Astragaloside IV alleviates post-traumatic cytotoxic edema via inhibition of AQP4 expression and subcellular localization.

The astroglial water channel aquaporin-4 (AQP4) facilitates water movement into and out of brain parenchyma. To investigate the role of AQP4 in meningitis-induced brain edema, Streptococcus pneumoniae was injected into cerebrospinal fluid (CSF) in wild type and AQP4 null mice. AQP4-deficient mice had remarkably lower intracranial pressure (9 +/- 1 versus 25 +/- 5 cm H2O) and brain water accumulation (2 +/- 1 versus 9 +/- 1 microl) at 30 h, and improved survival (80 versus 0% survival) at 60 h, through comparable CSF bacterial and white cell counts. Meningitis produced marked astrocyte foot process swelling in wild type but not AQP4 null mice, and slowed diffusion of an inert macromolecule in brain extracellular space. AQP4 protein was strongly up-regulated in meningitis, resulting in a approximately 5-fold higher water permeability (P(f)) across the blood-brain barrier compared with non-infected wild type mice. Mathematical modeling using measured P(f) and CSF dynamics accurately simulated the elevated lower intracranial pressure and brain water produced by meningitis and predicted a beneficial effect of prevention of AQP4 upregulation. Our findings provide a novel molecular mechanism for the pathogenesis of brain edema in acute bacterial meningitis, and suggest that inhibition of AQP4 function or up-regulation may dramatically improve clinical outcome.

Aquaporin 4 knockdown alleviates traumatic brain edema and reduces neuronal axonal growth cone collapse via the RhoA/ROCK pathway.

Objective To explore the neuroprotective effects of dexmedetomidine (Dex) combined with targeted temperature management (TTM) on brain edema in traumatic brain injury (TBI) mice. Methods A total of 132 male C57BL/6 mice were randomly divided into normal group, sham-operation (Sham) group, traumatic brain injury (TBI) group, targeted temperature management (TTM) group, dexmedetomidine (Dex) group, and combination with TTM and Dex (DT) group, respectively (n=22 per group). The TBI animal model was established by electric controlled cortical impactor (eCCI), and then settled promptly on a mild hypothermic blanket (targeted temperature of (32±1)℃) for 6 h, or intraperitoneal injection of Dex (20 μl/kg) at 0, 2 and 4 h after TBI. Neurological function and spatial learning and memory ability were evaluated by modified neurological severity scores (mNSS) and Morris water maze (MWM), respectively. Then, mice were sacrificed at 24 h after TBI and stained using hematoxylin and eosin (H & E). Additionally, the cerebral edema was evaluated from the water content of the brain tissue using the wet-to-dry weight ratio, and the expression of aquaporin 4 (AQP4) was measured by Western blot assay. Results Compared with the Sham group, TBI mice showed neurologic deficits ((13.2±3.0) vs (0.5±0.7))(P<0.01), spatial learning and memory capacity decline((2.9±1.0) vs (7.4±1.3))( P<0.05), and the brain water content and the expression of AQP4 were increased ((79.81±0.80)% vs (75.98±0.62)%): ((1.60±0.07) vs (0.73±0.03))(all P<0.01). However, Dex or TTM could improve the neurological function(Dex: (10.3±2.8), TTM: (10.0±2.9)), reduce the brain water content(Dex: (78.50±0.40)%, TTM: (78.10±0.45)%), and significantly decrease the expression of AQP4 compared with the TBI group(Dex: (1.40±0.04), TTM: (1.15±0.12)) (all P<0.05), especially that of DT group ((9.2±2.5) , (5.4±1.8) , (77.67±0.30)%, (0.93±0.09)) (all P<0.01). Conclusion These finding suggests that Dex combined with targeted temperature management can reduce cerebral edema and improve neurological outcome in mice after TBI to exert neuroprotection effect, which may be related with the down-regulation of AQP4 protein. Key words: Traumatic brain injury; Dexmedetomidine; Targeted temperature management; Brain edema

