Elsevier

Brain Research

Volume 1381, 24 March 2011, Pages 243-253
Brain Research

Research Report
Prevention and diminished expression of experimental autoimmune encephalomyelitis by low dose naltrexone (LDN) or opioid growth factor (OGF) for an extended period: Therapeutic implications for multiple sclerosis

https://doi.org/10.1016/j.brainres.2011.01.036Get rights and content

Abstract

Endogenous opioids inhibit the onset and progression of experimental autoimmune encephalomyelitis (EAE) with 30 days of treatment. This study examined the long term effects of the opioid growth factor (OGF, [Met5]-enkephalin) and a low dose of the opioid antagonist naltrexone (LDN) on expression of myelin oligodendrocyte glycoprotein (MOG)-induced EAE. C57BL/6 mice began receiving daily injections of 10 mg/kg OGF (MOG + OGF), 0.1 mg/kg naltrexone (MOG + LDN), or saline (MOG + Vehicle) at the time of EAE induction and continuing for 60 days. In contrast to 100% of the MOG + Vehicle group with behavioral symptoms of EAE, 63% and 68% of the MOG + OGF and MOG + LDN mice expressed disease. Both severity and disease indices of EAE in OGF- and LDN-treated mice were notably decreased from MOG + Vehicle cohorts. By day 60, 6- and 3-fold more animals in the MOG + OGF and MOG + LDN groups, respectively, had a remission compared to MOG + Vehicle mice. Neuropathological studies revealed i) astrocyte activation and neuronal damage as early as day 10 (prior to behavioral symptoms) in all MOG-injected groups, ii) a significant reduction of activated astrocytes in MOG + OGF and MOG + LDN groups compared to MOG + Vehicle mice at day 30, and iii) no demyelination on day 60 in mice treated with OGF or LDN and not displaying disease symptoms. These results indicate that treatment with OGF or LDN had no deleterious long-term repercussions and did not exacerbate EAE, but i) halted progression of disease, ii) reversed neurological deficits, and iii) prevented the onset of neurological dysfunction across a considerable span of time.

Research Highlights

► OGF and LDN have long-term effects on EAE. ► OGF and LDN prevent the onset of neurological behavioral symptoms of EAE. ► OGF and LDN can halt the expression of EAE. ► OGF and LDN can reverse neurological deficits of EAE. ► OGF and LDN may be utilized in translational studies for multiple sclerosis.

Introduction

Multiple sclerosis (MS) is a chronic and debilitating autoimmune disease of the central nervous system (CNS) that affects 400,000 people in the United States and 2 million individuals worldwide (Rizvi and Agius, 2004, Forte et al., 2007). While the etiology of MS is unclear, disease manifestation involves inflammation, demyelination, and axonal damage in the CNS (Bennett and Stuve, 2009, Weiner, 2009, Van der Walt et al., 2010). The first neurologic episode is termed “clinically isolated syndrome” (CIS), with an estimated 35,000 patients presenting each year, and 80% or more converting from CIS to clinically definite MS (Pandey and Lublin, 2009). The proliferation of immune cells and the production of myelin-specific antibodies at the site of active CNS lesions suggest that MS involves a dysregulation of the immune system, particularly T and B cells (Ercolini and Miller, 2006). Treatment/prevention/reparation of the neurodegeneration associated with the disease, particularly effective, inexpensive, non-toxic, and preferably orally delivered treatments, are needed.

