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Differential expressions of aquaporin subtypes in astroglia in the hippocampus of chronic epileptic rats
Neuroscience 163 (2009) 781–789
DIFFERENTIAL EXPRESSIONS OF AQUAPORIN SUBTYPES IN ASTROGLIA IN THE HIPPOCAMPUS OF CHRONIC EPILEPTIC RATS J.-E. KIM,a,b H. J. RYU,a,b S.-I. YEO,a,b C. H. SEO,c B. C. LEE,d I.-G. CHOI,d D.-S. KIMe AND T.-C. KANGa,b*
et al., 1999; Yamamoto et al., 2001). Among them, AQP1, AQP4 and AQP9 are identified by their protein expression levels and localizations in the rat brain. AQP1 protein expression is restricted to the ventricular-facing surface of the choroid plexus. AQP4 is expressed in astrocyte foot processes near blood vessels in rat and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, including ependymal cells and tanycytes, and astrocytes (for review, Badaut et al., 2002). Astrocytes play a role in maintenance of homeostasis in the brain by regulating local ion concentrations, pH and clearance of neurotransmitters released into the synaptic cleft. Furthermore, astrocytes release many neuroactive substances, such as chemical transmitters, cytokines, neuropeptides and growth factors (Anderson and Swanson, 2000; Ransom et al., 2003). Reactive astrogliosis represents high glial fibrillary acidic protein (GFAP) and their cell bodies hypertrophy, and begin to proliferate, migrate and form glial scars, which is frequently encountered in association with temporal lobe epilepsy in humans and with drug- or kindling-induced seizures in animal models (Bordey and Sontheimer, 1998; Mathern et al., 1998). This reactive astrogliosis is considered as a consequent healing process that produces pathological effects by interfering with the functions of residual neuronal circuits (Represa et al., 1995), or as a compensatory response, that provides trophic factor to survived neuronal populations (Ridet et al., 1997). Interestingly, Revuelta et al. (2005) reported astroglial death in the CA1 region and the amygdalar complex after kainic acid administration. We also found TUNEL positive astroglial death in the rat dentate gyrus after pilocarpine-induced status epilepticus (SE) (Kang et al., 2006) and TUNEL negative astroglial hypertrophy and vacuolization in the stratum radiatum of the CA1 region (Kim et al., 2008a), which is considered an early stage of necrosis (Deloncle et al., 2001; Sugawara et al., 2002; Tomimoto et al., 1997). Unlike that observed in the dentate gyrus and entorhinal cortex, furthermore, conventional anti-epileptic drugs prevented delayed necrotic astroglial degeneration in the CA1 region. Thus, these findings reveal that CA1 astroglial damage may be a consequence of prolonged seizure. Although AQPs are supposed to play a role in astroglial hypertrophy/vacuolization (for review, Badaut et al., 2002), the underlying molecules involved in delayed necrotic astroglial degeneration are still unclear. Therefore, the present study was designed to elucidate the relationship between astroglial responses and AQPs in the experimental epileptic hippocampus.
a Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea b Institute of Epilepsy Research, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea c Department of Rehabilitation Medicine, Hangang Sacred Heart Hospital, Hallym University, Seoul 150-719, South Korea d Department of Psychiatry, Hangang Sacred Heart Hospital, Hallym University, Seoul 150-719, South Korea e Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan, Chungcheongnam-Do 330-090, South Korea
Abstract—In order to elucidate the roles of aquaporins (AQPs) in astroglial responses, we investigated AQP expressions in the experimental epileptic hippocampus. In control animals, AQP1 protein expression was restricted to the ventricular-facing surface of the choroid plexus. AQP4 was expressed in astrocyte foot processes near blood vessels and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, and in astrocytes. Six to eight weeks after status epilepticus (SE), AQP1 expression was mainly, but not all, detected in vacuolized astrocytes, which were localized in the stratum radiatum of the CA1 region. AQP4 was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in non-vacuolized astrocytes was increased as compared to control level. AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes. Therefore, our findings suggest that AQP subunits may play differential roles in various astroglial responses (including astroglial swelling and astroglial loss) in the chronic epileptic hippocampus. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: aquaporin 1, aquaporin 4, aquaporin 9, astrocytes, epilepsy, hippocampus.
Aquaporins (AQPs) are water channels that provide the major route for water movement across plasma membranes in a variety of tissues including the brain (Agre et al., 2002; Verkman, 2002; Manley et al., 2000; Badaut et al., 2002; Amiry-Moghaddam and Ottersen, 2003). In normal rat brain, six AQPs subtypes have been described: AQP1, AQP3, AQP4, AQP5, AQP8 and AQP9 (Badaut et al., 2001; Elkjaer et al., 2000; Nielsen et al., 1997; Venero *Correspondence to: T.-C. Kang, Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chuncheon, Kangwon-do 200-702, South Korea. Tel: ⫹82-33-248-2524; fax: ⫹82-33256-1614. E-mail address: [email protected] (T.-C. Kang). Abbreviations: AQP, aquaporin; GFAP, glial fibrillary acidic protein; GFAP⫹, glial fibrillary acidic protein immunoreactive; PBS, phosphatebuffered saline; SD, Sprague–Dawley; SE, status epilepticus.
0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.07.028
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EXPERIMENTAL PROCEDURES Experimental animals This study utilized the progeny of Sprague–Dawley (SD) rats obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions (22⫾2 °C, 55⫾5% and a 12-h light/dark cycle with lights). Procedures involving animals and their care were conducted in accord with our institutional guidelines that comply with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, 1996). We have made all efforts to minimize the number of animals used and their suffering.
Seizure induction and drug treatments Male SD rats (9 weeks old, n⫽20) were treated with pilocarpine (Sigma-Aldrich Co., St. Louis, MO, USA; 380 mg/kg i.p.) at 20 min after atropine methylbromide (Sigma-Aldrich Co., St. Louis, MO, USA; 5 mg/kg i.p.). Among pilocarpine-treated rats, 17 rats showed acute behavioral features of SE (including akinesia, facial automatisms, limbic seizures consisting of forelimb clonus with rearing, salivation, masticatory jaw movements and falling). Diazepam (10 mg/kg i.p.; Valium, Hoffman la Roche, Neuilly sur-Seine, France) was administered 2 h after onset of SE and repeated, as needed. Rest animals (non-experienced SE animals, n⫽3) showed only acute seizure behaviors during 10 –30 min. Rats that did not experience SE were used as controls, acute or brief pilocarpine-induced seizure could not result in neuropathological changes in the rat brain (Kim et al., 2009; Bower and Buckmaster, 2008). Indeed, we could not observe changes in AQP expression in these animals (data not shown). In addition, 30 min before pilocarpine treatment, diazepam (10 mg/kg i.p.) was given to some animals. Diazepam pretreatment completely prevented SE. Diazepam-pretreated animals used immunohistochemical studies at designated time courses (3 days 1 week and 5 weeks after SE, n⫽3, respectively). Age-matched animals (n⫽8) were also used as controls. One week after SE, rats were observed 3– 4 h a day in the vivarium for general behavior and occurrence of spontaneous seizures. The onset of spontaneous complex partial seizure occurrence was approximately 4 weeks after SE. On average, these animals developed two seizures/day.