Paeoniflorin (PF) and astragaloside IV (AS-IV) have protective effects on cerebral ischemia. We aimed to test the effects of combined use of PF and AS-IV on ischemic brain edema and investigate whether the effects were dependent on connexin43 (Cx43). We detected the expression of Cx43 induced by PF and AS-IV after cerebral ischemia. We also examined the effects of combined use of PF and AS-IV on ischemic edema and further investigated the related pathways. We demonstrated PF and AS-IV decreased Cx43 and aquaporin4 (AQP4) associating with reduction of brain edema by dry-wet weight and brain-specific gravity methods after cerebral ischemia. Administration of PF and AS-IV displayed a further attenuation of brain edema with lower Cx43 levels. Meanwhile, Cx43 blockade inhibited AQP4 down-regulation by the two drugs. Moreover, phosphorylation of C-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) were increased by PF and AS-IV, respectively. The effects of PF and AS-IV to down-regulate Cx43 were suppressed by JNK and ERK inhibitors, respectively. Our data indicate that PF and AS-IV alleviate ischemic brain edema, which has close relation to Cx43 down-regulation causing decrease of AQP4 via JNK and ERK pathways activation, respectively. Combined administration elicits synergistic effects on brain edema reduction. Copyright © 2017 John Wiley & Sons, Ltd.

Alignment of human aquaporin 4 and ß‐amyloid proteins may indicate involvement of ß‐amyloid in brain water homeostasis and prevention of brain edema

Spinal cord edema, mainly including vasogenic and cytotoxic edema, influences neurological outcome after spinal cord contusion (SCC). Aquaporin 4 (AQP4) is the most ubiquitous water channel in the central nervous system (CNS), which is a rate-limiting factor in vasogenic edema expressing in brain injury, and it contributes to the formation of cytotoxic edema locating in astrocytes. However, little is known about the regulatory mechanism of AQP4 within vasogenic and cytotoxic edema in SCC, and whether the regulation mechanism of AQP4 is related to Cytochrome coxidase (COX5A) affecting energy metabolism. Therefore, the SCC model is established by Allen’s method, and the degree of edema and neuronal area is measured. The motor function of rats is evaluated by the Basso, Beattie, and Bresnahan (BBB) scoring system. Meanwhile, AQP4 and COX5A are detected by real-time quantitative PCR (qRT-PCR) and western blot (WB). The localization of targeted protein is exhibited by immunohistochemical staining (IHC) and immunofluorescence (IF). Additionally, the methodology of AQP4 lentivirus-mediated RNA interference (AQP4-RNAi) is used to reveal the effect on edema of SCC and the regulating molecular mechanism. Firstly, we observe that the tissue water content increases after SCC and decreases after the peak value of tissue water content at 3 days (P < 0.05) with abundant expression of AQP4 protein locating around vascular endothelial cells (VECs), which suggests that the increasing AQP4 promotes water reabsorption and improves vasogenic edema in the early stage of SCC. However, the neuronal area is larger than in the sham group in the 7 days (P < 0.05) with the total water content of spinal cord decrease. Meanwhile, AQP4 migrates from VECs to neuronal cytomembrane, which indicates that AQP4 plays a crucial role in aggravating the formation and development of cytotoxic edema in the middle stages of SCC. Secondly, AQP4-RNAi is used to elucidate the mechanism of AQP4 to edema of SCC. The neuronal area shrinks and the area of cytotoxic edema reduces after AQP4 downregulation. The BBB scores are significantly higher than in the vector group after AQP4-RNAi at 5, 7, and 14 (P < 0.05). There is a relationship between AQP4 and COX5A shown by bioinformatics analysis. After AQP4 inhibition, the expression of COX5A is significantly upregulated in the swelling astrocytes. Therefore, the inhibition of AQP4 expression reduces cytotoxic edema in SCC and improves motor function, which may be associated with upregulation of COX5A via affecting energy metabolism. Moreover, it is not clear how the inhibition of AQP4 directly causes the upregulation of COX5A.

Background/Aims: Traumatic brain injury (TBI) is a complex neurological injury in young adults lacking effective treatment. Emerging evidences suggest that inflammation contributes to the secondary brain injury following TBI, including breakdown of the blood brain barrier (BBB), subsequent edema and neurological deterioration. High mobility group box-1 (HMGB1) has been identified as a key cytokine in the inflammation reaction following TBI. Here, we investigated the therapeutic efficacy of HMGB1 A-box fragment, an antagonist competing with full-length HMGB1 for receptor binding, against TBI. Methods: TBI was induced by controlled cortical impact (CCI) in adult male mice. HMGB1 A-box fragment was given intravenously at 2 mg/kg/day for 3 days after CCI. HMGB1 A-box-treated CCI mice were compared with saline-treated CCI mice and sham mice in terms of BBB disruption evaluated by Evan’s blue extravasation, brain edema by brain water content, cell death by propidium iodide staining, inflammation by Western blot and ELISA assay for cytokine productions, as well as neurological functions by the modified Neurological Severity Score, wire grip and beam walking tests. Results: HMGB1 A-box reversed brain damages in the mice following TBI. It significantly reduced brain edema by protecting integrity of the BBB, ameliorated cell degeneration, and decreased expression of pro-inflammatory cytokines released in injured brain after TBI. These cellular and molecular effects were accompanied by improved behavioral performance in TBI mice. Notably, HMGB1 A-box blocked IL-1β-induced HMGB1 release, and preferentially attenuated TLR4, Myd88 and P65 in astrocyte cultures. Conclusion: Our data suggest that HMGB1 is involved in CCI-induced TBI, which can be inhibited by HMGB1 A-box fragment. Therefore, HMGB1 A-box fragment may have therapeutic potential for the secondary brain damages in TBI.

