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Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat
Molecular Brain Research 78 (2000) 131–137 www.elsevier.com / locate / bres
Research report
Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat a,b a,b , a b Masaaki Taniguchi , Toshihide Yamashita *, Eiji Kumura , Michio Tamatani , c c a a Akihiro Kobayashi , Takashi Yokawa , Motohiko Maruno , Amami Kato , Takanori Ohnishi a , Eiji Kohmura a , Masaya Tohyama b , Toshiki Yoshimine a a
b
Department of Neurosurgery, Osaka University Medical School, 2 -2 Yamadaoka, Suita, Osaka 565 -0871, Japan Department of Anatomy and Neuroscience, Osaka University School of Medicine, 2 -2 Yamadaoka, Suita, Osaka 565 -0871, Japan c Basic Research Institute, Nihon Schering K.K., Osaka, Japan Accepted 4 April 2000
Abstract Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4–mRNA is increased in the peri-infarcted cortex during the observation period (|7 days) after MCA-occlusion, maximally on day 3. The change corresponds to the generation and resolution of brain edema monitored by MRI. The signals for the mRNA are predominantly observed in glial cells in the molecular and outer granular layer of the peri-infarcted cortex. These results indicate that AQP4 plays a role in post-ischemic edema formation. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Uptake and transporters Keywords: Middle cerebral artery occlusion; Aquaporin-4; Brain edema
1. Introduction The brain water-content increases under various pathological conditions, which may cause diverse sequelae on the remaining neuronal function. Extensive edema formation is triggered after an ischemic insult. Brain edema is classified into two categories: Cytotoxic edema, resulting from disturbances in cell metabolism without disruption of the blood brain barrier (BBB), and vasogenic edema, which is caused by breakdown of the BBB to macromolecules [16]. In post ischemic brain edema, the cytotoxic edema
Abbreviations: AQP4, aquaporin-4; EDTA, ethylenediaminetetraacetate; MCA, middle cerebral artery; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; VSOAC, volume-sensitive organic osmolyte / anion channel *Corresponding author. Tel.: 181-6-6879-3652; fax: 181-6-68793659. E-mail address: [email protected] (T. Yamashita)
occurs first followed by the vasogenic edema [9,18]. The early astroglial swelling is seen prominently at processes around neurons and capillaries [15,25]. Under lactoacidosis, intracellular pH decreases by exchange of H 1 and bicarbonate ions against extracellular Na 1 and Cl 2 via the antiporters [27]. Uptake of extracellularly accumulated glutamate via the specific transporter also results in cotransporting Na 1 ions intracellularly [1,3]. This Na-shift into the cell and concomitant water influx causes swelling of the glial cell. The swelling under lactoacidosis may be considered as a self-protecting event to regulate the intracellular pH. Uptake of mediator compounds by glial cell may be considered as the housekeeping effect for the ideal environment to protect the neuron. To maintain these glial functions and to make the intracellular uptake of Na 1 feasible, osmotic gradient between plasma membrane has to be quickly canceled out. To enable this, specific route for robust shift of water across plasma membrane should be mandatory. In vasogenic brain edema, water movement across the
0169-328X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00084-X
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BBB into brain tissue presumably follows the Starling equation [14]. Many factors modify the edema process, including cerebrovascular permeability, capillary hydraulic conductivity, tissue compliance and conductivity as well as hydrostatic and osmotic pressure gradients. During the genesis of vasogenic edema, ion exchanges through the BBB are achieved by simple diffusion. The resulting increase in cations together with the macromolecules then generates osmotic forces which cause a net increase in water in the extracellular space [23]. Extravasated compounds with different molecular weights and diffusion coefficients travel together as a result of hydrostatic pressure. Once the fluid is in the brain extracellular space, further advancement of the edema front is primarily accomplished by bulk flow [17]. As the ‘pure’ form of cytotoxic or vasogenic edema is unlikely to exist, the water, cations and various mediators thus accumulating in the extracellular space are amenable to further shift into the glia via the mechanism mentioned above to augment astrocytic swelling [29]. The concomitant water influx into the extra- as well as intracellular space reaches its peak around 1–3 days after an ischemic insult and diminishes thereafter [5,20]. Though complete resolution of the edema takes more than a week, the osmotic gradient between brain and blood is not parallelly changed with the edema. Different studies provided an evidence of osmolality increase in the brain tissue under focal cerebral ischemia [8,24]. Their common finding was that the increase in osmolality was apparent only during the early stage of ischemia, indicating the presence of a mechanism enabling the robust water shift into the brain extracellular as well as intracellular space, which cancels out the increase in osmotic gradient. The aquaporins are a water-selective channel family consisting of as yet known seven members expressed in the variety of tissues, of which the type 4 (AQP4) is mainly expressed in the brain [7,12]. Its localization to the astroglia on the brain surface and subependymal lining, as well as in osmoreceptor region and adjacent to intracerebral vasculature indicates close functional relationship to the water balance regulation [12], and considered as a candidate for major pathway of a massive water shift across plasma membrane under various condition [22]. We show here that the transcription of AQP4 drastically changes after focal cerebral ischemia, suggesting the involvement in the evolution and resolution of brain edema.
