Research ArticleTraumatic Brain Injury

Repetitive blast exposure in mice and combat veterans causes persistent cerebellar dysfunction

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Science Translational Medicine  13 Jan 2016:
Vol. 8, Issue 321, pp. 321ra6
DOI: 10.1126/scitranslmed.aaa9585
  • Fig. 1. Number of blast exposures is associated with decreased FDG uptake in the cerebellum of veterans with mild TBI.

    (A) Distribution and relative magnitude of negative correlations between log10 number of blast exposures and FDG uptake by the human brain (normalized to global activity) overlaid on magnetic resonance template images for anatomical orientation. Shown are right lateral (RT LAT), left lateral (LT LAT), right medial (RT MED), left medial (LT MED), and inferior (INF) aspects of the human brain. Scale bar indicates z scores. A greater number of log10 blast exposures were associated with decreased metabolic activity in three adjacent loci within the right inferior semilunar lobule by whole-brain voxel-wise analyses. (B) Scatter plots of the mean activity in predefined cerebellar hemisphere volumes of interest (VOIs; left and right cerebellum) versus log10 number of blast exposures during military service. There was a statistically significant negative correlation between the number of blast exposures (log10) and FDG uptake in each cerebellar hemisphere VOI (Spearman r correlations, n = 33).

  • Fig. 2. A single blast causes inferior cerebellar BBB dysfunction and Purkinje cell injury in a mouse model of mild TBI.

    (A) Shock tube–generated highly reproducible battlefield-relevant blast overpressure. The red trace shows the time (ms) versus pressure (psi) measured 5 cm above the animal averaged among n = 32 blast overpressure waveforms that were collected over a randomly selected 2-day period (black bars indicate ±SEM). Superimposed blue Friedlander waveform illustrates the close correspondence of the shock tube–generated blast overpressure to the waveform expected from an open-field detonation of about 22.4 kg of trinitrotoluene (TNT) at a distance of 6.1m. (B) Blast-exposed mice were retro-orbitally loaded with 10-kD dextran labeled with Texas Red; they were allowed to recover for 1 or 4 hours after a single blast exposure, and brain slices were observed using confocal microscopy. Blast-specific dextran (red) labeling was most frequently observed in the cerebellum (white arrowheads). Shown is an example of a mouse brain slice (50 μm thickness) 1 hour after treatment. (C) Texas Red–labeled dextran in the blood that crossed the BBB into the cerebellar parenchyma was observed at both 1 and 4 hours after blast exposure (red highlights). Extravasated dextran was most noticeable in the cerebellar molecular layer. DAPI, 4′,6-diamidino-2-phenylindole. (D) Blast-exposed Purkinje cells that internalized dextran that had crossed the BBB were associated with morphologically distinct microglia (green) that appeared to be activated. (n = 20 blast-treated animals, 19 of 73 brain slices had dextran-labeled Purkinje cells with only one cell (a granule cell) labeled in the upper half of the cerebellum; n = 20 sham-treated animals, with 0 of 75 slices showing dextran-positive Purkinje cells. GFAP, glial fibrillary acidic protein. (E) To assess regional BBB breakdown, the linear pixel-by-pixel mean dextran fluorescence intensity within the molecular layer of the cerebellum was measured across each lobe and plotted. Results show the mean normalized with respect to values for seven sham-treated and seven blast-treated animals. Only lobule 9 showed evidence of increased dextran fluorescence (P ≤ 0.05, two-tailed t test; mean = 1.65 and 1.0 for blast- and sham-treated animals, respectively). (F) Confocal microscopy revealed elevated aberrant tau phosphorylated at Ser396 (detected with mouse monoclonal anti-tau396 antibody) in blast-treated versus sham-treated cerebellum. (G) Confocal microscopy revealed elevated aberrant tau phosphorylated at Ser396 (detected with rabbit anti-tau396 antibody) in blast-treated versus sham-treated cerebellum. (H) In mouse cerebellum, multiple pathological forms of phosphorylated tau were observed 1 to 4 hours after blast exposure. Representative Western blots illustrate blast-induced elevation of phosphorylated tau in mouse cerebellum. Each lane corresponds to one sham-treated or blast-exposed animal (1 hour: n = 4, 4 hours: n = 4; n = 3 sham-treated and n = 4 blast-exposed). Pyruvate kinase (PK) was the gel protein loading control. (I) Three days after experimental treatment, Purkinje cell injury (black arrowheads) was observed in the form of swollen cell bodies, cell membrane breakdown, and aberrant IP3R1 staining in two of three blast-exposed animals. Similar Purkinje cell dysmorphology was not observed in sham-treated animals (n = 0/3). Images are representative examples from lobule 9, taken at 20×. Slides were prepared using antibodies against IP3R1 and standard immunohistochemistry. Scale bars, 500 μm (B), 250 μm (C), and 50 μm (D, F, and G). (F) and (G) represent data from five sham-treated and five blast-exposed animals. Error bars represent SEM. *P ≤ 0.05, two-tailed t test.

