Merging pathology with biomechanics using CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration): a novel, surgery-free model of traumatic brain injury - PubMed (original) (raw)

Merging pathology with biomechanics using CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration): a novel, surgery-free model of traumatic brain injury

Dhananjay R Namjoshi et al. Mol Neurodegener. 2014.

Abstract

Background: Traumatic brain injury (TBI) is a major health care concern that currently lacks any effective treatment. Despite promising outcomes from many preclinical studies, clinical evaluations have failed to identify effective pharmacological therapies, suggesting that the translational potential of preclinical models may require improvement. Rodents continue to be the most widely used species for preclinical TBI research. As most human TBIs result from impact to an intact skull, closed head injury (CHI) models are highly relevant, however, traditional CHI models suffer from extensive experimental variability that may be due to poor control over biomechanical inputs. Here we describe a novel CHI model called CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) that fully integrates biomechanical, behavioral, and neuropathological analyses. CHIMERA is distinct from existing neurotrauma model systems in that it uses a completely non-surgical procedure to precisely deliver impacts of prescribed dynamic characteristics to a closed skull while enabling kinematic analysis of unconstrained head movement. In this study, we characterized head kinematics as well as functional, neuropathological, and biochemical outcomes up to 14d following repeated TBI (rTBI) in adult C57BL/6 mice using CHIMERA.

Results: Head kinematic analysis showed excellent repeatability over two closed head impacts separated at 24h. Injured mice showed significantly prolonged loss of righting reflex and displayed neurological, motor, and cognitive deficits along with anxiety-like behavior. Repeated TBI led to diffuse axonal injury with extensive microgliosis in white matter from 2-14d post-rTBI. Injured mouse brains also showed significantly increased levels of TNF-α and IL-1β and increased endogenous tau phosphorylation.

Conclusions: Repeated TBI using CHIMERA mimics many of the functional and pathological characteristics of human TBI with a reliable biomechanical response of the head. This makes CHIMERA well suited to investigate the pathophysiology of TBI and for drug development programs.

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Figures

Figure 1

Figure 1

Head kinematics during rTBI. Head kinematic parameters during impacts were assessed in 8 mice subjected to rTBI. Data are represented as the means for each impact. (A) Head trajectory during the maximum acceleration phase in the sagittal plane following impact. (B) Head displacement-time graph following impact. (C) Head deflection is measured as the angle between the snout, side marker and the horizontal plane. Linear head velocity and linear head acceleration are depicted in (D) and (E), respectively. (F) and (G) show angular head velocity and angular acceleration, respectively. Data in (A) are represented as mean ± 95% CI in both X- and Y- direction, respectively. Data in B-G are represented as mean ± 95% CI. (H) Summary of peak values of kinematic parameters averaged across all 8 rTBI mice. The coefficient of variation (CV) was calculated as the average of day 1 and day 2 peak values from all available recordings.

Figure 2

Figure 2

CHIMERA rTBI induces behavioral deficits. (A) Duration of loss of righting reflex (LRR) was assessed immediately following the sham or TBI procedure. Cohort size: Sham, N = 31; rTBI, N = 39. (B) Neurological severity score (NSS) was assessed at 1h, 1d, 2d, and 7d post-rTBI. Cohort size: Sham (1 h: N = 34, 1d: N = 31, 2d: N = 35, 7d: N = 21); rTBI (1 h, 1d and 2d: N = 42, 7d: N = 25). (C) Motor performance was assessed on an accelerating rotarod at 1d, 2d, 7d and 14d post-rTBI. Cohort size: Sham (1d and 2d: N = 35, 7d: N = 21, 14d: N = 15); rTBI (1d and 2d: N = 41, 7d: N = 25, 14d: N = 15). (D) Thigmotaxis was quantified at 1d, 7d and 14d post-rTBI. Cohort size: Sham (1d: N = 23, 7d: N = 16, 14d: N = 15); rTBI (1d: N = 24, 7d: N = 10, 14d: N = 15). Data in A-D were analyzed by repeated measures two-way ANOVA followed by the Holm-Sidak post-hoc test. (E) Working memory was assessed by the passive avoidance test from 7d to 10d post-rTBI.. (F) Spatial reference memory was assessed by Barnes maze from 9d to 13d post-rTBI. Data at each time point represent the mean of four trials. Data in E and F (Cohort size: N = 15/group) were analyzed by repeated measures two-way ANOVA. For all graphs, data are presented as mean ± SEM. For all graphs, * indicates a significant rTBI effect within a particular time point and # indicates a significant time effect within each group. ***: p < 0.001. ###: p < 0.001.

Figure 3

Figure 3

CHIMERA rTBI induces diffuse axonal injury. Axonal degeneration was assessed by silver staining at 2, 7, and 14d post-rTBI. (A) Coronal sections showing white matter areas including the olfactory nerve layer, corpus callosum, and optic tract with regions of prominent silver staining indicated by black rectangles. (B) Representative 40X-magnified images of the same brain regions in sham-operated (upper row) or rTBI-induced (lower three rows) animals at the indicated time points. (C) 100X-magnified images of the same brain regions in rTBI-induced animals. Axonal varicosities are indicated by arrows.

