Increased brain nitric oxide levels following ethanol administration.

Nitric oxide is a ubiquitous messenger molecule, which at elevated concentrations has been implicated in the pathogenesis of several neurological disorders. Its role in oxidative stress, attributed in particular to the formation of peroxynitrite, proceeds through its high affinity for the superoxide radical. Alcoholism has recently been associated with the induction of oxidative stress, which is generally defined as a shift in equilibrium between pro-oxidant and anti-oxidant species in the direction of the former. Furthermore, its primary metabolite acetaldehyde, has been extensively associated with oxidative damage related toxic effects following alcohol ingestion. The principal objective of this study was the application of long term in vivo electrochemistry (LIVE) to investigate the effect of ethanol (0.125, 0.5 and 2.0 g kg(-1)) and acetaldehyde (12.5, 50 and 200 mg kg(-1)) on NO levels in the nucleus accumbens of freely moving rats. Systemic administrations of ethanol and acetaldehyde resulted in a dose-dependent increases in NO levels, albeit with very differing time courses. Subsequent to this the effect on accumbal NO levels, of subjecting the animal to different drug combinations, was also elucidated. The nitric oxide synthase inhibitor L-NAME (20 mg kg(-1)) and acetaldehyde sequestering agent D-penicillamine (50 mg kg(-1)) both attenuated the increase in NO levels following ethanol (1 g kg(-1)) administration. Conversely, the alcohol dehydrogenase inhibitor 4-methylpyrazole (25 mg kg(-1)) and catalase inhibitor sodium azide (10 mg kg(-1)) potentiated the increase in NO levels following ethanol administration. Finally, dual inhibition of aldehyde dehydrogenase and catalase by cyanamide (25 mg kg(-1)) caused an attenuation of ethanol effects on NO levels. Taken together these data highlight a robust increase in brain NO levels following systemic alcohol administration which is dependent on NO synthase activity and may involve both alcohol- and acetaldehyde-dependent mechanisms.

Development and validation of a real-time microelectrochemical sensor for clinical monitoring of tissue oxygenation/perfusion.

Oxygen is of critical importance to tissue viability and there is increasing demand for its reliable real-time clinical monitoring in order to prevent, diagnose, and treat several pathological disorders, including hypoxia, stroke and reperfusion injury. Herein we report the development and characterisation of a prototype clinical O2 sensor, and its validation in vivo, including proof-of-concept monitoring in patients undergoing surgery for carpal tunnel release. An integrated platinum-based microelectrochemical device was custom designed and controlled using a miniaturised telemetry-operated single channel clinical potentiostat. The in vitro performance of different sensor configurations is presented, with the best sensor design (S2) displaying appropriate linearity (R2 = 0.994) and sensitivity (0.569 ± 0.022 nA µM-1). Pre-clinical validation of S2 was performed in the hind limb muscle of anaesthetised rats; tourniquet application resulted in a significant rapid decrease in signal (90 ± 27%, [ΔO2] ca. 140 ± 18 µM), with a return to baseline within a period of ca. 3 min following tourniquet release. Similar trends were observed in the clinical study; an immediate decrease in signal (39 ± 3%, [ΔO2] ca. 30 ± 20 µM), with basal levels re-established within 2 min of tourniquet release. These results confirm that continuous real-time monitoring of dynamic changes in tissue O2 can serve as an indicator of reperfusion status in patients undergoing carpal tunnel surgery, and suggests the potential usefulness of the developed microelectrochemical sensor for other medical conditions where clinical monitoring of O2 and perfusion is important.

Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors.

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

Pleiotropic activity of systemically delivered angiogenin in the SOD1G93A mouse model.

