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Manganese induces cell swelling in cultured astrocytes
NeuroToxicology 28 (2007) 807–812
Manganese induces cell swelling in cultured astrocytes K.V. Rama Rao b,*, P.V.B. Reddy b, A.S. Hazell d, M.D. Norenberg a,b,c a
Veterans Affairs Medical Center, University of Miami School of Medicine, Miami, FL 33125, United States b Department of Pathology, University of Miami School of Medicine, Miami, FL 33125, United States c Department of Biochemistry & Molecular Biology, University of Miami School of Medicine, Miami, FL 33125, United States d Department of Medicine, University of Montreal Saint-Luc Hospital, Montreal, Que. H2X 3J4, Canada Received 19 February 2007; accepted 1 March 2007 Available online 4 March 2007
Abstract Manganese in excess is neurotoxic and causes a CNS disorder that resembles Parkinson’s disease (manganism). Manganese highly accumulates in astrocytes, which renders these cells more vulnerable to its toxicity. Consistent with this vulnerability, manganese has been shown to cause histopathological changes in astrocytes (Alzheimer type II change), generates oxidative stress and bring about mitochondrial dysfunction, including the induction of the mitochondrial permeability transition (mPT) in astrocytes. In addition to manganism, increased brain levels of manganese have been found in hepatic encephalopathy, a chronic neurological condition associated with liver dysfunction, wherein Alzheimer type II astrocytic changes are also observed. As low-grade brain edema, possibly secondary to astrocyte swelling, has been reported in hepatic encephalopathy, we hypothesized that manganese may contribute to such edema. We therefore exposed cultured astrocytes to manganese (Mn3+) acetate (25 and 50 mM) for different time periods and examined for changes in cell volume. Manganese dose-dependently induced astrocyte swelling; such swelling was first observed at 12 h (28%), which further increased (54%) at later time points (24–48 h). Pretreatment of astrocyte cultures with antioxidants, including vitamin E, the spin trapping agent PBN, and the iron-chelating agent desferroximine, as well as the nitric oxide synthase inhibitor L-NAME, all significantly blocked (50–80%) astrocyte swelling caused by manganese, suggesting that oxidative/ nitrosative stress is involved in the mechanism of such swelling. Cyclosporin A, an inhibitor of mPT also blocked (90%) manganese-induced astrocyte swelling. The data indicate that manganese exposure results in astrocyte swelling and such swelling, at least in part, may be caused by oxidative stress and/or mPT. Astrocyte swelling by manganese may represent an important aspect of manganese neurotoxicity, and may be a factor in low-grade brain edema associated with chronic hepatic encephalopathy. # 2007 Elsevier Inc. All rights reserved. Keywords: Ammonia; Antioxidants; Astrocyte swelling; Brain edema; Cell volume; Cyclosporin A; Desferroximine; Hepatic encephalopathy; Manganese; Manganese neurotoxicity; Mitochondrial permeability transition; Oxidative stress
1. Introduction Manganese is an essential trace metal and is an integral component of key enzymes such as glutamine synthetase (Wedler et al., 1982) and mitochondrial superoxide dismutase (McCord, 1976). However, excess deposition of manganese in the CNS leads to neurological abnormalities (manganism) which is associated with hypokinesia, rigidity and tremor, symptoms that resemble those of Parkinson’s disease (Graham,
* Corresponding author at: Department of Pathology, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, United States. Tel.: +1 305 324 4455x6397; fax: +1 305 585 5311. E-mail address: [email protected] (K.V. Rama Rao). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.03.001
1981; Olanow, 2004). Manganese toxicity is also a potential occupational health hazard in workers in manganese ferro alloy plants and welding factories (Gorell et al., 1999). Additionally, manganese pollution has been a subject of environmental concern because of the wide use of methylcyclopentadienyl manganese tricarbonyl, a manganese derivative that is used as an antiknock agent in automobile fuels (Kaiser, 2003). Manganese in the brain is preferentially deposited in astrocytes because of the presence of high capacity transporters in these cells (Aschner et al., 1992, 1999). Such preferential accumulation suggests that astrocytes may be more vulnerable to manganese toxicity than other neural cells. Consistent with this possibility, primate models of manganese toxicity have shown astrocytic pathological alterations (Alzheimer type II change) (Olanow et al., 1996; Pentschew et al., 1963).
