Genes commonly regulated in glaucoma and after IONC. ↑ gene upregulation, ↓ gene downregulation. See text for further explanations.
After receiving their visual input from photoreceptors via the intermediate neurons (bipolar, horizontal and amacrine cells), retinal ganglion cells (RGCs) relay this intraretinally pre-processed visual information to the centres in the brain for further processing. Thus, RGCs are the only projecting neurons in the retina, and their axons form the optic nerve. These central nerve axons originate at the RGC bodies located in the neural retina, travel along the nerve fibre layer converging to the optic disk and finally exit the eye through the optic nerve head (ONH).
Glaucomatous optic neuropathy is a complex, multifactorial and heterogeneous human chronic neurodegenerative disease that characteristically affects theRGC population and their axons. It is a slowly progressive form of optic nerve damage and blindness that begins with loss of peripheral vision and is followed by gradual narrowing of the remaining central vision.
Glaucoma currently affects over 70 million people (Quigley, 1996). Second only to cataract is the leading cause of irreparable blindness. This diseaseaffects all age groups, but is more frequent in the elderly (Quigley & Vitale, 1997). Glaucoma is broadly classified into three main groups, i) primary open glaucoma (POAG); ii) primary acute closed angle glaucoma; and iii) primary congenital glaucoma. Among these, the most frequent is POAG (Shields et al., 1996). In this type of glaucoma the main risk factorsare age and an elevated intraocular pressure (IOP). In most cases, lowering the IOP has a beneficial effect on RGC survival. However, some individuals with low or even normal IOP develop this disease with associated RGC loss, yet others with high IOP do not develop glaucoma or lose these neurons. Because of this, it has been proposed that there are genetic variants in humans that affect the relative susceptibility or resilience of RGCs to the same insult. This hypothesis is based on recent works showing that RGCs from different mice strains have an intrinsically different resistance to optic nerve injury. Li et al (2007) analyzed the survival of RGCs in 15 mice strains after optic nerve crush, and found that RGCs from the DAB/2J strain were the most resistant, while the ones from BALB/cByJ were the most susceptible. Interestingly, this rank of relative vulnerability to optic nerve crush does not translate to other lesions or regions of the CNS, which means that the genetic background has a complex effect on the resistance to injury of specific neurons from specific areas. In addition to this putative implication of the different human genotypes on developing glaucoma, there are 25 loci that have been found linked to POAG. However, only 3 genes are known to cause glaucoma if mutated (
Two of the more popular hypotheses to explain the initial glaucomatous damage to the ONH are the mechanical and the vascular theory of glaucoma. The mechanical theory states that the raised IOP obstructs RGC axons, thus causing their degeneration and death. With respect to the vascular theory, there is evidence of vascular dysregulation in some forms of glaucoma (Flammer & Mozaffarieh, 2007) that may endanger ONH blood flow. Thus, this might induce an ischemia-reperfusion nerve injury. Additionally, the role of glial cells at the ONH has received increasing attention (Ramirez et al., 2010; Nguyen et al., 2011).
2. Animal models of glaucoma
The high incidence of glaucoma, and the lack of non-invasive approaches to study in humansthe subjacent neuronal responses, make necessary the use of animal models to understand the pathogenesis of this neuropathy (for a more detailed account of these models see Morrison et al., 2010).
2.1. Spontaneous, transgenic or deficient mice
Some mice strains have spontaneously developed mutations that produce a chronic elevation of the IOP, and result in a phenotype similar to chronic age-related glaucoma. The best studied is the DAB/2J inbred line (John et al., 1997, 1998). This strain is homozygous for two mutations related to melanosomes accumulation. Thus, it has been suggested that the initial pathology of the anterior chamber is due to an abnormal iris pigmentation and melanosome structure (John, 1998) though it is not the only cause as there is also an immune component involved (Chang et al., 1999). These mice start to exhibit increased IOP by 8-13 months old. The course of the disease, however, varies on individual colonies and environmental factors.
Mice can be genetically manipulated, and so transgenic mice have been generated targeting proteins implicated in the aqueous outflow or those for which human mutations related to glaucoma have been described. Examples of such transgenic mice are the ones with the targeted mutation in the α1 subunit of collagen type 1 (Aihara et al., 2003) or the ones expressing the Tyr437His mutation in myocilin(Zhou et al., 2008), associated with human patients developing early onset glaucoma (Alward et al., 1998). Recently, it has been reported that mice deficient of Vav 2 and/or Vav 3 proteins (guanine nucleotides exchange factors for Rho guanosine triphosphatases) show early onset of iridocorneal angle changes and elevated intraocular pressure (Fujikawa et al., 2010). Interestingly,
The majority of the induced animal models of glaucoma are based on experimental elevation the IOP. Most of these models are carried out in rats and in mice. There are some advantages of these experimental models over the spontaneous models. First, the onset of the elevated IOP is predictable, making possible to determine the temporal course of the degenerative events in retina and optic nerve. Second, unilateral IOP elevation leaves the fellow eye as internal control, hence accounting for inter-individual variability. Third, IOP increase occurs soon after the treatment, and thus it is not necessary to wait long periods for the animal to develop the symptoms. However, these models have in common a high variability, in terms of the IOP reached and the damage induced.
The goal of any experimental procedure to elevate the IOP is to inhibit aqueous humour outflow without interfering with the eye’s ability to produce this humour. There are four main approaches, scarring the anterior angle chamber by hypertonic saline injection (rats, Johnson et al., 1996; Morrison et al., 1997; mice, Kipfer-Kauer et al., 2010) laser photocoagulation (rats and mice, Salinas-Navarro et al., 2009a, 2010, Cuenca et al., 2010), venous cautery (Shareef et al., 1995) and injection of microbeads in the anterior chamber (mice, Cone et al., 2010; Chen et al., 2011; rats and mice, Sappington et al., 2010).
Laser photocoagulation, a method first described in monkeys (Gaasterland & Kupfer, 1974) and later in rats (Ueda et al., 1998), is based on the photocoagulation of the trabecular mesh alone or in combination with episcleral veins and/or perilimbar vessels (Wolde Mussie et al., 2001; Levkovitch-Verbin et al.,2007; Salinas-Navarro et al.,2009a,2010, Cuenca et al., 2010).The IOP increase occurs because the drainage of the aqueous humour is obstructed by the closure of the intratrabecular spaces and of the majority of drainage channels. In this model, RGC loss occurs mainly in pie-shaped sectors located in the dorsal retina, with their apex pointing to the optic nerve disk. It has been described that there is first an impairment of the anterograde active axonal transport, followed by a retrograde degeneration of RGCs (Salinas-Navarro et al., 2009a, 2010).
3. Intraorbital optic nerve crush (IONC)
Another good approach is to study separately the RGC response to axotomy. Intraorbital optic nerve crush (IONC) is based on crushing the entire retinofugal pathway at the exit of the eye. This model specifically injures all the RGC axons, avoiding any ischemic insult. Thus, as a model to study the underlying causes of RGC death by axonal injury, IONC is clean and predictable.
Traumatic axonal injury to the optic nerve, either by complete transection or crush, is a well established model (Bray et al., 1987) that can be evaluated morphologically (Nadal-Nicolas et al., 2009; Parrilla-Reverter et al., 2009a, 2009b), molecularly (Agudo et al., 2008, 2009) and functionally, and has been thoroughly analyzed by our group, both in rats and mice (Alarcon-Martinez et al., 2009, 2010). These lesions induce a quick and massive death of RGCs (Peinado-Ramon et al., 1996; Chidlow et al., 2005a; Sobrado-Calvo et al., 2007; Agudo et al., 2008, 2009; Galindo-Romero et al., 2011; Sanchez-Migallon et al., 2011). After IONC, the whole RGC population is injured and degenerates within a short period of time. In addition, as long as the lesion is inflicted at the same distance from the optic disk (Villegas-Perez et al., 1993), there is a small variability among different animals.
