Sildenafil ameliorates EAE by decreasing apoptosis in the spinal cord of C57BL/6 mice.

Apoptosis is one form of cell death that is intimately related to health and pathological conditions. In most neuroinflammatory and/or neurodegenerative diseases, apoptosis is associated with disease development and pathology and inhibition of this process leads to considerable amelioration. It is becoming evident that apoptosis also participates in the pathogenesis of Multiple Sclerosis (MS) and its animal model, Experimental Autoimmune Encephalomyelitis (EAE). Drugs such as Sildenafil, a Phosphodiesterase type 5 Inhibitor (PDE5I), have proven to be neuroprotective in MS models. However, it is not known whether Sildenafil is able to modulate cell death, specifically apoptosis, in EAE mice. Therefore, the aim of this study was to determine the effects of Sildenafil on extrinsic and intrinsic apoptosis pathways in the spinal cord of C57BL/6 mice with EAE. TUNEL analysis showed that EAE mice had elevated number of TUNEL+ cells and that treatment with Sildenafil led to reduced number of dying cells, indicating that Sildenafil was able to inhibit cell death. We observed that both extrinsic and intrinsic pathways of apoptosis were governing the dynamics of EAE progression. We showed that in EAE mice there were increased levels of extrinsic (Caspase-8, -3, TNF-α, FADD) and intrinsic (Caspase-9, Bax and Cytochrome C) apoptosis markers. Bcl-2, an anti-apoptotic protein, was downregulated in EAE mice. We also demonstrated that EAE mice had increased levels of non-caspase mediators of cell survival/cell death (p-IκBα and p-MAPK-p38). Besides, EAE mice presented augmented demyelination. Nevertheless, this is the first research to demonstrate that Sildenafil, when administered concomitant to disease induction, modulated the expression of pro- and anti-apoptotic proteins of the extrinsic and intrinsic pathways, as well as diminished the expression of non-caspase mediators and promoted remyelination in the spinal cord, indicating neuroprotective effects. Thus, the present study demonstrated that Sildenafil inhibits apoptosis by two distinct, although interconnected, mechanisms: directly by modulating caspase expression (through extrinsic and intrinsic pathways) and indirectly by modulating the expression of molecules involved in cell death and/or cell survival.

Phosphodiesterase-5 inhibition promotes remyelination by MCP-1/CCR-2 and MMP-9 regulation in a cuprizone-induced demyelination model.

While it has recently been shown that sildenafil (Viagra®) has a protective effect on myelination/remyelination, the mechanism of this protection is still unknown. In general, cytokines, chemokines and metalloproteinases have a pro-inflammatory action, but can also exert a role in modulating glial cell activation, contributing to the balance of cell response. Investigating these molecules can contribute to clarifying the mechanisms of sildenafil neuroprotection. In addition, it is not known whether sildenafil is able to restore an already installed neurodegenerative process or if the treatment period is critical for its action. The aim of the present study was to evaluate, in a cuprizone (CPZ)-induced demyelination model, the effects and mechanisms of time-dependent treatment with sildenafil (beginning 15 days after neurodegeneration and continuing for 15 days, or starting concomitantly with neurodegeneration and continuing for 30 days) on neuroinflammation and remyelination. Neuroinflammation and demyelination induced by CPZ in rodents has been widely used as a model of multiple sclerosis (MS). In the present study, five male C57BL/6 mice aged 7-10 weeks were used per group. For four weeks, the groups received either cuprizone (CPZ) 0.2% mixed in feed or CPZ combined with the administration of sildenafil (Viagra®, Pfizer, 25 mg/kg) orally in drinking water, starting concurrently with (sild-T0) or 15 days (sild-T15) after the start of CPZ treatment. Control animals received pure food and water. The cerebella were dissected and processed for immunohistochemistry, immunofluorescence (frozen), Western blotting, Luxol fast blue staining and transmission electron microscopy. Magnetic resonance was performed for live animals, after the same treatment, using CPZ 0.3%. CPZ induced an increase in the expression of IL-1ß and a decrease in MCP-1, CCR-2, MBP and GST-pi, as well as promoting damage in the structure and ultra-structure of the myelin sheath. Interestingly, the administering of sild-T0 promoted a further increase of MMP-9, MCP-1, and CCR-2, possibly contributing to changes in the microglia phenotype, which becomes more phagocytic, cleaning myelin debris. It was also observed that, after sild-T0 treatment, the expression of GST-pi and MBP increased and the myelin structure was improved. However, sild-T15 was not efficient in all aspects, probably due to the short treatment period and to starting after the installation of the degenerative process. Therefore, the present study shows that sildenafil modulates inflammation, with the involvement of MMP-9, MCP-1, and CCR-2, and also contributes to myelin repair. These protective effects were dependent on the therapeutic strategy used. This clarification can strengthen research proposals into the mechanism of action of sildenafil and contribute to the control of neurodegenerative diseases such as MS.

