Cerebral ischemia/reperfusion injury typically causes the disruption of water homeostasis, and an excess of water accumulates in the brain parenchyma, causing brain edema.1 The serious sequelae of brain edema include deficits in neurological function, increased intracranial pressure, and herniation.2 Currently, there are few therapeutic drugs available to treat this condition. The discovery of aquaporins provides a new basis for understanding the formation and resolution of brain edema, and novel potential therapies targeting aquaporins are of interest in current research.
Aquaporins are homologous water-transporting channel proteins. They provide the major means of water permeability across plasma membranes in secretory and absorptive cells that regulate or require rapid water movement. Among the family of aquaporin proteins, aquaporin 4 (AQP4) is the major water-selective channel in the central nervous system. It is extensively expressed in specialized cells that form the blood-brain and brain-cerebral spinal fluid interfaces, including astrocyte end-feet that surround cerebral blood vessels, ependymal cells, and microvascular endothelial cells.3–6 The dysfunction or damage of AQP4 may be associated with brain edema. Brain ischemia/reperfusion in mice induced an increase in AQP4 expression that coincided with the development of edema.7 Occlusion of the middle carotid artery was followed by changes in AQP4 mRNA that corresponded to the generation and resolution of brain swelling monitored by magnetic resonance imaging.8 Further investigation showed that after an ischemic stroke, mice with a deficit in AQP4 experienced reduced brain edema and less severe neurological deficits compared with wild-type mice.9 In addition, some reports showed that AQP4 participated in the pathogenesis of brain edema in traumatic brain injury10 and brain tumors.11 Further work has shown that protein kinase C (PKC) is implicated in the regulation of AQP4 expression. In cultured astrocytes, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, reduced AQP4 mRNA and inhibited its biological activity.12,13 An in vivo study showed that the activation of PKC relieved brain edema by reducing AQP4 expression.14 Because prolonged pretreatment of cultured astrocytes with PKC activators causes PKC depletion and prevents the subsequent decrease in AQP4 by TPA,13,15 we used this modality in our study.
Propofol (2,6-diisopropylphenol) is an IV anesthetic used extensively in clinical practice. Many reports have suggested that propofol has neuroprotective effects.16–19 However, research is limited on its role in edema. Chen et al.20 showed that propofol ameliorated edema induced by 3 h brain ischemia followed by 24 h reperfusion in rats. Ishii et al.21 used magnetic resonance imaging to illustrate that high-dose propofol inhibited edema induced by focal cerebral ischemia in hyperglycemic rats. Our previous work showed that propofol inhibited both the increase of AQP4 and brain edema in a rat ischemia/reperfusion injury model.22 There is a report showing that propofol regulates the PKC pathway in the brain.23 This suggested the hypothesis that propofol might ameliorate edema by regulating AQP4 expression via the PKC pathway.
In this study, we prepared an oxygen glucose deprivation (OGD)/reoxygenation model of cultured rat astrocytes to investigate the effects of propofol on edema, neuroprotection, AQP4 expression, and the PKC pathway. We found that AQP4 protein expression gradually decreased with OGD over time, and this was reversed and exceeded baseline levels after reoxygenation. Propofol dose-dependently improved cell viability, but only the maximal concentration of propofol (10 μM) significantly decreased AQP4 expression during OGD/reoxygenation, and the effect was reversed by prolonged pretreatment with TPA for 24 h.
The animal protocols were approved by the Zhejiang University Institutional Animal Care and Use Committee.
The effect of increasing time of exposure (1, 2, 4, 6, 8, and 12 h) to OGD on cultured astrocytes was first evaluated to determine a suitable timepoint for the subsequent reoxygenation experiments. An OGD duration of 6 h was identified as suitable for studies, and therefore studies were conducted on the effect of propofol at various concentrations (1, 5, and 10 μM) on AQP4 expression in astrocytes exposed to OGD 6 h/reoxygenation 24 h. To further investigate pathways involved in the effect of propofol on AQP4 in the OGD 6 h/reoxygenation 24 h model, astrocytes were pretreated with TPA for 24 h before OGD.