Brain edema is a major traumatic brain injury (TBI)-related neurological complication. In the initiation stage of TBI, brain edema is characterized by astrocyte swelling (cytotoxic edema). We studied the impact of a long noncoding RNA, Malat1, on the TBI-induced astrocyteswelling and brain edema. Our results showed thatMalat1 was downregulated in both the TBI rat model and the astrocytefluid percussion injury (FPI) model, which concurred with brain edema and astrocyteswelling. Overexpression of Malat1 significantly inhibited rat brain edema, meanwhile reducing interleukin-6 (IL-6), nuclear factor-κB (NF-κB), and aquaporin 4 (AQP4) expression after TBI. In addition, overexpression of Malat1 ameliorated FPI-induced astrocyteswelling and reduced IL-6 release. Quantitative real-time polymerase chain reaction and Western blot analysis also corroborated the inhibitory effects of Malat1 on NF-κB and AQP4 expression after FPI. Our results highlighted the protective effects of Malat1 on the TBI-induced brain edema, which were mediated through regulating IL-6, NF-κB, and AQP4 expression. Our study could provide a novel approach for TBI treatment.

Astragaloside IV protects blood-brain barrier integrity from LPS-induced disruption via activating Nrf2 antioxidant signaling pathway in mice

Increased cranial pressure due to development of edema contributes significantly to the pathology of traumatic brain injury (TBI). Induction of an astrocytic water channel protein, Aquaporin 4 (AQP4), is known to predominantly contribute to cytotoxic edema following TBI. However, the mechanism for the increase in AQP4 following 24 h of TBI is poorly understood. Here we show that transcriptional activation of a ubiquitously expressed mammalian forkhead transcription factor, Foxo3a, induces cerebral edema by increasing the AQP4 level in the controlled cortical impact model of TBI in mice. TBI stimulates nuclear translocation of Foxo3a in astrocytes and subsequently augments its binding to AQP4 promoter in pericontusional cortex. Nuclear accumulation of Foxo3a is augmented by a decrease in phosphorylation at its Ser256 residue due to inactivation of Akt after TBI. Depletion of Foxo3a in mice rescues cytotoxic edema by preventing induction of AQP4 as well as attenuates memory impairment after TBI in mice.

Transgenic mice overexpressing endothelin-1 (ET-1) in astrocytes (GET-1) displayed more severe brain edema and neurologic dysfunction after experimental ischemic stroke. However, it was not clear whether astrocytic ET-1 contributed to cytotoxic or vasogenic edema associated with stroke. In this study, the role of astrocytic ET-1 in cytotoxic edema and brain injury was investigated. Upon acute water intoxication, the GET-1 mice had a lower survival rate and more severe neurologic deficits. Such an exacerbated condition in the GET-1 mice may be a result of a significant increase in cerebral water content and increased expression of the water channel protein, aquaporin 4 (AQP-4). The GET-1 mice treated with OPC-31260, a nonpeptide arginine vasopressin V(2) receptor antagonist, were alleviated from the cerebral water accumulation and neurologic deficit during the early time period after water intoxication. In addition, a significant reduction of AQP-4 expression was observed in astrocytic end-feet AQP-4 in the hippocampus of the GET-1 mice treated with OPC-31260. Therefore, ET-1-induced AQP-4 expression and cerebral water accumulation are the key factors in brain edema associated with acute water intoxication. The V(2) receptor antagonist, OPC-31260, may be one of the effective drugs for the early treatment of ET-1-induced cytotoxic edema and brain injury.

