In vivo quantification of excitation and kilohertz frequency block of the rat vagus nerve.

OBJECTIVE: There is growing interest in treating diseases by electrical stimulation and block of peripheral autonomic nerves, but a paucity of studies on the excitation and block of small-diameter autonomic axons. We conducted in vivo quantification of the strength-duration properties, activity-dependent slowing (ADS), and responses to kilohertz frequency (KHF) signals for the rat vagus nerve (VN). APPROACH: We conducted acute in vivo experiments in urethane-anaesthetized rats. We placed two cuff electrodes on the left cervical VN and one cuff electrode on the anterior subdiaphragmatic VN. The rostral cervical cuff was used to deliver pulses to quantify recruitment and ADS. The caudal cervical cuff was used to deliver KHF signals. The subdiaphragmatic cuff was used to record compound action potentials (CAPs). MAIN RESULTS: We quantified the input-output recruitment and strength-duration curves. Fits to the data using standard strength-duration equations were qualitatively similar, but the resulting chronaxie and rheobase estimates varied substantially. We measured larger thresholds for the slowest fibres (0.5-1 m s-1), especially at shorter pulse widths. Using a novel cross-correlation CAP-based analysis, we measured ADS of ~2.3% after 3 min of 2 Hz stimulation, which is comparable to the ADS reported for sympathetic efferents in somatic nerves, but much smaller than the ADS in cutaneous nociceptors. We found greater ADS with higher stimulation frequency and non-monotonic changes in CV in select cases. We found monotonically increasing block thresholds across frequencies from 10 to 80 kHz for both fast and slow fibres. Further, following 25 s of KHF signal, neural conduction could require tens of seconds to recover. SIGNIFICANCE: The quantification of mammalian autonomic nerve responses to conventional and KHF signals provides essential information for the development of peripheral nerve stimulation therapies and for understanding their mechanisms of action.

Modeling the response of small myelinated axons in a compound nerve to kilohertz frequency signals.

OBJECTIVE: There is growing interest in electrical neuromodulation of peripheral nerves, particularly autonomic nerves, to treat various diseases. Electrical signals in the kilohertz frequency (KHF) range can produce different responses, including conduction block. For example, EnteroMedics' vBloc® therapy for obesity delivers 5 kHz stimulation to block the abdominal vagus nerves, but the mechanisms of action are unclear. APPROACH: We developed a two-part computational model, coupling a 3D finite element model of a cuff electrode around the human abdominal vagus nerve with biophysically-realistic electrical circuit equivalent (cable) model axons (1, 2, and 5.7 µm in diameter). We developed an automated algorithm to classify conduction responses as subthreshold (transmission), KHF-evoked activity (excitation), or block. We quantified neural responses across kilohertz frequencies (5-20 kHz), amplitudes (1-8 mA), and electrode designs. MAIN RESULTS: We found heterogeneous conduction responses across the modeled nerve trunk, both for a given parameter set and across parameter sets, although most suprathreshold responses were excitation, rather than block. The firing patterns were irregular near transmission and block boundaries, but otherwise regular, and mean firing rates varied with electrode-fibre distance. Further, we identified excitation responses at amplitudes above block threshold, termed 're-excitation', arising from action potentials initiated at virtual cathodes. Excitation and block thresholds decreased with smaller electrode-fibre distances, larger fibre diameters, and lower kilohertz frequencies. A point source model predicted a larger fraction of blocked fibres and greater change of threshold with distance as compared to the realistic cuff and nerve model. SIGNIFICANCE: Our findings of widespread asynchronous KHF-evoked activity suggest that conduction block in the abdominal vagus nerves is unlikely with current clinical parameters. Our results indicate that compound neural or downstream muscle force recordings may be unreliable as quantitative measures of neural activity for in vivo studies or as biomarkers in closed-loop clinical devices.

Recording evoked potentials during deep brain stimulation: development and validation of instrumentation to suppress the stimulus artefact.

