Myelin structure in unfixed, single nerve fibers: Scanning X-ray microdiffraction with a beam size of 200nm.

Previous raster-scanning with a 1µm X-ray beam of individual, myelinated fibers from glutaraldehyde-fixed rat sciatic nerve revealed a spatially-dependent variation in the diffraction patterns from single fibers. Analysis indicated differences in the myelin periodicity, membrane separations, distribution of proteins, and orientation of membrane lamellae. As chemical fixation is known to produce structural artifacts, we sought to determine in the current study whether the structural heterogeneity is intrinsic to unfixed myelin. Using a 200nm-beam that was about five-fold smaller than before, we raster-scanned individual myelinated fibers from both the peripheral (PNS; mouse and rat sciatic nerves) and central (CNS; rat corpus callosum) nervous systems. As expected, the membrane stacking in the internodal region was nearly parallel to the fiber axis and in the paranodal region it was perpendicular to the axis. A myelin lattice was also frequently observed when the incident beam was injected en face to the sheath. Myelin periodicity and diffracted intensity varied with axial position along the fiber, as did the calculated membrane profiles. Raster-scanning with an X-ray beam at sub-micron resolution revealed for the first time that the individual myelin sheaths in unfixed nerve are heterogeneous in both membrane structure and packing.

Plasmalogen phospholipids protect internodal myelin from oxidative damage.

Reactive oxygen species (ROS) are implicated in a range of degenerative conditions, including aging, neurodegenerative diseases, and neurological disorders. Myelin is a lipid-rich multilamellar sheath that facilitates rapid nerve conduction in vertebrates. Given the high energetic demands and low antioxidant capacity of the cells that elaborate the sheaths, myelin is considered intrinsically vulnerable to oxidative damage, raising the question whether additional mechanisms prevent structural damage. We characterized the structural and biochemical basis of ROS-mediated myelin damage in murine tissues from both central nervous system (CNS) and peripheral nervous system (PNS). To determine whether ROS can cause structural damage to the internodal myelin, whole sciatic and optic nerves were incubated ex vivo with a hydroxyl radical-generating system consisting of copper (Cu), hydrogen peroxide (HP), and ortho-phenanthroline (OP). Quantitative assessment of unfixed tissue by X-ray diffraction revealed irreversible compaction of myelin membrane stacking in both sciatic and optic nerves. Incubation in the presence of the hydroxyl radical scavenger sodium formate prevented this damage, implicating hydroxyl radical species. Myelin membranes are particularly enriched in plasmalogens, a class of ether-linked phospholipids proposed to have antioxidant properties. Myelin in sciatic nerve from plasmalogen-deficient (Pex7 knockout) mice was significantly more vulnerable to Cu/OP/HP-mediated ROS-induced compaction than myelin from WT mice. Our results directly support the role of plasmalogens as endogenous antioxidants providing a defense that protects ROS-vulnerable myelin.

Nanoemulsions of an anti-oxidant synergy formulation containing gamma tocopherol have enhanced bioavailability and anti-inflammatory properties.

The present study investigated whether MicroFluidizer Processor-based nanoemulsions of an antioxidant synergy formulation (ASF), containing delta, alpha and gamma tocopherol influenced inflammation and bioavailability in CD-1 mice. Croton oil was applied to all animals' right ear lobe to induce inflammation. Auricular thickness was measured after 2 and 6h after the various treatments. The animal plasma and ear lobes were collected and frozen for bioavailability and cytokine analyses. The ASF nanoemulsions of alpha, delta, or gamma tocopherol significantly reduced auricular thickness compared to control (57, -57, and -71%, respectively) and blank nanoemulsion (-50, -50, -67%, respectively). Relative to the suspensions of ASF, only the nanoemulsion of ASF containing gamma tocopherol significantly reduced auricular thickness (-60%), whereas the 40% reduction with nanoemulsions of delta tocopherol compared to suspension was not statistically significant. Auricular concentrations of cytokines TNF-alpha and IL-1 alpha were significantly reduced in mice treated only with ASF nanoemulsions of gamma tocopherol compared to control (-53, -46%, respectively) and blank nanoemulsion (-52, -46%, respectively). Auricular thickness was significantly associated with tissue TNF-alpha (r=0.539, p<0.001) and IL-1 alpha concentrations (r=0.404, p=0.01). Bioavailability for gamma and delta was dramatically enhanced (2.2- and 2.4-folds) with the nanoemulsion compared to suspensions. Only the plasma gamma tocopherol concentration was significantly associated with auricular thickness (r=-0.643, P=0.001). In conclusion, nanoemulsions of ASF containing gamma, alpha, and delta tocopherol, have enhanced anti-inflammatory properties and increased bioavailability, with gamma tocopherol, in particular compared to their suspensions.

Enhancement of anti-inflammatory property of aspirin in mice by a nano-emulsion preparation.

Aspirin, a non-steroidal anti-inflammatory drug, widely used for its anti-inflammatory properties is associated with several systemic side effects including gastro-intestinal discomfort. Inflammation can be mediated by pro-inflammatory cytokines and, along with various other host factors eventually give rise to edema at the inflamed site. Because of the adverse side effects oftentimes associated with systemic exposure to aspirin, the aim of the present study was to investigate whether the anti-inflammatory property of aspirin would enhance if delivered as nano-emulsion preparation. Nano-emulsion preparations of aspirin prepared with a Microfluidizer Processor were evaluated in the croton-oil-induced ear edema CD-1 mouse model using ear lobe thickness and the accumulation of specific in situ cytokines as biomarkers of inflammation. The results showed that particle size (90 nm) populations of nano-emulsion preparations of aspirin compared to an aspirin suspension (363 nm), significantly decreased (p<0. 05) ear lobe thickness approximately 2 fold greater than the aspirin suspension. In addition, the aspirin nano-emulsion further reduced the auricular levels of IL-1alpha (-37%) and TNFalpha (-69%) compared to the aspirin suspension preparation (p<0.05). The reductions in ear lobe thickness were also significantly associated with accumulated tissue levels of IL-1alpha (r=0.5, p<0.009) and TNFalpha (r=0.7, p<0.0004), respectively. In conclusion, these studies indicate that a nano-emulsion preparation of aspirin significantly improved the anti-inflammatory properties of an aspirin suspension in a CD-1 mouse model of induced inflammation.

A nanoemulsion of an anti-oxidant synergy formulation reduces tumor growth rate in neuroblastoma-bearing nude mice.

Neuroblastoma, the most common form of childhood cancer, may arise from a biochemical block of cellular differentiation and a resultant continuation of a proliferative state. Neuroblastoma often spontaneously reverts by undergoing partial differentiation and ultimate degeneration and may be associated with the generation of reactive oxygen species (ROS). We have recently reported in neuroblastoma cell culture studies that an anti-oxidant synergy formulation (ASF) can induce differentiation and buffer neuronal degeneration and oxidative stress in cultured cortical neurons and in central nervous system tissue of apolipoprotein E-deficient mice. The objective of the present study was to investigate whether a subcutaneous injection and/or transdermal application of a nanoemulsion preparation of ASF would reduce tumor growth rate in a neuroblastoma xenograph mouse model. The results indicate that whereas suspensions of ASF were ineffective in decreasing tumor growth rate in the neuroblastoma mouse model, tumor growth rate was similarly reduced an average 65% by either subcutaneous injection or transdermal application of an ASF nanoemulsion preparation to the tumor. In conclusion, the data suggest that subcutaneous and/or transdermal application of an ASF nanoemulsion preparation is effective in reducing tumor growth rate in this neuroblastoma mouse model.