Epicardial injection of nanoformulated calcium into cardiac ganglionic plexi suppresses autonomic nerve activity and postoperative atrial fibrillation.

BACKGROUND: Imbalanced activation of the cardiac autonomic nervous system triggers postoperative atrial fibrillation (POAF). Neuronal calcium overload induces apoptosis. We hypothesize that epicardial injection of timed-release nanoformulated CaCl2 (nCaCl2) into left atrial ganglionic plexi (GP) modulates autonomic function and suppresses POAF. OBJECTIVE: The purpose of this study was to determine whether nCaCl2 GP therapy suppresses POAF. METHODS: We used a novel canine model of POAF with implanted radiotelemetry to record nerve activity (NA) from the left stellate ganglion (SNA), left cardiac vagus nerve, and GP. At week 3, nCaCl2 (n = 7) or vehicle control (sham; n = 3) was injected into left pulmonary vein GP (LGP), followed by right pulmonary vein GP at week 4. Atrial effective refractory period (AERP) and atrial fibrillation vulnerability (AFV) were assessed in vivo. Resting and exercise NA and heart rate (HR) were assessed before and after LGP treatment. RESULTS: AERP decreased (P < .0001) and AFV increased (P = .008) at week 3 vs baseline. However, nCaCl2-LGP treatment reversed these changes and restored them to baseline after 1 week (P = .04). Subsequent nCaCl2-right pulmonary vein GP treatment further reduced AFV (P = .03). In contrast, AFV increased (P = .001) and AERP remained decreased (P = .01) 1 week after sham-LGP treatment vs baseline. nCaCl2-LGP treatment reduced NA from GP (P < .02) and NA from the left cardiac vagus nerve (P < .05) and increased SNA (P < .02). Despite increased SNA, HR was decreased (P < .01) with loss of HR-SNA correlation (R = 0.62). After sham-LGP treatment, NA was unchanged and HR-SNA remained correlated (R = 0.95). Histology confirmed nCaCl2-GP colocalization, apoptosis, and loss of immunoreactivity in nCaCl2-treated somas. CONCLUSION: Epicardial injection of nCaCl2 into left atrial GP induced neuroapoptosis and modulated autonomic function. This reversed a postoperative reduction in AERP and suppressed POAF.

Magnetic targeted delivery of dexamethasone acetate across the round window membrane in guinea pigs.

HYPOTHESIS: Magnetically susceptible PLGA nanoparticles will effectively target the round window membrane (RWM) for delivery of dexamethasone-acetate (Dex-Ac) to the scala tympani. BACKGROUND: Targeted delivery of therapeutics to specific tissues can be accomplished using different targeting mechanisms. One technology includes iron oxide nanoparticles, susceptible to external magnetic fields. If a nanocomposite composed of biocompatible polymer (PLGA), magnetite, and Dex-Ac can be pulled into and across the mammalian RWM, drug delivery can be enhanced. METHOD: In vitro targeting and release kinetics of PLGA-magnetite-Dex-Ac nanoparticles first were measured using a RWM model. Next, these optimized nanocomposites were targeted to the RWM by filling the niche in anesthetized guinea pigs. A permanent magnet was placed opposite the RWM for 1 hour. Cochlear soft tissues, perilymph, and RWM were harvested after euthanasia and steroid levels were measured using HPLC. RESULTS: Membrane transport, in vitro, proved optimal targeting using a lower particle magnetite concentration (1 versus 5 or 10 mg/ml). In vivo targeted PLGA-magnetite-Dex-Ac particles had an average size of 482.8 ± 158 nm (DLS) and an average zeta potential -19.9 ± 3.3 mV. In 1 hour, there was significantly increased cochlear targeted delivery of Dex or Dex-Ac, compared with diffusion alone. CONCLUSION: Superparamagnetic PLGA-magnetite-Dex-Ac nanoparticles under an external magnetic field (0.26 mT) for 1 hour significantly increased Dex-Ac delivery to the inner ear. The RWM was not completely permeated and also became loaded with nanocomposites, indicating that delivery to the cochlea would continue for weeks by PLGA degradation and passive diffusion.

Distribution of PLGA nanoparticles in chinchilla cochleae.