statically-controlled heating pad. The left MCA was exposed with a modified procedure of the method described by Tamura et al. [28]. Briefly, after a 2 cm vertical incision was made over the temporalis muscle, the muscle was split and its insertion into the mandible divided to reach the infratemporal fossa to expose the base of the skull from the foramen ovale to the foramen opticum. While preserving the zygoma, a drill was used to perform a small subtemporal craniectomy 2–3 mm rostral to the foramen ovale in order to expose the proximal part of the MCA, which is located under the dura. A 27-gauge needle was used to open the dura. The MCA was then irreversibly occluded by electrocoagulation over a distance of 2 mm, starting proximally at the medial margin of the olfactory tract. In sham-operated controls, the MCA was exposed but not coagulated (n54). Blood gases, pH, and mean arterial blood pressure (MABP) were monitored throughout the operation. Experiments were completed only if these physiological variables remained within normal limits. The normal values for MABP were set at 90–110 mmHg, those for Paco 2 at 30–50 mmHg, for Pao 2 at 80–110 mmHg, and for arterial blood pH at 7.25–7.45. After recovery from anesthesia, the animals were returned to their cages and allowed ad libitum normal rat chow and water. The rats were divided into two groups: in one group the MRI was acquired after 1, 3, and 7 days following MCAocclusion and the unilateral cortex was subjected for northern blot analysis. In the other group, the animals were deeply anesthetized by intraperitoneal injection of pentobarbital (60 mg / kg) at 1, 3, and 7 days after MCAocclusion and their brains were removed and quickly frozen at 2808C (n55, 5 and 4 for each time point per group, respectively). Serial coronal sections (8–10 mm thick) were obtained from the frozen brains with a cryostat and stored in a tightly closed case at 2808C.
2.2. MRI acquisition To elucidate the edema formation, brain MR images were obtained at 1, 3 and 7 days after MCA-occlusion (n52 for control, n54 for 1, 3, 7 days) using Omega CSI-2 system equipped with a 4.7-T magnet (Bruker GENMR Instruments, Fremont, CA, USA). The T2-weighted sagittal image was first obtained for positioning. Then, four slices of coronal brain spin-echo T2-weighted images were obtained with repetition time / echo time51000 / 80 ms, slice thickness of 2.5 mm, FOV of 30 mm and 1283128 pixels.
2. Materials and methods
2.3. Northern blot analysis
2.1. Animals with focal ischemia
The entire cortex of the affected side including ischemic tissue samples was dissected out from the brains of rats with MCA-occlusion at 1, 3 and 7 days (n54 for each time interval), as well as from sham-operated controls (n53), and immediately frozen at 2808C. Total RNA was isolated
Male Wistar rats weighing 250–300 g were anesthetized with intraperitoneal chloral hydrate (300 mg / kg). Rectal temperature was maintained at 36.5–37.58C with a thermo-
M. Taniguchi et al. / Molecular Brain Research 78 (2000) 131 – 137
by the acid guanidinium isothiocyanate method as described previously [4]. Aliquots (20–40 mg) of RNA were separated on 1% agarose formaldehyde gels and was transferred onto nylon membranes (Immobilion TM -N; Millipore Corp., Bedford, MA). A 385 bp rat AQP4 cDNA which was 93% identical to the published rat AQP4 cDNA (bases 21–405) [7,12] and a rat beta-actin oligonucleotide (Clontech Laboratories Inc., Palo Alto, CA) were labeled by random priming (Amersham Corp., Arlington Heights, IL) with [a-32P]dCTP (3000 Ci / mmol; Amersham). Hybridization with AQP4 probes was carried out at 688C and 1 h in ExpressHybE hybridization solution (Clontech). The blots were washed two times at room temperature for 30 min in 23SSC and 0.05% SDS, one time at 508C for 40 min in 0.13SSC and 0.1% SDS. The membranes were placed in contact with X-Ray film at 2808C for 1 day using intensifying screens. After quantification of the hybridized probe, it was removed from the membrane to be hybridized with the beta-actin probe. The hybridized probe was removed in a boiling solution of 0.5% SDS for 10 min and allowed to cool to room temperature. After the removal of the AQP4 probe, hybridization and washing for the beta-actin probe was carried out at 378C and room temperature, respectively, in the same manner. Finally, X-Ray film was placed on the membrane at 2808C for 16 h.