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While mechanisms of manganese neurotoxicity are not completely understood, oxidative stress has been implicated in its toxicity (Chen and Liao, 2002; Erikson et al., 2004; Jayakumar et al., 2004). Oxidative stress by manganese is enhanced by a transitional shift of manganese from one valancy to higher one which increases the prooxidant capacity of this metal as shown by the ability of Mn3+ causing more cellular damage than Mn2+ (Reaney et al., 2002). Such oxidative stress contributes to cell injury including the induction of the mitochondrial permeability transition (mPT) and cell swelling as discussed below. Manganese is also known to cause mitochondrial dysfunction (Gavin et al., 1990), including the inhibition of enzymes of TCA cycle (Malthankar et al., 2004; Zheng et al., 1998; Zwingmann et al., 2004), and a reduction in the activities of the electron transport chain (Malecki, 2001), ultimately resulting in ATP depletion (Brouillet et al., 1993; Verity, 1999). Some of these mitochondrial events are significantly blocked by antioxidants (Chen and Liao, 2002), suggesting the involvement of oxidative stress in the mechanism of mitochondrial dysfunction. Further, recent studies have shown that manganese induces the mPT in cultured astrocytes which may also contribute to mitochondrial dysfunction (Rao and Norenberg, 2004). One neurological disorder associated with an abnormal accumulation of manganese in brain is hepatic encephalopathy (HE), a condition occurring in patients with chronic liver disorders (Barron et al., 1994; Krieger et al., 1995; Layrargues et al., 1998). Brain manganese accumulation is believed to be due to elevated blood levels of manganese resulting from impaired biliary elimination as a consequence of cirrhosis. Similar to manganese toxicity, HE is also associated with astrocytic abnormalities, including the development of Alzheimer type II astrocytosis (Norenberg, 1981). While astrocyte swelling represents a well known component of the brain edema in the acute form of HE (fulminant hepatic failure), increased intracranial pressure and brain herniation associated with astrocytic swelling are not found in chronic HE. Nevertheless, employing proton magnetic resonance spectroscopic studies (1H-MRS), low levels of brain myo-inositol were reported in patients with HE (Ha¨ussinger and Gerok, 1994; Laubenberger et al., 1997; Co´rdoba et al., 2002), suggesting the presence of low-grade brain edema, presumably due to astrocyte swelling. In view of similar clinical and pathological features between manganese toxicity and hepatic encephalopathy, we examined whether astrocyte swelling is also a feature of manganese toxicity. Our studies demonstrate that exposure of cultured astrocytes to pathophysiologically relevant concentrations of manganese causes a dose- and time-dependent swelling of astrocytes, and that such swelling appears to be a consequence of oxidative stress and the mPT. 2. Experimental procedures 2.1. Astrocyte cultures Primary cultures of astrocytes were prepared from cerebral cortices of 1–2 day old rats by the method of Ducis et al. (1989). Briefly, cortices were freed of meninges, minced and dissociated
by trituration, passed through sterile nylon sieves and then placed in Dulbecco’s modified Eagle medium (DMEM) containing penicillin, streptomycin, and 15% fetal bovine serum. Approximately 0.5 106 cells were seeded in 35 mm culture plates and maintained at 37 8C in an incubator equilibrated with 5% CO2 and 95% air. After 2 weeks, cells were maintained with dibutyryl cAMP and 3–5 week old cells were used for experiments. Cultures consisted of 95–99% astrocytes based on immunohistochemical staining with glial fibrillary acidic protein. 