Fig 1.
PBBI model (A) Visualization of PBBI probe (B) PBBI probe with balloon inflation (C) anatomical location of injury tract with probe alone and probe with balloon inflation.
Fig 2.
Domain location of antibodies Tau1, 5E2, Tau5 and caspase cleavage at Asp421.
Fig 3.
Detection of FL APP and cleavage products following penetrating TBI.
Protein levels from sham, PrI and PBBI at 3d post-injury were evaluated for the detection of bands corresponding to APP-FL, βCTF, αCTF, and βactin and compared to positive controls including transgenic AD model mouse brain lysate (3XTg-AD) and cell culture lysate from N2A cells transfected with human APP (N2A wtAPP).
Fig 4.
Loss of APP and accumulation of βCTFs after TBI.
Protein levels from sham, PrI and PBBI were evaluated by Western blot at time points 4h, 24h, 3d and 7d days post injury. Bands were measured and normalized to corresponding βactin levels. A representative blot at 3d post injury is shown in (A). Quantification corresponding to (B) APP-FL, (C), APP βCTFs, and (D) APP αCTFs show significant loss of APP-FL with PBBI and significant increases in βCTFs at all time-points for PBBI and at 7d for PrI. APP αCTFs were significantly increased post-PrI and -PBBI at 24h and significantly decreased at 3d post-PBBI Statistical analysis of changes at each time point was conducted (K-W, * p<0.05, 2 way ANOVA # p<0.05; error bars: standard error of the mean, n = 8–10 per group).
Fig 5.
Increased Aβ40 and Aβ42 7 days post-TBI.
Amyloid levels from Sham, PrI and PBBI were evaluated by chemiluminescent ELISAs 7d post-injury. (A) Quantification of Aβ40 (B) Representative visualizing of Aβ40 chemiluminescent ELISA wells. (C) Quantification of Aβ42 (D) Representative visualizing of Aβ42 chemiluminescent ELISA wells. Both Aβ40 and Aβ42 were significantly upregulated by PrI and PBBI 7d post-injury with PBBI inducing significantly more Aβ40 and Aβ42 than PrI. Statistical analysis of changes was conducted (K-W, * p<0.05; error bars: standard error of the mean, n = 10 per group).
Fig 6.
Detection of FL tau and tau fragments following penetrating TBI.
Protein levels from Sham, PrI and PBBI were evaluated by Western blot at time points 4h, 24h, 3d and 7d post injury. Bands were measured and normalized to corresponding βactin levels. A representative blot at 7d post injury is shown in (A). Quantification corresponding to (B) tau-FL, (C) 40 kDa tau and (D) 22 kDa tau show significant loss of tau-FL and 40 kDa tau and significant increases in 22 kDa tau fragment through 7d. Statistical analysis of changes at each time point was conducted (K-W, * p<0.05, 2 way ANOVA # p<0.05; error bars: standard error of the mean, n = 8–10 per group).
Fig 7.
Pilot study: tau22 fragmentation in regions of interest.
Tissue from specific regions of interest (Frontal Cortex, Striatum, Hippocampus, and Residual Midbrain) were qualitatively evaluated by Western blot for the presence or absence of tau22 fragmentation (A) 24h following PBBI (n = 3) and (B) 1m following PrI and PBBI (n = 3).
Fig 8.
Epitope binding confirmed for Tau-FL and Tau22 using Tau1 and Tau5 antibodies.
Specific binding to proline rich domain of tau confirmed for Tau-FL and tau22 fragment in samples 24h post-injury using (A) Tau1 and (B) Tau5 antibodies against total tau and compared to positive control transgenic AD model mouse brain lysate (3XTg-AD). The 22 kDa tau fragment (*) was detected by both total tau antibodies. Experiment replicated 3 times for (sham, PBBI).
Fig 9.
22 kDa Tau fragment binding confirmation.
Protein levels from 7d post-injury PBBI brain lysate and 7d post-PBBI brain lysate that underwent immunoprecipitation with Tau 1 antibody (tau IP) were evaluated for evidence of (A) tau caspase cleavage at Asp421and (B) tau phosphorylation using AT8 antibody. Evidence of caspase cleavage of the 22 kDa tau fragment was seen using the tau Asp421 specific caspase antibody in both the original brain lysate as well as the Tau 1 immunoprecipitation product. Antibody AT8, specific for phosphorylation at sites S202, did not detect the 22 kDa tau fragment. Experiments replicated at least 3 times (sham, PBBI). AT8 phosphorylation experiments replicated twice (sham, PBBI).