IONC has several advantages: i) the insult affects the whole RGC population. This is important because the population of RGCs only represents approximately 1% of the retinal cells, thus, there is a dilution effect that may hinder the study of protein or transcript regulation in the affected RGCs. This dilution effect is higher if only part of the RGC population is affected by the lesion, as it happens in the increased IOP models (Guo et al., 2010); ii) the temporal course of RGC degeneration is well established; iii) there is a body of reports in which the molecular correlates associated to this injury have been studied(Agudo et al., 2008, 2009). Importantly, this lesion mimics the crush-like injury observed in the optic nerve after IOP increase (Salinas-Navarro et al., 2009a, 2010) but leaves aside the possible ischemic insult, thus easing the understanding of the obtained data.
3.1. Early signs of retinal ganglion cell degeneration
Degeneration of CNS axons after a traumatic insult was first described by Ramón y Cajal (Ramón y Cajal 1914) and his co-workers (Tello 1907; Leoz y Arcuate 1914). They observed many degenerative changes such as axons that broke up rapidly and axons ending in club-like shapes known as terminal swellings, ganglioform swellings or cytoid bodies. Later, in 1967, Reimer and Wolter (Reimer & Wolter, 1967) after analyzing the retinas of 3 patients with ocular or systemic diseases and the cordotomized spinal cord of a fourth patient, concluded that these degenerative events were the common reaction of CNS axons to damage. These authors stained the axons with the silver-carbonate or silver nitrate methods. Nowadays axons are identified by immunodetecting proteins specifically expressed by them. Furthermore, because specific proteins (or their isoforms) are expressed in specific neuronal compartments, it is possible to study them separately. Neurofilaments (NF) are the main cytoskeletal proteins in mature axons. They are assembled from three subunits, high (H), medium (M) or low (L). NF suffer post-translational modifications wherein the most relevant is phosphorylation. Phosphorylation of NF, particularly of NFH (pNFH) is considered to decrease their transport rate (for review see Perrot et al., 2008). Thus, highly phosphorylated isoforms of pNFH are found in the mature axons while dephosphorylated isoforms are expressed in the soma and dendrites (Perrot et al., 2008). Degenerating neurons insulted by either disease or trauma show an altered organisation and/or metabolism of pNFH that is associated with several human neurodegenerative diseases or experimental paradigms (for review see Al Chalabi & Miller, 2003). The levels of NF transcripts decrease after different retinal injuries, such as ischemia, excitotoxicity or optic nerve injury (Mc Kerracher et al., 1990a, 1990b; Chidlow et al., 2005a; Agudo et al., 2008). This regulation correlates, after optic nerve transection, with an impairment of the axonal transport if the RGCs are committed to death, but not if RGCs are allowed to regenerate along peripheral nerve grafts (Mc Kerracher et al., 1990a, 1990b; Vidal-Sanz et al., 1991, 2000). In healthy retinas pNFH expression is circumscribed to the mature portion of the intrarretinal axons, i.e. in the central-medial retina, while in the periphery few, thin pNFH positive axons (pNFH+) are observed. In fact, quantitative analyses show that pNFH+ axons occupy 61%, 14% and 0.37% of the central, medial and peripheral retinal surface, respectively (Parrilla-Reverter et al., 2009a). In addition, few or none RGC somas express this isoform. After optic nerve injury this pattern changes dramatically (Drager & Olsen, 1981; Vidal-Sanz et al., 1987; Villegas-Perez et al., 1988, Parrilla-Reverter et al., 2009 ). Three days after IONC the expression pattern of pNFH resembles that of a control retina when analyzed at low magnification. However, at high magnification two aberrant patterns emerge. Firstly, pNFH signal in the medial and peripheral retina increases significantly, occupying 24% and 9% of the middle and peripheral retinal surface, respectively. Secondly, some RGCs somas, preferentially in these retinal areas, become pNFH positive (pNFH+RGCs) (Figure 1A). With time, these abnormalities progress further (Figure 1B-D). The distribution of pNFH+RGCs widens, their number increases, peaking at 14 dpl, and the intensity of their staining augments. pNFH+RGCs appear first in the medial and peripheral retina, then they spread centripetally throughout the whole retinal surface. In general, pNFH+somas are weakly stained (Figure 1A arrow), but from day 14 onwards strongly stained ones appear, mainly in the temporal retina (Figures 1B-C arrows).
With respect to the axonal expression of pNFH, in the medial and peripheral retina is maintained above the control levels at least till 30dpl. In the central retina, where the RGC axonal bundles converge to exit the eye, pNFH expression decreases gradually. At late times post-lesion axonal degenerative events similar to those described by Ramón y Cajal are observed, such as rosary-like intra-axonal accumulations and club-like axonal ends (Figure 1D, arrows and arrowhead).
The aberrant expression of pNFH in the ganglion cell layer is common to other insults that affect the RGC either primarily, like optic nerve transection or IOP increase(Salinas-Navarro et al., 2009a, 2010; Parrilla-Reverter et al., 2009a; Nguyen et al., 2011), or secondarily, like phototoxicity (Villegas-Pérez et al., 1996,1998; Marco-Gomariz et al., 2006; Garcia-Ayuso et al., 2010).It is worth noting than even though all these insults cause the same aberrant patterns, their quantity and time course differs.For example after intraorbital nerve transection there are fewer pNFH+somas but in turn, early after the injury, there are more axons showing rosary-like accumulations of pNFH. These differences allow the correlation of different retinal diseases with a crush or transection-like temporal course of degeneration. In relation to this, our group has shown that after increasing the IOPin mice and rats, the RGCs located in the sectors of RGC loss express pNFH in their soma in a pattern closer to that observed after optic nerve crush than after optic nerve transection (Salinas-Navarro et al., 2009a; 2010). In conclusion, thepathological expression of pNFH is an early event that marks RGC degeneration and it is observed as early as 3 days following the insult.
3.2. Retinal ganglion cell loss after IONC
3.2.1. RGC identification and quantification
RGCs share their location in the ganglion cell layer with the equally numerous population of displaced amacrine cells (Drager& Olsen, 1981).In order to study the RGC population is then, necessary to distinguish them from the amacrine neurons. There are several techniques that specifically label RGCs. These are based either on tracing them from their projection areas, which in rodents are mainly the superior colliculi (SCi) (Linden & Perry, 1983; Thanos et al., 1987) or by detecting proteins or transcripts specifically expressed by these neurons (Barnstable & Drager, 1984; Chidlow et al., 2005). Retrograde tracing is based on applying onto the retinorecipient areas in the brain a fluorescent tracer, among which fluorogold is the most utilized. This tracer is taken up by the RGC terminals and transported actively through their axons till their somas in the retina (Figure 2A). In mice or rats, fluorogold applied onto the SCi detects 97.5% or 98.4% of the total RGC population, respectively (Salinas-Navarro et al.,2009b, 2009c).
Few proteins are known to be specifically expressed by RGCs, among them are γ-synuclein, Bex1/2, Thy1, NeuN or Brn3a(Barnstable & Drager, 1984; Quina et al.,2005; Bernstein et al.,2006; Soto et al.,2008; Buckingham et al.,2008; Nadal-Nicolas et al., 2009).Detection of γ-synuclein mRNA (Soto et al., 2008; Nguyen et al., 2011) has been used to investigate the fate of mice RGCs after ocular hypertension. Immunodetection of the transcription factor Brn3b has been used to estimate RGCs in a mice model of ocular hypertension (Fu & Sretavan, 2010). Detection of another member of this family of transcriptions factors, Brn3a, is a reliable method to identify the whole population of rats and mice RGCs in control retinas, after insults such as optic nerve axotomy, ocular hypertension or photoreceptor degeneration(Nadal-Nicolas et al.,2009; Salinas-Navarro et al.,2009a, 2010; Garcia-Ayuso et al.,2010; Galindo-Romero et al.,2011) and importantly, to test the efficacy of neuroprotective therapies (Sanchez-Migallon et al.,2011).