Sildenafil (Viagra) protective effects on neuroinflammation: the role of iNOS/NO system in an inflammatory demyelination model.

We recently demonstrated that sildenafil reduces the expression of cytokines, COX-2, and GFAP in a demyelinating model induced in wild-type (WT) mice. Herein, the understandings of the neuroprotective effect of sildenafil and the mediation of iNOS/NO system on inflammatory demyelination induced by cuprizone were investigated. The cerebella of iNOS(-/-) mice were examined after four weeks of treatment with cuprizone alone or combined with sildenafil. Cuprizone increased GFAP, Iba-1, TNF- α , COX-2, IL-1 ß , and IFN- γ expression, decreased expression of glutathione S-transferase pi (GSTpi), and damaged myelin in iNOS(-/-) mice. Sildenafil reduced Iba-1, IFN- γ , and IL-1 ß levels but had no effect on the expression of GFAP, TNF- α , and COX-2 compared to the cuprizone group. Sildenafil elevated GSTpi levels and improved the myelin structure/ultrastructure. iNOS(-/-) mice suffered from severe inflammation following treatment with cuprizone, while WT mice had milder inflammation, as found in the previous study. It is possible that inflammatory regulation through iNOS-feedback is absent in iNOS(-/-) mice, making them more susceptible to inflammation. Sildenafil has at least a partial anti-inflammatory effect through iNOS inhibition, as its effect on iNOS(-/-) mice was limited. Further studies are required to explain the underlying mechanism of the sildenafil effects.

Sildenafil (Viagra®) down regulates cytokines and prevents demyelination in a cuprizone-induced MS mouse model.

Sildenafil induces cGMP accumulation through phosphodiesterase-5 (PDE5) inhibition. cGMP-pathways protect oligodendrocytes and modulate astroglial and microglial reactions. Microglia and astrocytes play an important role in perpetuating multiple sclerosis (MS), a chronic inflammatory disease characterized by demyelination. Therefore, sildenafil can be a potential tool for MS treatment. The present study investigated the effects of sildenafil on the myelin structure and astrocyte/microglia-mediated neuroinflammation in an animal model of MS. Cuprizone-induced demyelination and neuroinflammation in rodents has been widely used as a model for MS. Herein, five male C57BL/6 mice (7-10 weeks old) were used per group. Over a 4-week period, the different groups received the following: (1) cuprizone (0.2%) mixed into the chow; (2) cuprizone in the chow and sildenafil (Viagra®; 3, 25 or 50mg/kg) in the drinking water; or (3) pure chow and water (control group). Cerebella were analyzed using transmission electron microscopy, western blotting, immunohistochemistry and luxol fast blue staining. Cuprizone induced tissue damage, with an increase in GFAP, Iba-1 and COX-2 and demyelination in comparison to the control group. However, cuprizone did not affect the expression of cytokines (TNF-α, IFN-γ, IL-1ß and IL-2). Sildenafil reduced GFAP (25 and 50mg/kg) and Iba-1 expression (25mg/kg) in comparison to the cuprizone group, indicating the modulation of astrocytes and microglia, respectively. Sildenafil preserved myelin and axons ultrastructure and strongly reduced IFN-γ, TNF-α, IL-1ß, IL-2 and COX-2 expression in comparison to the control and/or cuprizone groups. The results demonstrate a protective effect of sildenafil in the cerebellum. Thus, well-designed clinical trials may demonstrate that the oral administration of sildenafil can be suitable for individuals with MS and other neuroinflammatory/neurodegenerative diseases, providing additional benefits to current treatments.