The Neurotoxin DSP-4 Induces Hyperalgesia in Rats that is Accompanied by Spinal Oxidative Stress and Cytokine Production.

Central neuropathic pain (CNP) a significant problem for many people, is not well-understood and difficult to manage. Dysfunction of the central noradrenergic system originating in the locus coeruleus (LC) may be a causative factor in the development of CNP. The LC is the major noradrenergic nucleus of the brain and plays a significant role in central modulation of nociceptive neurotransmission. Here, we examined CNS pathophysiological changes induced by intraperitoneal administration of the neurotoxin DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride). Administration of DSP-4 decreased levels of norepinephrine in spinal tissue and cerebrospinal fluid (CSF) and led to the development of thermal and mechanical hyperalgesia over 21 days, that was reversible with morphine. Hyperalgesia was accompanied by significant increases in noradrenochrome (oxidized norepinephrine) and expression of 4-hydroxynonenal in CSF and spinal cord tissue respectively at day 21, indicative of oxidative stress. In addition, spinal levels of pro-inflammatory cytokines (interleukins 6 and 17A, tumor necrosis factor-α), as well as the anti-inflammatory cytokine interleukin10 were also significantly elevated at day 21, indicating that an inflammatory response occurred. The inflammatory effect of DSP-4 presented in this study that includes oxidative stress may be particularly useful in elucidating mechanisms of CNP in inflammatory disease states.

Simultaneous analysis of vascular norepinephrine and ATP release using an integrated microfluidic system.

BACKGROUND: Sympathetic nerves are known to release three neurotransmitters: norepinephrine, ATP, and neuropeptide Y that play a role in controlling vascular tone. This paper focuses on the co-release of norepinephrine and ATP from the mesenteric arterial sympathetic nerves of the rat. NEW METHOD: In this paper, a quantification technique is described that allows simultaneous detection of norepinephrine and ATP in a near-real-time fashion from the isolated perfused mesenteric arterial bed of the rat. Simultaneous detection is enabled with 3-D printing technology, which is shown to help integrate the perfusate with different detection methods (norepinephrine by microchip-based amperometery and ATP by on-line chemiluminescence). RESULTS: Stimulated levels relative to basal levels of norepinephrine and ATP were found to be 363nM and 125nM, respectively (n=6). The limit of detection for norepinephrine is 80nM using microchip-based amperometric detection. The LOD for on-line ATP detection using chemiluminescence is 35nM. COMPARISON WITH EXISTING METHOD: In previous studies, the co-transmitters have been separated and detected with HPLC techniques. With HPLC, the samples from biological preparations have to be derivatized for ATP detection and require collection time before analysis. Thus real-time measurements are not made and the delay in analysis by HPLC can cause degradation. CONCLUSIONS: In conclusion, the method described in the paper can be used to successfully detect norepinephrine and ATP simultaneously and in a near-real-time fashion.

Direct intranigral injection of dopaminochrome causes degeneration of dopamine neurons.

Parkinson's disease (PD) is characterized by progressive neurodegeneration of nigrastriatal dopaminergic neurons leading to clinical motor dysfunctions. Many animal models of PD have been developed using exogenous neurotoxins and pesticides. Evidence strongly indicates that the dopaminergic neurons of the substantia nigra pars compacta (SNpc) are highly susceptible to neurodegeneration due to a number of factors including oxidative stress and mitochondrial dysfunction. Oxidation of DA to a potential endogenous neurotoxin, dopaminochrome (DAC), may be a potential contributor to the vulnerability of the nigrostriatal tract to oxidative insult. In this study, we show that DAC causes slow and progressive degeneration of dopaminergic neurons in contrast to 1-methyl-4-phenylpyridinium (MPP(+)), which induces rapid lesions of the region. The DAC model may be more reflective of early stresses that initiate the progressive neurodegenerative process of PD, and may prove a useful model for future neurodegenerative studies.

Dopaminochrome induces caspase-independent apoptosis in the mesencephalic cell line, MN9D.