Quantification of RGCs can be done by sampling and manual counting, or by automated routines. Automated quantification is an objective routine that allows counting the whole population of a given cell (Soto et al., 2008; Nadal-Nicolas et al., 2009; Salinas-Navarro et al., 2009b, 2009c; Ortin-Martinez et al., 2010) while avoiding the tiresome and time consuming
effort of sampling and manual-counting. These routines have been shown to be successful to quantify RGCs identified by their Brn3a (Figure 2C) or γ-synuclein expression and can be applied to control or injured retinas (Soto et al., 2008; Nadal-Nicolas et al., 2009; Nguyen et al., 2011). In addition, automated routines allow the generation of detailed isodensity maps(Figure 2 B,D). These maps are useful to assess the distribution and densities of the studied cells in control retinas as well as to understand their topographical loss after an insult(Nadal-Nicolas et al., 2009; Salinas-Navarro et al., 2009a, 2009b, 2009c, 2010 Garcia-Ayuso et al., 2010; Ortin-Martinez et al., 2010; Galindo-Romero et al., 2011; Sanchez-Migallon et al., 2011).
By using this approach our group has reported that RGCs are not homogenously distributed in the retina (Figure 2 B,D). In fact, their lower densities are located in the periphery (blue colours) while their higher ones are found in the superior retina (red-oranges), along the nasotemporal axis, wherein the highest densities are located in the superotemporal quadrant. Furthermore, because in this retinal area L-cones reach their maximum densities, paralleling the RGC distribution, and the S cones reach their lowest, this area of high RGC density has been proposed to be the visual streak of rats (Nadal-Nicolas et al., 2009; Salinas-Navarro et al., 2009b, 2009c; Ortin-Martinez et al., 2010).
Both, tracing and immunodetection of RGCs have advantages and disadvantages. For instance, by retrograde tracing is possible to assess the number of RGCs that maintain a functional retrograde axonal transport. However, if the lesion impairs the axonal flow only the RGCs that have a competent axonal transport will be detected and the alive but impaired ones will be missed. On the other hand, while Brn3a orγ-synuclein detect those RGCs that are still alive independently of their axonal transport state, they are not useful to identify transport failures. Combination of tracing and Brn3a immunodetection has served to demonstrate that after IOP increase there is first a loss of the retrograde active transport that is followed by the death of the affected RGCs (Salinas-Navarro et al., 2009a, 2010).
3.2.2. Temporal course of RGC loss after IONC.
RGC loss after axonal injury depends on two main factors. The type of lesion, crush or transection and the distance from the eye where the lesion is inflicted (Villegas-Perez et al., 1993). In albino Sprague Dawley rats, when the optic nerve is completely crushed 3mm away from the eye, the loss of Brn3a+RGCs is first significant 5 days later (Nadal-Nicolas et al., 2009). At this time point the percentage of surviving RGCs is a 48% of the original population. This loss progresses quickly, decreasing to 28 and 14% of the original population at 9 and 14 days, respectively (Figure 3).
If the population of RGCs is identified by tracing, the loss of RGCs is significant later, at 7 dpl, and by day 14pl the percentage of survival is 29% (Nadal-Nicolas et al., 2009; Parrilla-Reverter et al., 2009b). This discrepancy is explained by the different nature of each marker.While Brn3a is an endogenous protein that is expressed as long as the RGC is alive (Sanchez-Migallon et al., 2011), fluorogold is an exogenous molecule that persists in the retina for at least 3-4 weeks after application (Selles-Navarro et al., 1996; Gomez-Ramirez et al., 1999). This means that fluorogold positive but already dead RGCs will be detected and will only disappear from the retina when the phagocytic microglia clears them.
Comparing these results with the time course of RGC loss in our model of IOP increase by laser photocoagulation (LP), it is observed that there is a massive death of these neurons
within the first 2 weeks post-LP, which accounts for 53% and 81% of the RGC population at 8 and 14 days post-LP, respectively (Nadal-Nicolas et al., 2009; Parrilla-Reverter et al., 2009b; Salinas-Navarro et al., 2010). Thus, the mean number and course of RGC loss in both models are similar. However there are two important differences. First, in terms of the damage caused IONC induces a consistent injury, while LP insult is highly variable among animals and so, in some retinas the RGC loss accounts for almost 80% of the RGCs, whilst in others only 40% of the original RGC population is affected. Second, after IONC, RGC death is diffuse and affects the whole retina (Figure 4) whereas after LP, RGC death occurs mainly in the dorsal retina in pie-like sectors which are devoid of RGCs, though it has been also observed a diffuse RGC loss in the rest of the retina.
3.3. Molecular causes underlying RGC loss after IONC
Why do RGC degenerate upon axonal injury? The main theory is that there is a withdrawal of trophic factors from the retinorecipient areas in the brain once the retina is deafferented. That is the reason why many neuroprotective therapies have been based on trophic factors delivery (see below). However, these therapies are only successful to a point, since the best protection only lasts up to 9 days post-lesion wherefrom the RGCs decay quickly.
This prompted us to do an extensive array analysis comparing the transcriptome regulation in naive retinas with retinas extracted at different times post-IONC (12h, 24h, 48h, 3d and 7d) (Agudo et al., 2008). This analysis rendered plenty of data, in fact over a thousand of genes along time showed an altered regulation compared to control retinas. Clustering of the regulated sequences revealed that they were related to several biological processes. Among them, the most significant were: cytoskeleton and associated processes(pEASE: 8,20E-13), primary metabolism (pEASE: 7,80E-14), protein metabolism (pEASE: 1,8E-29), immune response and inflammation (pEASE: 4,80E-11), RNA metabolism (processing and translation pEASE: 3,90E-25), cell cycle (pEASE: 1,20E-05), extracellular matrix remodelling (pEASE: 4,10E-07) and sensory perception of light (pEASE: 1,40E-09). Cell death, as expected, was highly regulated (97 genes, pEASE 2,20E-7). It was surprising to observe a transient down-regulation of genes related to phototransduction such as opsins, rhodopsins and phosducin, since IONC only affects RGCs. The down-regulation of photorreceptor genes may be a reflection of the arrest in transcription observed in the retina soon after injury (Lindqvist et al., 2002; Casson et al., 2004). Thus, whileIONC specifically injuresRGCs, also induces a retinal response that, even though is not lethal for other retinal neurons, has an effect on them.
Because the more dramatic effect of IONC is the RGC death, we focused our array analyses on the 97 death-related regulated genes. These were further clustered, and the most relevant sub-clusters were inflammation and death receptors, both implicated in triggering the extrinsic pathway of apoptosis and DNA damage, caspases, cell cycle deregulation and stress response, all of which are linked to the induction of the intrinsic pathway of apoptosis (Figure 5).There was a seventh group of death-related genes clustered under lysosomal cell death. A current body of evidence points to a role of lysosomes, and more specifically of cathepsins, in apoptosis. In fact, the “lysosomal pathway of apoptosis” is a phenomenon widely recognized (reviewed in Guicciardi et al., 2004) which would act through caspase activation, and hence activating the intrinsic pathway of apoptosis. However, it has been reported the role of autophagy in RGC death triggered by axotomy (Koch et al., 2010).
Autophagy is a highly regulated pathway that involves the degradation of cytoplasmic organelles or cytosolic components by the lysosomes, thus it is possible that the activation of the lysosomal cell death observed in our arrays analysis is in fact playing a part in autophagy and apoptosis (Figure 5). This is based on the fact that there is a crosstalk between both processes (Zhou et al., 2011) which is complex, and sometimes opposing. Indeed, autophagy can bea cell survival pathway to suppress apoptosis (Yang et al., 2010) or can lead to cell death, either in collaboration with apoptosis or as an alternative mechanism when apoptosis is defective. This crosstalk is mediated, in part, by calpains, which are activated in axotomized RGCs (Paquet-Durand et al., 2007; Agudo et al., 2008, 2009). Calpains cleave and activate Atg5, which is one of the autophagy effectors (Zhou et al., 2011).
