growth hormone (GH) and endothelin have been found to be implicated in diabetic retinopathy [Wilkinson-Berka JL, 2008]. Insulin stimulates anabolic functions and prevents the breakdown of skeletal muscle tissue. In diabetes, the loss of insulin signaling profoundly alters carbohydrate, lipids, amino acids and protein metabolism in a range of tissues including retina, altering nutrients pool and resulting in metabolic dysregulation that ultimately induces tissue damage. Also, the loss of insulin action in diabetic patients causes muscle loss [Serrarbassa PD, 2008]. Numerous studies towards understanding whether the role of insulin concise to its effect on blood level only or extend its role in maintaining retinal homoeostasis reveals the neurotrophic action of insulin [Meyer-Franke A, 1995] pointing to the possibilities that exogenous insulin have a role in the treatment of DR via its neurotrophic actions [Reiter CE, 2006]. Few studies also describe the role of insulin in inflammatory processes [Fort PE, 2009]. Data and research from the Diabetes Control and Complications Trial (DCCT, Diabetes, 1995), as a study by Barber et  ]. Systemic inhibition of GH or insulin like growth factor (IGF-1) or both, may have therapeutic potential in preventing some forms of retinopathy [Smith LE, 1997]. Thus growth hormone may play a major role in the progression of diabetic retinopathy in combination with IGF-I and VEGF. In diabetes induced TNF-knockout mice the BRB breakdown was completely suppressed showing that TNF is essential for progression BRB breakdown and would be a good therapeutic target to prevent BRB breakdown, retinal leukostasis, and apoptosis associated with DR [Huang H, 2011]. Increased level of IL-6 is detected in vitreous fluid of the patients with PDR and DME [Noma H, 2009;Murugeswari P, 2008]. Serum level of IL-6 in patients with both type 1 and type 2 diabetes were also found to be increased [Myśliwiec M, 2008;Bertoni AG, 2010]. Levels of soluble IL-6 receptor in the vitreous and serum of patients with PDR was found to be significantly higher than control [Kawashima M, 2007]. Increased level of IL-6 was found to be related to retinal vascular permeability and the severity of DME [Noma H, 2009;Noma H, 2010]. Up-regulation of IL-6 increase leukocyte-endothelial interaction which contributes to breakdown of BRB in diabetes [Adamis AP, 2008].

Inflammation and diabetic retinopathy
Chemokines such as MCP-1, IP-10, IL-8 and stromal derived factor-1 (SDF-1) have been also found to play a potential role in pathogenesis of diabetic retinopathy [Murugeswari P, 2008;Yoshimura T, 2009]. MCP-1 which is a strong activator of macrophages and monocytes, have been shown to be involved in the pathogenesis of DR where vitreous MCP-1 levels are 13 increased in PDR compared with those in controls [Maier R, 2008;Hernández C, 2005]. The angiogenic effect of MCP-1 was completely inhibited by a VEGF inhibitor, suggesting that MCP-1 induced angiogenesis is mediated through pathways involving VEGF [Hong KH, 2004].The increased MCP-1 expression contributes to the development of neovascularization and fibrosis in proliferative vitreoretinal disorders ]. Abu El-Asrar and others have found increased levels of IP-10 in the vitreous humor samples from eyes with PVR and PDR patients [Abu El-Asrar AM, 2006;Maier R, 2008] and IP-10 expression under both in vitro and in vivo conditions has been shown to be induced by VEGF, indicating a potent angiogenesis factor in PDR [Maier R, 2008]. VEGF induced augmentation of IP-10 expression is a major mechanism underlying its proinflammatory function. In age-related macular degeneration, IP-10 is also marked as early biomarkers to understand the regulation and neovascular response [Mo FM, 2010]. The work by Liu shows that diabetic tears exhibited elevated levels of pro-angiogenic cytokines such as IP-10 and MCP-1 than anti-angiogenic cytokines [Liu J, 2010]. IL-8 is angiogenic and inflammatory mediator which is elevated in vitreous of patients with PDR in comparison to control subjects [Murugeswari P, 2008;Petrovic MG, 2007]. It has been shown that IL-8 is produced by endothelial and glial cells in the retina with ischemic angiogenesis [Yoshida A, 1998] where it could act as a marker of ischaemic inflammatory reaction, and play a role in deteriorating visual acuity by DR progression [Petrovič MG, 2010].
In humans, vitreous SDF-1 concentration increases as proliferative diabetic retinopathy progresses [Butler JM, 2005 The role of various growth factors such as epidermal growth factor (EGF), VEGF, basic FGF, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colonystimulating factor (GM-CSF) in the retinal pathogenesis have been evaluated. Schallenberg and his group have shown that the hematopoietic cytokine, GM-CSF and its receptor are expressed within rat and human retina where GM-CSF reduced apoptosis and protected injured retinal ganglion cells by activating the ERK1/2 pathway [Schallenberg M, 2009].

