Cochlear Morphology and Sound-Induced Motion of the Apical Mammalian Inner Ear by Scott Lawrence Page Submitted to the Department of Electrical Engineering and Computer Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2016 @ Massachusetts Institute of Technology 2016. All rights reserved. Signature redacted A u th o r ............... ................... W .............................. Department of Electrical Engineering and Computer Science January 29, 2016 Signature redacted Certified by..... Dennis M. Freeman Professor Thesis Supervisor Signature redacted.......... Accepted by ...... U [kslie A. Kolodziejski Chairman, Department Committee on Graduate Theses MASSACHUSE S NSITUTE OF TECHNOLOGY co LU APR 15 2016 LIBRARIES 2 Cochlear Morphology and Sound-Induced Motion of the Apical Mammalian Inner Ear by Scott Lawrence Page Submitted to the Department of Electrical Engineering and Computer Science on January 29, 2016, in partial fulfillment of the requirements for the degree of Doctor of Philosophy Abstract Extraordinary sensitivity, frequency selectivity, and dynamic range are hallmarks of mammalian hearing. While a variety of cellular and molecular mechanisms are known to be critical to these properties, how the cellular and molecular mechanisms interact to generate the remarkable properties remains unclear. Direct observations of these interacts has proved to be difficult, in large part because the inner ear is fragile and has been difficult to probe with conventional measurement technologies. We have developed an Optical Coherence Tomography (OCT) system to use light to probe both the structure and mechanical responses of the inner ear to sound stimulation. The technique takes advantage of the interference of low coherence sources of light to detect even weakly scattering tissues in the inner ear. By sensing Doppler shifts of light scattered off moving structures in the inner ear, the OCT system can also detect sound-induced motions of cochlear structures with sub-nanometer resolution. This thesis demonstrates the use of the OCT system to study the structure of the inner ears of mice, gerbils, and guinea pigs, as well as the acoustic response of the apical turn of in vitro and in vivo apical mammalian cochleae to low frequency (100 to 1000 Hz) sounds - frequencies that are critical to our understanding of speech. Thesis Supervisor: Dennis M. Freeman Title: Professor 3 4 Acknowledgments I'd like to acknowledge my research group: Denny Freeman, Stan Hong, A. J. Aranyosi, Rooz Ghaffari, Shirin Farrahi, and Jon Sellon for all their help over the years. I'd also like to acknowledge Natasha Guha and Collin Kaufman for their help with ex- periments and experimental setup. Finally, I'd like to acknowledge everyone that has helped me along the way, including my friends and family. 5 6 Contents 1 Introduction 17 1.1 The mammalian cochlea . . . . . . . . . . . . . . . . . . . . . . . . .. 19 1.1.1 Cochlear anatomy . . . . . . . . . . . . . . . . . . . . . . . . . 19 1.1.2 Effects of sound . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1.1.3 Cochlear amplification and sensitivity . . . . . . . . . . . . . . 20 2 DOCM/DOCT Methodology 25 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2 DOCM /DOCT .............................. 26 3 Tectorial membrane electrokinetics 31 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 3.2 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.3 C onclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.4 M aterials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.4.1 Isolated TM Preparation . . . . . . . . . . . . . . . . . . . . . 44 3.4.2 Microaperture Chamber . . . . . . . . . . . . . . . . . . . . . 45 3.4.3 Measuring Fixed Charge Density cf . . . . . .. . . . . . . . . . 45 3.4.4 Measuring Electrokinetic Motion of the TM . . . . . . . . . . 47 3.4.5 Motion Analysis with Computer Vision System . . . . . . . . 47 3.4.6 Motion Analysis with Doppler Optical Coherence Microscopy. 47 3.5 Supplemental DOCM results . . . . . . . . . . . . . . . . . . . . . . . 48 7 4 Cochlear morphology 49 4.1 Methods . .... . .. . . . .. .... . .. . 49 4.1.1 In situ preparation . . . . . . . . . . . 49 4.1.1.1 Animal preparation . . . . . . 49 4.1.1.2 Health . . . . . . . . . . . . . 50 4.1.1.3 DOCM optical methods . . . 50 4.1.2 Image intensity resolution . . . . . . . 52 4.1.3 Image spatial resolution . . . . . . . . 54 4.1.3.1 Scanning apparatus radial and axial spatial resolution 54 4.1.3.2 Optical axial spatial resolution 56 4.2 Results and Discussion . . . . . . . . . . . . . 57 4.2.1 In situ apical Mongolian gerbil . . . . . 57 4.2.2 In situ apical mouse . . . . . . . . . . 63 4.2.3 In situ apical guinea pig . . . . . . . . 64 5 Sound-induced axial motion in the apex of the mammalian inner ear 69 5.1 Methods ...... ............... 69 5.1.1 In vitro preparation . . . . . . . . . . 69 5.1.1.1 Animal preparation . . . . . 69 5.1.1.2 Health . . . . . . . . . . . . 71 5.1.1.3 Stapes-driven excitation 71 5.1.1.4 DOCM optical methods 72 5.1.2 In vivo preparation . . . . . . . . . . 73 5.1.2.1 Animal preparation . . . . . 73 5.1.2.2 DOCT Optical alignment . 74 5.1.2.3 Acoustic sound delivery and calibration 74 5.1.2.4 Health/Viability . . . . . . 76 5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 5.2.1 In vitro stapes induced apical mechanics . . . . . . 78 5.2.1.1 DOCM imaging and axial motion analysis 78 8 5.2.1.2 Differential analysis . . . . . . . . . . . . . . . . . . 81 5.2.2 In vivo sound-induced apical mechanics . . . . . . . . . . . . . 84 5.2.2.1 DOCT imaging and axial motion analysis . . . . . . 84 5.2.2.2 Frequency-dependent motion analysis . . . . . . . . . 88 5.2.2.3 Differential analysis . . . . . . . . . . . . . . . . . . 91 5.2.2.4 Post-mortem axial motion and differential analysis 92 5.3 D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 5.4 Sum m ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 9 10