Primary Astrocyte Cultures
Acute cultures of neocortical astrocytes were prepared from 1- to 2-day-old Sprague Dawley rats, as described by Yamamoto et al.13,24 Briefly, cerebral cortices were isolated, minced with forceps, and passed through a stainless steel mesh. Dissociated cells were centrifuged, resuspended in Dulbecco’s modified Eagle medium (GIBCO BRL, Rockville, MD) containing 10% fetal calf serum (GIBCO BRL), and seeded on poly-l-lysine–coated flasks. Cells were maintained at 37°C and 5% CO2, with a change of medium every 2 days. At confluence (Days 8–10), the flasks were shaken at 200 rpm for 16 h at 37°C. After removal of microglia and oligodendrocytes, the astrocytes were trypsinized, resuspended, and seeded on 6-well plates for experiments. The purity of the astrocytes was >98%, as assessed by immunochemical staining with glial fibrillary acidic protein.
OGD Followed by Reoxygenation
For exposure to OGD, 3 × 105/well astrocytes were washed 3 times with glucose-free phosphate buffered saline, then placed in a preequilibrated glucose-free balanced salt solution (BSS0, pH 7.4), containing phenol red (10 mg/L) and the following (in mM): NaCl 116, CaCl2 1.8, MgSO4 0.8, KCl 5.4, NaH2PO4 1.0, NaHCO3 14.7, and N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid. The astrocytes were then placed into an airtight chamber, bubbled with an anaerobic gas mixture (92% N2, 5% CO2, and 3% O2; CO2 Incubator, Heraeus, Germany) at 37°C, and incubated for 1, 2, 4, 6, 8, and 12 h. After 6 h of OGD exposure, the astrocytes were reoxygenated by washing with a BSS containing 5.5 mM glucose (BSS5.5) and incubated under normoxic conditions for 24 h. The astrocyte cultures subjected to OGD 6 h/reoxygenation 24 h were called control cells. Sham-washed cell cultures not exposed to OGD were placed in the BSS5.5 and aerated with an aerobic gas mix (95% air and 5% CO2) for 10 min.
Propofol at concentrations of 1, 5, and 10 μM, identified in a previous report,17 was added just before the OGD exposure and continued until the astrocyte cultures were collected and analyzed.
The phorbol ester, TPA, was dissolved in water and added to the culture medium at a 1 μM final concentration 24 h before the cells were subjected to OGD/reoxygenation injury.
Cell morphology was assessed under a phase-contrast microscope. In the progressive duration of exposure to OGD, evaluation of astrocyte viability was quantitatively determined by measuring the reduction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and astrocyte injury by measuring the release of cytoplasmic lactate dehydrogenase (LDH) in the OGD 6 h/reoxygenation 24 h model. AQP4 expression was determined by Western blot analysis performed after reoxygenation.
MTT Reduction Test
Astrocyte viability was quantitatively assessed by the MTT reduction test. After exposure to OGD, cultures were incubated with 20 μL MTT (5 mg/mL) at 37°C in culture medium for 4 h. Cultures were then washed and incubated in dimethyl sulfoxide (DMSO) (100 μL). Cell viability was evaluated by optical density, read at 570 nm (background subtracted at 630 nm) with an auto-universal microplate spectrophotometer (Type 3550, BioRad, USA). Results are presented as the percent of the control optical density measured in sham-washed cells.
Astrocyte injury was estimated quantitatively as the amount of LDH released into the culture medium. We used the Cytotoxicity Detection Kit from Roche Diagnostic (Meylan, France). Background LDH concentrations (12%) were determined in sham-washed sister cultures and were subtracted from experimental values to yield the LDH activity that specifically corresponded to experimental injury. Results obtained from cells exposed to 6 h of OGD followed by 24 h of reoxygenation were expressed as the percent of the maximal LDH released by the control cells.
Western Blot Analysis
Western blot analysis was performed to investigate AQP4 expression. The astrocytes were harvested, and protein was extracted. Protein samples (50 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk for 2.5 h at room temperature then incubated with rabbit polyclonal anti-AQP4 (1:500, Chemicon, USA) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (1:1000, Zhongshan Biotechnology Co., Beijing, China) for 2 h at room temperature. Subsequently, immunoreactive signal bands were visualized with enhanced chemiluminescence (ECL Kit, Santa Cruz Biotechnology, USA) and exposed to radiograph film to show the AQP4 band (30 kDa). The integrated densities were analyzed with a computer-assisted image analysis system (Gel-pro Analyzer 4.0, Media Cybernetics, Silver Spring, MD).
