Neurodegeneration and neuroprotection in experimental models of retinal diseases

Retinal diseases like diabetic retinopathy (DR) and glaucoma are major causes of vision loss and blindness worldwide.DR has been classically defined as a vascular disease. Nevertheless, in the last decade, different studies highlighted that neuronal death and glial activation occur before any vascular changes. In particular, hyperglycaemia triggers metabolic pathways that lead to the onset of oxidative stress (OS). OS, in turn, enhances retinal metabolic dysfunction and leads to neurodegeneration. Moreover, OS induces the upregulation of vascular endothelial growth factor (VEGF).In DR, VEGF performs a double role. Indeed, in the early phases of the disease, it is released to protect retinal neurons, but, with the progression of DR, it undergoes an uncontrolled upregulation responsible for retinal pathological vascular changes and neoangiogenesis. The molecular mechanisms involved in this VEGF upregulation are not well known. I hypothesised that the OS-induced VEGF upregulation is due to an autocrine loop in which VEGF, mainly released by Müller cells, directly induces its own expression. The results confirmed that OS may induce VEGF expression through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor involved in the antioxidant response. VEGF, in turn, would promote its own expression and release through an autocrine loop in which the growth factor would bind to its receptor VEGFR2 and activate hypoxia-inducible factor 1, the main transcription factor involved in VEGF expression, thereby favouring the upregulation of the growth factor and then the progression of DR. Similar to DR, OS plays a key role also in glaucoma, even though with different mechanisms. Indeed, an increase in intraocular pressure would lead to OS, glial reactivity, retinal ganglion cell death, and functional deficits. Therefore, it appears that, although DR and glaucoma manifest different pathophysiological features, they are characterised by the same pathological trigger, that is OS. Considering the OS related mechanisms of neuroinflammation and neurodegeneration, counteracting and preventing retinal OS could be a good strategy for treating both DR and glaucoma. Different therapeutic approaches have been directed at modulating the retinal prooxidant/antioxidant equilibrium using nutraceuticals as natural antioxidant compounds. In particular, I evaluated the antioxidant and neuroprotective effects of Lisosan G (LG), a fermented powder obtained from whole grains and composed by different metabolites(including gallic acid, 4-hydroxybenzoic acid, quercetin, and nicotinamide), using an in vivo model of glaucoma represented by DBA/2J mice. In this context, the antioxidant, anti-inflammatory and neuroprotective effects of LG were highlighted. Moreover, a pharmacokinetic analysis demonstrated that the main LG metabolites can reach the retina in a relatively short time. These data, on the one hand, confirm and expand the notion of LG as a powerful natural compound with high therapeutic potential, while on the other provide important information for further studies aimed at improving the bioavailability of the active components of LG. Within the same research line, I also investigated the antioxidant efficacy of a nature inspired hybrid (NIH1) using an ex vivo model of retinal OS. NIH1 is a hybrid derived from curcumin and diallyl sulphide, which exerts its antioxidant effect by activating Nrf2. The hybridization strategy was aimed at increasing curcumin bioavailability and enhancing its antioxidant properties. Indeed, in previous studies, NIH1 emerged for its ability to activate Nrf2 to a higher extent than curcumin alone in in vitro models. At that point, it was essential to gather information about NIH1 efficacy in models that maintain the retinal complexity. For this reason, I tested the effect of NIH1 in organotypic retinal explants. In particular, I evaluated the impact of the hybrid on the activation of antioxidant enzyme expression, neuroprotection, and glial reactivity in response to OS. The results confirmed the antioxidant properties of NIH1 and provided a rationale for further in vivo studies. Taken together, the findings of this thesis contribute to characterize a mechanism involved in VEGF upregulation in response to OS and novel therapeutic strategies for early treatment of glaucoma and DR

"Espressione e rilascio di VEGF indotto da VEGF in modelli in vitro di stress retinico"