OBJECTIVES: To study the distribution of polylactic/glycolic acid-encapsulated iron oxide nanoparticles (PLGA-NPs) in chinchilla cochleae after application on the round window membrane (RWM). STUDY DESIGN AND SETTING: Six chinchillas (12 ears) were equally divided into controls (no treatments) and experimentals (PLGA-NP with or without magnetic exposure). After 40 minutes of PLGA-NP placement on the RWM, perilymph was withdrawn from the scala tympani. The RWM and cochleae were fixed with 2.5% glutaraldehyde and processed for transmission electron microscopy. RESULTS: Nanoparticles were found in cochleae with or without exposure to magnet forces appearing in the RWM, perilymph, endolymph, and multiple locations in the organ of Corti. Electron energy loss spectroscopy confirmed iron elements in nanoparticles. CONCLUSION: The nanoparticles were distributed throughout the inner ear after application on the chinchilla RWM, with and without magnetic forces. SIGNIFICANCE: PLGA-NP applied to the RWM may have potential for sustained therapy to the inner ear.

Magnetic characterization of superparamagnetic nanoparticles pulled through model membranes.

BACKGROUND: To quantitatively compare in-vitro and in vivo membrane transport studies of targeted delivery, one needs characterization of the magnetically-induced mobility of superparamagnetic iron oxide nanoparticles (SPION). Flux densities, gradients, and nanoparticle properties were measured in order to quantify the magnetic force on the SPION in both an artificial cochlear round window membrane (RWM) model and the guinea pig RWM. METHODS: Three-dimensional maps were created for flux density and magnetic gradient produced by a 24-well casing of 4.1 kilo-Gauss neodymium-iron-boron (NdFeB) disc magnets. The casing was used to pull SPION through a three-layer cell culture RWM model. Similar maps were created for a 4 inch (10.16 cm) cube 48 MGOe NdFeB magnet used to pull polymeric-nanoparticles through the RWM of anesthetized guinea pigs. Other parameters needed to compute magnetic force were nanoparticle and polymer properties, including average radius, density, magnetic susceptibility, and volume fraction of magnetite. RESULTS: A minimum force of 5.04 x 10(-16) N was determined to adequately pull nanoparticles through the in-vitro model. For the guinea pig RWM, the magnetic force on the polymeric nanoparticles was 9.69 x 10-20 N. Electron microscopy confirmed the movement of the particles through both RWM models. CONCLUSION: As prospective carriers of therapeutic substances, polymers containing superparamagnetic iron oxide nanoparticles were succesfully pulled through the live RWM. The force required to achieve in vivo transport was significantly lower than that required to pull nanoparticles through the in-vitro RWM model. Indeed very little force was required to accomplish measurable delivery of polymeric-SPION composite nanoparticles across the RWM, suggesting that therapeutic delivery to the inner ear by SPION is feasible.

Magnetic resonance imaging compatibility and safety of the SOUNDTEC Direct System.