2.4. In situ hybridization The antisense and sense probe for AQP4 was synthesized from a 385 bps rat AQP4 cDNA (bases 21–405) insert cloned in the pGEM-T vector (Promega Corporation, Madison, WI). To synthesize hybridization riboprobes by in vitro transcription, these sequences were first linearized by digestion with restriction endonucleases of Sal 1 for antisense RNA and of Nco 1 for sense RNA synthesis. The linearized cDNA was then incubated at 378C for 60 min with a mixture of reagents. This mixture consisted of 2 ml of transcription buffer (35), 0.25 ml of 100 mM dithiothreitol, 0.25 ml of RNase inhibitor, 0.25 ml of 10 mM ATP, CTP and GTP, 2.5 ml of [ 35 S]UTP (NEG-039H, New England Nuclear), 1 ml of DNA template (1 mg / ml) with 1 ml of appropriate RNA polymerase (T7 RNA polymerase for antisense probe; SP6 RNA polymerase for sense probe). DNA digestion was achieved by the addition of 2 ml of DNase and incubation at 378C for 20 min. Efficacy of labeling was estimated by quantification of radioactivity of the synthesized probes. In situ hybridization techniques for AQP4–mRNA (RNA probe) were based on those of Wilkinson et al. [30] with some modifications. The sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 min. After washing with PB, the sections were treated with 10 mg / ml of proteinase K in 50 mM Tris–HCl and 5 mM EDTA (pH 8.0) for 30 s at room temperature. They were fixed again in the same fixative, then acetylated with acetic
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anhydride in 0.1 M triethanolamine, rinsed with PB, dehydrated and air-dried. The 35 S-labeled RNA probes (sense or antisense) were diluted in hybridization buffer, placed over the sections and covered with siliconized coverslips. Hybridization was performed overnight in a humid chamber at 558C. The hybridization buffer consisted of 50% deionized formamide, 0.3 M NaCl, 20 mM Tris– HCl, 5 mM EDTA, 10 mM PB, 10% dextran sulfate, 13Denhardt’s solution, 0.2% sarcosyl, 500 mg / ml yeast tRNA, and 200 mg / ml Salmon Testis DNA (pH 8.0). The probe concentration was 5310 5 cpm / 200 ml per slide. After hybridization, the slides were immersed in 53SSC at 558C, and the coverslips were allowed to fall off. The sections were then incubated at 658C in 50% deionized formamide with 23SSC for 30 min. After rinsing with RNase buffer [0.5 M NaCl, 10 mM Tris–HCl, 5 mM EDTA (pH 8.0)] three times for 10 min each time at 378C, the sections were treated with 1 mg / ml of RNase A in RNase buffer for 10 min at 378C. After an additional wash in RNase buffer, the slides were incubated in 50% formamide with 23SSC for 30 min at 658C, rinsed with 23SSC and 0.13SSC for 10 min each at room temperature, dehydrated in an ascending alcohol series and airdried. X-Ray film was placed on the uncoated sections for 6 days. Next, the slides were coated with Ilford K-5 emulsion diluted in distilled water containing 2% glycerine (1:1). The slides were exposed for 3 weeks in a tightly sealed dark box at 48C, developed in Kodak D-19, fixed with photographic fixer, stained with hematoxylin, and coverslipped. After the X-Ray macroautoradiogram had been studied, the tissue sections were examined under a regular light microscope. For quantitative assessment of AQP4–mRNA expression on the macroautoradiograms, the optical density of the peri-infarcted cortex was measured. The film was first stored digitally and density was measured with the image analyzing software (NIH image ver. 1.6). Two areas adjacent to the infarction were measured (Fig. 5). After the background density was subtracted, the counts were used for further analysis. Optical density ratio (ODR) of the peri-infarcted cortex was calculated in comparison with the relevant area on the non-affected hemisphere. Statistical significance of results was determined using the nonparametric analysis of the Mann–Whitney U-test with two-tailed probability.
3. Results We first determined brain edema formation after cerebral ischemia using MR images. Typical MR images at each timepoint are shown in Fig. 1. The T2-prolonged region was already detected on the first day after MCA-occlusion at the left hemisphere MCA territory. The signal intensity increased until 3 days and became inhomogeneous at 7
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Fig. 1. Representative MRI images of rats on 1 (a), 3 (b) and 7 (c) days after MCA-occlusion. The focal ischemic lesion is clearly depicted as high intensity area in the left hemisphere, which is most prominent and largest on day 3. On day 7, the high intensity region became confound and inhomogenious.
days after arterial occlusion. The volume of high intensity region also increased until 3 days but rather confound at cerebral cortex at 7 days post-occlusion. As increase of signal intensity in T2-weighted MRI indicates an increase in water content within the damaged brain tissue [13,21], the post-ischemic brain edema developed already on the first day after MCA-occlusion, which became most prominent 3 days and diminished but persisted 7 days postischemia. To examine whether AQP4 is implicated in the pathological condition of focal ischemia, we first monitored expression of AQP4–mRNA after focal cerebral ischemia. Total RNA was isolated from the ipsilateral and contralateral cortex of the affected side following MCAocclusion, and hybridized with the rat AQP4 cDNA probe. Fig. 2 shows a Northern blot revealing the time course of changes in cortical AQP4–mRNA expression after MCAocclusion. The main band was clearly seen with a size of 5.5 kilobases, which is consistent with other reports [7,12]. The amount of this transcript was increased at the affected left hemisphere on 3 days after MCA-occlusion. AQP4 expression continued to be augmented but in a lesser degree on 7 days after surgery. Localization of AQP4–mRNA was assessed by in situ hybridization using a [ 35 S]-labeled cRNA probe. Specificity of hybridization signals was confirmed in a control study using the sense cRNA probe (Fig. 3c). Signals were observed only in the sections hybridized with the antisense probes. In sham-operated animals, expression of AQP4– mRNA was detected on the brain surface, ependymal lining and at suprachiasmatic and paraventricular nucleus (Fig. 3a, b). In animals with MCA-occlusion, the increased expression of AQP4–mRNA was weakly noticeable at 1 day in the peri-infarcted cortex of the affected side (Fig. 4). The signal increase became most prominent and widespread on 3 days after MCA-occlusion. Seven days after surgery, the increased signal was still noticeable in the peri-infarcted cortex but in a lesser degree than day 3. This signal increase, however, was statistically significant on day 3 and 7, but not on day 1, according to the ODR analysis (Fig. 5). No signals were observed in the core of
the infarct at any time during the observation period. Microautoradiography demonstrated marked induction of AQP4–mRNA signals mainly in cells with medium-sized nuclei in peri-infarcted cortex, most abundant in molecular and outer granular layer, suggesting that these AQP4– mRNA-positive cells are morphologically non-neuronal cells and the upregulation of the gene expression in these areas was mainly due to the increase in glial AQP4 expression (Fig. 6).
