Quantitative assessment of nociceptive processes in conscious dogs by use of the nociceptive withdrawal reflex

OBJECTIVE: To investigate the feasibility of evoking the nociceptive withdrawal reflex (NWR) from fore and hind limbs in conscious dogs, score stimulus-associated behavioral responses, and assess the canine NWR response to suprathreshold stimulations. ANIMALS: 8 adult Beagles. PROCEDURE: Surface electromyograms evoked by transcutaneous electrical stimulation of ulnaris and digital plantar nerves were recorded from the deltoideus, cleidobrachialis, biceps femoris, and tibialis cranialis muscles. Train-of-five pulses (stimulus(train)) were used; reflex threshold (I(t train)) was determined, and recruitment curves were obtained at 1.2, 1.5, and 2 x I(t train). Additionally, a single pulse (stimulus(single)) was given at 1, 1.2, 1.5, 2, and 3 x I(t train). Latency and amplitude of NWRs were analyzed. Severity of behavioral reactions was subjectively scored. RESULTS: Fore- and hind limb I(t train) values (median; 25% to 75% interquartile range) were 2.5 mA (2.0 to 3.6 mA) and 2.1 mA (1.7 to 2.9 mA), respectively. At I(t train), NWR latencies in the deltoideus, cleidobrachialis, biceps femoris, and cranial tibialis muscles were not significantly different (19.6 milliseconds [17.1 to 20.5 milliseconds], 19.5 milliseconds [18.1 to 20.7 milliseconds], 20.5 milliseconds [14.7 to 26.4 milliseconds], and 24.4 milliseconds [17.1 to 40.5 milliseconds], respectively). Latencies obtained with stimulus(train) and stimulus(single) were similar. With increasing stimulation intensities, NWR amplitude increased and correlated positively with behavioral scores. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, the NWR can be evoked from limbs and correlates with behavioral reactions. Results suggest that NWR evaluation may enable quantification of nociceptive system excitability and efficacy of analgesics in individual dogs.

In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.

One-Hertz transcranial magnetic stimulation over the frontal eye field induces lasting inhibition of saccade triggering

The aim of the study was to examine the effect of low-frequency repetitive transcranial magnetic stimulation on saccade triggering. In five participants, a train of 600 pulses with a frequency of 1 Hz was applied over the right frontal eye field and--as control condition--over the vertex. After repetitive transcranial magnetic stimulation application, oculomotor performance was evaluated with an overlap paradigm. The results show that the repetitive transcranial magnetic stimulation effect was specific for frontal eye field stimulation. Saccade latencies were found to be increased bilaterally for several minutes after the stimulation, and the time course of recovery was different for the ipsilateral and contralateral sides. The results are discussed in the light of possible local and remote repetitive transcranial magnetic stimulation effects on the oculomotor network.

BOLD correlates of edge detection in human auditory cortex

Edges are important cues defining coherent auditory objects. As a model of auditory edges, sound on- and offset are particularly suitable to study their neural underpinnings because they contrast a specific physical input against no physical input. Change from silence to sound, that is onset, has extensively been studied and elicits transient neural responses bilaterally in auditory cortex. However, neural activity associated with sound onset is not only related to edge detection but also to novel afferent inputs. Edges at the change from sound to silence, that is offset, are not confounded by novel physical input and thus allow to examine neural activity associated with sound edges per se. In the first experiment, we used silent acquisition functional magnetic resonance imaging and found that the offset of pulsed sound activates planum temporale, superior temporal sulcus and planum polare of the right hemisphere. In the planum temporale and the superior temporal sulcus, offset response amplitudes were related to the pulse repetition rate of the preceding stimulation. In the second experiment, we found that these offset-responsive regions were also activated by single sound pulses, onset of sound pulse sequences and single sound pulse omissions within sound pulse sequences. However, they were not active during sustained sound presentation. Thus, our data show that circumscribed areas in right temporal cortex are specifically involved in identifying auditory edges. This operation is crucial for translating acoustic signal time series into coherent auditory objects.

Calcium indicator loading of neurons using single-cell electroporation

Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.

Dendritic spikes in apical dendrites of neocortical layer 2/3 pyramidal neurons

Layer 2/3 (L2/3) pyramidal neurons are the most abundant cells of the neocortex. Despite their key position in the cortical microcircuit, synaptic integration in dendrites of L2/3 neurons is far less understood than in L5 pyramidal cell dendrites, mainly because of the difficulties in obtaining electrical recordings from thin dendrites. Here we directly measured passive and active properties of the apical dendrites of L2/3 neurons in rat brain slices using dual dendritic-somatic patch-clamp recordings and calcium imaging. Unlike L5 cells, L2/3 dendrites displayed little sag in response to long current pulses, which suggests a low density of I(h) in the dendrites and soma. This was also consistent with a slight increase in input resistance with distance from the soma. Brief current injections into the apical dendrite evoked relatively short (half-width 2-4 ms) dendritic spikes that were isolated from the soma for near-threshold currents at sites beyond the middle of the apical dendrite. Regenerative dendritic potentials and large concomitant calcium transients were also elicited by trains of somatic action potentials (APs) above a critical frequency (130 Hz), which was slightly higher than in L5 neurons. Initiation of dendritic spikes was facilitated by backpropagating somatic APs and could cause an additional AP at the soma. As in L5 neurons, we found that distal dendritic calcium transients are sensitive to a long-lasting block by GABAergic inhibition. We conclude that L2/3 pyramidal neurons can generate dendritic spikes, sharing with L5 pyramidal neurons fundamental properties of dendritic excitability and control by inhibition.

Riluzole-induced oscillations in spinal networks

We previously showed in dissociated cultures of fetal rat spinal cord that disinhibition-induced bursting is based on intrinsic spiking, network recruitment, and a network refractory period after the bursts. A persistent sodium current (I(NaP)) underlies intrinsic spiking, which, by recurrent excitation, generates the bursting activity. Although full blockade of I(NaP) with riluzole disrupts such bursting, the present study shows that partial blockade of I(NaP) with low doses of riluzole maintains bursting activity with unchanged burst rate and burst duration. More important, low doses of riluzole turned bursts composed of persistent activity into bursts composed of oscillatory activity at around 5 Hz. In a search for the mechanisms underlying the generation of such intraburst oscillations, we found that activity-dependent synaptic depression was not changed with low doses of riluzole. On the other hand, low doses of riluzole strongly increased spike-frequency adaptation and led to early depolarization block when bursts were simulated by injecting long current pulses into single neurons in the absence of fast synaptic transmission. Phenytoin is another I(NaP) blocker. When applied in doses that reduced intrinsic activity by 80-90%, as did low doses of riluzole, it had no effect either on spike-frequency adaptation or on depolarization block. Nor did phenytoin induce intraburst oscillations after disinhibition. A theoretical model incorporating a depolarization block mechanism could reproduce the generation of intraburst oscillations at the network level. From these findings we conclude that riluzole-induced intraburst oscillations are a network-driven phenomenon whose major accommodation mechanism is depolarization block arising from strong sodium channel inactivation.

Processing of temporal unpredictability in human and animal amygdala

The amygdala has been studied extensively for its critical role in associative fear conditioning in animals and humans. Noxious stimuli, such as those used for fear conditioning, are most effective in eliciting behavioral responses and amygdala activation when experienced in an unpredictable manner. Here, we show, using a translational approach in mice and humans, that unpredictability per se without interaction with motivational information is sufficient to induce sustained neural activity in the amygdala and to elicit anxiety-like behavior. Exposing mice to mere temporal unpredictability within a time series of neutral sound pulses in an otherwise neutral sensory environment increased expression of the immediate-early gene c-fos and prevented rapid habituation of single neuron activity in the basolateral amygdala. At the behavioral level, unpredictable, but not predictable, auditory stimulation induced avoidance and anxiety-like behavior. In humans, functional magnetic resonance imaging revealed that temporal unpredictably causes sustained neural activity in amygdala and anxiety-like behavior as quantified by enhanced attention toward emotional faces. Our findings show that unpredictability per se is an important feature of the sensory environment influencing habituation of neuronal activity in amygdala and emotional behavior and indicate that regulation of amygdala habituation represents an evolutionary-conserved mechanism for adapting behavior in anticipation of temporally unpredictable events.

