How Does Postmortem Interval and Other Processing Variables Impact Myelin Integrity in Human and Non-human Primate Brain Tissue?

Abstract Body : Introduction:

Myelin makes up >40% of brain volume in human and non-human primate brains and is crucial to normal brain connectivity and function. This lab has shown that in the normal aging monkey brain, neurons are not lost as monkeys do not get Alzheimer’s disease. Instead, there is age-related accumulation of myelin pathology that correlates with cognitive impairment. In addition, even limited brain damage in the motor cortex causes myelinated fibers to degenerate, impairing motor function. Monkey brain tissue is perfusion-fixed so tissue is harvested with almost no postmortem interval (PMI), whereas human brain is immersion fixed after autopsy with delays of 24 hours and up. Here we investigated how typical handling and processing steps from PMI to how long sections are allowed to dry on the slide affect myelin integrity and how a label-free imaging method, quantitative birefringence microscopy (qBRM), can avoid most processing degradation and still be combined with state of the art confocal immunohistochemistry (IHC).

Methods:

Subjects were adult male and female adult rhesus monkeys from ongoing studies of normally aging monkeys as well as stroke-afflicted monkeys ranging in age from 5 to 25+. All were behaviorally tested and MRI scanned after which they were euthanized by exsanguination during perfusion of the brain, first with ice-cold buffer for unfixed biopsy harvesting, then by 4% paraformaldehyde to fix the brain. Tissue from some monkeys was retrieved before and after buffer infusion and held in cold buffer solution for varying times to simulate PMI. The brain tssue was then cryoprotected, flash frozen, and stored at -80°C until cut into 30µm sections that were stored until processed. Tissue from immersion-fixed calorie-restricted monkey brains was also examined.

First, we mounted the sections in 85% glycerol and collected qBRM image montages for a whole hemisphere section either immediately or after various processing steps. After initial qBRM imaging, tissue was subjected to IHC labeling for degraded (dMBP) and intact (MBP) myelinn. On tissue from aging monkeys, the following variables were tested: drying time, mounting medium, and processing for IHC with detergents.

Results:

Section drying, IHC processing with detergent and PMI of anything beyond 5 minutes degrades myelin regardless of the etiology of myelin damage. Imaging with qBRM can be used to image both damaged and intact myelin, even in very dense white matter tracts, and does not require these processing steps.

Conclusion:

Standard tissue processing methods add to or exacerbate myelin degradation. As qBRM provides exceptionally clear high-resolution label-free images of intact myelin and myelin defects without processing steps that degrade myelin, it can accurately evaluate myelin integrity and still preserving tissue to be processed for specific markers with IHC..

Significance:

Standard tissue processing methods degrade myelin. We have developed methods so that degradation of myelin can be avoided while still providing high-resolution light microscopic images of myelin and myelin defects even in dense white matter tracts. Thus defects in images of myelin can be safely assumed to be inherent to the tissue and not an artifact of PMI or processing.