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Background Oxidative stress plays an important role in neurodegeneration, pain and inflammation. (R)-(-)-linalool (LIN) is endowed with neuroprotective, anti-nociceptive and anti-inflammatory properties. Purpose The present study aims at investigating the hypothesis that LIN's neuroprotective, antinociceptive and anti-inflammatory properties descend from its ability to act as antioxidant. The study challenges this hypothesis by verifying whether LIN may counteract hydrogen peroxide (H2O2)-induced oxidative stress in PC12 cells. Methods In H2O2-exposed PC12 cells, LIN was tested on a) cell viability, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), b) damage of plasma membrane, measured by lactate dehydrogenase (LDH) release, c) intracellular levels of reactive-oxygen-species (ROS), d) apoptosis and e) cell cycle distribution. Results Under H2O2-induced cell viability reduction, LIN protects PC12 cells. Likewise, LIN protects cells from oxidative damage by preventing the H2O2-dependent increase of LDH release, counteracts intracellular ROS overproduction and reduces H2O2-induced apoptosis. Finally, the results of the cell cycle analysis from cells exposed to H2O2 indicate that LIN incubation reduces the number of cells induced into quiescence by H2O2 in the G2/M phase. Conclusions These findings indicate that LIN protects PC12 cells from H2O2-induced oxidative stress. This mechanism could justify the neuroprotective, anti-nociceptive and anti-inflammatory effects of this compound and suggest LIN as a potential therapeutic agent for the management oxidative stress-mediated pain.

Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

Highly sensitive detection of ethanol concentrations in discrete brain regions of rats voluntarily accessing ethanol, with high temporal resolution, would represent a source of greatly desirable data in studies devoted to understanding the kinetics of the neurobiological basis of ethanol's ability to impact behavior. In the present study, we present a series of experiments aiming to validate and apply an original high-tech implantable device, consisting of the coupling, for the first time, of an amperometric biosensor for brain ethanol detection, with a sensor for detecting the microvibrations of the animal. This device allows the real-time comparison between the ethanol intake, its cerebral concentrations, and their effect on the motion when the animal is in the condition of voluntary drinking. To this end, we assessed in vitro the efficiency of three different biosensor designs loading diverse alcohol oxidase enzymes (AOx) obtained from three different AOx-donor strains: Hansenula polymorpha, Candida boidinii, and Pichia pastoris. In vitro data disclosed that the devices loading H. polymorpha and C. boidinii were similarly efficient (respectively, linear region slope [LRS]: 1.98 ± 0.07 and 1.38 ± 0.04 nA/mM) but significantly less than the P. pastoris-loaded one (LRS: 7.57 ± 0.12 nA/mM). The in vivo results indicate that this last biosensor design detected the rise of ethanol in the nucleus accumbens shell (AcbSh) after 15 minutes of voluntary 10% ethanol solution intake. At the same time, the microvibration sensor detected a significant increase in the rat's motion signal. Notably, both the biosensor and microvibration sensor described similar and parallel time-dependent U-shaped curves, thus providing a highly sensitive and time-locked high-resolution detection of the neurochemical and behavioral kinetics upon voluntary ethanol intake. The results overall indicate that such a dual telemetry unit represents a powerful device which, implanted in different brain areas, may boost further investigations on the neurobiological mechanisms that underlie ethanol-induced motor activity and reward.

The importance of physiological glutamate has been widely demonstrated in cognitive and memory processes, as well as in neurotransmission. The involvement of physiological glutamate in several pathologies has also been established. Therefore, analytical devices for studying variations in physiological glutamate are of fundamental importance, particularly in preclinical studies. The necessary knowledge to develop and characterize biosensors for glutamate detection is often restricted to only a few research groups. However, many more groups have sought to implant such analytical devices to study the glutamatergic system in vivo. On this basis, a series of studies was undertaken to explore the medium-term storage of biosensors, thereby allowing their usage results to be differentiated from their construction and characterization processes to facilitate the wider diffusion and use of such sensors. Therefore, it has become vital to determine the best storage conditions to extend the life and functionality of these biosensors, especially due to the diachronic instability of the enzyme present on the surface. In the present study, we analyzed the impact of glycols, such as glycerol and triethylene glycol, as enzyme stabilizers coupled with long-term storage at low temperatures (−20 and −80 °C) on biosensor performance. The biosensors were observed for 5 months and evaluated for their enzymatic activity by measuring the VMAX(app) and KM(app). The analytical features were also evaluated in terms of the Linear Region Slope, which is one the most important parameters for indicating the efficiency and the sensitivity of biosensors. Interestingly, both glycols proved to be capable of increasing enzymatic activity and maintaining good biosensor efficiency over time. Moreover, the combination with low-temperature storage highlighted the different behaviors of the two glycols. In particular, glycerol was more effective in stabilizing the enzyme and maintaining analytical performance when the biosensors were stored at −20 °C. Instead, triethylene glycol performed the same function as glycerol but when the biosensors were stored at −80 °C.

Analyte quantification in first generation electrochemical biosensors is threatened by electro-active molecules, such as ascorbic acid (AA). Electrochemical deposition of ortho-phenylendiamine (oPD) on transducer considerably reduces AA interfering. These properties of poly-oPD (PPD) are influenced by electro-polymerization conditions. Chronoamperometry (CA) is proposed as an alternative technique for oPD electro-deposition over Pt. Pt/PPD sensors are evaluated through functional parameters related to AA rejection and analytical performances. N, O, and air bubbled supporting electrolyte and several step duration times are used. Best performing CA-PPD sensor is obtained in N-purged phosphate solution containing 300 mM oPD by means of a 1 s step duration/120 steps CA. High performing PPD is achieved in shorter time compared to commonly used constant potential amperometry. Aging of polymeric features and scanning electron microscopy investigations are performed and optimized CA electro-polymerization conditions are used to build up an efficient interference blocking layer in a glucose oxidase biosensor.

