Rapamycin reduces clinical signs and neuropathic pain in a chronic model of experimental autoimmune encephalomyelitis

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Abstract

Current treatments used in Multiple Sclerosis (MS) are partly effective in the early stages of the disease but display very limited benefits in patients affected by progressive MS. One possible explanation is that these therapies are unable to target the inflammatory component most active during the progressive phase of the disease, and compartmentalized behind the blood–brain barrier. Our findings show that Rapamycin ameliorates clinical and histological signs of chronic EAE when administered during ongoing disease. Moreover, Rapamycin significantly reduced the hyperalgesia observed before clinical development of EAE which, in turn, is completely abolished by the administration of the drug.

Introduction

Multiple sclerosis (MS) is one of the most common neurological disorders among young adults; it occurs on a background of neuro-inflammatory reaction, sustained by lymphocytes and activated microglia and macrophages, leading to demyelination (Noseworthy et al., 2000), neurodegeneration and often to permanent disability. In the majority of MS patients, the disease begins with a relapsing course (relapsing/remitting MS, RRMS), followed (in roughly 15% of the cases) by a progressive phase after several years (secondary progressive MS, SPMS). In 10% of the cases, the relapsing phase is missing and the disease is progressive from the onset (primary progressive MS, PPMS) (Ghezzi, 2004). Secondary progression is characterized by the irreversibility of clinical deficits, as for example severe motor impairment and spasticity, caused by neurodegenerative processes. Neurodegeneration occurs as a consequence of axonal damage which can be induced directly by the inflammatory attacks and/or by the demyelination process (Fitzner and Simons, 2010). Moreover, neuronal lesions and dysfunction lead often to neuropathic pain, the most prevalent pain syndrome observed in MS patients as well as the most difficult to treat (O'Connor et al., 2008). Approximately 50% of MS patients experience clinically significant pain during the course of disease, with significant reduction of life quality.

Current anti-inflammatory, immunomodulatory or immunosuppressive treatments, including also newly available drugs (i.e. cladribine, alemtuzumab, rituximamb), are partly effective in the early stages of the disease but display very limited benefits in patients affected by progressive MS (for a recent review see Jones and Coles, 2010). These therapies are probably unable to target the inflammatory component, most active during the progressive phase of the disease, i.e. the activation of innate immune system sustained by microglia and dendritic cells (Lassmann, 2007). Moreover, there is also a compartmentalization of inflammatory cells behind the blood–brain barrier (BBB), which is not sufficiently permeable to most drugs at this stage of disease (Hochmeister et al., 2006). In this scenario, it might be postulated that more aggressive immunomodulatory or immunosuppressive treatments or a much earlier start of such therapies are required to substantially affect the timing and the severity of progression in MS. In particular, there is need of drugs that have the ability to easily cross the BBB and target both the adaptive as well as the innate immune system.

Rapamycin, also known as sirolimus, is a macrocyclic triene antibiotic displaying antitumor and immunosuppressive activities; it has been widely used in preventing clinical allograft rejection and in treating some autoimmune diseases (Kelly et al., 1997). Rapamycin potently blocks cytokine-driven T cell proliferation, by inhibiting the activity of the serine/threonine kinase mammalian target of Rapamycin (mTOR) (Jozwiak et al., 2006, Mondino and Mueller, 2007). However, Rapamycin and other mTOR inhibitors do not interfere with the ability of CD4+-CD25+ regulatory T cells (Tregs) to induce immunological tolerance (Game et al., 2005), which appears to be relevant to the MS pathology. It has been reported that Rapamycin can induce the expansion of both mouse and human CD4+CD25+FOXP3+ Tregs in vitro, the expansion of mouse CD4+CD25+FOXP3+ T cells in vivo, and it contributes to normalization of defective Treg function in type 1 diabetic patients (Inoki, 2008). Rapamycin readily crosses the BBB, thus exerting direct effects within the CNS (Pong and Zaleska, 2003). At this level, we have recently shown that Rapamycin is able to inhibit glial activation in vitro; in particular, the block of mTOR reduces the expression and activity of the inducible form of nitric oxide synthase (iNOS) in microglial cultures activated by proinflammatory cytokines, and selectively affects microglia viability and proliferation without any effect on the viability of primary astrocytes (Dello Russo et al., 2009). However, 10 nM Rapamycin is able to up-regulate iNOS mRNA levels without any significant effect on iNOS activity in astrocytes. This apparently contradictory result is explained by a parallel increase in the rate of iNOS mRNA degradation associated with the inhibition of mTOR kinase (Lisi et al., 2011), thus explaining the overall anti-inflammatory properties of this drug within the CNS. Interestingly, central administration of Rapamycin reduces neuropathic pain induced by ligation of peripheral nerves, by acting both on an mTOR-positive subset of A-nociceptors and on the lamina I neuronal projections (Géranton et al., 2009), as well as by reducing TNF-α production in the spinal cord following sciatic nerve injury (Orhan et al., 2010). All together these data suggest that Rapamycin has the potential to exert beneficial effects in the treatment of chronic forms of MS.

