The amount of SST produced by the human retina is significant as can be deduced by the strikingly high levels found in the vitreous fluid [35, 36]. Apart from SST, SSTRs are also expressed in the retina, with SSTR1 and SSTR2 being the most widely expressed [34, 37, 38]. The production of both SST and its receptors simultaneously suggests an autocrine action in the human retina.

The main functions of SST for retinal homeostasis are the following: (1) SST acts as a neuromodulator through multiple pathways, including intracellular Ca2+ signaling, nitric oxide function, and glutamate release from the photoreceptors. In addition, a loss of SST immunoreactivity occurs after degeneration of the ganglion cells. Therefore, the neuroretinal damage that occurs in DR might be the reason for the decreased SST levels detected in the vitreous fluid of these patients. In fact, we have recently found that low SST expression and production is an early event in DR and is associated with retinal neurodegeneration (apoptosis and glial activation) [8]. (2) SST is a potent angiostatic factor. SST may reduce endothelial cell proliferation and neovascularization by multiple mechanisms, including the inhibition of postreceptor signaling events of peptide growth factors such as IGF-I, VEGF, epidermal growth factor (EGF), and PDGF [39]. (3) SST has been involved in the transport of water and ions. Various ion/water transport systems are located on the apical side of the RPE, adjacent to the subretinal space, and indeed, a high expression of SST-2 has been shown in this apical membrane of the RPE [37].

In DR, there is a downregulation of SST (Fig. 3B) that is associated with retinal neurodegeneration [8]. The lower expression of SST in RPE and neuroretina is associated with a dramatic decrease of intravitreal SST levels in both PDR [35, 36] and DME [40]. As a result, the physiological role of SST in preventing both neovascularization and fluid accumulation within the retina could be reduced, and consequently, the development of PDR and DME is favored. In addition, the loss of neuromodulator activity could also contribute to neuroretinal damage. For all these reasons, intravitreal injection of SST analogues or gene therapy has been proposed as a new therapeutic approach in DR [41].

ERYTHROPOIETIN

Erythropoietin (Epo) was first described as a glycoprotein produced exclusively in fetal liver and adult kidney that acts as a major regulator of erythropoiesis. However, Epo expression has also been found in the human brain and in the human retina [42, 43]. In recent years, we have demonstrated that not only Epo but also its receptor (EpoR) are expressed in the adult human retina (Fig. 4) [44]. Epo and EpoR mRNAs are significantly higher in RPE than in the neuroretina [44]. In addition, intravitreal levels of Epo are ~3.5-fold higher that those found in plasma [43]. The role of Epo in the retina remains to be elucidated, but it seems that it has a potent neuroprotective effect [45, 46].

Epo is upregulated in DR [43, 44, 47, 48]. Epo overexpression has been found in both the RPE and neuroretina of diabetic eyes [43, 44]. This is in agreement with the elevated concentrations of Epo found in the vitreous fluid of diabetic patients (~30-fold higher than plasma and ~10-fold higher than in nondiabetic subjects) [43]. Hypoxia is a major stimulus for both systemic and intraocular Epo production. In fact, high intravitreous levels of Epo have recently been reported in ischemic retinal diseases such as

Fig. 4 Epo (green ) and Epo receptor (red ) immunofluorescence in the retinal pigment epithelium of human retina. In the merged image (lower panel ), the nuclei have been stained using DAPI (blue )

PDR [43, 47–49]. In addition, it has been reported that Epo has an angiogenic potential equivalent to VEGF [48, 50]. Therefore, Epo could be an important factor involved in stimulating retinal angiogenesis in PDR. However, intravitreal levels of Epo have been found at a similar range in PDR to that in DME (a condition in which hypoxia is not a predominant event). In addition, intravitreal Epo levels are not elevated in nondiabetic patients with macular edema secondary to retinal vein occlusion [51]. Finally, a higher expression of Epo has been detected in the retinas from diabetic donors at early stages of DR in comparison with nondiabetic donors, and this overexpression is unrelated to mRNA expression of hypoxic inducible factors (HIF-1a and HIF-1b) [44]. Therefore, stimulating agents other than hypoxia/ischemia are involved in the upregulation of Epo that exists in the diabetic eye.

The reason why Epo is increased in DR remains to be elucidated, but the bulk of the available information points to a protective effect rather than a pathogenic effect, at least in the early stages of DR. In addition, Epo is a potent physiological stimulus for the mobilization of endothelial progenitor cells (EPCs), and therefore, it could play a relevant role in regulating the traffic of circulating EPCs toward injured retinal sites [52]. In this regard, the increase of intraocular synthesis of Epo that occurs in DR can be contemplated as a compensatory mechanism to restore the damage induced by the diabetic milieu. In fact, exogenous Epo administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function in STZ-DM rats [53]. Nevertheless, in advanced stages, the elevated levels of Epo could potentiate the effects of VEGF, thus contributing to neovascularization and, in consequence, worsening PDR [52, 54].

The potential advantages of Epo or EpoR agonists in the treatment of DR include neuroprotection, vessel stability, and enhanced recruitment of EPCs to the pathological area. However, as mentioned above, timing is critical since if Epo is given at later hypoxic stages,

the severity of DR could even increase. However, in the case of the eye, disease progression is easy to follow without invasive investigation and allows timing of the administration of drugs to be carefully monitored, hopefully resulting in better clinical outcomes.

