Abstract
Background: Animal models of peripheral neuroinflammation are essential to understanding the pathogenesis of, and deducing new treatments for demyelinating polyneuropathies. This paper updates and describes adapted methods for the isolation, purification and verification of bovine peripheral nerve myelin used to reliably induce peripheral neuritis in susceptible mice. Methods: Myelin was isolated via discontinuous sucrose and continuous cesium chloride density gradient ultracentrifugation from frozen adult bovine cauda equina. Negatively stained electron microscopy was employed to confirm myelin structure. Phase contrast and routine light microscopy, gravimetric analyses, gel electrophoresis and lens culinaris fluorescent western blotting, analytical colorimetric assays and indirect immunohistochemistry were performed to analyze whole myelin or its protein and lipid subcomponents. Myelin isolates were used to induce experimental autoimmune neuritis in 8-12 Weeks old female Swedish Jim Lambert/ Jackson mice. Results: Negatively-stained electron microscopy demonstrated the two-dimensional repetitive lamella structure of myelin and a lack of organelle contamination. Lipids accounted for ~72% of the dry mass of bovine peripheral nerve myelin, while ~25% was protein/ proteolipid and <4% insoluble residue based on gravimetric analyses. Glycoproteins accounted for the majority of isolated myelin proteins, with myelin protein zero (~28 KDa) being the most prevalent protein. Phospholipids accounted for ~60% of the total dry weight of bovine peripheral nerve myelin. Surface expression of galactocerebroside and sulfatide, two major lipid subcomponents present in myelin, was detected by indirect fluorescent immunohistochemistry. Bovine peripheral nerve myelin reliably induced a severe macrophagepredominant demyelinating polyneuropathy with associated axonal loss in female mice, consistent with experimental autoimmune neuritis. Conclusions: These updated and adapted methods using readily available resources can be applied to rapidly isolate, purify and verify peripheral nerve myelin from animal sources, and determine its protein and lipid composition prior to use in peripheral neuritis animal models.
Highlights
Myelin, the complex biological membrane that facilitates saltatory transmission of electrical impulses along axons, is derived from the differentiation of Schwann cell plasma membranes in the peripheral nervous system (PNS) and from oligodendrocytes in the central nervous system (CNS) [1]
Mice immunized with purified myelin proteins or peptide fractions are often resistant or develop very mild forms of the disease, making these models inadequate to study PNS inflammation and demyelination seen in Guillain-Barré syndrome (GBS) [7]
Similarities in neural-immune responses between mice and humans make it essential to work with reliable mouse PNS inflammation models to understand the pathogenesis of demyelinating polyneuropathies and find novel molecular therapeutic targets
Summary
The complex biological membrane that facilitates saltatory transmission of electrical impulses along axons, is derived from the differentiation of Schwann cell plasma membranes in the peripheral nervous system (PNS) and from oligodendrocytes in the central nervous system (CNS) [1]. Studies using native myelin remain important despite advances in peptide biosynthesis. This is the case for a severe murine experimental autoimmune neuritis (EAN) model of GBS [3,4,5,6]. Mice immunized with purified myelin proteins or peptide fractions are often resistant or develop very mild forms of the disease, making these models inadequate to study PNS inflammation and demyelination seen in GBS [7]. Similarities in neural-immune responses between mice and humans make it essential to work with reliable mouse PNS inflammation models to understand the pathogenesis of demyelinating polyneuropathies and find novel molecular therapeutic targets. This paper updates and describes adapted methods for the isolation, purification and verification of bovine peripheral nerve myelin used to reliably induce peripheral neuritis in susceptible mice
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