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• W1968318618 abstract "Indirect evidence suggests that morphine-3-glucuronide, the major metabolite of morphine in man, may be primarily responsible for the neuroexcitatory behaviours (myoclonus and allodynia) observed in some cancer patients following chronic administration of high systemic doses of morphine for cancer pain management (Smith 2000). These findings are consistent with behavioural studies in rodents, whereby morphine-3-glucuronide has been shown to dose-dependently evoke neuro-excitatory behaviours when administered by the intracerebroventricular route (Bartlett et al. 1994). The steepness of the intracerebroventricular morphine-3-glucuronide dose-response curve in rats suggests the possibility that morphine-3-glucuronide may be neurotoxic. Calcium is known to play a crucial role in synaptic neurotransmission as well as neuronal excitability. Hence, agents that increase the cytosolic free calcium concentration, ([Ca2+]CYT), in neurones, have the potential to not only enhance synaptic transmission, but also to increase neuronal excitability and the risk of producing neurotoxicity (Sattler & Tymianski 2000). High-dose L-glutamate is well known to be neurotoxic in various cell types including neurones (Choi 1987; Choi et al. 1988). However, the potential neurotoxic effects of morphine-3-glucuronide have not been previously assessed. Ethical approval for this study was obtained from The University of Queensland Animal Experimentation Ethics Committee. Cultured hippocampal neurones have been used extensively as a model system of excitotoxicity (Shen & Thayer 1998) and apoptosis (Mattson et al. 1998). Our previous behavioural studies in rats have shown that the hippocampus has a range of subregional sensitivities to morphine-3-glucuronide with the ventral hippocampus being the most sensitive, the CA1 region being the least sensitive, and the CA2–CA3 region having intermediate sensitivity (Halliday et al. 1999). Hence, the purpose of the present study was to determine whether prolonged exposure (24 hr) of primary neuronal cultures to M3G (0.5 and 50 μM) or morphine (0.5 and 50 μM) produced neurotoxicity in a manner analogous to that produced by prolonged L-glutamate (500 μM) exposure. Primary cultures of embryonic rat hippocampal neurones were prepared according to the method reported previously by Brewer et al. (1993) with necessary modifications. Embryonic hippocampal neurones were obtained from timed pregnant Sprague-Dawley rats at 18–19 days gestation. Two pregnant rats were lightly anaesthetized with 50% O2:50% CO2 and killed by cervical dislocation. Briefly, hippocampi were dissected and dissociated by treatment with 2 mg/ml papain/Hibernate-E for 30 min. at 30 °, followed by trituration. Cells were pelleted and resuspended in Neurobasal plating medium containing L-glutamine (0.5 mM), L-glutamate (2.5 μM), 2% B-27®, and Neurobasal medium. Cultured cerebellar granule neurones are a widely used model system for the assessment of neurotoxicity, due to the ease of access to a large homogenous neuronal cell population, thereby facilitating the rapid generation of numerous neuronal cell cultures (Miller & Johnson 1996; Yamagishi et al. 2001). Primary cultures of cerebellar granule neurones were prepared as described by Smith et al. (2001). Hippocampal and cerebellar granule neurones were plated onto 96 well plates coated with poly-D-lysine (PDL) (0.05 mg/ml). In separate experiments, hippocampal neurones were plated onto 25 mm round glass PDL-coated coverslips. All cultures were grown in a humidified atmosphere of 5% CO2:95% O2 at 37 °. The culture medium was first changed on day three of culture by exchanging half of the medium with Neurobasal feeding medium containing 2% B-27®, and L-glutamine (0.5 mM). Thereafter, the culture medium was changed every third day for cultures grown in 96 well plates and weekly for cultures grown on coverslips. To compare qualitatively the acute effects of morphine-3-glucuronide, and L-glutamate on [Ca2+]CYT in cultured hippocampal neurones, fluo-3 fluorescence digital imaging techniques were used, in a manner similar to that utilized in our recent studies (Hemstapat et al. 2003). Coverslips containing dye-loaded neurones (14–18 days in culture) were transferred to the recording chamber of an inverted microscope, where neurones were superfused with HEPES Hank's Salt Solution (HHSS) containing an opioid receptor-saturating concentration of naloxone (Nalox) (1 μM). Morphine-3-glucuronide or L-glutamate was added to the chamber through a gravity-fed perfusion system. [Ca2+]CYT was measured in the somatic region of neurones and increases in fluo-3 fluorescence intensity were representative of increases in [Ca2+]CYT. Neuronal death was quantitatively assessed by measurement of the release of lactate dehydrogenase into the culture medium (Koh & Choi 1987). Lactate dehydrogenase assays are suitable for the assessment of both apoptotic and necrotic cell death in neurones (Koh & Choi 1987; Lee et al. 1998; Lobner 2000). Indeed, lactate dehydrogenase assays have been used by many investigators to assess cell death after activation of apoptosis in neuronal cultures, including those from the hippocampus (Suuronen et al. 2000; Yamaguchi et al. 2001; Zhao et al. 2001) and the cerebellum (Smith et al. 2001; Suuronen et al. 2000; Tabuchi et al. 2003). Cell death was evaluated using a Tox-7 lactate dehydrogenase-based in vitro toxicology assay kit (Sigma Aldrich, Castle Hill, Australia) following 24 hr of neuronal exposure. The