The clinical efficacy of deep brain stimulation (DBS) for the treatment of movement disorders depends on the identification of appropriate stimulation parameters. Since the mechanisms of action of DBS remain unclear, programming sessions can be time consuming, costly and result in sub-optimal outcomes. Measurement of electrically evoked compound action potentials (ECAPs) during DBS, generated by activated neurons in the vicinity of the stimulating electrode, could offer insight into the type and spatial extent of neural element activation and provide a potential feedback signal for the rational selection of stimulation parameters and closed-loop DBS. However, recording ECAPs presents a significant technical challenge due to the large stimulus artefact, which can saturate recording amplifiers and distort short latency ECAP signals. We developed DBS-ECAP recording instrumentation combining commercial amplifiers and circuit elements in a serial configuration to reduce the stimulus artefact and enable high fidelity recording. We used an electrical circuit equivalent model of the instrumentation to understand better the sources of the stimulus artefact and the mechanisms of artefact reduction by the circuit elements. In vitro testing validated the capability of the instrumentation to suppress the stimulus artefact and increase gain by a factor of 1000 to 5000 compared to a conventional biopotential amplifier. The distortion of mock ECAP (mECAP) signals was measured across stimulation parameters, and the instrumentation enabled high fidelity recording of mECAPs with latencies of only 0.5 ms for DBS pulse widths of 50 to 100 µs/phase. Subsequently, the instrumentation was used to record in vivo ECAPs, without contamination by the stimulus artefact, during thalamic DBS in an anesthetized cat. The characteristics of the physiological ECAP were dependent on stimulation parameters. The novel instrumentation enables high fidelity ECAP recording and advances the potential use of the ECAP as a feedback signal for the tuning of DBS parameters.

Advances in phase-sensitive acoustic microscopy studies of polymer blend films: annealing effects and micro-elastic characterization of PS/PMMA blends.

The unique phase-sensitive acoustic microscope is used for the structural and mechanical characterization of thin films of polystyrene/polymethylmethacrylate blends. The effect of annealing on blends of polystyrene/polymethylmethacrylate spin coated from different solvents unto a substrate is studied. Varying the solvents according to vapour pressure and spin coating at different speeds (for thickness variation) led to changes in phase domain distributions and overall structural properties before annealing. Annealing in vacuum at 190 degrees C for 48 h resulted in the elimination of solvent effects with all samples reverting to a similar morphology irrespective of common solvent and thickness. The Young's moduli at specific points on the film (E(polystyrene)= 3.4 +/- 0.3 GPa, E(polymethylmethacrylate)= 4.2 +/- 0.4 GPa) and over a given area (E(polystyrene/polymethylmethacrylate)= 3.9 +/- 0.4 GPa) were determined by combinatory use of the atomic force microscope and phase-sensitive acoustic microscope. These results demonstrate a minimally invasive method for the quantitative characterization of polymer blend films.

Mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration.

Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.

Modeling of Coulomb coupling and acoustic wave propagation in LiNbO3.

The angular spectrum propagation technique is applied to modeling of wave propagation through piezoelectric media. The calculations of the angular spectrum propagator are based on the relevant equation for the slowness surface resulting from the solution of the Christoffel equation with piezoelectrically stiffened elastic constants. A two-dimensional FFT algorithm is applied in the final field superposition. We concentrate on the case of Coulomb coupling through local electrical point contacts on both the excitation and detection side. To model that case we superpose solutions for acoustic Green's functions of different propagation modes convoluted with equivalent distributed effective sources. Calculated results are in good agreement with the measured ones.

On interference effects in the elastodynamic Green's functions G33(x, omega) of Si and GaAs.

In this paper, we analyze interference effects present in the elastodynamic Green's functions G33(x,omega) of the cubic crystals Si and GaAs, which are associated with folded portions of the wave surface of the slow transverse (ST) acoustic mode. G33(x,omega) represents the three dimensional extension of the amplitude distribution imaged in the transmission acoustic microscopy of these crystals. The intensity contrast for oscillations of a particular wave vector k in the interference pattern is determined essentially by the 3D Fourier transform of G33(x,omega)G33*(x,omega). According to the Fourier autocorrelation theorem, that transform is equivalent to the autocorrelation function of the corresponding distribution G(33)(k,omega) in k-space. We show that due to the linear mapping between k-space and the slowness vector s-space, the interference phenomena discussed here are related to geometrical features of the slowness surface of the ST mode. We present calculations of these effects based on the angular spectrum technique.

Topography of spinal neurons active during hindlimb withdrawal reflexes in the decerebrate cat.