Tissue processing and immunohistochemistry At designated time courses, animals were anesthetized (urethane, 1.5 g/kg, i.p.; Sigma-Aldrich Co., St. Louis, MO, USA) and perfused transcardially with phosphate-buffered saline (PBS, SigmaAldrich Co., St. Louis, MO, USA) followed by 4% paraformaldehyde (Sigma, MO, USA) in 0.1 M PB (pH 7.4; Sigma-Aldrich Co., St. Louis, MO, USA). The brains were removed, and postfixed in the same fixative for 4 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter the tissues were frozen and sectioned with a cryostat at 30 m and consecutive sections were collected in six-well plates containing PBS. The sections were first incubated with 3% bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) in PBS for 30 min at room temperature. Sections were then incubated in primary antibodies (listed below) in PBS containing 0.3% Triton X-100 (SigmaAldrich Co., St. Louis, MO, USA) overnight at room temperature: rabbit anti-AQP1 (Abcam, Cambridge, UK, diluted 1:200), AQP4 (Chemicon, CA, USA, diluted 1:200) or AQP9 IgG (LifeSpan, Seattle, WA, USA, diluted 1:200). The sections were washed three times for 10 min with PBS, incubated sequentially, in biotinylated horse anti-mouse or goat anti-rabbit IgG (Vector, Burlingame, CA, USA) and ABC complex (Vector, Burlingame, CA, USA), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with PBS three
times for 10 min each. The sections were visualized with 3,3=diaminobenzidine (DAB, Sigma-Aldrich Co., St. Louis, MO, USA) in 0.1 M Tris buffer and mounted on gelatin-coated slides. The immunoreactions were observed under the Axiophot microscope (Carl Zeiss, Munchen-Hallbergmoos, Germany). All images were captured using an Axiocam HRc camera and Axio Vision 3.1 software. Double immunofluorescent staining for AQP1, AQP4 or AQP9/GFAP was also performed. Brain tissues were incubated in mixture of rabbit anti-AQP1, AQP4 or AQP9 IgG (diluted 1:50)/ mouse anti-GFAP IgG (diluted 1:100) overnight at room temperature. After washing three times for 10 min with PBS, sections were also incubated in a mixture of FITC- and Cy3-conjugated secondary antisera (1:200, Amersham, PA, USA) for 1 h at room temperature. Sections were mounted in Vectashield mounting medium (Vector, Burlingame, CA, USA). All images were captured using an Axiocam HRc camera and Axio Vision 3.1 software. In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of the primary antibody (for GFAP) or a pre-absorption test was performed with control peptide (for AQPs). The control for immunohistochemistry resulted in the absence of immunoreactivity in any structure (data not shown). All experimental groups in the present study were included in each immunochemistry and were therefore processed under the same conditions.
Quantification of data and statistical analysis For quantification of GFAP/AQPs double immunofluorescence, we have performed the cell count. GFAP and AQP immunofluorescent images (10 sections/rat) were captured in the same region (500⫻500 m). Images were sampled from at least five different points within each hippocampal section. Thereafter, the number of GFAP positive cells that are each AQP positive was actually counted within the sampled images. All immunoreactive cells were counted regardless the intensity of labeling. The number of vacuolized GFAP positive cells was counted by the same method. The diameter of vacuoles in astrocytes was also measured by Axio Vision 3.1 software. Cell counts and the measurement of the diameter of vacuoles were performed by two different investigators who were blind to the classification of tissues. All data obtained from the quantitative measurements were analyzed using one-way ANOVA to determine statistical significance. Bonferroni’s test was used for post hoc comparisons. A P-value below 0.05 was considered statistically significant (Kim JE et al., 2008, 2009).
RESULTS Coagulative necrosis of astrocytes in the CA1 region Generally, vacuolization of cells may be considered as early stage of necrosis, because these cells often show necrotic features, such as eosinophilic cytoplasm, mitochondrial/nuclear membrane alterations, or TUNEL negativity (Deloncle et al., 2001; Sugawara et al., 2002). Similarly, astroglial hypertrophy and vacuolization are reported in various neurodegenerative diseases (Deloncle et al., 2001; Sugawara et al., 2002; Tomimoto et al., 1997). In our previous study (Kim DS et al., 2008), we had also reported that prolonged recurrent seizure resulted in TUNEL negative loss/vacuolization of CA1 astrocytes (presumably delayed necrotic astroglial damage). In the present study, at 6 – 8 weeks after SE we identified vacuolized astrocytes only in the stratum radiatum of the CA1 region by H & E staining. On the basis of the localization and nuclear size/ shape, we could detect astrocytes on H-E-stained slides.