La retinopatia diabetica (DR) è una patologia che colpisce la retina ed è considerata una delle principali complicanze del diabete di tipo I e II. Il decorso della patologia è lento e graduale. Infatti, si passa da una fase non proliferativa, contraddistinta da un’iniziale neurodegenerazione, fino ad arrivare ad una fase proliferativa in cui si assiste a eventi di neo angiogenesi. Sono molti i fattori di crescita in grado di provocare alterazioni patologiche a livello della glia, degli astrociti e dei vasi sanguigni, tra questi uno dei più importanti è il fattore di crescita endoteliale vascolare (VEGF). Il VEGF è di fondamentale importanza nella comunicazione neurovascolare. Tale fattore in condizioni di diabete va incontro ad un aumento anomalo e, nelle fasi tardive della patologia, è in grado di promuovere la formazione di vasi aberranti. Tali vasi a lungo andare eserciteranno una trazione meccanica in grado di provocare il distacco della retina con conseguente perdita della vista da parte del paziente. Inoltre, studi recenti hanno evidenziato una produzione di VEGF da parte della componente neuronale retinica durante le prime fasi della patologia. In queste fasi, il VEGF sembra essere in grado di fornire protezione da una serie di insulti derivanti dall’eccesso di glucosio, come eccitotossicità da glutammato, prodotti avanzati della glicazione (AGE) e soprattutto stress ossidativo (OS). È quindi chiaro che il VEGF svolge un ruolo chiave nella DR. Nonostante ciò ad oggi non si hanno informazioni chiare su quali siano i processi alla base dell’espressione e del rilascio di tale fattore durante le prime fasi della DR. Soprattutto non è ancora chiaro quali siano i meccanismi molecolari che permettono il continuo rilascio del fattore di crescita. La nostra ipotesi prevede che il VEGF inizialmente prodotto dalle cellule della retina sottoposte a stress sia in grado di influenzare il suo stesso rilascio, probabilmente attraverso un processo di feedback positivo. Quindi, l’oggetto del presente lavoro di tesi è stato quello di valutare il rilascio e l’espressione del VEGF indotti dallo stesso VEGF in presenza 9 di uno stress tipico delle prime fasi della DR, cioè lo OS. I modelli utilizzati sono stati due: un modello ex vivo di DR, rappresentato da espianti organotipici di retina, e un modello in vitro costituito da una linea derivata da cellule di Müller, le cellule MIO-M1. Espianti o cellule MIO-M1 sono stati incubati in un terreno contenete H2O2. Dopo 24h di incubazione, questo terreno (terreno condizionato, CM) è stato utilizzato per l’incubazione di altri espianti e cellule MIO-M1. Inoltre, per valutare come l'espressione di VEGF variasse in presenza di VEGF stesso sia le cellule MIO-M1 che gli espianti retinici sono stati trattati con diverse concentrazioni di VEGF esogeno. Dai risultati ottenuti si registra un aumento dell'espressione di VEGF in cellule MIO-M1 trattate con VEGF esogeno e con terreni condizionati. Al contrario, negli espianti retinici tale aumento di espressione viene registrato solo in seguito al trattamento con VEGF esogeno e non con terreni condizionati. Questo risultato potrebbe essere giustificato dal fatto che probabilmente gli espianti sottoposti a stress ossidativo non sono in grado di rilasciare una quantità adeguata di VEGF all'interno del terreno. Nonostante ciò, i risultati ottenuti sembrerebbero essere promettenti, quest'ultimi, uniti alla messa a punto di nuovi modelli sperimentali di coltura organotipica, potrebbero mettere in luce una nuova via di regolazione del VEGF in cui tale fattore potrebbe essere direttamente implicato nel favorire la sua stessa espressione e il suo stesso rilascio

DNA alteration induced by ultraviolet light in human metaphase chromosomes substituted with 5′-bromodeoxy uridine: monitoring by monoclonal antibodies to double-stranded and single stranded DNA

Fixed human metaphase chromosomes, whose DNA had been substituted with 5'-bromodeoxyuridine (BrdUrd) for two rounds of replication (TB/BB) or for one round in BrdUrd followed by another round in thymidine (TT/BT), were treated with ultraviolet light (UV), in the presence or in the absence of 33258 Hoechst, to produce sister chromatid differentiation (SCD). Giemsa staining was compared with staining with monoclonal antibodies to double-stranded or single-stranded DNA. We confirmed that UV acts by debrominating BrdUrd-stubstituted DNA but showed that debromination alone cannot explain all our findings. We postulated that UV-induced protein-protein cross-linking, occurring to a different extent in differently BrdUrd-substituted chromatids, may also be invoked in explaining our data. Lastly, the different behaviour of unifilarly substituted TB as opposed to BT chromatids in UV-treated chromosomes, allowed us to hypothesize that such chromatids may differ depending on whether or not newly synthesized DNA is formed on a BrdUrd-containing stran