There exists a spatial organization of receptive fields and a modular organization of the flexion withdrawal reflex system. However, the three dimensional location and organization of interneurons interposed in flexion reflex pathways has not been systematically examined. We determined the anatomical locations of spinal neurons involved in the hindlimb flexion withdrawal reflex using expression of the immediate early gene c-fos and the corresponding FOS protein. The flexion withdrawal reflex was evoked in decerebrate cats via stimulation of the tibial or superficial peroneal nerve. Animals that received stimulation had significantly larger numbers of cells expressing FOS-like immunoreactivity (42.7+/-2.3 cells/section, mean+/-standard error of the mean) than operated unstimulated controls (18.6+/-1.4 cells/section). Compared with controls, cells expressing FOS-like immunoreactivity were located predominantly on the ipsilateral side, in laminae IV-VI, at L6 and rostral L7 segments, and between 20% and 60% of the distance from the midline to the lateral border of the ventral gray matter. Labeled neurons resulting from tibial nerve stimulation were medial to neurons labeled following superficial peroneal nerve stimulation in laminae I-VI, but not VII. The mean mediolateral positions of labeled neurons from both nerves shifted medially as the transverse plane in which they were viewed was moved from rostral to caudal and as the coronal plane in which they were viewed was moved from dorsal to ventral. The mediolateral separation between populations of labeled cells was consistent with primary afferent projections and the location of reflex encoders. This topographical segregation corresponding to different afferent inputs is a possible anatomical substrate for a modular organization of the flexion withdrawal reflex system.

Temporal and spatial apodization in defocused acoustic transmission microscopy.

Two non-confocally adjusted spherical transducers are employed to implement an acoustic microscope operating in transmission with an approximately line-shaped point spread function (PSF). Such a PSF is of advantage in acoustic transmission line tomography and spatially resolved velocity measurements in solids. The foci of the transducers are viewed as diffraction-limited point transducers and appropriate time-selective signal acquisition is designed to restrict the ultrasound wave paths to the line connecting them. It is found that for typical commercially available transducers the largest contribution to the detected signal is not due to the direct ultrasound wave but due to the edge waves emanating from the rim of the focusing transducer. This poses constraints on achieving a line-shaped PSF in defocused acoustic transmission microscopy. It is shown that, due to the strong contribution from edge waves, it is impossible to achieve a line-shaped PSF in the case of application of a long exciting toneburst. The influence of the exciting pulse length, as well as the position of the time gate on the obtainable PSF is investigated.

Characterization of polymer thin films by phase-sensitive acoustic microscopy and atomic force microscopy: a comparative review.

The potential of phase-sensitive acoustic microscopy (PSAM) for characterizing polymer thin films is reviewed in comparison to atomic force microscopy (AFM). This comparison is based on results from three-dimensional vector contrast imaging and multimodal imaging using PSAM and AFM, respectively. The similarities and differences between the information that can be derived from the AFM topography and phase images, and the PSAM phase and amplitude micrographs are examined. In particular, the significance of the PSAM phase information for qualitative and quantitative characterization of the polymer films is examined for systems that generate surface waves, and those that do not. The relative merits, limitations and outlook of both techniques, individually, and as a complementary pair, are discussed.

Inversion of acoustic diffraction fields in anisotropic solids.

We show that the fast Fourier transform (FFT) technique provides a computationally efficient method of calculating 2D amplitude and phase images of complex wave fields generated and measured in elastically anisotropic solids by phase sensitive acoustic microscopy. Further, we discuss how this technique can be used to treat inverse problems such as source reconstruction, image quality assessment, and the determination of elastic constants.

Phase-sensitive acoustic microscopy of polymer thin films.

The three-dimensional images obtained by scanning acoustic microscopy with vector contrast (PSAM), contain significant qualitative and quantitative information that is not easily obtainable by other methods. We employ this technique to examine homopolymer and polymer blend thin films. The complex V(z) functions derived from the images, and the results obtained by image processing and meticulous analysis are employed to render the morphology, composition and micro-mechanical properties of the polymer films. In addition, ways by which the information inherent in the phase images can be extracted are examined. This is highly desirable, as the phase images contain very useful additional information.

Apertureless near-field optical microscopy of metallic nanoparticles.

Image formation in apertureless near-field optical microscopes employing evanescent-wave excitation is studied quantitatively as a function of the polarisation and the wavelength of the excitation. Aggregate Mie theory is used to describe the probe-sample interactions self-consistently, including retardation. Only p-polarised excitation yields images, which closely reproduce the sample, and the contrast is much higher in this case than for s-polarised waves. Particular attention is paid to the case of imaging of metallic nanoparticles, for which local and nonlocal versions of aggregate Mie theory are compared. Nonlocality arises from the excitation of longitudinal bulk plasmons at the particle surface. It is shown that this effect is essential in the imaging of such particles and implies comparatively rapid convergence, in contrast to the local theory. The converged images calculated within the nonlocal theory resemble the results of the local theory, when, arbitrarily, within the latter only dipole-dipole interactions are taken into account. Significant qualitative and quantitative differences, however, are shown to exist. Signal and contrast enhancements due to resonant excitation of surface plasmon polaritons are studied quantitatively using the results of the converged nonlocal theory.

Scanning acoustic Doppler microscopy and scanning acoustic correlation microscopy.

Acoustic microscopy with vector contrast at 100 MHz in a fluid with immersed particles is used to detect the flow profile in front of a microscopic orifice. The velocity profile concerning the component in axial direction of the focused beam is derived from the phase contrast. Possibilities to resolve the flow profile also for the components in normal direction with respect to the axis are demonstrated. The methods concerning measurement techniques and data evaluation for scanning acoustic Doppler microscopy are presented. For scanning acoustic correlation microscopy the time dependent phase and amplitude signals resulting from sound waves scattered by the immersed particles (aluminium flakes with a typical diameter of 10 microm) have been analysed by correlation procedures. From the obtained autocorrelation functions the velocity distribution can be derived. Both methods can be applied simultaneously. Data analysis is based on the information contained in the originally obtained images in vector contrast derived from temporal and spatial resolved analogue and digital processing of the acoustic signals.

Neural network classification of nerve activity recorded in a mixed nerve.

Whole-nerve cuff electrodes can be used to record electrical nerve activity in peripheral nerves and are suitable for chronic implantation in animals or humans. If the whole nerve innervates multiple target organs or muscles then the recorded activity will be the superposition of the activity of different nerve fibers innervating these organs. In certain cases it is desirable to monitor mixed nerve activity and to determine the origin (modality) of the recorded activity. A method using the autocorrelation function of recorded nerve activity and an artificial neural network was developed to classify the modality of nerve signals. The method works in cases where different end organs are innervated by nerve fibers having different diameter distributions. The electrical activity in the cat S1 sacral spinal root was recorded using a cuff electrode during the activation of cutaneous, bladder, and rectal mechanoreceptors. Using the classification method, 87.5% of nerve signals were correctly classified. This result demonstrates the effectiveness of the neural network classification method to determine the modality of the nerve activity arising from activation of different receptors.

Finite element analysis of the current-density and electric field generated by metal microelectrodes.

Electrical stimulation via implanted microelectrodes permits excitation of small, highly localized populations of neurons, and allows access to features of neuronal organization that are not accessible with larger electrodes implanted on the surface of the brain or spinal cord. As a result there are a wide range of potential applications for the use of microelectrodes in neural engineering. However, little is known about the current-density and electric field generated by microelectrodes. The objectives of this project were to answer three fundamental questions regarding electrical stimulation with metal microelectrodes using geometrically and electrically accurate finite elements models. First, what is the spatial distribution of the current density over the surface of the electrode? Second, how do alterations in the electrode geometry effect neural excitation? Third, under what conditions can an electrode of finite size be modeled as a point source? Analysis of the models showed that the current density was concentrated at the tip of the microelectrode and at the electrode-insulation interface. Changing the surface area of the electrode, radius of curvature of the electrode tip, or applying a resistive coating to the electrode surface altered the current-density distribution on the surface of the electrode. Changes in the electrode geometry had little effect on neural excitation patterns, and modeling the electric field generated by sharply tipped microelectrodes using a theoretical point source was valid for distances > approximately 50 microm from the electrode tip. The results of this study suggest that a nearly uniform current-density distribution along the surface of the electrode can be achieved using a relatively large surface area electrode (500-1000 microm2), with a relatively blunt tip (3-6 microm radius of curvature), in combination with a thin (approximately 1 microm) moderately resistive coating (approximately 50 omega m).

Optimal filtering of whole nerve signals.

Electroneurographic recordings suffer from low signal to noise (S/N) ratios. The S/N ratio can be improved by different signal processing methods including optimal filtering. A method to design two types of optimal filters (Wiener and Matched filters) was developed for use with neurographic signals, and the calculated filters were applied to nerve cuff recordings from the cat S1 spinal root that were recorded during the activation of cutaneous, bladder, and rectal mechanoreceptors. The S1 spinal root recordings were also filtered using various band-pass (BP) filters with different cut-off frequencies, since the frequency responses of the Wiener and Matched filters had a band-pass character. The mean increase in the S/N ratio across all recordings was 54, 89, and 85% for the selected best Wiener, Matched, and band-pass filters, respectively. There were no statistically significant differences between the performance of the selected filters when all three methods were compared. However, Matched filters yielded a greater increase in S/N ratio than Wiener filters when only two filtering techniques were compared. All three filtering methods have in most cases also improved the selectivity of the recordings for different sensory modalities. This might be important when recording nerve activity from a mixed nerve innervating multiple end-organs to increase the modality selectivity for the nerve fibers of interest. The mean Modality Selectivity Indices (MSI) over different receptor types and for the same selected filters as above were 1.12, 1.27, and 1.29, respectively, and indicate increases in modality selectivity (MSI>1). Improving the S/N ratio and modality selectivity of neurographic recordings is an important development to increase the utility of neural signals for understanding neural function and for use as feedback or control signals in neural prosthetic devices.

Detection and inhibition of hyperreflexia-like bladder contractions in the cat by sacral nerve root recording and electrical stimulation.

Detection of bladder volume and hyperreflexive bladder contractions would be useful in individuals with overactive bladders. We sought to determine whether bladder filling and/or reflex bladder contractions could be detected by electrical recording from the sacral nerve roots, and whether bladder contractions could be inhibited by stimulation of sacral afferents. Six male cats were anesthetized with alpha-chloralose and bipolar cuff electrodes were used to measure sacral nerve root electroneurograms (ENG) during slow bladder filling, during rapid injections of fluid into the bladder, and during hyperreflexia-like bladder contractions. The rectified and time-averaged activity of the S1 extradural root increased by 0-5 % above the baseline during bladder filling. Rapid injections caused a sudden increase in bladder pressure, and a 3-36 % increase over baseline in the S1 nerve activity. Withdrawal of the same volume caused a reduction in pressure and a decrease in recorded activity (4-14 %). At the onset of a bladder contraction, there was a 7-38 % increase over baseline in the S1 nerve activity. This activity increase was sustained for the duration of the contraction and decreased during bladder relaxation. The onset and duration of bladder contractions could be detected consistently from these nerve activity changes. Recording only afferent activity showed that the increased nerve activity was due to S1 sensory rather than motor fibers. In two cats, it was demonstrated that an ongoing bladder contraction could be inhibited by rectal distension. In one cat, the contractions could be terminated by electrical stimulation of the S1 dorsal root. The results demonstrate that afferent sacral root nerve activity can be used to detect hyperreflexive bladder contractions at low bladder pressures. Such a signal might be used to trigger bladder inhibition via electrical stimulation of specific sacral afferents.

Titanium powder sintering for preparation of a porous functionally graded material destined for orthopaedic implants.

This work focuses on basic research into a P/M processed, porous-surfaced and functionally graded material (FGM) destined for a permanent skeletal replacement implant with improved structural compatibility. Based on a perpendicular gradient in porosity the Young's modulus of the material is adapted to the elastic properties of bone in order to prevent stress shielding effects and to provide better long-term performance of the implant-bone system. Using coarse Ti particle fractions the sintering process was accelerated by silicon-assisted liquid-phase sintering (LPS) resulting in a substantial improvement of the neck geometry. A novel evaluation for the strength of the sinter contacts was proposed. The Young's modulus of uniform non-graded stacks ranged from 5 to 80 GPa as determined by ultrasound velocity measurements. Thus, the typical range for cortical bone (10-29 GPa) was covered. The magnitude of the Poisson's ratio proved to be distinctly dependent on the porosity. Specimens with porosity gradients were successfully fabricated and characterized using quantitative description of the microstructural geometry and acoustic microscopy.

At the interface: convergence of neural regeneration and neural prostheses for restoration of function.

The rapid pace of recent advances in development and application of electrical stimulation of the nervous system and in neural regeneration has created opportunities to combine these two approaches to restoration of function. This paper relates the discussion on this topic from a workshop at the International Functional Electrical Stimulation Society. The goals of this workshop were to discuss the current state of interaction between the fields of neural regeneration and neural prostheses and to identify potential areas of future research that would have the greatest impact on achieving the common goal of restoring function after neurological damage. Identified areas include enhancement of axonal regeneration with applied electric fields, development of hybrid neural interfaces combining synthetic silicon and biologically derived elements, and investigation of the role of patterned neural activity in regulating various neuronal processes and neurorehabilitation. Increased communication and cooperation between the two communities and recognition by each field that the other has something to contribute to their efforts are needed to take advantage of these opportunities. In addition, creative grants combining the two approaches and more flexible funding mechanisms to support the convergence of their perspectives are necessary to achieve common objectives.