| The purpose of this research was to determine if manipulation of an endogenous opioid system affects the progression of multiple sclerosis (MS) using an in vivo experimental autoimmune encephalomyelitis (EAE) animal model. The endogenous opioid system was manipulated using an opioid receptor agonist ([Met5]-enkephalin, Met-enk, opioid growth factor, OGF) or a high or low dose of an opioid receptor antagonist (naltrexone, NTX). Additional in vitro studies were conducted to determine if administration of exogenous OGF or NTX affects splenic-derived lymphocyte proliferation. OGF and NTX have been shown to decrease and increase the rate of cancer cell proliferation, respectively; the present studies focus on defining the unknown role of OGF and NTX in EAE progression.;For the in vivo studies, EAE was induced in 7--8 week old female C57Bl/6 mice with a series of myelin oligodendrocyte glycoprotein (MOG) injections on Days 0, 3, and 6. Beginning with the first MOG injection, mice received a daily intraperitoneal injection of either phosphate-buffered saline (vehicle), a high dose of NTX to cause 24-hour opioid receptor blockade (HDN, 10mg/kg), a low dose of NTX to cause intermittent 4--6 hour opioid receptor blockade (LDN, 0.1mg/kg), or a high dose of opioid growth factor (OGF, 10mg/kg). Treatment was administered at the same time each day. Behavioral symptoms were monitored daily and disease scores were assigned based on a 0--5 scale. A score of 0 was assigned to healthy animals free of EAE symptoms, and subsequent increasing scores correlated to increasing levels of disability. Animals were sacrificed 10, 20, 30, and 60 days post-EAE induction to monitor pre-clinical EAE, acute EAE, established EAE, and chronic EAE, respectively. Spinal cord demyelination was visualized with a luxol fast blue (LFB) stain, and additional spinal cord sections were stained with antibodies directed at astrocytes and damaged neurons to show changes in number or morphology of the cells.;In vitro studies were conducted to determine if OGF or NTX administration altered splenic-derived lymphocyte proliferation. First, the presence of both OGF and OGFr on splenic-derived mouse lymphocytes was confirmed with immunocytochemical analysis. Next, fresh lymphocytes were isolated from the spleens of normal mice, activated with phytohemagluttinin (PHA), treated in culture with OGF or NTX, and counted daily to measure proliferation over a 72 hour time period.;Results revealed that chronic LDN and OGF treatment decreased EAE disease severity, disease incidence, and disease index, and increased the average day of EAE disease onset compared to vehicle-treated mice. Continuous opioid receptor blockade with HDN had no effect on EAE disease onset or progression compared to vehicle treatment. Immunohistochemical analysis of astrocyte activation and neuronal damage showed that daily administration of LDN improved histological symptoms of EAE in comparison to vehicle treatment at 10, 20, and 30 days post-EAE induction. Similarly, daily injections of OGF decreased the level of astrocyte activation on Days 10, 20, 30, and 60 and the level of neuronal damage on Day 20 compared to vehicle-treated controls. Interestingly, mice given HDN had lower astrocyte counts on Days 30 and 60 and less neuronal damage on Day 30 compared to vehicle-treated mice. In vitro experiments demonstrated that OGF caused a decrease in lymphocyte proliferation compared to lymphocytes exposed to saline or NTX. It is possible that OGF is effective at treating EAE in vivo due to a downregulation of immune system function due to an inhibition of lymphocyte proliferation. Similarly, intermittent opioid receptor blockade with LDN, which causes upregulation in the production of endogenous opioid peptide and receptor, could inhibit the hyperproliferative immune response in EAE. It is possible that LDN is beneficial in vivo because it causes an upregulation in opioid-opioid receptor interactions with subsequent decreased immune system function.;Collectively, these results indicate that intermittent opioid receptor blockade with LDN and administration of exogenous OGF can prevent or decrease the behavioral and histopathological signs of EAE. Although EAE is not a perfect model of MS, hallmarks of both diseases - inflammation, demyelination, and neurodegeneration - were decreased in EAE following LDN and OGF administration. (Abstract shortened by UMI.)... |