2.2. Cell volume determination Cell volume (intracellular water space) was determined by [3H]-O-methylglucose (OMG) equilibration method (Kletzien et al., 1975), as modified for cell cultures by Norenberg et al. (1991). In brief, astrocytes at different time points of manganese treatment were incubated with [3H] OMG (1 mM containing 1 mCi of radioactive OMG), and at the end of incubation, a small aliquot of medium was saved for specific activity determination. Cultures were washed three times with ice-cold buffer containing 290 mM sucrose, 1 mM Tris–nitrate (pH 7.4), 0.5 mM calcium nitrate and 0.1 mM phloretin. Cells were then harvested in 0.5 ml of 1 N sodium hydroxide and the amount of radioactive OMG (mmol/mg protein) present in cells was determined. As the OMG in the intra- and extra-cellular space is in equilibrium, the data can be expressed as ml/mg cell protein. 2.3. Statistical analysis Data are presented as means S.E.M. of 3–4 separate experiments (consisting of 5–6 individual cultures plates in each experimental group) employing different batches of astrocyte cultures. The data were analyzed by ANOVA followed by Neuman–Keuls multiple test. A p-value <0.05 was considered statistically significant. 3. Results 3.1. Effect of manganese on astrocyte swelling Cultures were treated with manganese (Mn3+) acetate (25 and 50 mM) for 1 day, and cell volume was determined. Concentrations of Mn3+ employed in the present study are typically found in brains of patients dying from HE (Krieger et al., 1995; Layrargues et al., 1995). Manganese dose-dependently caused cell swelling in cultured astrocytes by 50–80% ( p < 0.01) (Fig. 1). We next examined the time course of astrocyte swelling after exposing the cells to 25 mM manganese. Such treatment resulted in a time-dependent increase in astrocyte cell volume beginning at 12 h (30%, p < 0.05) and increasing to 54% ( p < 0.05) at 24 and 48 h (Fig. 2). 3.2. Effect of antioxidants on manganese-induced astrocyte swelling Treatment of cultures with vitamin E (250 mM, 24 h), an inhibitor of lipid peroxidation, blocked manganese-induced
K.V. Rama Rao et al. / NeuroToxicology 28 (2007) 807–812
Fig. 1. Dose-dependent effect of manganese on astrocyte cell volume. Cultures were treated with manganese acetate (Mn3+) for 24 h. *vs. control ( p < 0.01); ** vs. Mn3+ (25 mM) ( p < 0.01).
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Fig. 3. Effect of antioxidants on manganese-induced astrocyte swelling. Cultures were pretreated with PBN (250 mM), vitamin E (Vit E, 250 mM) and desferroximine (DFX, 20 mM) and nitric oxide synthase inhibitor L-NAME (500 mM) 10 min before exposure to Mn3+ (25 mM) and cell volume determined after 24 h. *vs. control ( p < 0.05); yvs. Mn3+ ( p < 0.01–0.05).
3.3. Effect of cyclosporin A (CsA) on manganese-induced astrocyte swelling astrocyte swelling by (80%, p < 0.05). The iron chelating agent desferroximine (20 mM, 24 h) completely blocked the swelling, while the spin-trapping agent N-t-butyl-phenylnitrone (PBN, 250 mM) significantly (70%, p < 0.01) inhibited cell swelling. Similar to antioxidants, treatment of cultures with an inhibitor of nitric oxide synthase, N(G)-nitro-L-arginine methyl ester (L-NAME, 500 mM) also blocked (50%, p < 0.05) the astrocyte swelling (Fig. 3).
Fig. 2. Time-dependent effect of manganese on astrocyte cell volume. Cultures were treated with manganese acetate (Mn3+). *vs. control ( p < 0.01).
The mPT has been implicated in astrocyte swelling in ammonia toxicity (Rama Rao et al., 2003). Since manganese induces the mPT in cultured astrocytes (Rao and Norenberg, 2004), we examined whether CsA, an inhibitor of mPT (Broekemeier et al., 1989), is capable of mitigating astrocyte swelling by manganese. CsA (100 and 500 nM) significantly
Fig. 4. Effect of cyclosporin (CsA) on manganese-induced astrocyte swelling. Cultures were pretreated with CsA and FK 506, 10 min before exposure to Mn3+ and cell volume determined after 24 h. *vs. control ( p < 0.05); yvs. Mn3+ ( p < 0.01–0.05).
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(90%, p < 0.01) blocked cell swelling (Fig. 4). Since in addition to blocking the mPT, CsA also inhibits calcineurin, we examined the effect of FK506 (1 mM), a calcineurin inhibitor that has no effect on the mPT. Treatment with FK506 failed to inhibit manganese-induced astrocyte swelling (Fig. 4) indicating that the effect of CsA on cell swelling was due to its mPT inhibitory effect. 4. Discussion This study demonstrates that exposure of cultured astrocytes to pathophysiological concentrations of manganese results in swelling in a concentration- and time-dependent manner. The ability of antioxidants in attenuating such swelling suggests that oxidative stress plays an important role in the mechanism of swelling. The reduction of swelling by CsA further suggests that the mPT is significantly involved in the mechanism of such swelling. Alzheimer type II astrocytosis is a prominent feature of manganese neurotoxicity in primates (Olanow et al., 1996; Pentschew et al., 1963), and a recent studiy by Hazell et al. (2006) has also demonstrated Alzheimer type II astrocytosis in manganese-treated rats. In the latter study, swollen astrocyte cell bodies and processes were also observed, and such swelling was significantly blocked by antioxidant N-acetylcyteine, suggesting the involvement of oxidative stress in evolution of astrocytic swelling. It is of interest that the Alzheimer type II astrocytic change is also the histopathological characteristic in HE (Norenberg, 1981), wherein manganese accumulation has been reported (Barron et al., 1994; Krieger et al., 1995; Layrargues et al., 1998). The mechanisms by which manganese induces astrocyte swelling are not known. Manganese is known to exert oxidative stress (HaMai and Bondy, 2004). Reduced glutathione levels (Erikson et al., 2005), as well as increased free radical production (Brenneman et al., 1999) were reported in experimental models of manganese neurotoxicity. Similarly, manganese has been shown to produce free radicals in cultured astrocytes (Chen and Liao, 2002; Jayakumar et al., 2004), as well as to decrease the activities of antioxidant enzymes (Chen and Liao, 2002). Increased free radical production and associated oxidative stress can cause swelling in brain slices (Brahma et al., 2000), as well as in cultured astrocytes (Chan et al., 1982; Jayakumar et al., 2006). In the present study, we demonstrate the ability of antioxidants to block astrocyte swelling induced by manganese. Taken together, the data suggest that oxidative stress plays major role in the mechanism of astrocyte swelling. One consequence of oxidative stress is induction of the mitochondrial permeability transition (mPT) (Halestrap et al., 1997). The mPT is a Ca2+-dependent process, characterized by the opening of a permeability transition pore in the inner mitochondrial membrane resulting in a dissipation of the mitochondrial inner membrane potential and a subsequent decrease in oxidative phosphorylation leading to decreased ATP production (Zoratti and Szabo, 1995). Manganese has been shown to cause the mPT in cultured astrocytes (Rao and Norenberg, 2004).
We now demonstrate that treatment of cultured astrocytes with the mPT inhibitor CsA significantly blocked astrocyte swelling by manganese, suggesting the involvement of the mPT in this process. While the mechanism(s) by which mPT contributes to astrocyte swelling is not completely understood, one possibility includes mitochondria dysfunction and associated bioenergetic failure resulting from manganese-induce mPT. It is known that cell volume regulation requires the operation of various ionic pumps resulting in the extrusion of osmotically active amino acids (Kimelberg and Mongin, 1998), all of which require energy (Kimelberg, 1995). Earlier studies demonstrated a close correlation between energy metabolism and cell volume regulation in cultured astrocytes (Olson and Evers, 1992). Since manganese is known to cause mitochondrial dysfunction (Gavin et al., 1990; Malecki, 2001; Malthankar et al., 2004; Zwingmann et al., 2004), resulting in a depletion of cellular ATP (Brouillet et al., 1993; Verity, 1999), such impaired bioenergetics, likely resulting from the induction of the mPT, may contribute to the mechanism of astrocyte swelling. In patients with the chronic form of hepatic encephalopathy, a condition in which brain manganese levels are elevated, 1Hproton magnetic resonance spectroscopic studies have shown low levels of brain myo-inositol, suggesting the presence of cell swelling and low-grade brain edema (Ha¨ussinger et al., 1994; Laubenberger et al., 1997). Although factors associated with low-grade brain edema in HE remain to be determined, it is possible that manganese may be involved in its production. In support of this view, Mossakowski et al. (1983a, b) have shown astrocyte swelling in manganese-treated rats. Low-grade brain edema in chronic HE, presumably on the basis of astrocyte swelling, in contrast to acute HE, is not associated with increased intracranial pressure and brain herniation. Nevertheless, such astrocyte swelling may have profound effects on astrocyte properties since the state of cellular hydration represents a key regulator of cell function, including amino acid and protein synthesis, gene expression, and control of intracellular pH and ion concentrations (Ha¨ussinger et al., 2000). Such metabolic abnormalities in astrocytes may potentially impact on astrocyte–neuronal interactions leading to aberrant neuronal function and neurological symptoms. In summary, manganese exposure results in cell swelling in cultured astrocytes. Such swelling appears to be, at least in part, a consequence of oxidative stress and the mitochondrial permeability transition. Astrocyte swelling may represent an important pathological event associated with manganese toxicity, contributing to the pathogenesis of manganism and hepatic encephalopathy. Acknowledgements The authors thank K. Panneerselvam and A. Fernandez for the preparation of the astrocyte cultures. This work was supported by the Department of Veterans Affairs Merit Review and by NIH Grant No. DK063311. KVR is recipient of grant from The American Liver Foundation.
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Manganese induces cell swelling in cultured astrocytes K.V. Rama Rao b,*, P.V.B. Reddy b, A.S. Hazell d, M.D. Norenberg a,b,c a
Veterans Affairs Medical Center, University of Miami School of Medicine, Miami, FL 33125, United States b Department of Pathology, University of Miami School of Medicine, Miami, FL 33125, United States c Department of Biochemistry & Molecular Biology, University of Miami School of Medicine, Miami, FL 33125, United States d Department of Medicine, University of Montreal Saint-Luc Hospital, Montreal, Que. H2X 3J4, Canada Received 19 February 2007; accepted 1 March 2007 Available online 4 March 2007
Abstract Manganese in excess is neurotoxic and causes a CNS disorder that resembles Parkinson’s disease (manganism). Manganese highly accumulates in astrocytes, which renders these cells more vulnerable to its toxicity. Consistent with this vulnerability, manganese has been shown to cause histopathological changes in astrocytes (Alzheimer type II change), generates oxidative stress and bring about mitochondrial dysfunction, including the induction of the mitochondrial permeability transition (mPT) in astrocytes. In addition to manganism, increased brain levels of manganese have been found in hepatic encephalopathy, a chronic neurological condition associated with liver dysfunction, wherein Alzheimer type II astrocytic changes are also observed. As low-grade brain edema, possibly secondary to astrocyte swelling, has been reported in hepatic encephalopathy, we hypothesized that manganese may contribute to such edema. We therefore exposed cultured astrocytes to manganese (Mn3+) acetate (25 and 50 mM) for different time periods and examined for changes in cell volume. Manganese dose-dependently induced astrocyte swelling; such swelling was first observed at 12 h (28%), which further increased (54%) at later time points (24–48 h). Pretreatment of astrocyte cultures with antioxidants, including vitamin E, the spin trapping agent PBN, and the iron-chelating agent desferroximine, as well as the nitric oxide synthase inhibitor L-NAME, all significantly blocked (50–80%) astrocyte swelling caused by manganese, suggesting that oxidative/ nitrosative stress is involved in the mechanism of such swelling. Cyclosporin A, an inhibitor of mPT also blocked (90%) manganese-induced astrocyte swelling. The data indicate that manganese exposure results in astrocyte swelling and such swelling, at least in part, may be caused by oxidative stress and/or mPT. Astrocyte swelling by manganese may represent an important aspect of manganese neurotoxicity, and may be a factor in low-grade brain edema associated with chronic hepatic encephalopathy. # 2007 Elsevier Inc. All rights reserved. Keywords: Ammonia; Antioxidants; Astrocyte swelling; Brain edema; Cell volume; Cyclosporin A; Desferroximine; Hepatic encephalopathy; Manganese; Manganese neurotoxicity; Mitochondrial permeability transition; Oxidative stress
1. Introduction Manganese is an essential trace metal and is an integral component of key enzymes such as glutamine synthetase (Wedler et al., 1982) and mitochondrial superoxide dismutase (McCord, 1976). However, excess deposition of manganese in the CNS leads to neurological abnormalities (manganism) which is associated with hypokinesia, rigidity and tremor, symptoms that resemble those of Parkinson’s disease (Graham,
* Corresponding author at: Department of Pathology, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, United States. Tel.: +1 305 324 4455x6397; fax: +1 305 585 5311. E-mail address: [email protected] (K.V. Rama Rao). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.03.001
1981; Olanow, 2004). Manganese toxicity is also a potential occupational health hazard in workers in manganese ferro alloy plants and welding factories (Gorell et al., 1999). Additionally, manganese pollution has been a subject of environmental concern because of the wide use of methylcyclopentadienyl manganese tricarbonyl, a manganese derivative that is used as an antiknock agent in automobile fuels (Kaiser, 2003). Manganese in the brain is preferentially deposited in astrocytes because of the presence of high capacity transporters in these cells (Aschner et al., 1992, 1999). Such preferential accumulation suggests that astrocytes may be more vulnerable to manganese toxicity than other neural cells. Consistent with this possibility, primate models of manganese toxicity have shown astrocytic pathological alterations (Alzheimer type II change) (Olanow et al., 1996; Pentschew et al., 1963).
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While mechanisms of manganese neurotoxicity are not completely understood, oxidative stress has been implicated in its toxicity (Chen and Liao, 2002; Erikson et al., 2004; Jayakumar et al., 2004). Oxidative stress by manganese is enhanced by a transitional shift of manganese from one valancy to higher one which increases the prooxidant capacity of this metal as shown by the ability of Mn3+ causing more cellular damage than Mn2+ (Reaney et al., 2002). Such oxidative stress contributes to cell injury including the induction of the mitochondrial permeability transition (mPT) and cell swelling as discussed below. Manganese is also known to cause mitochondrial dysfunction (Gavin et al., 1990), including the inhibition of enzymes of TCA cycle (Malthankar et al., 2004; Zheng et al., 1998; Zwingmann et al., 2004), and a reduction in the activities of the electron transport chain (Malecki, 2001), ultimately resulting in ATP depletion (Brouillet et al., 1993; Verity, 1999). Some of these mitochondrial events are significantly blocked by antioxidants (Chen and Liao, 2002), suggesting the involvement of oxidative stress in the mechanism of mitochondrial dysfunction. Further, recent studies have shown that manganese induces the mPT in cultured astrocytes which may also contribute to mitochondrial dysfunction (Rao and Norenberg, 2004). One neurological disorder associated with an abnormal accumulation of manganese in brain is hepatic encephalopathy (HE), a condition occurring in patients with chronic liver disorders (Barron et al., 1994; Krieger et al., 1995; Layrargues et al., 1998). Brain manganese accumulation is believed to be due to elevated blood levels of manganese resulting from impaired biliary elimination as a consequence of cirrhosis. Similar to manganese toxicity, HE is also associated with astrocytic abnormalities, including the development of Alzheimer type II astrocytosis (Norenberg, 1981). While astrocyte swelling represents a well known component of the brain edema in the acute form of HE (fulminant hepatic failure), increased intracranial pressure and brain herniation associated with astrocytic swelling are not found in chronic HE. Nevertheless, employing proton magnetic resonance spectroscopic studies (1H-MRS), low levels of brain myo-inositol were reported in patients with HE (Ha¨ussinger and Gerok, 1994; Laubenberger et al., 1997; Co´rdoba et al., 2002), suggesting the presence of low-grade brain edema, presumably due to astrocyte swelling. In view of similar clinical and pathological features between manganese toxicity and hepatic encephalopathy, we examined whether astrocyte swelling is also a feature of manganese toxicity. Our studies demonstrate that exposure of cultured astrocytes to pathophysiologically relevant concentrations of manganese causes a dose- and time-dependent swelling of astrocytes, and that such swelling appears to be a consequence of oxidative stress and the mPT. 2. Experimental procedures 2.1. Astrocyte cultures Primary cultures of astrocytes were prepared from cerebral cortices of 1–2 day old rats by the method of Ducis et al. (1989). Briefly, cortices were freed of meninges, minced and dissociated
by trituration, passed through sterile nylon sieves and then placed in Dulbecco’s modified Eagle medium (DMEM) containing penicillin, streptomycin, and 15% fetal bovine serum. Approximately 0.5 106 cells were seeded in 35 mm culture plates and maintained at 37 8C in an incubator equilibrated with 5% CO2 and 95% air. After 2 weeks, cells were maintained with dibutyryl cAMP and 3–5 week old cells were used for experiments. Cultures consisted of 95–99% astrocytes based on immunohistochemical staining with glial fibrillary acidic protein. 2.2. Cell volume determination Cell volume (intracellular water space) was determined by [3H]-O-methylglucose (OMG) equilibration method (Kletzien et al., 1975), as modified for cell cultures by Norenberg et al. (1991). In brief, astrocytes at different time points of manganese treatment were incubated with [3H] OMG (1 mM containing 1 mCi of radioactive OMG), and at the end of incubation, a small aliquot of medium was saved for specific activity determination. Cultures were washed three times with ice-cold buffer containing 290 mM sucrose, 1 mM Tris–nitrate (pH 7.4), 0.5 mM calcium nitrate and 0.1 mM phloretin. Cells were then harvested in 0.5 ml of 1 N sodium hydroxide and the amount of radioactive OMG (mmol/mg protein) present in cells was determined. As the OMG in the intra- and extra-cellular space is in equilibrium, the data can be expressed as ml/mg cell protein. 2.3. Statistical analysis Data are presented as means S.E.M. of 3–4 separate experiments (consisting of 5–6 individual cultures plates in each experimental group) employing different batches of astrocyte cultures. The data were analyzed by ANOVA followed by Neuman–Keuls multiple test. A p-value <0.05 was considered statistically significant. 3. Results 3.1. Effect of manganese on astrocyte swelling Cultures were treated with manganese (Mn3+) acetate (25 and 50 mM) for 1 day, and cell volume was determined. Concentrations of Mn3+ employed in the present study are typically found in brains of patients dying from HE (Krieger et al., 1995; Layrargues et al., 1995). Manganese dose-dependently caused cell swelling in cultured astrocytes by 50–80% ( p < 0.01) (Fig. 1). We next examined the time course of astrocyte swelling after exposing the cells to 25 mM manganese. Such treatment resulted in a time-dependent increase in astrocyte cell volume beginning at 12 h (30%, p < 0.05) and increasing to 54% ( p < 0.05) at 24 and 48 h (Fig. 2). 3.2. Effect of antioxidants on manganese-induced astrocyte swelling Treatment of cultures with vitamin E (250 mM, 24 h), an inhibitor of lipid peroxidation, blocked manganese-induced
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Fig. 1. Dose-dependent effect of manganese on astrocyte cell volume. Cultures were treated with manganese acetate (Mn3+) for 24 h. *vs. control ( p < 0.01); ** vs. Mn3+ (25 mM) ( p < 0.01).
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Fig. 3. Effect of antioxidants on manganese-induced astrocyte swelling. Cultures were pretreated with PBN (250 mM), vitamin E (Vit E, 250 mM) and desferroximine (DFX, 20 mM) and nitric oxide synthase inhibitor L-NAME (500 mM) 10 min before exposure to Mn3+ (25 mM) and cell volume determined after 24 h. *vs. control ( p < 0.05); yvs. Mn3+ ( p < 0.01–0.05).
3.3. Effect of cyclosporin A (CsA) on manganese-induced astrocyte swelling astrocyte swelling by (80%, p < 0.05). The iron chelating agent desferroximine (20 mM, 24 h) completely blocked the swelling, while the spin-trapping agent N-t-butyl-phenylnitrone (PBN, 250 mM) significantly (70%, p < 0.01) inhibited cell swelling. Similar to antioxidants, treatment of cultures with an inhibitor of nitric oxide synthase, N(G)-nitro-L-arginine methyl ester (L-NAME, 500 mM) also blocked (50%, p < 0.05) the astrocyte swelling (Fig. 3).
Fig. 2. Time-dependent effect of manganese on astrocyte cell volume. Cultures were treated with manganese acetate (Mn3+). *vs. control ( p < 0.01).
The mPT has been implicated in astrocyte swelling in ammonia toxicity (Rama Rao et al., 2003). Since manganese induces the mPT in cultured astrocytes (Rao and Norenberg, 2004), we examined whether CsA, an inhibitor of mPT (Broekemeier et al., 1989), is capable of mitigating astrocyte swelling by manganese. CsA (100 and 500 nM) significantly
Fig. 4. Effect of cyclosporin (CsA) on manganese-induced astrocyte swelling. Cultures were pretreated with CsA and FK 506, 10 min before exposure to Mn3+ and cell volume determined after 24 h. *vs. control ( p < 0.05); yvs. Mn3+ ( p < 0.01–0.05).