  • Fig. 3. Purkinje cell loss increases with the number of blast exposures in mice.

    (A) Representative confocal images of cerebellar lobule 9 in sham- and 3× blast–treated mice 30 days after treatment. Images show a merged maximum projection of 22 scans acquired at 2.27-μm intervals in the z plane immunostained for neurofilament heavy chain (NF-HC)–positive basket cells (green) and IP3R1-positive Purkinje cells (red). Blast-exposed cerebellum displayed prominent microdomains of Purkinje cell loss (empty baskets) that were also apparent when viewed along the z plane for highlighted regions (white boxes) projected above and to the right of the merged x-y images. (B) For illustrative purposes, this sagittal section denotes cerebellar lobule anatomy (lobules 1/2 to 10). (C) Histogram shows empty basket frequencies in 1× blast– and 3× blast–exposed mice normalized with respect to the number of empty baskets observed in sham-treated mice (n = 10, 7, and 12 for sham-treated, 1× blast–exposed, and 3× blast–exposed mice, respectively). In both dorsal/superior (lobules 1 to 6) and ventral/inferior (lobules 7 to 10) regions of the cerebellum, blast exposure caused an overall increase in empty baskets [P < 0.01 and P < 0.0001, analyses of variance (ANOVAs), respectively]. 3× blast exposure caused marked Purkinje cell loss throughout the cerebellum. However, the ventral/inferior lobules appeared more vulnerable to Purkinje cell loss after a single blast exposure. **P < 0.01, Helmert test. (D) Histogram shows normalized empty baskets for each lobule in the same sham-treated, 1× blast–exposed, and 3× blast–exposed mice. *P < 0.05, **P < 0.01, ANOVA. n.s., nonsignificant. Scale bars, 50 μm (A) and 500 μm (B). Data represent mean normalized to values for sham-treated mice, with error bars indicating ±SEM.

  • Fig. 4. Repetitive blast exposure impairs motor performance in mice.

    (A) Single blast exposure (1×) did not alter rotarod performance in mice at 30 days after treatment (n = 20 sham-treated, n = 19 blast-exposed mice). (B) In contrast, mice exposed to 3× blast exposure showed significant impairment in rotarod performance compared to sham-treated mice (n = 4 sham-treated, n = 5 blast-exposed; P ≤ 0.034, ANOVA). Data represent mean, with error bars indicating ±SEM.

  • Fig. 5. Blast exposure reduces PSD-95 expression in mouse cerebellum.

    (A) Western blots for PSD-95 and GABAB-R1 performed at 30 days after treatment showed reduced PSD-95, but not GABAB-R1, in 1× blast-exposed mice compared to sham-treated animals. Each lane shows Triton X-100–soluble cerebellar protein lysates (20 μg), with one animal per lane using PK as a gel protein loading control. (B) Histograms show densitometric quantification of Western blots in (A) normalized to sham-treated mice, which are considered to be 1.0 (that is, 100%). Expression of PSD-95, but not GABAB-R1, was reduced by blast exposure (P ≤ 0.0003, t test; n = 5 sham-treated and n = 5 blast-exposed mice). (C) Histogram shows normalized PSD-95 expression from Western blot experiments. One day after 1× blast exposure, PSD-95 expression was not different from sham controls (ANOVA, n.s.; n = 5 sham-treated and n = 4 blast-exposed mice). However, 30 days after blast exposure, PSD-95 expression was significantly reduced in blast-exposed animals compared to sham controls [P ≤ 0.006, ANOVA; n = 14, 11, and 4, sham-treated, 1× blast–exposed, and 3× blast–exposed mice, respectively; includes data pooled from (B) and (C)]. (D) Confocal microscopy confirmed decreased PSD-95 immunostaining in cerebellum of 3× blast–exposed mice 4 months after treatment. (E) At higher magnification, different sections from (D) showed reduced PSD-95 immunopositive puncta in the cerebellar molecular layer (white arrows) of 3× blast–exposed mice 4 months after treatment compared to sham-treated mice. Results are representative of n = 5 blast-exposed and n = 5 sham-treated mice. Scale bars, 500 μm (D) and 50 μm (E).

