Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Naloxone is a highly effective antagonist of the G-protein-linked classic opioid receptors which are widely expressed on cells of the central nervous system as well as peripheral systems (Smith and Lee, 1988; Herz, 1993; Barron, 2000). Opioid receptors are involved in the mediation of a variety of cellular activities including nociceptive/analgesic effect, respiration, ionic channel activity, and immune responses (Roy and Loh, 1996). Binding of naloxone to opioid receptors is stereospecific where only ()-naloxone is effective (Iijima et al., 1978; Marcoli et al., 1989). The affinity of the (+)-enantiomer to opioid receptor, however, is 3 to 4 orders of magnitude less than that of ()-naloxone.

Although (+)-naloxone was synthesized as an inert control compound, several groups have reported equal efficacy for both ()-naloxone and (+)-naloxone in certain nonopiate receptor-mediated situations. For example, Dunwiddie et al. (1982) reported that the (+)- and ()-isomers of naloxone were equally effective in modulating spontaneous hippocampal activity in rats. In protecting cortical neurons against N-methyl-D-aspartate-mediated neurotoxicity, (+)-naloxone was actually more effective than the ()-enantiomer (Kim et al., 1987). Moreover, (+)-naloxone was found to be capable of reducing both cocaine- and amphetamine-induced hyperactivity in mice (Chatterjie et al., 1996, 1998). These studies raise the possibility that naloxone isomers bind to sites other than opioid receptors with no stereospecificity and exert activity that does not involve the opioid receptor system.

We have recently reported that both naloxone stereoisomers are equally potent in protecting nigral dopaminergic neurons against inflammation-mediated degeneration (Liu et al., 2000a,c). Loss of nigral dopaminergic neurons is a hallmark of Parkinson's disease (PD) for which the etiology remains unclear (Olanow and Tatton, 1999). On the other hand, inflammation in the brain has been increasingly associated with the pathogenesis of PD, as well as several other degenerative neurological disorders including Alzheimer's disease (AD) (McGeer and McGeer, 1995). Inflammation in the brain involves mainly the activity of brain microglial and astroglial cells. In response to environmental insults or brain injury, glia, especially microglia, become activated and secrete a host of factors including cytokines, nitric oxide, and reactive oxygen species that impact on neurons to induce neurodegeneration (Liu et al., 2002). Since dopaminergic neurons are known to be especially vulnerable to oxidative stress (Jenner and Olanow, 1996; Greenamyre et al., 1999), inhibition of microglial activation, especially the generation of superoxide by activated microglia, may be an effective strategy for the development of potential therapeutic agents.

We have recently reported that microglia significantly enhance the amyloid peptide A (1-42)-induced degeneration of both cortical and mesencephalic neurons (Qin et al., 2002). Degeneration of cortical and hippocampal neurons is a pathological hallmark of AD (Braak et al., 1996). More interestingly, in addition to AD, recent studies have indicated that amyloid peptides may also be involved in the pathogenesis of other neurodegenerative diseases such as PD (Puglielli and Kovacs, 2001; Small et al., 2001). Therefore, we set out to determine whether naloxone stereoisomers had any effect on A (1-42)-induced degeneration of both cortical and mesencephalic neurons. Mechanistically, the effect of naloxone on the production of reactive oxygen intermediates such as superoxide by A (1-42)-activated microglia was investigated. In this report, we show that naloxone stereoisomers protect both cortical and mesencephalic neurons against A (1-42)-induced degeneration through inhibition of superoxide production in microglia.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents. ()-Naloxone hydrochloride, naloxone methiodide, and partially acetylated ferricytochrome c were purchased from Sigma-Aldrich (St. Louis, MO). (+)-Naloxone was supplied by National Institute of Drug Abuse (Bethesda, MD). Cell culture ingredients were obtained from Invitrogen (Carlsbad, CA). [³H]DA (30 Ci/mmol) and [³H]GABA (90 Ci/mmol) were from PerkinElmer Life Sciences (Boston, MA). Monoclonal antibody against the neuron-specific nuclear protein (Neu-N) was obtained from Chemicon International (Temecula, CA). The polyclonal anti-tyrosine hydroxylase (TH) antiserum was a gift from Dr. John Reiherd of GlaxoSmithKlein (Research Triangle Park, NC). Vectastain avidin-biotinylated enzyme complex and biotinylated horse anti-mouse and goat anti-rabbit secondary antibodies were purchased from Vector Laboratories (Burlingame, CA).

