Temporal and Spatial Effects of Blast Overpressure on Blood-Brain Barrier Permeability in Traumatic Brain Injury

Abstract

Blast-induced traumatic brain injury (bTBI) is a "signature wound" in soldiers during training and in combat and has also become a major cause of morbidity in civilians due to increased insurgency. This work examines the role of blood-brain barrier (BBB) disruption as a result of both primary biomechanical and secondary biochemical injury mechanisms in bTBI. Extravasation of sodium fluorescein (NaF) and Evans blue (EB) tracers were used to demonstrate that compromise of the BBB occurs immediately following shock loading, increases in intensity up to 4 hours and returns back to normal in 24 hours. This BBB compromise occurs in multiple regions of the brain in the anterior-posterior direction of the shock wave, with maximum extravasation seen in the frontal cortex. Compromise of the BBB is confirmed by (a) extravasation of tracers into the brain, (b) quantification of tight-junction proteins (TJPs) in the brain and the blood, and (c) tracking specific blood-borne molecules into the brain and brain-specific proteins into the blood. Taken together, this work demonstrates that the BBB compromise occurs as a part of initial biomechanical loading and is a function of increasing blast overpressures.

Figure 1

The shock tube housed in the blast laboratory in the Center for Injury Biomechanics, Materials, and Medicine at NJIT. (A) The 9-inch, square cross-section, 6 meters long shock tube instrumented with pressure sensors along the top of the shock tube. (B) Representative pressure-time profiles acquired from pressure sensors in the shock tube at the four overpressures used in this study. (C) Rat holder mounted in the test section of the shock tube, with rat placed in the prone position (top) and (D) tightly wrapped in a harness to minimize head and body motion during the blast (bottom). (E) Control (left) and injured (right) brains following perfusion-fixation. All blood in the neurovasculature has been washed away, as seen from the white appearance of brains, confirming that all tracers measured has leaked from the vessels into the brain parenchyma.

Figure 2

Fluorescent images of Evans blue extravasation. Images show 10x macro-shots as well as zoomed in 40x images in representative regions in the frontal cortex (A), striatum (B), somatosensory barrel-field cortex (C), hippocampus (D), thalamus (E) and cerebellum (F), 15 minutes following 180 kPa blast exposure. Control images were dramatically enhanced, yet still show limited visibility, due to the absence of extravasated dye. Frontal cortex was taken as a representative control image (G). Quantitation of extravasation is shown using a semilog plot in order to capture magnitudinal differences (H). Absorption spectrophotometry results of Evans blue in control and injured rats (n = 5) (I). *Indicates a difference in intensity compared with control with a statistical significance of p < 0.05, **Indicates p < 0.01. Scale bar = 1 mm in coronal sections and 50 μm in 40x images.

Figure 3

Fluorescent images of sodium fluorescein extravasation. Images show 10x macro-shots as well as zoomed in 40x images in representative regions in the frontal cortex (A), striatum (B), somatosensory barrel-field cortex (C), hippocampus (D), thalamus (E) and cerebellum (F) 15 minutes following 180 kPa blast exposure. Quantitation of extravasation is shown using a semilog plot in order to capture magnitudinal differences (G). Control images were dramatically enhanced, yet still show limited visibility, due to the absence of extravasated dye. Frontal cortex was taken as a representative image (H). **Indicates a difference in intensity compared with control with a statistical significance of p < 0.01. Scale bar = 1 mm in coronal sections and 50 μm in 40x images.

Figure 4

Quantitation of extravasation of Evans blue for 15 minutes (t0), 4 (t4), and 24 (t24) hours post-180 kPa blast exposure in frontal cortex, striatum, somatosensory barrel-field cortex, hippocampus, and thalamus using a semilog plot in order to capture magnitudinal differences (A). The frontal cortex was chosen for illustrative purposes and to qualitatively depict the difference between 4 (B) and 24 hrs (C). Arrows indicate areas of leakage from the vessels, which are more pronounced in 4 hours than any other time point studied in this investigation. **Indicates a difference in intensity compared with control with a statistical significance of p < 0.01. Scale bar = 100 μm.

Figure 5

Quantitation of extravasation of sodium fluorescein for 15 minutes (t0), 4 (t4), and 24 (t24) hours post- 180 kPa blast exposure in frontal cortex, striatum, somatosensory barrel-field cortex, hippocampus, and thalamus using a semilog plot in order to capture magnitudinal differences (A). The frontal cortex was chosen for illustrative purposes and to qualitatively depict the difference between 4 (B) and 24 hrs (C). Arrows indicate areas of leakage from the vessels, which are more pronounced in 4 hours than any other time point studied in this investigation. **indicates a difference in intensity compared with control with a statistical significance of p < 0.01. Scale bar = 100 μm.

Figure 6

ELISA results for tight junction proteins occludin and claudin-5, respectively in brain (A,B) and blood serum (C,D). Assay conducted for blast (180 kPa BOP) samples fifteen minutes, four, and twenty four hours post-exposure and compared with controls. *Indicates a difference in intensity compared with control with a statistical significance of p < 0.05, **Indicates p < 0.01.

Figure 7

Quantitation of extravasation of Evans blue for 35, 70, 130, and 180 kPa blast overpressures, 15 minutes post-exposure in frontal cortex, striatum, somatosensory barrel-field cortex, hippocampus, and thalamus using a semilog plot in order to capture magnitudinal differences (A). The striatum was chosen for illustrative purposes and to qualitatively depict the difference between 70 (B) and 130 kPa (C). Leaks appear longer and more intense with increasing overpressure in the same brain regions. *Indicates a difference in intensity compared with control with a statistical significance of p < 0.05, **Indicates p < 0.01. Scale bar = 100 μm.

Figure 8

Quantitation of extravasation of sodium fluorescein for 35, 70, 130, and 180 kPa blast overpressures, 15 minutes post-exposure in frontal cortex, striatum, somatosensory barrel-field cortex, hippocampus, and thalamus using a semilog plot in order to capture magnitudinal differences (A). The striatum was chosen for illustrative purposes and to qualitatively depict the difference between 70 (B) and 130 kPa (C). Leaks appear longer and more intense with increasing overpressure in the same brain regions. *Indicates an intensity compared with control with a statistical significance of p < 0.05, **Indicates p < 0.01. Scale bar = 100 μm.

Figure 9

Concentration of s100-β in blood serum. Assay conducted for blast (180 kPa BOP) samples four and twenty four hours post-exposure and compared with controls. *Indicates a difference in intensity compared with control with a statistical significance of p < 0.05.

Figure 10

Double immunofluorescence of endothelial cell marker (RECA-1, red) and monocyte marker (CCL2, green) at four hours post-exposure in frontal cortex in animals from control (A) and moderate (180 kPa BOP) blast injury (B). Scale bar = 50 μm.