  • Fig. 6. Blast exposure induces persistent axonal injury in mice.

    (A) Bielschowsky silver staining of cerebellar slices from single blast-exposed mice at 24 hours shows thickened axonal varicosities evident in axons emanating from the Purkinje cell layer (n = 5/6 blast-exposed and n = 1/5 sham-treated mice). (B) Enlarged insets from (A) numbered 1 to 4 (arrowheads denote axonal varicosities). (C) Representative Western blot analyses from individual blast-treated and sham-exposed mice showed that blast exposure increased APP expression in cerebellum. Each lane represents Triton X-100–soluble protein lysates (20 μg total protein per lane) for a single animal using PK or SYPRO-Ruby (SR) as a total protein loading control. (D) Histogram indicates APP normalized to values for sham-treated mice, which are considered to be 100%. A two-factor ANOVA comparing all groups (sham-treated, 1× blast–exposed, and 3× blast–exposed mice) with respect to posttreatment delay (1, 7, and 30 days) confirmed that blast exposure increased APP in the cerebellum (ANOVA: treatment group, P ≤ 0.0005). Each group n = 5, except 1× blast–exposed and 1× sham–treated, which were n = 4. Error bars indicate ±SEM. Scale bars, 50 μm (A) and 10 μm (B).

  • Fig. 7. Blast exposure induces persistent reactive gliosis in mouse cerebellum.

    (A) Representative confocal images (20 merged scans at 2.5 μm along the z axis) show persistently elevated GFAP expression (red) in cerebellar white matter 30 days after 1× and 3× blast–exposure. White lines indicate areas of interest (AOI) delineated by the interior border of the cerebellar granule cell layer. (B) GFAP immunoreactivity (normalized with respect to sham-treated mice) in cerebellar AOIs circumscribing overall cerebellar white matter was increased in the blast-exposed animals (P ≤ 0.001, ANOVA; n = 8, 10, and 3 sham-treated, 1× blast–exposed and 3× blast–exposed mice, respectively). (C) In these slices, GFAP immunostaining in white matter surrounding deep cerebellar nuclei/dentate nuclei (DCN/DN) was increased (see insets). (D) GFAP immunoreactivity (normalized with respect to sham-treated) in cerebellum surrounding the deep cerebellar nuclei/dentate nuclei was increased in the blast-exposed animals (P ≤ 0.001, ANOVA; n = 8, 10, and 3 sham-treated, 1× blast–exposed, and 3× blast–exposed mice, respectively). (E) Fourteen days after treatment, Iba-1 staining (green) in the brains of 3× blast–exposed mice shows clusters of Iba-1–positive cells (green). Highlighted regions (white boxes) are shown at higher magnification (far right panels). Iba-1 immunoreactivity does not colocalize with GFAP-positive astrogliosis (red). (F) The cumulative distribution of microglial/macrophage clusters was observed in the brains of individual animals (with sample size noted in panels) at 14 and 30 days after exposure. At 14 days, microglial clusters were observed with a predominant distribution in the cerebellar nuclei and white matter, with occurrences increasing near the peduncle. The number of clusters increased with the number of blast exposures and diminished 30 days after exposure. (G) The overall volume of microglial processes was quantified by calculating the convex hull volume (right panels) of the Iba-1 immuno-positive cells in 285-μm2 (x, y plane) × 50-μm (z plane) images from deep cerebellar white matter from sham-treated, 1× blast–exposed, and 3× blast–exposed animals 14 days after treatment (left panel shows representative maximum field projections). (H) Histogram shows that microglial convex hull volume was reduced in blast-exposed animals compared to sham-treated mice (P ≤ 0.04; n = 8, 6, and 12, 1× blast, 3× blast, and sham-treated mice, respectively). (I) Histogram of Western blot densitometry of CD68 expression (an indication of microglial/macrophage activation) in cerebellar protein lysates in blast-exposed mice (ANOVA: P ≤ 0.01; n = 9, 5, and 5, sham-treated, 1× blast–exposed, and 3× blast–exposed mice, respectively) 14 days after exposure. Total protein was normalized with respect to reprobing of PK immunoreactivity. (J) Double-label confocal microscopy shows Iba-1–positive microglial cells (green) coexpressing CD68 (red). Data represent mean (normalized to sham values), with error bars indicating ± SEM. Scale bars, 250 μm (A and C), 100 μm (E, left panels, and G), 50 μm (E, right panels), and 25 μm (J). WM, white matter; ML, molecular layer; GCL, granule cell layer.

  • Fig. 8. Decreased mean diffusivity in human cerebellum correlates with an increase in the number of blast-related TBIs.

