Abstracts

Rationale: We have previously shown that a depth electrode implanted for direct brain stimulation therapy can only activate neurons within a 4mm radius of its surface. Extending its radius of influence in white matter by at least 1mm can increase the volume of tissue activation by over 95%. One method to increase the extent of brain activation is to enhance tissue conductivity (σ), and potentially reduce the impedance adjacent to the electrode. Our study demonstrates the influence of fluorescein-thiosemicarbazide-functionalized metallic carbon nanotubes (fCNTs) altering tissue impedance, their intracellular uptake with stable long-term neural localization in situ, and biocompatibility in vitro.Four experiments were carried out as follows: 1) electrical impedance spectroscopy to evaluate fCNT-induced changes in σ in a 0.6% agarose gel; 2) in vitro fCNT cytotoxicity assay; 3) in situ immunohistochemistry after fCNT injection, 4wks prior, to evaluate cellular uptake and stability; and 4) computational Hodgkin and Huxley (HH) cable modeling. Methods: Impedance SpectroscopyTo assess changes in σ, electrical impedance spectroscopy (50Hz-1MHz) was carried out in a 0.6% agarose gel around a pair of 8-platinum (Pt) cylindical contact depth contacts spaced 10mm center-to-center. Nine 2µl metallic fCNT 250 µg/ml injections into the gel were spaced perpendicularly at 1mm intervals. Gel impedances were measured again after injection. Impedance values between controls and fCNT samples were compared.Cytotoxicity TestingCortical human astrocytes (HAs) were obtained, cultured, and treated with raw and fCNTs of 90, 95, and 99% purity (NanoIntegris). Cell viability was measured after 72hrs via an alamar blue assay.Cellular Uptake TestImmunofluorescent staining was performed to observe the influence of 99% pure fCNTs injected stereotactically into the left hippocampal formation of F344 rats 4wks before sacrifice. Astrocytes and neurons were immunolabeled in fresh frozen 20µm thick brain tissue sections via fluorescent tagged GFAP/Cy5 (λ = 665nm), and anti-synaptophysin (λ = 405nm), respectively. fCNT clusters were visually localized using fluorescence microscopy (100x).Cable ModelingA computational HH impulse-propagation cable model was used to model the effect of fCNT-induced σ changes in the axonal intracellular medium. Changes in action potential production and propagation velocity were analyzed. Results: Impedance measurements showed a statistically significant difference between the fCNT sample and controls (p < 0.05). Furthermore, the cytotoxicity assay of 99% pure raw metallic CNTs (25 ng/mL) demonstrated maximal HA viability. No statistically significant difference in viability was found with fCNTs. Immunostaining revealed fCNT clusters in the hippocampal formation with fCNT uptake by both astrocytes and neurons near the injection point. The HH cable model revealed that intracellular σ changes enhance axon excitability and propagation speed. Conclusions: fCNTs are biocompatible following targeted injection into the brain, remain anchored long-term intracellularly, while chronically modifying the biophysical properties of neural cells. In particular, fCNT can impact the excitability properties of neural tissue by altering the local tissue σ, and augmenting the maximal extent of cortical activation by a depth lead placed in white matter for delivering direct neurostimulation therapy. Funding: Mary Keane Fund, Foglia Family Foundation, CONACYT.