Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Temporo-spatial evolution of prolonged clusterin immunoreactivity in the cortex, dentate gyrus, and thalamus ipsilateral to the injury. (A) A brightfield photomicrograph of a Nissl-stained coronal section from a rat perfused for immunohistochemistry at 1 month after TBI. The lesion core is indicated by an arrow. Dashed boxes indicate the brain regions with prominent clusterin immunoreactivity (ir) in layer IV of the ipsilateral cortex (Cx; panels B,E,H,K), molecular layer of the dentate gyrus (DG; panels C,F,I,L), and dorsal aspect of the thalamus (Th; panels D,G,J,M). Higher-power darkfield photomicrographs show that at 7 d post-TBI, punctate clusterin immunoreactivity (arrowheads) was prominently present in (B) layer IV of the perilesional cortex, (C) in the molecular layer of ipsilateral dentate gyrus, and faintly in (D) the ipsilateral dorsal thalamus. At 14 d post-TBI, (G) the thalamic immunostaining increased. Cortical (E,H) and hippocampal (F,I) staining persisted prominently for up to 1 month post-TBI, becoming substantially weaker by ( cortex K , hippocampus L) 12 months post-TBI. In contrast, (J,M) thalamic staining remained prominent from 1 month until 12 months post-TBI. No immunostaining was observed in the corresponding contralateral brain areas. Abbreviations: CA3, cornu Ammonis 3; d, days; DLG, dorsal lateral geniculate nucleus; G, granule cell layer of the dentate gyrus; H, hilus; LDVL, laterodorsal thalamic nucleus, ventrolateral part; M, molecular layer of the dentate gyrus; mo, months; Rt, reticular thalamic nucleus; TBI, traumatic brain injury; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bar equals 100 µm for all panels.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Staining, Immunohistochemistry, Immunostaining

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Post-TBI expression of clusterin is prominent in the extracellular space. Double-immunofluorescence revealed that in the perilesional cortex, ipsilateral dentate gyrus, and ipsilateral thalamus, clusterin-immunoreactivity did not colocalise with the (A–C) neuronal marker NeuN, (D–F) astrocyte marker GFAP, microglial markers (G–I) CD68 or (J–L) OX42, or (M–O) the mitochondrial marker MT-CO1. In the ipsilateral dorsal thalamus, robust clusterin immunoreactivity surrounded the cells labelled with microglial markers (I) CD68 and (L) OX42, and (O) mitochondria labelled with MT-CO1. All images were taken from rats perfused at 1 month post-TBI. Abbreviations: CD68, cluster of differentiation 68; Clu, clusterin; GFAP, glial fibrillary acidic protein; MT-CO1, mitochondrially encoded cytochrome c oxidase 1; NeuN, neuronal nuclei; OX42, antibody against CD11b/c; Scale bar = 50 µm for all panels.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Expressing, Immunofluorescence, Marker

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Elevated Clu mRNA expression was observed in the brain at 3 months post-TBI. TaqMan RT-qPCR analysis revealed increased Clu mRNA expression in the (A) perilesional cortex (FC 3.3, p < 0.01) and (B) ipsilateral thalamus (FC 2.4, p < 0.05) of the rats at 3 months post-TBI as compared to sham-operated controls (n = 9 TBI, 6 sham; each dot in panels A-B refers to one animal). Clusterin Ct values were normalised to the housekeeping gene GAPDH. Statistical significances: *p < 0.05; **p < 0.01 (Mann-Whitney U test). Abbreviations: Clu, clusterin; Ct, cycle threshold; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sham, sham-operated controls.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Acute reduction in plasma clusterin levels after TBI. ( A ) The dilution curve demonstrated linearity, indicating no significant matrix interference effect in the ELISA assay. (B) Clusterin levels in plasma derived from the cardiac puncture at very acute post-TBI time-points (<6 h, i.e ., 2 h-6 h post-TBI) were lower than that in controls (15%, p < 0.01) or that at 6 months post-TBI (21%, p < 0.01). At the other time-points, there was no difference between the TBI and control animals, or within the TBI groups. Moreover, at 6 months post-TBI, the plasma clusterin levels did not distinguish the rats with (open circles) or without (filled circles) spontaneous seizures (p > 0.05). (C) ROC analysis indicated that plasma clusterin levels sampled <6 h from TBI distinguished the animals with TBI from controls with an AUC of 0.851 (p < 0.05) and (D) from the 6 months post-TBI group with an AUC of 0.917 (p < 0.01). Statistical significances: **p < 0.01 compared with the control group; ##p < 0.01 compared to 6 months post-TBI (Mann-Whitney U test). Abbreviations: AUC, area under curve; C, controls; d, days; h, hours; mo, months; R 2 , coefficient of determination; wk, weeks.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Study design for clusterin analysis in brain tissue, plasma, and CSF. Spatiotemporal expression of clusterin protein in the brain after TBI was investigated in cohorts 1–2. Chronic expression of clusterin mRNA in the brain was assessed using RT-qPCR in cohort 3. Post-TBI plasma clusterin levels were assessed using ELISA in cohorts 4–5. In cohort 5, 5 of the 16 TBI rats had epilepsy. Consequently, the study was powered to diagnose post-traumatic epilepsy in the TBI group if the AUC was ≥0.900. Finally, clusterin levels in CSF was analysed using iTRAQ proteomics in the chronic cohort 2. Abbreviations: CSF, cerebrospinal fluid; d, days; ELISA, enzyme-linked immunosorbent assay; h, hours; IHC, immunohistochemistry; iTRAQ, isobaric tag for relative and absolute quantification; RNA-Seq, RNA sequencing; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sham, sham-operated controls; TBI, traumatic brain injury.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Clinical Proteomics, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Multiplex sample analysis, Immunohistochemistry, Quantitative Proteomics, RNA Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction