Elsevier

Brain Research Bulletin

Volume 98, September 2013, Pages 122-131
Brain Research Bulletin

Research report
Treatment of a relapse-remitting model of multiple sclerosis with opioid growth factor

https://doi.org/10.1016/j.brainresbull.2013.08.001Get rights and content

Highlights

  • First evidence to demonstrate therapeutic role for OGF in RR-EAE in mice.

  • OGF therapy mitigated initial clinical disease scores and prevented further relapse.

  • OGF therapy increased the number and length of remissions.

  • OGF therapy inhibited activation of Iba1 + microglia/macrophages and CD3+ T cells.

  • OGF inhibited astrocyte proliferation and repressed neuronal damage in spinal cord.

Abstract

Relapse-remitting multiple sclerosis (MS) is an immune-mediated disease of the central nervous system that affects more than 2.5 million individuals worldwide. While the etiology of MS is unclear, disease manifestation involves proliferation and activation of lymphocytes and astrocytes, leading to demyelination and neuronal damage. Current therapies are not completely effective, and few target the underlying pathophysiology of MS. The purpose of this study was to examine the therapeutic efficacy of a novel biological pathway, the opioid growth factor (OGF)–OGF receptor (OGFr) axis. OGF inhibits DNA synthesis and has been shown to repress proliferation of T lymphocytes, microglia, and astrocytes in other autoimmune disorders. An animal model for relapse-remitting experimental autoimmune encephalomyelitis (RR-EAE) was established by immunization of SJL/J mice with proteolipid protein. Treatment with OGF or saline was initiated simultaneously with immunization, and within 9 days, behavioral signs of RR-EAE were observed. OGF-treated RR-EAE animals had less severe clinical disease than mice receiving saline and exhibited 66% reductions in median cumulative disease scores, as well as prolonged periods of remission and diminished number and length of disease relapses. Neuropathological examination of lumbar spinal cord revealed reductions in the number of T lymphocytes, microglia/macrophages, and activated astrocytes, with cell proliferation being the mechanism targeted by OGF. Areas of demyelination and neuronal damage were markedly reduced during the 55-day observation period. These data are the first to demonstrate that OGF prevented relapses in RR-EAE and diminished underlying neuropathology, corroborating the potential of the OGF–OGF receptor pathway for treatment of MS.

Introduction

Multiple sclerosis (MS) is a chronic, debilitating immune-mediated disease of the central nervous system (CNS) that affects more than 2.5 million individuals worldwide, with nearly 85% of the patients afflicted with the relapse-remitting form (RR-MS) (NMSS, 2013). Disease manifestation involves proliferation and activation of T-lymphocytes, microglia, and astrocytes, leading to inflammation, demyelination, and axonal damage. Over a period of time, neurodegeneration in the spinal cord and brain are associated with disease progression. Current therapies are designed to target one or more of the symptoms of the disease, but few are disease-modifying in nature (Stoll et al., 2012). Despite some differences in the cause of relapse-remitting disease, the mouse model of relapse-remitting experimental autoimmune encephalomyelitis (RR-EAE) represents an animal model that responds to proteolipid protein immunizations by proliferation of T-cells and microglia, and activation of astrocytes (Pollinger et al., 2009, Summers De Luca et al., 2010). The behavioral course of disease can be charted and utilized as an endpoint for therapeutic interventions.

