Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model

Abstract

Blast exposure is associated with traumatic brain injury (TBI), neuropsychiatric symptoms, and long-term cognitive disability. We examined a case series of postmortem brains from U.S. military veterans exposed to blast and/or concussive injury. We found evidence of chronic traumatic encephalopathy (CTE), a tau protein-linked neurodegenerative disease, that was similar to the CTE neuropathology observed in young amateur American football players and a professional wrestler with histories of concussive injuries. We developed a blast neurotrauma mouse model that recapitulated CTE-linked neuropathology in wild-type C57BL/6 mice 2 weeks after exposure to a single blast. Blast-exposed mice demonstrated phosphorylated tauopathy, myelinated axonopathy, microvasculopathy, chronic neuroinflammation, and neurodegeneration in the absence of macroscopic tissue damage or hemorrhage. Blast exposure induced persistent hippocampal-dependent learning and memory deficits that persisted for at least 1 month and correlated with impaired axonal conduction and defective activity-dependent long-term potentiation of synaptic transmission. Intracerebral pressure recordings demonstrated that shock waves traversed the mouse brain with minimal change and without thoracic contributions. Kinematic analysis revealed blast-induced head oscillation at accelerations sufficient to cause brain injury. Head immobilization during blast exposure prevented blast-induced learning and memory deficits. The contribution of blast wind to injurious head acceleration may be a primary injury mechanism leading to blast-related TBI and CTE. These results identify common pathogenic determinants leading to CTE in blast-exposed military veterans and head-injured athletes and additionally provide mechanistic evidence linking blast exposure to persistent impairments in neurophysiological function, learning, and memory.

Fig. 1

CTE neuropathology in postmortem brains from military veterans with blast exposure and/or concussive injury and young athletes with repetitive concussive injury. (A and E) Case 1, phosphorylated tau (CP-13) neuropathology with perivascular neurofibrillary degeneration in the frontal cortex of a 45-year-old male military veteran with a history of single close-range blast exposure 2 years before death and a remote history of concussion. Whole-mount section. Scale bar (E), 100 μm. (B and F) Case 2, phosphorylated tau (CP-13) neuropathology with perivascular neurofibrillary degeneration in the frontal cortex of a 34-year-old male military veteran with history of two blast exposures 1 and 6 years before death and without a history of concussion. Whole-mount section. Scale bar (F), 100 μm. (C and G) Case 6, phosphorylated tau (CP-13) neuropathology with perivascular neurofibrillary degeneration in the frontal cortex of an 18-year-old male amateur American football player with a history of repetitive concussive injury. Whole-mount section. Scale bar (G), 100 μm. (D and H) Case 7, phosphorylated tau (CP-13) neuropathology with perivascular neurofibrillary degeneration in the frontal cortex of a 21-year-old male amateur American football player with a history of repetitive subconcussive injury. Whole-mount section. Scale bar (H), 100 μm. (I) Case 1, phosphorylated tau (CP-13) immunostaining in the parietal cortex revealed a string of perivascular foci demonstrating intense immunoreactivity (areas enclosed by hash lines). Whole-mount section. (J) Case 1, phosphorylated neurofilament (SMI-34) immunostaining in adjacent parietal cortex section demonstrating colocalization of multifocal axonal swellings and axonal retraction bulbs surrounding small blood vessels (black circles) relative to perivascular tau foci (areas enclosed by hash lines). Whole-mount section. (K) Case 1, human leukocyte antigen–DR (HLA-DR) (LN3) immunostaining in adjacent parietal cortex section demonstrating colocalization of microglial clusters (black circles) relative to perivascular tau foci (areas enclosed by hash lines). Whole-mount section. (L) Case 1, high-magnification micrograph of phosphorylated tau (CP-13) immunostaining in the parietal cortex demonstrating string of perivascular phosphorylated tau foci. Whole-mount section. (M) Case 1, phosphorylated tau (PHF-1, brown) and phosphorylated neurofilament (SMI-34, red) double immunostaining in parietal cortex demonstrating axonal swellings and a retraction bulb (arrow) in continuity with phosphorylated tau neuritic abnormalities. Whole-mount section. Scale bar, 100 μm. (N) Case 1, phosphorylated neurofilament (SMI-34) immunostaining showing diffuse axonal degeneration and multifocal irregular axonal swellings in subcortical white matter subjacent to cortical tau pathology. Whole-mount section. (O) Case 1, phosphorylated neurofilament (SMI-34) immunostaining demonstrating perivascular axonal pathology and axonal retraction bulbs near a small cortical blood vessel. Whole-mount section. (P) Case 1, activated microglia (LN3) immunostaining showing a large microglial nodule in the subcortical white matter subjacent to cortical tau pathology. LN3 immunostaining was not observed in brain areas devoid of tau pathology. Whole-mount section. Scale bar, 100 μm. (Q) Case 2, phosphorylated tau (CP-13) immunostaining showing diffuse neuronal tau pathology (pre-tangles) in the hippocampal CA1 field. Whole-mount section. (R) Case 2, phosphorylated tau (CP-13) pathology in temporal cortex. Whole-mount section. (S) Case 1, phosphorylated tau (AT8) immunostaining showing diffuse neuronal tau pathology (pre-tangles) in the hippocampal CA1 field. Whole-mount section. (T) Case 1, phosphorylation-independent total tau (Tau-46) immunostaining in the frontal cortex. Whole-mount section. (U) Case 3, phosphorylated tau (CP-13) immunostained axonal varicosities in the external capsule of a 22-year-old male military veteran with a history of a single close-range IED blast exposure and remote history of concussions. Whole-mount section. (V to X) Case 3, SMI-34 immunostained axonal varicosities and retraction bulbs in the thalamic fasiculus and external capsule. Whole-mount sections.