OBJECTIVE/HYPOTHESIS: The purpose of this study was to evaluate magnetic resonance imaging (MRI) compatibility and safety of an electromagnetic implanted hearing device (the SOUNDTEC Direct System; SOUNDTEC, Inc., Oklahoma City, OK) implant during a 0.3-Tesla open MRI imaging examination of the head and neck and to develop an MRI protocol that maximizes patient safety while minimizing the need for implant removal. The current literature regarding MRI compatibility of implantable hearing devices was reviewed. STUDY DESIGN: Linear and torsional forces, heating, and implant magnetization were evaluated in vitro. Implanted fresh-frozen human temporal bones were used to evaluate image distortion. A prospective study of 11 volunteers previously implanted with the SOUNDTEC Direct System was conducted to evaluate MRI compatibility and safety. A MEDLINE search of the literature between 1980 and July 2005 was reviewed to summarize MRI compatibility testing of implantable hearing devices. METHODS: Torsional and linear forces experienced by eight implant magnets were measured using calibrated neurologic Von Frey Hairs and compared with finite element analysis predictions as well as forces required to separate the incudostapedial joints of 12 fresh-frozen human temporal bones. Implant heating was determined by measuring the temperature change of eight implant vials compared with saline controls immediately after a head MRI scan. Implant magnetization was evaluated after repeated exposure to a 0.3-Tesla magnetic field. An 11-patient prospective study was performed to evaluate MRI compatibility in a 0.3-Tesla open MRI environment using adult volunteers previously implanted with the SOUNDTEC Direct System. A modified MRI protocol was developed to maximize patient safety. Each individual underwent an audiometric and otologic examination immediately before and after MRI. RESULTS: Peak linear force at the MRI entry measured 0.5 g +/- 0.2 standard deviation (SD). Maximum torque occurred at isocenter and measured 11.4 g-cm +/- 1.2 SD. The mean torque required to separate the incudostapedial joint was 33.8 g-cm +/- 20.4 SD. The average increase in temperature of the eight implant vials was 0.45 degrees C +/- 0.11 SD, whereas the increase in temperature of the three saline controls measured 0.47 degrees C +/- 0.11 SD. The average change in magnetic flux density of the 14 implant magnets tested was 22.0 gauss. Maximum image distortion occurred during the gradient echo sequence and measured 8.6 cm in diameter with a volume of 5,096 mm. Eleven patients completed a total of 12 head, one shoulder, and three lumbar 0.3-Tesla open MRI scans without patient- or device-related complications other than degradation of the MR image. There was no report of discomfort, tinnitus, dizziness, change in hearing, or change in device performance. All post-MRI changes in pure-tone thresholds, speech discrimination, soundfield thresholds, and aided soundfield thresholds were within the range of test-retest variability. CONCLUSION: When considering MRI of implantable ferromagnetic hearing devices, issues related to mechanical forces, implant heating, current induction, implant demagnetization, image degradation, and acoustic trauma must be considered. The SOUNDTEC Direct System is both MRI-compatible and safe in a 0.3-Tesla open MRI environment when a modified protocol is used. Degradation of the head MRI image may impair visualization of the ipsilateral temporal bone and adjacent structures within a 2.5- to 4.3-cm radius of the implant and is minimized by using a fast spin echo sequence.

The permeability of SPION over an artificial three-layer membrane is enhanced by external magnetic field.

BACKGROUND: Sensorineural hearing loss, a subset of all clinical hearing loss, may be correctable through the use of gene therapy. We are testing a delivery system of therapeutics through a 3 cell-layer round window membrane model (RWM model) that may provide an entry of drugs or genes to the inner ear. We designed an in vitro RWM model similar to the RWM (will be referred to throughout the paper as RWM model) to determine the feasibility of using superparamagnetic iron oxide (Fe3O4) nanoparticles (SPION) for targeted delivery of therapeutics to the inner ear. The RWM model is a 3 cell-layer model with epithelial cells cultured on both sides of a small intestinal submucosal (SIS) matrix and fibroblasts seeded in between. Dextran encapsulated nanoparticle clusters 130 nm in diameter were pulled through the RWM model using permanent magnets with flux density 0.410 Tesla at the pole face. The SIS membranes were harvested at day 7 and then fixed in 4% paraformaldehyde. Transmission electron microscopy and fluorescence spectrophotometry were used to verify transepithelial transport of the SPION across the cell-culture model. Histological sections were examined for evidence of SPION toxicity, as well to generate a timeline of the position of the SPION at different times. SPION also were added to cells in culture to assess in vitro toxicity. RESULTS: Transepithelial electrical resistance measurements confirmed epithelial confluence, as SPION crossed a membrane consisting of three co-cultured layers of cells, under the influence of a magnetic field. Micrographs showed SPION distributed throughout the membrane model, in between cell layers, and sometimes on the surface of cells. TEM verified that the SPION were pulled through the membrane into the culture well below. Fluorescence spectrophotometry quantified the number of SPION that went through the SIS membrane. SPION showed no toxicity to cells in culture. CONCLUSION: A three-cell layer model of the human round window membrane has been constructed. SPION have been magnetically transported through this model, allowing quantitative evaluation of prospective targeted drug or gene delivery through the RWM. Putative in vivo carrier superparamagnetic nanoparticles may be evaluated using this model.

Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant.

Superparamagnetic iron oxide nanoparticles (SNP) composed of magnetite (Fe(3)O(4)) were studied preliminarily as vehicles for therapeutic molecule delivery to the inner ear and as a middle ear implant capable of producing biomechanically relevant forces for auditory function. Magnetite SNP were synthesized, then encapsulated in either silica or poly (D,L,-Lactide-co-glycolide) or obtained commercially with coatings of oleic acid or dextran. Permanent magnetic fields generated forces sufficient to pull them across tissue in several round window membrane models (in vitrocell culture, in vivo rat and guinea pig, and human temporal bone) or to embed them in middle ear epithelia. Biocompatibility was investigated by light and electron microscopy, cell culture kinetics, and hair cell survival in organotypic cell culture and no measurable toxicity was found. A sinusoidal magnetic field applied to guinea pigs with SNP implanted in the middle ear resulted in displacements of the middle ear comparable to 90 dB SPL.