Rat Mesencephalic and Cortical Neuron-Glia Cultures. Primary mesencephalic neuron-glia cultures were prepared from the brains of embryonic day 14/15 Fischer 344 rats following our previously described protocol (Liu et al., 2000a) with modifications (Gao et al., 2002). Primary cortical neuron-glia cultures were prepared from cortical tissues of embryonic day 16/17 Fischer 344 rats as described (Qin et al., 2002). Briefly, mesencephalic or cortical tissues were obtained and dissociated by a mild mechanical trituration. Cells were seeded at 5 × 10⁵/well to 24-well culture plates precoated with poly(D-lysine) (20 µg/ml) and maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air in 0.5 ml/well maintenance medium. The medium consisted of minimum essential medium containing 10% heat-inactivated fetal bovine serum and 10% heat-inactivated horse serum, 1 g/l glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 U/ml penicillin and 50 µg/ml streptomycin. Three days after the initial seeding, 0.5 ml of fresh medium was added to each well. For superoxide assays, 10⁵ cells/0.1 ml of medium were seeded to poly(D-lysine)-coated 96-well culture plates with 0.1 ml/well of fresh medium supplemented 3 days later. Seven-day-old cultures were used for treatment. At the time of treatment, the composition of the mesencephalic neuron-glia cultures was 48% astrocytes, 11% microglia, 40% neurons, and 0.8% dopaminergic neurons. The cortical neuron-glia cultures were made up of 60% neurons, 3% microglia, and 37% astrocytes.

Primary Microglia-Enriched Cultures. Rat microglia-enriched cultures were prepared from whole brains of 1-day old Fischer 344 rat pups as described (Liu et al., 2001b). The cultures were >98% pure for microglia. For superoxide assays, 10⁵ cells/0.1 ml of medium were grown overnight in 96-well culture plates before use.

Treatment. A (1-42) was dissolved in sterile deionized and distilled water as a stock solution (1 mM) and stored in aliquots at -70°C. Before use, the stock solution was aged for 7 days at 37°C. For treatment, the A (1-42) stock solution was diluted to the desired final concentrations in treatment medium (minimum essential medium containing 2% fetal bovine serum, 2% horse serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin). Naloxone analogs were freshly prepared as stock solutions (10 mM) in water and diluted to the desired final concentrations in treatment medium. Neuron-glia cultures in 24-well plates were pretreated for 30 min at 37°C with vehicle control (water) or naloxone prior to treatment with A (1-42) in a final volume of 1 ml/well.

Analysis of Neurotoxicity. Multiple parameters were used to assess the degenerative effect of A (1-42) on neurons in the cultures (Liu et al., 2000a; Gao et al., 2002). Deterioration of dopaminergic and GABAergic neurons was determined by the uptake of [³H]DA and [³H]GABA, respectively. Quantification of neuronal loss was performed by visually counting the number of neurons following immunostaining with neuron-specific antibodies. In addition, degeneration of dopaminergic neuronal dendritic network was determined by measuring the total dendrite length for each neuron following immunostaining (Liu et al., 2001a).

Uptake Assays. [³H]DA or [³H]GABA uptake assays were performed as previously described (Liu et al., 2000a; Gao et al., 2002). Briefly, after washing twice with warm Krebs-Ringer buffer (KRB, 16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4), cultures were incubated for 20 min at 37°C with 1 µM [³H]DA and 5 µM [³H]GABA in KRB for DA uptake or GABA uptake, respectively. Afterward, cultures were washed (three times) with ice-cold KRB and cells were then collected in 1 N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific DA or GABA uptake observed in the presence of mazindol (10 µM) or 1-(2-[([diphenylmethylene]imino)oxy]ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (NO-711, 10 µM) was subtracted.

