Using quantitative birefringence microscopy to identify myelin in the aging primate brain
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Abstract
In the central nervous system, oligodendrocytes extend processes to wrap around axons, creating a lipid-rich, multilayer, insulative sheath that enables rapid signal conduction along the length of the axon. When axons lose their myelin sheaths, or when the sheath’s structural integrity is compromised, the axon is unable to efficiently transmit action potentials and conduction may fail. In the extreme, the axon ultimately dies. Without these axonal connections, cognitive abilities decline. This loss of myelin may be due to overt pathological processes or may be a consequence of normal biological aging. Quantifying the deterioration of myelin across the lifespan and understanding under what conditions myelin might degrade is a key part of understanding mechanisms of normal cognitive decline seen in aging.
Quantitative birefringence microscopy (qBRM) is a method that exploits the birefringent nature of myelin sheaths – the densely packed, multilayered phospholipid sheath surrounding the axon is capable of refracting polarized light in a way distinct from surrounding structures. This is a unique feature in the central nervous system and enables myelin to be selectively imaged (Blanke, Go, Rosene, & Bigio, 2021). The image produced renders myelin bright against darkfield background with few-to-no additional visible structures. This birefringent nature enables specific visualization of myelin without staining or otherwise manipulating the tissue. It provides tissue preservation benefits over standard brightfield microscopy, which requires permanent staining of tissue and over darkfield microscopy, which illuminates anything that refracts light. Other methods such as confocal microscopy, labels tissue irreversibly with unstable fluorescent antibody tags while electron microscopy requires plastic embedding and heavy metal staining, which destroys tissue properties. Additionally, the qBRM method can be automated to render an image of an entire sample of tissue in a relatively short time.
In order to optimize and validate the use of qBRM to identify and quantify normal and damaged myelin, standard neurohistological methods that demonstrate myelin have been modified and optimized for compatibility with birefringence microscopy while still allowing subsequent immunohistochemical labeling of myelin. First, this required adjusting the method of mounting tissue sections onto microscope slides to minimize myelin degradation while allowing the use of glycerol as a mounting medium to match the refractive index (RI) required for qBRM. Second, in order to use the same section for validation using immunohistochemical staining for myelin markers after imaging with qBRM, a method was worked out to allow removal of the coverslip and glycerol while preserving the tissue section. Finally, methods were optimized for imaging the labeled tissue in gray and/or white matter with qBRM and immunohistochemistry (IHC) with confocal microscopy.
These studies were conducted using archived and cryoprotected 30µm thick sections of brain tissue from behaviorally characterized rhesus monkeys. Glycerol mounted sections were imaged in their entirety with qBRM at low magnification, and then at high magnification in predetermined regions of interest. The coverslips were removed, and the tissue was labeled with anti-myelin basic protein antibody that is co-labeled with Alexa 568, and also stained with Fluromyelin which contains a red fluorescent tag. This allows the same regions imaged with qBRM to be imaged again with confocal epifluorescence.
Results show that qBRM, in its current stage of development, is useful for imaging myelinated fibers and has the potential to identify demonstrate myelin defects in gray matter where the myelin is only moderately dense. However, qBRM is unable to detect all myelin and to verify the presence or absence of myelin defects in dense white matter tracts in 30µm thick tissue sections reliably. The reason for this limitation is that the light scattering inherent in birefringence microscopy obscures individual myelinated axons in dense whiter matter where the packing density produces too much light scattering. To address this, pilot work has been initiated using specially prepared thinner 15µm thick sections. Preliminary results show that this improves resolution significantly in dense white matter but cutting, collection, handling and mounting is a major challenge as these sections are extremely fragile. Additionally, results with IHC-labeled tissue using the combination MBP + Fluoromyelin protocol do not yet allow a direct comparison of qBRM ‘defects’ as the IHC procedure results in substantial deterioration of tissue. Finally, some instances of apparent ballooning myelin degradation found in qBRM imaging may be not ballooning of myelin but instead the bending of myelinated axons around chains of oligodendrocytes. This could potentially be resolved with IHC for oligodendrocyte markers, ideally with neurofilament staining, but the issue of IHC damage to tissue must first be resolved. Additionally, the problem of preserving 15µm thick sections must also be resolved before full validation of qBRM can be done.
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