Studies of oxidative stress mechanisms using a morphine – ascorbate animal model and novel N-stearoyl cerebroside and laurate sensors
by P. A. Broderick
Summary. The field of oxidative stress, free radicals, cellular defense and antioxidants is a burgeoning field of research. An important biomarker of oxidative stress is ascorbate and alterations in ascorbate have been shown to be a reliable measure of oxidative stress mechanisms. The purpose of this pharmacological study was to assess changes in ascorbate in a morphine= ascorbate animal model using novel sensors which selectively detect electrochemical signals for ascorbate, dopamine (DA) and serotonin (5-HT). Studies were also performed to show reversal of morphine-induced effects by the opioid antagonist, naloxone. In vivo studies were modeled after (Enrico et al. 1997, 1998) in which the oxidative biomarker, ascorbate, was reported to compensate for free radicals produced by morphine-induced increases in DA and 5-HT. In vivo studies consisted of inserting the Laurate sensor in ventrolateral nucleus accumbens (vlNAcc), in anesthetized male, Sprague-Dawley rats. In separate studies, laboratory rats were injected with (1) ascorbate, (5–35 mg=kg, ip) or (2) dehydroascorbate (DHA) (20–100 mg=kg, ip). In another study, (3) morphine sulfate (10–20 mg=kg, sc) was injected followed by a single injection of naloxone (5 mg=kg, ip) in the
same animal. Results showed that in vlNAcc, (1) neither ascorbate nor DHA injections produced ascorbate release, (2) morphine significantly increased DA and 5-HT release but did not alter ascorbate release, and (3) naloxone significantly reversed the increased DA and 5-HT release produced by morphine. Moreover, the sensors, N-stearoyl cerebroside, and laurate were
studied in vitro, in separate studies, in order to assess selective and separate electrochemical detection of ascorbate, DA and 5-HT, neuromolecules involved in oxidative stress mechanisms. In vitro studies consisted of pretreatment of each sensor with a solution of phosphotidylethanolamine (PEA) and bovine serum albumin (BSA) which simulates the lipid=protein composition of the brain. Each new sensor was tested for stability, sensitivity, and selectivity by pipetting graduated increases in the concentration of ascorbate, DA and 5-HT into an electrochemical cell containing saline=phosphate buffer. Multiple and repetitive images of electrochemical signals from ascorbate, DA and 5-HT were recorded. Results showed that both sensors produced three well-defined cathodic, selective and separate electrochemical signals for ascorbate, DA and 5-HT at characteristic oxidation potentials. Dopamine and 5-HT were detected at nM concentrations while ascorbate was detected at mM concentrations. In summary, the data show that very low concentrations of ascorbate occurred in vlNAcc since novel sensors detected ascorbate at high concentrations in vitro. The data indicate that little or no change in oxidative stress mechanisms occurred in vlNAcc after morphine or naloxone administration since the oxidative biomarker, ascorbate, was not significantly altered. Thus, oxidative stress mechanisms and novel N-stearoyl cerebroside and laurate sensors, which selectively detect and separate neuromolecules involved in these mechanisms, may be potentially clinically relevant.
Keywords: Ascorbate; BRODERICK PROBE+ sensors; dehydroascorbic acid; dopamine; drug addiction; electrodes; laurate sensor; microelectrodes; microvoltammetry; morphine; naloxone; neuromolecular imaging (NMI); N-stearoyl cerebroside sensor; nucleus accumbens; sensors; serotonin; opioids; oxidative stress; pain; stroke
Previous studies have shown that morphine increased extracellular DA in nigrostriatal and mesolimbic pathways in the rat brain (Herz and Shippenberg 1989). These morphine effects have been postulated to be a factor in the reinforcing effect of morphine and the known increased locomotor effects produced by morphine (Wood 1993). A microdialysis study has shown that morphine increased extracellular DA in nucleus accumbens (NAcc) (Spanagel et al. 1990). Other reports have provided indirect evidence that morphine reduces pain by increasing extracellular DA in the NAcc. This evidence is derived from research with a new opioid peptide, nociceptin, which produces hyperalgesia (Meunier et al. 1995; Okuda-Ashitaka et al. 1996), and decreases morphine-induced extracellular DA in the NAcc shell (Di Giannuario and Pieretti 2000).