The novel finding that endogenous opioids are biological factors that target growth was first reported in 1983 (Zagon and McLaughlin, 1983a, Zagon and McLaughlin, 1983b). Using an innovative model that employed both an intermittent and continuous daily opioid receptor blockade with a low and high dose, respectively, of the opioid receptor antagonist naltrexone (NTX), native opioid peptides were hypothesized to function as tonically active inhibitory molecules for cell replication in a receptor-mediated manner. The duration of opioid receptor blockade proved critical in this concept (Zagon and McLaughlin, 1984, Zagon and McLaughlin, 1989a, Zagon and McLaughlin, 1989b). NTX administration results in a compensatory upregulation of endogenous opioids and opioid receptors, and interaction of these elements takes place provided that there is a sufficient interval after NTX is no longer bound to a receptor. Hence, a low dose of NTX (LDN) can inhibit growth when NTX is no longer present after an initial 4- to 6-h period, leaving a window of 18–20 h for interfacing of the elevated opioids and opioid receptors that produces an exaggerated response (e.g., inhibition of cell replication). Subsequent studies (Zagon and McLaughlin, 1989a, Zagon and McLaughlin, 1989b, Zagon and McLaughlin, 1991) revealed the endogenous opioid serving to tonically regulate cell proliferation through an inhibitory pathway was the pentapeptide [Met5]-enkephalin, termed opioid growth factor (OGF) to distinguish its distribution (neural and non-neural) and function (growth) from its original characterization as a neurotransmitter. The growth related function of OGF was found to be mediated by the non-classical opioid receptor, OGFr (originally termed zeta; Zagon et al., 1989, Zagon et al., 2000), and the mechanism of OGF–OGFr action involves upregulating the cyclin-dependent inhibitory pathway and delaying the G1-S phase of the cell cycle (Chen et al., 2007, 2009a).

A series of reports (Zagon et al., 2009, Zagon et al., 2010) reveal that endogenous opioids have a neuroprotective effect on encephalitogenic processes in mice with experimental autoimmune encephalomyelitis (EAE), a prototype used to study MS in animals. Exposure to OGF interferes with autoimmune events and suppresses the onset and progression of EAE. Similarly, daily treatment with LDN has marked anti-EAE activity. However, exposure to a daily high dosage of NTX (HDN) that is continuously present for 24 h did not change the course of EAE expression, suggesting that opioid-receptor interaction is critical to modulating disease processes.

These earlier preclinical studies have focused on a short window of evaluation in terms of the effects of OGF and LDN on EAE, with behavioral observations limited to 30 days and neuropathology assessed after 20 days of treatment. This raises the important question of the whether OGF and/or LDN action is(are) short-term and diminish(es) after a longer course of treatment, or if the modulations of myelin oligodendrocyte glycoprotein (MOG)-induced EAE by one or both of these treatments are sustained. In this study, mice were given injections of MOG and received daily injections of the opioid agonist OGF, or LDN to invoke an intermittent opioid receptor blockade, beginning at the time of EAE induction. Outcome measures included analysis of behavior and incidence of remission across a 60-day period, as well as neuropathological assessment at 10, 30, and 60 days.

Section snippets

General observations

No lesions or ulcerations at the injection sites were observed in any animal. Behavioral abnormalities were not noted in any group for the first week following MOG injections. There were no differences in body weight between any MOG-injected group, and no deaths were noted. Control + Vehicle, as well as Normal mice, did not present with behavioral signs of EAE.

LDN and OGF treatment and EAE: behavioral assessment

Daily administration of LDN and OGF significantly decreased disease scores compared to MOG + Vehicle mice (Fig. 1A). Average disease scores

Discussion

This study shows that modulation of opioid peptide–opioid receptor interactions by either daily administration of a native opioid peptide, OGF, or intermittent opioid receptor blockade with LDN, beginning at the time of EAE induction has long term repercussions in repressing the appearance and progression of disease. Over 60% of the MOG injected mice receiving OGF or LDN ever presented with behavioral symptoms of EAE and, in those mice that did, the severity of behavioral abnormality was

Animals and induction of experimental autoimmune encephalomyelitis

C57BL/6 female mice (7–10 weeks) were purchased from Harlan Laboratories (Indianapolis, IN) and maintained at the Penn State Hershey Medical Center, with food and water provided ad libitum. All experiments were conducted in accordance with the NIH guidelines on animal care and were approved by the Penn State Hershey Institutional Animal Care and Use Committee.

EAE was induced in mice following earlier reports (Suen et al., 1997, Zagon et al., 2009, Zagon et al., 2010). In brief, mice were given a

Acknowledgments

This work was supported in part by the National Multiple Sclerosis Society, the Paul K. and Anna E. Shockey Family Foundation, and the Zagon/Kostel families.

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