Neurodegeneration
A pathogenic mechanism of nerve damage in diabetic retinopathy begins shortly after the onset of diabetes. Several clinical tools such as multifocal electroretinography (ERG), flash ERG, contrast sensitivity, color vision, and short-wavelength automated perimetry, all detect neuronal dysfunction at early stages of diabetes Fletcher EL, 2007]. Occurrence of many functional changes in the retina can be identified before the development of vascular pathology, suggesting that they result from a direct effect of diabetes on the neural retina [Lieth E, 2004]. Diabetic mice develop capillary lesion that are characteristic of the early stages of DR and cause pathologic progression resulting due to neuronal loss or upregulation of glial fibrillary acidic protein (GFAP) in retinal glial cells [Feit-Leichman RA, 2005]. Van Dijk and his group has shown the gradual and selective thinning of mean ganglion cell/inner plexiform retinal layer in type 1 diabetic patients [van Dijk HW, 2009] which further supports the concept that early DR includes a neurodegenerative sign [van Dijk HW, 2010; Peng PH, 2009]. Retinal glial cells that play important roles in maintaining the normal function of the retina, after the onset of diabetes the normal function of these cells are altered and compromised. They are known to become gliotic displaying altered potassium siphoning, GABA uptake, glutamate excitoxicity and are also known to express several modulators of angiogenic factors. In addition to metabolic stress, there are many growth factors involved in process of neuronal death in DR suggesting further investigation into the mechanism of neurodegenaration [Whitmire W, 2011].

Apoptosis
Even before the emergence of the concept of programmed cell death (PCD)/apoptosis in diabetes, studies have identified a pyknotic bodies in histological sections of the retina of people with diabetes [Bloodworth JM Jr, 1962;Wolter JR, 1962]. Diabetes causes chronic loss of inner retinal neurons by increasing the frequency of apoptosis as studied in streptozotocin-induced diabetic mice [Martin PM, 2004]. Many findings suggest that the visual loss associated with DR could be associated not only to an early phase of photoreceptor loss but also to later microangiopathy [Park SH, 2003], so both retinal neurodegeneration and retinal microangiopathy should be considered as sign and onset of DR [Ning X, 2004]. Caspases, the enzymes involved in apoptosis are also elevated in retinas of diabetic rats thus making them as markers for apoptosis [Mohr S, 2002]. The role of proinflammatory cytokine (IL-1 ) and caspase-1 in diabetes-induced mice have shown that caspase-1/IL-1 signaling pathways play an important role in degeneration of retinal capillaries [Vincent JA, 2007] and its inhibition might represent a new strategy to inhibit capillary degeneration in diabetic retinopathy [Mohr S, 2008]. The increased expression of apoptotic mediators, Bcl-2 in the vascular endothelium inhibits the diabetes-induced degeneration of retinal capillaries and superoxide generation [Kern TS, 2010;Susnow N, 2009].
Several studies also demonstrate that the expression of Bax (Bcl-2 associate X protein), proapoptotic protein is associated with degenerative diseases and are increased in retinas of diabetic rats, confirming the increase in apoptosis within the inner retina as a component of DR [Podesta F, 2000]. Involvement of TNF-and AGE, in retinal pericyte apoptosis through activation of the pro-apoptotic transcription factor Forkhead box O1 (FOXO1) establishes the possible mechanism of apoptosis in DR [Alikhani M, 2010].