Moreover, the antioxidant, ascorbate, when administered orally in high doses, has been shown to relieve pain, reduce opioid use, inhibit endogenous opioid degrading metalloenzymes, increase endorphin levels, and ameliorate withdrawal symptoms from heroin addiction (Evangelou et al. 2000). Chronically, but not acutely administered ascorbate has been shown to inhibit morphine withdrawal symptoms (Johnston and Chahl 1992). Agus et al. (1997) has shown that it is dehydroascorbate (DHA), the oxidized form of ascorbate, that enters the brain by a mechanism termed facilitative transport. Increased ascorbate concentrations in the brain may provide neuroprotection during cerebral ischemia and stroke (Huang et al. 2001; Cherubini et al. 2005). Acute tryptophan depletion has been reported to block morphine analgesia, thus implicating a role for serotonin (5-HT) in pain-relieving effects since l-tryptophan is a precursor to 5-HT (Abbott et al. 1992). Moreover, morphine increased striatal extracellular 5-HT levels as well as striatal DA levels in laboratory rats (Enrico et al. 1997, 1998).
In these previous studies, morphine was also reported to increase free radicals produced by DA and 5-HT as shown by increased ascorbate in nigrostriatal sites; the authors further suggested that these specific oxidative stress mechanisms are mopiate mediated.
In the present paper oxidative stress mechanisms were tested by studying ascorbate, DA and 5-HT in this morphine=ascorbate animal model, using novel sensors inserted in a mesolimbic site, vlNAcc, in anesthetized, male, Sprague-Dawley laboratory rats.
Pharmacological studies of oxidative stress mechanisms, specifically, the oxidative biomarker, ascorbate, were performed in a morphine=ascorbate animal model described by (Enrico et al. 1997, 1998). Novel electrochemical sensors were used to selectively detect ascorbate, DA and 5-HT. The neuroanatomic site targeted was vlNAcc. Signals for ascorbate, DA and 5-HT were studied in vitro and in vivo. Signals for ascorbate, DA and 5-HT were also studied after ascorbate, DHA, morphine and naloxone administration in vivo. The electrochemical methodologies are described first because electrochemistry is used in both in vitro and in vivo studies.
Conventional microvoltammetry began almost three decades ago (Adams and Marsden 1982, review) wherein an indicator=working electrode was inserted into a neuroanatomic region of brain, and a reference was placed in contact with cortex. The use of an auxiliary electrode provided an additional ground and was optional. Summarized,
- Electroactive molecules undergo redox reactions at the surface of the indicator electrode.
- The reference provides a relative zero point for measurement of current
produced by the indicator.
- The formula is: O þ ne () R, wherein, ne1⁄4 number of electrons, O 1⁄4 oxidation, R1⁄4 reduction.
- The amount of current that is produced is proportional to concentration according to the Cottrell equation (Kissinger et al. 1996).
Neuromolecular imaging (NMI)
NMI, a term recently introduced into the literature (Broderick and Pacia 2005), is based on, but different from, conventional microvoltammetry because in NMI, sensors rather than electrical circuits are emphasized. NMI includes the development of several novel sensors to image neurotransmitters at more than one site, within seconds, over long periods of time, in vivo, in vitro, and in situ. New formulations of sensors include biochemical classes of lipid, glycolipid, lipoprotein, saturated and unsaturated fatty acids. Detection capabilities include, in addition to the monoamines and ascorbate, l-tryptophan, dynorphin, and somatostatin. A schematic generic diagram of the BRODERICK PROBE+ sensor is shown in Fig. 1.
N-Stearoyl cerebroside sensors
N-Stearoyl cerebroside sensors are manufactured on site. N-Stearoyl cerebroside is a complex lipid wherein the Cerebroside portion is derived from bovine brain (formula: (C42H81NO8). N-Stearoyl cerebroside sensors, for these studies, were comprised of <1 mg aliquot of stock, a mixture of ultra-pure carbon (0.75 g), oil (Nujol) containing a-tocopherol (0.062 g), and N-stearoyl cerebroside (5.0 mg). The detailed construction of the N-stearoyl cerebroside sensor is published (Broderick 1995, 1999). NMI oxidation potentials in vitro, are in the order of detection from lowest to highest potential, ascorbate [0.07 V]; DA [0.12 V]; 5-HT [0.27 V]. Oxidation potentials for these sensors, in vivo, are, in the order of detection from lowest to highest oxidation potential, ascorbate [0.08 V], DA [0.14 V], 5-HT [0.28 V].
Laurate sensors are manufactured on site. Lauric acid is a saturated fatty acid (formula: CH3(CH2)10COOH). Laurate sensors, for these studies, were comprised of <1 mg aliquot of stock, a mixture of ultra-pure carbon (1.5 g), oil (Nujol) containing a-tocopherol (1.24 g), and lauric acid (100 mg). The detailed construction of the Laurate sensor is published (Broderick 1995, 1999). NMI oxidation potentials for these sensors, in vitro, are, in the order of detection from lowest to highest potential, ascorbate [0.08 V]; DA [0.13 V]; 5-HT [0.29 V]. Oxidation potentials for these sensors, in vivo, are, in the order of detection from lowest to highest oxidation potential, ascorbate [0.09 V], DA [0.15 V], 5-HT [0.3 V].