Objective To investigate the effect of hypertonic saline on the expressions of aquaporin 4 (AQP4) and caspase-3 in the brain edema area after traumatic brain injury (TBI) in rats Methods Seventy-two male SD rats weighing 220-250 g were selected and randomly divided into three groups (24 rats per group): sham operation group (Group A), traumatic brain injury + normal saline group (Group B) and traumatic brain injury + hypertonic saline group (Group C). Moderate TBI model was induced by Feeney's free falling method. Normal saline and hypertonic saline were delivered respectively. The neurological score was measured at 6, 24, and 48 hours after operation. The brain water content was measured, and the blood brain barrier stability was detected by Evans blue staining. AQP4 positive cells was detected by immunohistochemistry. The expressions of AQP4 and caspase-3 protein in brain tissue were detected by Western blot, and the apoptosis of neurons in brain tissue by TUNEL method. Results Compared with Group A, the neurological score of Group B were obviously decreased, while the water content in the brain tissue, Evans blue staining, AQP4 positive cells, AQP4(6 hours: 1.73±0.31 vs. 0.33±0.13; 24 hours: 2. 47±0. 27 vs. 0. 33±0. 14; 48 hours: 2.18±0.19 vs. 0.33±0.12), caspase-3 protein expression(6 hours: 0.53±0.18 vs. 0.34±0.07; 24 hours: 0.58±0.16 vs. 0.33±0.08; 48 hours: 0.59±0.11 vs. 0.33±0.07) and apoptosis index in brain tissue in Group B were significantly increased (all P<0.05). Compared with Group B, the neurological score of Group C were obviously increased, while the water content in the brain tissue, Evans blue staining, AQP4 positive cells, AQP4 (6 hours: 1.51±0.27 vs. 1.73±0.31; 24 hours: 2.13±0.13 vs. 2.47±0.27; 48 hours: 1.84±0.22 vs. 2.18±0.19) and Caspase-3 protein expression (6 hours: 0.44±0.09 vs. 0.53±0.18; 24 hours: 0.46±0.10 vs. 0.58±0.16; 48 hours: 0.48±0.12 vs. 0.59±0.11) and apoptosis index in brain tissue of Group C were significantly decreased (all P<0.05). Conclusion Hypertonic saline can attenuate TBI-induced brain edema and have a significant neuroprotective effect, possibly by down-regulating the expressions of AQP4 and caspase-3. Key words: Brain injuries; Saline solution, hypertonic; Brain edema; Aquaporin 4; Caspase-3

Brain edema is a complication of radiation used to treat brain metastasis (BM) in which the brain parenchyma accumulates fluid and ions, often leading to the suspension of systemic anticancer treatment. While brain edema is often attributed to disruption of the Blood Brain Barrier (BBB), RTx induces cytotoxic-edema, a premorbid cellular process whereby extracellular Na+ and other cations enter into neurons and astrocytes and accumulate intracellularly, resulting in osmotic expansion of the cells and necrotic cell death. Aquaporin 4 (AQP4) is a main regulator of osmotic expansion (water intake) in astrocytes and we have shown that RTx upregulates AQP4 in astrocytes and leads to astrocytic swelling in vitro. However, whether pharmacological modulation of AQP4 could be used to prevent cytotoxic brain edema in RTx-treated BM and its impact on metastatic tumor progression remains unknown. Goal: To determine if the FDA-approved drug Topiramate (TPM), an anti-epileptic drug able to inhibit AQP4) can prevent astrocytic swelling in vitro, reduce RTx-induced brain edema and modulate brain metastatic progression. Results: Electron microscopy of brain cortex from mice treated with 35 Gy RTx showed acute astrocytic end-feet swelling and increase in AQP4 expression compared with non-irradiated mice. A single 8 Gy dose increased astrocytic cell area of human astrocytes by 4.8 fold compared with non-irradiated cells 24 h after RTx. This increased cell-swelling did not result from senescence-associated cellular hypertrophy, as staining of senescent β-galactosidase positive (SA-β-Gal+) cells showed that Rtx-induced astrocytic area only increased significantly in non-senescent (SA-β-Gal- cells). shAQP4s reduced AQP4 levels by 60% and 50%, respectively, and significantly reduced RTx-induced astrocytic swelling. Since there are no FDA-approved AQP4 inhibitors, we tested whether the AQP4-blocking function of TPM could be sufficient to prevent cytotoxic edema, prevent BBB dysfunction and protect from necrotic cell death in vitro. TPM pretreatment did not alter radiation-induced ERK1/2 or AKT activation (a known maker of radioprotection) in astrocytes, but TPM decreased radiation-induced PARP-cleavage, pP38 and pJNK levels. TPM prevented loss of Trans-electric epithelial resistance (TEER) of Rtx-treated astrocytes, but was not able to protect astrocytes from ultimate cell death. Immunohistochemical analysis of a cohort of breast cancer BM showed heterogeneous AQP4 expression in cancer cells ranging from 1.6% to 91% AQP4+ tumoral areas and from 0.6% to 86.9% in stroma. AQP4 inhibition using shRNAs decreased proliferation and survival of AQP4 + 231BR, and EO711 cells in vitro. However, TPM did not alter survival of AQP4+ or AQP4- cells in vitro, suggesting that while AQP4 expression is important for survival of AQP4+ cells in vitro, the inhibition of AQP4 function by TPM is not sufficient to decrease their growth. To determine if TPM could decrease brain edema without negatively impacting tumor progression, female NSG mice were injected intracardially with JmT1BR3 AQP4-cells and ten days later randomized to (1) RTx + vehicle, (2) RTx + TPM (2 days prior to irradiation), (3) Non-RTx + vehicle, and (4) Non-RTx + TPM. TPM decreased brain-water content (a marker of brain edema) in irradiated mice as compared with vehicle-treated mice, without alteration of metastatic burden 21 days post-injection. However, a similar study using AQP4+ E0711 cells in C57Bl6 mice showed TPM was less effective in decreasing brain water content and resulted in a significant increase in extracranial metastatic tumor burden, suggesting that TPM can promote tumor progression by non-tumor intrinsic mechanisms. Conclusions: while TPM shows promise in preventing RTx-induced brain edema, our results show a potential pro-tumorigenic mechanism for TPM that warrants further investigation. Citation Format: Maria J. Contreras-Zarate, Karen ALvarez-Eraso, Nicole Tsuji, Peter Kabos, D.Ryan Ormond, Sana Karam, Diana Cittelly. AQP4 inhibition prevents cytotoxic edema of AQP4+ astrocytes but promotes tumor growth of AQP4+ breast cancer brain metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-10-09.

