The retina is the light-sensitive tissue that lines the inner surface of the eye. Phototransduction, or the transformation of light signals into electrical impulses, takes place in photoreceptors, thus making the retina the tissue where the processing of visual information begins. The structural and functional complexity of the retina, and its direct exposure to light, makes this tissue one of the most vulnerable to the molecular alterations derived from any sort of pathological injury or disease, leading to blindness.

Whatever the origin of the retinal damage, oxidative stress, inflammation and apoptosis pathways are common features and are controlled by a plethora of cell mediators, some of them protective, some of them harmful, and several having a dual role depending on the particular conditions present.¹  Therapy for retinal diseases include preventive strategies to neutralize the underlying disease mechanisms and the administration of anti-oxidants, anti-apoptotic and anti-inflammatory compounds, as well as neurotrophic and growth factors, which slow the neurodegeneration of the retina by delaying retinal cell death.^1,2  Due to the tight connection of all these processes and their parallel running, one drug is likely to act in more than one pathway and mechanisms involving more than one effect are common.

Oxidative stress plays an important role in the development and progression of retinal degeneration, with implications in the pathogenesis of age-related macular degeneration (AMD), diabetic retinopathy, retinitis pigmentosa³  and glaucoma.^1,2,4,5  Photoreceptor cells are continuously exposed to light photons, have a high metabolic rate and are great consumers of oxygen, mainly due to a large accumulation of mitochondria in the ellipsoid and the aerobic metabolism in the membranous disks of photoreceptor outer segments. Hence the retina is a perfect target for reactive oxygen species (ROS) damage. Both overproduction of ROS and/or a reduced ability to neutralize normally produced ROS (due to deficiencies in anti-oxidant enzymes) can bring oxidative stress which, in turn, can induce cell death by different mechanisms: (i) triggering apoptosis by damage in DNA, lipids and proteins; (ii) by oxidizing and inhibiting phosphatases and/or kinases, among other proteins, and altering their downstream signaling pathways. If the levels of oxidative stress are severe the cells undergo necrosis.^2,6  The administration of anti-oxidants has shown good results in degeneration models and has been proven to preserve functional vision for longer in animal models and in patients.^7–11  Supplementation with anti-oxidants such as lutein, zeaxanthin and meso-zeaxanthin are recommended to AMD patients at significant risk for visual loss, although its utility for other retinal degenerative processes as well as the therapeutic potential of other anti-oxidants remain to be determined.¹²  Impairment of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling increases the vulnerability to oxidative damage and is associated with oxidative stress in several degenerative diseases, such as AMD or diabetic retinopathy (reviewed in Cuenca et al.¹ ). Activation of the redox-sensitive transcription factor Nrf2 is one of the critical defensive mechanisms against oxidative stress in many species, as it binds to anti-oxidant response elements located in the promoter region of genes encoding many anti-oxidant enzymes and phase II detoxifying enzymes. Nrf2 activators have been tested in a number of retinal pathological models and several studies suggest that Nrf2 activation could be a therapeutic option for oxidative stress-related retinal diseases such as diabetic retinopathy or glaucoma.^13–15 

A common feature of retinal degenerative diseases is neuroinflammation, which is mainly mediated by microglia that become activated in the course of the degeneration and release noxious factors which produce a plethora of inflammatory mediators.^1,3,16–20  Therapy with anti-inflammatory agents seems to be a good strategic point, and several anti-inflammatory compounds have been proven effective against retinal degeneration; among them, natural compounds with anti-oxidant and anti-inflammatory properties such as curcumin or quercetin.¹  Anti-inflammatory drugs as corticosteroids, that suppress multiple pathways of inflammation, have been proposed for the treatment of diabetic macular edema, particularly in cases refractory to laser photocoagulation.^21,22  However, it is necessary to take into consideration the fact that the inflammatory process has a dual effect: it may be pathogenic but also has reparative properties, and some authors have suggested that immunomodulation, rather than an anti-inflammatory approach, may be a more effective therapeutic strategy in retinal pathologies.²³ 

The cannabinoid (CB) system has also been suggested as a useful target for the treatment of retinal degenerative diseases due to its implication in different aspects of the degenerative process: inflammation, apoptosis, neurodegeneration or angiogenesis. In this sense, CB1 agonists have showed neuroprotective actions and CB2 agonists neurogenic and anti-inflammatory properties in neurodegenerative diseases.^24,25  Norgestrel, a synthetic form of progesterone, can inhibit apoptosis and inflammation in cells and animal models,^2,26–28  likely through progesterone receptor membrane component 1 (PGRMC1), a key regulator of apoptosis.²⁹ 