Immunostaining. Neurons in general were stained with an antibody against Neu-N, a neuron-specific nuclear protein. Dopaminergic neurons were stained with an antibody against TH, the rate-limiting enzyme in dopamine synthesis. Immunostaining was performed following protocols previously described in detail (Liu et al., 2000a). Briefly, formaldehyde (3.7%)-fixed cultures were treated with 1% hydrogen peroxide (10 min) followed by sequential incubation with blocking solution (30 min), primary antibody (overnight, 4°C), biotinylated secondary antibody (2 h), and avidin-biotinylated enzyme complex reagents (40 min). Color was developed with 3,3'-diaminobenzidine. For morphological analysis, the images were recorded with a Nikon inverted microscope connected to a charge-coupled device camera (Dage-MTI, Michigan City, IN) operated through the MetaMorph software (Universal Imaging Corp., West Chester, PA). For visual counting of Neu-N- or TH-immunoreactive (IR) neurons, nine representative areas per well of the 24-well plate were counted under the microscope at 100× magnification. The overall dendrite length of TH-IR neurons was determined as described (Liu et al., 2001a).

Superoxide Assay. The production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (Liu et al., 2000a; Gao et al., 2002). Briefly, cultures were washed (two times) with Hanks' balanced salt solution without phenol red (HBSS). After pretreatment for 30 min with vehicle control or naloxone analogs in 100 µl/well HBSS, the cultures received 50 µl/well of vehicle control or A (1-42) in HBSS followed by 50 µl/well of ferricytochrome c (100 µM) in HBSS with and without SOD (600 U/ml).

Statistical Analysis. The data are expressed as the mean ± S.E.M. Statistical significance was assessed with an analysis of variance followed by Bonferroni's t test using the StatView program (Abacus Concepts, Inc., Berkeley, CA). A value of p < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

A (1-42)-Induced Neurodegeneration Is Concentration- and Time-Dependent. We have recently reported that sub- to low micromolar concentrations of A (1-42) activated microglia to induce degeneration of both dopaminergic and GABAergic neurons (Qin et al., 2002). To characterize the dose and time dependence of A (1-42)-induced degeneration of dopaminergic neurons, rat primary mesencephalic neuron-glia cultures were treated for 9 days with 0.1 to 1 µM A (1-42) or with 0.5 µM A (1-42) for 5 to 11 days. Degeneration of dopaminergic neurons was determined by [³H]DA uptake and counting of TH-IR neurons following immunostaining with the anti-TH antibody. As shown in Fig. 1A, A (1-42)-induced degeneration of dopaminergic neurons was concentration-dependent. Significant damage to dopaminergic neurons was observed in cultures treated with 0.3 µM A (1-42) for 9 days (Fig. 1A). At the highest concentration used (1 µM), A (1-42) resulted in a 75% decrease in [³H]DA uptake and a 45% decrease in the number of TH-IR neurons. Furthermore, A (1-42)-induced dopaminergic neurodegeneration was also time dependent. In cultures treated with 0.5 µM A (1-42), significant decrease in [³H]DA uptake was observed 5 days after treatment and by 11 days, a 50% reduction in [³H]DA uptake was observed (Fig. 1B). Meanwhile, significant loss of TH-IR neurons was detected 9 days after treatment with 0.5 µM A (1-42).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Concentration dependence and time course for A (1-42)-induced degeneration of dopaminergic neurons in mesencephalic neuron-glia cultures. Cultures were treated for 9 days with vehicle or indicated concentrations of A (1-42) (A) or with 0.5 µM A (1-42) for the indicated number of days (B). Afterward, neurotoxicity was assessed by either DA uptake or counting of TH-IR neurons following immunostaining with an anti-TH antibody, as described under Materials and Methods. Results are expressed as a percentage of the control cultures and are mean ± S.E.M. of three experiments performed in triplicate. , p < 0.05 compared with the control cultures.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of A (1-42) on GABA uptake of cortical neuron-glia cultures. Cultures were treated for 11 days with vehicle or the indicated concentrations of A (1-42) (A) or with 1.5 µM A (1-42) for the indicated number of days (B). Afterward, GABA uptake was performed as described under Materials and Methods. Results are expressed as a percentage of the control cultures and are mean ± S.E.M. of three experiments performed in triplicate. , p < 0.05 compared with the control cultures.