Parkinson's disease is characterized by a deficiency in motor cortex modulation due to degeneration of pigmented dopaminergic neurons of the substantia nigra projecting to the striatum. These neurons are particularly susceptible to oxidative stress, perhaps because of their dopaminergic nature. Like all catecholamines, dopamine is easily oxidized, first to a quinone intermediate and then to dopaminochrome (DAC), a 5-dihydroxyindole tautomer, that is cytotoxic in an oxidative stress-dependent manner. Here we show, using the murine mesencephalic cell line MN9D, that DAC causes cell death by apoptosis, illustrated by membrane blebbing, Annexin V, and propidium iodide labeling within 3 h. In addition, DAC causes oxidative damage to DNA within 3 h, and positive terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence by 24 h. DAC, however, does not induce caspase 3 activation and its cytotoxic actions are not prevented by the pan-caspase inhibitor, Z-VAD-fmk. DAC-induced cytotoxicity is limited by the PARP1 inhibitor, 5-aminoisoquinolinone, supporting a role for apoptosis-inducing factor (AIF) in the apoptotic process. Indeed, AIF is detected in the nuclear fraction of MN9D cells 3 h after DAC exposure. Taken together these results demonstrate that DAC induces cytotoxicity in MN9D cells in a caspase-independent apoptotic manner, likely triggered by oxidative damage to DNA, and involving the translocation of AIF from the mitochondria to the nucleus.

Cytotoxicity of dopaminochrome in the mesencephalic cell line, MN9D, is dependent upon oxidative stress.

Parkinson disease is a specific form of neurodegeneration characterized by a loss of nigra-striatal dopaminergic neurons in the midbrain of humans. The disease is also characterized by an increase in oxidative stress and a loss of glutathione in the midbrain region. A potential link between all these factors is the oxidation of dopamine to dopaminochrome (DAC). Using the murine mesencephalic cell line MN9D, we have shown that DAC [50-250 microM] leads to cell death in a concentration-dependent manner, whereas oxidized l-dopa, dopachrome [50-250 microM], is only toxic at the highest concentration used. Furthermore, chronic exposure of MN9D cells to low concentrations of DAC [50-100 microM] is cytotoxic between 48 and 96 h. DAC also increases superoxide production within MN9D cells as indicated by dihydroethidium fluorescence, that can be prevented by co-administration with the antioxidant, N-acetylcysteine [5 mM]. Moreover, the cytotoxicity induced by DAC can also be prevented by administration of N-acetylcysteine [1-5mM]. Finally, depletion of reduced glutathione in MN9D cells by buthionine sulfoximine [50-100 microM] administration significantly enhances the cytotoxic effect of low concentrations of DAC [50-100 microM] and DAC [175 microM] itself reduces the proportion of oxidized glutathione in total glutathione within 30 min of administration in MN9D cells. Overall, we have shown that DAC causes MN9D cell death in an oxidatively dependent manner that appears closely linked with a rapid loss of reduced glutathione. These findings have implications for understanding the pathogenesis of neurodegenerative pathways in Parkinson disease.

Oxidative stress attenuates NO-induced modulation of sympathetic neurotransmission in the mesenteric arterial bed of spontaneously hypertensive rats.

Current evidence suggests that hyperactivity of the sympathetic nervous system and endothelial dysfunction are important factors in the development and maintenance of hypertension. Under normal conditions the endothelial mediator nitric oxide (NO) negatively modulates the activity of the norepinephrine portion of sympathetic neurotransmission, thereby placing a "brake" on the vasoconstrictor ability of this transmitter. This property of NO is diminished in the isolated, perfused mesenteric arterial bed taken from the spontaneously hypertensive rat (SHR), resulting in greater nerve-stimulated norepinephrine and lower neuropeptide Y (NPY) overflow from this mesenteric preparation compared with that of the normotensive Wistar-Kyoto rat (WKY). We hypothesized that increased oxidative stress in the SHR contributes to the dysfunction in the NO modulation of sympathetic neurotransmission. Here we demonstrate that the antioxidant N-acetylcysteine reduced nerve-stimulated norepinephrine and increased NPY overflow in the mesenteric arterial bed taken from the SHR. Furthermore, this property of N-acetylcysteine was prevented by inhibiting nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester, demonstrating that the effect of N-acetylcysteine was due to the preservation of NO from oxidation. Despite a reduction in norepinephrine overflow, the nerve-stimulated perfusion pressure response in the SHR mesenteric bed was not altered by the inclusion of N-acetylcysteine. Studies including the Y(1) antagonist BIBO 3304 with N-acetylcysteine demonstrated that this preservation of the perfusion pressure response was due to elevated NPY overflow. These results demonstrate that the reduction in the bioavailability of NO as a result of elevated oxidative stress contributes to the increase in norepinephrine overflow from the SHR mesenteric sympathetic neuroeffector junction.