Several of these death-effectors have been shown to be expressed by the primarily injured neurons, the RGCs (Cheung et al., 2004; Agudo et al., 2009). Among them, Tnfr1, Caspases 3 and 11, Calpain 1 and Cathepsins B and C are of special interest because each is linked to a different pathway that ends in cell death. The majority of these proteins showed an up-regulation as soon as 12h post-IONC, peaking at 48h, well before the time point when the anatomical RGC loss is first significant (5 days, see above). Thus, these data indicate that axotomized RGCs enter the path to death quickly after the injury, and even though pro-survival or protective mechanisms are concomitantly activated (Schwartz 2004; Levkovitch-Verbin et al., 2007) they are not enough to overcome the death signals. It is worth highlighting that Tnfr1 is up-regulated in our IONC model and in glaucomatous retinas from human patients (Tezel et al., 2001).
Finally, we have compared our array results with the array analyses of Guo et al, 2010 (Table 1). These data show that there is a common response to both types of injuries. In fact all the altered genes follow the same trend (up or down-regulation). These genes are mainly related to inflammation, apoptosis, cytoskeleton and extracellular matrix remodeling.Guo et al, (2010) analyzed the gene expression patterns occurring in the retina in response to elevated IOP induced by hypertonic saline injection. Their big contribution has been that the analysis was performed in extracts from whole retinas and in extracts from cells isolated from the ganglion cell layer. They observed that several genes were regulated in the whole retina and in the isolated cells (both, in the table) while others where only detected in the whole-retinal extracts (retina, in the table) and
In conclusion, even though IONC only mimics part of the pathogenesis of glaucoma only by compiling data from different models will be possible to understand the retinal response to injury. Given the complexity of these molecular events, the development of neuroprotectivetherapies must, probably, be combinatory. This panorama might be further complicated because basic cellular functions, such as primary metabolism and cytoskeleton maintenance are highly altered after IONC and IOP increase, opening up the possibility that the failure to preserve the cellular homeostasis might be the actual trigger of this complex regulation.
|Growth arrest and DNA-damage-inducible 45 gamma||Gadd45g||Activation p38/JNK pathway||↑ (both)||↑|
|Clusterin||Clu||Apoptosis and cell survival||↑ (retina)||↑|
|Suppressor of cytokine signaling 3||Socs3||Apoptosis and cell survival||↑ (both)||↑|
|Tumor necrosis factor receptor superfamily, member 12a||Tnfrsf12a||Apoptosis and cell survival||↑ (both)||↑|
|Lysozyme||Lyz||Cell wall catabolism, cytolysis||↑ (both)||↑|
|Ceruloplasmin||Cp||CNS iron homeostasis and neuroprotection||↑ (retina)||↑|
|Neurofilament, light polypeptide||Nfl||Cytoskeleton||↓ (both)||↓|
|Transgelin 2||Tagln2||Development||↑ (retina)||↑|
|Olfactomedin 1||Olfm1||Development||↓ (both)||↓|
|Secreted acidic cysteine rich glycoprotein||Sparc||Extracellular matrix||↑ (both)||↑|
|Alpha-2-macroglobulin||A2m||Immune response and inflammation||↑ (retina)||↑|
|Beta-2 microglobulin||B2m||Immune response and inflammation||↑ (retina)||↑|
|Interferon-inducible protein variant 10||Ifitm3||Immune response and inflammation||↑ (retina)||↑|
|Signal transducer and activator of transcription 3||Stat3||JAK/STAT cascade||↑ (both)||↑|
|Signal transducer and activator of transcription 1||Stat1||JAK/STAT cascade||↑ (retina)||↑|
|Cd63 antigen||Cd63||Lysosomal protein||↑ (both)||↑|
|Synuclein, gamma||Sncg||Nervous system development||↓ (retina)||↓|
|Carbonic anhydrase 14||Car14||Primary metabolism||↓ (retina)||↓|
|Carnitine o-octanoyltransferase||Crot||Primary metabolism||↑ (retina)||↑|
|Udp glycosyltransferase 1 family, polypeptide a1||Ugt1a||Primary metabolism||↑ (both)||↑|
|Lipocalin 2||Lcn2||Protection of MMP-9, iron transport||↑ (both)||↑|
|Arginyl aminopeptidase||Rnpep||Proteolysis||↑ (both)||↑|
|Corneal wound healing related protein||Mak10||Response to injury||↑ (retina)||↑|
|Hnrnp-associated with lethal yellow||Raly||RNA metabolism||↓ (retina)||↓|
|Tax1 binding protein 3||Tax1bp3||Signalling||↑ (both)||↑|
|Potassium voltage gated channel, member 2||Kcnd2||Synapsis||↓ (both)||↓|
|Solute carrier family 6, member 6||Slc6a6||Transport||↓ (retina)||↓|
|ATPAse, NA+/K+ transporting, beta 1 polypetide||Atp1b1||Transport||↓ (both)||↓|
4. RGC neuroprotection
Neuroprotection refers to therapies aimed to prevent, stop or at least delay the degeneration that follows CNS trauma. For a neuroprotective agent to be successful it must be directed to a target in the retina, it must be applied before the death of the neurons, it should be safe and it should work on animal models. Once fulfilled these criteria, the next steps are to design the pharmaceutical forms to deliver the compound to the human retina at the appropriate concentrations and to conduct the clinical trials.
After the injury, which cells are the candidates for neuroprotection? Obviously, not the neurons already dead, thus, it is important to know the temporal course of RGC degeneration to narrow the temporal window to intervene. Those cells in the process of degeneration are candidates if their somas are still healthy. In this case, function will only be restored if protection of the cell bodies is followed by regeneration and reconnection. Having this in mind, neuroprotection can be attained by preventing the degeneration of the healthy fibres, or by delaying it in the cell bodies of the damaged fibres.
Neuroprotection can be divided into two main groups: therapies that target specific genes or proteins related to cell death or therapies that increase the resistance of the injured neurons to the trauma. The former approach, though more specific, is very difficult for several reasons: i) it would be necessary to identify all the implicated signals; ii) some of these signals are pleiotropic, playing roles in survival or death depending on the delicate balance of other proteins; iii) some proteins while pathological if over-expressed, are essential for survival; thus, blocking their expression might be deleterious for the neurons that are not yet compromised; iv) signalling pathways are interconnected. Consequently, to tailor an effective therapy all this must be taken into account, and probably should imply the manipulation of several proteins. This requires careful analyses of the pathways and their interactions. Furthermore, the targeted proteins/genes should be head of pathways, provided that the downstream signals do not bifurcate into survival or death. Alternative targets could be the effectors, either proteins (i.e. caspases) or metabolites (i.e. ceramide).
4.1. Targeting specific signals
One way of targeting specific pathways is the use of inhibitors.Rho Kinase (ROCK) isa serine-threonine kinase that regulates the organization of the actin cytoskeleton (Mueller et al., 2005). Its activation is linked to the morphological changes observed during the execution phase of apoptosis (Coleman & Olson, 2002). Tura et al, (2009) showed that after IONC, the intravitreal administration of the ROCK inhibitor H-1152P produced two effects: a significant reduction of apoptosis in the ganglion cell layer anda reduction of the reactive gliosis.
Among the best characterized pro-death proteins is BAX. This protein associates with the permeability transition pore of the mitochondrial inner membrane opening it and thus inducing a series of events such as a disruption of the electrochemical membrane gradient, a disruption in ATP production and release of cytochrome C (Marzo et al., 1998). The released cytochrome C in turn, activates the caspase cascade. Li et al. (2000) demonstrated that in Bax deficient mice 2 weeks after optic nerve crush, there was less than a 10% of cell loss in the ganglion cell layer, which was significantly lower than the 41.3% observed in wild type mice. This survival was not due to a slower death-rate in the Bax-/- mice, since 4 weeks after the lesion there was not further cell loss. However, the lack of BAX did not prevent the ganglion cells to undergoearly changes in response to optic nerve crush, such as a decrease in the average size oftheir nuclei. It is worth noting, that the deficiency in this gene protected cells in the ganglion layer after optic nerve crush but not after experimentally induced excitotoxicity. This means that in response to different insults, ganglion cells activate different pathways of cell death.
The discovery of RNA interference (RNAi) has opened new avenues to investigate in neuroscience. RNAi is an endogenous mechanism that silences gene expression after translation. Silencing is highly sequence-specific and ends with the targeted mRNA cleaved into smaller fragments which results in the inhibition of protein synthesis (for review see Sontheimer, 2005). RNAi is mediated by small interference RNA (siRNA). The use of RNAi to knock-down specific pro-death proteins in axotomized RGCs has been only used after complete transection of the optic nerve (Lingor et al., 2005). Lingor et al. showed that in retinas treated with siRNAs against Apaf-1 (component of the apoptosome) or c-Jun (immediate response gene) there was a significant RGC survival compared to control ones. However, anti-Bax siRNAs did not increase RGC survival; this does not correlate with the experiments carried out on Bax-deficient mice. This discrepancy might be due to the different species (rats
In glaucoma, RNAi has been directed mainly to lower the IOP (for review see Mediero et al., 2009) rather than to induce a direct RGC survival. Targeting adrenergic receptors, acetylcholinesterase and ATPases, decreases the IOP. This approach has minimal side effects and the reduction of IOP lasts almost 5 days. This is important at the clinical level because the regime is simpler than pharmacological treatments, particularly eye drops that may require several administrations at day. In addition to lower the IOP, RNAi is been tested in vitro to knock-down the gain of function of mutated genes associated to glaucoma, such as myocilin(Li et al., 2009).
4.2. Therapies toincrease the resistance of the injured neurons
Administration of neurotrophins to the injured retina is one of the most successful therapies. Our group has shown that a single intravitreal injection of neurotrophin 4 (NT-4), ciliary neurotrophic factor (CNTF), or brain derived neurotrophic factor (BDNF) at the moment of the injury delays IONC-induced RGC death (Parrilla-Reverter et al., 2009b). At 7dpl all of them prevent almost completely the loss of RGCs. However, while at 12 dpl NT-4 and BDNF still protect, CNTF does not. Nevertheless, none of these factors were able to rescue the injured RGCs permanently. It is possible that a single injection is not sufficient. To solve this there are strategies to achieve a sustained expression of neurotrophins: transfection by viral vectors, cell based delivery approaches or microspheres loaded with the neurotrophin of choice (reviewed in Dahlmann-Noor et al., 2010). Another approach consists on the selective stimulation of trophic factor receptors using specific agonists. Thus, it has been shown that a selective agonist of TrkB (BDNF receptor) causes a long term TrKB activation and significantly delays RGC degeneration after IOP increase and after optic nerve transection (Bai et al., 2010).
Brimonidine, an α-2 adrenergic agonist, has been shown to neuroprotect RGCs after retinal ischemia (Lafuente et al.,2001,2002; Lafuente Lopez-Herrera et al., 2002; Vidal-Sanz et al.,2001a,b,2007; Aviles-Trigueros et al.,2003; Mayor-Torroglosa et al.,2005; Lonngren et al.,2006), after optic nerve crush (Wheeler et al., 1999) and after laser-induced ocular hypertension (Wheeler & WoldeMussie, 2001; Lambert et al., 2011). A randomized trial comparing brimonidine and timolol (β-adrenergic antagonist) in low tension glaucomatous patients, has shown that the loss of visual field is statistically lower in brimonidine treated-patients than in those treated with timolol, thus documenting, for the first time, its neuroprotective effect in human diseases (Krupin et al., 2011).
Optic nerve injury induced either by IOP increase or by direct trauma, causes profound structural, functional and molecular alterations in the primarily injured neurons, the RGCs, as well as in the rest of the retina. The consequences of these alterations are permanent as the CNS neurons die upon injury and the surviving ones fail to spontaneously regenerate their axons till their targets (Aguayo et al., 1987). Research is being focused in understanding the network of changes occurring in the traumatized retina and, more importantly, the effect of one upon another. To date, it is clear that numerous mechanisms are involved; some of them are common to different insults while others are injury-specific, some of them depend on inherited tracts, others implicate the glial and immune response and many reflect the commitment to death of the injured neurons. Only by gathering and unifying all these data we will be able to understand the common responses of the CNS to injury and to decipher the specific ones. With this knowledge it will be possible to design broad-spectrum and tailored therapies to successfully rescue the wounded system.
This work was supported by research grants from Fundación Séneca 04446/GERM/07; Spanish Ministry of Education and Science SAF-22010-10385; Spanish Ministry of Science and Innovation and ISCIII-FEDER: PI10/00187, PI006/0780 and Red Temática de Investigación Cooperativa en Oftalmología RD07/0062/0001.
Aguayo A. J. Vidal-Sanz M. Villegas-Perez M. P. Bray G. M. 1987Growth and connectivity of axotomized retinal neurons in adult rats with optic nerves substituted by PNS grafts linking the eye and the midbrain. Ann N Y Acad Sci 495 1 9
Agudo M. Perez-Marin M. C. Lonngren U. Sobrado P. Conesa A. Canovas I. Salinas-Navarro M. Miralles-Imperial J. Hallbook F. Vidal-Sanz M. 2008Time course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush. Mol Vis 14 1050 63
Agudo M. Perez-Marin M. C. Sobrado-Calvo P. Lonngren U. Salinas-Navarro M. Canovas I. Nadal-Nicolas F. M. Miralles-Imperial J. Hallbook F. Vidal-Sanz M. 2009Immediate upregulation of proteins belonging to different branches of the apoptotic cascade in the retina after optic nerve transection and optic nerve crush. Invest Ophthalmol Vis Sci 50 1 424 31
Aihara M. JD Lindsey Weinreb. RN 2003Ocular hypertension in mice with a targeted type I collagen mutation. Invest Ophthalmol Vis Sci 44 4 1581 5
Al Chalabi. A. Miller C. C. 2003Neurofilaments and neurological disease. Bioessays 25 4 346 55
Alarcon-Martinez L. Aviles-Trigueros M. Galindo-Romero C. Valiente-Soriano J. Agudo-Barriuso M. de la Villa P. Villegas-Perez M. P. Vidal-Sanz M. 2010ERG changes in albino and pigmented mice after optic nerve transection. Vision Res 50 21 2176 87
Alarcon-Martinez L. de la Villa P. Aviles-Trigueros M. Blanco R. Villegas-Perez M. P. Vidal-Sanz M. 2009Short and long term axotomy-induced ERG changes in albino and pigmented rats. Mol Vis 15 2373 83
Alward W. L. Fingert J. H. MA Coote Johnson. A. T. Lerner S. F. Junqua D. Durcan F. J. Mc Cartney P. J. Mackey D. A. Sheffield V. C. Stone E. M. 1998Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A). N Engl J Med 338 15 1022 7
Aviles-Trigueros M. Mayor-Torroglosa S. Garcia-Aviles A. Lafuente M. P. ME Rodriguez Miralles. d. I. Villegas-Perez M. P. Vidal-Sanz M. 2003Transient ischemia of the retina results in massive degeneration of the retinotectal projection: long-term neuroprotection with brimonidine. Exp Neurol 184 2 767 77