Figure 4

Figure 4

Quantitative analysis of silver stain images. Silver stained images were quantified by calculating the % of region of interest (ROI) in the white matter tract area that was stained with silver. Bars indicate mean ± SD percent of ROI showing positive signal in sham and rTBI-induced animals in (A) olfactory nerve layer, (B) corpus callosum and (C) optic tract. Data were analyzed using two-way ANOVA followed by a Tukey post-hoc test. Cohort size: olfactory nerve layer: Sham (2d: N = 4, 7d: N = 5, 14d: N = 6); rTBI (2d: N = 8, 7d and 14 d: N = 5); corpus callosum: Sham (2d and 7d: N = 5, 14d: N = 6); rTBI (2d: N = 16, 7d: N = 5, and 14 d: N = 6); optic tract: Sham (2d: N = 5, and 7d: N = 4, 14d: N = 6); rTBI (2d: N = 17, 7d and 14d: N = 5). For all graphs, * indicates a significant rTBI effect within a particular time point and # indicates a significant time effect within rTBI group. **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. ###: p < 0.001, ####: p < 0.0001.

Figure 5

Figure 5

CHIMERA rTBI induces widespread microglial activation. Microglial activation was assessed using Iba-1 immunohistochemistry at 2, 7, and 14d post-rTBI. (A) Representative images of Iba-1 stained coronal sections of olfactory bulb and brain. Areas with prominent microglial activation are indicated by black rectangles. (B) Representative 40X-magnified images of the same white matter tract regions showing resting microglia in sham brains (upper row) and activated microglia in injured brains (lower three rows) at the indicated time points. (C) Representative 100X-magnified images showing the morphology of Iba-1-stained resting microglia in sham (upper row) and activated microglia in rTBI (lower row) brains.

Figure 6

Figure 6

Quantitative analysis of microglial response to rTBI. Bar graphs in the left column (A-D) indicate mean ± SD fractal dimension for microglial morphology in (A) olfactory nerve layer, (B) corpus callosum, (C) brachium of superior colliculus, and (D) optic tract. Bar graphs in the right column (E-H) show mean ± SD number of Iba-1 positive cells per mm2 in the same white matter regions. Data were analyzed by two-way ANOVA followed by a Tukey post-hoc test. Numbers inside the bars indicate sample size. For all graphs, * indicates a significant rTBI effect within a particular time point while # indicates a significant time effect within rTBI group. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. ##: p < 0.01, ####: p < 0.0001.

Figure 7

Figure 7

CHIMERA rTBI increases proinflammatory cytokine levels. Bar graphs represent mean ± SD % fold change in TNFα (A) and IL-1β (B) levels in rTBI brain lysates compared to the levels in the sham brain lysates at the respective time points. For both graphs, ** indicates p < 0.01 in comparison of rTBI vs sham, using multiple t-tests with Bonferroni corrections for multiple comparisons (p = 0.05/5 = 0.01).

Figure 8

Figure 8

CHIMERA rTBI increases endogenous tau phosphorylation. Tau phosphorylation was analyzed using the Simple Western system (ProteinSimple). The graphs in the left column (A-C) depict fold change in endogenous phosphorylated tau levels in rTBI half-brain homogenates compared to the sham brains using antibodies CP13 (pSer202 and pThr205, Panel A), RZ3 (pThr231, Panel B) and PHF1 (pSer396 and pSer404, Panel C), respectively. Graphs in the middle column (D-F) depict quantitation of phosphorylated tau as a proportion of total tau (DA9). Representative digital immunoblots of phosphorylated and corresponding total tau are depicted in the right column (G-I). Arrows on the left of the blots indicate molecular weight marker at 66 kDa. Data are presented as the mean ± SD fold change in rTBI compared to the respective shams at each time point. For all graphs, *, ** and *** indicate p < 0.01 for the comparison of rTBI vs respective sham values, using multiple t-tests with Bonferroni correction for multiple comparison (p = 0.05/5 = 0.01).

Figure 9

Figure 9

CHIMERA device and mouse head positioning. (A) The picture depicts the CHIMERA device. Various parts are labeled with numbers as follows: 1. head plate, 2. body plate, 3. animal bed, 4. Velcro straps, 5. air tank, 6. air pressure regulator, 7. digital pressure gauge, 8. two-way solenoid valve, 9 vertical piston barrel. (B) Close-up view of animal strapped on the holding platform. (C) Location of impact relative to the mouse head and brain. P: impact piston. (D) Air pressure-energy calibration curve was obtained by driving a 50 g piston at increasing air pressure values and calculating the resultant impact energy. The graph depicts three measurements for each air pressure value.

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References

    1. Langlois JA, Rutland-Brown W, Wald MM. The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil. 2006;21:375–378. doi: 10.1097/00001199-200609000-00001. - DOI - PubMed
    1. Faul MM, Xu L, Wald MM, Coronado VG. Traumatic Brain Injury In The United States: Emergency Department Visits, Hospitalizations And Death 2002–2006. Atlanta: Centers for Disease Control and Prevention, National Center for Injury Prevention and Control; 2010.
    1. Gerberding JL, Binder S. Report To Congress On Mild Traumatic Brain Injury In The United States: Steps To Prevent A Serious Public Health Problem. Atlanta: Centers for Disease Control and Prevention; 2003.
    1. Cassidy JD, Carroll LJ, Peloso PM, Borg J, von Holst H, Holm L, Kraus J, Coronado VG. Incidence, risk factors and prevention of mild traumatic brain injury: results of the WHO Collaborating Centre Task Force on Mild Traumatic Brain Injury. J Rehabil Med. 2004;Suppl. 43:28–60. doi: 10.1080/16501960410023732. - DOI - PubMed
    1. Tagliaferri F, Compagnone C, Korsic M, Servadei F, Kraus J. A systematic review of brain injury epidemiology in Europe. Acta Neurochir (Wien) 2006;148:255–268. doi: 10.1007/s00701-005-0651-y. - DOI - PubMed

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