Cell death is a major determinant of inflammatory disease severity. During inflammation, the pro-survival process of autophagy and the pro-death process of apoptosis interact and influence each other, tilting the balance towards life or death.³⁰  To date, the whole routes of cell death and survival are not fully known. New unexpected roles for cytokines and other mediators emerge each day, showing more connexions between death and survival mechanisms. In this context, the three major cell death pathways, apoptosis, necrosis and autophagy, are involved in retinal degenerative diseases and are mutually interconnected in such a way that some mediators can influence more than one pathway.^30–34  So, as a therapeutic strategy, it could be essential to act in different steps to avoid the activation of alternative dying routes or to favour the survival side. Whenever a harmful stimulus threatens the cell, its machinery starts working in a way that in the best case will rescue the cell or the tissue from a greater damage, killing injured organelles or cells in a precise, organized way; and in the worst case will end with irreparable tissue damage. The balance between cell death and survival signals will determine the final outcome. Certain genetic mutations, age and environmental factors can trigger specific pathways to induce apoptosis in retinal cells, contributing to the development of many diseases.^1,35  The changes responsible for dystrophic and degenerative retinal diseases, which cause structural and functional damage, may occur at any level of the signal transduction cascade or in any of the morphological components of these differentiated cells. The life-or-death decision seems to be the result of a complex balance between pro- and anti-apoptotic signals at several levels: extracellular, mitochondrial, nuclear and cytoplasmic.^36,37  The final common pathway of cell death in retinal diseases is apoptosis, which initially affects only certain retinal cells, such as photoreceptors, followed by the apoptosis of all remaining cells in the retina.¹  Hence, the pharmacologic inhibition of cell death through the use of anti-apoptotic agents may prevent disease-associated retinal degeneration. In this sense, several anti-apoptotic agents as tauroursodeoxycholic acid (TUDCA) or pro-insulin have shown good results in retinal neurodegenerative diseases (reviewed in Cuenca et al.¹ ). Other agents, as the anti-oxidant melatonin, could improve retinal degeneration through the attenuation of apoptosis, but also avoiding the reactive gliosis and microglial activation in rd10 mice, and could have therapeutic interest improving photoreceptors survival in human retinitis pigmentosa.³⁸  The neuroprotective activity of FAS apoptotic inhibitory molecule 2 (FAIM2), which is an inhibitor of the FAS signaling pathway that is activated by stress in photoreceptors, has been reported recently. Therefore, modulation of the FAS signaling pathway to increase FAIM2 expression may be a potential therapeutic option to prevent photoreceptor death.³⁹ 

Nevertheless, the inhibition of the apoptotic process induced by a noxious stimulus does not completely prevent retinal cell death, thus indicating that many cells can enter other mechanisms of cell death, such as necroptosis. Additional pathways including autophagy and inflammation can also contribute to the loss of retinal cells, as shown in different disease models.³²  In retinal degenerative diseases such as retinitis pigmentosa, both apoptosis and necroptosis can be triggered simultaneously and sometimes by the same death stimuli, but with distinct biochemical and morphological features.^40–43  Necroptosis is the regulated form of necrotic death, is caspase-independent and is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which translocates to membranes and promotes cell lysis. It is considered a pro-inflammatory form of cell death, because rupturing of the cell releases intracellular contents that can stimulate innate immune cells, so it is believed to be a more potent inducer of inflammation than apoptosis, although additional in vivo studies are needed to confirm this statement. The precise mechanisms determining the decision whether a cell will die by apoptosis or necroptosis are not yet fully understood.^40,44–47  To date, no suitable inhibitors of the necroptosis pathway with activity in the CNS of mice or humans have been identified and more research in this field is needed. Blocking necroptotic pathways with synthetic inhibitors or by genetic manipulation relieves neurodegenerative disease both in vitro and in vivo, which suggests a promising therapeutic strategy for neurodegenerative diseases.⁴⁸ 

