Spinal Cord

Postnatal day 30 (P30) TWI mice and their WT littermates (5 for each experimental group processed in 5 different experimental sessions, every TWI with its WT littermate) and one P15 TWI mouse versus its WT littermate were perfused with a fixative solution (4% paraformaldehyde and 0.1%–1%–2.5% glutaraldehyde in phosphate buffer, pH 7.4). Sciatic nerves, spinal cords and gastrocnemius muscles were dissected and post-fixed for 4 hours at room temperature in the same fixative solution.
Spinal cords were dissected in the lumbar region, isolating four 1-mm-thick sections in the lumbar enlargement region and the gastrocnemius muscles were cut in small portions, approximately 1 mm³ in volume. Sciatic nerves were processed without further sectioning.
The selected tissues were further treated for epoxy resin embedding as previously described⁴³ . Briefly, the samples were deeper fixed in 2–2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4). After rinsing, specimens were post-fixed with osmium tetroxide (1%)-potassium ferricyanide (1%) in cacodylate buffer, rinsed again, en bloc stained with 3% uranyl acetate in ethanol, dehydrated and embedded in epoxy resin, that was baked for 48 h at 60 °C. Thin sections were obtained with an ultramicrotome (UC7, Leica Microsystems, Vienna, Austria) and collected on G300Cu grids (EMS). Finally, sections were examined with a Zeiss LIBRA 120 plus transmission electron microscope equipped with an in-column omega filter.

Authorizations for reporting these three cases were granted by the Eastern Ontario Regional Forensic Unit and the Laboratoire de Sciences Judiciaires et de Médecine Légale du Québec.
The sampling followed a relatively standardized protocol for all TBI cases: samples were collected from the cortex and underlying white matter of the pre-frontal gyrus, superior and middle frontal gyri, temporal pole, parietal and occipital lobes, deep frontal white matter, hippocampus, anterior and posterior corpus callosum with the cingula, lenticular nucleus, thalamus with the posterior limb of the internal capsule, midbrain, pons, medulla, cerebellar cortex and dentate nucleus. In some cases, gross pathology (e.g. contusions) mandated further sampling along with the dura and spinal cord if available. The number of available sections for these three cases was 26 for case1, and 24 for cases 2 and 3.
For the detection of ballooned neurons, all HE or HPS sections, including contusions, were screened at 200×.
Representative sections were stained with either hematoxylin–eosin (HE) or hematoxylin-phloxin-saffron (HPS). The following histochemical stains were used: iron, Luxol-periodic acid Schiff (Luxol-PAS) and Bielschowsky. The following antibodies were used for immunohistochemistry: glial fibrillary acidic protein (GFAP) (Leica, PA0026,ready to use), CD-68 (Leica, PA0073, ready to use), neurofilament 200 (NF200) (Leica, PA371, ready to use), beta-amyloid precursor-protein (β-APP) (Chemicon/Millipore, MAB348, 1/5000), αB-crystallin (EMD Millipore, MABN2552 1/1000), ubiquitin (Vector, 1/400), β-amyloid (Dako/Agilent, 1/100), tau protein (Thermo/Fisher, MN1020 1/2500), synaptophysin (Dako/Agilent, ready to use), TAR DNA binding protein 43 (TDP-43) ((Protein Tech, 10,782-2AP, 1/50), fused in sarcoma binding protein (FUS) (Protein tech, 60,160–1-1 g, 1/100), and p62 (BD Transduc, 1/25). In our index cases, the following were used for the evaluation of TAI: β-APP, GFAP, CD68 and NF200; for the neurodegenerative changes: αB-crystallin, NF200, ubiquitin, tau protein, synaptophysin, TDP-43, FUS were used.
For the characterization of the ballooned neurons only, two cases of fronto-temporal lobar degeneration, FTLD-Tau, were used as controls. One was a female aged 72 who presented with speech difficulties followed by neurocognitive decline and eye movement abnormalities raising the possibility of Richardson’s disorder. The other was a male aged 67 who presented with a primary non-fluent aphasia progressing to fronto-temporal demαentia. In both cases, the morphological findings were characteristic of a corticobasal degeneration.