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, 77 (9), 782-792

Intensity Specific Repetitive Mild Traumatic Brain Injury Evokes an Exacerbated Burden of Neocortical Axonal Injury

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Intensity Specific Repetitive Mild Traumatic Brain Injury Evokes an Exacerbated Burden of Neocortical Axonal Injury

Yasuaki Ogino et al. J Neuropathol Exp Neurol.

Abstract

Mild traumatic brain injury (mTBI) has been linked to enduring neurological damage following repetitive injury. Previously, we reported that intensity-specific, repetitive mTBI exacerbated microvascular and axonal damage in brainstem. For a more rigorous and global assessment, we assessed the burden of neocortical diffuse axonal injury (DAI) evoked by repetitive mTBI. Mice were subjected to mild central fluid percussion injuries at 1.4 and 1.6 atm with or without repetitive insult at a 3-hour interval and killed at 24 hours postinjury. Neocortical DAI within layer V was quantitatively assessed by double-labeling p-c-Jun and NeuN to identify both the axotomized and total neuronal population. Both confocal and electron microscopic findings revealed no apparent evidence of neuronal death. Repetitive mTBI of 1.6 atm group, but not of 1.4 atm group, demonstrated a significantly higher proportion of axotomized neurons. These results demonstrate that different intensities of mTBI induced different burdens of DAI after repetitive insult. Interestingly, the parallel loss of the righting reflex reflected differences in injury intensity, yet the duration of this reflex was not elongated by the repetitive insult. These data highlight some of the complex issues surrounding repetitive mTBI and its associated morbidity, mandating the need for continued exploration.