The third major route of cell death, autophagy, also has a main role in sustaining retinal and eye function, and alterations in autophagy and lysosomal pathways are involved in many, if not all, diseases of the eye. Autophagy is evolutionarily a well conserved process from early eukaryotes to mammals. It is generally considered an anti-inflammatory and pro-survival process. It is a lysosome-mediated degradation process that inhibits the accumulation of misfolded proteins and damaged cytoplasmic organelles and removes microorganisms, contributes to the maintenance of metabolic homeostasis and provides energy and recycling macromolecules in response to nutrient and environmental stress.^30,49–52  In CNS diseases, autophagy is a key point in the control of inflammation and production of cytokines by activated microglial cells. Autophagy modulates inflammatory responses through interactions with immune signaling pathways and regulates the secretion of molecular mediators of inflammation in a context-dependent manner.^53,54  Autophagy is also involved in the regulation of proteins relevant for cellular anti-oxidative defence, including Nrf2. In this context, it has been proposed that autophagy is an adaptive response that might confer protection against persistent inflammation in the retina during aging.⁵⁵  The decline in lysosomal activity associated with age exacerbates alterations in autophagy, potentially aggravating related conditions. While appropriate autophagy contributes to neuroprotection, inappropriate autophagy could induce cell death and its dysfunction has been related to several neurodegenerative diseases such as Parkinson's or Alzheimer's diseases, among other neuron-affecting pathologies.^54,56,57  In a neurodegenerative process, a mild induction of autophagy should protect cells from damaged proteins and organelles.^57,58  Autophagy is active in the retina from the developmental stage, during which it is primarily implicated in cell death processes. Autophagy plays an important role in recycling photoreceptor outer segments, avoiding lipofuscin accumulation, preventing aggregation and minimizing oxidative and endoplasmic reticulum (ER)-stress, preserving mitochondrial function, attenuating inflammasome activation in the retinal pigment epithelium (RPE) and minimizing ROS levels and sustaining mitochondrial function in retinal ganglion cells (RGCs).⁵¹  Autophagy is involved in AMD pathogenesis, and it has been proposed that autophagy-related genes and proteins can be molecular targets for prevention and therapy of this disease.⁵⁹ 

ROS prompt apoptosis and autophagy pathways, and stress-specific recruitment of autophagy pathways with cytoprotective function.⁶⁰  mTOR, the mammalian target of rapamycin, has a main role in many degenerative diseases as one of the main regulators of autophagy.³  In this context, work has been published showing that pharmacological treatment with the mTOR inhibitor rapamycin preserved photoreceptor function in metabolic and oxidative stress mouse models that exhibited RPE mitochondrial dysfunction accompanied by activation of the AKT/mTOR pathway.⁶¹  It has also been shown that phosphoinositide 3 kinase (PI3K)/Akt and mTOR/p70S6K pathways play a major role in protection against oxidative stress-induced apoptosis in the ARPE-19 cell line.⁶²  The mTOR pathway plays an exquisitely complex role in the regulation of retina protein biosynthesis, RNA translation and ER stress-induced apoptosis. mTOR signaling can be suppressed by the inhibition of serine/threonine kinase Akt, which leads to the induction of autophagy.³³  Degradation and inhibition of mTOR attenuates retinal degeneration.⁶³  Thus, mTOR pathway inhibition has been suggested as a therapeutic strategy for retinal degenerative diseases involving oxidative stress, although it has showed a different response according to the degree of oxidative stress imposed.^2,64  However, clinical trials to assess the effect of rapamycin, an mTOR inhibitor, for the treatment of geographic atrophy reported no clear efficacy and some doubts concerning its safety.^65,66  Although rapamycin's side effects, such as immuno-suppression at early stages of treatment prevented its use in treating neurodegenerative diseases, small molecule enhancers of rapamycin (SMERs) which increase autophagy independently of mTOR pathway have been suggested as potential therapeutic agents to treat neurodegenerative disorders.^3,67 

Other molecules can increase pro-survival signals and have therapeutic potential for retinal degeneration. Calcium channels inhibitors such as amlodipine or amiloride activate the pro-survival kinase Akt, decrease calpain activity, attenuate JNK/c- Jun activation and can improve RGC survival after optic nerve crush, so they could be useful tools for therapeutic interventions in traumatic and degenerative CNS disorders.⁶⁸ 

Another regulator of autophagy is p53. While the apoptotic activity of p53 is well recognized, several reports have highlighted the functional link between p53 and autophagy. The role of this protein varies depending on its subcellular location.^34,45  Moreover, p53 has been demonstrated to have a neuroprotective role in a Drosophila model of tauopathy, by controlling synaptic genes, so it has been proposed that p53-related molecules could have potential therapeutic value in Alzheimer's disease and related neurodegenerative disorders.⁶⁹ 

Glycogen synthase kinase 3 (GSK3) is a critical central figure in many cellular signaling pathways. It is a multitasking serine/threonine kinase that regulates many cellular functions, such as structure, gene expression, mobility and apoptosis, with more than 100 known substrates to deal with. GSK3 promotes p53-induced apoptosis forming a complex with nuclear p53.^70,71  The inhibition of GSK3 was proved to be one of the mechanisms by which PI3K activation protects neurons from programmed cell death, and it has been proposed that GSK3 be included to the list of potential drug targets for pharmacotherapy of neurodegenerative disorders.⁷²  There are strong connections between GSK3 and pathophysiology and/or therapeutics of a large number of prevalent diseases that share the common feature of chronic inflammation, as GSK3 is an important positive regulator of the inflammatory process.⁷³  In this sense, inhibitors of GSK3 may provide a therapeutic strategy to control inflammation⁷⁴  and could also be useful therapeutic agents for the treatment of degenerative diseases such as retinitis pigmentosa.⁷⁵ 

Retinal degenerative diseases entail a non-specific disease-remodeling phenomenon accompanied by common mechanisms of cell stress, harmful and pro-survival signals. The ability of the cell to increase life stimuli and to control death ones tilts the balance towards survival or death. Our help in any of these aspects can have potential therapeutic value to prevent retinal degeneration.

All vertebrate retinas consist of the neural retina (or neuroretina) and the pigment epithelium, which separates the neural retina from the choroid. The neural retina is made up of three nuclear layers, which contain the cell bodies (or somas) of retinal neurons, and two plexiform layers, in which the synaptic contacts are established.⁷⁶  The outer nuclear layer (ONL) is formed by the cell bodies of photoreceptors (cones and rods); the inner nuclear layer contains the cell bodies of bipolar, horizontal and amacrine cells; and the ganglion cell layer consists of the cell bodies of ganglion and displaced amacrine cells. Between these three layers of cell bodies there are two plexiform layers where the synaptic contacts take place: the outer plexiform layer (OPL),⁷⁷  where the synaptic contacts between the axon terminals of photoreceptors and the dendrites of their two postsynaptic neurons, bipolar and horizontal cells, are established; and the inner plexiform layer (IPL), with synaptic connections between bipolar, amacrine and ganglion cells. In the IPL there is a horizontal flow of information through a large variety of amacrine cells, which together with the bipolar cells integrate the information that reaches the ganglion cells that transmit the information received from bipolar and amacrine cells to the brain via the optic nerve.^76,78 

Animal models of retinitis pigmentosa have an early onset of retinal degeneration. Retinitis pigmentosa-associated genes are primarily expressed in rods. Consequently, this disease is characterized by an initial loss of rods followed by a progressive mutation-independent cone death. In this context, the extensive downstream of neuronal changes that accompany and follow photoreceptor degeneration has been well-established.¹ 

One of the first signs of photoreceptor cell impairment is the mislocation of proteins with specific subcellular distribution. Rhodopsin, the rod visual pigment, is localized in the outer segments of rod photoreceptors in healthy retinas. However, during retinal degeneration it is common to observe an abnormal accumulation of this protein in the inner segments, cell bodies and axon terminals of rods (Figure 1.1A and B). A possible explanation for this irregular distribution is that rhodopsin synthesis continues after photoreceptor outer segments are lost due to the degenerative process.⁷⁹  Thus, the newly synthesized rhodopsin cannot be translocated to its normal cellular destination, therefore accumulates near the site of synthesis, in the photoreceptor cell soma. The same event can be observed in proteins involved in neurotransmission and synaptic vesicle formation and trafficking, such as synaptophysin and the vesicular glutamate transporter 1 (VGluT1) whose normal distribution is throughout photoreceptor axon terminals (spherules and pedicles) and under degenerative conditions can be found in the cellular bodies.⁸⁰  Another prominent feature of photoreceptor degeneration is the progressive outer segment shortening (Figure 1.1A–C), which is also observed in retinal histologic studies in retinitis pigmentosa patients.^81–83  Sprouting of rod processes has also been noted in human retinas with retinitis pigmentosa⁸³  and in animal models of this disease, i.e. rhodopsin transgenic pigs⁸⁴  and cats with autosomal dominant retinal dysplasia.⁸⁵ 

As mentioned earlier, as rod degeneration evolves, cone morphology undergoes progressive changes, including the shortening of outer and inner segments and axons (Figure 1.1D–G).^1,86,87  Additionally, the progressive loss of photoreceptors within the ONL causes the appearance of hypertrophied side branches of Müller cells into the outermost photoreceptor layer. Apical Müller cell processes rearrange in clusters forming firework-like structures. This peculiar disposition of cones and apical Müller cell processes has been described in the S334ter-line-3 ^88,89  and the P23H^90,91  rat models of retinitis pigmentosa.

The previously described early onset of morphological retinal changes correlate well with altered electroretinogram (ERG) measurements. In concordance, abnormal rod ERG function was detected at an early age in P23H rats⁹²  and ERG impairment is an early clinical manifestation in autosomal dominant retinitis pigmentosa patients, even without subjective symptoms.^1,93 

As death of rods and cones progresses, bipolar and horizontal cells, the second-order neurons in the retinal circuitry, become deafferented and display early retraction and loss of dendrites^1,80,94,95  (Figure 1.2). Additionally, in the P23H animal model of retinitis pigmentosa, it has been observed that bipolar and horizontal cells seek out new functional photoreceptors with which to make contacts, thus extending their dendrites (sprouting).^1,94,96  These results resemble closely those seen in the retina of the rd-mutant mice.⁹⁷  Therefore, the loss of synaptic connections with photoreceptor cells evokes atrophy and connective re-patterning in their postsynaptic cells.^80,86 