Fig. 2

Free-field pressure (FFP) and intracranial pressure (ICP) dynamics and head kinematics during single-blast exposure in a blast neurotrauma mouse model. (A) Measured incident static blast pressure (blue line) and blast impulse (red line) are compared to equivalent explosive blast waveform expected from 5.8 kg of TNT at a standoff distance of 5.5 m (black line) calculated according to software analysis using ConWep (44). The positive phase terminates at 4.8 ms (t₊ = 4.8 ms; black hash line). Blast characteristics and waveform structure are comparable to a typical IED fabricated from a 120-mm artillery round (4.53 kg of TNT equivalent charge weight). The measured blast waveform and equivalent TNT blast waveform are in close agreement with a leading shock wavefront followed by a smooth decay. Note that ConWep presents an idealized blast resulting from an above-ground spherical charge and does not model negative-phase pressure transients or modulating factors commonly encountered in military blast scenarios. Reflecting surfaces, bounding structures (for example, crew compartments in armored vehicles, rooms within buildings, walled streets, and alleyways), local geometry, device and deployment characteristics (for example, encapsulation, internal reflectors, and open versus buried deployment), ambient environmental conditions, and other factors strongly influence blast pressure amplitude (positive and negative), phase duration, impulse history, waveform structure, and target interactions (, –86). (B and C) ICP waveform and impulse profile in the brain of an intact living mouse (B) and isolated mouse head severed at the cervical spine (C) subjected to the same blast conditions as in (A). Blast waveforms recorded in the brains of living mice (B) and isolated heads (C) were similar in amplitude to each other and to the measured free-field static pressure. Small differences in the ICP signal waveforms were within the expected range given differences in frequency-dependent transducer response characteristics and experimental preparations. (D) Kinetographic representation of projected Cartesian motion of a representative mouse head during blast exposure as determined by high-speed videography acquired at 100,000 frames per second. Cartesian motion of the head was calculated by tracking a reflective paint mark on the snout. Labeled time points identify corresponding time points in (A) and (E) to (G). (E to G) Relative position (E), angular velocity (F), and angular acceleration (G) of the mouse head referenced to the horizontal (blue) and sagittal (red) planes of motion as determined by analysis of high-speed videographic records obtained during blast exposure. Head acceleration was most significant during the positive phase of the blast shock wave.

