Histopathological tests were performed using standard histological methods as previously described.78 (link),80 (link)-82 (link) Briefly, on the day of histological assessments, Drosophila melanogaster flies were anesthetized with CO2 (Fly CO2 anesthesia setup; Genesee Scientific, 59-114/54-104M, USA), and placed on a CO2 perfused pad for collecting. The flies were decapitated under a dissecting stereo microscope (Leica Microsystems, Leica M60 CMO), and the heads were fixed in 10% neutral buffered formalin (NBF) fixative at room temperature (22- 30°C) for 24 h, after which tissues were processed using routine histology techniques by dehydrating with graded alcohols, clearing with xylene, and embedding into paraffin wax using an automated tissue processor (Histokinette-SLEE MAINZ, MTP type). Every tissue was randomly sectioned into six, 6 μm-thick transverse histological sections using a rotary microtome (SLEE MAINZ, CUT4062), and the sections were then placed on slides and stained with hematoxylin and eosin (H &E) and combined Luxol fast blue (LFB) and Nissl (Klüver's) staining techniques following standard protocols.78 (link),80 (link)-82 (link) The stained sections were mounted in mounting media and qualitative histological examination of the sections was done and photographed with a light microscope (Nikon Eclipse Ci-L Upright Microscope, New York, USA), at a magnification of 200x or 400x, digital camera (Nikon DS-Fi1c Digital Camera, New York, USA), and imaging software (Nikon NIS- NIS-Elements F Ver4.60.00 Imaging software), for image analysis and documentation.
A qualitative examination of the tissues was done using previously described methods64 ,83 (link)-85 (link) where brain neurodegeneration was categorized as normal, moderate, or severe based on the size and frequency of brain vacuolations in H and E stained-sections following previous methods.84 (link),85 (link) The non-myelinating Schwann cells in mammals are comparable to the ‘ensheathing’ glial cells within the CNS of Drosophila which encase axons and neuropil of the flies,86 (link),87 (link) therefore, LFB in Klüver LFB stain was used to demonstrate axonal degeneration of neurons rather than axonal demyelination.88 (link) The nature of the Nissl substance and nerve tracts (axons) were demonstrated using the Klüver LFB-stained tissues following previous methods,64 ,83 (link) where the nerve tracts were shown by blue color and the Nissl substance was shown by magenta (violet) colour. A weak LFB stain (light blue patchy areas) indicated axonal degeneration of nerve tracts, while a weak cresyl echt violet stain (light violet) indicated abnormalities in Nissl substance,64 ,83 (link)supplementary file 2 .
A qualitative examination of the tissues was done using previously described methods64 ,83 (link)-85 (link) where brain neurodegeneration was categorized as normal, moderate, or severe based on the size and frequency of brain vacuolations in H and E stained-sections following previous methods.84 (link),85 (link) The non-myelinating Schwann cells in mammals are comparable to the ‘ensheathing’ glial cells within the CNS of Drosophila which encase axons and neuropil of the flies,86 (link),87 (link) therefore, LFB in Klüver LFB stain was used to demonstrate axonal degeneration of neurons rather than axonal demyelination.88 (link) The nature of the Nissl substance and nerve tracts (axons) were demonstrated using the Klüver LFB-stained tissues following previous methods,64 ,83 (link) where the nerve tracts were shown by blue color and the Nissl substance was shown by magenta (violet) colour. A weak LFB stain (light blue patchy areas) indicated axonal degeneration of nerve tracts, while a weak cresyl echt violet stain (light violet) indicated abnormalities in Nissl substance,64 ,83 (link)