Bai Y. Xu J. Brahimi F. Zhuo Y. Sarunic M. V. Saragovi H. U. 2010An agonistic TrkB mAb causes sustained TrkB activation, delays RGC death, and protects the retinal structure in optic nerve axotomy and in glaucoma. Invest Ophthalmol Vis Sci 51 9 4722 31
Barnstable CJ, Drager UC. 1984Thy-1 antigen: a ganglion cell specific marker in rodent retina. Neuroscience 11 4 847 55
Bernstein S. L. Koo J. H. Slater B. J. Guo Y. Margolis F. L. 2006Analysis of optic nerve stroke by retinal Bex expression. Mol Vis 12 147 55
Bray G. M. Villegas-Perez M. P. Vidal-Sanz M. Carter D. A. Aguayo A. J. 1991Neuronal and nonneuronal influences on retinal ganglion cell survival, axonal regrowth, and connectivity after axotomy. Ann N Y Acad Sci 633 214 28
Buckingham B. P. Inman D. M. Lambert W. Oglesby E. Calkins D. J. Steele M. R. Vetter M. L. Marsh-Armstrong N. Horner P. J. 2008Progressive ganglion cell degeneration precedes neuronal loss in a mouse model of glaucoma. J Neurosci 28 11 2735 44
Casson R. J. Chidlow G. Wood J. P. Vidal-Sanz M. Osborne N. N. 2004The effect of retinal ganglion cell injury on light-induced photoreceptor degeneration. Invest Ophthalmol Vis Sci 45 2 685 93
Chang B. Smith R. S. Hawes N. L. Anderson M. G. Zabaleta A. Savinova O. Roderick T. H. Heckenlively J. R. Davisson M. T. John S. W. 1999Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice. Nat Genet 21 4 405 9
Chen H. Wei X. Cho K. S. Chen G. Sappington R. Calkins D. J. Chen D. F. 2011Optic neuropathy due to microbead-induced elevated intraocular pressure in the mouse. Invest Ophthalmol Vis Sci 52 1 36 44
Cheung Z. H. Chan Y. M. Siu F. K. Yip H. K. Wu W. Leung M. C. So K. F. 2004Regulation of caspase activation in axotomized retinal ganglion cells. Mol Cell Neurosci 25 3 383 93
Chidlow G. Casson R. Sobrado-Calvo P. Vidal-Sanz M. Osborne N. N. 2005aMeasurement of retinal injury in the rat after optic nerve transection: an RT-PCR study. Mol Vis 11 387 96
Coleman ML, Olson MF. 2002Rho GTPase signalling pathways in the morphological changes associated with apoptosis. Cell Death Differ 9 5 493 504
Cone FE, Gelman SE, Son JL, Pease ME, Quigley HA. 2010Differential susceptibility to experimental glaucoma among 3 mouse strains using bead and viscoelastic injection. Exp Eye Res 91 3 415 24
Cuenca N. Pinilla I. Fernandez-Sanchez L. Salinas-Navarro M. Alarcon-Martinez L. Aviles-Trigueros M. de Miralles l. V. Villegas-Perez d. I. Vidal-Sanz M. P. M. 2010Changes in the inner and outer retinal layers after acute increase of the intraocular pressure in adult albino Swiss mice. Exp Eye Res 91 2 273 85
Dahlmann-Noor A. Vijay S. Jayaram H. Limb A. Khaw P. T. 2010Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve. Can J Ophthalmol 45 4 333 41
Drager UC, Olsen JF. 1981Ganglion cell distribution in the retina of the mouse. Invest Ophthalmol Vis Sci 20 3 285 93
Flammer J. Mozaffarieh M. 2007What is the present pathogenetic concept of glaucomatous optic neuropathy? Surv Ophthalmol 52 Suppl 2: S 162S173.
Fu C. T. Sretavan D. 2010Laser-induced ocular hypertension in albino CD-1 mice. Invest Ophthalmol Vis Sci 51 2 980 90
Fujikawa K. Iwata T. Inoue K. Akahori M. Kadotani H. Fukaya M. Watanabe M. Chang Q. Barnett E. M. Swat W. 2010VAV2 and VAV3 as candidate disease genes for spontaneous glaucoma in mice and humans. PLoS One 5 (2): e9050.
Fuse N. 2010Genetic bases for glaucoma. Tohoku J Exp Med 221 1 1 10
Gaasterland D. Kupfer C. 1974Experimental glaucoma in the rhesus monkey. Invest Ophthalmol 13 6 455 7
Galindo-Romero C. Aviles-Trigueros M. Jimenez-Lopez M. Valiente-Soriano F. J. Salinas-Navarro M. Nadal-Nicolas F. Villegas-Perez M. P. Vidal-Sanz M. Agudo-Barriuso M. 2011Axotomy-induced retinal ganglion cell death in adult mice: Quantitative and topographic time course analyses. Exp Eye Res. 92 5 377 87
Garcia-Ayuso D. Salinas-Navarro M. Agudo M. Cuenca N. Pinilla I. Vidal-Sanz M. Villegas-Perez M. P. 2010Retinal ganglion cell numbers and delayed retinal ganglion cell death in the 23Hrat retina. Exp Eye Res 91 (6): 800-10.
Gomez-Ramirez A. M. Villegas-Perez M. P. Miralles d. I. Salvador-Silva M. Vidal-Sanz M. 1999Effects of intramuscular injection of botulinum toxin and doxorubicin on the survival of abducens motoneurons. Invest Ophthalmol Vis Sci 40 2 414 24
ME Guicciardi Leist. M. Gores G. J. 2004Lysosomes in cell death. Oncogene 23 16 2881 90
Guo Y. Cepurna W. O. Dyck J. A. Doser T. A. Johnson E. C. Morrison J. C. 2010Retinal cell responses to elevated intraocular pressure: a gene array comparison between the whole retina and retinal ganglion cell layer. Invest Ophthalmol Vis Sci 51 6 3003 18
John S. W. Hagaman J. R. Mac Taggart. T. E. Peng L. Smithes O. 1997Intraocular pressure in inbred mouse strains. Invest Ophthalmol Vis Sci 38 1 249 53
Johnson E. C. Morrison J. C. Farrell S. Deppmeier L. Moore C. G. Mc Ginty M. R. 1996The effect of chronically elevated intraocular pressure on the rat optic nerve head extracellular matrix. Exp Eye Res 62 6 663 74
Kipfer-Kauer A. Mc Kinnon S. J. Frueh B. E. Goldblum D. 2010Distribution of amyloid precursor protein and amyloid-beta in ocular hypertensive C57BL/6 mouse eyes. Curr Eye Res 35 9 828 34
Koch J. C. Knoferle J. Tonges L. Ostendorf T. Bahr M. Lingor P. 2010Acute axonal degeneration in vivo is attenuated by inhibition of autophagy in a calcium-dependent manner. Autophagy 6 (5).
Krupin T. Liebmann J. M. DS Greenfield Ritch. R. Gardiner S. 2011A Randomized Trial of Brimonidine Versus Timolol in Preserving Visual Function: Results From the Low-pressure Glaucoma Treatment Study. Am J Ophthalmol 151 4 671 81
Lafuente-Herrera Lopez. Mayor-Torroglosa M. P. Miralles S. Villegas-Perez d. I. Vidal-Sanz M. P. M. 2002Transient ischemia of the retina results in altered retrograde axoplasmic transport: neuroprotection with brimonidine. Exp Neurol 178 2 243 58
Lafuente M. P. Villegas-Perez M. P. Mayor S. ME Aguilera Miralles. d. I. Vidal-Sanz M. 2002Neuroprotective effects of brimonidine against transient ischemia-induced retinal ganglion cell death: a dose response in vivo study. Exp Eye Res 74 2 181 9
Lafuente M. P. Villegas-Perez M. P. Sobrado-Calvo P. Garcia-Aviles A. Miralles d. I. Vidal-Sanz M. 2001Neuroprotective effects of alpha (2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death. Invest Ophthalmol Vis Sci 42 9 2074 84
Lambert W. S. Ruiz L. Crish S. D. Wheeler L. A. Calkins D. J. 2011Brimonidine prevents axonal and somatic degeneration of retinal ganglion cell neurons. Mol Neurodegener 6(1): 4.
Leoz O. Arcuate L. R. 1914Procesos regenerativos del nervio óptico y retina, con ocasión de injertos nerviosos. Trab Lab Invest Biol 11 239 54
Levkovitch-Verbin H. Harizman N. Dardik R. Nisgav Y. Vander S. Melamed S. 2007Regulation of cell death and survival pathways in experimental glaucoma. Exp Eye Res 85 2 250 8
Li M. Xu J. Chen X. Sun X. 2009RNA interference as a gene silencing therapy for mutant MYOC protein in primary open angle glaucoma. Diagn Pathol 4: 46.