Fig. 3

Single-blast exposure induces CTE-like neuropathology in wild-type C57BL/6 mice. (A to F) Absence of macroscopic tissue damage (contusion, necrosis, hematoma, or hemorrhage) 1 day (A to C) or 2 weeks (D to F) after exposure to a single blast. Experimental blast conditions were compatible with 100% survival and full recovery of gross locomotor function. (G) Normal astrocytic glial fibrillary acidic protein (GFAP) immunoreactivity in a mouse brain 2 weeks after exposure to sham blast. Whole-mount sections. (H) Increased astrocytic GFAP immunoreactivity in the ipsilateral cortex (area enclosed by white hash line), bilateral thalamus (white asterisks), and bilateral hypothalamus (black asterisks) 2 weeks after single-blast exposure. Parenchymal atrophy with ventricular dilation was also observed (white arrowhead). Whole-mount sections. (I) Background phosphorylated tau (CP-13) immunostaining in superficial layers of the cerebral cortex 2 weeks after exposure to sham blast. (J) Phosphorylated tau (CP-13) immunostaining in superficial layers of the cerebral cortex 2 weeks after exposure to a single blast. Increased accumulation of phosphorylated tau in the brains of blast-exposed mice was confirmed by quantitative immunoblot analysis (Fig. 5). (K and P) Background phosphorylated neurofilament (SMI-31) immunostaining in the hippocampus 2 weeks after exposure to sham blast demonstrating normal-appearing CA1 pyramidal neurons with no detectable axonal pathology. (L and Q) Increased phosphorylated neurofilament (SMI-31) immunostaining in the hippocampus 2 weeks after exposure to single blast demonstrating pyknotic CA1 pyramidal neurons with nuclear smudging and injured axons with beaded, irregular swellings [arrowhead, (Q); enlargement shown in inset]. (M and R) Faint total tau (Tau-46) immunoreactivity in the soma and processes of pyramidal neurons in the hippocampal CA1 field 2 weeks after exposure to sham blast. (N and S) Increased total tau (Tau-46) immunoreactivity in the soma and processes of pyramidal neurons [arrowheads, (S)] in the hippocampal CA1 field 2 weeks after exposure to single blast. Biochemical abnormalities in total tau expression in the brains of blast-exposed mice were confirmed by quantitative immunoblot analysis (Fig. 5). (O) Faint activated microglial [Ricinus communis agglutinin (RCA)] immunoreactivity in the cerebellum 2 weeks after exposure to sham blast. (T) Increased activated microglial RCA immunoreactivity in the cerebellum indicative of brisk microgliosis [arrowheads, (T)] 2 weeks after exposure to single blast.