Pure propofol (2,6-diisopropylphenol) was obtained from AstraZeneca (Rueil Malmaison, France) and was dissolved in DMSO shortly before use; the final concentration in the culture medium was limited to 0.005–10 μM. Propofol was added at the beginning of OGD exposure, and the final DMSO concentration was <0.1%. The propofol vehicle control experiments contained the same amounts of DMSO and indicated that the vehicle alone did not affect culture activities (data not shown).
Data were expressed as the mean ± sd. Statistical analyses were performed using 1-way analysis of variance followed by the least significant difference or Fisher’s post hoc tests (SPSS 16.0 for Windows, SPSS, Chicago, IL). Differences with a P value <0.05 were considered significant.
The Effects of OGD on Astrocytes
The cultured astrocytes were first identified by positive immunofluorescence staining with glial fibrillary acidic protein. Bright green spots were observed in the cytoplasm under a fluorescence microscope, and the cultured cell bodies were surrounded by numerous apophyses. The purity was ≥98% (Fig. 1).
Astrocyte damage was assessed under the light microscope by an observer blinded to the experimental groups. In the sham-washed group, astrocytes grew vigorously and exhibited different phases of division. They grew in a variety of shapes and strongly refracted light. Numerous apophyses formed close connections between cells (Fig. 2A). After exposure to OGD, the morphology of astrocytes changed (Fig. 2, B and C). Cells became large, round, and swollen. After 6 h of OGD exposure, the ratio of the nucleus to the cytoplasm decreased and light refraction weakened markedly. After 12 h of OGD exposure, most astrocytes appeared shrunken and necrotic (Fig. 2D).
The MTT reduction test closely correlated with the morphological observations. Cell viability decreased with the time of exposure to OGD. Cell viability decreased to about 85% after 4 h of OGD exposure, about 60% after 6 h, and about 10% after 12 h OGD (P < 0.05, Fig. 3).
The Western blot analysis showed that, compared with sham-washed cells, the expression of AQP4 decreased significantly after 6 h of OGD exposure (P < 0.05; Fig. 5, A and B) and was gradually attenuated with OGD exposure time (data not shown).
The Effects of OGD 6 h/Reoxygenation 24 h on Astrocytes
Upon removal from the anaerobic chamber, osmotic swelling of the cell bodies was more apparent in astrocytes exposed to 6 h of OGD. Under the light microscope, we observed obvious astrocyte swelling that was associated with marked accumulation of cellular debris after 24 h of reoxygenation (Fig. 2E). The measurements of LDH release into the culture medium confirmed the morphological assessments. After 6 h of OGD exposure and 24 h of reoxygenation, almost all of the astrocytes died compared with the sham-washed cells (P < 0.01, Fig. 4). The Western blot analysis showed that the AQP4 protein expression level was reversed and exceeded baseline levels dramatically in astrocytes after 6 h of OGD and 24 h of reoxygenation (P < 0.01; Fig. 5, A and B).
The Effects of Propofol on Astrocytes After OGD 6 h/Reoxygenation 24 h Injury
When propofol (10 μM) was added before OGD, we observed a preservation of the number of viable cells under the light microscope (Fig. 2F). Cell swelling was attenuated, and the death of astrocytes was lessened. According to the LDH release test, propofol dose-dependently reduced cell death (P < 0.05, Fig. 4). At propofol 10 μM, the cell death rate was <60% (P < 0.05, Fig. 4).
In Western blot analysis, astrocyte AQP4 protein expression seemed to be inhibited by propofol in a dose-dependent manner after 6 h OGD/24 h reoxygenation, but a significant inhibitory effect was observed at propofol 10 μM (P < 0.01; Fig. 5, A and C).
Effect of the PKC Activator, TPA, on the Effects of Propofol on AQP4 Expression in Astrocytes
To investigate whether the down-regulation of AQP4 expression by propofol was mediated by the PKC pathway, astrocytes were pretreated with TPA (1 μM) for 24 h, then 10 μM propofol was added before the OGD/reoxygenation treatment. The results from the Western blot analysis showed AQP4 expression increased significantly in cells pretreated with TPA compared with the 10 μM propofol alone group (P < 0.01; Fig. 5, A and D).