Effect of Naloxone Analogs on A (1-42)-Induced Neurodegeneration. Two lines of evidence prompted us to test whether naloxone had any effect on A (1-42)-induced neurodegeneration. First, naloxone attenuates dopaminergic neurodegeneration induced by an inflammagen, lipopolysaccharide (LPS; Liu et al., 2000a,c; Lu et al., 2000). Second, A (1-42) activates microglia to facilitate the degeneration of both dopaminergic and cortical neurons (Qin et al., 2002). Therefore, mesencephalic neuron-glia cultures were pretreated for 30 min with vehicle, ()-naloxone, or (+)-naloxone prior to treatment with 0.75 µM A (1-42) for 9 days. As shown in Fig. 3, both naloxone stereoisomers, in a dose-dependent manner, significantly attenuated A (1-42)-induced decrease in DA uptake. In addition, treatment with the quaternary amine-carrying analog of naloxone, naloxone methiodide (5 µM), also afforded significant neuroprotection (Fig. 3). Treatment with 5 µM ()-naloxone, (+)-naloxone, or naloxone methiodide alone did not significantly affect the DA uptake of cultures (Fig. 3). Immunocytochemical analysis of dopaminergic neurons with an anti-TH antibody revealed that, compared with the control cultures, TH-IR neurons in A (1-42)-treated cultures (0.75 µM; 11 days) were less abundant, and the remaining ones had lost most of the elaborate dendrite network (Fig. 4A). However, TH-IR neurons in cultures treated with 5 µM ()-naloxone or (+)-naloxone prior to A (1-42) treatment were markedly more healthy and had better preserved dendrite network than those in cultures treated with A (1-42) alone (Fig. 4A). Counting of TH-IR neurons in the cultures indicated that a 50% decrease was induced by A (1-42) treatment, and both ()-naloxone and (+)-naloxone significantly attenuated the A (1-42)-induced loss of TH-IR neurons (Fig. 4B). Compared with the loss of TH-IR neurons, the shortening of TH-IR dendrites was even more dramatic, and both ()-naloxone and (+)-naloxone significantly attenuated A (1-42)-induced shortening of TH-IR dendrites (Fig. 4B).

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Effect of naloxone analogs on A (1-42)-induced decrease in DA uptake in mesencephalic neuron-glia cultures. Cultures were pretreated with the indicated concentrations of ()-naloxone, (+)-naloxone, or naloxone methiodide for 30 min before treatment for 9 days with 0.75 µM A (1-42). Afterward, DA uptake was performed. Results are expressed as a percentage of the control cultures and are mean ± S.E.M. of three to four experiments performed in triplicate. A, A (1-42); ()-Nal, ()-naloxone; (+)-Nal, (+)-naloxone; ()-Nal-Met, ()-naloxone methiodide. , p < 0.05 compared with the control cultures; +, p < 0.05 compared with the A (1-42)-treated cultures.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Immunocytochemical analysis and quantification of the effect of naloxone stereoisomers on A (1-42)-induced degeneration of TH-IR neurons. Mesencephalic neuron-glia cultures were pretreated with 5 µM ()-naloxone or (+)-naloxone prior to treatment for 9 days with 0.75 µM A (1-42). Afterward, the cultures were immunostained with an anti-TH antibody. A, morphology. In control cultures, TH-IR neurons were healthy, with an intricate network of TH-IR dendrites. However, TH-IR neurons in A (1-42)-treated cultures were less numerous, with the remainder exhibiting dramatically shortened and damaged dendrites. Significant improvement in the morphology of TH-IR neurons, as judged by the intactness of cell bodies and extensiveness of dendrites, was observed in cultures pretreated with either naloxone isomer prior to A (1-42) treatment. B, quantitative analysis. After immunostaining, the number of TH-IR neurons or the average length of TH-IR dendrites was quantified as described under Materials and Methods. Results are mean ± S.E.M. from two to three experiments performed in duplicate. ()-Nal, ()-naloxone; (+)-Nal, (+)-naloxone. , p < 0.05 compared with the control cultures; +, p < 0.05 compared with the A (1-42)-treated cultures.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   Effect of ()-naloxone on A (1-42)-induced degeneration of cortical neurons. Cortical neuron-glia cultures were pretreated with 1 to 5 µM ()-naloxone prior to treatment for 11 days with 1.5 µM A (1-42). Neurotoxicity was assessed by GABA uptake and counting of Neu-N-IR neurons, as described under Materials and Methods. Results are mean ± S.E.M. from three to four experiments performed in triplicate. , p < 0.05 compared with the control cultures; +, p < 0.05 compared with the A (1-42)-treated cultures.

