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: Antibodies Used for Cell Type and Synapse Identification in Combination with Antibodies to Toxic Conformer Proteins.

Article Snippet: Synapsin 1 and 2 , Presynaptic terminals , Rabbit polyclonal , Synaptic Systems, 106002.

Techniques: Drug discovery, Ubiquitin Proteomics, Transduction

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: Synucleinopathy occurs in three different piglet models of HI brain damage as demonstrated by the accumulation of aggregated α-Syn. ( A ) Piglets were exposed to hypoxia–ischemia (HI) and treatment with hypothermia (HT) for 20 h followed by rewarming at 0.5 °C/hours or left at normothermia (NT) or treated with the sham procedure (with and without HT) and survived for 29 h. Western blotting of forebrain extracts show that aggregated α-Syn (oligomer-specific antibody Syn33) accumulated subacutely after HI. Ponceau S staining shows protein loading. ( B ) Graph of Western blot densitometry quantification of aggregated α-Syn in forebrain of sham-NT ( n = 4), sham-HT ( n = 4), HI-NT ( n = 4), and HI-HT ( n = 4) piglets. Box plot showing mean values (with IQR and 5–95 percentile whiskers). ( C ) Piglets were exposed to HI, but no hypothermia treatment, or were exposed to sham procedure and survived for 4 days. Western blotting of somatosensory cortex extracts shows that aggregated α-Syn (oligomer-specific antibody Syn33) accumulated days after HI. Brain sample from human mutant α-Syn-A53T transgenic mouse was the + control. Ponceau staining shows protein loading. ( D ) Graph of Western blot densitometry quantification for aggregated α-Syn at 4 days after HI (sham n = 4, HI n = 4). Box plot showing mean values (with IQR and 5–95 percentile whiskers). ( E ). Immunohistochemical localization of aggregated α-Syn in HI-QA piglets and 14 days after injury. Aggregated α-Syn was found enriched in attritional neurons in striatum (black arrow) while other neurons were lesser-enriched (gray arrow), and other neurons were negative (white arrow). Presynaptic bouton-like structures in the neuropil (thin arrows) were also positive. Scale bar = 10 µm (same for F ). ( F ) Sham piglet neurons were largely negative for aggregated α-Syn suggesting that the antibody is detecting a pathological form of α-Syn. ( G ) Counts of positive aggregated α-Syn boutons in striatum of HI-QA and sham piglets at 15 days after injury (sham piglets n = 5, HI-QA piglets n = 6; 3 sections/piglet). Box plots show mean values (with IQR and 5–95 percentile whiskers). Statistically significant p values are indicated. ( H ) Immunofluorescence demonstrated that some aggregated α-Syn immunoreactivity (green) localizes to presynaptic terminals (white arrows, yellow) identified by SV2 (red) in the neuropil and in perineuronal/peridendritic regions of the HI piglet neocortex. Scale bar = 3 µm. ( I ) Immunofluorescence shows that aggregated α-Syn (green) colocalizes (white arrows, yellow) with synaptophysin (red) in the striatum of HI-QA piglets. Scale bar = 5 µm. ( J ) In a favorably preserved sample of the human HIE neocortex, immunofluorescence also showed that aggregated α-Syn (green) colocalized with SV2 (red) seen as yellow (white arrows). Some presynaptic terminals appeared swollen (left most white arrow). Scale bar = 6 µm.

Article Snippet: Synapsin 1 and 2 , Presynaptic terminals , Rabbit polyclonal , Synaptic Systems, 106002.

Techniques: Western Blot, Staining, Mutagenesis, Transgenic Assay, Control, Immunohistochemical staining, Immunofluorescence