Sequential immunosuppressive therapy in progressive IgA nephropathy

BACKGROUNDS: Cyclophosphamide and high-dose steroids have been used as limited induction therapy in progressive IgA nephropathy (IgAN) to reduce the loss of renal function and proteinuria. We evaluated the effect of cyclophosphamide pulses (CyP) and mycophenolic acid (MPA) as sequential therapy on renal function in patients with progressive IgAN. METHODS: Twenty patients with progressive IgAN and advanced renal failure (median GFR 22 ml/min per 1.73 m2) and further disease activity (triangle downGFR -0.8 ml/min per month) after cyclophosphamide (CyP; n = 18) or steroid pulse therapy (n = 2) were treated with mycophenolate mofetil 1 g per day for a median of 27 months. RESULTS: The monthly loss of renal function was significantly reduced in linear regression analysis from -2.4 ml/min before CyP to -0.12 ml/min with CyP/MPA (p = 0.0009). Estimated renal survival time was significantly prolonged by a median of 65 months (p = 0.0014). Proteinuria decreased significantly from 1.7 to 0.4 g/l during MPA treatment (p = 0.015). In Cox regression analysis, only proteinuria >1.0 g/l was an independent risk factor for doubling of creatinine during CyP/MPA treatment (p = 0.03). CONCLUSION: A sequential therapy with CyP/MPA may arrest or slow down the loss of renal function and reduces proteinuria even in patients who passed the so called 'point of no return' with progressive IgAN.

Introduction of a novel dose saving acquisition mode for the PortalVision aS500 EPID to facilitate on-line patient setup verification

In external beam radiotherapy, electronic portal imaging becomes more and more an indispensable tool for the verification of the patient setup. For the safe clinical introduction of high dose conformal radiotherapy like intensity modulated radiation therapy, on-line patient setup verification is a prerequisite to ensure that the planned dosimetric coverage of the tumor volume is actually realized in the patient. Since the direction of setup fields often deviates from the direction of the treatment beams, extra dose is delivered to the patient during the acquisition of these portal images which may reach clinical relevance. The aim of this work was to develop a new acquisition mode for the PortalVision aS500 electronic portal imaging device from Varian Medical Systems that allows one to take portal images with reduced dose while keeping good image quality. The new acquisition mode, called RadMode, selectively enables and disables beam pulses during image acquisition allowing one to stop wasting valuable dose during the initial acquisition of "reset frames." Images of excellent quality can be taken with 1 MU only. This low dose per image facilitates daily setup verification with considerably reduced extra dose.

Theta burst transcranial magnetic stimulation is associated with increased EEG synchronization in the stimulated relative to unstimulated cerebral hemisphere

Theta burst transcranial magnetic stimulation (TBS) may induce behavioural changes that outlast the stimulation period. The neurophysiological basis of these behavioural changes are currently under investigation. Given the evidence that cortical information processing relies on transient synchronization and desynchronization of neuronal assemblies, we set out to test whether TBS is associated with changes of neuronal synchronization as assessed by surface EEG. In four healthy subjects one TBS train of 600 pulses (200 bursts, each burst consisting of 3 pulses at 30 Hz, repeated at intervals of 100 ms) was applied over the right frontal eye field and EEG synchronization was assessed in a time-resolved manner over 60 min by using a non-overlapping moving window. For each time step the linear cross-correlation matrix for six EEG channels of the right and for the six homotopic EEG channels of the left hemisphere were computed and their largest eigenvalues used to assess changes of synchronization. Synchronization was computed for broadband EEG and for the delta, theta, alpha, beta and gamma frequency bands. In all subjects EEG synchronization of the stimulated hemisphere was significantly and persistently increased relative to EEG synchronization of the unstimulated hemisphere. This effect occurred immediately after TBS for the theta, alpha, beta and gamma frequency bands and 10-20 min after TBS for broadband and delta frequency band EEG. Our results demonstrate that TBS is associated with increased neuronal synchronization of the cerebral hemisphere ipsilateral to the stimulation site relative to the unstimulated hemisphere. We speculate that enhanced synchronization interferes with cortical information processing and thus may be a neurophysiological correlate of the impaired behavioural performance detected previously.

