The Emergence of Hierarchical Somatosensory Processing in Late Prematurity.

The somatosensory system has a hierarchical organization. Information processing increases in complexity from the contralateral primary sensory cortex to bilateral association cortices and this is represented by a sequence of somatosensory-evoked potentials recorded with scalp electroencephalographies. The mammalian somatosensory system matures over the early postnatal period in a rostro-caudal progression, but little is known about the development of hierarchical information processing in the human infant brain. To investigate the normal human development of the somatosensory hierarchy, we recorded potentials evoked by mechanical stimulation of hands and feet in 34 infants between 34 and 42 weeks corrected gestational age, with median postnatal age of 3 days. We show that the shortest latency potential was evoked for both hands and feet at all ages with a contralateral somatotopic source in the primary somatosensory cortex (SI). However, the longer latency responses, localized in SI and beyond, matured with age. They gradually emerged for the foot and, although always present for the hand, showed a shift from purely contralateral to bilateral hemispheric activation. These results demonstrate the rostro-caudal development of human somatosensory hierarchy and suggest that the development of its higher tiers is complete only just before the time of normal birth.

The Development of Nociceptive Network Activity in the Somatosensory Cortex of Freely Moving Rat Pups.

Cortical perception of noxious stimulation is an essential component of pain experience but it is not known how cortical nociceptive activity emerges during brain development. Here we use continuous telemetric electrocorticogram (ECoG) recording from the primary somatosensory cortex (S1) of awake active rat pups to map functional nociceptive processing in the developing brain over the first 4 weeks of life. Cross-sectional and longitudinal recordings show that baseline S1 ECoG energy increases steadily with age, with a distinctive beta component replaced by a distinctive theta component in week 3. Event-related potentials were evoked by brief noxious hindpaw skin stimulation at all ages tested, confirming the presence of functional nociceptive spinothalamic inputs in S1. However, hindpaw incision, which increases pain sensitivity at all ages, did not increase S1 ECoG energy until week 3. A significant increase in gamma (20-50 Hz) energy occurred in the presence of skin incision at week 3 accompanied by a longer-lasting increase in theta (4-8 Hz) energy at week 4. Continuous ECoG recording demonstrates specific postnatal functional stages in the maturation of S1 cortical nociception. Somatosensory cortical coding of an ongoing pain "state" in awake rat pups becomes apparent between 2 and 4 weeks of age.

A Simple fMRI Compatible Robotic Stimulator to Study the Neural Mechanisms of Touch and Pain.

This paper presents a simple device for the investigation of the human somatosensory system with functional magnetic imaging (fMRI). PC-controlled pneumatic actuation is employed to produce innocuous or noxious mechanical stimulation of the skin. Stimulation patterns are synchronized with fMRI and other relevant physiological measurements like electroencephalographic activity and vital physiological parameters. The system allows adjustable regulation of stimulation parameters and provides consistent patterns of stimulation. A validation experiment demonstrates that the system safely and reliably identifies clusters of functional activity in brain regions involved in the processing of pain. This new device is inexpensive, portable, easy-to-assemble and customizable to suit different experimental requirements. It provides robust and consistent somatosensory stimulation, which is of crucial importance to investigating the mechanisms of pain and its strong connection with the sense of touch.

Multi-modal pain measurements in infants.

A non-invasive integrated method was developed to measure neural and behavioural responses to peripheral sensory and noxious stimulation in human infants. The introduction of a novel event-detection interface allows synchronous recording of: (i) muscle and central nervous system activity with surface electromyography (EMG), scalp electroencephalography (EEG) and near-infrared spectroscopy (NIRS); (ii) behavioural responses with video-recording and (iii) autonomic responses (heart rate, oxygen saturation, respiratory rate and cardiovascular activity) with electrocardiography (ECG) and pulse oximetry. The system can detect noxious heel lance and touch stimuli with precision (33 µs and 624 µs respectively) and accuracy (523 µs and 256 µs) and has 100% sensitivity and specificity for both types of stimulation. Its ability to detect response latencies accurately was demonstrated by a shift in latency of the vertex potential of 20.7 ± 15.7 ms (n=6 infants), following touch of the heel and of the shoulder, reflecting the distance between the two sites. This integrated system has provided reliable and reproducible measurements of responses to sensory and noxious stimulation in human infants on more than 100 test occasions.

Electrophysiological measurements and analysis of nociception in human infants.

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns. The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord. This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

A method for removing artefacts from continuous EEG recordings during functional electrical impedance tomography for the detection of epileptic seizures.

