Müller glial cells (MGCs) are known to participate actively in retinal development and to contribute to homoeostasis through many intracellular mechanisms. As there are no homologous cells in other neuronal tissues, it is certain that retinal health depends on MGCs. These macroglial cells are located at the centre of the columnar subunit and have a great ability to interact with neurons, astrocytes, microglia and endothelial cells in order to modulate different events. Several investigations have focused their attention on the role of MGCs in diabetic retinopathy, a progressive pathology where several insults coexist. As expected, data suggest that MGCs display different responses according to the severity of the stimulus, and therefore trigger distinct events throughout the course of the disease. Here, we describe physiological functions of MGCs and their participation in inflammation, gliosis, synthesis and secretion of trophic and antioxidant factors in the diabetic retina. We invite the reader to consider the protective/deleterious role of MGCs in the early and late stages of the disease. In the light of the results, we open up the discussion around and ask the question: Is it possible that the modulation of a single cell type could improve or even re-establish retinal function after an injury?

Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As retinal impairment involves abnormal neovascularization and neuronal degeneration, we evaluated here the involvement of galectin-1 in vascular and non-vascular alterations associated with retinopathies, using the oxygen-induced retinopathy (OIR) model. Postnatal day 17 OIR mouse retinas showed the highest neovascular profile and exhibited neuro-glial injury as well as retinal functional loss, which persisted until P26 OIR. Concomitant to VEGF up-regulation, galectin-1 was highly expressed in P17 OIR retinas and it was mainly localized in neovascular tufts. In addition, OIR induced remodelling of cell surface glycophenotype leading to exposure of galectin-1-specific glycan epitopes. Whereas VEGF returned to baseline levels at P26, increased galectin-1 expression persisted until this time period. Remarkably, although anti-VEGF treatment in P17 OIR improved retinal vascularization, neither galectin-1 expression nor non-vascular and functional alterations were attenuated. However, this functional defect was partially prevented in galectin-1-deficient (Lgals1−/−) OIR mice, suggesting the importance of targeting both VEGF and galectin-1 as non-redundant independent pathways. Supporting the clinical relevance of these findings, we found increased levels of galectin-1 in aqueous humor from patients with proliferative diabetic retinopathy and neovascular glaucoma. Thus, using an OIR model and human samples, we identified a role for galectin-1 accompanying vascular and non-vascular retinal alterations in neovascular retinopathies.

It has been shown that a single exposure to amphetamine is sufficient to induce long-term behavioral, neurochemical, and neuroendocrine sensitization in rats. Dopaminergic neurotransmission in the nucleus accumbens and the caudate-putamen plays a critical role in the addictive properties of drugs of abuse. Angiotensin (Ang) II receptors are found on the soma and terminals of mesolimbic dopaminergic neurons and it has been shown that Ang II acting through its AT₁ receptors facilitates dopamine release. The hypothesis was tested that Ang II AT₁ receptors are involved in the neuroadaptative changes induced by a single exposure to amphetamine and that such changes are related to the development of behavioral and neurochemical sensitization. For this purpose, the study examined the expression of amphetamine-enhanced (0.5 mg kg⁻¹ i.p.) locomotor activity in animals pretreated with candesartan, an AT₁ blocker, (3 mg kg⁻¹ p.o. x 5 days), 3 weeks after an amphetamine injection (5 mg kg⁻¹ i.p.). Dopaminergic hyperreactivity was tested by measuring the 3H-DA release in vitro from caudate-putamen and nucleus accumbens slices, induced by K+ stimulus. It was confirmed the behavioral sensitization in the two-injection protocol and candesartan pretreatment attenuate this response. It was also found that AT₁ blockade pretreatment did not affect the locomotor response to dopamine agonists. In respect to the neurochemical sensitization tested using ex vivo 3H-DA release experiments it was found that AT₁ receptor pretreatment blunted the enhanced response induced by K+ stimulus. The results support the idea that the development of neuroadaptive changes induced by amphetamine involves brain AT₁ Ang II receptor activation.