Analysis of nonlinear gain-induced effects on short-pulse amplification in doped fibers by use of an extended power equation

Optical pulse amplification in doped fibers is studied using an extended power transport equation for the coupled pulse spectral components. This equation includes the effects of gain saturation, gain dispersion, fiber dispersion, fiber nonlinearity, and amplified spontaneous emission. The new model is employed to study nonlinear gain-induced effects on the spectrotemporal characteristics of amplified subpicosecond pulses, in both the anomalous and the normal dispersion regimes.

Differentiating normal hyaline cartilage from post-surgical repair tissue using fast gradient echo imaging in delayed gadolinium-enhanced MRI (dGEMRIC) at 3 Tesla

The purpose was to evaluate the relative glycosaminoglycan (GAG) content of repair tissue in patients after microfracturing (MFX) and matrix-associated autologous chondrocyte transplantation (MACT) of the knee joint with a dGEMRIC technique based on a newly developed short 3D-GRE sequence with two flip angle excitation pulses. Twenty patients treated with MFX or MACT (ten in each group) were enrolled. For comparability, patients from each group were matched by age (MFX: 37.1 +/- 16.3 years; MACT: 37.4 +/- 8.2 years) and postoperative interval (MFX: 33.0 +/- 17.3 months; MACT: 32.0 +/- 17.2 months). The Delta relaxation rate (DeltaR1) for repair tissue and normal hyaline cartilage and the relative DeltaR1 were calculated, and mean values were compared between both groups using an analysis of variance. The mean DeltaR1 for MFX was 1.07 +/- 0.34 versus 0.32 +/- 0.20 at the intact control site, and for MACT, 1.90 +/- 0.49 compared to 0.87 +/- 0.44, which resulted in a relative DeltaR1 of 3.39 for MFX and 2.18 for MACT. The difference between the cartilage repair groups was statistically significant. The new dGEMRIC technique based on dual flip angle excitation pulses showed higher GAG content in patients after MACT compared to MFX at the same postoperative interval and allowed reducing the data acquisition time to 4 min.

Optimized spectrally selective steady-state free precession sequences for cartilage imaging at ultra-high fields

OBJECT: Fat suppressed 3D steady-state free precession (SSFP) sequences are of special interest in cartilage imaging due to their short repetition time in combination with high signal-to-noise ratio. At low-to-high fields (1.5-3.0 T), spectral spatial (spsp) radio frequency (RF) pulses perform superiorly over conventional saturation of the fat signal (FATSAT pulses). However, ultra-high fields (7.0 T and more) may offer alternative fat suppression techniques as a result of the increased chemical shift. MATERIALS AND METHODS: Application of a single, frequency selective, RF pulse is compared to spsp excitation for water (or fat) selective imaging at 7.0 T. RESULTS: For SSFP, application of a single frequency selective RF pulse for selective water or fat excitation performs beneficially over the commonly applied spsp RF pulses. In addition to the overall improved fat suppression, the application of single RF pulses leads to decreased power depositions, still representing one of the major restrictions in the design and application of many pulse sequences at ultra-high fields. CONCLUSION: The ease of applicability and implementation of single frequency selective RF pulses at ultra-high-fields might be of great benefit for a vast number of applications where fat suppression is desirable or fat-water separation is needed for quantification purposes.

Using transcranial magnetic stimulation to probe decision-making and memory

Decision-making and memory are fundamental processes for successful human behaviour. For eye movements, the frontal eye fields (FEF), the supplementary eye fields (SEF), the dorsolateral prefrontal cortex (DLPFC), the ventrolateral frontal cortex and the anterior cingulum are important for these cognitive processes. The online approach of transcranial magnetic stimulation (TMS), i.e., the application of magnetic pulses during planning and performance of saccades, allows interfering specifically with information processing of the stimulated region at a very specific time interval (chronometry of cortical processing). The paper presents studies, which showed the different roles of the FEF and DLPFC in antisaccade control. The critical time interval of DLPFC control seems to be before target onset since TMS significantly increased the percentage of antisaccade errors at that time interval. The FEF seems to be important for the triggering of correct antisaccades. Bilateral stimulation of the DLPFC could demonstrate parallel information-processing transfer in spatial working memory during memory-guided saccades.