As described earlier, in the context of retinal degeneration bipolar cells undergo dramatic morphological changes accompanying photoreceptor loss. The first obvious changes are the underdevelopment of rod bipolar and horizontal cells dendrites. This is not surprising, since rod bipolar cells receive direct input from rod photoreceptors. These cells are presumably more vulnerable to photoreceptor degeneration at the time of synaptogenesis. As retinal degeneration progresses, rod bipolar cells axonal endings become atrophied, cell bodies lose their normal lamination pattern, become disorganized and, eventually, die (Figure 1.2A and B). The loss of the depolarizing ON rod bipolar cell population is well reflected by the absence of the ERG b-wave in the P23H rat.⁸⁶  In a similar way, cone bipolar cells undertake morphological impairment during degeneration (Figure 1.2C and D).

Horizontal cells also show early signs of degeneration as rod photoreceptors are lost. Horizontal cell dendrites connect with cones and the axon terminals contact with rods in the OPL. Thus, morphological changes in horizontal cell axon terminals would be expected. The dendritic arborization of horizontal cells is abnormal and form a weaker network in P23H rat retinas, compared to normal retinas (Figure 1.2E and F). In addition, horizontal cell dendrites appear compromised and seem to become condensed with aberrant rod bipolar cell dendrites.⁸⁶  With the progression of retinal degeneration, most of the horizontal cell dendrites are lost, cell bodies become inverted and disorganized and the axon terminals appear to sprout into the ONL as though looking for any remaining degenerate rod spherule. These changes are very similar to those seen in transgenic rd mice by Strettoi et al.⁹⁷ 

The number of retinal neurons decreases progressively as the retinal degeneration proceeds. In concordance, the surviving neurons interconnect in clusters that are uniformly distributed throughout the OPL, establishing new contacts as part of the remodeling process. In this context, the scarce dendrites of rod bipolar cells, which express the metabotropic glutamate receptor mGluR6, partake in these synapses. The remaining dendrites of horizontal cells associated with the presynaptic protein Bassoon are confined within the aforementioned clusters of synaptic contacts, as well as the contacts between cones and rod bipolar cell dendrites. In the absence of rods, rod bipolar cells receive ectopic synapses from cones, as shown in electron microscopic studies performed in different animal models of photoreceptor degeneration, including the P347L pig,⁹⁸  rd mouse⁹⁸  and RCS rat.⁹⁹  Bassoon and synaptophysin, two presynaptic proteins, also downregulate their expression during retinal degeneration (Figure 1.3).

The cartoon in Figure 1.4 summarizes the changes that take place in horizontal and bipolar cells at the OPL level after photoreceptor degeneration.

There is an obvious reduction in the thickness of the IPL after the onset of rod photoreceptor degeneration. This is almost certainly due to the shrinking of the rod bipolar axon terminal in the stratum S5 of the IPL. Cone bipolar cell axons also reduce their size and show morphological impairments. The AII amacrine cells undergo morphological modifications as photoreceptors degenerate. The AII amacrine cells relay rod information from rod bipolar cells to cone bipolar cells and ganglion cells, so the early modification of rod bipolar cells would naturally affect AII amacrine cells.⁸⁰  With the progression of retinal degeneration, their lobular appendages get smaller and disappear, and eventually there is complete cell dropout. Like the attenuation of the b-wave of the ERG with the loss of rod bipolar cells, the loss of AII amacrine cells might be reflected in diminution of any scotopic oscillatory potentials.¹⁰⁰ 

The decrease in density of AII amacrine processes, with a simultaneous loss of certain populations of amacrine cells and, eventually, even ganglion cell dendrites, induces the decrease of thickness of this layer. The IPL becomes filled with sprouting Müller cell processes to replace the lost neural synaptic complexes. Simultaneously, the blood vessels become distorted, possibly by Müller and glial cell proliferation. The blood vessels rearrange the previously ordered cell and neuropil layering and, eventually, pull ganglion cell axons into the inner retina. These changes in the IPL due to neuron and Müller cell atrophy and blood vessel attenuation are seen in human specimens of advanced retinitis pigmentosa¹⁰¹  and have been described in other animal models of retinal dystrophies.¹⁰²  In addition, ganglion cells undergo a series of changes after the loss of photoreceptors, and their dysfunction and death has been linked to altered retinal glial cell function.¹⁰³ 

Virtually all forms of retinal injury or disease trigger reactive gliosis (reviewed in Cuenca et al.¹ ). Retinal neurodegenerative diseases are associated with chronic microglial activation and neuroinflammation. In the degenerating retina, endogenous signals activate microglial cells, which in turn can proliferate, migrate, enhance phagocytosis and secrete cytokines, chemokines and neurotoxins (Figure 1.5A and B). These immunological responses and the loss of limiting control mechanisms may contribute significantly to retinal tissue damage and pro-apoptotic events in retinal dystrophies.^1,104–106 

