ABSTRACT
Visual deficits are among the most prevalent symptoms in patients with multiple sclerosis (MS). To understand deficits in the visual pathway during MS and potential treatment effects, we used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The afferent visual pathway was assessed in vivo using optical coherence tomography (OCT), electroretinography (ERG), and visually evoked cortical potentials (VEPs). Inflammation, demyelination, and neurodegeneration were examined by immunohistochemistry ex vivo. In addition, an immunomodulatory, remyelinating agent, the estrogen receptor ß ligand chloroindazole (IndCl), was tested for its therapeutic potential in the visual pathway. EAE produced functional deficits in visual system electrophysiology, including suppression of ERG and VEP waveform amplitudes and increased signal latencies. Therapeutic IndCl rescued overall visual system latency by VEP but had little impact on amplitude or ERG findings relative to vehicle. Faster VEP conduction in IndCl-treated mice was associated with enhanced myelin basic protein signal in all visual system structures examined. IndCl preserved retinal ganglion cells (RGCs) and oligodendrocyte density in the prechiasmatic white matter, but similar retinal nerve fiber layer thinning by OCT was noted in vehicle and IndCl-treated mice. Although IndCl differentially attenuated leukocyte and astrocyte staining signal throughout the structures analyzed, axolemmal varicosities were observed in all visual fiber tracts of mice with EAE irrespective of treatment, suggesting impaired axonal energy homeostasis. These data support incomplete functional recovery of VEP amplitude with IndCl, as fiber tracts displayed persistent axon pathology despite remyelination-induced decreases in latencies, evidenced by reduced optic nerve g-ratio in IndCl-treated mice. Although additional studies are required, these findings demonstrate the dynamics of visual pathway dysfunction and disability during EAE, along with the importance of early treatment to mitigate EAE-induced axon damage.
Subject(s)
Azo Compounds/pharmacology , Encephalomyelitis, Autoimmune, Experimental/pathology , Naphthalenes/pharmacology , Remyelination/drug effects , Visual Pathways/drug effects , Visual Pathways/pathology , Animals , Evoked Potentials, Visual/drug effects , Inflammation/pathology , Mice , Mice, Inbred C57BL , Multiple Sclerosis , Nerve Degeneration/pathologyABSTRACT
Pharmaceutical agents currently approved for the treatment of multiple sclerosis reduce relapse rates, but do not reverse or prevent neurodegeneration nor initiate myelin repair. The highly selective estrogen receptor (ER) ß ligand chloroindazole (IndCl) shows particular promise promoting both remyelination while reducing inflammatory cytokines in the central nervous system of mice with experimental autoimmune encephalomyelitis. To optimize these benefits, we developed and screened seven novel IndCl analogues for their efficacy in promoting primary oligodendrocyte (OL) progenitor cell survival, proliferation, and differentiation in vitro by immunohistochemistry. Two analogues, IndCl-o-chloro and IndCl-o-methyl, induced proliferation and differentiation equivalent to IndCl and were selected for subsequent in vivo evaluation for their impact on clinical disease course, white matter pathology, and inflammation. Both compounds ameliorated disease severity, increased mature OLs, and improved overall myelination in the corpus callosum and white matter tracts of the spinal cord. These effects were accompanied by reduced production of the OL toxic molecules interferon-γ and chemokine (C-X-C motif) ligand, CXCL10 by splenocytes with no discernable effect on central nervous system-infiltrating leukocyte numbers, while IndCl-o-methyl also reduced peripheral interleukin (IL)-17. In addition, expression of the chemokine CXCL1, which is associated with developmental oligodendrogenesis, was upregulated by IndCl and both analogues. Furthermore, callosal compound action potential recordings from analogue-treated mice demonstrated a larger N1 component amplitude compared to vehicle, suggesting more functionally myelinated fibers. Thus, the o-Methyl and o-Chloro IndCl analogues represent a class of ERß ligands that offer significant remyelination and neuroprotection as well as modulation of the immune system; hence, they appear appropriate to consider further for therapeutic development in multiple sclerosis and other demyelinating diseases.