Fig. 4

Single-blast exposure induces hippocampal ultrastructural pathology in wild-type C57BL/6 mice. (A to G) Normal histology and ultrastructure in the hippocampal CA1 field 2 weeks after sham-blast exposure. (A) Tolui-dine blue–stained semithick section of the hippocampal CA1 field after sham blast. The CA1 field exhibits normal histological structure with a densely compacted layer of intact pyramidal neurons in the stratum pyramidale (pyr) and profuse dendritic profiles (black arrowheads) in the stratum radiatum (rad). (B to G) Electron micrographs of adjacent ultrathin sections demonstrating normal neuronal, axonal, and perivascular ultrastructure in the hippocampal CA1 field 2 weeks after sham-blast exposure. (B) CA1 pyramidal neurons in proximity to a capillary (asterisk) and endothelial cell. Scale bar, 10 μm. (C) Hippocampal CA1 field with normal stratum pyramidale (above white hash line) and stratum radiatum (below white hash line). Numerous dendrites are evident in the stratum radiatum. Scale bar, 10 μm. (D) Axon field in the stratum alveus demonstrating normal neuropil ultrastructure. Scale bar, 500 nm. (E) Capillary (asterisk) with endothelial cell nucleus (e) in a field of myelinated axons demonstrating normal ultrastructure in the stratum alveus. Scale bar, 500 nm. (F) Pyramidal neurons with normal ultrastructure in the hippocampal CA1 field. Scale bar, 2 μm. (G) Myelinated axons in transverse section in proximity to a capillary (asterisk) and endothelial cell (e). Scale bar, 500 nm. (H to N) Histological and ultrastructural pathology in the hippocampal CA1 field 2 weeks after single-blast exposure. (H) Toluidine blue–stained semithick section of hippocampus. Clusters of chromatolytic and pyknotic neurons (asterisks) are evident throughout the stratum pyramidale (pyr). Note the marked paucity of dendrites in the stratum radiatum (rad). A tortuous axon (white arrowhead) is present at the boundary between the stratum pyramidale and the stratum oriens. (I to N) Electron micrographs of adjacent ultrathin cryosections demonstrating widespread ultrastructural pathology in the hippocampal CA1 field 2 weeks after single-blast exposure. (I) Hydropic perivascular astrocytic end-feet (a_e) surround an abnormal capillary (asterisk) and endothelial cell (e). The astrocytic end-feet are grossly distended and edematous. Numerous vacuoles are scattered throughout the pale cytoplasm. The capillary exhibits an abnormal shape and grossly thickened, tortuous basal lamina (white arrow). A pericyte (p) and numerous electron-dense inclusion bodies are also present. Scale bar, 2 μm. (J) Degenerating pyramidal neurons (n_x) in proximity to a capillary (asterisk), endothelial cell (e), and swollen, hydropic processes of a perivascular astrocyte in the stratum pyramidale. A neighboring pyramidal neuron (n₁) appears normal. Scale bar, 2 μm. An enlarged field of this same region is shown in fig. S20. (K) Degenerating myelinated nerve fiber (black star) in the stratum alveus. Scale bar, 500 nm. (L) Swollen, hydropic perivascular astrocyte end-feet (a_e) surrounding a dysmorphic capillary (asterisk) in the hippocampal CA1 field. Note the abnormal endothelial cell (e) with irregularly shaped nucleus and nearby perivascular pericyte (p). The capillary basal lamina (white arrow) is grossly thickened. Lipofuscin granules (white star) are present in an adjacent process. Scale bar, 500 nm. A micrographic montage (fig. S11; corresponding high-magnification micrographs, fig. S12) of this same region reveals the soma and communicating processes of this perivascular astrocyte. (M) Degenerating CA1 pyramidal neuron (n_x) in the stratum pyramidale of the hippocampal CA1 field. The electron-dense cytoplasm and condensed nucleus of this “dark neuron” correspond to the pyknotic neurons observed in toluidine blue–stained semithick sections (Fig. 4H). A neighboring neuron (n₁) appears normal. Scale bar, 2 μm. (N) Presumptive autophagic vacuoles (v₁, v₂) in a perivascular astrocyte in the hippocampal CA1 field. Scale bar, 500 nm.

Fig. 5

Single-blast exposure induces increased brain tau protein phosphorylation in wild-type C57BL/6 mice. (A and B) Immunoblots of brain extracts from the left and right hemispheres of mice probed with monoclonal antibody CP-13 directed against phosphorylated tau protein (pS²⁰²/pT²⁰⁵) 2 weeks after exposure to sham blast (lanes 1 to 4) or single blast (lanes 5 to 8). Note the single broad band that migrated with an apparent molecular mass of 53 kD (arrows) in brains from mice in both groups. (C and D) Immunoblots of brain extracts from the left and right hemispheres of mice probed with monoclonal antibody AT270 directed against phosphorylated tau protein (pT¹⁸¹) using the same homogenates as in (A) and (B). (E and F) Immunoblots of brain extracts from the left and right hemispheres of mice probed with monoclonal antibody Tau 5 directed against total tau protein using the same homogenates as in (A) to (D). Unlike the results shown in the preceding panels, Tau 5 immunoblots revealed an apparent blast-related alteration in tau protein isoform distribution. (G) Densitometric quantitation of CP-13 phosphorylated tau protein (pS²⁰²/pT²⁰⁵) immunolabel in brain homogenates from mice exposed to single blast or sham blast 2 weeks before euthanizing. Mean values ± SEM in arbitrary densitometric units (a.u.). P < 0.005, two-tailed Student’s t test. (H) Densitometric quantitation of CP-13 phosphorylated tau protein (pS²⁰²/pT²⁰⁵) immunolabel in brain homogenates as a proportion of total tau protein (Tau 5) in brain homogenates from mice exposed to single blast or sham blast 2 weeks before euthanizing. Mean values ± SEM in arbitrary densitometric units. P < 0.05, two-tailed Student’s t test. (I) Densitometric quantitation of AT270 phosphorylated tau protein (pT¹⁸¹) immunolabel in brain homogenates from mice exposed to single blast or sham blast 2 weeks before euthanizing. Mean values ± SEM in arbitrary densitometric units. P < 0.001, two-tailed Student’s t test. (J) Densitometric quantitation of AT270 phosphorylated tau protein (pT¹⁸¹) immunolabel in brain homogenates as a proportion of total tau protein (Tau 5) in brain homogenates from mice exposed to single blast or sham blast 2 weeks before euthanizing. Mean values ± SEM in arbitrary densitometric units. P < 0.001, two-tailed Student’s t test.