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Briefly, most neurons have euchromatic nucleus with a prominent nucleolus. The hippocampal neurons are usually localized in the stratum pyramidale, while a few neurons are localized in the stratum radiatum. Astrocytes have small nuclei (approximate 10 m in diameter) among the larger nuclei of neurons. In addition, astrocytes are abundantly localized in the stratum radiatum. The nuclei of oligodendroglia are smaller and more intensely stained than are the nuclei of astrocytes. Furthermore, oligodendroglia localized mainly close to the neuronal cell body. Microglia have small, dense and elongated shapes. Their nuclei show highly condensed chromatin and an elongated shape along the axis of the cell body. Furthermore, vacuolized cells showed only GFAP immunoreactivity (see below). These immunostaining results permit identification of astrocytes in H-E staining. Vacuolized astrocytes showed coagulative necrosis that is characterized by preservation of cell outline, watery cytoplasmic staining and nuclear dissolution. The diameter of vacuoles in these astrocytes was 2.3⫾1.49 m (Fig. 1A). The derivations of vacuoles in CA1 astrocytes In control animals, we observed light GFAP immunoreactivity in astrocytes over the hippocampus (Fig. 1B). One week after SE the processes of glial fibrillary acidic protein immunoreactive (GFAP⫹) astrocyte became unevenly thick with ragged edges in the CA1 region (data not shown). Six to eight weeks after SE, vacuolized GFAP⫹ astrocytes were observed only in the stratum radiatum of the CA1 region (Fig. 1C–I). Since GFAP immunohistochemistry clearly showed astroglial vacuolization rather than classic histological stains, we could detect small vacuoles (diameter, ⬍1.5 m) in hypertrophic GFAP⫹ astrocytes, which could not be observed by H&E stain. The fractions of vacuolized astrocytes in total astrocytes were 17.6, 23.4 and 21.4% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 1J). Based on the size of vacuoles and the GFAP immunoreactivity, vacuolized GFAP⫹ astrocytes were divided into three groups: one had largesized vacuoles (diameter ⬎2 m), round-shaped cell body, short blunt processes and GFAP tangles in the cytoplasm. GFAP immunoreactivity was unevenly detected in the edematous cell bodies. Based on these morphological evidences, astrocytes containing large vacuoles may be degenerative astrocytes. GFAP immunoreactivity in these cells was lower than that in other astrocytes (Fig. 1E). Another group had medium-sized vacuoles (1 m⬍ diameter⬍2 m) that were evenly widespread through cytoplasm. Medium-sized vacuoles were in the peripheral regions and processes. Some vacuoles were attached to the cell membrane. GFAP immunoreactivity in these cells was higher than that in astrocytes containing large vacuoles (Fig. 1H). The third group had small-sized vacuoles (diameter ⬍0.5 m) that were clearly observed in longcurled processes. GFAP immunoreactivity was observed frequently in the cell bodies rather than processes (Fig. 1I). In the dentate gyrus, GFAP⫹ astrocytes showed typical reactive gliosis (hypertrophy and hyperplasia of cell bodies
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and processes of astrocytes). There was no vacuolized astrocyte in this region (data not shown). AQP1 immunoreactivity In control animals, AQP1 immunoreactivity was not observed in the hippocampus (Fig. 2A), whereas AQP1 immunoreactivity was strongly detected in choroid plexus (Fig. 2B). This AQP1 labeling was also observed in experimental groups and was independent of SE until 4 weeks after SE (data not shown). Six to 8 weeks after SE, AQP1 immunoreactivity was mainly, but not always, detected in vacuolized astrocytes that were localized in the stratum radiatum of the CA1 region (Figs. 2C–E and 3A). The fractions of AQP1 immunoreactive astrocytes in total astrocytes were 14.5, 21.2 and 17.8% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 5). AQP4 immunoreactivity In controls, AQP4 immunoreactivity was diffusely observed in the hippocampus (Fig. 4A). Double immunofluorescent study revealed that AQP4 immunoreactivity was obviously observed in the end-feet of all astrocytes, leaving unstained the neuronal somata (Fig. 3B). Six to 8 weeks after SE, AQP4 immunoreactivity was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in nonvacuolized astrocytes was increased as compared to the control level (Figs. 3C and 4B). Therefore, the fractions of AQP4 immunoreactive astrocytes in total astrocytes were reduced to 70.3, 65.3 and 53.6% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 5). However, vessellike structures showed strong AQP4 immunoreactivity without GFAP immunoreactivity. AQP9 immunoreactivity Few AQP9 immunoreactive astrocytes were observed in the hippocampus of control group (data not shown). Six to eight weeks after SE, AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes (Figs. 3D and 4C). The fractions of AQP9 immunoreactive astrocytes in total astrocytes were reduced to 27.4, 28.4 and 29.3% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 5).
DISCUSSION Pilocarpine-induced SE results in hypotension, hyperpyrexia, hypoglycemia, acidosis and hypoxia (Turski et al., 1989; Walker et al., 2002). Since coagulative necrosis is typically seen after hypoxic damages, it is likely that astroglial coagulative necrosis would be simply implied as the result from SE. Vacuolar degeneration is considered as early stage of necrosis. Vacuolized cells show necrotic features, such as eosinophilic cytoplasm, mitochondrial/ nuclear membrane alterations or TUNEL negativity (StruysPonsar et al., 1994; Tomimoto et al., 1997; Deloncle et al., 2001; Sugawara et al., 2002; Kim DS et al., 2008). Consistent with these previous studies, we could observe that the astrocytes in the CA1 region had pyknotic nucleus,
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Fig. 1. Vacuolized astrocytes in the CA1 region at 6 – 8 weeks following SE (A, H &E staining; B–I, GFAP immunostaining) As compared to control (B), vacuolized astrocytes are observed in the CA1 region at 6 – 8 weeks after SE (C–I). The insets in C, D, F and G indicate the high magnifications of the rectangles. (J) The fractions of vacuolized astrocytes in total astrocytes following SE (% of control). Arrows indicate vacuoles in astrocytes. Scale bar⫽5 m (A, E, H, I) and 25 m (B–D, F, G).
edematous eosinophilic cytoplasm and large-sized vacuoles at 6 weeks after SE. Immunostaining for GFAP showed that these astrocytes had a round-shaped cell body, short blunt processes and GFAP tangles in the cytoplasm. In the present study, however, histopathological changes were not observed prior to 6 weeks after SE. Therefore, our findings suggest that in necrotic CA1 astroglial damage may be a consequence of prolonged seizure
activity, not of SE (Kim DS et al., 2008). Indeed, Fabene et al. (2007) reported that the cerebral cortex responds in different ways to pilocarpine stimulation, with ischemic, necrotic processes in the subgranular layers and excitotoxic, apoptotic cell death in the supragranular layers. In addition, these degenerative pathways happen at different time points, with a delayed degeneration in the supragranular layers. Thus, the present findings suggest that the
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Fig. 2. AQP1 immunoreactivity in the rat hippocampus. In control animals (A, B), AQP1 immunoreactivity is strongly detected only in choroid plexus (B). As compared to control (A), AQP1 immunoreactivity is mainly detected in vacuolized astrocytes at 6 – 8 weeks after SE (C–E). Arrows indicate vacuolized astrocytes. The insets in C indicate the high magnification of the rectangle (D, E). Scale bar⫽200 m (A, C) and 25 m (B, D, E).
