Macroscopic changes in aquaporin-4 underlie blast traumatic brain injury-related impairment in glymphatic function

Abstract

Mild traumatic brain injury (mTBI) has emerged as a potential risk factor for the development of neurodegenerative conditions such as Alzheimer's disease and chronic traumatic encephalopathy. Blast mTBI, caused by exposure to a pressure wave from an explosion, is predominantly experienced by military personnel and has increased in prevalence and severity in recent decades. Yet the underlying pathology of blast mTBI is largely unknown. We examined the expression and localization of AQP4 in human post-mortem frontal cortex and observed distinct laminar differences in AQP4 expression following blast exposure. We also observed similar laminar changes in AQP4 expression and localization and delayed impairment of glymphatic function that emerged 28 days following blast injury in a mouse model of repetitive blast mTBI. In a cohort of veterans with blast mTBI, we observed that blast exposure was associated with an increased burden of frontal cortical MRI-visible perivascular spaces, a putative neuroimaging marker of glymphatic perivascular dysfunction. These findings suggest that changes in AQP4 and delayed glymphatic impairment following blast injury may render the post-traumatic brain vulnerable to post-concussive symptoms and chronic neurodegeneration.

Keywords: aquaporin-4; blast; glymphatic; perivascular space; sleep; traumatic brain injury.

Figure 1

Altered AQP4 expression at the white matter–grey matter interface in human post-mortem blast tissue. Representative images of AQP4 and GFAP immunoreactivity in frontal cortex tissue from Young Control, Blast TBI and Older Control cases. Young Controls (19–37 years), Blast TBI (27–45 years), Older Controls (54–69 years), n = 5 per group. Top: Wide-field images extending from the crests of the gyri and depths of the sulci. High AQP4 and GFAP immunoreactivity at the white matter–grey matter interface and in the white matter of the Blast TBI case compared with Young or Older Controls, scale bar = 2 mm. Middle: Enlarged images of intensely immunoreactive astrocytes at the interface and white matter, scale bar = 1 mm. Bottom: Representative images at the depths of the sulci showing increased subpial and interface AQP4 labelling after blast injury, scale bar = 1 mm. Arrows indicate the junction between the white matter and grey matter. GM = grey matter; WM = white matter.

Figure 2

Laminar differences in GFAP and AQP4 immunoreactivity in human blast TBI. (A) Representative image of the five laminar regions of interest (ROIs) analysed, with the outermost ROI being the superficial grey matter, followed by the middle grey matter, deep grey matter, superficial white matter and the innermost ROI, the deep white matter. (B) Quantification of AQP4 and (C) GFAP across grey and white matter layers expressed as normalized mean fluorescence intensity. Data are mean ± SEM from n = 5 per group and were analysed with two-way repeated measure ANOVA followed by Sidak’s post hoc test (*P < 0.05, **P < 0.01 for Blast versus Older Controls). (D) Representative image of drawn line ROI extending from the cortical surface into the subcortical white matter to generate intensity projection plots of GFAP and AQP4 across the tissue at the sulcus (S). (E) Averaged intensity plots of normalized fluorescence intensity of AQP4 and GFAP at the sulcal depth are shown for n = 5 cases per group. Increased GFAP and AQP4 was observed at the white matter–grey matter interface and throughout the white matter in blast-injured post-mortem human brain tissue. (F and G) Averaged fluorescence intensity plots of AQP4 immunostaining surrounding capillaries and large vessels. Vessel diameter was measured and lines were drawn through the perivascular endfoot, continuing through the surrounding astrocytes and parenchyma. Both intensity projection plots were averaged for a single intensity plot per vessel and then averaged across groups. (F) Quantification of binned AQP4 intensity plot segments surrounding capillaries and large vessels at the junction of the white matter and grey matter. For graphing of perivascular and non-perivascular segments, the first five pixels were averaged for the perivascular endfoot segment and the next 30 pixels were averaged for the non-perivascular segment. Data are mean ± SEM from an average of 7–10 vessels per vessel type, per region, per case. Data were analysed with mixed effect analysis followed by Sidak’s post hoc test to control for within case correlation (*P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001). GM = grey matter; WM = white matter.

