Aldehydic load as an objective imaging biomarker of mild traumatic brain injury

Abstract

Mild traumatic brain injury (mTBI) is neurological impairment induced by biomechanical forces without structural brain damage, currently without an objective diagnostic tool. Downstream injury stems from oxidative damage leading to the production of neurotoxic aldehydes. A collagen-based 3D corticomimetic in vitro model of concussion was developed, confirming aldehyde production following impact. Total aldehyde levels were mapped in vivo following mTBI using a novel CEST-MRI contrast agent, ProxyNA₃, in a new model of closed-head, awake, single-impact concussion in aged and young mice with aldehyde dehydrogenase 2 (ALDH2) deficiency. ProxyNA₃-MRI was performed before impact, and on days two- and seven- post-impact. MRI signal enhancement significantly increased at two days post-injury prior to astrocyte activation at seven days post-injury. The data suggest that advanced age and ALDH2 deficiency contribute to increased aldehydic load following mTBI. Overall, ProxyNA₃ was capable of mapping concussion-associated aldehydes, supporting its application as an objective diagnostic tool for concussion.

Fig. 1. Aldehydes are produced following concussion-like impact in primary neurons growing in a corticomimetic scaffold.

A Formulation of corticomimetic scaffold with crosslinked collagen exterior, and collagen interior containing primary mouse neurons. The scaffold was impacted with a concussion-like force followed by confocal microscopy with an aldehyde-binding fluorophore 5MeONA₃. B Young’s Modulus of the corticomimetic scaffold with two different sized interior plugs compared to a murine cortical punch. Data are shown in a box plot with individual values, n = 5 per group, p < 0.05. C Confocal microscope images showing NeuO fluorescent staining (top) of primary mouse cortical neurons seven days after seeding into corticomimetic scaffold to confirm differentiation of neurons. Differential interference contrast (DIC; bottom) shows elongation of cells within 3D scaffold. Scale bar = 50 µM. D Evaluation of cell death at three- and seven-days post-seeding of primary mouse cortical neurons into corticomimetic scaffold. Scale bar = 200 µM. E Representative confocal images of primary neurons pre- (left) and within 5 min post- (right) concussion-like impact after incubation with 5MeONA₃ for fluorescent staining of aldehydes (top) and DIC of cells showing morphological changes pre- and post-impact. Scale bar = 20 µM.

Fig. 2. Mice exhibit changes in behaviour but not blood-brain barrier permeability after mTBI.

A Mice underwent behavioural testing for neurological severity score (NSS) comprised of 5 pass-or-fail tests including beam walk, hindlimb reflex, startle reflex, escape paradigm, and balance test. A point was awarded for every test failed, with a score of 5 representing the more severe behavioural deficit. Individual data points (n = 5 per group) and box plots are shown. Sham mice were restrained, but not impacted. *p < 0.05. B, C Results from 3-min grid walk test for pre- and post-impact mice (n = 5 per group). B Percentage of foot faults of total forelimb steps before and after ACHI. C Time active including walking, grooming, and exploratory behaviour during the 3-min grid walk challenge is shown. Statistical analysis was performed using a One-Tailed Wilcoxon Test, * denotes p < 0.05. D Regions of interest (brain, ventricles, meninges, and blood) were drawn to evaluate contrast enhancement following intravenous gadolinium-based contrast agent pre- and post- impact. E–I Dynamic contrast enhanced MRI was performed pre-and post-impact at various timepoints. Percent change in voxel intensity after gadobutrol injection in each of the ROIs from images acquired (E) five days pre-impact and (F–I) at timepoints of 6 h-, 30 h-, and 54 h post-impact. Data are shown as means +/− standard deviation. Each data point represents the analysis of ROIs on 6 slices per brain MRI. P > 0.05 using GLM repeated measures, n = 5 mice.

Fig. 3. Experimental procedure for evaluation of mTBI in ALDH2^−/− mouse model using behavioural tests, histology, and ProxyNA₃ CEST-MRI.