Fig. 2. Northern blot analysis of AQP4–mRNA-induction in the ischemic cortex following left MCA-occlusion (each blot represents tissue specimen from single animal). The main band was clearly seen at a size of 5.5 kilobases (kb). The level of this transcript was markedly increased in the left hemisphere on 3 days after surgery. Increased AQP4 expression continued until day 7 but in a lesser degree.
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Fig. 3. Macroautoradiogram of in situ hybridization for AQP4–mRNA. Antisense probe (a, b) and sense probe (c) were hybridized with the tissue sections of sham-operated controls. Signals were observed only in sections hybridized with the antisense probe (a). Moderate expression of AQP4–mRNA was detected on the brain surface, ependymal lining and at suprachiasmatic and paraventricular nuclei.
4. Discussion During the early stage of ischemia, lactic acid leaked out from necrotic tissue accumulates in the surrounding penumbra zone. In addition, incomplete ischemia promotes anaerobic glycolysis and further advance the lactic
acidosis. This leads to formation of carbonic acid, which dissociates immediately into CO 2 and water. The CO 2 enters the cell freely and converted to H 2 CO 3 . The resulting H 1 and bicarbonate ions are exchanged against extracellular Na 1 and Cl 2 via the Na 1 / H 1 and Cl 2 / HCO 2 3 antiporters [27]. Beside this, increased glutamate in
Fig. 4. Macroautoradiogram of in situ hybridization obtained at different levels of the brain sections of animals with MCA-occlusion with relevant sense controls on top of each row. Expression of AQP4–mRNA was weakly increased on 1 day (n55) after MCA-occlusion in the peri-infarcted cortex of the affected side which became most prominent on 3 days (n55) and lasted until 7 days after surgery (n54). There were no signals present in the core of the infarct.
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Fig. 5. Time-dependent AQP4–mRNA expression after focal cerebral ischemia (n54, 5, 5 and 4 for sham-operated and 1, 3 and 7 days after MCA-occlusion, respectively). Optical density ratio (ODR) of the periinfarcted cortex as indicated in the inset was calculated in comparison with the corresponding area in the non-affected side. Data are presented as means6S.D. for each time point. Non-parametric analysis of the Mann–Whitney U-test with two-tailed probability was performed to demonstrate differences between affected and non-affected hemispheres. Significant (*P,0.01) increase in AQP4–mRNA level was observed in the affected side on three and 7 days after MCA-occlusion.
the extracellular space, leaked out from neurons as well as from surrounding necrotic tissue, is uptaken by the glial cells where the sodium is co-transported intracellularly [1,3]. Thus, glial cells regulates extracellular as well as intracellular pH and eliminates excitotoxic glutamate, and may so strive to maintain ideal extracellular environment for neurons to recover from the ischemic insult [2,26,27]. To enable cellular uptake of osmoles, glial cells has to allow simultaneous influx of significant amount of water to buffer the increased intracellular osmotic pressure. It is a well known feature that the plasma membrane, despite its
lipid bilayer configuration, is water transparent. Recently, channels selective to the water were identified in the various tissue where certain water transport is required [7,12]. AQP4 is a member of such a channel family which is mainly expressed in the glial cells in the brain. In the present study, the AQP4–mRNA was already weakly upregulated at 1 day after MCA-occlusion, became significantly increased on day 3 and attenuated thereafter. These changes are parallel to the edema formation and resolution monitored by MRI, and strongly suggest that AQP4 is a major channel enabling water to shift across the astrocyte plasma membrane when osmoles are accumulating intracellularly due to ischemic insult. That the expression was documented predominantly in non-neuronal cells in the peri-infarcted cortex also agrees with the previous finding that the astrocyte was the major cell type which became swollen under ischemia [15]. As the increased AQP4–mRNA expression continued over 7 days when the edema began to resolve, it may also suggest that the same channel is employed when the water exits the cell during that period. In neurons, sodium influx triggers upregulation of Na 1 / myo–inositol co-transporter and protect itself from preturbating effect of high intracellular sodium accumulation by replacing it with organic osmolytes [31]. The glial cells, however, is sparse of expression of such transporter. Recently, volume-sensitive organic osmolyte / anion channel (VSOAC) is identified in human glial cells which mediates organic osmolyte efflux in response to cellular swelling [10]. This channel requires intracellular ATP binding for its activation [11]. It may be hypothesized that the astrocytes respond to extracellular environmental changes under ischemia by uptaking various osmoles to provide an ideal environment for neurons to survive. The AQP4 may hereby act as a major water pathway to suppress osmotic gradient across cellular membrane to
Fig. 6. Dark-field microautoradiogram of in situ hybridization for AQP4–mRNA. Three days after MCA-occlusion, intense signals were found mainly in cells with medium-sized nuclei in the peri-infarcted cortex. The signals were most abundant in the molecular, (a) and layer 2, (b). Bar550 mm.
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make further influx of the osmoles feasible. The subsequent cellular volume increase may activate VSOAC under normal conditions. However, under ischemic conditions with an insufficient amount of ATP, the cell may fail to regulate its own volume and expand ultimately to raise intracranial pressure which causes critical adverse sequelae. Han and colleagues reported inhibition of AQP4 by phorbol ester via protein kinase C up to 92% in vitro [6]. Functional alteration of the channel would also contribute to the pathogenesis in vivo, and the regulation of the AQP4 provides the possibility of a novel approach to the pharmacological edema treatment. The AQP4 knockout mouse demonstrated almost no impairment in the development and neuromuscular function but only a small defect in urinary concentrating ability [19]. It suggests that AQP4 may have a more substantial role in the regulation of water transport under pathological conditions. These animals would be useful for clear elucidation of edema mechanisms.