Glutamate is a major excitatory neurotransmitter in the brain. It is involved in many normal physiological brain activities, but also neurological disorders and excitotoxicity. Hence, glutamate measurement is important both in clinical and pre-clinical studies. Pre-clinical studies often use amperometric biosensors due to their low invasiveness and the relatively small size of the devices. These devices also provide fast, real-time measurements because of their high sensitivity. In the present study, diethylene glycol (DEG), neopentyl glycol (NPG), triethylene glycol (TEG), and glycerol (GLY) were used to increase the long-term stability of glutamate biosensors. The evaluation was made by measuring variations of the main enzymatic (VMAX and KM) and analytical (Linear Region Slope (LRS)) parameters. Of the glycols tested, TEG was the most promising stabilizer, showing about twice as high VMAX maintained over a greater duration than with other stabilizers tested. It is also yielded the most stable linear region slope (LRS) values over the study duration. Moreover, we highlighted the ability of glycols to interact with enzyme molecules to form a containment network, able to maintain all the layered components of the biosensor adhering to the transducer.

Interference rejection in amperometric biosensors can be more effective introducing some modifiers during electro-deposition of permselective film. Addition of β-cyclodextrin (βCD), a cyclic oligosaccharide composed of seven glucose units, to the ortho-phenylendiamine (oPD) monomer were already demonstrated to provide an enhancement in ascorbic acid (AA) rejection. Here we evaluated the improvement in permselectivity of poly-eugenol and poly-magnolol films electro-polymerized in presence of different amounts of βCD or eugenol-βCD inclusion complex for amperometric biosensor application. Starting from Pt-Ir wire as transducer several microsensors were covered with polymeric films doped with βCD-based modifiers through constant potential amperometry. Characterization of modified polymers was achieved by scanning electron microscopy and permselectivity analysis. Poly-magnolol film in combination with βCD showed a worsening in permselectivity compared to poly-magnolol alone. In contrast, the introduction of βCD-based modifier enhanced the interference rejection toward the archetypal interferent AA, while slightly affecting permeability toward H2O2 compared to the poly-eugenol without modifier. The AA rejection seems to be influenced by the availability of βCD cavity as well as film performance due to concentration of βCD-Eugenol inclusion complex. A poly-eugenol film co-polymerized with 2 mM βCD-eugenol inclusion complex showed a permselectivity equal to poly-orthophenylendiamine film (PPD), with a lower permeability to AA, likely to be related with a self-blocking mechanism. Based on these results, a biosensor for glutamate was constructed with a poly-eugenol doped with βCD-eugenol as permselective layer and its permselectivity, stability and lifetime were determined. © 2018 This work was supported by the Qatar National Research Fund (a member of Qatar Foundation) [NPRP grant # 8-392-4-003 ]. Scopus

The oxidative metabolism of ethanol into acetaldehyde involves several enzymes, including alcohol dehydrogenase (ADH) and catalase-hydrogen peroxide (H 2 O 2 ). In this regard, while it is well known that 4-methylpyrazole (4-MP) acts by inhibiting ADH in the liver, little attention has been placed on its ability to interfere with fatty acid oxidation-mediated generation of H 2 O 2 , a mechanism that may indirectly affect catalase whose enzymatic activity requires H 2 O 2 . The aim of our investigation was twofold: 1) to evaluate the effect of systemic (i.p. [intraperitoneal]) and local (into the posterior ventral tegmental area, pVTA) administration of 4-MP on oral ethanol self-administration, and 2) to assess ex vivo whether or not systemic 4-MP affects liver and brain H 2 O 2 availability. The results show that systemic 4-MP reduced ethanol but not acetaldehyde or saccharin self-administration, and decreased the ethanol deprivation effect. Moreover, local intra-pVTA administration of 4-MP reduced ethanol but not saccharin self-administration. In addition, although unable to affect basal catalase activity, systemic administration of 4-MP decreased H 2 O 2 availability both in liver and in brain. Overall, these results indicate that 4-MP interferes with ethanol self-administration and suggest that its behavioral effects could be due to a decline in catalase-H 2 O 2 system activity as a result of a reduction of H 2 O 2 availability, thus highlighting the role of central catalase-mediated metabolism of ethanol and further supporting the key role of acetaldehyde in the reinforcing properties of ethanol.

The use of amperometric biosensors has attracted particular attention in recent years, both from researchers and from companies, as they have proven to be low-cost, reliable, and very sensitive devices, with a wide range of uses in different matrices. The continuous development of amperometric biosensors, since their use involves an enzyme, is specifically aimed at keeping and increasing the catalytic properties of the loaded protein, so as to be able to use the same device over time. The present study aimed to investigate the impact of glycerol and polysaccharides, in the presence of polycationic substances to constitute a hydrogel, in enhancing the enzymatic and analytic performance of a glucose biosensor. Initially, it was possible to verify how the deposition of the starch-based hydrogel, in addition to allowing the electropolymerization of the poly(p-phenylenediamine) polymer and the maintenance of its ability to shield the ascorbic acid, did not substantially limit the permeability towards hydrogen peroxide. Moreover, different biosensor designs, loading a mixture containing all the components (alone or in combination) and the enzyme, were tested in order to evaluate the changes of the apparent enzyme kinetic parameters, such as VMAX and KM, and analytical response in terms of Linear Region Slope, highlighting how the presence of all components (starch, glycerol, and polyethyleneimine) were able to substantially enhance the performance of the biosensors. The surface analysis of the biosensors was performed by scanning electron microscope (SEM). More, it was shown that the same performances were kept unchanged for seven days, proving the suitability of this biosensor design for short- and mid-term use.

Molecular biomarkers are very important in biology, biotechnology and even in medicine, but it is quite hard to convert biology-related signals into measurable data. For this purpose, amperometric biosensors have proven to be particularly suitable because of their specificity and sensitivity. The operation and shelf stability of the biosensor are quite important features, and storage procedures therefore play an important role in preserving the performance of the biosensors. In the present study two different designs for both glucose and lactate biosensor, differing only in regards to the containment net, represented by polyurethane or glutharaldehyde, were studied under different storage conditions (+4, &minus, 20 and &minus, 80 &deg, C) and monitored over a period of 120 days, in order to evaluate the variations of kinetic parameters, as VMAX and KM, and LRS as the analytical parameter. Surprisingly, the storage at &minus, C yielded the best results because of an unexpected and, most of all, long-lasting increase of VMAX and LRS, denoting an interesting improvement in enzyme performances and stability over time. The present study aimed to also evaluate the impact of a short-period storage in dry ice on biosensor performances, in order to simulate a hypothetical preparation-conservation-shipment condition.