repeated recurrent seizure activity, not only SE, may play a role in the mechanism of CA1 astroglial loss. Brain edema after injury initially involves astroglial swelling occurring in both gray and white matter (Kimelberg, 1995). This activity-induced astroglial swelling is due to water influx (Walz, 1987, 1992). AQP4 is a key molecule for maintaining water balance, and its dysfunction or structural damage causes cytotoxic edema (Kimelberg, 1995; Kimelberg and Ransom, 1986; Mongin and Kimelberg, 2004). In the present study, AQP4 immunoreactivity was markedly reduced in vacuolized CA1 astrocytes. Astroglial vacuolization is an early stage of necrosis (Deloncle et al., 2001; Sugawara et al., 2002). Similar to in the present study, we reported TUNEL negative vacuolization of astrocytes (presumably delayed necrotic astroglial damage) in the CA1 region (Kim DS et al., 2008). Furthermore, Eid et al. (2005) reported that the loss of perivascular AQP4 in mesial temporal lobe epilepsy patients results in a perturbed flux of water through astrocytes leading to an impaired buffering of extracellular K⫹ and an increased propensity for seizures, although Lee et al. (2004) reported that a significant increase in AQP4 is observed in sclerotic, but not in non-sclerotic, hippocampi obtained from patients with medically intractable temporal lobe epilepsy. AmiryMoghaddam et al. (2003) also reported that the anchoring of AQP4 to ␣-syntrophin may be a target for treatment of brain edema. With respect to previous reports described above, reduced AQP4 immunoreactivity in vacuolized astrocytes may be correlated to irreversible cytotoxic edema of astrocytes. Astroglial swelling subsequently or simultaneously leads to dysfunction of astroglial membrane permeability and impairment of re-uptake of excitatory neurotransmitters or ions by astrocytes (Kimelberg, 2000), which can synchronize reverberant epileptiform discharges of
neurons in the CA1 region (Walz, 1989; Gabriel et al., 1998; D’Antuono et al., 2002; Xu et al., 2007). Indeed, Zeng et al. (2007) reported that in AQP4 knockout mice astroglial glutamate transporter, GLT-1, was downregulated as compared to wild-type animals. In addition, AQP4 knockout astrocytes showed a lower uptake capability of glutamate. Therefore, reduced AQP4 immunoreactivity in astrocytes may be one of the phenotypes of coagulative necrosis of astrocytes or at least of astroglial dysfunctions. However, non-vacuolized astrocytes showed strong AQP4 immunoreactivity as compared to control level. Therefore, upregulated AQP4 immunoreactivity in remaining nonvacuolized reactive astrocytes (particularly in endfeet) may be a compensatory response for loss of astrocytes. In the present study, furthermore, AQP4 immunoreactivity was increased in the background that was GFAP-negative vessel-like structures. This increased AQP4 immunoreactivity may be due to neovascularization. This is because neovascularization is one of the phenomena in chronic epileptic hippocampus (Marcon et al., 2009; Rigau et al., 2007), and endothelial cells in neovasculatures showed overexpression of AQP4, unlike normal endothelial cells (Sawada et al., 2007). Further research is needed to elucidate the cause of increased AQP4 immunoreactivity in non-GFAP structures. AQP1 and AQP9 are upregulated in astrocytes following various CNS damage including subarachnoid hemorrhage, transient focal ischemia and brain tumor, although their expression is rarely observed in the normal brain (Badaut et al., 2001, 2003; Ribeiro et al., 2006). In the present study, AQP1 and AQP9 immunoreactivity was upregulated in the CA1 region. Unexpectedly, upregulated AQP1 immunoreactivity in CA1 astrocyte was correlated to vacuolization in astroglial cytoplasm, while increased
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Fig. 3. Double immunofluorescent staining for AQPs (red) and GFAP (green) in the hippocampus. Six to eight weeks after SE, AQP1 immunoreactivity is mainly detected in vacuolized GFAP⫹ astrocytes (A). In control animals, AQP4 immunoreactivity is observed in the perivascular GFAP⫹ astrocyte endfeet (B). AQP4 immunoreactivity is decreased in vacuolized astrocytes (arrows), while its immunoreactivity is increased in non-vacuolized astrocytes (arrowheads) at 6 – 8 weeks after SE (C). Six to eight weeks after SE, AQP9 immunoreactivity is shown to be mainly induced in non-vacuolized CA1 astrocytes (D). The insets in C indicate the high magnification of the rectangle. (A3–D3) Merged-images of A1–D1 and A2–D2. Scale bar⫽20 and 10 m (insets). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
AQP9 immunoreactivity was observed in non-vacuolized CA1 astrocytes. Similar to the present study, AQP1 immunoreactivity was markedly increased in the astrocytes of patients with intractable epilepsy (Zhou et al., 2008). In glioma cells, Endo et al. (1999) suggested that AQP1 might allow to glioma cells to shrink by extruding water, thereby permitting invasion of the surrounding brain through the extracellular matrix. Therefore, over-expression of AQP1 immunoreactivity in vacuolized astrocytes
may be a compensatory response for maintenance of hydrostatic pressure in swollen astrocytes, when AQP4 was absent. Alternatively, over-expression of AQP1 immunoreactivity in vacuolized astrocytes may be an apoptotic event. This is because Jablonski et al. (2004) reported that AQP1-mediated water loss is important for the apoptotic volume decrease and downstream apoptotic events, and that the water permeability of the plasma membrane can control the rate of apoptosis.
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Fig. 4. AQP4 (A, B) and AQP9 (C) immunoreactivity in the hippocampus. As compared to control (A), AQP4 immunoreactivity decreased in small areas (presumably vacuolized astrocytes, arrows) at 6 – 8 weeks after SE (B). However, AQP9 immunoreactivity is detected only in non-vacuolized CA1 astrocytes (C). The insets in A1–C1 indicate the high magnification of the rectangle. Scale bar⫽200 m (A1–C1) and 25 m (A2–C2, A3–C3).
On the other hand, AQPs have been subdivided into three groups according to permeability characteristics (Verkman and Mitra, 2000): AQP0, AQP1, AQP2, AQP4, AQP5 and AQP6 are permeable to water; AQP3, AQP7 and AQP8 are permeable to water, glycerol, and urea
(aquaglyceroporins); AQP9 is permeable to water, glycerol, urea, purines, pyrimidines, and monocarboxylates (neutral solute channel). Thus, AQP9 is not only implicated in water movements during edema formation, but also plays a role as a metabolite channel in the brain, facilitating
Fig. 5. The fractions of AQP1, AQP4 and AQP9 immunoreactive astrocytes in total astrocytes (% of control). As compared to controls, the numbers of AQP1 and AQP9 immunoreactive astrocytes are increased in the hippocampus, while that of AQP4 immunoreactive astrocytes is reduced.
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the diffusion of glycerol and lactate (Badaut et al., 2004; Badaut and Regli, 2004). In the ischemic condition, AQP9 in reactive astrocytes is involved in the elimination of excess glycerol and lactate from extracellular space (Ribeiro et al., 2006). AQP9 permeability to lactate increases fourfold when pH decreases to 5.5 (Tsukaguchi et al., 1998). Therefore, lactic acidosis during ischemia increases the permeability of AQP9 and enables uptake of the excess lactate by the astrocytes (Ribeiro et al., 2006). Similar to the ischemic condition, metabolic disturbances are commonly observed in chronic epilepsy rats (Melø et al., 2005; Dubé et al., 2001). In particular, Pereira de Vasconcelos et al. (2002) reported that chronic epilepsy rats showed pronounced mismatch between blood supply and metabolic demand. In this condition, an accumulation of CO2 and metabolic production of lactic acid from active cells (Chesler and Kaila, 1992) result in extracellular acidification in the CA1 region (Xiong and Stringer, 2000). Based on these previous studies, the present findings indicate that upregulated AQP9 in non-vacuolized astrocytes may play a role in inhibition of extracellular lactic acidosis induced by repeated spontaneous seizures.
CONCLUSION In conclusion, AQP subunits may play differential roles in various astroglial responses including astroglial swelling and astroglial loss in the chronic epileptic hippocampus. Therefore, our findings suggest that the selective regulation of AQP subunit functions may provide new therapeutic approaches to epilepsy. Acknowledgments—This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (grant number A084589).
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(Accepted 13 July 2009) (Available online 18 July 2009)
DIFFERENTIAL EXPRESSIONS OF AQUAPORIN SUBTYPES IN ASTROGLIA IN THE HIPPOCAMPUS OF CHRONIC EPILEPTIC RATS J.-E. KIM,a,b H. J. RYU,a,b S.-I. YEO,a,b C. H. SEO,c B. C. LEE,d I.-G. CHOI,d D.-S. KIMe AND T.-C. KANGa,b*
et al., 1999; Yamamoto et al., 2001). Among them, AQP1, AQP4 and AQP9 are identified by their protein expression levels and localizations in the rat brain. AQP1 protein expression is restricted to the ventricular-facing surface of the choroid plexus. AQP4 is expressed in astrocyte foot processes near blood vessels in rat and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, including ependymal cells and tanycytes, and astrocytes (for review, Badaut et al., 2002). Astrocytes play a role in maintenance of homeostasis in the brain by regulating local ion concentrations, pH and clearance of neurotransmitters released into the synaptic cleft. Furthermore, astrocytes release many neuroactive substances, such as chemical transmitters, cytokines, neuropeptides and growth factors (Anderson and Swanson, 2000; Ransom et al., 2003). Reactive astrogliosis represents high glial fibrillary acidic protein (GFAP) and their cell bodies hypertrophy, and begin to proliferate, migrate and form glial scars, which is frequently encountered in association with temporal lobe epilepsy in humans and with drug- or kindling-induced seizures in animal models (Bordey and Sontheimer, 1998; Mathern et al., 1998). This reactive astrogliosis is considered as a consequent healing process that produces pathological effects by interfering with the functions of residual neuronal circuits (Represa et al., 1995), or as a compensatory response, that provides trophic factor to survived neuronal populations (Ridet et al., 1997). Interestingly, Revuelta et al. (2005) reported astroglial death in the CA1 region and the amygdalar complex after kainic acid administration. We also found TUNEL positive astroglial death in the rat dentate gyrus after pilocarpine-induced status epilepticus (SE) (Kang et al., 2006) and TUNEL negative astroglial hypertrophy and vacuolization in the stratum radiatum of the CA1 region (Kim et al., 2008a), which is considered an early stage of necrosis (Deloncle et al., 2001; Sugawara et al., 2002; Tomimoto et al., 1997). Unlike that observed in the dentate gyrus and entorhinal cortex, furthermore, conventional anti-epileptic drugs prevented delayed necrotic astroglial degeneration in the CA1 region. Thus, these findings reveal that CA1 astroglial damage may be a consequence of prolonged seizure. Although AQPs are supposed to play a role in astroglial hypertrophy/vacuolization (for review, Badaut et al., 2002), the underlying molecules involved in delayed necrotic astroglial degeneration are still unclear. Therefore, the present study was designed to elucidate the relationship between astroglial responses and AQPs in the experimental epileptic hippocampus.
a Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea b Institute of Epilepsy Research, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea c Department of Rehabilitation Medicine, Hangang Sacred Heart Hospital, Hallym University, Seoul 150-719, South Korea d Department of Psychiatry, Hangang Sacred Heart Hospital, Hallym University, Seoul 150-719, South Korea e Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan, Chungcheongnam-Do 330-090, South Korea
Abstract—In order to elucidate the roles of aquaporins (AQPs) in astroglial responses, we investigated AQP expressions in the experimental epileptic hippocampus. In control animals, AQP1 protein expression was restricted to the ventricular-facing surface of the choroid plexus. AQP4 was expressed in astrocyte foot processes near blood vessels and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, and in astrocytes. Six to eight weeks after status epilepticus (SE), AQP1 expression was mainly, but not all, detected in vacuolized astrocytes, which were localized in the stratum radiatum of the CA1 region. AQP4 was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in non-vacuolized astrocytes was increased as compared to control level. AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes. Therefore, our findings suggest that AQP subunits may play differential roles in various astroglial responses (including astroglial swelling and astroglial loss) in the chronic epileptic hippocampus. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: aquaporin 1, aquaporin 4, aquaporin 9, astrocytes, epilepsy, hippocampus.
Aquaporins (AQPs) are water channels that provide the major route for water movement across plasma membranes in a variety of tissues including the brain (Agre et al., 2002; Verkman, 2002; Manley et al., 2000; Badaut et al., 2002; Amiry-Moghaddam and Ottersen, 2003). In normal rat brain, six AQPs subtypes have been described: AQP1, AQP3, AQP4, AQP5, AQP8 and AQP9 (Badaut et al., 2001; Elkjaer et al., 2000; Nielsen et al., 1997; Venero *Correspondence to: T.-C. Kang, Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chuncheon, Kangwon-do 200-702, South Korea. Tel: ⫹82-33-248-2524; fax: ⫹82-33256-1614. E-mail address: [email protected] (T.-C. Kang). Abbreviations: AQP, aquaporin; GFAP, glial fibrillary acidic protein; GFAP⫹, glial fibrillary acidic protein immunoreactive; PBS, phosphatebuffered saline; SD, Sprague–Dawley; SE, status epilepticus.
0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.07.028
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EXPERIMENTAL PROCEDURES Experimental animals This study utilized the progeny of Sprague–Dawley (SD) rats obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions (22⫾2 °C, 55⫾5% and a 12-h light/dark cycle with lights). Procedures involving animals and their care were conducted in accord with our institutional guidelines that comply with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, 1996). We have made all efforts to minimize the number of animals used and their suffering.
Seizure induction and drug treatments Male SD rats (9 weeks old, n⫽20) were treated with pilocarpine (Sigma-Aldrich Co., St. Louis, MO, USA; 380 mg/kg i.p.) at 20 min after atropine methylbromide (Sigma-Aldrich Co., St. Louis, MO, USA; 5 mg/kg i.p.). Among pilocarpine-treated rats, 17 rats showed acute behavioral features of SE (including akinesia, facial automatisms, limbic seizures consisting of forelimb clonus with rearing, salivation, masticatory jaw movements and falling). Diazepam (10 mg/kg i.p.; Valium, Hoffman la Roche, Neuilly sur-Seine, France) was administered 2 h after onset of SE and repeated, as needed. Rest animals (non-experienced SE animals, n⫽3) showed only acute seizure behaviors during 10 –30 min. Rats that did not experience SE were used as controls, acute or brief pilocarpine-induced seizure could not result in neuropathological changes in the rat brain (Kim et al., 2009; Bower and Buckmaster, 2008). Indeed, we could not observe changes in AQP expression in these animals (data not shown). In addition, 30 min before pilocarpine treatment, diazepam (10 mg/kg i.p.) was given to some animals. Diazepam pretreatment completely prevented SE. Diazepam-pretreated animals used immunohistochemical studies at designated time courses (3 days 1 week and 5 weeks after SE, n⫽3, respectively). Age-matched animals (n⫽8) were also used as controls. One week after SE, rats were observed 3– 4 h a day in the vivarium for general behavior and occurrence of spontaneous seizures. The onset of spontaneous complex partial seizure occurrence was approximately 4 weeks after SE. On average, these animals developed two seizures/day.
