Cuff electrodes for very small diameter nerves -- prototyping and first recordings in vivo.

A fabrication method for cuff electrodes to interface small nerves was developed. Medical grade silicone rubber conforms the body of the cuff and insulation of the wires, platinum was used as metal for the embedded wiring and contacts. Planar electrode arrays where fabricated using a picosecond laser and then positioned into a carrying tube to provide the third dimension with the desired inner diameter (Ø 0.3-0.5 mm). The post preparation of the cuffs after structuring allows the fabrication of a stable self-closing flap that insulates the opening slit of the cuff without the need of extra sutures. Basic for the success of the cuff is the laser-based local thinning of both the silicone rubber and the metal at defined sections. This is critical to permit the PDMS' body to dominate the mechanical properties. Finite element modeling was applied to optimize the displacement ability of the cuff, leading to design capable of withstanding multiple implantation procedures without wire damage. Furthermore, the contact's surface was roughened by laser patterning to increase the charge injection capacity of Pt to 285 µC/cm(2) measured by voltage transient detection during pulse testing. The cuff electrodes were placed on a small sympathetic nerve of an adult female Sprague-Dawley rat for recording of spontaneous and evoked neural activity in vivo.

First long term in vivo study on subdurally implanted micro-ECoG electrodes, manufactured with a novel laser technology.

A novel computer aided manufacturing (CAM) method for electrocorticography (ECoG) microelectrodes was developed to be able to manufacture small, high density microelectrode arrays based on laser-structuring medical grade silicone rubber and high purity platinum. With this manufacturing process, we plan to target clinical applications, such as presurgical epilepsy monitoring, functional imaging during cerebral tumor resections and brain-computer interface control in paralysed patients, in the near future. This paper describes the manufacturing, implantation and long-term behaviour of such an electrode array. In detail, we implanted 8-channel electrode arrays subdurally over rat cerebral cortex over a period of up to 25 weeks. Our primary objective was to ascertain the electrode's stability over time, and to analyse the host response in vivo. For this purpose, impedance measurements were carried out at regular intervals over the first 18 weeks of the implantation period. The impedances changed between day 4 and day 7 after implantation, and then remained stable until the end of the implantation period, in accordance with typical behaviour of chronically implanted microelectrodes. A post-mortem histological examination was made to assess the tissue reaction due to the implantation. A mild, chronically granulated inflammation was found in the area of the implant, which was essentially restricted to the leptomeninges. Overall, these findings suggest that the concept of the presented ECoG-electrodes is promising for use in long-term implantations.

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis.

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.

Quartz crystal microbalances for quantitative biosensing and characterizing protein multilayers.

The use of quartz crystal microbalances (QCMs) for quantitative biosensing and characterization of protein multilayers is demonstrated in three case studies. Monolayers of QCM-based affinity biosensors were investigated first. Layers of a thiol-containing synthetic peptide constituting an epitope of the foot-and-mouse-disease virus were formed on gold electrodes via self-assembly. The binding of specific antibodies to epitope-modified gold electrodes was detected for different concentrations of antibody solutions. Oligolayers were studied in a second set of experiments. Dextran hydrogels were modified by thrombin inhibitors. The QCM response was used in a competitive binding assay to identify inhibitors for thrombin at different concentrations. Multilayers of proteins formed by self-assembly of a biotin-conjugate and streptavidin were investigated next. The QCM frequency response was monitored as a function of layer thickness up to 20 protein layers. A linear frequency decay was observed with increasing thickness. The decay per layer remained constant, thus indicating perfect mass coupling to the substrate. Frequency changes a factor of four higher were obtained in buffer solution as compared to measurements in dry air. This indicates a significant incorporation of water (75% weight) in the protein layers. This water behaves like a solid concerning the shear mode coupling to the substrate. The outlook discusses briefly the need for controlled molecular engineering of overlayers for subsequent QCM analysis, and the importance of an additional multiparameter analysis with other transducer principles and with additional techniques of interface analysis to characterize the mechanical coupling of overlayers as biosensor coatings. A promising trend concerns the use of QCM-arrays for screening experiments.

QCM Operation in Liquids: Constant Sensitivity during Formation of Extended Protein Multilayers by Affinity.

The quartz crystal microbalance (QCM) is a well-established tool in mass-sensitive detection. Due to recent improvements in experimental procedures, QCMs are finding increasing attention for applications in liquids. One important application is bioaffinity measurements for analytical or research purposes. The effect of the formation of solid films at a QCM surface, especially in gases or vacuum, is well understood. However, the situation is more complex in bioaffinity applications due to the comparably high viscosity of the liquid and the softness of the biological overlayer. Typically frequency responses found for protein layers exceed the values expected from simple models. The use of a hydrogel extending several hundred nanometers from the transducer surface as interacting matrix is common in bioaffinity applications and further increases complexity. Pure mass-related effects as well as viscosity-mediated effects may contribute to the overall frequency response observed experimentally. To improve our understanding of the effects during the formation of extended biological overlayers we have investigated systematically the formation of protein multilayers with a QCM in situ. The attenuation of the QCM oscillation by the liquid leads to a broadening of the resonance frequency. We have overcome this limitation by frequency-dependent admittance analysis and by curve fitting of the resulting admittance. A time resolution of 5 s and a noise of 0.2 Hz has been achieved with 6-MHz AT-cut quartz crystals operating in liquids. Protein multilayers were formed by successive incubations with a biotin-albumin conjugate and streptavidin. Frequency responses for dry protein layers in air were in agreement with mass changes estimated from the Sauerbrey equation. However, in water, the corresponding frequency decrease was increased by a factor of 4, thereby indicating that significant amounts of water are embedded in the hydrated protein layer. Unexpectedly a constant frequency decrease per layer was found during the successive formation of up to 20 protein layers (∼400 nm). Neither noise nor drift increased with the number of protein layers. These results indicate that, despite the high hydration of the protein layers, viscosity-induced effects play a negligible role and that the frequency decrease reflects primarily mass changes at the surface.

A 'mixed' self-assembled monolayer for an impedimetric immunosensor.

A synthetic peptide with the amino acid sequence 135-154 of the capsid protein VP1 of the foot-and-mouth-disease virus was modified with omega-hydroxyundecanethiol and applied together with non-derivatised omega-hydroxyundecanethiol for consecutive adsorption onto gold electrodes according to self-assembling procedures. The binding of a specific antibody to prepared recognition layers could be monitored by measurement of impedance or capacitance. In order to avoid non-specific effects, all measurements were performed in the presence of BSA. The complex between the antigenic peptide and the antibody was split by applying 6 M urea solution. The gold electrodes were mounted into an optimised flow-through system in order to perform capacitance-time measurements. The immobilised peptide can be recognised repeatedly by specific antibodies.

Results of spinal instrumentation of adolescent idiopathic scoliosis by King type.

The purpose of this study is to determine the usefulness of the King classification in predicting decompensation in adolescent idiopathic scoliosis. Fifty-one patients were reviewed with a mean follow-up of 25 months. Five patients had Type 1 adolescent idiopathic scoliosis: four were treated with Zielke/Cotrel-Dubousset instrumentation or Zielke instrumentation alone. Correction was greater than 51% in these cases and there was no decompensation. Twenty-three patients had Type II scoliosis. Nineteen of whom were treated with Cotrel-Dubousset instrumentation; 3 with Zielke and Cotrel-Dubousset instrumentation, and 1 with Zielke. The best correction occurred with anterior/posterior instrumentation. Decompensation occurred in 9 patients, all of whom were treated with Cotrel-Dubousset instrumentation alone. Fourteen patients had Type III scoliosis. All were treated with Cotrel-Dubousset instrumentation with correction of 65%. Decompensation occurred in 4 patients, all of whom were fused to or beyond the stable vertebra. Four patients had Type IV scoliosis; all were fused short of the stable vertebra with Cotrel-Dubousset instrumentation, resulting in correction of 52% and no decompensation. Five patients had Type V instrumentation; four were treated with Cotrel-Dubousset instrumentation and 1 with Zielke. There was no relationship between level of fusion and decompensation. Based on this study, the authors contend that the King classification is a valuable tool in the selection of type of instrumentation and fusion level.