Journal: Neuroscience

Article Title: Blast Exposure Causes Dynamic Microglial/Macrophage Responses and Microdomains of Brain Microvessel Dysfunction

doi: 10.1016/j.neuroscience.2016.01.022

Figure Lengend Snippet: (A) A single mild blast exposure caused microglia/macrophages to rapidly adopt morphology consistent with a state of activation. In vivo two-photon microscopy was performed on sham and blast-exposed CX3CR1-GFP+/− mice expressing GFP in microglia/macrophages (green). Microglia/macrophages were imaged within the cortical CNS parenchyma 45 minutes post-treatment. Figure depicts reconstructed 3-D microglial/macrophage morphology derived from in vivo two-photon image stacks analyzed by Imaris software (see Methods) to identify individual cells, then determine cellular, morphology, process length, and calculate an encapsulating convex hull volume (red). (B) Results of this analysis revealed that blast significantly decreased filament length (p<0.006, N=6 and 4, BOP and sham, respectively) and convex hull volume in blast-exposed mice compared to shams (p<0.022, N=6 and 4, BOP and sham, respectively). (C) Microglia in sham-treated CX3CR1-GFP+/− animal (upper panel) demonstrated mostly thin, ramified processes with no evidence of aberrant peripherally injected QDots655 escaping microvessels. Middle panel shows that mild blast caused vascular disruption with QDots655 escaping into surrounding parenchyma and accompanied by microglia process retraction and microglia/macrophages adopting rounded amoeboid morphology consistent with an activated state. Lower panel shows images from the same blast-exposed animal approximately 100μm from the microdomain of vascular disruption shown above with less activated-appearing microglia/macrophages compared to middle panel. Scale bars: 40 μm.

Article Snippet: Microglial convex hull surfaces were generated with Imaris using the ‘convex hull’ add-on written with Matlab (MathWorks, Natick, MA).

Techniques: Activation Assay, In Vivo, Microscopy, Expressing, Derivative Assay, Software, Injection, Disruption

Journal: Neuroscience

Article Title: Blast Exposure Causes Dynamic Microglial/Macrophage Responses and Microdomains of Brain Microvessel Dysfunction

doi: 10.1016/j.neuroscience.2016.01.022

Figure Lengend Snippet: (A) In CX3CR1-GFP+/− mice, QDots655 (red) remained predominantly in microvessels in sham-treated mice. In 1X blast-exposured animals, QDots655 crossing the endothelium accumulated in juxtavascular processes and somas of microglia/macrophages (green). QDots655 colocalized with microglia/macrophages denoted by arrowheads. Other red puncta not colocalizing with GFP-positive cells is likely indicative of damaged/fragmented cells. (B) Mild blast caused a significant increase in Qdots655 internalized in microglia/macrophages compared to sham treated animals (p< 0.02). (C) Time lapse image series (60–140 min post-blast) show phagocytosed QDots655 accumulating in enlarged perivascular processes extending from a microglial cell body. (D) Lack of QDots655 emissions (645 nm to 670 nm) in CX3CR1-GFP+/− mice (emissions at 505nm to 530nm) without QDots655 confirms that QDot colocalization in GFP-expressing cells is specific and not due to non-specific auto-flourescence. Scale bars: (A, C) 20 μm, (D) 30 μm.

Article Snippet: Microglial convex hull surfaces were generated with Imaris using the ‘convex hull’ add-on written with Matlab (MathWorks, Natick, MA).

Techniques: Expressing

Journal: Neuroscience

Article Title: Blast Exposure Causes Dynamic Microglial/Macrophage Responses and Microdomains of Brain Microvessel Dysfunction

doi: 10.1016/j.neuroscience.2016.01.022

Figure Lengend Snippet: All images show maximum-field projections of 40 serially acquired images scanned at 1 μm intervals in the Z-plane (40 μm total tissue thickness) in cortex of fixed, immunostained sections from wild-type C57BL6 mice. (A) Triple-label confocal microscopy shows tight junctional claudin-5 (white pseudo color) and microglial/macrophage Iba-1 (green) immunoreactivity, as well as 10 kDa dextran (red) that had crossed the BBB one hour post blast. In contrast to sham (upper panel), 1X blast exposure caused focal vascular disruption evidenced by escape of 10 kDa dextran into the surrounding paranchyma (arrowheads) associated with aberrant tight junction claudin-5 morphology and activated-appearing microglia/macrophages. (B) Claudin-5 immunostaining in sham-treated animals (24 hours post-treatment) revealed normal appearing tight junction morphology in cortical penetrating vessels. At 24 hours after a single (1X) blast exposure, disturbed tight junction morphology was markedly less pronouced than at 4 hours after 1X blast-exposure as in Panel A. However, following repetitive 3X blast exposure claudin-5 immunostained tight junctions appear discontinuous and irregular compared to shams. (C) Even though 1X blast induced transiently disturbed tight junction morphology, within 1 hour peripherally-injected 10kDa dextran escapes into the cortical parenchyma (images representative of 3/4 blast-exposed animals and 4/4 shams). Scale bars: (A) 20 μm, (B) 10 μm. (C) 40 μm.

Article Snippet: Microglial convex hull surfaces were generated with Imaris using the ‘convex hull’ add-on written with Matlab (MathWorks, Natick, MA).

Techniques: Confocal Microscopy, Disruption, Immunostaining, Injection

Journal: Neuroscience

Article Title: Blast Exposure Causes Dynamic Microglial/Macrophage Responses and Microdomains of Brain Microvessel Dysfunction

doi: 10.1016/j.neuroscience.2016.01.022

Figure Lengend Snippet: (A) Confocal microscopy of fixed cortical tissue shows that Iba-1 immunostained microglia (green) in wild-type C57BL6 1X blast-exposed cortex have fewer processes compared to sham controls. (B) In cortex (150–500 μm below cortical surface) of wild-type C57BL6 mice, reconstructed 3-D microglial/macrophage morphology derived from confocal image stacks of fixed tissue shows Iba-1 immuno-positive (green) cellular morphology, process length, and convex hull volume (red). Microglia-encapsulating complex hull volume was reduced in blast exposed C57BL6 mice compared to shams. (C) Histogram shows microglia filament length was significantly reduced at 1 hour (p≤ 0.04, N= 5 and 5, BOP and sham respectively) and 4 hours (p≤ 0.03, N= 4 and 4, BOP and sham, respectively) after 1X blast exposure compare to shams. (D) Convex hull volume was significantly reduced at 1 hour (p≤ 0.04, N=5 and 5, BOP and sham, respectively) and 4 hours (p≤ 0.02, N=4 and 4, BOP and sham, respectively) after 1X blast compare to controls. Sholl analysis of Iba-1 immunostained cortical microglia evaluated at 1 (E) and 4 (F) hours post-treatment revealed a significant decrease in the mean number of Sholl crossings in 1X blast-exposed mice compared to shams (p≤0.022, N=5 animals per group). Error bars denote ±s.e.m. Scale bars: 20 μm.

Article Snippet: Microglial convex hull surfaces were generated with Imaris using the ‘convex hull’ add-on written with Matlab (MathWorks, Natick, MA).

Techniques: Confocal Microscopy, Derivative Assay