Li Y. Schlamp C. L. Poulsen K. P. Nickells R. W. 2000Bax-dependent and independent pathways of retinal ganglion cell death induced by different damaging stimuli. Exp Eye Res 71 2 209 13
Li Y. Semaan S. J. Schlamp C. L. Nickells R. W. 2007Dominant inheritance of retinal ganglion cell resistance to optic nerve crush in mice. BMC Neurosci 8: 19.
Linden R. Perry V. H. 1983Massive retinotectal projection in rats. Brain Res 272 (1): 145 9
Lindqvist N. Vidal-Sanz M. Hallbook F. 2002Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching. Brain Res Brain Res Protoc 10 2 75 83
Lingor P. Koeberle P. Kugler S. Bahr M. 2005Down-regulation of apoptosis mediators by RNAi inhibits axotomy-induced retinal ganglion cell death in vivo. Brain 128 (Pt 3): 550-8.
Lonngren U. Napankangas U. Lafuente M. Mayor S. Lindqvist N. Vidal-Sanz M. Hallbook F. 2006The growth factor response in ischemic rat retina and superior colliculus after brimonidine pre-treatment. Brain Res Bull 71 (1-3): 208-18.
MA Marco-Gomariz-Montalban Hurtado. Vidal-Sanz N. Lund M. Villegas-Perez R. D. M. P. 2006Phototoxic-induced photoreceptor degeneration causes retinal ganglion cell degeneration in pigmented rats. J Comp Neurol 498 2 163 79
Marzo I. Brenner C. Zamzami N. Jurgensmeier J. M. Susin S. A. Vieira H. L. Prevost M. C. Xie Z. Matsuyama S. Reed J. C. Kroemer G. 1998Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281 5385 2027 31
Mayor-Torroglosa S. de la Villa P. ME Rodriguez-Herrera Lopez. Aviles-Trigueros M. P. Garcia-Aviles M. de Imperial A. Villegas-Perez J. M. Vidal-Sanz M. P. M. 2005Ischemia results 3 months later in altered ERG, degeneration of inner layers, and deafferented tectum: neuroprotection with brimonidine. Invest Ophthalmol Vis Sci 46 10 3825 35
Mc Kerracher L. Vidal-Sanz M. Aguayo A. J. 1990aSlow transport rates of cytoskeletal proteins change during regeneration of axotomized retinal neurons in adult rats. J Neurosci 10 2 641 8
Mc Kerracher L. Vidal-Sanz M. Essagian C. Aguayo A. J. 1990bSelective impairment of slow axonal transport after optic nerve injury in adult rats. J Neurosci 10 8 2834 41
Mediero A. Alarma-Estrany P. Pintor J. 2009New treatments for ocular hypertension. Auton Neurosci 147 (1-2): 14-9.
Morrison J. C. Cepurna W. Guo Y. Johnson E. C. 2010Pathophysiology of human glaucomatous optic nerve damage: Insights from rodent models of glaucoma. Exp Eye Res. E-pub ahead of print doi:10.1016/j.exer.2010.08.005.
Morrison JC, Moore CG, Deppmeier LM, Gold BG, Meshul CK, Johnson EC. 1997A rat model of chronic pressure-induced optic nerve damage. Exp Eye Res 64 1 85 96
Mueller B. K. Mack H. Teusch N. 2005Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov 4 5 387 98
Nadal-Nicolas F. M. Jimenez-Lopez M. Sobrado-Calvo P. Nieto-Lopez L. Canovas-Martinez I. Salinas-Navarro M. Vidal-Sanz M. Agudo M. 2009Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci 50 8 3860 8
Nguyen J. V. Soto I. Kim K. Y. Bushong E. A. Oglesby E. Valiente-Soriano F. J. Yang Z. Davis C. H. Bedont J. L. Son J. L. Wei J. O. Buchman V. L. Zack D. J. Vidal-Sanz M. Ellisman M. H. Marsh-Armstrong N. 2011Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma. Proc Natl Acad Sci USA 108 3 1176 81
Ortin-Martinez A. Jimenez-Lopez M. Nadal-Nicolas F. M. Salinas-Navarro M. Alarcon-Martinez L. Sauve Y. Villegas-Perez M. P. Vidal-Sanz M. Agudo-Barriuso M. 2010Automated quantification and topographical distribution of the whole population of S- and L-cones in adult albino and pigmented rats. Invest Ophthalmol Vis Sci 51 6 3171 83
Paquet-Durand F. Johnson L. Ekstrom P. 2007Calpain activity in retinal degeneration. J Neurosci Res 85 4 693 702
Parrilla-Reverter G. Agudo M. Nadal-Nicolas F. Alarcon-Martinez L. Jimenez-Lopez M. Salinas-Navarro M. Sobrado-Calvo P. Bernal-Garro J. M. Villegas-Perez M. P. Vidal-Sanz M. 2009aTime-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: a comparative study. Vision Res 49 23 2808 25
Parrilla-Reverter G. Agudo M. Sobrado-Calvo P. Salinas-Navarro M. Villegas-Perez M. P. Vidal-Sanz M. 2009bEffects of different neurotrophic factors on the survival of retinal ganglion cells after a complete intraorbital nerve crush injury: a quantitative in vivo study. Exp Eye Res 89 1 32 41
Peinado-Ramon P. Salvador M. Villegas-Perez M. P. Vidal-Sanz M. 1996Effects of axotomy and intraocular administration of NT-4, NT-3, and brain-derived neurotrophic factor on the survival of adult rat retinal ganglion cells. A quantitative in vivo study. Invest Ophthalmol Vis Sci 37 4 489 500
Perrot R. Berges R. Bocquet A. Eyer J. 2008Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Mol Neurobiol 38 1 27 65
Quigley HA. 1996Number of people with glaucoma worldwide. Br J Ophthalmol 80 5 389 93
Quigley H. A. Vitale S. 1997Models of open-angle glaucoma prevalence and incidence in the United States. Invest Ophthalmol Vis Sci 38 1 83 91
Quina L. A. Pak W. Lanier J. Banwait P. Gratwick K. Liu Y. Velasquez T. O’Leary D. D. Goulding M. EE Turner 2005Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways. J Neurosci 25 50 11595 604
Ramirez A. I. Salazar J. J. de Hoz R. Rojas B. Gallego B. I. Salinas-Navarro M. Alarcon-Martinez L. Ortin-Martinez A. Aviles-Trigueros M. Vidal-Sanz M. Trivino A. Ramirez J. M. 2010Quantification of the effect of different levels of IOP in the astroglia of the rat retina ipsilateral and contralateral to experimental glaucoma. Invest Ophthalmol Vis Sci 51 11 5690 6
Ramón y. Cajal S. 1914Estudios sobre la degeneración y regeneración del sistema nervioso. In: Hijos de Nicolás Moyá M, editor. 203 217
Ray K. Mookherjee S. 2009Molecular complexity of primary open angle glaucoma: current concepts. J Genet 88 4 451 67
Reimer J. MD Wolter 1967Axonal enlargments in the nerve-fiber layer of the human retina. American Journal of Ophthalmology 65 (1): 12.
Salinas-Navarro M. Alarcon-Martinez L. Valiente-Soriano F. J. Jimenez-Lopez M. Mayor-Torroglosa S. Aviles-Trigueros M. Villegas-Perez M. P. Vidal-Sanz M. 2010Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration. Exp Eye Res 90 1 168 83
Salinas-Navarro M. Alarcon-Martinez L. Valiente-Soriano F. J. Ortin-Martinez A. Jimenez-Lopez M. Aviles-Trigueros M. Villegas-Perez M. P. de Vidal-Sanz l. V. M. 2009aFunctional and morphological effects of laser-induced ocular hypertension in retinas of adult albino Swiss mice. Mol Vis 15 2578 98
Salinas-Navarro M. Jimenez-Lopez M. Valiente-Soriano F. J. Alarcon-Martinez L. Aviles-Trigueros M. Mayor S. Holmes T. Lund R. D. Villegas-Perez M. P. Vidal-Sanz M. 2009bRetinal ganglion cell population in adult albino and pigmented mice: a computerized analysis of the entire population and its spatial distribution. Vision Res 49 6 637 47
Salinas-Navarro M. Mayor-Torroglosa S. Jimenez-Lopez M. Aviles-Trigueros M. Holmes T. M. Lund R. D. Villegas-Perez M. P. Vidal-Sanz M. 2009cA computerized analysis of the entire retinal ganglion cell population and its spatial distribution in adult rats. Vision Res 49 1 115 26
Samsel P. A. Kisiswa L. Erichsen J. T. Cross S. D. Morgan J. E. 2010A novel method for the induction of experimental glaucoma using magnetic microspheres. Invest Ophthalmol Vis Sci.
