Publication Date

2014-12-02

Availability

Open access

Embargo Period

2014-12-02

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biomedical Engineering (Engineering)

Date of Defense

2014-10-16

First Committee Member

Suhrud M. Raguru

Second Committee Member

Fabrice Manns

Third Committee Member

Ozcan Ozdamar

Fourth Committee Member

Claus-Peter Richter

Fifth Committee Member

Fred F. Telischi

Abstract

Cochlear implants are currently the most effective solution for profound sensorineural hearing loss, and vestibular prostheses are under development to treat bilateral vestibulopathies. Electrical current spread in these neuroprostheses limits channel independence, and in some cases may impair device performance. In comparison to conventional electrical stimulation, optical stimuli that are spatially confined may result in a significant functional improvement. Pulsed infrared radiation (IR) allows direct stimulation of tissue without genetic or pharmacological manipulation, and has been shown to excite neurons. This study analyzes if pulsed IR (λ = 1863 nm) elicits intracellular Ca2+ ([Ca2+]i) transients in cultured neonatal rat spiral and vestibular ganglion neurons. The neurons responded consistently with [Ca2+]i transients that matched the low frequency IR pulses applied (4 ms, 0.25-1 pps, 398-796 mJ cm-2). While blockage of Na+, K+, and Ca2+ plasma membrane channels did not alter the IR-evoked [Ca2+]i response, blocking of mitochondrial Ca2+ cycling with CGP-37157 or Ruthenium Red reversibly inhibited the IR-evoked response. Additionally, the magnitude of the IR-evoked [Ca2+]i transients was dependent on Ca2+ extrusion from the endoplasmic reticulum (ER). To clarify the role of mitochondria in these intracellular responses, we characterized the IR modulation of mitochondrial transmembrane potential, ΔΨm. Two ΔΨm sensors, TMRE and Rhodamine 123, showed transient mitochondrial hyperpolarization in response to pulsed IR stimuli (100 μs, 100 pps, 178-374 mJ/cm2; 4 ms, 0.25-1 pps, 398-796 mJ/cm2) in the neurons. Drugs targeting either mitochondrial Ca2+ cycling, intracellular Ca2+ or the mitochondrial electron transport chain (ETC) inhibited the IR-evoked increases in ΔΨm. Intracellular pH (pHi) was acidified in the neurons during stimulation. Regarding long-term effects of IR (1 ms, 30 Hz, 661 mJ/cm2, 10 minutes) on the neurons, reactive oxygen species (ROS) levels measured at 6 hours were higher than in non-radiated neurons. However, colocalization of mitochondria and cytochrome c suggested no permanent mitochondrial permeability transition pore (mPTP) opening. Accordingly, JC-1 fluorescence indicated that ΔΨm was also observed at normal resting level 24 hours after radiation. Minimal active caspase-3 was detected further supporting that IR modulation of ΔΨm was transient and did not result in irreversible damage. Finally, the present study also examined whether IR-evoked [Ca2+]i events contribute to plasma membrane depolarization. Results indicate that pulsed IR stimuli (100 us, 100 pps, 178-374 mJ/cm2) delivered to the neurons resulted in an increase in fluorescence of plasma membrane potential (ΔΨp) sensor FluoVolt™. These increases in fluorescence suggest ΔΨp depolarization. Drugs targeting either mitochondrial Ca2+ cycling, intracellular Ca2+ or Ca2+ extrusion from the ER decreased the IR-evoked ΔΨp depolarization. This pharmacological analysis suggests correlation between the depolarization observed and IR induced intracellular Ca2+ release. Overall, the results suggest that pulsed IR stimulation provides a novel optical tool to study the role of intracellular organelles in manipulating Ca2+ cycling and related events. The selective excitation of neurons in the IR beam path suggests the feasibility of infrared neural stimulation (INS) in cochlear and vestibular implants.

Keywords

Infrared Radiation; Calcium; Mitochondria; pH; Cochlea; Vestibular

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