Naloxone Analogs Inhibit A (1-42)-Induced Production of Superoxide Free Radical in Microglia. It has recently been demonstrated that the release of superoxide from microglia is a contributor to the microglia-enhanced degeneration of cortical and mesencephalic neurons induced by A (1-42) (Qin et al., 2002). Therefore, the effect of naloxone on A (1-42)-induced production of superoxide in neuron-glia- and microglia-enriched cultures was determined. Cultures were pretreated for 30 min with 1 to 5 µM ()-naloxone prior to stimulation with 0.3 µM A (1-42). The production of superoxide was measured as the SOD-inhibitable reduction of cytochrome c. As shown in Fig. 6A, ()-naloxone inhibited, in a concentration-dependent manner, A (1-42)-stimulated superoxide production. Next, we compared the effect of naloxone analogs on A (1-42)-induced superoxide production in microglia and mesencephalic neuron-glia cultures. Significant inhibition of A (1-42) (0.3 µM)-induced superoxide production was observed with 5 µM ()-naloxone, (+)-naloxone, or naloxone methiodide in both microglia-enriched and neuron-glia cultures (Fig. 6B).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of naloxone analogs on A (1-42)-induced production of superoxide. A, mesencephalic neuron-glia cultures were pretreated for 30 min with the indicated concentrations of ()-naloxone prior to stimulation with 0.3 µM A (1-42). Production of superoxide was measured as SOD-inhibitable reduction of cytochrome c, as described under Materials and Methods. B, mesencephalic neuron-glia cultures or microglia-enriched cultures were pretreated for 30 min with a 5 µM concentration of the indicated naloxone analogs prior to stimulation with 0.3 µM A (1-42). Superoxide generation was then measured. The results are expressed as a percentage of the control cultures and are the mean ± S.E.M. of three to four experiments performed in triplicate. , p < 0.05 compared with the control cultures; +, p < 0.05 compared with the A (1-42)-treated cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have demonstrated that the degeneration of cortical GABAergic and mesencephalic dopaminergic neurons induced by A (1-42) can be significantly attenuated by both ()-naloxone and (+)-naloxone, as well as the quaternary amine-bearing naloxone methiodide. The underlying mechanism of action responsible for the neuroprotective effect of naloxone analogs may be related to their inhibition of A (1-42)-induced production of superoxide free radical by activated microglia.

The observations that naloxone stereoisomers are capable of reducing microglial activation and subsequent degeneration of both cortical and mesencephalic neurons induced by either the inflammagen LPS (Liu et al., 2000a,b) or the amyloid peptide A (1-42) (this study) have several potentially important implications. First, since neuroprotection and inhibition of microglial activation are obtained with both the opioid receptor antagonists ()-naloxone and (+)-naloxone, these observed effects are unlikely mediated through interaction with the opioid receptor system, but rather through yet undefined novel mechanisms of action.

Second, the neuroprotective effect of naloxone is not limited to neurodegeneration induced by the inflammagen LPS alone. Previously, we have suggested that interference with the assembly of cell surface receptor complex, which is a prerequisite for the initiation of the LPS signaling pathway, may be partially responsible for the inhibition by naloxone stereoisomers of the LPS-stimulated microglial activation (Liu et al., 2000a). The ability of naloxone to attenuate A (1-42)-induced microglial activation suggests the existence of a unique target for naloxone since LPS and A (1-42) activate microglia by interacting with distinct cell surface molecules. Although multiple pathways for LPS signaling have been suggested, the best characterized is the pathway that requires the binding of LPS to a soluble LPS-binding protein which, in turn, binds a cell surface protein (CD14). Transduction of signal across the cell membrane is accomplished by a recently discovered Toll-like protein that initiates the production of a variety of immune modulators (Hoffmann et al., 1999). Less is known regarding the activation of microglia by amyloid peptides. However, several recent studies have provided important insights. For example, Tan et al. (1999) reported that interaction of A with the cell surface molecule CD40 can lead to the activation of microglia. Therefore, naloxone most likely intercepts at a converging point downstream of the binding of LPS or A (1-42) to the cell surface receptor(s). The fact that the charged naloxone methiodide is also capable of inhibiting A (1-42)-induced microglial activation implies that the target(s) of interaction is at the cell surface.

