Journal: Cells
Article Title: Hypothermia Shifts Neurodegeneration Phenotype in Neonatal Human Hypoxic–Ischemic Encephalopathy but Not in Related Piglet Models: Possible Relationship to Toxic Conformer and Intrinsically Disordered Prion-like Protein Accumulation
doi: 10.3390/cells14080586
Figure Lengend Snippet: Misfolded/oxidized SOD1 accumulates in human neonatal HIE brain revealing nuclear, cytoplasmic, and possible connectome pathology. ( A ) Many cells are positive for misfolded/oxidized SOD1 (black arrows), but other cells are not positive (open arrows) in the HIE frontal cortex. Blue is CV counterstain. Shown is representative of n = 12 HIE cases and 3–4 sections from each brain region from each case. Scale bar = 56 µm. ( B ) Higher magnification shows the nuclear (black broad arrow) and neuropil (black thin arrows) localization of misfolded/oxidized SOD1. Some cells are negative (open arrow). The misfolded/oxidized SOD1 immunoreactivity in the neuropil is seen as granules and round structures (black thin arrows), possibly end stage apoptotic cells or degenerating terminals. Scale bar = 8 µm. ( C ) Misfolded/oxidized SOD1 immunoreactivity forms large nuclear inclusions in putative apoptotic cells in the cerebral cortex of neonatal HIE cases. Scale bar = 3 µm. ( D ) Colocalization of aberrant SOD1 (brown) in TBR1-positive (blue-green) cortical neurons. SOD1 single labeling (open broad arrow). TBR1 single labeling (hatched thin arrows). Double-labeled cells (solid black arrows). Scale bar = 10 µm. ( E ) Colocalization of aberrant SOD1 (blue-gray) in GLAST-positive (brown) cortical astrocytes. SOD1 single labeling (thin hatched arrow). Double-labeled normal appearing astrocyte (solid black broad arrow). Double-labeled astrocyte with condensed nucleus (solid black thin arrow). Scale bar = 10 µm. ( F ) Immunofluorescence confirms the colocalization of misfolded/oxidized SOD1 in astrocytes identified by GLAST (seen as yellow). Misfolded/oxidized SOD1 appeared compartmentalized within the cells. Scale bar = 5 µm. ( G ) Immunofluorescent colocalization of aberrant SOD1 (green) in Olig2-positive (red) cortical oligodendrocytes. Scale bar = 10 µm. ( H ) Misfolded/oxidized SOD1 is nuclear (black arrows) in the caudate nucleus but some cells are negative (open arrows). Scale bar = 8 µm. ( I ) Hippocampal CA1 neurons in HIE cases are positive for misfolded/oxidized SOD1 (black arrow), while some cells are completely negative (open arrow). CA1 neurons can form misfolded/oxidized SOD1 inclusions that are round and perinuclear (I, lower left inset, black arrows) or rectangular crystals in the cytoplasm (I, upper right inset, black arrow). Blue is CV counterstain. Scale bar = 6 µm (insets 10 µm). ( J ) Subsets of large cerebellar cells, possibly Purkinje cells, are strongly positive for misfolded/oxidized SOD1 (black arrow), while adjacent Purkinje cells were not positive (open arrow). Scale bar = 7 µm. ( K ) Colocalization of aberrant SOD1 (brown) in NF68-positive (blue-green) large Purkinje cells. Scale bar = 5 µm. ( L ) Degenerating end stage Purkinje cells (arrow) are faintly positive for misfolded/oxidized SOD1 while other cells in the external granule cell layer (EGL) molecular layer (ML), and internal granule cell layer are negative. Scale bar = 10 µm. ( M ) Aberrant SOD1 (brown, thin solid arrows) localizes to the dendritic foci of Purkinje cells (asterisk) identified by morphology and NF68 (green-black, BDHC). Other dendrite segments are negative for aberrant SOD1 (hatched thin arrows). Scale bar = 20 µm. ( N ) Purkinje cell that are undergoing classic ischemic necrotic cell death (open arrow, Purkinje cell is delineated by the elliptical hatched line). Scale bar = 8 µm. ( O ) Misfolded/oxidized SOD1-positive neuritic structures with a quasi-vertical arrangement reflective of passing climbing fibers in the granule cell layer and molecular layer can be seen in the HIE cerebellum. Scale bar = 10 µm. ( P ). Misfolded/oxidized SOD1-positive neurites can also have a horizontal organization passing through the molecular layer of cerebellum reminiscent of parallel fibers. Scale bar = 10 µm. ( Q ) Aberrant SOD1 (brown, black arrow) localizes to NF68-positive axons (blue-gray) in the cerebellar molecular layer (ML). Scale bar = 8 µm.
Article Snippet: This was performed using antibodies to PDGF receptor-α (PDGFRα, mouse monoclonal IgG, clone JF104-6, Novus Biologicals, Centennial, CO, USA), oligodendrocyte marker O4 (mouse monoclonal IgM, clone O4, R&D Systems, Minneapolis, MN, USA), 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNPase, mouse monoclonal IgG, clone 11.5B, Chemicom-Millipore, St. Louis, MO, USA), Olig2 (rabbit polyclonal IgG, Chemicon-Millipore), bridging integrator-1 (BIN1, rabbit polyclonal IgG, Proteintech, Rosemont, IL, USA), class 3 β-tubulin (mouse monoclonal IgG, clone TUJ1, Covance, Dedham, MA, USA) and glial fibrillary acidic protein (GFAP, rabbit polyclonal IgG, Dako, Santa Clara, CA, USA).
Techniques: Labeling, Immunofluorescence
