Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 15;38(8):1193-1201.
doi: 10.1089/neu.2011.2114.

Aquaporin-4 Reduces Post-Traumatic Seizure Susceptibility by Promoting Astrocytic Glial Scar Formation in Mice

Daniel C Lu  1 , Zsolt Zador  1 , Jinghua Yao  1 , Farbod Fazlollahi  1 , Geoffrey T Manley  1
Affiliations

Aquaporin-4 Reduces Post-Traumatic Seizure Susceptibility by Promoting Astrocytic Glial Scar Formation in Mice

Daniel C Lu et al. J Neurotrauma. .

Abstract

Seizures are important neurological complications after traumatic brain injury (TBI) and are reported for up to 50% of patients with TBI. Despite several studies, no drug strategy has been able to alter the biological events leading to epileptogenesis. The glial water channel, aquaporin-4 (AQP4), was shown to facilitate cytotoxic cell swelling in ischemia and glial scar formation after stab wound injury. In this study, we examined post-traumatic seizure susceptibility of AQP4-deficient mice (AQP4-/-) after injection of pentylenetetrazole (PTZ) 1 month after controlled cortical impact (CCI) and compared them to wild-type sham injury controls. After PTZ injection, AQP4-/- mice demonstrated dramatically shortened seizure latency (120 ± 40 vs. 300 ± 70 sec; p < 0.001) and increased seizure severity (grade 7.5 ± 0.4 vs. 5.8 ± 0.4; p < 0.001) compared to their wild-type counterparts. Morphometric analysis demonstrated a significant 2-fold reduction in astrocytosis, with a concomitant increase in microgliosis in injured AQP4-null mice compared to their injured wild-type counterparts (44 ± 2 vs. 24 ± 3 cells per high power field [cells/hpf], respectively; p < 0.0001). Minocycline, an inhibitor of microglia, reversed the post-TBI epilepsy phenotype of AQP4-null mice. After minocycline treatment, AQP4-/- mice demonstrated similar latency of seizures evoked by PTZ (723 ± 35 vs. 696 ± 38 sec; p > 0.05) and severity of seizures evoked by PTZ (grade 4.0 ± 0.5 vs. 3.81 ± 0.30; p > 0.05) compared to wild-type counterparts. Immunohistochemical analysis demonstrated decreased immunostaining of microglia to levels comparable to wild-type (12 ± 2 vs. 11 ± 4 cells/hpf, respectively; p > 0.05). Taken together, these results suggest a protective role of AQP4 in post-traumatic seizure susceptibility by promoting astrogliosis, formation of a glial scar, and preventing microgliosis.

Keywords: aquaporin; astrocyte; glial scar; seizure epilepsy.