The purpose of the present review is to give an update regarding the classification, the epidemiology, the clinical manifestation, the diagnoses, and the treatment of the Rickettsiae species present in the Mediterranean area. We performed a comprehensive search, through electronic databases (Pubmed – MEDLINE) and search engines (Google Scholar), of peer-reviewed publications (articles, reviews, and books). The availability of new diagnostic tools, including Polymerase Chain Reaction and nucleotide sequencing has significantly modified the classification of intracellular bacteria, including the order Rickettsiales with more and more new Rickettsia species recognized as human pathogens. Furthermore, emerging Rickettsia species have been found in several countries and are often associated with unique clinical pictures that may challenge the physician in the early detection of the diseases. Rickettsial infections include a wide spectrum of clinical presentations ranging from a benign to a potentially life treating disease that requires prompt recognition and proper management. Recently, due to the spread of SARS-CoV-2 infection, the differential diagnosis with COVID-19 is of crucial importance. The correct understanding of the clinical features, diagnostic tools, and proper treatment can assist clinicians in the management of Rickettsioses in the Mediterranean area.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein&ndash, ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20&ndash, 423 nM for tyrosinase and 23&ndash, 2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 &micro, M. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

L-glutamate is one the most important excitatory neurotransmitter at central nervous system level and it is implicated in several pathologies. So, it is very important to monitor its variations, in real time in animal models’ brain. Amperometric biosensors have been used because of their very high temporal and spatial resolution, and because suitable for a short to mid-term implantation. The present study aimed to develop and characterize a new glutamate biosensor design that exploits the selectivity of Glutamate Oxidase (GluOx) for l-glutamate, and the capability of a small molecule as propylene glycol (PG) to influence and extend the stability and the activity of enzyme. Different designs were evaluated by modifying the main components in their concentrations to find the most suitable design. Moreover, enzyme concentrations from 100 U/ml up to 200 U/ml were verified and different PG concentrations (1%, 0.1% and 0.05%) were tested. The most suitable selected design was Ptc/PPD/PEI(1%)2/GlutOx5/PG(0.1%) and it was compared to the same already described design loading PEDGE, instead of PG, in terms of over-time performances. PG has proved to be capable of determining an over-time stability of the glutamate biosensor in particular in terms of linear region slope (LRS) up to 21 days.

Ceria nanoparticles with controlled size have been studied as antioxidant agents for the in vitro protection of catecholaminergic cells (PC12) exposed to manganese, which is responsible for an occupational form of Parkinson-like disease. The nanoparticle internalization has been followed by Raman and confocal microscopy while the effect of nanoceria concentration in the cell metabolism has been assessed by MTT and trypan blue assay. With the perspective to develop an innovative combined treatment, nanoceria has been tested either alone or in association with L-DOPA showing a significant effect in reducing the oxidative stress due to manganese chloride. Finally, to study the protective role of nanoceria on the metabolism of catecholamines, the intracellular concentration of dopamine and its metabolites have been monitored by liquid chromatography with electro-chemical detection in control and nanoparticle-exposed cells as a function of the nanoceria dosing. The results show a protective role of nanoceria both on PC12 cells survival and dopamine metabolism, which makes this class of nanoparticles a potential candidate for the treatment of Parkinson-like diseases induced by chronic manganese intoxication.

The aim of the present study was to investigate the usefulness of molecular breast imaging (MBI) in predicting complete tumor response to treatment and residual tumor extent following neoadjuvant therapy. A consecutive series of 43 female patients with large or locally advanced primary breast cancer, scheduled for surgery following neoadjuvant therapy, was retrospectively reviewed. Prior to surgery, all patients underwent MBI using a high‑resolution semiconductor‑based device for image acquisition. MBI data were correlated with surgical histopathological findings. Spearman's correlation coefficient was calculated to assess differences in residual tumor size with MBI and histopathological examination. From the images obtained using MBI, 7 patients were negative for residual tumors with pathological complete response (specificity, 100%) and positive in 34/36 patients with residual disease (sensitivity, 94.4%), including 26/27 patients with unifocal and 8/9 patients with multicentric/multifocal tumors, 5 of which exhibited multiple microscopic foci scattered in a fibrotic area. Overall accuracy was 95.3% and the positive predictive value (PPV) and negative predictive value (NPV) were 100 and 77.8%, respectively. MBI was false‑negative in one patient with a 2.5‑cm invasive ductal carcinoma located close to the chest wall and in one patient with microscopic foci of epithelial carcinoma. In the patients with unifocal residual tumors, correlation of tumor size between MBI and histopathology was r=0.981 (P

This study investigated the mechanisms and kinetics of nitric oxide (NO) generation by derivatives of Piloty’s acid (NO-donors) under physiological conditions. In order to qualitatively and quantitatively measure NO release, electron paramagnetic resonance (EPR) was carried out with NO spin trapping. In addition, voltammetric techniques, including cyclic voltammetry and constant potential amperometry, were used to confirm NO release from Piloty’s acid and its derivatives. The resulting data showed that Piloty’s acid derivatives are able to release NO under physiological conditions. In particular, electron-withdrawing substituents favoured NO generation, while electron-donor groups reduced NO generation. In vitro microdialysis, performed on PC12 cell cultures, was used to evaluate the dynamical secretion of dopamine induced by the Piloty’s acid derivatives. Although all the studied molecules were able to induce DA secretion from PC12, only those with a slow release of NO have not determined an autoxidation of DA itself. These results confirm that the time-course of NO-donors decomposition and the amount of NO released play a key role in dopamine secretion and auto-oxidation. This information could drive the synthesis or the selection of compounds to use as potential drugs for the therapy of Parkinson’s disease (PD).

The neurotoxin MPTP is known to induce dopamine release and depletion of ATP in the striatum of rats. Therefore, we studied the changes induced by MPTP and pargyline protection both on striatal dopamine release and on extracellular energy metabolites in freely moving rats, using dual asymmetric-flow microdialysis. A dual microdialysis probe was inserted in the right striatum of rats. MPTP (25 mg/kg, 15 mg/kg, 10 mg/kg) was intraperitoneally administered for three consecutive days. MAO-B inhibitor pargyline (15 mg/kg) was systemically administered before neurotoxin administration. The first MPTP dose induced an increase in dialysate dopamine and a decrease of DOPAC levels in striatal dialysate. After the first neurotoxin administration, increases in striatal glucose, lactate, pyruvate, lactate/pyruvate (L/P) and lactate/glucose (L/G) ratios were observed. Subsequent MPTP administrations showed a progressive reduction of dopamine, glucose and pyruvate levels with a concomitant further increase in lactate levels and L/P and L/G ratios. At day 1, pargyline pre-treatment attenuated the MPTP-induced changes in all studied analytes. Starting from day 2, pargyline prevented the depletion of dopamine, glucose and pyruvate while reduced the increase of lactate, L/P ratio and L/G ratio. These in vivo results suggest a pargyline neuroprotection role against the MPTP-induced energetic impairment consequent to mitochondrial damage. This neuroprotective effect was confirmed by TH immunostaining of the substantia nigra.