Oxidative Stress Induces a VEGF Autocrine Loop in the Retina: Relevance for Diabetic Retinopathy

Background: Oxidative stress (OS) plays a central role in diabetic retinopathy (DR), triggering expression and release of vascular endothelial growth factor (VEGF), the increase of which leads to deleterious vascular changes. We tested the hypothesis that OS-stimulated VEGF induces its own expression with an autocrine mechanism. Methods: MIO-M1 cells and ex vivo mouse retinal explants were treated with OS, with exogenous VEGF or with conditioned media (CM) from OS-stressed cultures. Results: Both in MIO-M1 cells and in retinal explants, OS or exogenous VEGF induced a significant increase of VEGF mRNA, which was abolished by VEGF receptor 2 (VEGFR-2) inhibition. OS also caused VEGF release. In MIO-M1 cells, CM induced VEGF expression, which was abolished by a VEGFR-2 inhibitor. Moreover, the OS-induced increase of VEGF mRNA was abolished by a nuclear factor erythroid 2-related factor 2 (Nrf2) blocker, while the effect of exo-VEGF resulted Nrf2-independent. Finally, both the exo-VEGF- and the OS-induced increase of VEGF expression were blocked by a hypoxia-inducible factor-1 inhibitor. Conclusions: These results are consistent with the existence of a retinal VEGF autocrine loop triggered by OS. This mechanism may significantly contribute to the maintenance of elevated VEGF levels and therefore it may be of central importance for the onset and development of DR

Importance of the hippocampus for the learning of route fidelity in homing pigeons

The avian hippocampal formation (HF) is thought to regulate map-like memory representations of visual landmarks/landscape features and has more recently been suggested to be similarly important for the perceptual integration of landmarks/landscapes. Aspects of spatial memory and perception likely combine to support the nowwell-documented ability of homing pigeons to learn to retrace the same routewhen homing fromfamiliar locations, leading to the prediction that damage to the HF would result in a diminished ability to repeatedly fly a similar route home. HF-lesioned homing pigeons were repeatedly released from three sites to assess the importance of the hippocampus as pigeons gradually learn a familiar route home guided by familiar landmark and landscape features. As expected, control pigeons displayed increasing fidelity to a familiar route home, and by inference, successful perceptual and memory processing of familiar landmarks/landscape features. By contrast, the impoverished route fidelity of the HF-lesioned pigeons indicated an impaired sensitivity to the same landmark/landscape features

GPS-profiling of retrograde navigational impairments associated with hippocampal lesion in homing pigeons.

The avian hippocampal formation (HF) is homologous to the mammalian hippocampus and plays a central role in the control of spatial cognition. In homing pigeons, HF supports navigation by familiar landmarks and landscape features. However, what has remained relatively unexplored is the importance of HF for the retention of previously acquired spatial information. For example, to date, no systematic GPS-tracking studies on the retention of HF-dependent navigational memory in homing pigeons have been performed. Therefore, the current study was designed to compare the pre- and post-surgical navigational performance of sham-lesioned control and HFlesioned pigeons tracked from three different sites located in different directions with respect to home. The pre- and post-surgical comparison of the pigeons’ flight paths near the release sites and before reaching the area surrounding the home loft (4 km radius from the loft) revealed that the control and HF-lesioned pigeons displayed similarly successful retention. By contrast, the HF-lesioned pigeons displayed dramatically and consistently impaired retention in navigating to their home loft during the terminal phase of the homing flight near home, i.e., where navigation is supported by memory for landmark and landscape features. The data demonstrate that HF lesions lead to a dramatic loss of pre-surgically acquired landmark and landscape navigational information while sparing those mechanisms associated with navigation from locations distant from home

Lisosan G Protects the Retina from Neurovascular Damage in Experimental Diabetic Retinopathy

Lisosan G (LG), a fermented powder obtained from whole grains, is a recognized antioxidant compound that improves the bioactivity and survival of different cell types. The purpose of this study was to investigate whether LG ameliorates both the neural and the vascular damage characterizing early stages of diabetic retinopathy (DR). The effects of LG were studied in cultured explants of mouse retinas challenged with oxidative stress (OS) or in retinas of streptozotocin (STZ)-treated rats. Apoptosis, vascular endothelial growth factor (VEGF) expression, OS markers, blood-retinal barrier (BRB) integrity, and inflammation were assessed, while retinal function was evaluated with electroretinogram (ERG). LG extensively inhibited apoptosis, VEGF expression, and OS both in retinal explants and in STZ rats. In addition, STZ rats treated with LG displayed an almost total BRB integrity, reduced levels of inflammatory markers and a partially restored visual function as evaluated with ERG. In summary, we demonstrated that LG exhibits antioxidant and anti-inflammatory effects that exert powerful protective actions against neural and vascular defects characteristic of DR. Therefore, LG-containing foods or supplements may be considered to implement DR treatments

The potential of Lisosan G as a possible treatment for glaucoma

Lisosan G (LG), a fermented powder obtained from whole grains, is a nutritional supplement containing a variety of metabolites with documented antioxidant properties. We have recently demonstrated that orally administered LG protects diabetic rodent retinas from oxidative stress, inflammation, apoptosis, blood-retinal barrier disruption, and functional damage. Here, we investigated whether LG may exert protective effects in a model of glaucoma and measured the amounts of selected LG components that reach the retina after oral LG administration. Six-month-old DBA/2J mice were given an aqueous LG solution in place of drinking water for 2 mo. During the 2 mo of treatment with LG, the intraocular pressure (IOP) was monitored and the retinal ganglion cell (RGC) functional activity was recorded with pattern-electroretinography (PERG). At the end of the 2-mo period, the expression of oxidative stress and inflammatory markers was measured with qPCR, and RGC survival or macroglial activation were assessed with immunofluorescence. Alternatively, LG was administered by gavage and the concentrations of four of the main LG components (nicotinamide, gallic acid, 4- hydroxybenzoic acid, and quercetin) were measured in the retinas in the following 24 h using mass spectrometry. LG treatment in DBA/2J mice did not influence IOP, but it affected RGC function since PERG amplitude was increased and PERG latency was decreased with respect to untreated DBA/2J mice. This improvement of RGC function was concomitant with a significant decrease of both oxidative stress and inflammation marker expression, of RGC loss, and of macroglial activation. All four LG metabolites were found in the retina, although with different proportions with respect to the amount in the dose of administered LG, and with different temporal profiles in the 24 h following administration. These findings are consistent with neuroenhancing and neuroprotective effects of LG in glaucoma that are likely to derive from its powerful antioxidant properties. The co-occurrence of different metabolites in LG may provide an added value to their beneficial effects and indicate LG as a basis for the potential treatment of a variety of retinal pathologies

A Nature-Inspired Nrf2 Activator Protects Retinal Explants from Oxidative Stress and Neurodegeneration

Oxidative stress (OS) plays a key role in retinal dysfunctions and acts as a major trigger of inflammatory and neurodegenerative processes in several retinal diseases. To prevent OS-induced retinal damage, approaches based on the use of natural compounds are actively investigated. Recently, structural features from curcumin and diallyl sulfide have been combined in a nature-inspired hybrid (NIH1), which has been described to activate transcription nuclear factor erythroid-2-related factor-2 (Nrf2), the master regulator of the antioxidant response, in different cell lines. We tested the antioxidant properties of NIH1 in mouse retinal explants. NIH1 increased Nrf2 nuclear translocation, Nrf2 expression, and both antioxidant enzyme expression and protein levels after 24 h or six days of incubation. Possible toxic effects of NIH1 were excluded since it did not alter the expression of apoptotic or gliotic markers. In OS-treated retinal explants, NIH1 strengthened the antioxidant response inducing a massive and persistent expression of antioxidant enzymes up to six days of incubation. These effects resulted in prevention of the accumulation of reactive oxygen species, of apoptotic cell death, and of gliotic reactivity. Together, these data indicate that a strategy based on NIH1 to counteract OS could be effective for the treatment of retinal diseases