Human middle ear transfer function measured by double laser interferometry system.

HYPOTHESIS: Simultaneous measurements of vibrations on the stapes footplate, incudostapedial (IS) joint, and tympanic membrane (TM) can be made in both normal and drained cochleae, and the stapes displacement transfer function (S-DTF) and TM displacement transfer function (TM-DTF) are derived. BACKGROUND: A single laser Doppler interferometer previously has been used for measuring movement of the stapes or TM in temporal bones. However, there may be a limitation to optimally describing acoustic-mechanical transmission when the interferometer and temporal bone are moved frequently during experimental recordings. Simultaneous measurements of vibrations of the TM and stapes footplate, or TM and IS joint may reveal different acoustic-mechanical characteristics of the middle ear. METHODS: Dual laser interferometers simultaneously measured vibrations of the TM, IS joint, and stapes in 10 temporal bones with both intact and drained cochleae. From these measurements, the middle ear transfer function was expressed as the S-DTF, TM-DTF, and displacement transmission ratio (DTR). RESULTS: Simultaneous displacements of the TM, IS joint, and stapes footplate induced by sound pressure in the ear canal were recorded in both amplitude and phase. The middle ear transfer functions in terms of displacement ratio confirmed published single interferometer data but provided new information from drained cochlea. CONCLUSION: Stapes and TM displacement transfer functions were determined using dual interferometry, provided accurate amplitude and phase relationships from stapes footplate, IS joint, and TM, with new data from drained and normal cochlea.

An advanced computer-aided geometric modeling and fabrication method for human middle ear.

This paper presents a practical and systematic method for reconstructing accurate computer and physical models of the entire human middle ear. The proposed method starts with the histological section preparation of human temporal bone. Through tracing outlines of the middle ear components on the sections, a set of discrete points is obtained and employed to construct B-spline curves that represent the exterior contours of the components using a curve-fitting technique. The surface-skinning technique is then employed to quilt the B-spline curves for smooth boundary surfaces of the middle ear components using B-spline surfaces. The solid models of the middle ear components are constructed using these surfaces and then assembled to create the entire middle ear in a computer-aided design environment. This method not only provides an effective way to visualize and measure the three-dimensional structure of the middle ear, but also provides a detailed knowledge of middle ear geometry that is required for finite element analysis or multibody dynamic analysis of the human middle ear. In addition, the geometric model constructed using the proposed method is smooth and can be fabricated in various scales using solid freeform fabrication technology. The physical model of the human middle ear is extremely effective in realizing the middle ear anatomy and enhancing discussion and collaboration among researchers and physicians.

Three-dimensional modeling of middle ear biomechanics and its applications.

HYPOTHESIS: This study investigated whether combined technologies of finite element (FE) analysis and three-dimensional reconstruction of human temporal bones could be used to construct a computational model, useful in describing normal and pathologic middle ear sound conduction. BACKGROUND: FE models for biologic systems have been used in ear biomechanics. Three-dimensional reconstructions have also been made, but not in combination with FE modeling and laser interferometry measuring of human temporal bones. Furthermore, an FE model for the human middle ear with its ossicular attachments has not been reported on the basis of temporal bone histologic sections and morphometric reconstruction, to the authors' best knowledge. Because of the size, variability, and complexity of the middle ear, accurate morphologic data and boundary conditions are necessary for accurate FE modeling. METHODS: A fresh temporal bone was decalcified, embedded in celloidin, sectioned and stained, scanned, and digitized, and the normal middle ear was reconstructed. The histologic sections were used to construct a computer-aided design model with ligaments, muscles, and tendons as boundary conditions. The data thus obtained were converted into an FE mechanical model that was validated by comparison with displacements obtained by laser Doppler interferometry on 17 fresh human temporal bones. RESULTS: An FE model was generated, demonstrating dynamic behavior that moderately approximated the laser interferometric data from human temporal bones receiving 90-dB sound pressure level auditory frequencies at the tympanic membrane. CONCLUSION: Accurate FE modeling, incorporating both morphometric and interferometric performance data, predicted both normal and pathologic mechanical performance of the human ossicular chain.