Transcranial amelioration of inflammation and cell death after brain injury

Abstract

Traumatic brain injury (TBI) is increasingly appreciated to be highly prevalent and deleterious to neurological function. At present, no effective treatment options are available, and little is known about the complex cellular response to TBI during its acute phase. To gain insights into TBI pathogenesis, we developed a novel murine closed-skull brain injury model that mirrors some pathological features associated with mild TBI in humans and used long-term intravital microscopy to study the dynamics of the injury response from its inception. Here we demonstrate that acute brain injury induces vascular damage, meningeal cell death, and the generation of reactive oxygen species (ROS) that ultimately breach the glial limitans and promote spread of the injury into the parenchyma. In response, the brain elicits a neuroprotective, purinergic-receptor-dependent inflammatory response characterized by meningeal neutrophil swarming and microglial reconstitution of the damaged glial limitans. We also show that the skull bone is permeable to small-molecular-weight compounds, and use this delivery route to modulate inflammation and therapeutically ameliorate brain injury through transcranial administration of the ROS scavenger, glutathione. Our results shed light on the acute cellular response to TBI and provide a means to locally deliver therapeutic compounds to the site of injury.

Figures

Figure 1. Pathology associated with compression injury

a, MRI of a patient’s brain 19 hrs after fall from 6 feet, with reported loss-of-consciousness, post-traumatic amnesia, a Glasgow Coma Scale of 15 on arrival, and a negative CT scan. Following administration of Gd-DTPA contrast agent, FLAIR MRI (grayscale images) revealed focal enhancement in the meninges along convexity underlying the area of blunt trauma, better visualized on surface-rendered 3D-FLAIR (pseudo-colored 3D images) as involving the frontal and temporal lobes as well as anterior aspects of the cerebral falx. b–d, f,g, Maximum projections (5 µm wide) are shown in the xz plane of two-photon z-stacks captured through a thinned skull. b, Images of skull bone (blue) and underlying meninges show sequential skull thinning from 50 to 10 µm. Dead cells (red) were labeled by transcranial propidium iodide (PI) administration. c, Intravenously injected Q-dots (red) leak from blood vessels into the meninges 15 min following compression injury, indicative of vascular damage. d, ROS (red) labeled with Amplex Red appears in the meninges 30 min following compression injury of CX3CR1gfp/+ (green) mice relative to uncompressed controls. (white dotted line = glial limitans) e, xy maximal projections (25 µm in depth) captured in GFAP-GFP mice show holes (white arrows) in the glial limitans as a result of astrocyte (green) death, which starts to occur 5 min after compression injury. f, Transcranially applied SR101 (red) diffuses into the brain parenchyma 30 min after injury, but is largely excluded from the parenchyma in an uncompressed control mouse. g, Cell death (PI+ cells; red) becomes apparent in the brain parenchyma 12 hrs following compression injury and is not observed in uncompressed controls. h, Quantification of cell death (mean + SD) in the meninges and parenchyma following compression injury. All data in the figure are representative of 3 mice per group (4 mice, g) and at least three independent experiments.

Figure 2. Innate immune response to a compression injury

25 µm xy maximum projections from CX3CR1gfp/+ mice (a–c) or LysMgfp/+ mice (d) captured at 30 min (a), 1 hr (b), 2 hrs (c), or 6 hrs (d) following a normal thinned skull preparation (uncompressed) or compression injury. a, Meningeal macrophages (green) visualized in CX3CR1gfp/+ mice burst and die within 30 min of compression injury relative to uncompressed controls. Blood vessels are red. b, Microglia (green) retract their ramified processes and form a highly connected “honeycomb” network at the glial limitans following compression injury. c, A subset of microglia (green) after compression injury retract ramified processes and generate a single, flat, motile, phagocytotic process at base of glial limitans, resembling a “jellyfish” (examples denoted with white asterisks). Blood vessels are red. d, LysMgfp/+ neutrophils (green) are recruited to the site of injury, but not to an uncompressed thinned skull window. Green cells residing in the uncompressed window represent meningeal macrophages. Data are representative of 3 mice per group and at least three independent experiments.