Glutamate excitotoxicity
Glutamate is the excitatory neurotransmitter in the retina, but it is neurotoxic when present in excessive amounts. Crucial role in the disruption of glutamate homeostasis in diabetic retina is due to decrease in the ability of Müller cells to remove the excess amount of glutamate from the extracellular space causing excitotoxicity leading to neurodegeneration [Li Q, 2002;Diederen RM, 2006]. Extracellular glutamate is transported into Müller cells by glutamate transporters (GLAST) and amidated by glutamine synthetase (GS) to the non-toxic amino acid, glutamine. Yu XH and coworkers have shown a linear correlation between time-dependent reduction in GS expression and the time course of diabetic retinopathy, making GS as a possible biomarker for evaluating the severity of diabetic retinopathy [Yu XH, 2009]. At postsynaptic neurons, two major classes of receptors referred to as amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) are activated by excess glutamate. The major causes for cell death following activation of NMDA receptors are the influx of calcium and sodium into cells, the generation of free radicals linked to the formation of AGEs and/or advanced lipoxidation endproducts (ALEs) as well as defects in the mitochondrial respiratory chain. Thus, glutamate may play an important role in the progression of disease and treatment by glutamate inhibitors may decrease neurotoxicity [Ola MS, 2011].

Role of neurotrophic factors
Neurotrophic factors play important roles in regulating growth, maintenance and survival of neurons [Mattson MP, 2004]. The role of brain derived neurotrophic factors (BDNF) in metabolism is supported by studies on BDNF-deficient mice which develop obesity and hyperphagia in early adulthood [Kernie SG, 2000] whereas, when it administered to normal mice or rats, it has no effect on blood glucose levels, indicating that BDNF exerts its effects by enhancing insulin sensitivity [Ono M, 1997] and activates several signaling pathways including phosphatidylinositol-3 kinase/Akt [Cotman CW, 2005]. Plasma levels of BDNF were decreased in humans with type 2 diabetes accompany impaired glucose metabolism [Krabbe KS, 2007] and act like a biomarkers of insulin resistance [Fujinami A, 2008]. Recently to understand the mechanism of action of BDNF under normal and hypoxic condition in Müller cells, BDNF treated cells increased glutamate uptake and also up regulated glutamine synthetase (GS) during hypoxia which may underlie neuroprotective effects of BDNF [Min D, 2011]. The therapeutic merit of BDNF was also evaluated by injecting it in diabetic mice, which not only ameliorated glucose metabolism ] but also prevented the development of diabetes in pre-diabetic mice [Yamanaka M, 2008 (b)]. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is shown to rescue photoreceptors in retinal explants, conveying its neuroprotective effects [Azadi S, 2007].
Several studies have shown an elevated level of Nerve Growth Factor (NGF), another potent neurotrophic factor, which contributes to neurogenic inflammation [Barhwal K, 2008]. NGF level was significantly elevated in the PDR samples as compared to controls, indicating that NGF might be a potent angiogenic factor in the pathogenesis of PDR [Chalam KV, 2003].
Another neurotrophic includes Basic Fibroblast Growth Factor (bFGF), which is important for survival and maturation of both glial cells and neurons and play an important role in regeneration after neural injury [Bikfalvi A, 1997;Molteni R, 2001]. Study found an increase in bFGF concentration in vitreous samples from patients with PDR [Sivalingam A, 1990] revealing that bFGF is a potent angiogenic factor playing an important role in the pathogenesis of neovascularization in DR [Wong CG, 2001]. Studies also suggest that bFGF have a therapeutic value for diabetic neuropathy when injected with cross-linked gelatin hydrogel in streptozotocin-induced diabetic rats [Nakae M, 2006].
Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-(TGF-)-related neurotrophic factor family. GDNF promotes photoreceptor survival during retinal degeneration mediated by interaction of the neurotrophic factors via receptors in Müller glial cells that in turn release secondary factors that act directly to rescue photoreceptors [Harada C, 2003].

Conclusions
As described in this chapter, extensive research progress has been made in investigating the pathophysiology of the disease, however, due to non availability of human retinal samples and also due to lack of proper animal model of DR, the exact molecular mechanism has not been elucidated, making therapeutic a difficult task. Therefore, research using large diabetic animal models which develop clinical signs of retinopathy are needed which may provide a correlation of the systemic metabolic profiles and retinal pathology with human studies to better understand the exact molecules and pathway(s) involved in DR. In addition, neurodegeneration and loss of neuronal functions as early signs of DR have been detected which may implicate later in vascular pathology. Precise molecular studies are required towards understanding the neurovascular damage in DR. These insights would be helpful in better understanding of the biochemical and molecular changes especially early in the diabetic retina for effective therapies towards prevention and amelioration of DR.