Figures

FIGURE 1.
FIGURE 1.
Group assignment and experimental paradigm. Animals were assigned to 4 experimental groups defined by the following 2 factors: Injury intensity (low or high), and the number of injuries (single or repetitive). Target intensities of injury were 1.4 atm for the lower intensity and 1.6 atm for the higher intensity. After postsurgical recovery, all animals were subjected to central fluid percussion injury (cFPI) at the target intensity. Three hours later, the second cFPI at the same intensity for the repetitive injury groups, or sham injury for the single injury groups, was initiated. Animals were killed and perfused at 24 hours after the second injury for immunocytochemical assessments. LS: lower intensity single mild injury, LR: lower intensity repetitive mild injury, HS: higher intensity single mild injury, HR: higher intensity repetitive mild injury.
FIGURE 2.
FIGURE 2.
Duration of loss of righting reflex. (A) Comparison of loss of righting reflex time after the first injury (first LORR) between the 2 different injury intensities. In this analysis, the L group represents the lower intensity mild injury groups, which consists of both the LS and the LR groups, and the H group represents the higher intensity mild injury groups, which consists of both the HS and the HR groups. The first LORR showed a significant difference between L and H groups, suggesting injury intensity was reflected on the LORR. (B) Comparison of the first LORR and the second LORR of in each experimental group. Note that the second LORR in the LS and HS groups were comparable to that observed after sham injuries. The LR and HR groups, both of which were employed mild injuries at the same intensity twice, did not demonstrated significant difference between the first and the second LORRs, suggesting that the second insult did not alter the second LORR. Pairwise comparisons of the first LORR did not show significant difference between groups. (A) One-sided Mann-Whitney U test. (B) Paired t test for the LS, LR, and HS groups, and Welch t test for HR group. *p < 0.05, **p < 0.01, ***p < 0.001. LS; lower intensity single mild injury, LR; lower intensity repetitive mild injury, HS; higher intensity single mild injury, HR; higher intensity repetitive mild injury.
FIGURE 3.
FIGURE 3.
Representative immunofluorescent images demonstrating p-c-Jun/NeuN colocalization. Representative immunofluorescent images that underwent quantitative assessments of the burden of diffuse axonal injury in layer V neurons. All presented images originate from the same animal in the HR group and show representative findings after double labeling with p-c-Jun (magenta) and NeuN (cyan), which were used as markers for the axotomized neuronal populations and the total neuron populations, respectively. (A) Tiled, 10× magnification image labeling p-c-Jun and NeuN. The white rectangle in the figure presents the location of region of interest (ROI) centering on the layer V neurons. Note that, p-c-Jun signals are distributed primarily in neocortical layer V, consistent with this well-established animal TBI model that generates axotomized neurons in this domain. Scale bar = 500μm. (B) A 20× magnification image of the ROI in neocortical layer V delineated by a white rectangle in panel (A). Fluorescent signals labeling p-c-Jun (magenta) or NeuN (cyan) are distributed broadly in the ROI, showing colocalization. Note, however, that colocalization did not show one to one correspondence. Scale bar = 100 μm. Panels (C–E) are enlarged images of one portion of panel (B), in which p-c-Jun and NeuN are presented. Each panel (C) and (D) presents single channel image for p-c-Jun and NeuN, respectively. Panel (E) presents their merged image. Arrowheads indicate the neurons with p-c-Jun and NeuN colocalization. Scale bars = 50 μm.
FIGURE 4.
FIGURE 4.
Electron micrograph of repetitively injured mouse. (A) Electron micrograph taken from a repetitively injured mouse confirms the mild nature of the traumatic injuries. Note that despite the presence of p-c-Jun immunoreactivity within scattered neuronal nuclei (arrows) that reflect the occurrence of axonal injury, the balance of the brain parenchyma is intact, showing normal detail in both its neuronal, glial and vascular (V) constituents. Scale bar = 5 μm. (B) Electron micrograph harvested from the same case shown in panel (A) again demonstrates the integrity of the brain tissue and its vascular components. Note that a normal/nonreactive neuronal soma (N) can be seen in the field, which also shows a damaged axonal profile (arrowheads) terminating in a reactive axonal swelling (asterisk) all of which represent diffuse axonal injury. Such damaged axons can be traced back to neuronal soma displaying nuclear p-c-Jun. Scale bar = 5 μm.
FIGURE 5.
FIGURE 5.
Quantitative assessment of p-c-Jun+/NeuN+ neurons. Quantitative assessments of p-c-Jun+/NeuN+ neurons. (A) The density of p-c-Jun+ neurons in the ROI. HR group demonstrated significant difference compared with LS and LR groups, but not to HS group (vs LS; d =3.09, p = 0.012. vs LR; d =2.11, p = 0.017. vs HS; d =1.37, p = 0.0528). (B) The density of NeuN+ neurons in the same ROI. Significant difference was observed in comparison between HS and LS groups (r =0.84, p = 0.045). (C) The density of p-c-Jun/NeuN colocalization. HR group demonstrated significant difference compared with LS and LR groups, but not to HS group (vs LS; d =2.60, p = 0.025. vs LR; d =2.00, p = 0.024. vs HS; d =1.31, p = 0.071). (D) The proportions of p-c-Jun/NeuN colocalization in the total NeuN+ neuron population. Estimated proportion of axotomy was 14.3% (95% CI; 13.1%–15.6%) in HR group, 7.4% (95% CI; 6.5%–8.4%) in HS, and they were significantly different. The OR of HR group to HS was 1.93 (95% CI; 1.63–2.28). In contrast, LR and LS group did not show significant difference and the OR was 1.01 (95% CI; 0.81–1.24). (E) The proportion of p-c-Jun/NeuN colocalization within p-c-Jun+ neuron population. Approximately 70% of p-c-Jun+ neurons were positive for NeuN. There was no difference between the groups. Error bars indicate the standard error of the means in (A–C) and 95% CI of the estimated proportions in (D) and (E). Pairwise comparisons were assessed by Tukey’s HSD test in (A–C), and by Chi-square test followed by pairwise Chi-square test with Holm’s correction in (D) and (E). d; Cohen’s d with Hedges correction, r; effect size for comparison of nonparametric variables, 95% CI; 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.; not significant. LS; lower intensity single mild injury, LR; lower intensity repetitive mild injury, HS; higher intensity single mild injury, HR; higher intensity repetitive mild injury.

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