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