Figure 3

Increased AQP4 in the murine brain following mild repetitive blast traumatic brain injury. (A) Representative whole brain slice images of AQP4 immunostaining in mouse brains of Sham group and 7 (7D Blast) and 28 days (28D Blast) post mild repetitive blast traumatic brain injury (mTBI). (B) Representative dorsal cortex images of AQP4 immunostaining in Sham, 7D Blast and 28D Blast mouse brains. Scale bar = 200 μm. Increased AQP4, particularly at the cortical surface, was observed at 28 days post mTBI. (C) Representative images of the regions of interest drawn for regional shell analysis of AQP4 in outer grey, inner grey and white matter and representative dorsal and ventral lines drawn for AQP4 line analysis generate intensity plots of AQP4 across the tissue. (D) AQP4 fluorescence intensity from regional shell analysis showing AQP4 immunoreactivity was significantly increased in the outer grey matter at 28 days post mTBI. Data are mean ± SEM from n = 8–10 per group and were analysed with two-way repeated measure ANOVA followed by Sidak’s post hoc test (*P < 0.05, 28 days versus 7 days post mTBI; *P < 0.05, 28 days post mTBI versus Sham). (E) Averaged fluorescence intensity plots from the dorsal surface through the grey matter (as shown in D) and into the subcortical white matter. Data are mean ± SEM from n = 9–10 mice/group. (F) Averaged fluorescence intensity plots extending from the ventral surface through the grey matter (as shown in D) for n = 9 per group. (G) Quantification of AQP4 expression and localization in the dorsal cortex. AQP4 was co-stained with lectin to label blood vessels, a vessel mask was generated and AQP4 fluorescence intensity was measured inside or outside the vessel mask to quantify perivascular (PV) and non-PV AQP4, respectively. AQP4 ratios were calculated as PV/non-PV. (H) Quantification and localization of AQP4 in the ventral cortex. Data were analysed with two-sided t-test. GM = grey matter; WM = white matter.

Figure 4

Delayed impairment of glymphatic influx and sleep-wake disruption in the murine blast-injured brain. (A) Quantification of the average cortical fluorescence intensity of in vivo dynamic transcranial images acquired every 2 min from 20 to 42 min following intracisternal tracer injection. Data are mean ± SEM from n = 9–12 mice/group and were analysed with two-way repeated measures ANOVA followed by Tukey’s post hoc test. Main effects of Time (****P < 0.0001) and Time × Treatment interaction (***P < 0.001). (B) Representative ex vivo whole brain images of dorsal surface fluorescence taken following perfusion and removal from the skull. 7D Blast and 28D Blast = 7 and 28 days post mild repetitive blast traumatic brain injury. (C) Quantification of average fluorescence intensity of whole brain dorsal surface images. Data are mean ± SEM from n = 9–12 mice/group and were analysed with one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05). IR = infrared. (D) Diagram showing the range of the quantified coronal sections relative to bregma. (E) Diagram of the coronal sections and regions of interest across three quantified brain slices: dorsal cortex, ventral cortex, corpus callosum, hippocampus and subcortical regions. (F) Representative images of coronal brain sections at +0.25 mm from bregma showing Texas Red-conjugated dextran (3 kD) distribution within the brain. (G) Quantification of average fluorescence intensity of the dorsal cortex region across the three coronal sections between +0.25 mm to −2.75 mm from bregma. Data are mean ± SEM from n = 10 mice/group and were analysed with two-way ANOVA followed by Tukey’s post hoc test (**P < 0.01). (H) Activity counts of Sham and blast-injured (Blast) mice measured using Comprehensive Lab Animal Monitoring (CLAM) chambers. (I) Heat map showing activity across time for Sham and Blast mice, starting at 6 a.m. on the second day in the CLAM chambers through 6 a.m. on the day they were removed. (J) Cosinor-based rhythmicity of sham and blast mice (K) Circadian rhythm acrophase shift of Blast versus Sham mice. (L) Quantification of total sleep, per cent sleep and sleep bout count for the dark ‘waking’ period measured from data collected in CLAM chambers. Data were analysed with two-sided t-test and are the mean ± SEM from n = 16 mice/group.

Figure 5

MRI visible perivascular spaces in human blast-injured veterans. T1-weighted MRI axial cut through the centrum semiovale (1-cm thick) in two age-matched subjects representative of the cohort. (A) Subject A had one mild traumatic brain injury (mTBI), whereas (B) Subject B had eight mTBIs. MRI-visible perivascular space (MV-PVS) burden was assessed using an automated algorithm. Frontal MV-PVSs, anterior to the central sulcus (asterisk) and parietal MV-PVSs, posterior to the central sulcus, are shown. Note the higher MV-PVS number in the frontal region detected by the algorithm in the subject with high blast mTBI exposure.