It remains unknown whether microglia activation is a cause or a consequence of neuronal damage. In the CNS, microglia have protective functions and contribute to the secretion of trophic factors, anti-oxidants and cytokines and to the removal of cellular debris. In the first stages of retinal neurodegeneration, microglia trigger repair mechanisms such as glial scar formation, which demarcates the lesion area and separates the injured tissue from its surroundings, promoting neuronal survival. But microglia chronically activate release noxious factors that mediate neuroinflammation and toxicity.^{1,104,105,107}  Microglia produce a plethora of inflammatory mediators, including cytokines, chemokines, trophic factors and small molecules, that promote and perpetuate the inflammatory response, potentially leading to neurodegeneration and photoreceptor death.^1,3,16–18  In this context, it has been published that cytokines play roles as mediators and modulators of diverse forms of neurodegeneration in the CNS, including the retina.^19,20  Due to the dual role of microglia activation in the progression of neurodegenerative diseases, both protecting and harmful, doubts remain regarding the optimal therapeutic intervention to halt or reduce the degeneration. On one hand, the selective inhibition of the overactive microglial activity and the preservation of their trophic and homeostatic functions seems a promising treatment for degenerative diseases. In this sense, minocycline, a semi-synthetic tetracycline analog, is an inhibitor of microglial activation, it counter-regulates microgliosis and is proven to have potent anti-inflammatory and neuroprotective effects in mouse models of retinal degeneration, indicating a promising concept for the treatment of retinal pathologies.^108–111  Clinical trials are under development to evaluate minocycline safety and efficacy on AMD patients (ClinicalTrials.gov identifier: NCT02564978). However, the inhibition of the protective effect of microglial activation could have harmful consequences because reactive microglia also participate in regenerative processes by removing dendritic structures, cell debris and glutamate, as well as releasing protective molecules (reviewed by Cuenca et al.,¹  Langmann¹⁰⁴  and Polazzi and Monti¹¹² ).

In the early stages of degeneration, macroglial cells, which include astrocytes and Müller cells, also become activated as part of the gliosis process (Figure 1.5C–F). Activated microglia induce macroglia, including astrocytes and Müller cells, and RPE to secrete cytotoxic factors such as tumor necrosis factor (TNF)-α or interleukin (IL)-1β, which act synergistically, promoting chronic neuroinflammation and collaborate to neuronal cell death and retinal degeneration.^113,114  While reactive gliosis has a direct neuroprotective effect on the retina, its chronicity exacerbates disease progression, increasing vascular permeability, infiltration of toxic compounds and neovascularization.^115,116 

The fractalkine (CX3CL1)/CX3CR1 signaling pathway has a relevant role in the control of retinal inflammation and hence in the pathogenesis of retinal inflammatory and neurodegenerative diseases of the retina.^117–119  This pathway protects neurons by regulating retinal microglial activation and migration. Fractalkine modulates TNF-α and IL-6 secretion and neurotoxicity induced by microglial activation^120–123  and can reduce neurotoxicity and microglial activation in neurodegenerative models.¹²⁴  In rd10 mice, a synthetic progesterone analog (norgestrel) upregulates fractalkine-CX3CR1 signaling and has dual actions as a neuroprotective and anti-inflammatory agent in the retina, becoming a promising treatment for retinitis pigmentosa.^25,125 

Current therapeutic approaches used to protect photoreceptors and slow down degeneration include the administration of neurotrophic factors. Some of them, such as methyl 3,4-dihydroxybenzoate (MDHB), can exert their protective actions at least in part by reducing reactive gliosis,¹²⁶  apart from other anti-angiogenic, neuroprotective and anti-inflammatory activities,^1,127  and can have a role in retinal therapy.¹²⁸ 

Due to the high metabolic activity of the retina and its great oxygen demand, the supply of blood and the ability to regulate blood flow is a key point for retinal health. All degenerative diseases affect retinal vascularization.¹  In pathological conditions, astrocytes and Müller cells are involved in the development of new vascularization by releasing angiogenic factors, such as the vascular endothelial growth factor (VEGF), in response to pathogenic stimuli.^8,129–131  Anti-VEGF factors such as bevacizumab have been proposed and tested as therapeutic tools in degenerative diseases, and clinical trials with aflibercept, bevacizumab and ranibizumab have shown good results when treating patients with retinal degeneration.^22,132  Unfortunately, to date, anti-VEGF factors cannot rescue the normal retinal vasculature and have serious drawbacks, for example in the treatment of infants affected by retinopathy of prematurity.^133–135  For patients with a suboptimal response to anti-VEGF therapy, a multimodality approach has been proposed, as the addition of the corticoid dexamethasone can induce better results.¹³⁶  It has been proposed that revascularization by using proangiogenic cells can be an option for therapeutic revascularization in the treatment of retinopathy of prematurity.¹³⁷ 