Third, the ability of naloxone to inhibit microglial activation and attenuate neurodegeneration induced by LPS or A (1-42) suggests that microglial activation is a key component of the neurodegenerative process. Furthermore, inhibition of microglial activation by naloxone can be a very promising strategy in the search for potential therapeutic regimens. Microglia, under physiological conditions, serve a role of immune surveillance. Under pathological conditions such as infection and brain injury, microglia become readily activated (Kreutzberg, 1996). Increasing evidence suggests that microglial activation plays an active role in a variety of neurodegenerative processes. In neuron-glia cultures, microglial activation has been associated with neurodegeneration induced by LPS, the human immunodeficiency virus-1 coat protein gp120, -amyloid peptides, and the pesticide rotenone (Boje and Arora, 1992; Chao et al., 1992; Dawson et al., 1993; Dickson et al., 1993; Ii et al., 1996; Jeohn et al., 1998; Liu et al., 2000a; Gao et al., 2002; Qin et al., 2002). In animal models and humans, microglial activation is frequently observed during the development of neurodegenerative diseases including PD, AD, amyotrophic lateral sclerosis, and multiple sclerosis, as well as reperfusion brain ischemia (McGeer et al., 1988; Compston, 1992; Rogers et al., 1992; Benveniste et al., 2001). Therefore, naloxone may have a broader than previously thought range of efficacy as a potential agent for the treatment of multiple disorders.

Fourth, among the wide spectrum of proinflammatory and potentially neurotoxic factors that can be produced by activated microglia, reactive oxygen species (ROS) are a major contributor to neurodegeneration. In mesencephalic neuron-glia cultures stimulated with LPS, inhibition of superoxide production by naloxone seemed to correlate best with reduction in neurodegeneration (Liu et al., 2000a). Similarly, in either mesencephalic or cortical neuron-glia cultures, inhibition of superoxide production by naloxone or neutralization of the reactivity of ROS afforded significant neuroprotection (this study; Gao et al., 2002; Qin et al., 2002). Furthermore, addition of SOD and catalase significantly attenuated the A (1-42)-induced neurotoxicity (Qin et al., 2002). Although neurons in general are significantly more susceptible than other neural cells such as glia to injuries induced by oxidative stress, dopaminergic neurons are particularly sensitive to oxidative damage (Jenner and Olanow, 1996). Overproduction of free radicals may lead to an imbalance in the oxidation/reduction capacity of a cell that is primarily maintained by high intracellular levels of glutathione, which is known to be low in dopaminergic neurons (Greenamyre et al., 1999). Depletion of glutathione can either render cells more susceptible to insults or result in activation of key death pathways (Liu et al., 1998). Therefore, inhibition of superoxide production by agents such as naloxone can be an effective approach to prevent damage induced by proinflammatory and cytotoxic factors.

It should be pointed out that results from this study do not attempt to rule out the involvement of astrocytes in inflammation-mediated neurodegeneration. Astrocytes play an important role in neuronal survival by secreting of neurotrophic factors and buffering the action of neurotransmitters. On the other hand, astrocytes appear to play a less prominent role than microglia in contributing to inflammation-mediated neurodegeneration due to production of a limited repertoire and quantity of proinflammatory and neurotoxic factors (Giulian, 1993). For example, it is known that microglia, but not astrocytes, are the primary source of TNF (Giulian, 1999). Similarly, the quantities of nitric oxide produced by astrocytes, at least when stimulated with LPS, are far less than that by microglia (Liu et al., 2002). Furthermore, superoxide released from microglia, but not nitric oxide or TNF, appears to be the predominant effector of A (1-42)-induced neurodegeneration; and addition of microglia, but not astrocytes, to neuron-enriched cultures potentiated the A (1-42)-induced neurotoxicity (Qin et al., 2002; this study). The recent reports on the involvement of neuronal ROS-generating enzymes in neuronal death induced by zinc and growth factor deprivation are very interesting (Noh and Koh, 2000; Tammariello et al., 2000). However, compared with microglia, little superoxide is produced by A (1-42)-stimulated astrocytes and neurons (Qin et al., 2002). Therefore, microglia, through the production of superoxide, appear to play a major role in A (1-42)-induced neurodegeneration.