Electrical impedance tomography (EIT) is a portable, non-invasive medical imaging method, which could be employed to image the seizure onset in subjects undergoing assessment prior to epilepsy surgery. Each image is obtained from impedance measurements conducted with imperceptible current at tens of kHz. For concurrent imaging with video electroencephalogram (EEG), the EIT introduces a substantial artefact into the EEG due to current switching at frequencies in the EEG band. We present here a method for its removal, so that EIT and the EEG could be acquired simultaneously. A low-pass analogue filter for EEG channels (-6 dB at 48 Hz) and a high-pass filter (-3 dB at 72 Hz) for EIT channels reduced the artefact from 2-3 mV to 50-300 microV, but still left a periodic artefact at about 3 Hz. This was reduced to less than 10 microV with a software filter, which subtracted an artefact template from the EEG raw traces. The EEG was made clinically acceptable at four times its acquisition speed. This method could enable EIT to become a technique for imaging on telemetry units alongside EEG, without interfering with routine EEG reporting.

An electrode addressing protocol for imaging brain function with electrical impedance tomography using a 16-channel semi-parallel system.

Electrical impedance tomography of brain function poses special problems because applied current is diverted by the resistive skull. In the past, image resolution was maximized with the use of an electrode addressing protocol with widely spaced drive electrode pairs and use of a multiplexer so that many electrode pairs could be flexibly addressed. The purpose of this study was to develop and test an electrode protocol for a 16-channel semi-parallel system which uses parallel recording channels with fixed wiring, the Kyung Hee University (KHU) Mk1. Ten protocols were tested, all addressing pairs of electrodes for recording or current drive, based on recording with a spiral, spiral with suboccipital electrodes (spiral s-o) and zig-zag configurations, and combinations of current injection from electrode pairs at 180 degrees , 120 degrees and 60 degrees . These were compared by assessing the image reconstruction quality of five simulated perturbations in a homogenous model of the human head and of four epileptic foci in an anatomically realistic model in the presence of realistic noise, in terms of localization error, resolution, image distortion and sensitivity in the region of interest. The spiral s-o with current injection at 180 degrees + 120 degrees + 60 degrees gave the best image quality and permitted reconstruction with a localization error of less than 10% of the head diameter. This encourages the view that it might be possible to obtain satisfactory images of focal abnormalities in the human brain with 16 scalp electrodes and improved instrumentation avoiding multiplexers on recording circuits.

A comparison of two EIT systems suitable for imaging impedance changes in epilepsy.

Electrical impedance tomography (EIT) has the potential to produce functional images of the conductivity changes associated with epilepsy to help localization of epileptic foci. Scalp voltage changes associated with internal conductivity changes due to focal seizures have been shown at the limit of detectability for present EIT systems. The performances of two EIT systems, which may be employed in clinical recordings during presurgical assessment of intractable epilepsy, were compared. Those were the 32-channel serial UCH Mk2.5 and the 16-channel semi-parallel KHU Mk1. Images of three conductivity perturbations, simulating epileptic foci, in a head-shaped saline tank without and with a real human skull were recorded using 31-channel and 16-channel protocols with the UCH Mk2.5, while only 16-channel protocols with the KHU Mk1. The UCH Mk2.5 employing the 31-channel protocol had better overall performance with a localization error of 12.7% of the tank diameter, which would be sufficient for lateralization of the epileptic activity. More blurred images, but with similar localization, were obtained using 16 electrodes.

Effectiveness of continuous wound infusion of 0.5% ropivacaine by On-Q pain relief system for postoperative pain management after open nephrectomy.

BACKGROUND: Block of parietal nociceptive afferent nerves using continuous wound infiltration with local anaesthetics may be beneficial in multimodal postoperative pain management. The effectiveness of continuous wound infusion of ropivacaine for postoperative pain relief after open nephrectomy was analysed in a prospective, randomized, double-blinded, placebo-controlled trial. METHODS: One hundred and sixty-eight patients were randomized to either 0.5% ropivacaine (ON-Q group) or 0.9% NaCl (control group), using an elastomeric pump which delivered 4 ml h(-1) over 48 h through two multiholed Soaker catheters placed between the transverse and the internal oblique muscles and the s.c. space. All patients received a standard postoperative pain management protocol, including patient-controlled analgesic morphine and ketorolac. Outcomes measured over 48 h after operation were visual analogue scale (VAS) and incident (i) VAS pain scores, morphine consumption, and side-effects; time to bowel function recovery; and mean length of hospitalization. RESULTS: Side-effects were similar between the two groups. VAS and i-VAS pain scores, morphine consumption [11.5 (0.27) vs 21.8 (0.37) mg; P<0.001], time to bowel recovery [21.8 (0.4) vs 33.6 (0.9) h; P<0.001], and mean length of hospitalization [2.1 (0.03) vs 3.2 (0.1) days; P<0.001] were significantly reduced in the ON-Q group. Cost analysis revealed an overall savings of approximately 273 euros per patient in the ON-Q group. CONCLUSIONS: Continuous surgical wound infusion with ropivacaine improved pain relief and accelerated recovery and discharge reducing overall costs of care.

