Sildenafil (Viagra®) down regulates cytokines and prevents demyelination in a cuprizone-induced MS mouse model
Highlights
► Sildenafil effects on cerebellum were studied in a multiple sclerosis mice model. ► CPZ increased GFAP and Iba-1 expression, and sildenafil inhibited this effect. ► Sildenafil had an anti-inflammatory effect, decreasing cytokines/COX-2 levels. ► Sildenafil inhibited CPZ-induced demyelination in the cerebellum. ► The data suggest a beneficial effect of PDE5 inhibition in CNS injury.
Introduction
Demyelination in the central nervous system (CNS) is often associated with acute and chronic inflammatory events involving the recruitment–activation of microglia/macrophages, astrocytes and leukocytes, with the release of pro-inflammatory cytokines and nitric oxide (NO) [1], [2], [3]. This neuroinflammatory mechanism can lead to neurodegenerative diseases, such as multiple sclerosis (MS), which is the most common neurological disorder in young adults in the Western world [4], [5], [6]. In addition to the well-characterized inflammatory white matter, studies have suggested axon injury, changes in the blood–brain barrier (BBB) and reactive astrogliosis and microgliosis [7], [8].
Neuroinflammatory responses seem to begin prior to any significant loss of neuronal populations in the progression of MS [9]. The sustained inflammatory mediators released by the microglia perpetuate the inflammatory cycle, activating additional microglia, promoting their proliferation and resulting in the further release of inflammatory factors. Persistent activation of brain-resident microglia may increase the permeability of the BBB and promote the increased infiltration of peripheral macrophages, the phenotype of which is critically determined by the CNS environment (peripheral immune cells are cloned and reactivated in the brain by the stimulus of resident cells) [10]. Moreover, recent evidence indicates that demyelination and oligodendrocyte degeneration follow inflammation induced by astrocyte dysfunction [9]. Therefore, neuroinflammation mediated by glial cells (astrocytes and microglia) seems to be the main phenomenon perpetuating neural damage in MS.
Current treatment options for relapsing–remitting MS are only partially effective and require a parenteral route of administration, as oral therapy is not available. Although the autoimmune and inflammatory nature of MS is well elucidated, the clinical response to immunosuppressors has been disappointing and the control of the disease by these drugs is insufficient [11]. The recent introduction of immunomodulators, such as interferon-beta, has led to a decrease in the frequency and severity of relapses [12]. As MS is the result of an imbalance in immune/inflammatory mechanisms, the identification and combination of drugs that can modulate inflammatory and immune responses through different mechanisms may offer beneficial effects. Moreover, the identification of drugs that directly act on resident inflammatory cells of the CNS may improve the treatment of MS, contributing to the control of relapses.
Mice deficient of inducible nitric oxide synthase (iNOS) develop more severe MS [13], suggesting that NO may be neuroprotective. NO is the main activator of soluble guanylyl cyclase (sGC), which is an enzyme that synthesizes cyclic GMP (cGMP) [14]. The NO-cGMP pathway plays an important role in the CNS. It participates in learning and memory mechanisms (to which cerebellum is involved) by protecting olygodendrocytes and modulating microglia/macrophage activation [15], [16], [17]. cGMP levels are regulated by specific phosphodiesterases (PDEs), enzymes that breakdown the phosphodiesteric bond of cGMP and cAMP. Several PDE inhibitors have been developed and used as therapeutic agents because they increase cyclic nucleotide levels by blocking the PDE function, thereby improving NO-cGMP signaling [18]. Thus, the modulation of NO-cGMP signaling could be a new pathway for intervention in cases of MS.
Sildenafil (Viagra, Pfizer) induces cGMP accumulation through selective PDE5 inhibition. This drug was approved for therapeutic use in erectile dysfunction and is also currently used for the treatment of pulmonary hypertension. Despite its excellent tolerability profile, these are the only diseases currently treated with sildenafil. Studies have shown that the selective PDE5 inhibitors sildenafil and vardenafil (Levitra; Bayer) and the PDE4 inhibitor ibudilast raise cGMP/cAMP levels in the brain and offer protective effects, such as improved memory in elderly rats [19] and mice [20] and decreased white matter damage in ischemic cerebrovascular injury [21]. Patients with MS report that sildenafil improves their general clinical state; these effects were initially attributed to better gray matter perfusion in the brain [22]. However, it is possible that sildenafil protects the CNS in patients with MS through the modulation of neuroinflammation [16].
Some characteristics of MS in the central nervous tissue have been induced in mouse by cuprizone ingestion. The cuprizone model is characterized by primary and reversible demyelination due to peripheral immune system-independent myelin injury [4]. Demyelination is accompanied by a well-characterized sequence of events involving the depletion of mature oligodendrocytes, microglia activation and astrocyte proliferation [7]. Therefore, cuprizone-induced demyelination and neuroinflammation in rodents has been widely used as a model for MS [23], [24]. Recently, it was demonstrated that subcutaneous injection of sildenafil produced beneficial effects in MS induced by EAE-(experimental autoimmune encephalomyelitis) [16]. However, none study was carried out using cuprizone-demyelination and daily oral administration of sildenafil so far. The cuprizone model may contribute to advance in the understanding of the sildenafil effects. The usefulness of cuprizone model gains importance because allows evaluating the role of the NO-cGMP pathway in the inflammation mediated by resident glial cells of the CNS (astrocytes and microglia), since peripheral immune cells are not mobilized by cuprizone [4]. Furthermore, an effective oral treatment may improve the quality of life of MS patients.
The present study demonstrates that sildenafil (Viagra®) administration ameliorates cuprizone-induced MS by modulating the innate neuroinflammatory response (mediated by glial cells) and prevents demyelination. The data shown here suggest a beneficial effect of cGMP-PDE inhibition in CNS injury through the regulation of glial inflammatory responses. The Food and Drug Administration (FDA) has recently approved the daily use of the PDE5 inhibitor tadalafil (Cialis; Lilly) (see http://www.clinicaspace.com/news) and, in 2005, the FDA approved sildenafil (Revatio, Pfizer) for the treatment of pulmonary hypertension with daily doses. Moreover, PDE5 inhibitors are widely used to treat erectile dysfunction and pulmonary hypertension. Thus, it is possible that well-designed clinical trials may demonstrate that the oral administration of sildenafil is suitable for individuals with neurodegenerative diseases.
Section snippets
Experimental design
Five C57BL/6 mice aged 7–10 weeks and weighing 15–20 g were used per group. The mice were examined for health status, acclimated to the laboratory environment at 25 °C and 12-h light/dark photoperiod and housed in metal cages. The control group received standard laboratory diet and pure water. Over a 4-week period, the experimental groups received either 0.2% cuprizone (oxalic-bis-cyclohexylidenehydrazide – Sigma–Aldrich Inc., St. Louis, MO, USA) mixed into the chow and pure water or 0.2%
Results
The clinical analysis revealed that cuprizone-treated animals had motor limitations, such as tremors and abnormal walking and posture. The group treated with 3 mg/kg of sildenafil exhibited no clinical improvement, but the animals treated with 25 and 50 mg/kg of sildenafil exhibited normal walking and posture as well as no or mild tremors.
Clinical signs were observed and recorded by three observers. The control animals exhibited normal motor function, exploring the environment normally. Posture
Discussion
The present study evaluated the effects of sildenafil (Viagra®, Pfizer) on cerebellar neuroinflammation and demyelination in a multiple sclerosis (MS) mice model. Cerebellum has been reported as an important affected region of CNS in MS patients, showing severe white matter atrophy [28], [29]. Despite the importance of this region, few studies have been conducted in animal models to elucidate the role of the cerebellum in MS. In the cuprizone model, demyelination is present not only in the
Acknowledgments
The authors are grateful to Ms. Josineide Correia, Anne Gabrielle Vasconcelos and Maria da Conceição Carvalho for their excellent technical assistance, Denise Rocha Gomes for careful animal care, the Department of Pharmacology (FCM, UNICAMP) and CPqAM for the animal lodging facilities. The study was funded by grants from the Brazilian fostering agencies FACEPE (Processes No. DCR-0010-2.06/10 and APQ-0158-2.06/10) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Process
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Contributed equally to the development of this study.