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(90%, p < 0.01) blocked cell swelling (Fig. 4). Since in addition to blocking the mPT, CsA also inhibits calcineurin, we examined the effect of FK506 (1 mM), a calcineurin inhibitor that has no effect on the mPT. Treatment with FK506 failed to inhibit manganese-induced astrocyte swelling (Fig. 4) indicating that the effect of CsA on cell swelling was due to its mPT inhibitory effect. 4. Discussion This study demonstrates that exposure of cultured astrocytes to pathophysiological concentrations of manganese results in swelling in a concentration- and time-dependent manner. The ability of antioxidants in attenuating such swelling suggests that oxidative stress plays an important role in the mechanism of swelling. The reduction of swelling by CsA further suggests that the mPT is significantly involved in the mechanism of such swelling. Alzheimer type II astrocytosis is a prominent feature of manganese neurotoxicity in primates (Olanow et al., 1996; Pentschew et al., 1963), and a recent studiy by Hazell et al. (2006) has also demonstrated Alzheimer type II astrocytosis in manganese-treated rats. In the latter study, swollen astrocyte cell bodies and processes were also observed, and such swelling was significantly blocked by antioxidant N-acetylcyteine, suggesting the involvement of oxidative stress in evolution of astrocytic swelling. It is of interest that the Alzheimer type II astrocytic change is also the histopathological characteristic in HE (Norenberg, 1981), wherein manganese accumulation has been reported (Barron et al., 1994; Krieger et al., 1995; Layrargues et al., 1998). The mechanisms by which manganese induces astrocyte swelling are not known. Manganese is known to exert oxidative stress (HaMai and Bondy, 2004). Reduced glutathione levels (Erikson et al., 2005), as well as increased free radical production (Brenneman et al., 1999) were reported in experimental models of manganese neurotoxicity. Similarly, manganese has been shown to produce free radicals in cultured astrocytes (Chen and Liao, 2002; Jayakumar et al., 2004), as well as to decrease the activities of antioxidant enzymes (Chen and Liao, 2002). Increased free radical production and associated oxidative stress can cause swelling in brain slices (Brahma et al., 2000), as well as in cultured astrocytes (Chan et al., 1982; Jayakumar et al., 2006). In the present study, we demonstrate the ability of antioxidants to block astrocyte swelling induced by manganese. Taken together, the data suggest that oxidative stress plays major role in the mechanism of astrocyte swelling. One consequence of oxidative stress is induction of the mitochondrial permeability transition (mPT) (Halestrap et al., 1997). The mPT is a Ca2+-dependent process, characterized by the opening of a permeability transition pore in the inner mitochondrial membrane resulting in a dissipation of the mitochondrial inner membrane potential and a subsequent decrease in oxidative phosphorylation leading to decreased ATP production (Zoratti and Szabo, 1995). Manganese has been shown to cause the mPT in cultured astrocytes (Rao and Norenberg, 2004).
We now demonstrate that treatment of cultured astrocytes with the mPT inhibitor CsA significantly blocked astrocyte swelling by manganese, suggesting the involvement of the mPT in this process. While the mechanism(s) by which mPT contributes to astrocyte swelling is not completely understood, one possibility includes mitochondria dysfunction and associated bioenergetic failure resulting from manganese-induce mPT. It is known that cell volume regulation requires the operation of various ionic pumps resulting in the extrusion of osmotically active amino acids (Kimelberg and Mongin, 1998), all of which require energy (Kimelberg, 1995). Earlier studies demonstrated a close correlation between energy metabolism and cell volume regulation in cultured astrocytes (Olson and Evers, 1992). Since manganese is known to cause mitochondrial dysfunction (Gavin et al., 1990; Malecki, 2001; Malthankar et al., 2004; Zwingmann et al., 2004), resulting in a depletion of cellular ATP (Brouillet et al., 1993; Verity, 1999), such impaired bioenergetics, likely resulting from the induction of the mPT, may contribute to the mechanism of astrocyte swelling. In patients with the chronic form of hepatic encephalopathy, a condition in which brain manganese levels are elevated, 1Hproton magnetic resonance spectroscopic studies have shown low levels of brain myo-inositol, suggesting the presence of cell swelling and low-grade brain edema (Ha¨ussinger et al., 1994; Laubenberger et al., 1997). Although factors associated with low-grade brain edema in HE remain to be determined, it is possible that manganese may be involved in its production. In support of this view, Mossakowski et al. (1983a, b) have shown astrocyte swelling in manganese-treated rats. Low-grade brain edema in chronic HE, presumably on the basis of astrocyte swelling, in contrast to acute HE, is not associated with increased intracranial pressure and brain herniation. Nevertheless, such astrocyte swelling may have profound effects on astrocyte properties since the state of cellular hydration represents a key regulator of cell function, including amino acid and protein synthesis, gene expression, and control of intracellular pH and ion concentrations (Ha¨ussinger et al., 2000). Such metabolic abnormalities in astrocytes may potentially impact on astrocyte–neuronal interactions leading to aberrant neuronal function and neurological symptoms. In summary, manganese exposure results in cell swelling in cultured astrocytes. Such swelling appears to be, at least in part, a consequence of oxidative stress and the mitochondrial permeability transition. Astrocyte swelling may represent an important pathological event associated with manganese toxicity, contributing to the pathogenesis of manganism and hepatic encephalopathy. Acknowledgements The authors thank K. Panneerselvam and A. Fernandez for the preparation of the astrocyte cultures. This work was supported by the Department of Veterans Affairs Merit Review and by NIH Grant No. DK063311. KVR is recipient of grant from The American Liver Foundation.
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