The traumatic penumbra (TP) is a secondary injury zone surrounding the core area of traumatic brain injury (TBI) and plays a critical role in determining TBI outcomes. The primary pathological change in the TP is brain edema, which includes both vasogenic and intracellular edema. Brain edema is closely associated with the expression of aquaporin-4 (AQP4). Bevacizumab, a vascular endothelial growth factor (VEGF) inhibitor, reduces vascular permeability. However, the effect of bevacizumab on traumatic brain edema remains unclear, as does its potential molecular mechanism in relation to AQP4. To examine the pathological changes and alterations in AQP4 expression following bevacizumab treatment in the TP. A total of 70 Wistar rats were randomly divided into five groups: a control group, a sham group, a TBI group, a TBI + normal saline group, and a TBI + bevacizumab group. Twenty-four hours after treatment, TP tissue samples were collected for analysis. Histopathological structural changes were examined using hematoxylin-eosin staining and transmission electron microscopy. Western blot was employed to assess changes in AQP4 protein levels, and double-labeling immunofluorescence was utilized to observe the AQP4 and VEGF. After TBI, the primary pathological changes in the TP were characterized by cerebral edema, which included both vasogenic and intracellular edema. Bevacizumab treatment reduced both types of edema. After TBI, AQP4 and VEGF expression in the TP was upregulated, and depolarization of AQP4 distribution was observed. However, bevacizumab treatment led to a downregulation of AQP4 and VEGF expression, and suppression of the AQP4 depolarized distribution. Bevacizumab alleviates cerebral edema in the TP after TBI by modulating the expression and polarized distribution of AQP4. Thus, bevacizumab may serve as a potential therapeutic agent for the clinical treatment of TP.

Traumatic brain injury (TBI) is one of the leading causes of mortality and permanent disabilities worldwide. Brain edema following TBI remains to be the predominant cause of mortality and disability in patients worldwide. Previous studies have reported that brain edema is closely associated with aquaporin-4 (AQP4) expression. AQP4 is a water channel protein and mediates water homeostasis in a variety of brain disorders. In the current study, a rat TBI model was established, and the features of brain edema following TBI were assessed using multimodal MRI. The results of the multimodal MRI were useful, reliable and were used to evaluate the extent and the type of brain edema following TBI. Brain edema was also successfully alleviated using an intracerebral injection of AQP4 small interfering (si)RNA. The expression of AQP4 and its role in brain edema were also examined in the present study. The AQP4 siRNA was demonstrated to downregulate AQP4 expression following TBI and reduced brain edema at the early stages of TBI (6 and 12 h). The current study revealed the MRI features of brain edema and the changes in AQP4 expression exhibited following TBI, and the results provide important information that can be used to improve the early diagnosis and treatment of brain edema.