Opioid growth factor (OGF), chemically termed [Met5]-enkephalin, and its receptor, OGFr, form a physiological pathway that maintains homeostasis and can be modulated to shift the course of disease (Zagon et al., 2002, McLaughlin and Zagon, 2012). Modulation of the OGF–OGFr axis either by chronic treatment with the endogenous peptide OGF, or by upregulation of OGF and OGFr following low dosages of naltrexone (LDN), in mice immunized with myelin oligodendrocytic glycoprotein (MOG) to establish progressive EAE was neuroprotective against encephalitogenic processes (Zagon et al., 2009b, Zagon et al., 2010, Rahn et al., 2011). Signs of behavioral deficits are delayed in appearance, reduced in severity, or reversed in EAE mice receiving 10 mg/kg OGF beginning at the time of disease induction in comparison to mice receiving daily injections of saline (Zagon et al., 2010, Rahn et al., 2011, Campbell et al., 2012). Evaluation of lumbar spinal cord sections revealed significant reductions in the number of activated astrocytes and regions of demyelination (Zagon et al., 2010, Rahn et al., 2011). Treatment of mice with exogenous OGF initiated at the time of established EAE reversed the progression of clinical disease within 6 days (Campbell et al., 2012). Mice with MOG-induced EAE and receiving OGF treatment initiated with established disease exhibited a reduced number of activated astrocytes and damaged neurons, decreased areas of demyelination, and repressed T cell proliferation (Campbell et al., 2012). Within 3 weeks of MOG immunization, EAE mice treated with saline had 3.5-fold elevated numbers of Iba1+ cells in the lumbar spinal cord in comparison to normal mice. OGF-treated EAE mice had 30% reductions in the number of microglia/macrophages relative to EAE mice receiving saline (Campbell et al., 2012). OGF therapy reduced the number of T lymphocytes in the spinal cord (detected by CD3 staining) by 56% relative to EAE mice receiving saline. The mechanism targeted by OGF was cell proliferation, with Ki67 staining markedly reduced in spinal cord sections from OGF-treated EAE mice. Sections stained with both Ki67 and GFAP revealed only 3% of cells in OGF-treated EAE mice being double labeled in comparison to ∼14% of cells in spinal cord sections from saline-injected EAE mice. OGF has been shown in a number of models to up-regulate cyclin-dependent inhibitory kinases and protract cell passage from G0G1 to S (Cheng et al., 2009). This mechanism to reduce cell proliferation has been documented for T and B lymphocytes stimulated in vitro to replicate (Zagon et al., 2011a, Zagon et al., 2011b).

These observations were demonstrated using an animal model of MOG-induced EAE that most resembles chronic, progressive MS; however, most patients have RR-MS (NMSS, 2013). In the present study, we established a mouse model of RR-EAE using proteolipid protein (PLP) immunization of SJL/J mice (Summers De Luca et al., 2010), and determined the efficacy of daily injections of OGF initiated at the time of disease induction. Mice were observed daily over a 55 day period of time, and lumbar spinal cord tissue was collected on 10, 14, and 55 days after initiation of treatment in order to assess expression and proliferation of astrocytes, T lymphocytes, microglia/macrophages, as well as demyelination and neuronal damage.

Section snippets

Mice

Female SJL/JOrlCRL mice (Charles River Labs, Wilmington, MA) were housed 5 per cage under standard conditions in a separate room from other rodents and acclimated for one week prior to disease induction; food and water were available ad libitum. As the course of EAE disease progressed, soft food and water packets were placed on the floor of the cages. All experiments were conducted in accordance with the National Institute of Health guidelines on animal care, and were approved by the

General observations and behavior

Two separate experiments were conducted and all mice inoculated with PLP developed signs of EAE in both studies. Injections of PLP139–151 resulted in redness and swelling at the site of injection in some SJL mice; however, no mouse died from immunization procedures (i.e. first 11 days). In one study, 3 of 30 mice died at or near the peak disease (i.e. days 12–14), with no additional deaths recorded. No normal mouse developed any neurological abnormality or died over the course of 2 months.

Discussion

Relapse-remitting multiple sclerosis is the most common form of this autoimmune-related disorder and presents in patients as a wide spectrum of behavioral and pathological signs (NMSS, 2013, Fitzner and Simons, 2010). This study demonstrates for the first time data that exogenous OGF treatment can prevent or mitigate relapses in a mouse model of RR-EAE when OGF treatment is initiated at the time of disease induction. OGF treatment initiated at the time of disease induction was effective at

Conflict of interest statement

There are no financial conflicts for any of the authors regarding this manuscript. There are no issues requiring disclosure.

Acknowledgement

This work was supported by the Paul K. and Anna M. Shockey Foundation.

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