Tyrosinase [EC 1.14.18.1] is a copper-containing enzyme involved in melanin biosynthesis, in unfavourable enzymatic browning of plant-derived foods, in pathological melanogenesis and in insects moulting process]. It catalyses the oxidation of both monophenols (monophenolase or cresolase activity) and o-diphenols (diphenolase or catecholase activity) to o-quinones (Fig. 1A). Investigation of tyrosinase inhibitors may lead to development of novel skin whitening agents, medicinal products, antibrowning substances or insect control compounds]. Many natural occurring compounds have been identified for those targets whose structure has inspired the preparation of several collections of synthetic tyrosinase inhibitors. 4,40-Dihydroxy-biphenyl 1, a natural biphenol, was found to be a potent tyrosinase inhibitor, more effective than kojic acid and arbutin. Hydroxylated biphenyls are a class of polyphenols widely present in nature , some of them manifest high biological activity like ellagitannins, vancomicin, biphenomicins. Compared to phenols, hydroxylated biphenyls manifest higher antioxidant activity and generally they are less toxic than the corresponding phenolic monomer .

The development and applications of biosensors in the food industry has had a rapid grown due to their sensitivity, specificity and simplicity of use with respect to classical analytical methods. In this study, glucose and ethanol amperometric biosensors integrated with a wireless telemetry system were developed and used for the monitoring of top and bottom fermentations in beer wort samples. The collected data were in good agreement with those obtained by reference methods. The simplicity of construction, the low cost and the short time of analysis, combined with easy interpretation of the results, suggest that these devices could be a valuable alternative to conventional methods for monitoring fermentation processes in the food industry. (C) 2016 Elsevier Ltd. All rights reserved.

Ethanol is one of the most widespread psychotropic agents in western society. While its psychoactive effects are mainly associated with GABAergic and glutamatergic systems, the positive reinforcing properties of ethanol are related to activation of mesolimbic dopaminergic pathways resulting in a release of dopamine in the nucleus accumbens. Given these neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF) is of great importance. In this study, we describe the development and characterization of an implantable biosensor for the amperometric detection of brain ethanol in real time. Ten different designs were characterized in vitro in terms of Michaelis-Menten kinetics (V(MAX) and K(M)), sensitivity (linear region slope, limit of detection (LOD), and limit of quantification (LOQ)), and electroactive interference blocking. The same parameters were monitored in selected designs up to 28 days after fabrication in order to quantify their stability. Finally, the best performing biosensor design was selected for implantation in the nucleus accumbens and coupled with a previously developed telemetric device for the real-time monitoring of ethanol in freely moving, untethered rats. Ethanol was then administered systemically to animals, either alone or in combination with ranitidine (an alcohol dehydrogenase inhibitor) while the biosensor signal was continuously recorded. The implanted biosensor, integrated in the low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF and represents a new generation of analytical tools for studying ethanol toxicokinetics and the effect of drugs on brain ethanol levels.

The classical animal models of Parkinson's disease (PD) rely on the use of neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine and, more recently, the agricultural chemicals paraquat and rotenone, to deplete dopamine (DA). These neurotoxins elicit motor deficits in different animal species although MPTP fails to induce a significant dopaminergic neurodegeneration in rats. In the attempt to better reproduce the key features of PD, in particular the progressive nature of neurodegeneration, alternative PD models have been developed, based on the genetic and neuropathological links between -synuclein ( -syn) and PD. In vivo microdialysis was used to investigate extracellular striatal DA dynamics in MPTP- and -syn-generated rodent models of PD. Acute and sub-acute MPTP intoxication of mice both induce prolonged release of striatal DA. Such DA release may be considered the first step in MPTP-induced striatal DA depletion and nigral neuron death, mainly through reactive oxygen species generation. Although MPTP induces DA reduction, neurochemical and motor recovery starts immediately after the end of treatment, suggesting that compensatory mechanisms are activated. Thus, the MPTP mouse model of PD may be unsuitable for closely reproducing the features of the human disease and predicting potential long-term therapeutic effects, in terms of both striatal extracellular DA and behavioral outcome. In contrast, the -syn-generated rat model of PD does not suffer from a massive release of striatal DA during induction of the nigral lesion, but rather is characterized by a prolonged reduction in baseline DA and nicotine-induced increases in dialysate DA levels. These results are suggestive of a stable nigrostriatal lesion with a lack of dopaminergic neurochemical recovery. The -syn rat model thus reproduces the initial stage and slow development of PD, with a time-dependent impairment in motor function. This article will describe the above experimental PD models and demonstrate the utility of microdialysis for their characterization.

A miniaturized biotelemetric device for the amperometric detection of brain tissue oxygen is presented. The new system, derived from a previous design, has been coupled with a carbon microsensor for the real-time detection of dissolved O(2) in the striatum of freely moving rats. The implantable device consists of a single-supply sensor driver, a current-to-voltage converter, a microcontroller, and a miniaturized data transmitter. The oxygen current is converted to a digital value by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC). The digital data is sent to a personal computer using a six-byte packet protocol by means of a miniaturized 434 MHz amplitude modulation (AM) transmitter. The receiver unit is connected to a personal computer (PC) via a universal serial bus. Custom developed software allows the PC to store and plot received data. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption, and good linear response in the nanoampere current range. The in vivo results confirmed previously published observations on oxygen dynamics in the striatum of freely moving rats. The system serves as a rapid and reliable model for studying the effects of different drugs on brain oxygen and brain blood flow and it is suited to work with direct-reduction sensors or O(2)-consuming biosensors.

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous “treatment–control” experimental design was performed to detect K+-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC–EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment–control experiments performed on the same initial PC12 cell population.

Amperometric polymer/enzyme composite (PEC) biosensors, incorporating a poly(o-phenylenediamine) ultra-thin permselective barrier, possess a variety of characteristics that make them suitable for monitoring brain energy and neurotransmitter dynamics in vivo. This review highlights PEC sensitivity and selectivity parameters, which allow development of the basic design in a systematic way in order to improve their performance and to diversify the analyte range of these novel probes of brain function.

Reports that globular proteins could enhance the interference blocking ability of the PPD (poly(o-phenylenediamine) layer used as a permselective barrier in biosensor design, prompted this study where a variety of modifying agents were incorporated into PPD during its electrosynthesis on Pt-Ir electrodes. Trapped molecules, including fibrous proteins and β-cyclodextrin, altered the polymer/modifier composite selectivity by affecting the sensitivity to both H2O2 (signal molecule in many enzyme-based biosensors) and the archetypal interference species, ascorbic acid. A comparison of electrochemical properties of Pt and a Pt-Ir alloy suggests that the benefits of the latter, more rigid, metal can be exploited in PPD-based biosensor design without significant loss of backward compatibility with studies involving pure Pt.

Biosensors were fabricated at neutral pH by sequentially depositing the polycation polyethyleneimine (PEI), the stereoselective enzyme L-glutamate oxidase (GluOx) and the permselective barrier poly-ortho-phenylenediamine (PPD) onto 125-microm diameter Pt wire electrodes (Pt/PEI/GluOx/PPD). These devices were calibrated amperometrically at 0.7 V versus SCE to determine the Michaelis-Menten parameters for enzyme substrate, l-glutamate (Glu) and co-substrate, dioxygen. The presence of PEI produced a 10-fold enhancement in the detection limit for Glu (approximately 20 nM) compared with the corresponding PEI-free configurations (Pt/GluOx/PPD), without undermining their fast response time (approximately 2 s). Most remarkable was the finding that, although some designs of PEI-containing biosensors showed a 10-fold increase in linear region sensitivity to Glu, their oxygen dependence remained low.

Research for the use of physical means, in order to induce cell differentiation for new therapeutic strategies, is one of the most interesting challenges in the field of regenerative medicine and then in the treatment of neurodegenerative diseases, Parkinson’s disease (PD) included. The aim of this work is to verify the effect of the radio electric asymmetric conveyer (REAC) technology on the PC12 rat adrenal pheochromocytoma cell line, as they display metabolic features of PD. PC12 cells were cultured with a REAC regenerative tissue optimization treatment (TO-RGN) for a period ranging between 24 and 192 hours. Gene expression analysis of specific neurogenic genes, as neurogenin-1, beta3-tubulin and Nerve growth factor, together with the immunostaining analysis of the specific neuronal protein beta3-tubulin and tyrosine hydroxylase, shows that the number of cells committed toward the neurogenic phenotype was significantly higher in REAC treated cultures, as compared to control untreated cells. Moreover, MTT and Trypan blue proliferation assays highlighted that cell proliferation was significantly reduced in REAC TO-RGN treated cells. These results open new perspectives in neurodegenerative diseases treatment, particularly in PD. Further studies will be needed to better address the therapeutic potential of the REAC technology.

Biosensors for glutamate (Glu) were fabricated from Teflon-coated Pt wire (cylinders and disks), modified with the enzyme glutamate oxidase (GluOx) and electrosynthesized polymer PPD, poly(o-phenylenediamine). The polymer/enzyme layer was deposited in two configurations: enzyme before polymer (GluOx/PPD) and enzyme after polymer (PPD/GluOx). These four biosensor designs were characterized in terms of response time, limit of detection, Michaelis-Menten parameters for Glu (J max and K(M)(Glu)), sensitivity to Glu in the linear response region, and dependence on oxygen concentration, K(M)(O2). Analysis showed that the two polymer/enzyme configurations behaved similarly on both cylinders and disks. Although the two geometries showed different behaviors, these differences could be explained in terms of higher enzyme loading density on the disks; in many analyses, the four designs behaved like a single population with a range of GluOx loading. Enzyme loading was the key to controlling the K(M)(O2) values of these first generation biosensors. The counterintuitive, and beneficial, behavior that biosensors with higher GluOx loading displayed a lower oxygen dependence was explained in terms of the effects of enzyme loading on the affinity of GluOx for its anionic substrate. Some differences between the properties of surface immobilized GluOx and glucose oxidase are highlighted.

The apparent Michaelis constant, K(M), for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOx-based amperometric biosensors in the linear response region (100+/-11 nA cm(-2)microM(-1) at pH 7.4; SD, n=23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design.

Alterations in developmental programming of neuroendocrine and immune system function may critically modulate vulnerability to various diseases. In particular, genetic factors, including gender, may interact with early life events such as exposure to hormones, endotoxins, or neurotoxins, thereby influencing disease predisposition and/or severity, but little is known about the role of the astroglial cell compartment and its mediators in this phenomenon. Indeed, in the context of innate inflammatory mechanisms, a dysfunction of the astroglial cell compartment is believed to contribute to the selective degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta in Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. Hence, in response to brain injury the roles of astrocytes and microglia are very dynamic and cell type-dependent, in that they may exert the known proinflammatory (harmful) effects, but in certain circumstances they can turn into highly protective cells and exert anti-inflammatory (beneficial) functions, thereby facilitating neuronal recovery and repair. Here, we summarize our work suggesting a chief role of hormonal programming of glial response to inflammation and oxidative stress in MPTP-induced loss of DA neuron functionality and demonstrate that endogenous glucocorticoids and the female hormone estrogen (E(2)) inhibit the aberrant neuroinflammatory cascade, protect astrocytes and microglia from programmed cell death, and stimulate recovery of DA neuron functionality, thereby triggering the repair process. The overall results highlight glia as a final common pathway directing neuroprotection versus neurodegeneration. Such recognition of endogenous glial protective pathways may provide a new insight and may contribute to the development of novel therapeutic treatment strategies for PD and possibly other neurodegenerative disorders.

The Ca2+ sensitizer levosimendan (LEV) improves myocardial contractility by enhancing the sensitivity of the contractile apparatus to Ca2+. In addition, LEV promotes Ca2+ entry through L-type channels in human cardiac myocytes. In this study, which was performed using microdialysis, infusion of LEV at 0.25 μM for 160 min increased dopamine (DA) concentrations (up to fivefold baseline) in dialysates from the striatum of freely moving rats. Ca2+ omission from the perfusion fluid abolished baseline DA release and greatly decreased LEV-induced DA release. Reintroduction of Ca2+ in the perfusion fluid restored LEV-induced DA release. Chelation of intracellular Ca2+ by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) did not affect basal DA release and scarcely affected LEV-induced increases in dialysate DA. In addition, co-infusion of the L-type (Cav 1.1 – 1.3) voltage-sensitive Ca2+-channel inhibitor nifedipine failed to inhibit LEV-induced increases in dialysate DA, which, in contrast, was inhibited by co-infusion of the N-type (Cav 2.2) voltage-sensitive Ca2+-channel inhibitor ω-conotoxin GVIA. We conclude that LEV promotes striatal extracellular Ca2+ entry through N-type Ca2+ channels with a consequent increase in DA release. Keywords:: levosimendan, microdialysis, striatal dopamine, calcium entry

We showed previously, using in vitro microdialysis, that activation of the nitric oxide (NO)/cyclic GMP pathway was the underlying mechanism of exogenous NO-induced dopamine (DA) secretion from PC12 cells. In this study, infusion of the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mM for 60 min) induced a long-lasting decrease in dialysate DA+3-methoxytyramine (3-MT) in dialysates from PC12 cell suspensions. Ascorbic acid (0.2 mM) co-infusion allowed SIN-1 to increase dialysate DA+3-MT. SIN-1+ascorbic acid effects were abolished by Ca 2+ omission. Infusion of high K + (75 mM) induced a 2.5-fold increase in dialysate DA+3-MT. The increase was inhibited by SIN-1 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mM) with SIN-1+high K + resulted in a 3.5 fold increase in dialysate DA+3-MT. The L-type Ca 2+ channel inhibitor nifedipine selectively inhibited the DA+3-MT increase pertaining to high K + , while the soluble guanylate cyclase (sGC) inhibitor 1 H -[1,2,4]-oxadiazolo[4,3]quinoxalin-1-one selectively inhibited the increase pertaining to SIN-1 effects. These results suggest that activation of the NO/sGC/cyclic GMP pathway is the underlying mechanism of extracellular Ca 2+ -dependent effects of SIN-1 on DA secretion from PC12 cells. Extracellular Ca 2+ entry occurs through nifedipine-insensitive channels. Ascorbic acid is a key determinant in modulating the distinct profiles of SIN-1 effects.

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitrosoN-acetylpenicillamine (SNAP, 1.0 mM) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mM) or by Ca 2+ omission. Ca 2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the L-type Ca 2+ channel inhibitor nifedipine. The NO-donor (+ / –)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro3-hexenamide (NOR-3, 1.0 mM) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mM) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K + (75 mM) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mM) with NOR-3 + high K + restored high K + effects. Co-infusion of nifedipine inhibited high K + -induced DA + 3-MT increases. These results suggest that activation of the NO / sGC / cyclic GMP pathway may be the underlying mechanism of extracellular Ca 2+ -dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca 2+ entry may occur through nifedipine-insensitive channels.

The effects of either intraperitoneally (i.p.) or intrastriatally administered sufentanil on the release and metabolism of dopamine (DA) in the rat striatum were evaluated using in vivo microdialysis. Dialysate concentrations of DA and its acidic metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were increased following i.p. administration of either clinical anesthetic (20 μg/kg) or clinical analgesic (1 μg/kg) sufentanil doses. In addition, sufentanil also increased uric acid concentrations. In contrast, dialysate ascorbic acid and glutamate concentrations were unaffected. Intrastriatal infusion of sufentanil (250 nM) induced only a short lasting decrease in dialysate DA. Subcutaneous naloxone (1.0 mg/kg) abolished sufentanil-induced increases in dialysate DA, DOPAC+HVA and uric acid; however, naloxone (0.1 mM) failed to affect these increases when infused intrastriatally. These results demonstrate that sufentanil, at clinical doses, increases striatal DA release and oxidative metabolism of both DA and xanthine acting at extrastriatal sites with a μ-receptor-mediated mechanism.

Swiss mice were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 25 mg/kg/day, for 5 consecutive days and killed at different days after MPTP discontinuance. Decreases in striatal tyrosine hydroxylase activity and levels of dopamine and its metabolites were observed 1 day after MPTP discontinuance. Ascorbic acid and glutamate levels had increased, dehydroascorbic acid and GSH decreased, whereas catabolites of high-energy phosphates (inosine, hypoxanthine, xanthine, and uric acid) were unchanged. In addition, gliosis was observed in both striatum and substantia nigra compacta (SNc). Sections of SNc showed some terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)-positive cells. Neurochemical parameters of dopaminergic activity showed a trend toward recovery 3 days after MPTP discontinuance. At this time point, TUNEL-positive cells were detected in SNc; some of them showed nuclei with neuronal morphology. A late (days 6-11) increase in striatal dopamine oxidative metabolism, ascorbic acid oxidative status, and catabolites of high-energy phosphates were observed concomitant with nigral neuron and nigrostriatal glial cell apoptotic death, as revealed by TUNEL, acridine orange, and Hoechst staining, and transmission electron microscopy. These data suggest that MPTP-induced activation/apoptotic death of glial cells plays a key role in the sequential linkage of neurochemical and cellular events leading to dopaminergic nigral neuron apoptotic death.

We evaluated the clinical usefulness of the combined use of 123I-ioflupane brain single photon emission computed tomography (SPECT) and 123I-metaiodobenzylguanidine (MIBG) cardiac scintigraphy in discriminating uncertain parkinsonism with vascular lesions in striatal nuclei at magnetic resonance imaging (MRI). Forty-three consecutive patients with uncertain parkinsonism and vascular lesions at MRI in striatal nuclei were retrospectively evaluated; the uncertain differential diagnosis was between Parkinson's disease and vascular parkinsonism (PD/VP) in 22 patients, between PD and other neurodegenerative parkinsonism (PD/PS) in 11 patients and between Lewy body dementia and Alzheimer disease (LBD/AD) in the remaining 10 cases. All patients underwent 123I-ioflupane SPECT with striatal dopaminergic activity determination as binding potentials (BP; cut-off: 3.3). 123I-MIBG cardiac planar scintigraphy was performed 2 weeks later, in early (15 minutes) and delayed (240 minutes) phases also calculating heart to mediastinum (H/M) ratio (cut-off: 1.56). 123I-Ioflupane uptake was normal in 9 patients with BP values >3.3, while it was reduced in 34/43 cases with BP values 1.56 in all cases; the uptake was reduced in 22/43 cases, (4 of whom were normal and 18 were with 123I-ioflupane striatal defects) with the H/M ratio

1 We showed previously that interaction between NO and iron (II), both released following the decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats; in addition, we showed that coinfusion of iron (II) with the NO-donor S-nitroso-N-acetylpenicillamine mimicked SNP eAects on striatal DA release. 2 In the present study, intrastriatal co-infusion of iron (II) (given as FeSO4 ,1m M for 40 min) with the NO-donor and potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1) (0.2, 0.5, 1.0 or 5.0 mM for 180 min), potentiated the SIN-1-induced increase in DA concentration in dialysates from the striatum of freely moving rats. Neither alone nor associated with iron (II) did SIN-1 induce changes in dialysate ascorbic acid or uric acid concentrations. 3 Neither co-infusion of a superoxide dismutase mimetic nor uric acid aAected SIN-1-induced increases in dialysate DA concentration. 4 Infusion of the iron chelator deferoxamine (0.2 mM for 180 min) decreased dialysate DA and attenuated SIN-1-induced increases in dialysate DA concentrations. 5 These results suggest that iron plays a key role in SIN-1-induced release of striatal DA and do not support any role for either peroxynitrite or superoxide anion in SIN-1-induced release of striatal DA. British Journal of Pharmacology (2001) 134, 275‐282

We showed previously that interaction between NO and iron(II), both released following decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats. In this study, intrastriatal infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (0.2 mM for 180 min) induced a moderate increase in dialysate DA and decreases in ascorbic acid dialysate concentrations; in contrast, SNAP 1 mM infusion induced a long-lasting decrease in both DA and ascorbic acid dialysate concentrations. 3-Methoxy-tyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and uric acid levels were unaffected. Co-infusion of ferrous sulphate [iron(II), 1 mM for 40 min] with SNAP either 1 or 0.2 mM (for 180 min), produced a significant increase in both DA and 3-MT dialysate concentrations, but it did not affect decreases in dialysate ascorbic acid levels. All other dialysate neurochemicals were unaffected. Co-infusion of ascorbic acid (0.1 mM) with SNAP (1 mM) for 180 min did not modify SNAP-induced decreases in dialysate DA levels. In contrast, co-infusion of uric acid (1 mM) reversed SNAP-induced decreases in dialysate DA; co-infusion of a superoxide dismutase mimetic delayed SNAP-induced DA decreases for a short period, while co-infusion of the antioxidant N-acetylcysteine (NAC, 0.1 mM) significantly increased dialysate DA. The results of this study show that SNAP induces concentration-related changes in DA dialysate levels. At higher concentrations, SNAP induces non-enzymatic DA oxidation, which is inhibited by uric acid and NAC; ascorbic acid failed to protect dialysate DA from oxidation, probably owing to its promoting effect on SNAP decomposition; exogenous iron(II) may react with NO generated from SNAP decomposition, with a consequent increase in dialysate DA and 3-MT, therefore mimicking SNP effects on striatal DA release. British Journal of Pharmacology (2001) 132, 941–949; doi:10.1038/sj.bjp.0703887

Aging is a factor known to increase neuronal vulnerability to oxidative stress, which is widely accepted as a mechanism of manganese-induced neuronal damage. We previously showed that subchronic exposure to manganese induced greater energy impairment (as revealed by increases in hypoxanthine, xanthine and uric acid levels) in the striatum and brainstem of aged rats vs young rats. This study shows that inhibition of glutathione (GSH) synthesis, by means of buthionine (SR) sulfoximine, decreased GSH levels and increased the ascorbic acid oxidation status in the striatum and limbic forebrain of both young and aged rats. In addition, inhibition of GSH synthesis greatly potentiated the manganese-induced increase in inosine, hypoxanthine, xanthine and uric acid levels in both regions of aged rats; moreover, inhibition of GSH synthesis significantly increased inosine, hypoxanthine, xanthine and uric acid levels in both regions of young rats, compared with the manganese-treated group. These results suggest that an impairment in the neuronal antioxidant system renders young rats susceptible to manganese-induced energetic impairment, and further support the hypothesis that an impairment in this system plays a permissive role in the increase of neuronal vulnerability that occurs with aging.

The degradation of high-energy phosphates was recently shown to precede manganese-induced cellular death. We evaluated hypoxanthine, xanthine, uric acid and glutamate levels in the striatum and brainstem of 3- and 20-month-old rats after subchronic oral exposure to manganese (MnCl2, 200 mg/kg/day in young rats, and 50-100 or 200 mg/kg/day in aged rats). Aged rats had higher basal levels of hypoxanthine, xanthine, and glutamate both in the striatum and brainstem than young rats; conversely, basal uric acid levels were lower in the striatum, but higher in the brainstem. Manganese induced a significantly greater increase in hypoxanthine, xanthine, uric acid and glutamate levels in aged rats than in young rats in both brain regions. These findings depict a greater manganese-induced energetic impairment (increases in hypoxanthine and xanthine levels), xanthine oxidase-induced free radical generation (increases in xanthine and uric acid levels), and excitotoxic status (increases in glutamate levels) in aged rats than in young rats. In addition, these findings may also account for a greater manganese toxicity to the nigro-striatal dopaminergic system in aged than in young rats, as shown in a previous work.