In this study of acutely cultured astrocytes, we found that cell viability was reduced with increasing exposure to OGD. After 6 h of OGD and 24 h of reoxygenation, significant astrocyte edema and cell death were observed compared with sham-washed cells.
Our results agree with previous reports25 showing that astrocytes could maintain their basic morphology and relatively high cell viability (about 60%) after 6 h of OGD exposure. Therefore, we chose this OGD exposure time for the subsequent reoxygenation studies. We found that the cell death rate significantly increased after 6 h of OGD exposure followed by 24 h of reoxygenation; this was observed under a light microscope and confirmed with the LDH release test. In addition, the Western blot analysis showed AQP4 expression was attenuated after 6 h of OGD exposure, similar to observations reported by Yamamoto et al.24 and Fu et al.,25 and the down-regulation of AQP4 expression was reversed after 24 h of reoxygenation.
The AQP4 protein is regarded as an important molecule for regulating water balance in the brain. It is abundantly expressed in the dense astrocyte processes located in blood-brain and brain-cerebral spinal fluid interfaces and acts as the principal bidirectional pathway for water movement. Thus, AQP4 plays an important role in the development of cytotoxic edema. In view of the pathophysiology that gives rise to cerebral edema, the OGD/reoxygenation injury model in our study imitated cytotoxic edema. With cytotoxicity, the blood-brain barrier is intact and fluid does not escape from the blood vessels. Thus, it is logical to assume that AQP4-rich astrocyte foot processes must be an essential pathway for fluid to enter and accumulate in the brain. However, we found that with mild OGD exposure (6 h), AQP4 expression did not increase substantially and may have even decreased. The result is comparable with that found by Fu et al.25 This may represent a self-protective response for astrocytes to mitigate rapid water influx and maintain basic morphology and cell viability in the face of a mild challenge. Findings from this in vitro model study also support similar clinical observations10,26–30 showing that the peak of edema occurs after the 6-h timepoint, regardless of the duration of ischemia and reperfusion. At that time, the self-protective mechanism gives way, and AQP4 expression is up-regulated to promote an influx of water. Eventually, cytotoxic edema occurs.
Our study indicated that propofol dose-dependently reduced the cell death rate after OGD/reoxygenation, and 10 μM propofol reduced the cell death rate to <60%. Propofol simultaneously reduced cell edema, cell death, and AQP4 up-regulation. Moreover, prolonged pretreatment with TPA (a PKC activator) reversed the effect of propofol on AQP4 expression after OGD/reoxygenation. This suggests that the PKC pathway is involved in the propofol inhibition of AQP4 expression. Our results are consistent with previous studies showing that AQP4 can be regulated by reversible protein phosphorylation.31 PKC was found to be one of the short-term regulators of AQP4 protein. Water permeability was altered after activation of PKC by phosphorylation.13,25,32,33 In a middle cerebral artery occlusion model, the activation of PKC caused the reduction of AQP4 expression; this activation down-regulated water permeability and alleviated ischemia-induced brain swelling.14 However, prolonged pretreatment with a PKC activator eliminated the subsequent decrease in AQP4 protein or its mRNA, because of PKC depletion.13,15 In another study, propofol anesthesia protected myocardial cells from ischemia/reperfusion injury by stimulating the transposition of PKC subunits (PKC-α, PKC-δ, PKC-ε, and PKC-ζ) from the cytoplasm to the cell membrane to activate PKC.34 Our study also demonstrated that the PKC pathway was involved in the effect of propofol on AQP4 expression.
As always, the use of cultured astrocytes exposed to OGD/reoxygenation injury as a model for human ischemia/reperfusion is a study limitation. In addition, the ability of astrocytes to oxidize glutamate as a sign of astrocyte viability was not used in the study.
In conclusion, we found that propofol exerted a protective effect on OGD/reoxygenation-injured astrocytes, and this protection was partly correlated with a down-regulation of AQP4 expression. Moreover, propofol-induced down-regulation of AQP4 expression might be mediated partly by the PKC pathway. Our results confirm and extend previous studies in in vivo ischemia/reperfusion models, in which we showed that propofol could ameliorate brain edema and down-regulate AQP4 levels.
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