    (A) Approximate spatial locations of three VOIs (1 to 3) used for quantification of DTI parameters are shown on a reference T1-weighted MRI scan (Montreal Neurological Institute −32 mm in z axis). (B) Scatter plot reveals a statistically significant negative correlation between log10 number of blast-related mild TBIs and mean diffusivity calculated as the average of VOI 1 to 3 (Spearman r, P ≤ 0.034, n = 19). (C) Examples of DTI fiber tracts arising from the seed point defined near the dentate nucleus for one VOI in a subject with 5 reported blast exposures (upper panel) and a subject with >100 reported blast exposures (lower panel). Color scale represents the maximum eigenvalue of the tensor that is related to mean diffusivity (MD). Compared to the subject with five blasts, the greater amount of green color in the highlighted regions (white arrows) in the >100 blast-exposed subject indicates a lower mean diffusivity. (D) Mean diffusivity axial images from the same two subjects shown in (C) depicted as a mean diffusivity map. The ovals highlight the same regions shown in (C), demonstrating lower mean diffusivity (more green) in the subject with >100 blast-related mild TBIs compared to the subject with 5 blast exposures. Color bars represent mean diffusivity values (mm2/s).

  • Table 1. FDG-PET (n = 33). PSQI, Pittsburgh Sleep Quality Index; NSI, Neurobehavioral Symptom Inventory.
    DemographicsMean (SD), range
      Age (years)31.5 (9.4), 23–60
      Education (years)13.7 (1.5), 11–16
      Race, nonwhite, n (%)8 (24.2%)
      APOE-ε4–positive (%)8 (25.8%)
    Blast exposures
      Number of blast-related mild TBIs
    during military service (lifetime)
    21.2 (26.7), 1–102
    Median = 11
      Number of lifetime mild TBIs with
    loss of consciousness
    2.3 (2.5), 0–11
    Median = 2
      Time since last blast-related mild
    TBI (years)
    3.6 (1.5), 1–7
    Median = 4
    Behavioral and neurological measures
      CAPS score (3 missing)55.6 (30.5), 4–106
      PHQ-9 score9.6 (7.4), 0–25
      PSQI score (4 missing)9.3 (5.1), 1–19
      AUDIT-C score4.8 (2.4), 0–10
      NSItotal score29.4 (17.8), 2–77
  • Table 2. DTI tractography (n = 19).
    DemographicsMean (SD), range
      Age (years)33.2 (7.7), 22–52
      Education (years)14.6 (1.6), 12–18
      Race, nonwhite, n (%)5 (26.3%)
      APOE-ε4 positive (%)6 (33.3%)
    Blast exposures
      Number of blast-related mild TBIs
    during military service (lifetime)
    27.1 (28.3), 3–102 Median = 15
      Number of lifetime mild TBIs with
    loss of consciousness
    2.6 (2.7), 0–11 Median = 2
      Time since last blast-related mild
    TBI (years)
    5.7 (2.2), 1–8 Median = 6
    Behavioral and neurological measures
      CAPS score64.1 (28.7), 13–111
      PHQ-9 score10.6 (7.9), 0–25
      PSQI score10.5 (4.3), 3–17
      AUDIT-C score3.4 (2.3), 0–9
      NSItotal score29.5 (13.4), 4–57
  • Table 3. Correlation between number (log10) blast exposures and symptoms related to sensorimotor integration (n = 41).

    D, dizziness; LB, loss of balance; PC, poor coordination.

    SymptomSpearman rP
    Dizziness0.3740.016
    Loss of balance0.4310.005
    Poor coordination0.3650.019
    Total (D + LB + PC)0.4380.004

Supplementary Materials

  • Supplementary Material for:

    Repetitive blast exposure in mice and combat veterans causes persistent cerebellar dysfunction

    James S. Meabon, Bertrand R. Huber, Donna J. Cross, Todd L. Richards, Satoshi Minoshima, Kathleen F. Pagulayan, Ge Li, Kole D. Meeker, Brian C. Kraemer, Eric C. Petrie, Murray A. Raskind, Elaine R. Peskind, David G. Cook*

    *Corresponding author. E-mail: dgcook{at}u.washington.edu

    Published 13 January 2016, Sci. Transl. Med. 8, 321ra6 (2016)
    DOI: 10.1126/scitranslmed.aaa9585

    This PDF file includes:

    • Methods
    • Tabulated data for Figs. 1B, 2E, 3 (C and D), 4 (A and B), 5 (B and C), 6D, 7 (B, D, H, and I), and 8.
    • References (92101)

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