The effects of intrastriatal infusion of 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP) on dopamine (DA), 3-methoxytyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), L-dihydroxyphenylalanine (L-DOPA), ascorbic acid and uric acid concentrations in dialysates from the striatum of freely moving rats were evaluated using microdialysis. SIN-1 (1 mM) infusion for 180 min increased microdialysate DA and 3-MT concentrations, while L-DOPA, DOPCA+HVA, ascorbic acid and uric acid levels were unaffected. Co-infusion with ascorbic acid (0.1 mM) inhibited SIN-1-induced increases in DA and 3-MT dialysate concentration. SNP (1 mM) infusion for 180 min increased greatly the dialysate DA concentration to a peak (2950% of baseline) at the end of the infusion, while increases in 3-MT were negligible. In addition, SNP decreased ascorbic acid and L-DOPA but increased uric acid concentration in the dialysate. Co-infusion with deferoxamine (0.2 mM) inhibited the late SNP-induced increase in DA dialysate concentration, but did not affect the decrease in ascorbic acid and increase uric acid dialysate concentrations. SNP (1 mM) infusion for 20 min moderately increased uric acid, DA and 3-MT, but decreased L-DOPA levels in the dialysate. Ascorbic acid concentration increased at the end of SNP infusion. Co-infusion with ascorbic acid (0.1 mM) inhibited the SNP-induced increase in DA and 3-MT, but did not affect the decrease in L-DOPA and increase in uric acid dialysate concentrations. These results suggest that NO released from SIN-1 may account for the increase in the dialysate DA concentration. NO released following decomposition of SNP may account for the early increase in dialysate DA, while late changes in microdialysate composition following SNP may result from an interaction between NO and the ferrocyanide moiety of SNP. Exogenous ascorbic acid inhibits the effect of exogenous NO on DA release probably by scavenging NO, suggesting that endogenous ascorbic acid may modulate the NO control of DA release from 300 striatal dopaminergic terminals.

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

A new telemetry system for simultaneous detection of extracellular brain glucose and lactate and motion is presented. The device consists of dual-channel, single-supply miniature potentiostat–I/V converter, a microcontroller unit, a signal transmitter, and a miniaturized microvibration sensor. Although based on simple and inexpensive components, the biotelemetry device has been used for accurate transduction of the anodic oxidation currents generated on the surface of implanted glucose and lactate biosensors and animal microvibrations. The device was characterized and validated in vitro before in vivo experiments. The biosensors were implanted in the striatum of freely moving animals and the biotelemetric device was fixed to the animal’s head. Physiological and pharmacological stimulations were given in order to induce striatal neural activation and to modify the motor behavior in awake, untethered animals.

Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.

Brain microdialysis is an analytical technique used for the dynamic monitoring of brain neurochemistry in awake, freely moving animals. This technique requires the insertion of a small dialysis catheter, called a microdialysis probe, into a specific brain region, and its perfusion with an artificial extracellular fluid. The microdialysate samples, obtained from the probe outlet, can be analysed using high-performance liquid chromatography with electrochemical detection for the quantification of oxidizable molecules recovered from the extracellular space. In this chapter, we describe a protocol for performing a microdialysis setup and experiment in freely moving rats and mice. Furthermore, the high-performance liquid chromatographic determination of ascorbic acid, uric acid, catecholamines, indolamines and derivatives is described in detail.

Microdialysis is an extensively used technique for both in vivo and in vitro experiments, applicable to animal and human studies. In neurosciences, the in vivo microdialysis is usually performed to follow changes in the extracellular levels of substances and to monitor neurotransmitters release in the brain of freely moving animals. Catecholamines, such as dopamine and their related compounds, are involved in the neurochemistry and in the physiology of mental diseases and neurological disorders. It is generally supposed that the brain's energy requirement is supplied by glucose oxidation. More recently, lactate was proposed to be the metabolic substrate used by neurons during synaptic activity. In our study, an innovative microdialysis approach for simultaneous monitoring of catecholamines, indolamines, glutamate and energy substrates in the striatum of freely moving rats, using an asymmetric perfusion flow rate on microdialysis probe, is described. As a result of this asymmetric perfusion, two samples are available from the same brain region, having the same analytes composition but different concentrations. The asymmetric flow perfusion could be a useful tool in neurosciences studies related to brain's energy requirement, such as toxin-induced models of Parkinson's disease.

Ascorbic acid (AA), one of the principal micronutrients in horticultural crops, plays a key role in the human metabolism, and its determination in food products has a great significance. Citrus fruits are rich in AA, but its content is highly susceptible to change during postharvest processing and storage. We present a new ultralow-cost system, constituted of an amperometric microsensor composed of three rod carbon electrodes connected to a telemetric device, for online detection of AA in orange juice, as an alternative to conventional analytical methods. The in vitro calibration, ranged from 0 to 5 mM, and AA juice content was calculated by adding low volumes of sample into an acetate buffer solution at a constant potential of +120 mV vs carbon pseudoreference. This new approach, which is simple, expandable, and inexpensive, seems appropriate for large scale commercial use.

Pt electrodes of different sizes (2 x 10(-5)-2 x 10(-2) cm(2)) and geometries (disks and cylinders) were coated with the ultrathin non-conducting form of poly(o-phenylenediamine), PPD, using amperometric electrosynthesis. Analysis of the ascorbic acid (AA) and H(2)O(2) apparent permeabilities for these Pt/PPD sensors revealed that the PPD deposited near the electrode insulation (Teflon or glass edge) was not as effective as the bulk surface PPD for blocking AA access to the Pt substrate. This discovery impacts on the design of implantable biosensors where electrodeposited polymers, such as PPD, are commonly used as the permselective barrier to block electroactive interference by reducing agents present in the target medium. The undesirable "edge effect" was particularly marked for small disk electrodes which have a high edge density (ratio of PPD-insulation edge length to electrode area), but was essentially absent for cylinder electrodes with a length of0.2 mm. Sample biosensors, with a configuration based on these findings (25 microm diameter Pt fiber cylinders) and designed for brain neurotransmitter L-glutamate, behaved well in vitro in terms of Glu sensitivity and AA blocking.

In this study we present the real-time monitoring of three key brain neurochemical species in conscious rats using implantable amperometric electrodes interfaced to a biotelemetric device. The new system, derived from a previous design, was coupled with carbon-based microsensors and a platinum-based biosensor for the detection of ascorbic acid (AA), O2 and glucose in the striatum of untethered, freely-moving rats. The miniaturized device consisted of a single-supply sensor driver, a current-to-voltage converter, a microcontroller and a miniaturized data transmitter. The redox currents were digitized to digital values by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC), and sent to a personal computer by means of a miniaturized AM transmitter. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption and good linear response in the nanoampere current range. The in-vivo results confirmed ON ACCESSpreviously published observations on striatal AA, oxygen and glucose dynamics recorded in tethered rats. This approach, based on simple and inexpensive components, could be used as a rapid and reliable model for studying the effects of different drugs on brain neurochemical systems.