degree of neurotoxicity was determined using a multiwell plate reader (BioRad, Hercules, CA, USA), using a spectrophotometric method with the absorbance determined at a wavelength of 490 nM. Neurotoxicity experiments were performed after 14–18 days and 10–12 days in culture for hippocampal and cerebellar granule neurones, respectively. The medium within each well was exchanged for medium supplemented with antioxidant free B-27® containing either morphine-3-glucuronide (0.5 and 50 μM) or morphine (0.5 and 50 μM) in the presence or absence of an opioid receptor-saturating concentration of naloxone (1 μM). As previous in vivo (Halliday et al. 1999) and in vitro (Hemstapat et al. 2003) studies in our laboratory have shown that morphine-3-glucuronide mediates its neuroexcitatory effects predominantly via a non-opioid mechanism, naloxone was used to facilitate characterization of the possible neurotoxic consequences of this non-opioid mechanism. L-Glutamate (500 μM, a concentration previously shown to cause neurotoxicity in cultured neurones) was introduced into one row of each plate (Cheung et al. 1998; Smith et al. 2001) as the positive control and for quantitative comparison of the morphine-3-glucuronide data. N values are number of wells tested; 3 separate experiments were performed for each compound examined. As expected, exposure (3 min.) of hippocampal neurones to L-glutamate (500 μM) evoked increases in [Ca2+]CYT (fig. 1A). Similarly, the acute exposure (3 min.) of neurones to morphine-3-glucuronide (50 μM) resulted in a rapid increase in [Ca2+]CYT (fig. 1B) and this effect was reversible upon subsequent morphine-3-glucuronide washout (data not shown), in accordance with our previous findings (Hemstapat et al. 2003). Despite the ability of morphine-3-glucuronide to cause a sustained increase in [Ca2+]CYT, it does not appear to exhibit neurotoxicity in contrast to the marked neurotoxicity produced by L-glutamate, as indicated by the data described below. Effect of (A) L-glutamate (500 μM) and (B) M3G (50 μM) on [Ca2+]CYT in cultured embryonic rat hippocampal neurones, assessed using Fluo-3 AM fluorescence digital imaging techniques. After prolonged (24 hr) exposure to either morphine (0.5 and 50 μM) or morphine-3-glucuronide (0.5 and 50 μM) in both hippocampal (n=23–28; fig. 2A) and cerebellar granule neurones (n=29–43; fig. 2B), irrespective of the presence or absence of naloxone (1 μM), the degree of inherent toxicity (if any) induced was significantly less (P<0.0001, one-way ANOVA) than that elicited by the well established excitotoxin, L-glutamate (Smith et al. 2001; Uryu et al. 2002). Indeed, L-glutamate was the only treatment that produced pronounced release of lactate dehydrogenase from both neuronal cultures. However, it must be noted that lactate dehydrogenase release may not detect changes in gene expression or other more subtle changes associated with neurotoxicity. Effect of prolonged (24 hr) morphine-3-glucuronide (M3G) and morphine (Mor) exposure (0.5 and 50 μM) on lactate dehydrogenase (LDH) release (index of neurotoxicity) from (A) hippocampal neurones (n=23–28) and (B) cerebellar granule neurones (n=29–43) in the presence and absence of naloxone (1 μM). Values are mean (±S.E.M.), and were standardized relative to the LDH release produced by L-glutamate (500 μM). The amount of LDH release evoked by L-glutamate (500 μM) was significantly greater than (P<0.0001) than that evoked by each of the other substances (or vehicle) tested. It should also be noted that conclusive elimination of morphine-3-glucuronide as a mediator of neuronal cell death in the clinic would require whole animal experiments, where markers for apoptosis in neurones exposed to morphine-3-glucuronide would be assessed. However, our current findings of the relatively low degree of neurotoxicity produced by prolonged exposure of cultured neurones to a relatively high concentration of morphine-3-glucuronide (50 μM) are consistent with observations in the clinical setting. Specifically, for patients exhibiting neuroexcitatory behaviours such as myoclonus and allodynia, implementation of procedures that facilitate morphine-3-glucuronide clearance from the patient CNS (e.g. marked dose reduction or cessation of morphine treatment, rotation of opioid from morphine to fentanyl or methadone), results in complete patient recovery without any apparent long-term CNS deficits. In summary, although indirect evidence has implicated the major morphine metabolite, morphine-3-glucuronide, in the production of the neuroexcitatory behaviours observed in some patients after chronic high-dose systemic morphine administration, and our recent in vitro findings suggest that morphine-3-glucuronide produces its neuroexcitatory effects via indirect activation of the NMDA receptor, the results of our present study indicate that morphine-3-glucuronide is unlikely to be neurotoxic in vivo. However, the possibility that morphine-3-glucuronide may augment established neurotoxicity or neurotoxicity initiated via another cause, remains to be investigated. KH is supported by an International Postgraduate Student Scholarship (IPRS) and a University of Queensland IPRS. This research was supported financially by the Queensland Cancer Fund (#2001000155)." @default.
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• W1968318618 title "The Neuroexcitatory Morphine Metabolite, Morphine-3-glucuronide (M3G), is not Neurotoxic in Primary Cultures of either Hippocampal or Cerebellar Granule Neurones" @default.
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