PubMed Disclaimer

Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
AQP4-null mice exhibited worse seizure profile after CCI. (A) Seizure severity in response to PTZ in wild-type and AQP4-null mice were measured before and after CCI. CCI increased seizure severity in each genotype (*p < 0.05 in wild-type and ***p < 0.001 in AQP4-null mice). Seizure was more severe in AQP4-null mice (grade 7.5) compared to wild-type mice (grade 4.2; ***p < 0.001). (B) Seizure latency was measured in the same cohort. Seizure latency was shortened after CCI in both wild-type and AQP4-null mice (**p < 0.01 in wild-type and ***p < 0.001 in AQP4-null mice). Seizure latency was increased in AQP4-null mice (720 ± 120 sec) compared to wild-type (480 ± 80 sec) before injury. In contrast, after injury, seizure latency was shortened in AQP4-null mice (120 ± 40 sec) compared to wild-type mice (300 ± 70 sec; p < 0.0001). *p < 0.05; **p < 0.001; ***p < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, controlled cortical impact.
FIG. 2.
FIG. 2.
Similar injury volume in wild-type and AQP4-null mice after CCI. (A) Representative brightfield images of Nissl-stained coronal images of wild-type and AQP4-null mice before and after injury are shown (injured region under dotted line). (B) Injury volume was quantified and assessed. Genotype did not contribute to differences in injury volume (p > 0.05); however, there was a significant CCI effect (p < 0.001). In both genotypes, CCI induced an injury volume of ∼40%. *p < 0.05; **p < 0.001; ***p < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, cpnmtrolled cortical impact.
FIG. 3.
FIG. 3.
Increased AQP4 immunoreactivity in injury zone after CCI. (A) Representative immunofluorescence images, taken from injury penumbra (immediately adjacent to injury area), showing AQP4 positive cells (red) in wild-type mice. Injury induced clustering and migration of AQP4-positive astrocytes to the injury area. Background immunoreactivity was observed in AQP4-null mice. (B) Quantification of AQP4-positive cells surrounding the injury area. Injury strongly increased the number of AQP4-reactive cells in wild-type animals (3.0 ± 0.3 cells/hpf before injury to 24 ± 3 cells/hpf after injury; p < 0.0001). This was not observed in AQP4-null mice. As expected, there was a significant difference in AQP4 staining in wild-type compared to AQP4-null mice (**p < 0.001 in sham and ***p < 0.0001 in CCI comparisons). *p < 0.05; **p < 0.001; ***p < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, controlled cortical impact.
FIG. 4.
FIG. 4.
Reactive astrocytes in injury penumbra after CCI. (A) Representative brightfield images showing a dramatic increase in GFAP reactivity in the injury penumbra after CCI in wild-type mice. This increase was observed in cortical regions above the hippocampus. (B) Quantification of GFAP-positive cells demonstrated a 2-fold difference in GFAP-positive cells after injury in wild-type versus AQP4-null mice (82 ± 10 vs. 40 ± 8 cells/hpf, respectively; ***p < 0.0001). This difference between the genotype was not observed in uninjured control mice (p > 0.05). *p < 0.05; **p < 0.001; ***p < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, controlled cortical impact; GFAP, glial fibrillary acidic protein.
FIG. 5.
FIG. 5.
Microgliosis in injury penumbra after CCI. (A) Representative brightfield images showing an increase in CD11b reactivity in the injury penumbra after CCI in AQP4-null mice. (B) Quantification of CD11b-positive cells demonstrated a significant difference in CD11b-positive cells after injury in wild-type versus AQP4-null mice (24 ± 3 vs. 44 ± 2 cells/hpf, respectively; ***p < 0.0001). This difference in genotype was not observed in uninjured control mice (2.0 ± 0.2 vs. 3.0 ± 0.4 cells/hpf, respectively; p > 0.05). *p < 0.05; **p < 0.001; ***p < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, controlled cortical impact.
FIG. 6.
FIG. 6.
Astrocytic scar morphology after CCI. Expression pattern of GFAP and AQP4 after cortical contusion. Immunolabeling of GFAP (white) in the cerebral cortex of uninjured AQP4+/+ (A) and AQP4–/– mice (B) demonstrated similar astrocyte morphology. Expression of GFAP in the glial scar lining the injury cavity in AQP4+/+ (C) and AQP4–/– (D) mice. After injury, there was robust GFAP immunoreactivity in (C) wild-type mice compared to (D) weaker GFAP immunostaining of AQP4-deficient mice. (E) Expression of AQP4 (red) in GFAP-positive (white) astrocyte endfeet in non-injured AQP4+/+ mice (inset 1). Note the predominant pericapillary AQP4 expression pattern of astrocytic foot processes. Expression pattern AQP4 in GFAP-positive astrocytes in the glial scar after injury (F) adjacent to the injury cavity (inset 2): note the AQP4 positive astrocyte cell bodies (arrows). In regions distal from the injury core (inset 3), note the perivascular pattern of AQP4 staining. Scale bars: A,B: 40 μm; C,D,E: 120 μm; F, inset 1, 2, and 3: 10 μm. Asterisk (“*”) denotes injury core. *p < 0.05; **p < 0.001; ***p < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, controlled cortical impact; GFAP, glial fibrillary acidic protein.
FIG. 7.
FIG. 7.
Minocycline treatment reverses seizure profile of AQP4-null mice after CCI. (A) Seizure severity in response to PTZ in wild-type and AQP4-null mice was measured in saline- and minocycline-treated animals subjected to CCI. Minocycline reduced the seizure severity in wild-type and AQP4-null cohorts compared to saline control (**p < 0.001). There was no genotype difference in seizure severity of minocycline-treated animals (p > 0.05). (B) Seizure latency was increased after minocycline treatment in AQP4-null and wild-type mice (analysis of variance treatment effect, **p < 0.001). There was no genotype difference in seizure latency of minocycline-treated animals (p > 0.05). *P < 0.05; **P < 0.001; ***P < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, cpnmtrolled cortical impact; PTZ, pentylenetetrazole.
FIG. 8.
FIG. 8.
Minocycline treatment inhibits microgliosis. (A) Injury volume was quantified and assessed by Nissel staining. Minocycline did not have an effect on injury volume (p > 0.05). In both genotypes, CCI induced an injury volume of ∼40%. (B) Quantification of AQP4-positive cells surrounding injury area. There was no difference in AQP4 immunoreactivity in the wild-type cohort with saline or minocycline treatment (p > 0.05). As expected, there was background AQP4 staining in AQP4-null mice with robust staining in the wild-type cohort (***p < 0.001). (C) Quantification of GFAP-positive cells demonstrated an ∼2-fold difference in GFAP-positive cells in wild-type versus AQP4-null injured mice in both saline- and minocycline-treated animals (**p < 0.001). There was no observed GFAP cell count difference between the saline- and minocycline-treated cohorts (p > 0.05). (D) Quantification of CD11b-positive cells demonstrated a significant increase in CD11b-positive cells in saline-treated wild-type vs. AQP4-null mice (25 ± 6 vs. 45 ± 4 cells/hpf, respectively; **p < 0.001). This difference in genotype was not observed in minocycline-treated mice (11 ± 4 vs. 12 ± 2 cells/hpf, respectively; p > 0.05) given that minocycline-treatment decreased CD11b-positive cells regardless of genotype. *p < 0.05; **p < 0.001; ***p < 0.0001; error bars, ± standard error of the mean. AQP4, aquaporin-4; CCI, controlled cortical impact; GFAP, glial fibrillary acidic protein.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Annegers, J.F., and Coan, S.P. (2000). The risks of epilepsy after traumatic brain injury. Seizure 9, 453–457 - PubMed
    1. Jennett, B., Teather, D., and Bennie, S. (1973). Epilepsy after head injury. Residual risk after varying fit-free intervals since injury. Lancet 2, 652–653 - PubMed
    1. Semah, F., Picot, M.C., Adam, C., Broglin, D., Arzimanoglou, A., Bazin, B., Cavalcanti, D., and Baulac, M. (1998). Is the underlying cause of epilepsy a major prognostic factor for recurrence? Neurology 51, 1256–1262 - PubMed
    1. Iudice, A., and Murri, L. (2000). Pharmacological prophylaxis of post-traumatic epilepsy. Drugs 59, 1091–1099 - PubMed
    1. Teasell, R., Bayona, N., Lippert, C., Villamere, J., and Hellings, C. (2007). Post-traumatic seizure disorder following acquired brain injury. Brain Inj. 21, 201–214 - PubMed

Publication types