Following photoreceptor degeneration, the inner retina becomes hyperoxic. In consequence, there is a reduction of VEGF expression and a loss of the deep capillary plexus.^1,138  The disruption of the deep capillary plexus alters the normal nutrient and oxygen supply to retinal cells, and thereby accelerates the progress of retinal degeneration. Retinal vasculature remodeling has been evidenced in retinitis pigmentosa models.^1,138  In conclusion, the loss of photoreceptors in retinitis pigmentosa induces changes in retinal vasculature, which in turn may induce the activation of astrocytes and Müller cells, which can induce inner retinal remodeling,^{1,86,139–141}  including ganglion cell degeneration.^101,142,143 

Independently of the etiology of the damage, retinal neurodegenerative diseases result in morphological and functional impairment of retinal cells that lead to well-established physiological changes affecting vision. Reliable assessment of visual capacity can provide key information about the mechanisms and progression of the degenerative events associated with retinal diseases.

The recording of electrical responses of the eye to light stimulus constitutes the ERG. The negative a-wave mainly represents light-evoked hyperpolarization of photoreceptors in the outer retina,^144,145  whereas the positive b-wave indirectly represents the activity of the on-bipolar cells.^146–150  The oscillatory potentials are rapid oscillatory responses superimposed on the ascending b-wave of the ERG.^151,152  Although the specific cellular origins of the oscillatory potentials have yet to be established, early oscillatory potentials have been associated with the activity of photoreceptors and bipolar cells in the outer retina, whereas later oscillatory potentials are attributed to activity in amacrine and ganglion cells in the inner retina.^100,153,154 

Even though considerable variability exists in the onset and evolution of retinal diseases, ERG recordings can be used to distinguish common abnormalities. The amplitude and implicit time of the ERG components reflect morphological alterations occurring during degenerative processes, including the progressive loss of photoreceptors and synaptic dysfunction in both the OPL and IPL. In patients with retinitis pigmentosa, ERGs characteristically show reduced a- and b-wave amplitudes, as well as delayed rod and/or cone b-wave implicit times.^155,156  In contrast, the ERGs of patients with cone dystrophy characteristically exhibit normal, albeit slower, rod b-waves and reduced or absent cone ERG responses.¹⁵⁷  In addition, ERG recordings provide a means to assess the progression of retinal degeneration in animal models^86,94,158  and to evaluate the therapeutic effects of neuroprotective agents on them.^159–163  In this context, it has been shown that the number of rows of photoreceptor nuclei positively correlates with the amplitude of the scotopic b-waves recorded in P23H rats,^160,162  a model of retinitis pigmentosa. Furthermore, the thickness of the ONL was found to be proportional to the scotopic a- and b-wave amplitudes in a rotenone rat model of Parkinson's disease.¹⁶⁴ 

The analysis of oscillatory potentials has been shown to be very useful in the study of retinal diseases.^{100,165–167}  Oscillatory potentials are a good indicator of the functional integrity of the microcirculation of the inner retina and are drastically affected by acute disturbances occurring in areas supplied by central retinal vessels.^167,168  Thereby, abnormal oscillatory potential responses reveal a pathological microcirculation in the inner retina.¹⁰⁰  However, oscillatory potentials have also been found to be aberrant in outer retinal diseases. In retinitis pigmentosa, characterized by a primary degeneration of the outer retina, diminished or delayed oscillatory potentials have been detected.^169,170 

Ganglion cells have very little contribution to the scotopic ERG responses to bright stimuli. However, the dark-adapted ERG response to very weak light flashes (scotopic threshold response; STR) depends directly upon ganglion cell function.^171,172  The negative component of these scotopic responses (nSTR) likely reflects primarily amacrine cells function, while the positive component (pSTR) has been postulated to reflect primarily retinal ganglion cell function.^173,174  Thus, STRs are reduced or extinguished after substantial retinal ganglion cell loss associated with glaucomatous damages^{171,172,175,176}  or excitotoxic insults.¹⁷⁷ 

The main limitation of the global or full-field ERG is that the recordings do not permit efficient mapping of retinal responsiveness for the detection of small dysfunctional areas. In this sense, the multifocal (mf)ERG allows concurrent testing of ERG activity in a large number of retinal locations, which enables functional mapping of the retina.^178,179  The mfERG is mainly used in the clinic to localize damage spatially, in such a way that variations in the topographic array of signals are more important than the absolute signal size. The mfERG is particularly valuable in cases in which the fundus appears normal and it is difficult to distinguish between diseases.^180,181  Thereby, the mfERG has been shown to be useful to evaluate maculopathies, Best disease, Stargardt disease, diabetic retinopathy and AMD.¹⁸² 

