Giancarlo Caramenti

Cooperative N-methyl-d-aspartate (NMDA) receptor antagonism and μ-opioid receptor agonism mediate the methadone inhibition of the spinal neuron pain-related hyperactivity in a rat model of neuropathic pain

Methadone (Racemic methadone) exerts its antinociceptive effect by activation of mu-opioid receptors and/or blockade of NMDA receptors. The aim of this study is to determine whether the methadone analgesic effect on neuropathic pain is achieved only by the agonism of the mu-opioid receptors or cooperatively with the antagonism of the NMDA receptors. To this purpose, in rats with neuropathic pain model of chronic constriction of one sciatic nerve (CCI rats), we administered methadone before or after opioid receptor blockade with naloxone and checked its effects on the spinal Wide Dynamic Range (WDR) neuron dynamics in three experimental conditions: on the spontaneous and noxious evoked neuronal activities in control rats (sham operated and naïve); on iontophoretic NMDA induced neuronal hyperactivity in intact rats; on pain-related spontaneous and noxious evoked hyperactivities in CCI rats. The results, as from the spike-frequency analysis, show that: (i) in control rats, methadone inhibits the noxious evoked neuronal activity and naloxone prevents or reverses about 94% of methadone inhibitory effect; (ii) in intact rats, pretreated with naloxone, methadone reduces the NMDA induced neuronal hyperactivity; (iii) in CCI rats, methadone inhibits the neuronal spontaneous and noxious evoked hyperactivities, and naloxone prevents or reverses about 60% of methadone inhibitory effect. These findings allow to conclude that methadone inhibition of the noxious evoked activity in normal rats is achieved predominantly through the agonism of the mu-opioid receptors, while the inhibition of the pain-related hyperactivity in rats with signs of neuropathic pain (CCI rats), involves also the NMDA receptors antagonism.

Disposable plastic microreactors for genomic analyses

We show the design, development and assessment of disposable, biocompatible, fully plastic microreactors, which are demonstrated to be highly efficient for genomic analyses, such as amplification of DNA, quantitative analyses in real time, multiplex PCR (both in terms of efficiency and selectivity), as compared to conventional laboratory equipment for PCR. The plastic microreactors can easily be coupled to reusable hardware, enabling heating/cooling processes and, in the case of qPCR applications, the real-time detection of the signal from a suitable fluorescent reporter present in the reaction mixture during the analysis. The low cost production of these polymeric microreactors, along with their applicability to a wide range of biochemical targets, may open new perspectives towards practical applications of biochips for point of care diagnostics.

Modeling neuronal ensemble firing activity through intermittent Chaos

While a large body of experimental works provided results about stimulus evoked activities in primary sensory cortices, the functional role and the dynamics of spontaneous activity (SA) have been less widely addressed. From the dynamical perspective, a major open problem is represented by the evolution of SA, most modeling works tacitly assuming SA activity to be essentially random. The alternative possibility that SA is, at least for a significant part, deterministic, although highly chaotic, did not gain much attention. We addressed the problem by a novel synthetic approach. We first classified the multi-unit spike patterns into a reasonable number of classes. Through the use of symbolic dynamics we described SA by characterizing its long range correlations and maximum residence times for a pattern class. We found that we could exploit the nonlinear dynamics of a logistic map tuned in the region of Type I Intermittency. We finally applied our analyses on recordings taken both from normal and neuropathic rats and we found a great variety of behaviors. Some data exhibited complex dynamics. Others were more regular with intermittent-like phases constituted by long reiteration of the same pattern class. By combining a random noisy component with a logistic map we could generate class sequences that faithfully reproduced the long range correlations and maximum residence times measured on the dataset.

Addressable electrodes microarray for biological analysis system

Multidisciplinary efforts, combining microfabrication, chemistry and molecular biology, have been recently focused on the development of large electrode arrays loaded with oligonucleotide probe to allow rapid analysis of nucleic acid samples. Different micromachining techniques can be used for obtaining the inlet, outlet and main reservoirs for the analyte. In the present work silicon wafers are used as substrates for the microarrays, patterned by means of direct writing or optical lithography. Three methods are developed in order to obtain reservoirs with depths ranging from 5 microns to 200 microns, allowing an analyte volume in the range of 1 nl to 1 ml: reactive ion etching of a polyimide layer, wet anisotropic etching of silicon, respectively deep wet isotropic etching of the glass cover. The glass cover is bonded at low temperature, using spin-on glass as adhesive and ensures a protection of the analyte, as well as a rapid entering of the analyte in the reservoirs, increasing thus the speed of the analysis. A custom laser induced fluorescence set-up is used in order to perform the analysis. The fluorescent DNA molecules are concentrated and localized during an observation time of 60 seconds, proving the functionality of the device.

Steps in miniaturizing analytical systems

The completion of the DNA sequence of several genomes, including the human one, has opened completely new scientific and technological frontiers. The huge amount of genetic information available requires the development of faster and cheaper analytical tools. This can be possible by miniaturising the analytical system itself and by the development of proper analytical procedures, involving fluidic processes. A precise genetic identifying technique is hybridization, that can be accomplished in an array format on very small bidimensional surfaces. In order to automate the fluidic process involved in the DNA hybridization, three micromachining techniques are approached by the authors team, for obtaining reservoirs with volumes ranging from 1nl to 2μl using different materials as polyimide, silicon and glass. Several configurations were proposed targeting a turbulence free fluid flow. A qualitatively fluid flow study was performed and the influence of the reservoir shape was revealed. One obtained device was tested in a Laser Induced Fluorescence detection set-up.