DOCOSAHEXAENOIC ACID AND NEUROPROTECTIN D1

Delivery of fatty acids such as docosahexaenoic acid (DHA) to the photoreceptors is important for visual function [55]. DHA is an essential omega-3 fatty acid that cannot be synthesized by neural tissue but is required as structural protein by the membranes of neurons and photoreceptors. DHA is synthesized from its precursor, linolenic acid, in the liver and transported in the blood bound to plasma lipoprotein where it is taken up in a concentration-dependent manner. Apart from the RPE’s functional integrity, DHA is the precursor of NPD1, a docosatriene that is required for the functional integrity of RPE. NPD1 protects RPE cells from oxidative stress, has an antiapoptotic effect, and inhibits the expression of IL-b-stimulated expression of COX-2 [56, 57]. Therefore, NPD1 can be postulated as a retinal neuroprotective factor.

BRAIN-DERIVED NEUROTROPHIC FACTOR

BDNF is a neurotrophin expressed in RGCs, Müller cells, and amacrine cells (both cholinergic and dopaminergic) in the retina [58]. BDNF expression is upregulated by noradrenaline [59] and is important for the survival of RGCs and amacrine cells [60]. In addition, BDNF acts as a synaptic modulator and is essential for the development of the dopaminergic network in the rodent retina [61]. Dopaminergic amacrine cell degeneration is accompanied by a reduction in BDNF levels in the retina of STZ-DM rats, and BDNF intravitreal administration can rescue these cells from neurodegeneration [62]. Furthermore, induction of BDNF expression by adrenergic agonists may provide a therapeutic approach to retinal neurodegenerative disorders including DR.

GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR

GDNF is a 20-kDa glycosylated homodimer belonging to the TGF-b superfamily that has been recognized for its ability to increase the survival of dopaminergic cells in animal models of Parkinson’s disease [63].

GDNF signals directly through the cell surface receptors (GFR-a1 and GFR-a2) and indirectly through the transmembrane Ret receptor, tyrosine kinase [64]. Both receptors have been identified on embryonic chick RGCs, as well as on amacrine and horizontal cells [65]. GFR-a2 overexpression has also been found in the epiretinal membranes of patients with PDR [66]. In addition, high levels of GFR-a2 have been detected in the vitreous fluid of PDR patients [67]. Finally, several experimental studies support the concept that GDNF exerts a neuroprotective effect in the retina.

CILIARY NEUROTROPHIC FACTOR

CNTF was first identified as a survival factor in studies involving ciliary ganglion neurons in the chick eye. CNTF is a member of the IL-6 family of cytokines and acts through a heterodimeric receptor complex composed of CNTF receptor a plus two

signal-transducing transmembrane subunits, leukemia inhibitory factor receptor b (LIFR), and glycoprotein gp130 (gp-130) [68]. The CNTF-a receptor is located on Müller glial membranes [69] and practically on all retinal layers [70]. CNTF is effective in retarding retinal degeneration in several experimental models of retinitis pigmentosa, amyotrophic lateral sclerosis, and in Huntington’s disease. CNTF administered as eyedrops prevents retinal neurodegeneration in STZ-DM rats [71].

ADRENOMEDULLIN

Adrenomedullin (AM) is a multifunctional protein with neuroprotective actions [72]. Administration of AM is neuroprotective in cerebral ischemia through an increase in astrocyte survival which is attributed to the inhibition of oxidative stress signaling pathways [73]. Recently, it has been demonstrated that the AM gene is one of those retinal genes differentially expressed in the neuroprotection conferred by hypoxic preconditioning [74] and, therefore, could be a new therapeutic target in retinal ischemic diseases such as DR.

CONCLUDING REMARKS AND THERAPEUTIC IMPLICATIONS

Neurodegeneration is an early event in the pathogenesis of DR and, apart from its own deleterious effects, participates in the microcirculatory abnormalities that occur in DR. Whereas the role of neuropathy is essential at early stages of DR, in advanced stages of DR, microangiopathy will be the main protagonist from the pathophysiological point of view.

The two capital findings of retinal neurodegeneration are apoptosis and glial activation. Although the bulk of the information on this issue has been drawn from experimental models, it has also been demonstrated in the human diabetic retina. The experimental model currently used for studying retinal neurodegeneration is the STZ-DM rat. However, STZ has neurotoxic effects, thus hampering our ability to elucidate whether the neurotoxic effects are due to the diabetic milieu or to STZ. In this regard, the use of genetically modified mice with spontaneous diabetes such as C57BL/KsJ-db/db seems to be more appropriate.

Elevated levels of glutamate play an essential role in the neurodegenerative process that occurs in the diabetic retina, and recent evidence suggests that overexpression of the RAS system is also an important contributing factor. Among the neuroprotective factors, PEDF, SST, and Epo seem to play a critical role, but the effect of other neurotrophic factors such as NPD1, BDNF, GDNF, CNTF, and AM should also be taken into account. In fact, the balance between neurotoxic and neuroprotective factors rather than the levels of neurotoxic factors alone is determinant for the presence or not of retinal neurodegeneration in the diabetic eye.

Intravitreal injection permits neurotrophic drugs to effectively reach the retina and overcome the potential adverse effects related to systemic administration. However, this is an invasive procedure, with the potential for blinding sequelae such as endophthalmitis and retinal detachment. Although the incidence of these serious complications is low,