Tissue processing and immunohistochemistry At designated time courses, animals were anesthetized (urethane, 1.5 g/kg, i.p.; Sigma-Aldrich Co., St. Louis, MO, USA) and perfused transcardially with phosphate-buffered saline (PBS, SigmaAldrich Co., St. Louis, MO, USA) followed by 4% paraformaldehyde (Sigma, MO, USA) in 0.1 M PB (pH 7.4; Sigma-Aldrich Co., St. Louis, MO, USA). The brains were removed, and postfixed in the same fixative for 4 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter the tissues were frozen and sectioned with a cryostat at 30 m and consecutive sections were collected in six-well plates containing PBS. The sections were first incubated with 3% bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) in PBS for 30 min at room temperature. Sections were then incubated in primary antibodies (listed below) in PBS containing 0.3% Triton X-100 (SigmaAldrich Co., St. Louis, MO, USA) overnight at room temperature: rabbit anti-AQP1 (Abcam, Cambridge, UK, diluted 1:200), AQP4 (Chemicon, CA, USA, diluted 1:200) or AQP9 IgG (LifeSpan, Seattle, WA, USA, diluted 1:200). The sections were washed three times for 10 min with PBS, incubated sequentially, in biotinylated horse anti-mouse or goat anti-rabbit IgG (Vector, Burlingame, CA, USA) and ABC complex (Vector, Burlingame, CA, USA), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with PBS three
times for 10 min each. The sections were visualized with 3,3=diaminobenzidine (DAB, Sigma-Aldrich Co., St. Louis, MO, USA) in 0.1 M Tris buffer and mounted on gelatin-coated slides. The immunoreactions were observed under the Axiophot microscope (Carl Zeiss, Munchen-Hallbergmoos, Germany). All images were captured using an Axiocam HRc camera and Axio Vision 3.1 software. Double immunofluorescent staining for AQP1, AQP4 or AQP9/GFAP was also performed. Brain tissues were incubated in mixture of rabbit anti-AQP1, AQP4 or AQP9 IgG (diluted 1:50)/ mouse anti-GFAP IgG (diluted 1:100) overnight at room temperature. After washing three times for 10 min with PBS, sections were also incubated in a mixture of FITC- and Cy3-conjugated secondary antisera (1:200, Amersham, PA, USA) for 1 h at room temperature. Sections were mounted in Vectashield mounting medium (Vector, Burlingame, CA, USA). All images were captured using an Axiocam HRc camera and Axio Vision 3.1 software. In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of the primary antibody (for GFAP) or a pre-absorption test was performed with control peptide (for AQPs). The control for immunohistochemistry resulted in the absence of immunoreactivity in any structure (data not shown). All experimental groups in the present study were included in each immunochemistry and were therefore processed under the same conditions.
Quantification of data and statistical analysis For quantification of GFAP/AQPs double immunofluorescence, we have performed the cell count. GFAP and AQP immunofluorescent images (10 sections/rat) were captured in the same region (500⫻500 m). Images were sampled from at least five different points within each hippocampal section. Thereafter, the number of GFAP positive cells that are each AQP positive was actually counted within the sampled images. All immunoreactive cells were counted regardless the intensity of labeling. The number of vacuolized GFAP positive cells was counted by the same method. The diameter of vacuoles in astrocytes was also measured by Axio Vision 3.1 software. Cell counts and the measurement of the diameter of vacuoles were performed by two different investigators who were blind to the classification of tissues. All data obtained from the quantitative measurements were analyzed using one-way ANOVA to determine statistical significance. Bonferroni’s test was used for post hoc comparisons. A P-value below 0.05 was considered statistically significant (Kim JE et al., 2008, 2009).
RESULTS Coagulative necrosis of astrocytes in the CA1 region Generally, vacuolization of cells may be considered as early stage of necrosis, because these cells often show necrotic features, such as eosinophilic cytoplasm, mitochondrial/nuclear membrane alterations, or TUNEL negativity (Deloncle et al., 2001; Sugawara et al., 2002). Similarly, astroglial hypertrophy and vacuolization are reported in various neurodegenerative diseases (Deloncle et al., 2001; Sugawara et al., 2002; Tomimoto et al., 1997). In our previous study (Kim DS et al., 2008), we had also reported that prolonged recurrent seizure resulted in TUNEL negative loss/vacuolization of CA1 astrocytes (presumably delayed necrotic astroglial damage). In the present study, at 6 – 8 weeks after SE we identified vacuolized astrocytes only in the stratum radiatum of the CA1 region by H & E staining. On the basis of the localization and nuclear size/ shape, we could detect astrocytes on H-E-stained slides.
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Briefly, most neurons have euchromatic nucleus with a prominent nucleolus. The hippocampal neurons are usually localized in the stratum pyramidale, while a few neurons are localized in the stratum radiatum. Astrocytes have small nuclei (approximate 10 m in diameter) among the larger nuclei of neurons. In addition, astrocytes are abundantly localized in the stratum radiatum. The nuclei of oligodendroglia are smaller and more intensely stained than are the nuclei of astrocytes. Furthermore, oligodendroglia localized mainly close to the neuronal cell body. Microglia have small, dense and elongated shapes. Their nuclei show highly condensed chromatin and an elongated shape along the axis of the cell body. Furthermore, vacuolized cells showed only GFAP immunoreactivity (see below). These immunostaining results permit identification of astrocytes in H-E staining. Vacuolized astrocytes showed coagulative necrosis that is characterized by preservation of cell outline, watery cytoplasmic staining and nuclear dissolution. The diameter of vacuoles in these astrocytes was 2.3⫾1.49 m (Fig. 1A). The derivations of vacuoles in CA1 astrocytes In control animals, we observed light GFAP immunoreactivity in astrocytes over the hippocampus (Fig. 1B). One week after SE the processes of glial fibrillary acidic protein immunoreactive (GFAP⫹) astrocyte became unevenly thick with ragged edges in the CA1 region (data not