VEPs consist of electrophysiological signals recorded from the electroencephalographic activity in the visual cortex in response to visual stimulation.^183,184  Since the visual cortex is activated primarily by visual inputs, VEPs depend on the functional integrity of each element of the visual pathway, including the retina, optic nerve, optic radiations and occipital cortex.¹⁸⁴  In addition to detecting disorders of the anterior visual pathway, posterior visual pathway dysfunctions can be assessed by analyzing the VEPs.¹⁸⁵  Thus, VEPs can be valuable in the diagnosis of optic neuropathies and non-organic visual loss, as well as in the assessment of visual function in infants or children.¹⁸⁶  Moreover, VEP results can be predictive of visual recovery in traumatic optic neuropathy¹⁸⁶  and VEP evaluation is used to detect subclinical demyelinating lesions in multiple sclerosis.¹⁸⁷ 

The multifocal (mf)VEPs provide local VEP responses from the visual field. Thus, mfVEPs allow the identification of spatially localized damage and pathologies that may be missed with a traditional single VEP, and facilitates investigations on structural–functional correlations.^180,188,189  The mfVEP is used to study visual field defects caused by ganglion cell or optic nerve damage^190–193  and has been considered a powerful tool for the identification of small, peripheral lesions in glaucoma, for example.^194–197  Additionally, mfVEP can be applied as a valuable method to detect visual pathway involvement in multiple sclerosis.¹⁹⁸ 

Visual acuity is the most frequently performed measure of visual function in clinical practice. Visual acuity measures the ability of the visual system to discriminate between two stimuli separated in space, with a high contrast in relation to the background.^199,200  Thus, visual acuity represents a practical tool for tracing the course of ocular disorders and therapy.²⁰¹ 

More recently, contrast sensitivity testing has been proposed as a valuable tool for psychophysical assessment of visual function. Contrast sensitivity measures how much contrast a person requires to see a target²⁰²  and concerns many aspects of vision, including motion detection, visual field, pattern recognition, adaptation to darkness and visual acuity.²⁰³  Low-contrast sensitivity is not specific to any particular disease, as many visual disturbances have similar effects on the contrast sensitivity function. Nevertheless, the testing of contrast sensitivity is a valuable tool for detecting eye disease and assessing the efficacy of treatment.²⁰³  Contrast sensitivity tests have been useful for evaluating intraocular lenses, cataract surgery and ophthalmic pathologies such as glaucoma, optic neuritis, diabetic retinopathy or AMD.^203–209  Conversely, the correlation between impaired contrast sensitivity and degenerative dementias has been demonstrated;^210,211  hence contrast sensitivity tests may be a useful biomarker for neurodegenerative diseases.

Visual function in animals has been evaluated using different approaches. The optomotor test enables the generation of a psychophysical threshold in a reduced period of time, and does not involve the failure of older animals to learn a task.^212–214  Pigmented animal responses are stronger and easier to recognize than those of albino mice or rats, which do not show clear responses to the optomotor test.²¹⁵  The optomotor test has been used successfully as a visual test in different animal models of retinal degeneration.^209,216 

Morphological and functional changes associated with retinal degeneration affect neuronal cells, glia and retinal vasculature, a process that ends in a generalized retinal remodeling that ultimately leads to blindness (Figure 1.6).^{1,86,94,97,99,217}  The remodeling process is independent of the initial death stimulus affecting rod cells and has been observed in many models of retinitis pigmentosa. In several studies performed in our lab we have observed that none of these changes in the inner retina or the subsequent synaptic remodeling are exclusive features of retinitis pigmentosa. Instead, this set of changes has also been found in other retinal diseases, such as metabolic disorders,^218,219  animal models of ocular hypertension^220,221  and even in human organotypic cultures,²²²  where retinal remodeling had also occurred at the OPL level. Several reviews about retinal remodeling are available.^{1,101,139,141} 

All the aforementioned mechanisms of photoreceptor cell death trigger a vast repertoire of cellular responses of other retinal cells. Understanding these responses may be extremely helpful in improving and orienting current therapies.

Development of treatments that provide novel ways of replacing photoreceptors assumes that the inner retina remains intact, and that ganglion cells (the output neurons of the retina) remain capable of transmit the electrical signals to higher cortical areas. However, animal models of retinal degeneration and retinitis pigmentosa patients show significant alterations and plastic neuronal changes in the inner retina. Preserving the inner retina degeneration after photoreceptor loss is crucial for the optimal success of new therapeutic strategies such as electronic retinal implants (bionic eye), optogenetics, gene therapy or stem cell transplantation.

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness-FEDER (BFU2015-67139-R), Instituto de Salud Carlos III RETICS-FEDER RD16/0008/0016, Organización Nacional de Ciegos Españoles (ONCE), FUNDALUCE and Generalitat Valenciana, PROMETEO/2016/158.