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. 2013 Jun;33(6):834-45.
doi: 10.1038/jcbfm.2013.30. Epub 2013 Feb 27.

'Hit & Run' model of closed-skull traumatic brain injury (TBI) reveals complex patterns of post-traumatic AQP4 dysregulation

Zeguang Ren  1 , Jeffrey J Iliff, Lijun Yang, Jiankai Yang, Xiaolin Chen, Michael J Chen, Rebecca N Giese, Baozhi Wang, Xuefang Shi, Maiken Nedergaard
Affiliations

'Hit & Run' model of closed-skull traumatic brain injury (TBI) reveals complex patterns of post-traumatic AQP4 dysregulation

Zeguang Ren et al. J Cereb Blood Flow Metab. 2013 Jun.

Abstract

Cerebral edema is a major contributor to morbidity associated with traumatic brain injury (TBI). The methods involved in most rodent models of TBI, including head fixation, opening of the skull, and prolonged anesthesia, likely alter TBI development and reduce secondary injury. We report the development of a closed-skull model of murine TBI, which minimizes time of anesthesia, allows the monitoring of intracranial pressure (ICP), and can be modulated to produce mild and moderate grade TBI. In this model, we characterized changes in aquaporin-4 (AQP4) expression and localization after mild and moderate TBI. We found that global AQP4 expression after TBI was generally increased; however, analysis of AQP4 localization revealed that the most prominent effect of TBI on AQP4 was the loss of polarized localization at endfoot processes of reactive astrocytes. This AQP4 dysregulation peaked at 7 days after injury and was largely indistinguishable between mild and moderate grade TBI for the first 2 weeks after injury. Within the same model, blood-brain barrieranalysis of variance permeability, cerebral edema, and ICP largely normalized within 7 days after moderate TBI. These findings suggest that changes in AQP4 expression and localization may not contribute to cerebral edema formation, but rather may represent a compensatory mechanism to facilitate its resolution.