In this regard, Rapamycin has been shown to prevent the induction and the progression of the relapsing–remitting experimental autoimmune encephalomyelitis (RR-EAE), a widely used animal model to study RR-MS pathology. This beneficial effect has been associated to suppression of effector T cell function and simultaneous increase of the percentage of Treg cells (Esposito et al., 2010). In addition, RR-EAE rats treated with Rapamycin exhibited a milder inflammatory infiltration of the spinal cord with smaller areas of demyelination and increased number of splenic Tregs in comparison to control animals (Donia et al., 2009). In the present study, we tested the effects of Rapamycin in a chronic model of EAE. Active immunization with myelin oligodendrocyte glycoprotein (MOG) or MOG35–55 peptide in C57BL6 mice yields a chronic monophasic disease, characterized by sustained central inflammation, demyelination and axonal damage (Iglesias et al., 2001). In our experience, animals usually develop a long-lasting disease, showing clinical symptoms 5–7 after the MOG35–55 booster injection and reaching the peak of disease at 10–14 days. The disease tends to remain stable or progress over the time; in fact animals barely recover unless effectively treated (Feinstein et al., 2002, Murphy et al., 2002). Therefore, this model appears to be a better experimental paradigm to study chronic forms of MS. Interestingly, we observed that Rapamycin ameliorates clinical and histological signs of chronic EAE mice when administered to already ill animals, at the peak of disease (therapeutic approach). Moreover, the drug significantly reduced the hyperalgesia, which can be detected at the clinical onset of disease. For this evaluation, Rapamycin was injected in a prophylactic manner (after the second MOG35–55 booster injection), and it completely abolished the development of EAE. In conclusion, the current findings suggest that Rapamycin has the potential to exert beneficial effects in chronic forms of MS (PPMS and SPMS). The exact mechanism of action has not been fully elucidated, but since the peripheral inflammatory component appears to be modest in the later stage of disease (Juedes et al., 2000), the therapeutic actions of Rapamycin may be probably due to its ability to cross the BBB as well as to its anti-inflammatory effects on CNS glia. Our data suggest that Rapamycin may also be useful in MS patients who experience pain symptoms, such as neuropathic pain.

Section snippets

Mice

Female C57BL/6 mice, aged 8–12 weeks were maintained in a controlled 12:12 h light/dark environment and provided food and water. The study was revised by local Institutional Animal Care and Use Committee, and approved by the Italian Ministry of Heath (protocol licensed to CDR).

EAE induction and clinical scoring assessment

EAE was actively induced in mice using the rat/mouse synthetic myelin oligodendrocyte glycoprotein peptide 35–55, (MOG35–55) (Sigma-Aldrich, St. Louis MO), as previously described (Feinstein et al., 2002). Briefly mice were

Rapamycin ameliorates clinical and histological signs of chronic EAE

Female C57BL/6 mice were immunized as described in the Methods section. At the peak of disease, mice were randomized into two groups (8 animals for each group) that had similar patterns of disease development. One group received the vehicle solution and the other one received 1 mg/kg of Rapamycin solution i.p according to the ‘therapeutic schedule’, described in the Methods section. Rapamycin displayed a significant therapeutic effect, reducing the severity of disease as shown by reduction of

Discussion

In the present study, we have characterized the beneficial effects of Rapamycin in a chronic model of EAE; such effects were observed both in a therapeutic schedule as well as in a prophylactic manner of administration. Moreover, Rapamycin was able to reduce signs of neuropathic pain, increasing the sensitivity threshold for mechanical allodynia. The EAE induced by immunization with the rodent MOG35–55 peptide in C57BL/6 mice is a chronic monophasic disease that well mimics the progressive form

Acknowledgment

The authors would like to thank Giulio Lisi for his precious help in building the plexiglass chambers used for the assessment of mechanical allodynia; Paul E Polak and Laura Bui for their assistance with immunohistochemistry analysis.

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