A Chemical structure of ProxyNA₃ in its unbound, silent state (left), and its CEST-MRI active state, upon reaction with aldehyde (right). B Western blots showing protein levels of ALDH2 in the liver (top) and brain (bottom) tissues from transgenic mice with ALDH2 ^+/+, ^+/−, and ^−/− genotypes. Vinculin was used as a loading control protein. C Experimental design for all testing done pre- and post-impact for in vivo mouse model of mTBI. CEST-MRI with ProxyNA₃ was performed pre- and at two- and seven days post-impact. Behavioural tests, denoted by grid-walk symbol, was performed pre- and immediately post-impact. A separate cohort of mice were used for immunohistological examination at the same timepoints as MRI. D Results of neurological severity score (NSS) comprised of 5 pass-or-fail tests pre-impact (black) and post-impact (red). A point was awarded for every test failed, with a score of 5 representing the most severe behavioural deficit. E, F Immediately after NSS test, mice were placed on a raised grid with video recording for 3 min. A researcher blindly scored (E) the fraction of time spent active and (F) the total number of foot faults divided by total number of forelimb steps. Groups of mice were chosen by age (young <16 weeks; aged >65 weeks) and by genotype. Individual data points (n = 4 per group) and box plots are shown. *p < 0.05, 2-way ANOVA followed by Tukey-corrected multiple comparisons test.

Fig. 4. Mapping of brain aldehydes by ProxyNA₃ in a mouse model of mTBI.

CEST-MRI (average %MTR_asym normalized to pre-contrast signal) 45 min after ProxyNA₃ injection mapped onto axial T₂-weighted image of mice of identified age group and ALDH2 deficiency (left). A representative image of a mouse from each cohort is shown longitudinally across timepoints (pre-impact, two days post-impact, and 7 days post-impact). Arrowheads indicate vascular signal derived from ProxyNA₃-aldehyde adducts. Violin plots showing voxel-wise intensity distribution for the corresponding mouse at each timepoint. Solid line = median, dashed lines = 25^th and 75^th %ile.

Fig. 5. Aldehyde production post-concussion is affected by age and ALDH2 genotype.

Box plots of voxel-wise intensity distribution of aldehyde signal following ProxyNA₃ CEST-MRI at day two post-mTBI. Mice are grouped by age and genotype. Data are presented as modes of normalized %MTR_asym. A Comparison of aldehyde signal two days post-impact between all groups of mice, n = 4 per group. B Comparison of young (<16 weeks) and aged (>65 weeks) mice, n = 12 per group. C Comparison of aldehyde signal between groups of either wild type (ALDH2^+/+) or transgenic mice with lower ALDH2 expression (ALDH2^+/−), or complete knockouts (ALDH2^−/−). *<0.05, A, C Brown-Forsythe ANOVA and Dunnett’s T3 multiple comparison, B two-tailed Mann–Witney U-test.

Fig. 6. Astroglial activation increases 7 days post-impact, following increase in aldehydic load during the neuroinflammatory cascade driving mTBI.

The brains of mice were excised at day two- and day seven- post-impact, or without impact (sham). Brain sections were fluorescently probed with anti-GFAP antibody to visualize astrocyte activation and DAPI. A representative image is shown for a mouse in each cohort based on genotype, age, and timepoint. A Young mice (<16 weeks) and B old mice (>65 weeks) all show an increase in GFAP signal in the hippocampus at seven days post-injury, but not at two days post-injury compared to sham mice. C Quantification of GFAP density in a region of the hippocampus from mice (pooled across age and ALDH2 genotype) with no impact, or two- and seven-days post-impact (n = 12 per group; **p < 0.01, ns non-significant, one-way ANOVA with post-hoc Tukey’s test). D Proposed causative role of aldehydes, produced from oxidative events, in the pathogenesis of mTBI: initial creation of aldehyde-rich tissue, followed by astrogliosis (Nf-κB-driven GFAP expression), and progression of the neuroinflammatory cascade.

Scheme 1

Synthesis of ProxyNA₃.