Figure 3. Metrics of transcranial diffusion through the skull bone

a, SR101 (red) was applied to an intact mouse skull for the indicated time and then the skull (blue) was quickly thinned and imaged. Five micron xz maximum projections show that SR101 is detectable in the meninges beginning 10 minutes after application and fully saturates the space within 15 min. (white dotted line = glial limitans) b, The size dependence of diffusion through an intact skull bone was evaluated 30 min following continuous transcranial application of the indicated MW dextrans (red). Dextrans that passed successfully through the skull generated fluorescence in the meninges. The skull bone is shown in blue, and the glial limitans is denoted with a white dotted line. c, A color coded table summarizing the imaging results shown in panels a and b denotes the presence (green) or absence (red) of fluorescent dye in the meninges at the indicated MW and time. Gray = not tested. d, Fluorescent compounds of increasing molecular weights were passed transcranially through a thinned skull window during imaging. Steady state concentrations (mean + SD) of the fluorescent compounds in the meninges and parenchyma were quantified from normalized fluorescence intensities. See associated Figure S4. e, Manganese chloride (500 mM solution) applied transcranially to an intact rat skull (~1 mm thick) is visible by MRI in the brain parenchyma 2 hrs after application (white arrow). The mean parenchymal manganese concentration ± SD is provided. All data in the figure are representative of 3 mice (or rats) per group and at least three independent experiments.

Figure 4. Purinergic receptor signaling mediates the innate immune response to compression injury

a, Honeycomb microglia were quantified 3 hrs following compression injury in mice treated transcranially with P2X7, P2Y6, P2X4, and P2Y12 antagonists or vehicle. Uncompressed mice served as a negative control. Blue dots represent individual microglia, and the horizontal red line denotes the mean. b, Quantification of jellyfish microglia was performed 3 hrs following compression injury. Because two compression injuries were generated per mouse, data are represented as a ratio (mean±SD) of purinergic receptor antagonist / vehicle and compared to vehicle / vehicle. A ratio of one signifies no difference between the two hemispheres. c, Quantification of neutrophils per mm3 tissue (mean±SD) was performed at 1, 3, and 6 hrs post-compression injury. d–f, Bar graphs (mean±SD) show quantification of CX3CR1gfp/+ microglia with a honeycomb (d) or jellyfish (e) morphology as well as the number LysMgfp/+ neutrophils (f) following transcranial administration of CBX or probenecid. f, Glial limitans permeability was quantified by generating two compression injuries per mouse. The mean SR101 fluorescence in the parenchyma beneath each injury was calculated and expressed as a ratio (CBX / vehicle or vehicle / vehicle). A ratio larger than one signifies increased permeability in the experimental group. Asterisks in all figures denote statistical significance (p < 0.05) relative to the vehicle control group. Data are representative of 3 mice per group and at least three independent experiments.

Figure 5. Transcranial administration of glutathione reduces inflammation and cell death following compression injury

25 µm xy maximum projections (a, c) and 5 µm xz projections (b, d) were captured in CX3CR1gfp/+ (a), B6 (b, d), or LysMgfp/+ (c) mice after compression injury (n=3, a–c, e–g; n=4, d, h–i). a, GSH pretreatment prevented jellyfish / honeycomb microglia formation (green) 1 hr following a compression injury that resulted in a cracked skull (blue). GSH administration also promoted survival of meningeal macrophages (green; white arrows). b, Relative to the vehicle control group, GSH pretreatment prevented glial limitans breakdown observed 1 hr post-injury. SR101 (red) localizes above the glial limitans (white dotted line) in the GSH treated group. c, In GSH pretreated mice, no neutrophil response (green) to compression injury was observed at 6 hrs. d, GSH administered 15 min or 3 hrs post-compression injury significantly reduced parenchymal cell death observed at 12 hrs. PI+ dead cells (red) reside primarily in the meninges of GSH-treated mice. (white dotted line = glial limitans). e–g, Bar graphs (mean±SD) show quantification of honeycomb / jellyfish microglia (e), glial limitans permeability (f), and neutrophil recruitment (g) in vehicle vs. GSH-treated mice following compression injury at the time points denoted above. h, The number of PI+ dead cells per mm3 (mean±SD) was quantified in the meninges at 30 min or 12 hrs post-compression injury. GSH significantly reduced meningeal cell death if applied before (pre-treatment), but not following (0 min, 15 min, 3 hrs) compression injury. P2X7 blockade increased meningeal cell death when administered 15 min after compression injury. i, Parenchymal cell death was quantified 12 hrs following compression injury in the denoted groups. All data in this figure are representative of three independent experiments, and asterisks denote a statistically significant difference (p < 0.05) from the vehicle control group.

METHOD REFERENCES