Sanchez-Migallon M. C. Nadal-Nicolas F. M. Jimenez-Lopez M. Sobrado-Calvo P. Vidal-Sanz M. Agudo-Barriuso M. 2011Brain derived neurotrophic factor maintains Brn3a expression in axotomized rat retinal ganglion cells. Exp Eye Res 92 (4): 260 7
Sappington RM, Carlson BJ, Crish SD, Calkins DJ. 2010The microbead occlusion model: a paradigm for induced ocular hypertension in rats and mice. Invest Ophthalmol Vis Sci 51 1 207 16
Schwartz M. 2004Optic nerve crush: protection and regeneration. Brain Res Bull 62 6 467 71
Selles-Navarro I. Villegas-Perez M. P. Salvador-Silva M. Ruiz-Gomez J. M. Vidal-Sanz M. 1996Retinal ganglion cell death after different transient periods of pressure-induced ischemia and survival intervals. A quantitative in vivo study. Invest Ophthalmol Vis Sci 37 10 2002 14
Shareef S. R. Garcia-Valenzuela E. Salierno A. Walsh J. Sharma S. C. 1995Chronic ocular hypertension following episcleral venous occlusion in rats. Exp Eye Res 61 3 379 82
Shields M. Ritch R. Krupin T. 1996Classfication of the glaucomas. Mosby, St Louis, USA.
Sobrado-Calvo P. Vidal-Sanz M. Villegas-Perez M. P. 2007Rat retinal microglial cells under normal conditions, after optic nerve section, and after optic nerve section and intravitreal injection of trophic factors or macrophage inhibitory factor. J Comp Neurol 501 6 866 78
Sontheimer EJ. 2005Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol 6 2 127 38
Soto I. Oglesby E. Buckingham B. P. Son J. L. Roberson E. D. Steele M. R. Inman D. M. Vetter M. L. Horner P. J. Marsh-Armstrong N. 2008Retinal ganglion cells downregulate gene expression and lose their axons within the optic nerve head in a mouse glaucoma model. J Neurosci 28 2 548 61
Tello F. 1907La régéneration dans les voies optiques. Trabajos de Laboratorio en Investigación Biológica: 237 48
Tezel G. Li L. Y. Patil R. V. Wax M. B. 2001TNF-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes. Invest Ophthalmol Vis Sci 42 8 1787 94
Thanos S. Vidal-Sanz M. Aguayo A. J. 1987The use of rhodamine-B-isothiocyanate (RITC) as an anterograde and retrograde tracer in the adult rat visual system. Brain Res 406 (1-2): 317-21.
Tura A. Schuettauf F. Monnier P. P. Bartz-Schmidt K. U. Henke-Fahle S. 2009Efficacy of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina. Invest Ophthalmol Vis Sci 50 1 452 61
Ueda J. Sawaguchi S. Hanyu T. Yaoeda K. Fukuchi T. Abe H. Ozawa H. 1998Experimental glaucoma model in the rat induced by laser trabecular photocoagulation after an intracameral injection of India ink. Jpn J Ophthalmol 42 5 337 44
Urcola J. H. Hernandez M. Vecino E. 2006Three experimental glaucoma models in rats: comparison of the effects of intraocular pressure elevation on retinal ganglion cell size and death. Exp Eye Res 83 2 429 37
Vidal-Sanz M. Aviles-Trigueros M. Whiteley S. J. Sauve Y. Lund R. D. 2002Reinnervation of the pretectum in adult rats by regenerated retinal ganglion cell axons: anatomical and functional studies. Prog Brain Res 137 443 52
Vidal-Sanz M. Bray G. M. Villegas-Perez M. P. Thanos S. Aguayo A. J. 1987Axonal regeneration and synapse formation in the superior colliculus by retinal ganglion cells in the adult rat. J Neurosci 7 9 2894 909
Vidal-Sanz M. de la Villa P. Aviles-Trigueros M. Mayor-Torroglosa S. Alarcon-Martinez L. Villegas-Perez M. P. 2007Neuroprotection of retinal ganglion cell function and their central nervous system targets. Eye 21:S 42S45.
Vidal-Sanz M. Lafuente M. P. Mayor S. de Imperial J. M. Villegas-Perez M. P. 2001aRetinal ganglion cell death induced by retinal ischemia. neuroprotective effects of two alpha-2 agonists. Surv Ophthalmol 45 Suppl 3: S 261S267.
Vidal-Sanz M. Lafuente M. P. Mayor-Torroglosa S. ME Aguilera Miralles. d. I. Villegas-Perez M. P. 2001bBrimonidine’s neuroprotective effects against transient ischaemia-induced retinal ganglion cell death. Eur J Ophthalmol 11 Suppl 2: S 36S40.
Vidal-Sanz M. Villegas-Perez M. P. Carter D. A. Julien J. P. Peterson A. Aguayo A. J. 1991Expression of Human Neurofilament-light Transgene in Mouse Neurons Transplanted into the Brain of Adult Rats. Eur J Neurosci 3 8 758 63
Villegas-Perez M. P. Lawrence J. M. Vidal-Sanz M. MM Lavail Lund. R. D. 1998Ganglion cell loss in RCS rat retina: a result of compression of axons by contracting intraretinal vessels linked to the pigment epithelium. J Comp Neurol 392 1 58 77
Villegas-Perez M. P. Vidal-Sanz M. Bray G. M. Aguayo A. J. 1988Influences of peripheral nerve grafts on the survival and regrowth of axotomized retinal ganglion cells in adult rats. J Neurosci 8 1 265 80
Villegas-Perez M. P. Vidal-Sanz M. Rasminsky M. Bray G. M. Aguayo A. J. 1993Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats. J Neurobiol 24 1 23 36
Villegas-Perez M. P. Vidal-Sanz M. Lund R. D. 1996Mechanism of retinal ganglion cell loss in inherited retinal dystrophy. Neuroreport 7 12 1995 9
Wheeler L. A. Lai R. Wolde Mussie. E. 1999From the lab to the clinic: activation of an alpha-2 agonist pathway is neuroprotective in models of retinal and optic nerve injury. Eur J Ophthalmol 9 Suppl 1: S 17S21.
Wheeler L. A. Wolde Mussie. E. 2001Alpha-2 adrenergic receptor agonists are neuroprotective in experimental models of glaucoma. Eur J Ophthalmol 11 Suppl 2: S 30S35.
Wolde Mussie. E. Ruiz G. Wijono M. Wheeler L. A. 2001Neuroprotection of retinal ganglion cells by brimonidine in rats with laser-induced chronic ocular hypertension. Invest Ophthalmol Vis Sci. 2001 Nov; 42 12 2849 55
Yang Y. D. Zhou F. Chen Q. 2010Mitochondrial autophagy protects against heat shock-induced apoptosis through reducing cytosolic cytochrome c releaseand downstream caspase-3 activation. Biochem Biophys Res Commun 395 2 190 5
Zhou F. Yang Y. D. 2011Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis. FEBS J 278 3 403 13
Zhou Y. Grinchuk O. Tomarev S. I. 2008Transgenic mice expressing the Tyr437His mutant of human myocilin protein develop glaucoma. Invest Ophthalmol Vis Sci 49 5 1932 9