Analysis of resting noise characteristics of three EIT systems in order to compare suitability for time difference imaging with scalp electrodes during epileptic seizures.

Electrical impedance tomography measurements in clinical applications are limited by an undesired noise component. We have investigated the noise in three systems suitable for imaging epileptic seizures, the UCH Mark1b, UCH Mark2.5 and KHU Mark1 16 channel, at applied frequencies in three steps from 1 to 100 kHz, by varying load impedance, single terminal or multiplexed measurements, and in test objects of increasing complexity from a resistor to a saline filled tank and human volunteer. The noise was white, and increased from about 0.03% rms on the resistor to 0.08% on the human; it increased with load but was independent of use of the multiplexer. The KHU Mark1 delivered the best performance with noise spectra of about 0.02%, which could be further reduced by averaging to a level where reliable imaging of changes of about 0.1% estimated during epileptic seizures appears plausible.

Factors limiting the application of electrical impedance tomography for identification of regional conductivity changes using scalp electrodes during epileptic seizures in humans.

Electrical impedance tomography (EIT) has the potential to produce images during epileptic seizures. This might improve the accuracy of the localization of epileptic foci in patients undergoing presurgical assessment for curative neurosurgery. It has already been shown that impedance increases by up to 22% during induced epileptic seizures in animal models, using cortical or implanted electrodes in controlled experiments. The purpose of this study was to determine if reproducible raw impedance changes and EIT images could be collected during epileptic seizures in patients who were undergoing observation with video-electroencephalography (EEG) telemetry as part of evaluation prior to neurosurgery to resect the region of brain causing the epilepsy. A secondary purpose was to develop an objective method for processing and evaluating data, as seizures arose at unpredictable times from a noisy baseline. Four-terminal impedance measurements from 258 combinations were collected continuously using 32 EEG scalp electrodes in 22 seizure episodes from 7 patients during their presurgical assessment together with the standard EEG recordings. A reliable method for defining the pre-seizure baseline and recording impedance data and EIT images was developed, in which EIT and EEG could be acquired simultaneously after filtering of EIT artefact from the EEG signal. Fluctuations of several per cent over minutes were observed in the baseline between seizures. During seizures, boundary voltage changes diverged with a standard deviation of 1-54% from the baseline. No reproducible changes with the expected time course of some tens of seconds and magnitude of about 0.1% could be reliably measured. This demonstrates that it is feasible to acquire EIT images in parallel with standard EEG during presurgical assessment but, unfortunately, expected EIT changes on the scalp of about 0.1% are swamped by much larger movement and systematic artefact. Nevertheless, EIT has the unique potential to provide invaluable neuroimaging data for this purpose and may still become possible with improvements in electrode design and instrumentation.

Mechanistic aspects of organophosphorothionate toxicity in fish and humans.

Metabolic transformation plays a major role in the mechanism of toxicity of organophosphorous (OP) pesticides. The modulation of their toxicity by oxonases and monooxygenases, alone or in combination, has been shown in mammals and fish. Very limited information exists for the identification of the metabolic factors relevant in the human toxicology of such chemicals. In this paper, we develop a simple algorithm, based on in vitro data, for the identification of fish species more susceptible to diazinon (D). Similar algorithms are likely to be applicable to other organophosphothionate (OPT) pesticides. We also report on preliminary studies on the OPT substrate specificity of human liver cytochromes P450 (CYPs): such information may be useful to understand the role of sulphoxidation in OPT toxicity to humans and to identify individuals with increased susceptibility to OPT toxicity. Studies of the mechanism of OPT toxicity may provide useful tools for a more detailed characterisation of these chemicals, with reference to the risk for the human population and to the impact on the fish species present in specific environments.

Time dependence of chloroform-induced metabolic alterations in the liver and kidney of B6C3F1 mice.