Although studies have shown that -amyloid peptides are capable of directly killing neurons through mechanisms including interaction with the nicotinic acetylcholine receptor (Wang et al., 2000), the neurotoxicity of A (1-42) observed in the neuron-glia cultures in this study probably was not due to direct neurotoxicity because at the highest concentrations used (1 or 3 µM; Figs. 1 and 2), A (1-42) was not toxic to dopaminergic or GABAergic neurons in the absence of glia (Qin et al., 2002). Therefore, the neuroprotective effect of naloxone observed in this study most likely reflected the consequence of inhibition by naloxone of A (1-42)-induced microglial activation, although the possibility that interaction between naloxone and neurons plays a role in neuroprotection cannot be completely ruled out.

The possibility that naloxone may directly bind A (1-42), hence interfering with its interaction with microglia/neurons, was also examined. Preincubation (30 min, 37°C) of naloxone (5 µM) with radiolabeled A (1-42) (0.75 µM; Garzon-Rodriguez et al., 1997) did not significantly alter the partition of A (1-42) over an appropriate sizing membrane. Conversely, preincubation of A (1-42) with radiolabeled naloxone did not affect the behavior of naloxone in passing through a suitable sizing membrane. These results indicated that there was a lack of direct interaction between naloxone and A (1-42) and implied that naloxone and A (1-42) interacted with distinct sites on microglia to exert the respective activating or inhibiting effect.

Since its initial synthesis, ()-naloxone has been used as a nonselective antagonist to study the involvement of opioid receptors in various experimental systems. For potential therapeutic purposes, naloxone has been tested in animal models and clinical trials to treat a wide range of disease conditions, including drug abuse, alcohol addiction, eating disorder, spinal cord injury, shock, and cerebral and cardiac ischemia (Hosobuchi et al., 1982; Fallis et al., 1983; Holaday and Malcolm, 1986; de Zwaan and Mitchell, 1992; Kan et al., 1992; Ward et al., 1999; Napolitano, 2000; Seidl, 2000; Anton, 2001; Chen et al., 2001). Although the efficacy of naloxone in treating drug abuse is clearly related to its activity as an opioid antagonist, the exact mechanism of action responsible for the efficacy of naloxone in other cases remains poorly understood.

An intriguing characteristic of at least some of those disease conditions in which naloxone has been shown to be beneficial may be the involvement of the inflammatory process. For example, the role of inflammation in ischemic brain injury is beginning to be defined (Stoll et al., 1998; Dirnagl et al., 1999). Initial neuronal death due to excitotoxicity triggers inflammation defined by the activation of microglia and production of cytotoxic factors that result in possibly a greater secondary neuronal death. In the case of myocardial tissue injuries, in addition to the potential involvement of local inflammatory process, systemic microbial infection has been suspected to be a risk factor (Vallance et al., 1997; Mehta and Li, 1999). A key component of the inflammatory process is the generation of ROS such as superoxide by microglia in central nervous system or neutrophils/macrophages in the peripheral system. Neurons, as well as cardiac cells, are particularly sensitive to oxidative damage induced by ROS alone or in combination with other inflammatory mediators such as nitric oxide. More interestingly, generation of ROS may be an upstream event that participates in the production of additional mediators such as TNF, as recently demonstrated by Sanlioglu et al. (2001). Therefore, it is plausible to speculate that inhibition of superoxide generation by naloxone and related compounds in either central nervous system (Chang et al., 2000; Liu et al., 2000a; this study) or peripheral system (Simpkins et al., 1985) may be part of the mechanisms of action responsible for the observed protective effects.

The fact that (+)-naloxone is an inert opioid receptor antagonist but an effective inhibitor of microglial activation and protector against neurodegeneration may be of particular interest from a therapeutic standpoint. The side effects reported for ()-naloxone have been minimal. Devoid of interaction with the opioid receptor system, (+)-naloxone stands as an even better candidate for potential therapeutic purposes.