It was already found that Ang II AT1 receptors are involved in the neuroadaptative changes induced by a single exposure to amphetamine, and such changes are related to the development of behavioral and neurochemical sensitization. The induction of the immediately early gene c-fos has been used to define brain activated areas by amphetamine. Our aim was to evaluate the participation of AT1 receptors in the neuronal activation induced by amphetamine sensitization. The study examined the c-fos expression in mesocorticolimbic areas induced by amphetamine challenge (0.5 mg/kg i.p) in animals pretreated with candesartan, a selective AT1 receptor blocker (3 mg/kg p.o × 5 days), and amphetamine (5 mg/kg i.p) 3 weeks before the challenge. Increased c-fos immunoreactivity was found in response to the amphetamine challenge in the dorsomedial caudate-putamen and nucleus accumbens, and both responses were blunted by the AT1 receptor blocker pretreatment. In the infralimbic prefrontal cortex, increased c-fos immunoreactivity was found in response to amphetamine and saline challenge, and both were prevented by the AT1 receptor blocker. No differences were found neither in ventral tegmental area nor prelimbic cortex between groups. Our results indicate an important role for brain Ang II in the behavioral and neuronal sensitization induced by amphetamine.

In ischemic proliferative diseases such as retinopathies, persistent hypoxia leads to the release of numerous neovascular factors that participate in the formation of abnormal vessels and eventually cause blindness. The upregulation and activation of metalloproteinases (MMP-2 and MMP-9) represent a final common pathway in this process. Although many regulators of the neovascular process have been identified, the complete role of the insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) appears to be significantly more complex. In this study, we used an oxygen-induced retinopathy (OIR) mouse model as well as an in vitro model of hypoxia to study the role of MMP-2 derived from Müller glial cells (MGCs) and its relation with the IGF-1/IGF-1R system. We demonstrated that MMP-2 protein expression increased in P17 OIR mice, which coincided with the active phase of the neovascular process. Also, glutamine synthetase (GS)-positive cells were also positive for MMP-2, whereas IGF-1R was expressed by GFAP-positive cells, indicating that both proteins were expressed in MGCs. In addition, in the OIR model a single intravitreal injection of the IGF-1R blocking antibody (αIR3) administered at P12 effectively prevented pathologic neovascularization, accelerated physiological revascularization, and improved retinal functionality at P17. Finally, in MGC supernatants, the blocking antibody abolished the IGF-1 effect on active MMP-2 under normoxic and hypoxic conditions without affecting the extracellular levels of pro-MMP-2. These results demonstrate, for the first time, that the IGF-1/IGF-1R system regulates active MMP-2 levels in MGCs, thus contributing to MEC remodeling during the retinal neovascular process.

Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, but it is also a cell survival mechanism. Here, we analyzed the role of autophagy at the three characteristic time periods in the oxygen-induced retinopathy (OIR) mouse model and determined if its modulation can improve vascular and non-vascular alterations. Experiments were performed with chloroquine (CQ) in order to monitor autophagosome accumulation by lysosomal blockade. Post natal day (P)17 OIR mouse retinas showed a significant increase in autophagy flux. In particular, an intense LC3B and p62 staining was observed in inner layers of the retina, mainly proliferating endothelial cells. After a single intraocular injection of Rapamycin at P12 OIR, a decreased neovascular area and vascular endothelial growth factor (VEGF) protein expression were observed at P17 OIR. In addition, whereas the increased expression of glial fibrillary acidic protein (GFAP) was reversed at P26 OIR, the functional alterations persisted. Using a similar therapeutic schedule, we analyzed the effect of anti-VEGF therapy on autophagy flux. Like Rapamycin, VEGF inhibitor treatment not only reduced the amount of neovascular tufts, but also activated autophagy flux at P17 OIR, mainly in ganglion cell layer and inner nuclear layer. Finally, the effects of the disruption of autophagy by Spautin-1, were evaluated at vascular, glial, and neuronal levels. After a single dose of Spautin-1, Western blot analysis showed a significant decrease in LC3B II and p62 protein expression at P13 OIR, returning both autophagy markers to OIR control levels at P17. In addition, neither gliosis nor functional alterations were attenuated. In line with these results, TUNEL staining showed a slight increase in the number of positive cells in the outer nuclear layer at P17 OIR. Overall, our results demonstrate that all treatments of induction or inhibition of the autophagic flux reduced neovascular area but were unable to completely reverse the neuronal damage. Besides, compared to current treatments, rapamycin provides a more promising therapeutic strategy as it reduces both neovascular tufts and persistent gliosis.

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