An immunohistochemical characterization of neurofilament loss from axons in a mouse model of Charcot-Marie-Tooth disease
Abstract:
“Charcot-Marie-Tooth Disease (CMT) is a progressive peripheral neurodegenerative disease. CMT type 2E (CMT2E) is caused by mutations in the light chain neurofilament protein (NFL) and presents as a denervation neuropathy. NFL forms neurofilaments which are microscopic protein polymers that provide structural support to nerve axons and facilitate increased velocity of nerve conduction. Biopsy analyses from affected human patients have revealed both neurofilament loss and neurofilament accumulations within axons. Physical defects in CMT2E can range from mild to debilitating, with earlier onset symptoms resulting in more severe disease later in life. This study used a mouse model of CMT2E, which has a disease-causing mutation in the Nefl gene, to compare proximal and distal axons by imaging dorsal root and tibial nerve cross-sections.
Tibial nerves and dorsal roots were dissected out of euthanized wild and mutated littermate mice and subsequently fixed. These were then embedded in hydrophilic acrylic resin (LR White) blocks. The hardened blocks were cut into one-micron thick sections which were immunostained with antibodies to detect NFL and markers for microtubules and myelin in subsequent fluorescent light microscopy imaging. This method allowed for visualizing specific components of peripheral nerves in cross-section in order to study their distribution.
The ratio of neurofilament positive (NFL+) myelinated axons between mutant and wild type mice–both in the tibial nerve and dorsal root–reveal striking differences. Wild type mice are NFL+ for nearly all myelinated axons. Whereas, mutant mice exhibit an extreme lack of NFL+ myelinated axons in the distal, tibial nerve. More proximal sections from the dorsal roots of mutant mice do show significant NFL+ loss, however, this drop-off is less striking than in the periphery.
Analysis of peripheral nerves revealed there are fewer NFL+ axons in the mutants, especially in the distal regions of peripheral nerves. This suggests increasing loss of neurofilament as the axon projects away from the spinal cord. Neurofilament loss leads to decreased axonal caliber and subsequent nerve atrophy. This translates to delayed nerve conduction and muscle weakness. The hoped application is this cross-sectional analysis allows us to better target regions of the tissue contributing to neurofilament loss.”
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Very nice poster, Alex! The voice-over really helped me understand what I was looking at in the figures. I am interested in how you quantitate neurofilament loss in dorsal vs peripheral nerves. (Is there any way to show statistical significance; how many samples did you stain; how consistent were your results when you compare different tissue sections?)