shown). Six to eight weeks after SE, vacuolized GFAP⫹ astrocytes were observed only in the stratum radiatum of the CA1 region (Fig. 1C–I). Since GFAP immunohistochemistry clearly showed astroglial vacuolization rather than classic histological stains, we could detect small vacuoles (diameter, ⬍1.5 m) in hypertrophic GFAP⫹ astrocytes, which could not be observed by H&E stain. The fractions of vacuolized astrocytes in total astrocytes were 17.6, 23.4 and 21.4% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 1J). Based on the size of vacuoles and the GFAP immunoreactivity, vacuolized GFAP⫹ astrocytes were divided into three groups: one had largesized vacuoles (diameter ⬎2 m), round-shaped cell body, short blunt processes and GFAP tangles in the cytoplasm. GFAP immunoreactivity was unevenly detected in the edematous cell bodies. Based on these morphological evidences, astrocytes containing large vacuoles may be degenerative astrocytes. GFAP immunoreactivity in these cells was lower than that in other astrocytes (Fig. 1E). Another group had medium-sized vacuoles (1 m⬍ diameter⬍2 m) that were evenly widespread through cytoplasm. Medium-sized vacuoles were in the peripheral regions and processes. Some vacuoles were attached to the cell membrane. GFAP immunoreactivity in these cells was higher than that in astrocytes containing large vacuoles (Fig. 1H). The third group had small-sized vacuoles (diameter ⬍0.5 m) that were clearly observed in longcurled processes. GFAP immunoreactivity was observed frequently in the cell bodies rather than processes (Fig. 1I). In the dentate gyrus, GFAP⫹ astrocytes showed typical reactive gliosis (hypertrophy and hyperplasia of cell bodies
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and processes of astrocytes). There was no vacuolized astrocyte in this region (data not shown). AQP1 immunoreactivity In control animals, AQP1 immunoreactivity was not observed in the hippocampus (Fig. 2A), whereas AQP1 immunoreactivity was strongly detected in choroid plexus (Fig. 2B). This AQP1 labeling was also observed in experimental groups and was independent of SE until 4 weeks after SE (data not shown). Six to 8 weeks after SE, AQP1 immunoreactivity was mainly, but not always, detected in vacuolized astrocytes that were localized in the stratum radiatum of the CA1 region (Figs. 2C–E and 3A). The fractions of AQP1 immunoreactive astrocytes in total astrocytes were 14.5, 21.2 and 17.8% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 5). AQP4 immunoreactivity In controls, AQP4 immunoreactivity was diffusely observed in the hippocampus (Fig. 4A). Double immunofluorescent study revealed that AQP4 immunoreactivity was obviously observed in the end-feet of all astrocytes, leaving unstained the neuronal somata (Fig. 3B). Six to 8 weeks after SE, AQP4 immunoreactivity was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in nonvacuolized astrocytes was increased as compared to the control level (Figs. 3C and 4B). Therefore, the fractions of AQP4 immunoreactive astrocytes in total astrocytes were reduced to 70.3, 65.3 and 53.6% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 5). However, vessellike structures showed strong AQP4 immunoreactivity without GFAP immunoreactivity. AQP9 immunoreactivity Few AQP9 immunoreactive astrocytes were observed in the hippocampus of control group (data not shown). Six to eight weeks after SE, AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes (Figs. 3D and 4C). The fractions of AQP9 immunoreactive astrocytes in total astrocytes were reduced to 27.4, 28.4 and 29.3% at 6 weeks 7 weeks and 8 weeks after SE, respectively (Fig. 5).
DISCUSSION Pilocarpine-induced SE results in hypotension, hyperpyrexia, hypoglycemia, acidosis and hypoxia (Turski et al., 1989; Walker et al., 2002). Since coagulative necrosis is typically seen after hypoxic damages, it is likely that astroglial coagulative necrosis would be simply implied as the result from SE. Vacuolar degeneration is considered as early stage of necrosis. Vacuolized cells show necrotic features, such as eosinophilic cytoplasm, mitochondrial/ nuclear membrane alterations or TUNEL negativity (StruysPonsar et al., 1994; Tomimoto et al., 1997; Deloncle et al., 2001; Sugawara et al., 2002; Kim DS et al., 2008). Consistent with these previous studies, we could observe that the astrocytes in the CA1 region had pyknotic nucleus,
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Fig. 1. Vacuolized astrocytes in the CA1 region at 6 – 8 weeks following SE (A, H &E staining; B–I, GFAP immunostaining) As compared to control (B), vacuolized astrocytes are observed in the CA1 region at 6 – 8 weeks after SE (C–I). The insets in C, D, F and G indicate the high magnifications of the rectangles. (J) The fractions of vacuolized astrocytes in total astrocytes following SE (% of control). Arrows indicate vacuoles in astrocytes. Scale bar⫽5 m (A, E, H, I) and 25 m (B–D, F, G).
edematous eosinophilic cytoplasm and large-sized vacuoles at 6 weeks after SE. Immunostaining for GFAP showed that these astrocytes had a round-shaped cell body, short blunt processes and GFAP tangles in the cytoplasm. In the present study, however, histopathological changes were not observed prior to 6 weeks after SE. Therefore, our findings suggest that in necrotic CA1 astroglial damage may be a consequence of prolonged seizure
activity, not of SE (Kim DS et al., 2008). Indeed, Fabene et al. (2007) reported that the cerebral cortex responds in different ways to pilocarpine stimulation, with ischemic, necrotic processes in the subgranular layers and excitotoxic, apoptotic cell death in the supragranular layers. In addition, these degenerative pathways happen at different time points, with a delayed degeneration in the supragranular layers. Thus, the present findings suggest that the
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Fig. 2. AQP1 immunoreactivity in the rat hippocampus. In control animals (A, B), AQP1 immunoreactivity is strongly detected only in choroid plexus (B). As compared to control (A), AQP1 immunoreactivity is mainly detected in vacuolized astrocytes at 6 – 8 weeks after SE (C–E). Arrows indicate vacuolized astrocytes. The insets in C indicate the high magnification of the rectangle (D, E). Scale bar⫽200 m (A, C) and 25 m (B, D, E).