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Figures

Figure 1
Figure 1
The ‘Hit & Run' model of murine traumatic brain injury (TBI). (A) Schematic of Hit & Run TBI model. (B) Time to arousal after piston impact. ***P<0.001 vs. sham; †P<0.01 vs. mild TBI (ANOVA, n=6–13 animals per group). (C) Gross pathology of mild (upper) and moderate (lower) TBI 24 hours post injury. Tetrazolium (TTC) staining (D) showed infarcts in animals subjected to moderate TBI, but not in mild TBI animals. (E) Evans Blue extravasation indicated low-grade and transient blood–brain barrier (BBB) disruption in moderate TBI animals, while mild TBI animals did not exhibit appreciable BBB disruption. (F) Quantification of TTC infarct and Evans Blue BBB disruption analysis (n=3 animals per group). (G) Brain water content was evaluated in ipsilateral (I) and contralateral (C) hemispheres 1, 3, and 7 days post TBI. When compared with control values (blue line), ipsilateral moderate TBI brains exhibited cerebral edema at all time points. Edema was not apparent in contralateral hemisphere of moderate TBI brain, nor was it observed in either hemisphere of mild TBI brains. ***P<0.001, **P<0.01 vs. Control (analysis of variance (ANOVA), n=3–6 per group). (H, I) Intracranial pressure (ICP) was monitored daily in animals for 6 days post injury. ICP (both peak and nadir values) were elevated over the first 3 days post injury, then returned to baseline by day 6. Pulse wave amplitude was elevated at 1 day post injury. *P<0.05 vs. value at time=0 (repeated-measures two-way ANOVA, n=6 animals).
Figure 2
Figure 2
Long-term behavioral deficits in mice undergoing traumatic brain injury (TBI). Behavioral deficits were evaluated in control (sham), mild, and moderate TBI mice for 28 days post injury. (A) Gross neuroscore revealed a graded effect of TBI severity between mild and moderate TBI that resolved within the first 7 days post injury. *P<0.05 vs. mild TBI (repeated-measures two-way ANOVA, n=10–12 per time point). (B) Rotarod testing revealed that mild and moderate TBI animals performed significantly worse than control animals. No difference was detected between mild and moderate groups. *P<0.05 vs. control (repeated-measures two-way analysis of variance (ANOVA), n=11–28 animals per group). (C) In the novel object recognition test, moderate TBI animals were significantly impaired compared with controls, while mild TBI animals exhibited no change. *P<0.05 vs. control (repeated-measures two-way ANOVA, n=11–28 animals per group). (D) Animals underwent Barnes maze testing 28 days after TBI. Mild TBI did not significantly affect Barnes maze performance, while performance was significantly impaired in moderate TBI animals compared with controls. *P<0.01 vs. control (repeated-measures two-way ANOVA, n=11-18 animals per group).
Figure 3
Figure 3
Differing patterns of reactive astrogliosis after mild and moderate traumatic brain injury (TBI). Reactive astrogliosis (glial fibrillary acidic protein (GFAP), red) and microgliosis (CD68, green) were evaluated after mild and moderate TBI. (A, C, G) Whole-slice montages demonstrate the extent of reactive gliosis in control animals and 3 days after TBI. (D–F) Within the cerebral cortex, reactive gliosis declines between 7 and 14 days after mild TBI. Control (no TBI) cortex exhibits little GFAP immunoreactivity (B). (H–J) After moderate TBI, cortical reactive gliosis does not resolve for more than 14 days post injury. (K-L) Quantification of reactive astrogliosis 3, 7, 14, and 28 days after TBI shows that astrogliosis after mild TBI is transient within both the ipsilateral cerebral cortex (K) and striatum (L), yet is more persistent after moderate TBI. Peak reactive gliosis at 7 days post injury does not significantly differ between mild and moderate injury. **P<0.01, ***P<0.001 ipsilateral vs. contralateral; #P<0.05 moderate vs. mild TBI; †P<0.05, ††P<0.01, †††P<0.001 between ipsilateral time points; ‡P<0.01 between contralateral time points (two-way analysis of variance (ANOVA)). (M–O) Post-traumatic reactive astrogliosis was evident in both the ipsilateral and contralateral hippocampus 7 days after both mild (N) and moderate (P) TBI. Hippocampal reactive astrogliosis persisted at least 28 days after Hit & Run TBI (O, Q). Scale bars: 25 μm.
Figure 4
Figure 4
Axonal degeneration and demyelination after mild and moderate traumatic brain injury (TBI). (A–C) Ipsilateral cortical, striatal, and subcortical white matter area fractions (expressed as a percentage of the overall slice area) were evaluated at 3, 7, 14, and 28 days after mild and moderate TBI. No cortical (A) or striatal (B) atrophy was observed in mild TBI animals. Delayed cortical atrophy was observed at 7 days post injury in moderate TBI animals, while striatal atrophy was comparably minor. (C) Transient subcortical white matter atrophy was observed after both mild and moderate TBI. *P<0.05 vs. control; (analysis of variance (ANOVA), n=3–5 animals per group). (D, E) Changes in myelination (myelin basic protein, MBP; green) were observed in cortical regions of reactive astrogliosis (GFAP, red) surrounding moderate TBI lesions in the ipsilateral cortex 7 days after injury. (F) Post-traumatic cortical demyelination was quantified from mild and moderate TBI animals at 7 and 14 days post injury. Significant loss of myelination was observed in the ipsilateral cortex after moderate TBI. *P<0.05 vs. control (ANOVA, n=12 regions from 2 animals per group). (G–J) Immunolabeling for phosphorylated neurofilament (SMI-34) 14 days post injury revealed sparse axon degeneration (arrows) in the ipsilateral cortex of mild TBI mice (G), and extensive axonal pathology (arrows) in moderate TBI mice (I). Scale bars: 25 μm.
Figure 5
Figure 5
Evolution of post-traumatic AQP4 dysregulation. Astroglial AQP4 expression was evaluated by immunofluorescence in control cortex (A) and after mild (B) or moderate traumatic brain injury (TBI) (C). Insets depict AQP4-GFAP co-localization. Normalized global (D) and perivascular (E) AQP4 expression, and AQP4 polarity (F) were quantified in control, mild TBI, and moderate TBI cortex at 3, 7, 14, and 28 days post-injury. *P<0.05, **P<0.01, ***P<0.001 vs. control (two-way analysis of variance (ANOVA)). Striatal AQP4 expression was evaluated similarly in control animals (G), and after mild (H) and moderate (I) TBI. Normalized global (J) and perivascular (K) AQP4 expression, and AQP4 polarity (L) were quantified at 3, 7, 14, and 28 days after mild or moderate TBI. *P<0.05, **P<0.01, ***P<0.001 vs. control; #P<0.05 moderate vs. mild TBI (2-way ANOVA). Scale bars: 25 μm.
Figure 6
Figure 6
Post-traumatic redistribution of AQP4 localization. Localization of AQP4 was assessed by high-power confocal microscopy 14 days after moderate TBI. (A) On the contralateral side, AQP4 immunoreactivity is confined to perivascular endfeet (arrowheads) and generally does not co-localize with large glial fibrillary acidic protein (GFAP)-positive astrocytic processes (arrows). Insets depict XZ and YZ projections at planes indicated by white lines. (B) After moderate traumatic brain injury (TBI), a large proportion of AQP4 immunoreactivity has shifted to the membrane of GFAP-positive reactive astrocyte soma and coarse processes (arrows). AQP4 immoreactivity is also evident diffusely surrounding GFAP-positive processes, presumably corresponding to fine astrocytic processes. Insets depict XZ and YZ projections at planes indicated by white lines. Scale bars: 25 μm.
Figure 7
Figure 7
AQP4 expression in the developing glial scar. AQP4 expression was evaluated within control cortex (A) and within the developing cortical glial scar after moderate TBI (C–H). (B) Overall, AQP4 expression was quantified at 3, 7, and 14 days post traumatic brain injury (TBI). ***P<0.001 vs. control (one-way ANOVA). (C, E, G) Representative confocal images depict glial fibrillary acidic protein (GFAP) (red) and AQP4 (green) co-localization in the evolving glial scar. (D, F, H) Isolation of the AQP4 emission channel demonstrates dramatic perturbation of AQP4 localization in the evolving glial scar. Scale bars: 25 μm.
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