Loss-of-function mutations in the angiogenin (ANG) gene have been identified in familial and sporadic ALS patients. Previous work from our group identified human ANG (huANG) to protect motoneurons in vitro, and provided proof-of-concept that daily intraperitoneal (i.p.) huANG injections post-symptom onset increased lifespan and delayed disease progression in SOD1G93A mice. huANG's mechanism of action remains less well understood. Here, we implemented a preclinical in vivo design to validate our previous results, provide pharmacokinetic and protein distribution data after systemic administration, and explore potential pleiotropic activities of huANG in vivo. SOD1G93A mice (n = 45) and non-transgenic controls (n = 31) were sex- age- and litter-matched according to the 2010 European ALS/MND group guidelines, and treated with huANG (1 µg, i.p., 3 times/week) or vehicle from 90 days on. huANG treatment increased survival and delayed motor dysfunction as assessed by rotarod in SOD1G93A mice. Increased huANG serum levels were detectable 2 and 24 h after i.p. injection equally in transgenic and non-transgenic mice. Exogenous huANG localized to spinal cord astrocytes, supporting a glia-mediated, paracrine mechanism of action; uptake into endothelial cells was also observed. 1 µg huANG or vehicle were administered from 90 to 115 days of age for histological analysis. Vehicle-treated SOD1G93A mice showed decreased motoneuron numbers and vascular length per ventral horn area, while huANG treatment resulted in improved vascular network maintenance and motoneuron survival. Our data suggest huANG represents a new class of pleiotropic ALS therapeutic that acts on the spinal cord vasculature and glia to delay motoneuron degeneration and disease progression.

Continuous real-time in vivo measurement of cerebral nitric oxide supports theoretical predictions of an irreversible switching in cerebral ROS after sufficient exposure to external toxins.

BACKGROUND: Mathematical models of the interactions between alphasynuclein (αS) and reactive oxygen species (ROS) predict a systematic and irreversible switching to damagingly high levels of ROS after sufficient exposure to risk factors associated with Parkinson's disease (PD). OBJECTIVES: We tested this prediction by continuously monitoring real-time changes in neurochemical levels over periods of several days in animals exposed to a toxin known to cause Parkinsonian symptoms. METHODS: Nitric oxide (NO) sensors were implanted in the brains of freely moving rats and the NO levels continuously recorded while the animals were exposed to paraquat (PQ) injections of various amounts and frequencies. RESULTS: Long-term, real-time measurement of NO in a cohort of animals showed systematic switching in levels when PQ injections of sufficient size and frequency were administered. The experimental observations of changes in NO imply a corresponding switching in endogenous ROS levels and support theoretical predictions of an irreversible change to damagingly high levels of endogenous ROS when PD risks are sufficiently large. CONCLUSIONS: Our current results only consider one form of PD risk, however, we are sufficiently confident in them to conclude that: (i) continuous long-term measurement of neurochemical dynamics provide a novel way to measure the temporal change and system dynamics which determine Parkinsonian damage, and (ii) the bistable feedback switching predicted by mathematical modelling seems to exist and that a deeper analysis of its characteristics would provide a way of understanding the pathogenic mechanisms that initiate Parkinsonian cell damage.

Brain nitric oxide: regional characterisation of a real-time microelectrochemical sensor.

A reliable method of directly measuring endogenously generated nitric oxide (NO) in real-time and in various brain regions is presented. An extensive characterisation of a previously described amperometric sensor has been carried out in the prefrontal cortex and nucleus accumbens of freely moving rats. Systemic administration of saline caused a transient increase in signal from baseline levels in both the prefrontal cortex (13 ± 3pA, n=17) and nucleus accumbens (12 ± 3pA, n=8). NO levels in the prefrontal cortex were significantly increased by 43 ± 9pA (n=9) following administration of l-arginine. A similar trend was observed in the nucleus accumbens, where an increase of 44 ± 9pA (n=8) was observed when compared against baseline levels. Systemic injections of the non-selective NOS inhibitor l-NAME produced a significant decrease in current recorded in the prefrontal cortex (24 ± 6pA, n=5) and nucleus accumbens (17 ± 3pA, n=6). Finally it was necessary to validate the sensors functionality in vivo by investigating the effect of the interferent ascorbate on the oxidation current. The current showed no variation in both regions over the selected time interval of 60 min, indicating no deterioration of the polymer membrane. A detailed comparison identified significantly greater affects of administrations on NO sensors implanted in the striatum than those inserted in the prefrontal cortex and the nucleus accumbens.