Subject(s)
Corpus Callosum , Estrogen Receptor beta/agonists , Immunologic Factors/pharmacology , Indazoles/pharmacology , Multiple Sclerosis , Remyelination/drug effects , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Corpus Callosum/metabolism , Corpus Callosum/pathology , Cytokines/metabolism , Estrogen Receptor beta/metabolism , Female , Ligands , Male , Mice , Multiple Sclerosis/drug therapy , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathologyABSTRACT
Estrogen receptor ß (ERß) ligands promote remyelination in mouse models of multiple sclerosis. Recent work using experimental autoimmune encephalomyelitis (EAE) has shown that ERß ligands induce axon remyelination, but impact peripheral inflammation to varying degrees. To identify if ERß ligands initiate a common immune mechanism in remyelination, central and peripheral immunity and pathology in mice given ERß ligands at peak EAE were assessed. All ERß ligands induced differential expression of cytokines and chemokines, but increased levels of CXCL1 in the periphery and in astrocytes. Oligodendrocyte CXCR2 binds CXCL1 and has been implicated in normal myelination. In addition, despite extensive immune cell accumulation in the CNS, all ERß ligands promoted extensive remyelination in mice at peak EAE. This finding highlights a component of the mechanism by which ERß ligands mediate remyelination. Hence, interplay between the immune system and central nervous system may be responsible for the remyelinating effects of ERß ligands. Our findings of potential neuroprotective benefits arising from the presence of CXCL1 could have implications for improved therapies for multiple sclerosis.
Subject(s)
Axons/metabolism , Chemokine CXCL1/metabolism , Estrogen Receptor beta/metabolism , Myelin Sheath/metabolism , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Axons/drug effects , Cells, Cultured , Central Nervous System/drug effects , Central Nervous System/metabolism , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Female , Ligands , Mice , Mice, Inbred C57BL , Multiple Sclerosis/drug therapy , Multiple Sclerosis/metabolism , Myelin Sheath/drug effects , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Receptors, Interleukin-8B/metabolismABSTRACT
BACKGROUND: While many groups use experimental autoimmune encephalomyelitis (EAE) as a model to uncover therapeutic targets and understand the pathology underlying multiple sclerosis (MS), EAE protocol variability introduces discrepancies in central nervous system (CNS) pathogenesis and clinical disease, limiting the comparability between studies and slowing much-needed translational research. OPTIMIZED METHOD: Here we describe a detailed, reliable protocol for chronic EAE induction in C57BL/6 mice utilizing two injections of myelin oligodendrocyte glycoprotein (35-55) peptide mixed with complete Freund's adjuvant and paired with pertussis toxin. RESULTS: The active MOG35-55-EAE protocol presented here induces ascending paralysis in 80-100% of immunized mice. We observe: (1) consistent T cell immune activation, (2) robust CNS infiltration by peripheral immune cells, and (3) perivascular demyelinating lesions concurrent with axon damage in the spinal cord and various brain regions, including the optic nerve, cortex, hippocampus, internal capsule, and cerebellum. COMPARISON WITH EXISTING METHOD(S): Lack of detailed protocols, combined with variability between laboratories, make EAE results difficult to compare and hinder the use of this model for therapeutic development. We provide the most detailed active MOG35-55-EAE protocol to date. With this protocol, we observe high disease incidence and a consistent, reliable disease course. The resulting pathology is MS-like and includes optic neuritis, perivascular mononuclear infiltration, CNS axon demyelination, and axon damage in both infiltrating lesions and otherwise normal-appearing white matter. CONCLUSIONS: By providing a detailed active MOG35-55-EAE protocol that yields consistent and robust pathology, we aim to foster comparability between pre-clinical studies and facilitate the discovery of MS therapeutics.