Modulation of serum cytokine levels by a novel superoxide dismutase mimetic, M40401, in an Escherichia coli model of septic shock: correlation with preserved circulating catecholamines.

OBJECTIVES: We have shown previously that inactivation of catecholamines by superoxide anions contributes to the loss of vascular reactivity to norepinephrine and the subsequent hypotension that develops in Gram-negative endotoxic shock. In addition to their vasopressor actions, catecholamines, via beta-adrenoceptor activation, are important regulators of cytokine production. Here we examined if maintenance of serum catecholamine levels by the superoxide dismutase mimetic, M40401, modulates serum cytokine levels and arterial hypotension in an Escherichia coli-infected conscious rat model of septic shock. DESIGN: Controlled laboratory animal study. SETTING: University animal research laboratory. SUBJECTS: Pathogen-free male Sprague-Dawley rats (n = 51). INTERVENTIONS: Conscious, antibiotic-treated animals with chronic in-dwelling carotid arterial and jugular venous catheters were intravenously infected with 10(10) live E. coli bacteria (O55:B5, n = 51) over 30 mins, ending at time = 0 hrs. At 0.5 or 3 hrs, infected rats were administered an intravenous infusion of either M40401 (n = 33) or 0.9% saline (n = 18) for 6 hrs at a rate of 1 mL/h. In additional experiments, anesthetized animals with catheterized left femoral arteries and veins were administered a dose-range of norepinephrine (0.1-1 microg/kg) as bolus intravenous injections. Thereafter, E. coli lipopolysaccharide (4 mg/kg, n = 6) was administered as a 0.3-mL slow bolus intravenous injection. One hour later, the norepinephrine protocol was repeated, after which the rats were administered an intravenous infusion of either M40401 or 0.9% saline for 15 mins. At 2 hrs, the dose response to norepinephrine was repeated. MEASUREMENTS AND MAIN RESULTS: Rats infected with live E. coli exhibited a biphasic fall in mean arterial pressure, with mortality reaching 83% by 24 hrs. Rats treated with M40401 (0.25, 2.5, or 25 microg x kg-1 x hr-1 ) 3 hrs after bacteremic sepsis maintained a normal mean arterial pressure, and mortality was dose-dependently reduced to 44, 33, and 22%, respectively, at 24 hrs. Furthermore, serum catecholamine levels were diminished in E. coli-infected rats treated with saline compared with rats treated with M40401. In separate experiments, E. coli-infected rats were administered M40401 (25 microg x kg-1 x hr-1 ) 0.5 hr after bacterial challenge. Blood samples taken at 0, 1.5, 3.5, and 6 hrs were analyzed for tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, and IL-10 and for norepinephrine and epinephrine. Serum levels of tumor necrosis factor-alpha and IL-1 beta were significantly depressed in M40401-treated septic rats, whereas IL-10 was elevated. Moreover, serum catecholamine levels were greater in M40401-treated septic rats at the same time points. IL-6 levels were unaffected by M40401 treatment. Finally we examined whether treatment with M40401 could reverse the hyporeactivity to norepinephrine typifying early septic shock. Using the E. coli lipopolysaccharide (4 mg/kg) challenged anesthetized rat model of shock, we demonstrated that the vasoconstrictor ability of norepinephrine was indeed restored after M40401 treatment (25 microg/kg). CONCLUSION: Postinfection treatment with the superoxide dismutase mimetic M40401 protects against hypotension, vascular hyporeactivity to catecholamines, and mortality associated with septic shock. Such beneficial effects correlate with both reduced oxidative inactivation of serum catecholamines and a reduction in canonical cytokine mediators of inflammation.