repeated recurrent seizure activity, not only SE, may play a role in the mechanism of CA1 astroglial loss. Brain edema after injury initially involves astroglial swelling occurring in both gray and white matter (Kimelberg, 1995). This activity-induced astroglial swelling is due to water influx (Walz, 1987, 1992). AQP4 is a key molecule for maintaining water balance, and its dysfunction or structural damage causes cytotoxic edema (Kimelberg, 1995; Kimelberg and Ransom, 1986; Mongin and Kimelberg, 2004). In the present study, AQP4 immunoreactivity was markedly reduced in vacuolized CA1 astrocytes. Astroglial vacuolization is an early stage of necrosis (Deloncle et al., 2001; Sugawara et al., 2002). Similar to in the present study, we reported TUNEL negative vacuolization of astrocytes (presumably delayed necrotic astroglial damage) in the CA1 region (Kim DS et al., 2008). Furthermore, Eid et al. (2005) reported that the loss of perivascular AQP4 in mesial temporal lobe epilepsy patients results in a perturbed flux of water through astrocytes leading to an impaired buffering of extracellular K⫹ and an increased propensity for seizures, although Lee et al. (2004) reported that a significant increase in AQP4 is observed in sclerotic, but not in non-sclerotic, hippocampi obtained from patients with medically intractable temporal lobe epilepsy. AmiryMoghaddam et al. (2003) also reported that the anchoring of AQP4 to ␣-syntrophin may be a target for treatment of brain edema. With respect to previous reports described above, reduced AQP4 immunoreactivity in vacuolized astrocytes may be correlated to irreversible cytotoxic edema of astrocytes. Astroglial swelling subsequently or simultaneously leads to dysfunction of astroglial membrane permeability and impairment of re-uptake of excitatory neurotransmitters or ions by astrocytes (Kimelberg, 2000), which can synchronize reverberant epileptiform discharges of
neurons in the CA1 region (Walz, 1989; Gabriel et al., 1998; D’Antuono et al., 2002; Xu et al., 2007). Indeed, Zeng et al. (2007) reported that in AQP4 knockout mice astroglial glutamate transporter, GLT-1, was downregulated as compared to wild-type animals. In addition, AQP4 knockout astrocytes showed a lower uptake capability of glutamate. Therefore, reduced AQP4 immunoreactivity in astrocytes may be one of the phenotypes of coagulative necrosis of astrocytes or at least of astroglial dysfunctions. However, non-vacuolized astrocytes showed strong AQP4 immunoreactivity as compared to control level. Therefore, upregulated AQP4 immunoreactivity in remaining nonvacuolized reactive astrocytes (particularly in endfeet) may be a compensatory response for loss of astrocytes. In the present study, furthermore, AQP4 immunoreactivity was increased in the background that was GFAP-negative vessel-like structures. This increased AQP4 immunoreactivity may be due to neovascularization. This is because neovascularization is one of the phenomena in chronic epileptic hippocampus (Marcon et al., 2009; Rigau et al., 2007), and endothelial cells in neovasculatures showed overexpression of AQP4, unlike normal endothelial cells (Sawada et al., 2007). Further research is needed to elucidate the cause of increased AQP4 immunoreactivity in non-GFAP structures. AQP1 and AQP9 are upregulated in astrocytes following various CNS damage including subarachnoid hemorrhage, transient focal ischemia and brain tumor, although their expression is rarely observed in the normal brain (Badaut et al., 2001, 2003; Ribeiro et al., 2006). In the present study, AQP1 and AQP9 immunoreactivity was upregulated in the CA1 region. Unexpectedly, upregulated AQP1 immunoreactivity in CA1 astrocyte was correlated to vacuolization in astroglial cytoplasm, while increased
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Fig. 3. Double immunofluorescent staining for AQPs (red) and GFAP (green) in the hippocampus. Six to eight weeks after SE, AQP1 immunoreactivity is mainly detected in vacuolized GFAP⫹ astrocytes (A). In control animals, AQP4 immunoreactivity is observed in the perivascular GFAP⫹ astrocyte endfeet (B). AQP4 immunoreactivity is decreased in vacuolized astrocytes (arrows), while its immunoreactivity is increased in non-vacuolized astrocytes (arrowheads) at 6 – 8 weeks after SE (C). Six to eight weeks after SE, AQP9 immunoreactivity is shown to be mainly induced in non-vacuolized CA1 astrocytes (D). The insets in C indicate the high magnification of the rectangle. (A3–D3) Merged-images of A1–D1 and A2–D2. Scale bar⫽20 and 10 m (insets). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
AQP9 immunoreactivity was observed in non-vacuolized CA1 astrocytes. Similar to the present study, AQP1 immunoreactivity was markedly increased in the astrocytes of patients with intractable epilepsy (Zhou et al., 2008). In glioma cells, Endo et al. (1999) suggested that AQP1 might allow to glioma cells to shrink by extruding water, thereby permitting invasion of the surrounding brain through the extracellular matrix. Therefore, over-expression of AQP1 immunoreactivity in vacuolized astrocytes
may be a compensatory response for maintenance of hydrostatic pressure in swollen astrocytes, when AQP4 was absent. Alternatively, over-expression of AQP1 immunoreactivity in vacuolized astrocytes may be an apoptotic event. This is because Jablonski et al. (2004) reported that AQP1-mediated water loss is important for the apoptotic volume decrease and downstream apoptotic events, and that the water permeability of the plasma membrane can control the rate of apoptosis.
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Fig. 4. AQP4 (A, B) and AQP9 (C) immunoreactivity in the hippocampus. As compared to control (A), AQP4 immunoreactivity decreased in small areas (presumably vacuolized astrocytes, arrows) at 6 – 8 weeks after SE (B). However, AQP9 immunoreactivity is detected only in non-vacuolized CA1 astrocytes (C). The insets in A1–C1 indicate the high magnification of the rectangle. Scale bar⫽200 m (A1–C1) and 25 m (A2–C2, A3–C3).
On the other hand, AQPs have been subdivided into three groups according to permeability characteristics (Verkman and Mitra, 2000): AQP0, AQP1, AQP2, AQP4, AQP5 and AQP6 are permeable to water; AQP3, AQP7 and AQP8 are permeable to water, glycerol, and urea
(aquaglyceroporins); AQP9 is permeable to water, glycerol, urea, purines, pyrimidines, and monocarboxylates (neutral solute channel). Thus, AQP9 is not only implicated in water movements during edema formation, but also plays a role as a metabolite channel in the brain, facilitating
Fig. 5. The fractions of AQP1, AQP4 and AQP9 immunoreactive astrocytes in total astrocytes (% of control). As compared to controls, the numbers of AQP1 and AQP9 immunoreactive astrocytes are increased in the hippocampus, while that of AQP4 immunoreactive astrocytes is reduced.
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the diffusion of glycerol and lactate (Badaut et al., 2004; Badaut and Regli, 2004). In the ischemic condition, AQP9 in reactive astrocytes is involved in the elimination of excess glycerol and lactate from extracellular space (Ribeiro et al., 2006). AQP9 permeability to lactate increases fourfold when pH decreases to 5.5 (Tsukaguchi et al., 1998). Therefore, lactic acidosis during ischemia increases the permeability of AQP9 and enables uptake of the excess lactate by the astrocytes (Ribeiro et al., 2006). Similar to the ischemic condition, metabolic disturbances are commonly observed in chronic epilepsy rats (Melø et al., 2005; Dubé et al., 2001). In particular, Pereira de Vasconcelos et al. (2002) reported that chronic epilepsy rats showed pronounced mismatch between blood supply and metabolic demand. In this condition, an accumulation of CO2 and metabolic production of lactic acid from active cells (Chesler and Kaila, 1992) result in extracellular acidification in the CA1 region (Xiong and Stringer, 2000). Based on these previous studies, the present findings indicate that upregulated AQP9 in non-vacuolized astrocytes may play a role in inhibition of extracellular lactic acidosis induced by repeated spontaneous seizures.
CONCLUSION In conclusion, AQP subunits may play differential roles in various astroglial responses including astroglial swelling and astroglial loss in the chronic epileptic hippocampus. Therefore, our findings suggest that the selective regulation of AQP subunit functions may provide new therapeutic approaches to epilepsy. Acknowledgments—This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (grant number A084589).
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(Accepted 13 July 2009) (Available online 18 July 2009)