The time course of some biochemical changes in the liver and in the kidney was studied in B6C3F1 male mice dosed with a single i.p. injection of 150 mg/kg body weight (b.w.) CHCl(3). Hepatic and renal microsomal cytochrome P450 (P450) content and some related monooxygenase activities, CHCl(3) oxidative and reductive metabolism, cytosolic reduced glutathione (GSH) content and serum markers of nephrotoxicity were measured. In the liver no biochemical changes were produced up to a week after chloroform treatment. On the contrary, the drug-metabolizing enzyme system in the kidney was dramatically and rapidly inactivated by chloroform treatment. Maximum loss of GSH (50%), P450 (80%) and of different enzymatic activities, including CHCl(3) bioactivation, occurred during the first 5 h. These biochemical alterations are early effects, not secondary to morphological tissue changes. Kidney parameters, altered by chloroform treatment, returned to control values at different times: renal function markers became normal in 48 h; GSH levels were recovered at 96 h and the drug-metabolizing enzyme activities at longer times. The present results clearly show that repeated daily doses of chloroform, as those used in carcinogenicity tests, find renal tubular cells not at their physiological status, due to the changes produced by the first chloroform dose. Therefore the similarity in P450-dependent chloroform metabolism shown in vitro by hepatic and renal microsomes from untreated B6C3F1 male mice or in vivo in animals treated once, is lost during repeated treatments. These features should be considered in understanding the different susceptibility of the liver and the kidney to chloroform-induced tumours.

Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver.

The metabolism of diazinon, an organo-phosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague-Dawley (SD) rats or SD rats treated with different P450-specific inducers (phenobarbital, dexamethasone, beta-napthoflavone, and pyrazole). Results obtained indicate an involvement of CYP2C11, CYP3A2, and CYP2B1/2, whereas CYP2E1 and CYP1A1 do not contribute to the pesticide oxidative metabolism. Indeed, diazinon was metabolized by microsomes from control rats; among the inducers, phenobarbital and dexamethasone only increased the production of either metabolites, although to different extents. The production of the two metabolites is self-limiting, due to P450 inactivation; therefore, the inhibition of CYP-specific monooxygenase activities after diazinon preincubation has been used to selectively identify the competent CYPs in diazinon metabolism. Results indicate that, after diazinon preincubation, CYP3A2-catalyzed reactions (2beta- and 6beta-testosterone hydroxylation) are very efficiently inhibited; CYP2C11- and CYP2B1/2-catalyzed reactions (2alpha- and 16beta-testosterone hydroxylation, respectively) are weakly inhibited, while CYP2E1-, CYP2A1/2-, and CYP1A1/2-related activities were unaffected. Results obtained by using chemical inhibitors or antibodies selectively active against specific CYPs provide a direct evidence for the involvement of CYP2C11, CYP3A2, and CYP2B1/2, indicating that each of them contributed about 40-50% of the diazinon metabolism, in hepatic microsomes from untreated, phenobarbital-, and dexamethasone-treated rats, respectively. The higher diazoxon/pyrimidinol ratio observed after phenobarbital-treatment together with the significantly more effective inhibition toward diazoxon production exerted by metyrapone in microsomes from phenobarbital-treated rats supports the conclusion that CYP2B1/2 catalyze preferentially the production of diazoxon.

Pre- and postprandial pyridostigmine and oxiracetam effects on growth hormone secretion in anorexia nervosa.

Previous studies have shown that food ingestion is not capable of inhibiting the GHRH-induced GH release in anorexia nervosa, at variance with what is observed in normal subjects. Moreover, a cholinergic alteration has been hypothesized in this disorder. In a group of 24 anorectic patients in a stabilized phase of the illness, we tested, before and after a standard meal, the GH response to GHRH alone and after pre-treatment with pyridostigmine, an inhibitor of acetylcholinesterase, and, on a different day, with oxiracetam, which stimulates the central cholinergic neurones. The GH response to GHRH was significantly increased by both drugs in a fasting state. The postprandial response was not significantly modified by pyridostigmine nor by oxiracetam. Neither of these compounds was able to enhance the postprandial GH 'paradoxical' response to GHRH in anorectic patients. The lack of effect of both groups postprandially also suggests a suppression of somatostatinergic activity.

The role of different cytochrome P450 isoforms in in vitro chloroform metabolism.

The two CHCl3 activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and beta-naphthoflavone). The present results provide direct evidence that CHCl3 concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl3 concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10-15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl3 only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl3 oxidative bioactivation do not participate in CHCl3 reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl3 activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity.