Biofilm development in fixed bed biofilm reactors: experiments and simple models for engineering design purposes.

Biofilm development in a fixed bed biofilm reactor system performing municipal wastewater treatment was monitored aiming at accumulating colonization and maximum biofilm mass data usable in engineering practice for process design purposes. Initially a 6 month experimental period was selected for investigations where the biofilm formation and the performance of the reactors were monitored. The results were analyzed by two methods: for simple, steady-state process design purposes the maximum biofilm mass on carriers versus influent load and a time constant of the biofilm growth were determined, whereas for design approaches using dynamic models a simple biofilm mass prediction model including attachment and detachment mechanisms was selected and fitted to the experimental data. According to a detailed statistical analysis, the collected data have not allowed us to determine both the time constant of biofilm growth and the maximum biofilm mass on carriers at the same time. The observed maximum biofilm mass could be determined with a reasonable error and ranged between 438 gTS/m(2) carrier surface and 843 gTS/m(2), depending on influent load, and hydrodynamic conditions. The parallel analysis of the attachment-detachment model showed that the experimental data set allowed us to determine the attachment rate coefficient which was in the range of 0.05-0.4 m d(-1) depending on influent load and hydrodynamic conditions.

Decreased plasma nociceptin/orphanin FQ levels after acute coronary syndromes.

Foregoing researches made on the N/OFQ system brought up a possible role for this system in cardiovascular regulation. In this study we examined how N/OFQ levels of the blood plasma changed in acute cardiovascular diseases. Three cardiac patient groups were created: enzyme positive acute coronary syndrome (EPACS, n = 10), enzyme negative ACS (ENACS, n = 7) and ischemic heart disease (IHD, n = 11). We compared the patients to healthy control subjects (n = 31). We found significantly lower N/OFQ levels in the EPACS [6.86 (6.21-7.38) pg/ml], ENACS [6.97 (6.87-7.01) pg/ml and IHD groups [7.58 (7.23-8.20) pg/ml] compared to the control group [8.86 (7.27-9.83) pg/ml]. A significant correlation was detected between N/OFQ and white blood cell count (WBC), platelet count (PLT), creatine kinase (CK), glutamate oxaloacetate transaminase (GOT) and cholesterol levels in the EPACS group.Decreased plasma N/OFQ is closely associated with the presence of acute cardiovascular disease, and the severity of symptoms has a significant negative correlation with the N/OFQ levels. We believe that the rate of N/OFQ depression is in association with the level of ischemic stress and the following inflammatory response. Further investigations are needed to clarify the relevance and elucidate the exact effects of the ischemic stress on the N/OFQ system.

Facilitation of spike-wave discharge activity by lipopolysaccharides in Wistar Albino Glaxo/Rijswijk rats.

In normal rats the proinflammatory cytokines like interleukin-1beta, interleukin-6, which are induced by bacterial lipopolysaccharides, are able to control thalamo-cortical excitability by exerting strong effects on physiological synchronization such as sleep and on pathological synchronization like that in epileptic discharges. To investigate whether proinflammatory cytokines or lipopolysaccharides could modulate absence seizures resulting from a very different generator mechanism than the already investigated bicuculline-, kindling- and kainate-induced seizures, we used a genetically epileptic Wistar Albino Glaxo/Rijswijk rat strain, which is spontaneously generating high voltage spike-wave discharges. Wistar Albino Glaxo/Rijswijk rats responded with an increase of the number of spike-wave discharges to lipopolysaccharide injection (from 10 microg/kg to 350 microg/kg). Repetitive administration of 350 microg/kg lipopolysaccharides daily for 5 days increased the number of spike-wave discharges on the first, second and third days but the number of spike-wave discharges returned to the control value on day 5, at the 5th injection of lipopolysaccharides, showing a tolerance to lipopolysaccharides. The lipopolysaccharide-induced increase in spike-wave discharges was not directly correlated with the elevation of the core body temperature, as it is in febrile seizures, although lipopolysaccharide induced prostaglandin and is clearly pyrogenic at the doses used. Indomethacin, the prostaglandin synthesis inhibitor, efficiently blocked lipopolysaccharide-induced enhancement of spike-wave discharge genesis suggesting that the spike-wave discharge facilitating effect of lipopolysaccharides involves induction of cyclooxygenase 2 and subsequent synthesis and actions of prostaglandin E2. Low dose (40 mg/kg, i.p.) of competitive N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid, and low dose of lipopolysaccharide (20 microg/kg) showed a synergistic interaction to increase the number of spike-wave discharges, whereas at supramaximal doses of lipopolysaccharide and the N-methyl-D-aspartate antagonist no synergy was present. The data reveal a functional connection between absence epileptic activity and lipopolysaccharide induction of prostaglandin synthesis and prostaglandin action and suggest some common cellular targets in epilepsy and lipopolysaccharide-induced inflammation.

The electroretinogram and visual evoked potential of freely moving rats.

The vascularised rat retina could be one of the most useful experimental objects in visual neuroscience to understand human visual physiological and pathological processes. We report here on a new method of implantation for studying the visual system of freely moving rats that provides a rat model for simultaneous recording at corneal and cortical level and is stable enough to record for months. We implanted light emitting diodes onto the skull behind the eyeball to stimulate the eye with flashes and to light adapt the retina with constant light levels. A multistrand, stainless steel, flexible fine wire electrode placed on the eyeball was used for electroretinogram recording and screw electrodes (left/right visual and parietal cortical) were used to record the visual evoked potential and the electroencephalogram. In the present report we focus on the new method of implantation for recording the corneal flash electroretinogram of normal, freely moving rats simultaneously with the visual evoked cortical potential showing examples in various visual experiments. We also introduce a program for retinogram and visual evoked potential analysis, which defines various measures (latencies, areas, amplitudes, and durations) and draw attention to the benefits of this method for those involved in visual, functional genomic, pharmacological, and human ophthalmologic research.

Sleep modifies retinal ganglion cell responses in the normal rat.

Recordings were obtained from the visual system of rats as they cycled normally between waking (W), slow-wave sleep (SWS), and rapid eye movement (REM) sleep. Responses to flashes delivered by a light-emitting diode attached permanently to the skull were recorded through electrodes implanted on the cornea, in the chiasm, and on the cortex. The chiasm response reveals the temporal order in which the activated ganglion cell population exits the eyeball; as reported, this triphasic event is invariably short in latency (5--10 ms) and around 300 ms in duration, called the histogram. Here we describe the differences in the histograms recorded during W, SWS, and REM. SWS histograms are always larger than W histograms, and an REM histogram can resemble either. In other words, the optic nerve response to a given stimulus is labile; its configuration depends on whether the rat is asleep or awake. We link this physiological information with the anatomical fact that the brain dorsal raphe region, which is known to have a sleep regulatory role, sends fibers to the rat retina and receives fibers from it. At the cortical electrode, the visual cortical response amplitudes also vary, being largest during SWS. This well known phenomenon often is explained by changes taking place at the thalamic level. However, in the rat, the labile cortical response covaries with the labile optic nerve response, which suggests the cortical response enhancement during SWS is determined more by what happens in the retina than by what happens in the thalamus.

Coupled intra- and extracellular Ca2+ dynamics in recurrent seizure-like events.

We considered the evolution of Ca2+ oscillation dynamics in recurrent seizure-like events. Dynamic system behaviour was characterized in the state space reconstructed from intra- and extracellular [Ca2+] fluctuations simultaneously measured in cultured rat hippocampal slices under low-[Mg2+] conditions. When associated in the seizure-like event, these fluctuations occurred on a restricted set, the attractor, embedded in the full state space with less than five degrees of freedom. Instantaneous relative phase differences indicated field potential-driven phase jumps locked onto seizure-like events. To account for recurrent dynamics, calculations were performed on different extensions of a model for Ca2+ oscillation. These identified bidirectional, asymmetrical coupling of extracellular with intracellular (cytosolic, Ca2+ store, mitochondrial) Ca2+ dynamics as critical in its development.

Temporal distribution of the ganglion cell volleys in the normal rat optic nerve.

We describe experiments on behaving rats with electrodes implanted on the cornea, in the optic chiasm, and on the visual cortex; in addition, two red light-emitting diodes (LED) are permanently attached to the skull over the left eye. Recordings timelocked to the LED flashes reveal both the local events at each electrode site and the orderly transfer of visual information from retina to cortex. The major finding is that every stimulus, regardless of its luminance, duration, or the state of retinal light adaptation, elicits an optic nerve volley with a latency of about 10 ms and a duration of about 300 ms. This phenomenon has not been reported previously, so far as we are aware. We conclude that the retina, which originates from the forebrain of the developing embryo, behaves like a typical brain structure: it translates, within a few hundred milliseconds, the chemical information in each pattern of bleached photoreceptors into a corresponding pattern of ganglion cell neuronal information that leaves via the optic nerve. The attributes of each rat ganglion cell appear to include whether the retinal neuropile calls on it to leave after a stimulus and, if so when, within a 300-ms poststimulus epoch. The resulting retinal analysis of the scene, on arrival at the cortical level, is presumed to participate importantly in the creation of visual perceptual experiences.

Low-[Mg2+]-induced Ca2+ fluctuations in organotypic hippocampal slice cultures.

We show here by whole field monitoring of free intracellular Ca2+ ([Ca2+]i), locally recorded field potential (fp) and external [Ca2+], that low-[Mg2+] induces seizure like events (SLEs) accompanied by simultaneous fluctuations of [Ca2+]i and [Ca2+]e in cultured hippocampal slices. Within a SLE, complex [Ca2+]e fluctuations are seen throughout phases of Ca2+ depletion (tonic) and Ca2+ recovery (clonic) of the extracellular space. Information theory entropy-based analyses revealed strong asymmetric associations of [Ca2+]i and [Ca2+]e kinetics. By contrast, signal-associations between SLEs were found to be weak and of symmetric nature distinguishing seizure-like and interictal events by extensive coupling and decoupling of [Ca2+]i and [Ca2+]e fluctuations, respectively.

Persistent depolarization and Glu uptake inhibition operate distinct osmoregulatory mechanisms in the mammalian brain.

The ways of coupling neuronal with glial compartments in natural physiology was investigated in microdialysis experiments by monitoring extracellular concentration of amino acids in the brain of anaesthetized rats. We hypothesized that extracellular [Glu], [Gln] and [Tau] patterns would be state-dependent. This was tested by stimulation of N-methyl-D-aspartate (NMDA) receptors, by inhibition of Glu uptake or by local depolarization with a high-K(+) dialysate, coupled with the addition of Co(2+) to block Ca(2+) influx. The results showed that (1) extracellular [Gln] was low whereas [Glu] and [Tau] were high during infusion of NMDA (0.5-1.0 mM) or high-K(+) (80 mM) in the hippocampus and ventrobasal thalamus, (2) hippocampal extracellular [Glu], [Gln] and [Tau] were increased in response to the Glu uptake inhibitor, L-trans-pyrrolidine-2, 4-dicarboxilic acid (tPDC, 0.5-3.0 mM), in a concentration-dependent manner, (3) high-K(+)-induced increase of extracellular [Glu] was partially blocked by the addition of 10 mM CoCl(2) with the high-K(+) dialysate in the hippocampus. Searching for main correlations between changes in [Glu], [Gln] and [Tau] by calculating partial correlations and with the use of factor analyses we found, the primary response of the mammalian brain to persistent depolarization is the neuronal uptake of [Gln] and release of [Tau] thereupon, acting independently of Glu changes. When glial and neuronal uptake of Glu is blocked, releases of Tau occur from neuronal as well as glial compartments accompanied by increases of [Gln] in the mammalian brain.

Axonal changes in chronic demyelinated cervical spinal cord plaques.

Imaging and pathomorphological studies in multiple sclerosis suggest that axonal injury and axonal loss are playing a crucial role in those with persistent disability and long-standing disease. Although the existence of axonal injury in multiple sclerosis is proven, especially in the zone of active inflammation, the effect of chronic inflammation on the axons remains elusive. The aim of this study was to perform a quantitative morphometrical analysis, estimating axonal loss and evaluating axonal degenerative changes in cervical spinal cord samples of patients suffering from secondary progressive multiple sclerosis. Completely demyelinated plaques, normal appearing white matter (NAWM) and control material from anatomically identical regions of the cord have been compared. Neurofilament immunostaining was used for identification of the axons. We observed a significant reduction of axonal density (number of axons/mm(2)) in multiple sclerosis, both in the plaque and in the NAWM compared with the control cases. Axons under approximately 3.3 microm diameter seemed to be more affected. The intensity of the immunostaining was significantly reduced in the plaque compared with either NAWM or control. Our results on the cervical cord combined with other observations support the concept of slow axonal degeneration rather than acute damage as a cause of chronic disability in multiple sclerosis.

Glucocorticoids alter recovery processes in the rat retina.

The effect of glucocorticoids on the electroretinogram (ERG) b-wave was studied in freely moving rats. b-Waves were evoked by flashes delivered by a light emitting diode implanted under the skin above the left eye. I.v. corticosterone and dexamethasone injection induced a transient increase in b-wave amplitude at 90 min. Retinal oscillatory potentials (OPs) were similarly enhanced. Pretreatment with a glucocorticoid receptor antagonist (RU 38486) abolished both increases. These results suggest that enhancements in retinal potentials may be the result of a glucocorticoid-induced facilitation of the processes under way in normal Müller cells following retinal excitation.

Analysis of purine and pyrimidine bases, nucleosides and deoxynucleosides in brain microsamples (microdialysates and micropunches) and cerebrospinal fluid.

A new chromatographic method is reported for the synchronous analysis of endogenous purine and pyrimidine bases, ribonucleosides, and deoxyribonucleosides in brain samples. An optimized gradient chromatography system with a cooled reversed-phase column allows the detection of these compounds in very low concentrations in microsamples (microdialysates and micropunches). Chromatographic peaks were identified via the retention times of known standards, with detection at two wavelengths, and also by electrospray tandem mass spectrometry, which permits the identification of certain compounds at extremely low concentrations. The method was tested on in vivo brain microdialysis samples, micropunch tissue sample and cerebrospinal fluid of rats. Extracellular concentrations of pyrimidine metabolites in brain samples and of various purine metabolites in thalamic samples are reported here first. A comparison of the results on microdialysis and cerebrospinal fluid samples suggests that the analysis of cerebrospinal fluid provides limited information on the local extracellular concentrations of these compounds. Basic dialysis experiments revealed temporarily stable baseline levels one hour after implantation of the microdialysis probes. An elevated potassium concentration in the perfusion solution caused increases in the extracellular levels of adenosine and its metabolites, and of guanosine and the pyrimidine nucleoside uridine.

Spectral components of cytosolic [Ca2+] spiking in neurons.

We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved into evolutionary spectra of a characteristic set of frequencies. Non-delayed small spikes on top of sustained [Ca2+]c were synthesized by a main component frequency, 0.132+/-0.012 Hz, showing its maximal amplitude in phase with the start of depolarization (25 mM KCI) combined with caffeine (10 mM) application. Delayed complex responses of large [Ca2+]c spiking observed in cells from a different set of cultures were synthesized by a set of frequencies within the range 0.018-0.117 Hz. Differential frequency patterns are suggested as characteristics of the [Ca2+]c spiking responses of neurons under different conditions.

Natural sleep modifies the rat electroretinogram.

We show here electroretinograms (ERGs) recorded from freely moving rats during sleep and wakefulness. Bilateral ERGs were evoked by flashes delivered through a light-emitting diode implanted under the skin above one eye and recorded through electrodes inside each orbit near the optic nerve. Additional electrodes over each visual cortex monitored the brain waves and collected flash-evoked cortical potentials to compare with the ERGs. Connections to the stimulating and recording instruments through a plug on the head made data collection possible at any time without physically disturbing the animal. The three major findings are (i) the ERG amplitude during slow-wave sleep can be 2 or more times that of the waking response; (ii) the ERG patterns in slow-wave and REM sleep are different; and (iii) the sleep-related ERG changes closely mimic those taking place at the same time in the responses evoked from the visual cortex. We conclude that the mechanisms that alter the visual cortical-evoked responses during sleep operate also and similarly at the retinal level.

Computer determination of systolic time intervals based on impedance cardiography.

A computerized system has been developed based on impedance cardiography for processing systolic time intervals (STI) in psychophysiological experiments. The conventional method of STI determination is based on transducing three signals: electrocardiogram, phonocardiogram and pulse tracing (carotid pulse). Because of its negligible pulse transmission time the first derivative impedance cardiogram (dZ/dt) is more reliable than other pulse signals. Since the sharply demarcated points of the dZ/dt waveform occur simultaneously with the cardiac events the dZ/dt makes phonocardiogram processing unnecessary. Computer algorithms for STI assessment are based on processing two signals: the ECG and dZ/dt. The pre-ejection period (PEP), the left ventricular ejection time (LVET) and electromechanical systole (QS2) are derived by recognizing the B and X points on the dZ/dt signal as the endpoints of PEP and LVET respectively. X point identification consists of two steps: (i) the estimation of the QS2 based on the regression relationship between QS2 and heart rate (HR) and (ii) a filtering procedure for exact localization of the X point. The B point is determined by calculating the curvature function of the dZ/dt and employing a clustering procedure. The accuracy and reliability of the software were tested by processing data from 40 subjects under stress condition (cold pressor and mental arithmetic).

The influence of monocular viewing on heart period variability.

Monocular viewing seems to differentially activate the cerebral hemispheres. Viewing with only one eye is accompanied by a relatively greater activation of the contralateral hemisphere. In this study heart periods were measured during binocular, left and right monocular viewing in 29 right-handed males. Power spectral analysis of heart period variability showed that the amount of heart rate fluctuations in the 0.07-0.13 Hz frequency range (the mid-frequency peak of the spectrum) depends on the viewing eye. Only viewing with the left eye produced a significant increase of the mid-frequency peak. It is known that sympathetic activity contributes strongly to the mid-frequency component of the heart rate. Therefore, it is concluded that the effects of left monocular viewing result in an increased sympathetic influence on the sinoatrial node.

Significance and assessment of autonomic indices in cardiovascular reactions.

The purpose of this review was to integrate recent evidence supporting the reliability of noninvasive measures of parasympathetic and sympathetic activity. Literature concerning spectral analysis of heart period (HP) variability is reviewed with special emphasis on works revealing neural mediation of high-frequency and mid-frequency components of HP power spectrum and suggesting their use as a tool to assess autonomic balance. Problems of derivations of autonomic indices based on impedance cardiography and HP variance analysis are discussed. Advantages of parametric time series (autoregressive-AR) models are described with the objective of providing an informed basis for choosing among methodological alternatives. Two original approaches developed in our laboratory are outlined, namely the algorithms for systolic time interval assessment based on impedance cardiogram as well as the AR method developed for heart period power spectral density estimation.

Effects of mental load on the spectral components of heart period variability in twins.

The contribution of genetic and environmental control to stress-related cardiovascular reactions was investigated in 10 monozygotic and 10 dizygotic twin pairs during mental arithmetics. Non-invasive indices reflecting vagal and sympathetic activity were used, namely: indices of myocardial contractility based on impedance cardiogram, and spectral components of heart period variance. Autoregressive algorithms were developed for heart period power spectral density estimation providing automatic decomposition of heart period spectra into individual spectral components. During the mental task spectral energy of the mid-frequency (central frequency approximately 0.1 Hz) and high frequency (around respiratory frequency) components of heart period variance significantly decreased indicating vagal withdrawal. A task-related increase of the mid-frequency component relative to the high-frequency component was obtained. This change in the ratio of the two components as well as the considerable shortening of the contractility indices are pointing to sympathetic activation. When comparing intraclass correlations computed separately for monozygotic and dizygotic twins highly significant correlations were found for the mid-frequency component in monozygotic but not in dizygotic twin pairs in resting condition indicating a substantial genetic contribution to the control mechanisms involved in the baroreflex. Contribution of genetic factors to the control of stress-related interplay of autonomic outflows has been shown.