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Research report
Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat a,b a,b , a b Masaaki Taniguchi , Toshihide Yamashita *, Eiji Kumura , Michio Tamatani , c c a a Akihiro Kobayashi , Takashi Yokawa , Motohiko Maruno , Amami Kato , Takanori Ohnishi a , Eiji Kohmura a , Masaya Tohyama b , Toshiki Yoshimine a a
b
Department of Neurosurgery, Osaka University Medical School, 2 -2 Yamadaoka, Suita, Osaka 565 -0871, Japan Department of Anatomy and Neuroscience, Osaka University School of Medicine, 2 -2 Yamadaoka, Suita, Osaka 565 -0871, Japan c Basic Research Institute, Nihon Schering K.K., Osaka, Japan Accepted 4 April 2000
Abstract Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4–mRNA is increased in the peri-infarcted cortex during the observation period (|7 days) after MCA-occlusion, maximally on day 3. The change corresponds to the generation and resolution of brain edema monitored by MRI. The signals for the mRNA are predominantly observed in glial cells in the molecular and outer granular layer of the peri-infarcted cortex. These results indicate that AQP4 plays a role in post-ischemic edema formation. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Uptake and transporters Keywords: Middle cerebral artery occlusion; Aquaporin-4; Brain edema
1. Introduction The brain water-content increases under various pathological conditions, which may cause diverse sequelae on the remaining neuronal function. Extensive edema formation is triggered after an ischemic insult. Brain edema is classified into two categories: Cytotoxic edema, resulting from disturbances in cell metabolism without disruption of the blood brain barrier (BBB), and vasogenic edema, which is caused by breakdown of the BBB to macromolecules [16]. In post ischemic brain edema, the cytotoxic edema
Abbreviations: AQP4, aquaporin-4; EDTA, ethylenediaminetetraacetate; MCA, middle cerebral artery; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; VSOAC, volume-sensitive organic osmolyte / anion channel *Corresponding author. Tel.: 181-6-6879-3652; fax: 181-6-68793659. E-mail address: [email protected] (T. Yamashita)
occurs first followed by the vasogenic edema [9,18]. The early astroglial swelling is seen prominently at processes around neurons and capillaries [15,25]. Under lactoacidosis, intracellular pH decreases by exchange of H 1 and bicarbonate ions against extracellular Na 1 and Cl 2 via the antiporters [27]. Uptake of extracellularly accumulated glutamate via the specific transporter also results in cotransporting Na 1 ions intracellularly [1,3]. This Na-shift into the cell and concomitant water influx causes swelling of the glial cell. The swelling under lactoacidosis may be considered as a self-protecting event to regulate the intracellular pH. Uptake of mediator compounds by glial cell may be considered as the housekeeping effect for the ideal environment to protect the neuron. To maintain these glial functions and to make the intracellular uptake of Na 1 feasible, osmotic gradient between plasma membrane has to be quickly canceled out. To enable this, specific route for robust shift of water across plasma membrane should be mandatory. In vasogenic brain edema, water movement across the
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BBB into brain tissue presumably follows the Starling equation [14]. Many factors modify the edema process, including cerebrovascular permeability, capillary hydraulic conductivity, tissue compliance and conductivity as well as hydrostatic and osmotic pressure gradients. During the genesis of vasogenic edema, ion exchanges through the BBB are achieved by simple diffusion. The resulting increase in cations together with the macromolecules then generates osmotic forces which cause a net increase in water in the extracellular space [23]. Extravasated compounds with different molecular weights and diffusion coefficients travel together as a result of hydrostatic pressure. Once the fluid is in the brain extracellular space, further advancement of the edema front is primarily accomplished by bulk flow [17]. As the ‘pure’ form of cytotoxic or vasogenic edema is unlikely to exist, the water, cations and various mediators thus accumulating in the extracellular space are amenable to further shift into the glia via the mechanism mentioned above to augment astrocytic swelling [29]. The concomitant water influx into the extra- as well as intracellular space reaches its peak around 1–3 days after an ischemic insult and diminishes thereafter [5,20]. Though complete resolution of the edema takes more than a week, the osmotic gradient between brain and blood is not parallelly changed with the edema. Different studies provided an evidence of osmolality increase in the brain tissue under focal cerebral ischemia [8,24]. Their common finding was that the increase in osmolality was apparent only during the early stage of ischemia, indicating the presence of a mechanism enabling the robust water shift into the brain extracellular as well as intracellular space, which cancels out the increase in osmotic gradient. The aquaporins are a water-selective channel family consisting of as yet known seven members expressed in the variety of tissues, of which the type 4 (AQP4) is mainly expressed in the brain [7,12]. Its localization to the astroglia on the brain surface and subependymal lining, as well as in osmoreceptor region and adjacent to intracerebral vasculature indicates close functional relationship to the water balance regulation [12], and considered as a candidate for major pathway of a massive water shift across plasma membrane under various condition [22]. We show here that the transcription of AQP4 drastically changes after focal cerebral ischemia, suggesting the involvement in the evolution and resolution of brain edema.
statically-controlled heating pad. The left MCA was exposed with a modified procedure of the method described by Tamura et al. [28]. Briefly, after a 2 cm vertical incision was made over the temporalis muscle, the muscle was split and its insertion into the mandible divided to reach the infratemporal fossa to expose the base of the skull from the foramen ovale to the foramen opticum. While preserving the zygoma, a drill was used to perform a small subtemporal craniectomy 2–3 mm rostral to the foramen ovale in order to expose the proximal part of the MCA, which is located under the dura. A 27-gauge needle was used to open the dura. The MCA was then irreversibly occluded by electrocoagulation over a distance of 2 mm, starting proximally at the medial margin of the olfactory tract. In sham-operated controls, the MCA was exposed but not coagulated (n54). Blood gases, pH, and mean arterial blood pressure (MABP) were monitored throughout the operation. Experiments were completed only if these physiological variables remained within normal limits. The normal values for MABP were set at 90–110 mmHg, those for Paco 2 at 30–50 mmHg, for Pao 2 at 80–110 mmHg, and for arterial blood pH at 7.25–7.45. After recovery from anesthesia, the animals were returned to their cages and allowed ad libitum normal rat chow and water. The rats were divided into two groups: in one group the MRI was acquired after 1, 3, and 7 days following MCAocclusion and the unilateral cortex was subjected for northern blot analysis. In the other group, the animals were deeply anesthetized by intraperitoneal injection of pentobarbital (60 mg / kg) at 1, 3, and 7 days after MCAocclusion and their brains were removed and quickly frozen at 2808C (n55, 5 and 4 for each time point per group, respectively). Serial coronal sections (8–10 mm thick) were obtained from the frozen brains with a cryostat and stored in a tightly closed case at 2808C.
2.2. MRI acquisition To elucidate the edema formation, brain MR images were obtained at 1, 3 and 7 days after MCA-occlusion (n52 for control, n54 for 1, 3, 7 days) using Omega CSI-2 system equipped with a 4.7-T magnet (Bruker GENMR Instruments, Fremont, CA, USA). The T2-weighted sagittal image was first obtained for positioning. Then, four slices of coronal brain spin-echo T2-weighted images were obtained with repetition time / echo time51000 / 80 ms, slice thickness of 2.5 mm, FOV of 30 mm and 1283128 pixels.
2. Materials and methods
2.3. Northern blot analysis
2.1. Animals with focal ischemia
The entire cortex of the affected side including ischemic tissue samples was dissected out from the brains of rats with MCA-occlusion at 1, 3 and 7 days (n54 for each time interval), as well as from sham-operated controls (n53), and immediately frozen at 2808C. Total RNA was isolated
Male Wistar rats weighing 250–300 g were anesthetized with intraperitoneal chloral hydrate (300 mg / kg). Rectal temperature was maintained at 36.5–37.58C with a thermo-
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by the acid guanidinium isothiocyanate method as described previously [4]. Aliquots (20–40 mg) of RNA were separated on 1% agarose formaldehyde gels and was transferred onto nylon membranes (Immobilion TM -N; Millipore Corp., Bedford, MA). A 385 bp rat AQP4 cDNA which was 93% identical to the published rat AQP4 cDNA (bases 21–405) [7,12] and a rat beta-actin oligonucleotide (Clontech Laboratories Inc., Palo Alto, CA) were labeled by random priming (Amersham Corp., Arlington Heights, IL) with [a-32P]dCTP (3000 Ci / mmol; Amersham). Hybridization with AQP4 probes was carried out at 688C and 1 h in ExpressHybE hybridization solution (Clontech). The blots were washed two times at room temperature for 30 min in 23SSC and 0.05% SDS, one time at 508C for 40 min in 0.13SSC and 0.1% SDS. The membranes were placed in contact with X-Ray film at 2808C for 1 day using intensifying screens. After quantification of the hybridized probe, it was removed from the membrane to be hybridized with the beta-actin probe. The hybridized probe was removed in a boiling solution of 0.5% SDS for 10 min and allowed to cool to room temperature. After the removal of the AQP4 probe, hybridization and washing for the beta-actin probe was carried out at 378C and room temperature, respectively, in the same manner. Finally, X-Ray film was placed on the membrane at 2808C for 16 h.
2.4. In situ hybridization The antisense and sense probe for AQP4 was synthesized from a 385 bps rat AQP4 cDNA (bases 21–405) insert cloned in the pGEM-T vector (Promega Corporation, Madison, WI). To synthesize hybridization riboprobes by in vitro transcription, these sequences were first linearized by digestion with restriction endonucleases of Sal 1 for antisense RNA and of Nco 1 for sense RNA synthesis. The linearized cDNA was then incubated at 378C for 60 min with a mixture of reagents. This mixture consisted of 2 ml of transcription buffer (35), 0.25 ml of 100 mM dithiothreitol, 0.25 ml of RNase inhibitor, 0.25 ml of 10 mM ATP, CTP and GTP, 2.5 ml of [ 35 S]UTP (NEG-039H, New England Nuclear), 1 ml of DNA template (1 mg / ml) with 1 ml of appropriate RNA polymerase (T7 RNA polymerase for antisense probe; SP6 RNA polymerase for sense probe). DNA digestion was achieved by the addition of 2 ml of DNase and incubation at 378C for 20 min. Efficacy of labeling was estimated by quantification of radioactivity of the synthesized probes. In situ hybridization techniques for AQP4–mRNA (RNA probe) were based on those of Wilkinson et al. [30] with some modifications. The sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 min. After washing with PB, the sections were treated with 10 mg / ml of proteinase K in 50 mM Tris–HCl and 5 mM EDTA (pH 8.0) for 30 s at room temperature. They were fixed again in the same fixative, then acetylated with acetic
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anhydride in 0.1 M triethanolamine, rinsed with PB, dehydrated and air-dried. The 35 S-labeled RNA probes (sense or antisense) were diluted in hybridization buffer, placed over the sections and covered with siliconized coverslips. Hybridization was performed overnight in a humid chamber at 558C. The hybridization buffer consisted of 50% deionized formamide, 0.3 M NaCl, 20 mM Tris– HCl, 5 mM EDTA, 10 mM PB, 10% dextran sulfate, 13Denhardt’s solution, 0.2% sarcosyl, 500 mg / ml yeast tRNA, and 200 mg / ml Salmon Testis DNA (pH 8.0). The probe concentration was 5310 5 cpm / 200 ml per slide. After hybridization, the slides were immersed in 53SSC at 558C, and the coverslips were allowed to fall off. The sections were then incubated at 658C in 50% deionized formamide with 23SSC for 30 min. After rinsing with RNase buffer [0.5 M NaCl, 10 mM Tris–HCl, 5 mM EDTA (pH 8.0)] three times for 10 min each time at 378C, the sections were treated with 1 mg / ml of RNase A in RNase buffer for 10 min at 378C. After an additional wash in RNase buffer, the slides were incubated in 50% formamide with 23SSC for 30 min at 658C, rinsed with 23SSC and 0.13SSC for 10 min each at room temperature, dehydrated in an ascending alcohol series and airdried. X-Ray film was placed on the uncoated sections for 6 days. Next, the slides were coated with Ilford K-5 emulsion diluted in distilled water containing 2% glycerine (1:1). The slides were exposed for 3 weeks in a tightly sealed dark box at 48C, developed in Kodak D-19, fixed with photographic fixer, stained with hematoxylin, and coverslipped. After the X-Ray macroautoradiogram had been studied, the tissue sections were examined under a regular light microscope. For quantitative assessment of AQP4–mRNA expression on the macroautoradiograms, the optical density of the peri-infarcted cortex was measured. The film was first stored digitally and density was measured with the image analyzing software (NIH image ver. 1.6). Two areas adjacent to the infarction were measured (Fig. 5). After the background density was subtracted, the counts were used for further analysis. Optical density ratio (ODR) of the peri-infarcted cortex was calculated in comparison with the relevant area on the non-affected hemisphere. Statistical significance of results was determined using the nonparametric analysis of the Mann–Whitney U-test with two-tailed probability.
3. Results We first determined brain edema formation after cerebral ischemia using MR images. Typical MR images at each timepoint are shown in Fig. 1. The T2-prolonged region was already detected on the first day after MCA-occlusion at the left hemisphere MCA territory. The signal intensity increased until 3 days and became inhomogeneous at 7
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Fig. 1. Representative MRI images of rats on 1 (a), 3 (b) and 7 (c) days after MCA-occlusion. The focal ischemic lesion is clearly depicted as high intensity area in the left hemisphere, which is most prominent and largest on day 3. On day 7, the high intensity region became confound and inhomogenious.
days after arterial occlusion. The volume of high intensity region also increased until 3 days but rather confound at cerebral cortex at 7 days post-occlusion. As increase of signal intensity in T2-weighted MRI indicates an increase in water content within the damaged brain tissue [13,21], the post-ischemic brain edema developed already on the first day after MCA-occlusion, which became most prominent 3 days and diminished but persisted 7 days postischemia. To examine whether AQP4 is implicated in the pathological condition of focal ischemia, we first monitored expression of AQP4–mRNA after focal cerebral ischemia. Total RNA was isolated from the ipsilateral and contralateral cortex of the affected side following MCAocclusion, and hybridized with the rat AQP4 cDNA probe. Fig. 2 shows a Northern blot revealing the time course of changes in cortical AQP4–mRNA expression after MCAocclusion. The main band was clearly seen with a size of 5.5 kilobases, which is consistent with other reports [7,12]. The amount of this transcript was increased at the affected left hemisphere on 3 days after MCA-occlusion. AQP4 expression continued to be augmented but in a lesser degree on 7 days after surgery. Localization of AQP4–mRNA was assessed by in situ hybridization using a [ 35 S]-labeled cRNA probe. Specificity of hybridization signals was confirmed in a control study using the sense cRNA probe (Fig. 3c). Signals were observed only in the sections hybridized with the antisense probes. In sham-operated animals, expression of AQP4– mRNA was detected on the brain surface, ependymal lining and at suprachiasmatic and paraventricular nucleus (Fig. 3a, b). In animals with MCA-occlusion, the increased expression of AQP4–mRNA was weakly noticeable at 1 day in the peri-infarcted cortex of the affected side (Fig. 4). The signal increase became most prominent and widespread on 3 days after MCA-occlusion. Seven days after surgery, the increased signal was still noticeable in the peri-infarcted cortex but in a lesser degree than day 3. This signal increase, however, was statistically significant on day 3 and 7, but not on day 1, according to the ODR analysis (Fig. 5). No signals were observed in the core of
the infarct at any time during the observation period. Microautoradiography demonstrated marked induction of AQP4–mRNA signals mainly in cells with medium-sized nuclei in peri-infarcted cortex, most abundant in molecular and outer granular layer, suggesting that these AQP4– mRNA-positive cells are morphologically non-neuronal cells and the upregulation of the gene expression in these areas was mainly due to the increase in glial AQP4 expression (Fig. 6).
Fig. 2. Northern blot analysis of AQP4–mRNA-induction in the ischemic cortex following left MCA-occlusion (each blot represents tissue specimen from single animal). The main band was clearly seen at a size of 5.5 kilobases (kb). The level of this transcript was markedly increased in the left hemisphere on 3 days after surgery. Increased AQP4 expression continued until day 7 but in a lesser degree.
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Fig. 3. Macroautoradiogram of in situ hybridization for AQP4–mRNA. Antisense probe (a, b) and sense probe (c) were hybridized with the tissue sections of sham-operated controls. Signals were observed only in sections hybridized with the antisense probe (a). Moderate expression of AQP4–mRNA was detected on the brain surface, ependymal lining and at suprachiasmatic and paraventricular nuclei.
4. Discussion During the early stage of ischemia, lactic acid leaked out from necrotic tissue accumulates in the surrounding penumbra zone. In addition, incomplete ischemia promotes anaerobic glycolysis and further advance the lactic
acidosis. This leads to formation of carbonic acid, which dissociates immediately into CO 2 and water. The CO 2 enters the cell freely and converted to H 2 CO 3 . The resulting H 1 and bicarbonate ions are exchanged against extracellular Na 1 and Cl 2 via the Na 1 / H 1 and Cl 2 / HCO 2 3 antiporters [27]. Beside this, increased glutamate in
Fig. 4. Macroautoradiogram of in situ hybridization obtained at different levels of the brain sections of animals with MCA-occlusion with relevant sense controls on top of each row. Expression of AQP4–mRNA was weakly increased on 1 day (n55) after MCA-occlusion in the peri-infarcted cortex of the affected side which became most prominent on 3 days (n55) and lasted until 7 days after surgery (n54). There were no signals present in the core of the infarct.
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Fig. 5. Time-dependent AQP4–mRNA expression after focal cerebral ischemia (n54, 5, 5 and 4 for sham-operated and 1, 3 and 7 days after MCA-occlusion, respectively). Optical density ratio (ODR) of the periinfarcted cortex as indicated in the inset was calculated in comparison with the corresponding area in the non-affected side. Data are presented as means6S.D. for each time point. Non-parametric analysis of the Mann–Whitney U-test with two-tailed probability was performed to demonstrate differences between affected and non-affected hemispheres. Significant (*P,0.01) increase in AQP4–mRNA level was observed in the affected side on three and 7 days after MCA-occlusion.
the extracellular space, leaked out from neurons as well as from surrounding necrotic tissue, is uptaken by the glial cells where the sodium is co-transported intracellularly [1,3]. Thus, glial cells regulates extracellular as well as intracellular pH and eliminates excitotoxic glutamate, and may so strive to maintain ideal extracellular environment for neurons to recover from the ischemic insult [2,26,27]. To enable cellular uptake of osmoles, glial cells has to allow simultaneous influx of significant amount of water to buffer the increased intracellular osmotic pressure. It is a well known feature that the plasma membrane, despite its
lipid bilayer configuration, is water transparent. Recently, channels selective to the water were identified in the various tissue where certain water transport is required [7,12]. AQP4 is a member of such a channel family which is mainly expressed in the glial cells in the brain. In the present study, the AQP4–mRNA was already weakly upregulated at 1 day after MCA-occlusion, became significantly increased on day 3 and attenuated thereafter. These changes are parallel to the edema formation and resolution monitored by MRI, and strongly suggest that AQP4 is a major channel enabling water to shift across the astrocyte plasma membrane when osmoles are accumulating intracellularly due to ischemic insult. That the expression was documented predominantly in non-neuronal cells in the peri-infarcted cortex also agrees with the previous finding that the astrocyte was the major cell type which became swollen under ischemia [15]. As the increased AQP4–mRNA expression continued over 7 days when the edema began to resolve, it may also suggest that the same channel is employed when the water exits the cell during that period. In neurons, sodium influx triggers upregulation of Na 1 / myo–inositol co-transporter and protect itself from preturbating effect of high intracellular sodium accumulation by replacing it with organic osmolytes [31]. The glial cells, however, is sparse of expression of such transporter. Recently, volume-sensitive organic osmolyte / anion channel (VSOAC) is identified in human glial cells which mediates organic osmolyte efflux in response to cellular swelling [10]. This channel requires intracellular ATP binding for its activation [11]. It may be hypothesized that the astrocytes respond to extracellular environmental changes under ischemia by uptaking various osmoles to provide an ideal environment for neurons to survive. The AQP4 may hereby act as a major water pathway to suppress osmotic gradient across cellular membrane to
Fig. 6. Dark-field microautoradiogram of in situ hybridization for AQP4–mRNA. Three days after MCA-occlusion, intense signals were found mainly in cells with medium-sized nuclei in the peri-infarcted cortex. The signals were most abundant in the molecular, (a) and layer 2, (b). Bar550 mm.
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make further influx of the osmoles feasible. The subsequent cellular volume increase may activate VSOAC under normal conditions. However, under ischemic conditions with an insufficient amount of ATP, the cell may fail to regulate its own volume and expand ultimately to raise intracranial pressure which causes critical adverse sequelae. Han and colleagues reported inhibition of AQP4 by phorbol ester via protein kinase C up to 92% in vitro [6]. Functional alteration of the channel would also contribute to the pathogenesis in vivo, and the regulation of the AQP4 provides the possibility of a novel approach to the pharmacological edema treatment. The AQP4 knockout mouse demonstrated almost no impairment in the development and neuromuscular function but only a small defect in urinary concentrating ability [19]. It suggests that AQP4 may have a more substantial role in the regulation of water transport under pathological conditions. These animals would be useful for clear elucidation of edema mechanisms.
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