sildenafil-citrate and Demyelinating-Diseases

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-1β 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. Therefor

Topics: Animals; Chemokine CCL2; Cuprizone; Demyelinating Diseases; Matrix Metalloproteinase 9; Mice; Multiple Sclerosis; Myelin Sheath; Phosphodiesterase 5 Inhibitors; Receptors, CCR2; Signal Transduction; Sildenafil Citrate

Sildenafil (Viagra®) has recently been found to have a neuroprotective effect, which occurs through the inhibition of inflammation and demyelination in the cerebellum. However, the mechanism of action of sildenafil remains unknown. AMPK, the regulatory protein of the lipid and glucose metabolism, plays a protective role by activating the eNOS enzyme. The production of a nanomolar concentration of NO by eNOS has an anti-inflammatory effect through the cGMP signaling pathway and plays an important role in the regulation of the nuclear transcription factor (NFkB), preventing the expression of inflammatory genes. The present study investigated whether AMPK-eNOS-NO-cGMP-IКβα-NFkB is involved in the mechanism of action of sildenafil in a cuprizone-demyelination model. Neuroinflammation and demyelination induced by cuprizone in rodents have been widely used as a model of MS. In the present study, five male C57BL/6 mice (7-10 weeks old) were used. Over a four week period, the groups received: cuprizone (CPZ) 0.2% mixed in feed; CPZ in the diet, combined with the administration of sildenafil (Viagra®, Pfizer, 25mg/kg) orally in drinking water, starting concurrently (sild-T0) or 15 days (sild-T15) after the start of CPZ. Control animals received pure food and water. The cerebella of the mice were dissected and processed for immunohistochemistry, immunofluorescence (frozen), western blotting and dosage of cytokines (Elisa). CPZ induced an increase in the expression of GFAP, IL-1β TNF-α, total NFkB and inactive AMPK, and prompt microglia activation. CPZ also induced a reduction of IKβα. The administration of sildenafil reduced the expression of the pro-inflammatory cytokines IL-1β and TNF-α and increased the expression of the anti-inflammatory cytokine IL-10. In addition, the administration of sildenafil reduced expression of GFAP, NFkB, inactive AMPK and iNOS, and increased IKβα. Interestingly, sildenafil also reduced levels of NGF. In general, the sild-T0 group was more effective than sild-T15 in improving clinical status and promoting the control of neuroinflammation. The present study offers evidence that sildenafil has anti-inflammatory and neuroprotective effects, which are probably achieved through modulation of AMPK-IKβα-NFκB signaling. In addition, eNOS may play a role in the sildenafil neuroprotective mechanism, contributing to the activation of AMPK. However, other pathways such as MAPK-NFkB and the downstream proteins AMPK (AMPK-SIRT1-NFκB) should also be

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Chelating Agents; Cuprizone; Cytokines; Demyelinating Diseases; Disease Models, Animal; Encephalitis; Enzyme Inhibitors; Glial Fibrillary Acidic Protein; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Nitric Oxide Synthase Type III; Sildenafil Citrate

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 β , 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 β 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.

Topics: Animals; Cuprizone; Cyclic GMP; Demyelinating Diseases; Glutathione S-Transferase pi; Inflammation; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Myelin Sheath; Neurons; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Sildenafil Citrate; Sulfones

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-1β 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-1β, 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.

Topics: Animals; Cerebellum; Cuprizone; Cyclooxygenase 2; Cytokines; Demyelinating Diseases; Disease Models, Animal; Down-Regulation; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Immunoblotting; Male; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Piperazines; Purines; Sildenafil Citrate; Sulfones

Cyclic GMP (cGMP)-mediated pathways regulate inflammatory responses in immune and CNS cells. Recently, cGMP phosphodiesterase inhibitors such as sildenafil, commonly used to treat sexual dysfunction in humans including multiple sclerosis (MS) patients, have been reported to be neuroprotective in animal models of stroke, Alzheimer's disease, and focal brain lesion. In this work, we have examined if sildenafil ameliorates myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show for the first time that treatment with sildenafil after disease onset markedly reduces the clinical signs of EAE by preventing axonal loss and promoting remyelination. Furthermore, sildenafil decreases CD3+ leukocyte infiltration and microglial/macrophage activation in the spinal cord, while increasing forkhead box transcription factor 3-expressing T regulatory cells (Foxp3 Tregs). However, sildenafil treatment did not significantly affect MOG₃₅₋₅₅-stimulated proliferation or release of Th1/Th2 cytokines in splenocytes but decreased ICAM-1 in spinal cord infiltrated cells. The presence of reactive astrocytes forming scar-like structures around infiltrates was enhanced by sildenafil suggesting a possible mechanism for restriction of leukocyte spread into healthy parenchyma. These results highlight novel actions of sildenafil that may contribute to its beneficial effects in EAE and suggest that treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention to ameliorate MS neuropathology.

Topics: Animals; CD3 Complex; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gliosis; Glycoproteins; Intercellular Adhesion Molecule-1; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurofilament Proteins; Peptide Fragments; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Sildenafil Citrate; Spinal Cord; Sulfones; Time Factors