It is estimated that worldwide there are around 285 million people of all ages with visual impairment; among these, 39 million are blind. There is a strong association between increasing age and visual impairment. Diabetic retinopathy (DR) and glaucoma are among the most important causes of vision loss in high-income regions of Central/Eastern Europe. Glaucoma affects more than 70 million people worldwide and it leads to progressive optic nerve degeneration, with a gradual loss of retinal ganglion cells (RGCs). DR is one of the most frequent complications of diabetes mellitus and it is now acknowledged as a neurodegenerative disease of the retina. In the early stages of DR, selective RGCs loss is observed without evident micro-vascular changes. Thus, glaucoma and DR share the development of progressive neurodegeneration. Therefore, a clear knowledge and understanding of the mechanism of RGCs death and how to compensate these events is essential for preserving the sight of those who are affected by these diseases. In recent decades, with the purpose to give an effective answer and solution to this issue, there was a strong boost in the field of neuroprotection. The term neuroprotection refers to any therapy that prevents, retards, or reverses apoptosis associated to neuronal cell death following primary neuronal lesions. The aim of our work was to prove whether cotreatment with citicoline and homotaurine has neuroprotective effects on cell survival in primary retinal cultures under experimental conditions mimicking retinal neurodegeneration similar to those occurring in glaucoma and in DR. In addition, the study aimed to establish the levels of pro-inflammatory cytokines and soluble mediators (TNF-α, IL6, IL2, and PDGF-AB) in 28 vitreous biopsies taken from patients with proliferative diabetic retinopathy and treated with increasing doses of curcumin (0. 5 and 1µM), with or without homotaurine (100µM) and vitamin D3 (50 nM). Primary cultures were derived from the retina of fetal rats and tested with citicoline plus homotaurine (100 μM). Thereafter, neurotoxicity was triggered using excitotoxic levels of glutamate and high glucose concentrations. The effects on retinal cultures were evaluated by cell viability [(3,4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide -MTT reduction assay] and immunodetection of apoptotic oligonucleosomes (Cell Death Detection ELISAPLUS kit). Then, in order to evaluate the anti-inflammatory effect of curcumin, homotaurine, and vitamin D3, ELISA tests were conducted on the supernatants from the 28 vitreous biopsies that were incubated with the bioactive molecules at 37◦C for 20 h. It was also tested, in a subset of four vitreous, the expression of pro-inflammatory genes and mitogen-activated genes. The concentration of the soluble mediators was calculated from a calibration curve and expressed in pg/mL. Shapiro-Wilk test was used to verify the normality of distribution of the residuals. Continuous variables among groups were compared using the General Linear Model. Homoscedasticity was verified using Levene and Brown-Forsythe tests. Post- hoc analysis was also performed with the Tukey test. A p≤0.05 was considered statistically significant. The results proved that a combination of citicoline and homotaurine synergistically decreases proapoptotic effects associated with glutamate- and high glucose-treated retinal cultures. In addition, it was shown that pro-inflammatory cytokines are associated with inflammation and angiogenesis, although there is a discrete variability in the doses of the mediators investigated among the different vitreous samples. Curcumin, homotaurine, and vitaminD3 individually have a slightly appreciable anti-inflammatory effect. However, when used in association, these substances modify the average levels of the soluble mediators of inflammation and retinal damage. Therefore, in the future, a multi-target treatment taking neuroprotective compounds may provide a therapeutic strategy for neurodegenerative eye diseases such as glaucoma and DR.
Nel mondo ci sono circa 285 milioni di persone con disabilità visive (di cui 39 milioni sono cieche). Un fattore importante è dato dall’invecchiamento della popolazione. Nelle regioni ad alto reddito dell'Europa centrale/orientale, la retinopatia diabetica (RD) ed il glaucoma sono tra le più importanti cause di perdita della vista. Il glaucoma colpisce più di 70 milioni di persone in tutto il mondo e causa la progressiva degenerazione del nervo ottico, con una graduale perdita delle cellule gangliari della retina (RGCs). La RD è tra le complicanze più frequenti del diabete mellito ed è ora riconosciuta come una malattia neurodegenerativa della retina. Nelle prime fasi della RD, si verifica la perdita selettiva di RGCs senza evidenti cambiamenti micro-vascolari. Quindi, glaucoma e RD condividono lo sviluppo di una progressiva neurodegenerazione. Pertanto, una profonda comprensione del meccanismo neurodegenerativo e di come compensarlo è essenziale per preservare la vista di coloro che sono colpiti da queste patologie. Negli ultimi decenni, con l'obiettivo di dare una risposta effettiva a questo problema, c'è stato un forte impulso nel campo della neuroprotezione. Il termine neuroprotezione indica qualsiasi terapia che prevenga, ritardi o inverta l'apoptosi delle cellule neuronali in seguito a lesioni neuronali primarie. Lo scopo del nostro lavoro è stato quello di valutare se il cotrattamento con citicolina ed omotaurina ha effetti neuroprotettivi sulla sopravvivenza cellulare nelle colture retiniche primarie poste in condizioni sperimentali che mimano una neurodegenerazione retinica simile a quella che si verifica nel glaucoma e nella RD. Inoltre, lo studio mirava a stabilire i livelli TNF-α, IL6, IL2, e PDGF-AB in 28 biopsie vitreali prelevate da pazienti con retinopatia diabetica proliferante e trattati con dosi crescenti di curcumina (0.5 e 1µM), con o senza omotaurina (100µM) e vitamina D3 (50 nM). Le colture primarie sono state ottenute dalla retina di ratti fetali e testate con citicolina più omotaurina (100 μM). Successivamente, la neurotossicità è stata innescata utilizzando livelli eccitotossici di glutammato e alte concentrazioni di glucosio. Gli effetti sulle colture retiniche sono stati valutati attraverso la vitalità cellulare (saggio MTT) e attraverso l’immunorilevazione di oligonucleosomi apoptotici (kit ELISAPLUS). Successivamente, per valutare l'effetto antinfiammatorio della curcumina, dell'omotaurina e della vitamina D3, sono stati condotti test ELISA sui supernatanti delle 28 biopsie vitreali che sono state incubate con le molecole bioattive a 37 °C per 20 ore. È stata inoltre testata, in un sottoinsieme di quattro vitrei, l'espressione di geni pro-infiammatori e geni attivati dai mitogeni. La concentrazione dei mediatori solubili è stata calcolata sulla base di una curva di taratura ed espressa in pg/mL. Per verificare la normalità è stato utilizzato il test Shapiro-Wilk. Le variabili continue tra i gruppi sono state confrontate utilizzando il Modello Lineare Generale. L'omoschedasticità è stata verificata utilizzando i test di Levene e Brown-Forsythe. L'analisi post-hoc è stata eseguita anche con il test Tukey. Un p≤0.05 è stato considerato statisticamente significativo. I risultati hanno dimostrato che una combinazione di citicolina e omotaurina riduce sinergicamente gli effetti proapoptotici nello colture retiniche trattate con glutammato ed elevate concentrazioni di glucosio. Inoltre, è stato dimostrato che nei vitrei analizzati, nonostante una discreta variabilità, le citochine pro-infiammatorie sono associate all'infiammazione e all'angiogenesi. Curcumina, omotaurina e vitaminD3 singolarmente hanno un effetto anti-infiammatorio leggermente apprezzabile. Tuttavia, quando utilizzate in associazione, queste sostanze modificano i livelli medi dei mediatori solubili di infiammazione/danno retinico. Pertanto, in futuro, un trattamento multi-target con neuroprotettori fornirà una strategia terapeutica per le malattie degenerative oculari.
Neuroprotection in degenerative eye diseases
FILIPPELLI, Mariaelena
2022-05-04
Abstract
It is estimated that worldwide there are around 285 million people of all ages with visual impairment; among these, 39 million are blind. There is a strong association between increasing age and visual impairment. Diabetic retinopathy (DR) and glaucoma are among the most important causes of vision loss in high-income regions of Central/Eastern Europe. Glaucoma affects more than 70 million people worldwide and it leads to progressive optic nerve degeneration, with a gradual loss of retinal ganglion cells (RGCs). DR is one of the most frequent complications of diabetes mellitus and it is now acknowledged as a neurodegenerative disease of the retina. In the early stages of DR, selective RGCs loss is observed without evident micro-vascular changes. Thus, glaucoma and DR share the development of progressive neurodegeneration. Therefore, a clear knowledge and understanding of the mechanism of RGCs death and how to compensate these events is essential for preserving the sight of those who are affected by these diseases. In recent decades, with the purpose to give an effective answer and solution to this issue, there was a strong boost in the field of neuroprotection. The term neuroprotection refers to any therapy that prevents, retards, or reverses apoptosis associated to neuronal cell death following primary neuronal lesions. The aim of our work was to prove whether cotreatment with citicoline and homotaurine has neuroprotective effects on cell survival in primary retinal cultures under experimental conditions mimicking retinal neurodegeneration similar to those occurring in glaucoma and in DR. In addition, the study aimed to establish the levels of pro-inflammatory cytokines and soluble mediators (TNF-α, IL6, IL2, and PDGF-AB) in 28 vitreous biopsies taken from patients with proliferative diabetic retinopathy and treated with increasing doses of curcumin (0. 5 and 1µM), with or without homotaurine (100µM) and vitamin D3 (50 nM). Primary cultures were derived from the retina of fetal rats and tested with citicoline plus homotaurine (100 μM). Thereafter, neurotoxicity was triggered using excitotoxic levels of glutamate and high glucose concentrations. The effects on retinal cultures were evaluated by cell viability [(3,4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide -MTT reduction assay] and immunodetection of apoptotic oligonucleosomes (Cell Death Detection ELISAPLUS kit). Then, in order to evaluate the anti-inflammatory effect of curcumin, homotaurine, and vitamin D3, ELISA tests were conducted on the supernatants from the 28 vitreous biopsies that were incubated with the bioactive molecules at 37◦C for 20 h. It was also tested, in a subset of four vitreous, the expression of pro-inflammatory genes and mitogen-activated genes. The concentration of the soluble mediators was calculated from a calibration curve and expressed in pg/mL. Shapiro-Wilk test was used to verify the normality of distribution of the residuals. Continuous variables among groups were compared using the General Linear Model. Homoscedasticity was verified using Levene and Brown-Forsythe tests. Post- hoc analysis was also performed with the Tukey test. A p≤0.05 was considered statistically significant. The results proved that a combination of citicoline and homotaurine synergistically decreases proapoptotic effects associated with glutamate- and high glucose-treated retinal cultures. In addition, it was shown that pro-inflammatory cytokines are associated with inflammation and angiogenesis, although there is a discrete variability in the doses of the mediators investigated among the different vitreous samples. Curcumin, homotaurine, and vitaminD3 individually have a slightly appreciable anti-inflammatory effect. However, when used in association, these substances modify the average levels of the soluble mediators of inflammation and retinal damage. Therefore, in the future, a multi-target treatment taking neuroprotective compounds may provide a therapeutic strategy for neurodegenerative eye diseases such as glaucoma and DR.File | Dimensione | Formato | |
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