Fig. 6

Single-blast exposure induces persistent impairments in axonal conduction velocity and LTP of synaptic transmission in wild-type C57BL/6 mice. (A) Conduction velocity measurements of first peak compound action potential delay as a function of distance between recording electrodes in CA1 pyramidal cell axons in the stratum alveus of hippocampal slices from mice exposed to single blast (red circles, n = 13) compared to sham blast (black circles, n = 11). Mean ± SEM for each group. (B) Representative stimulus-evoked compound action potentials at proximal and distal recording sites (solid and hash lines, respectively) in hippocampal slices from mice exposed to single blast (red) and sham blast (black). Arrows indicate peak negativities used to calculate conduction velocity. (C) Time course of LTP at Schaffer collateral–CA1 synapses evoked by TBS in hippocampal slices from mice exposed to single blast (red circles, n = 17) compared to sham blast (black circles, n = 11). Each point mean ± SEM fEPSP slope of n slices. (D) Time course of LTP at Schaffer collateral–CA1 synapses evoked by bath application of the adenylate cyclase stimulant forskolin (50 μM) plus the type II phosphodiesterase inhibitor rolipram (10 μM; bar, FOR+ROL) in hippocampal slices from mice exposed to single blast (red circles, n = 27) compared to sham blast (black circles, n = 19). Each point mean ± SEM fEPSP slope of n slices. (E) Time course of LTP at Schaffer collateral–CA1 synapses evoked by TBS in hippocampal slices from mice 2 weeks (blue squares, n = 10) and 4 weeks after exposure to single blast (red circles, n = 7) compared to each other and to sham blast (black circles, n = 11). Each point mean ± SEM fEPSP slope of n slices. (F) Time course of long-lasting potentiation at Schaffer collateral–CA1 synapses evoked by bath application of the adenylate cyclase stimulant forskolin (50 μM) plus the type II phosphodiesterase inhibitor rolipram (10 μM; bar, FOR+ROL) in hippocampal slices from mice 2 weeks (squares, n = 12) and 4 weeks after exposure to single blast (red circles, n = 15) compared to each other and to sham blast (black circles, n = 19). Each point mean ± SEM fEPSP slope of n slices.

Fig. 7

Single-blast exposure in wild-type C57BL/6 mice induces persistent hippocampal-dependent learning and memory deficits that are prevented by head fixation (immobilization) during blast exposure. (A to C) Open-field testing showed no effect of blast exposure on gross locomotor function, explorative activity, or thigmotaxis as measured by total distance traveled (A), mean velocity (B), and number of central zone entries (C), respectively, in mice exposed to single blast (red bars, single blast, head free, n = 10; blue bars, single blast, head fixed, n = 10) or sham blast (black bars, sham blast, n = 20). (D to F) Barnes maze testing demonstrated significant impairments in hippocampal-dependent spatial learning acquisition measured by decreasing latency to find the escape box across 4 days of training (D) (two-way ANOVA, P = 0.020) and long-term memory assessed by escape box location recall assessed 24 hours after the last training session (E) (**P = 0.004, Student’s t test). Mice exposed to single blast (red squares, single blast, head free, n = 10) are compared to pooled sham-blast control mice (circles, sham blast, n = 20). Fixation (immobilization) of the head during blast exposure (blue squares, single blast, head fixed, n = 10) reversed blast-induced learning and memory deficits. Arrowhead in (E) represents 5% level predicted by chance selection of the escape box from among the 20-hole choices. (F) Representative Barnes maze tracks obtained on trials 1, 8, and 16 for mice exposed to a single blast (bottom row) compared to sham blast (top row).