MAPPING THE OCEAN SOUND SPEED AT THE ALOHA CABLED OBSERVATORY USING RELIABLE ACOUSTIC PATH TOMOGRAPHY

M.S.M.S. Thesis. University of Hawaiʻi at Mānoa 201

Development of a non-invasive electrophysiological system for measuring the auditory capability of marine animals

Merged with duplicate record 10026.1/1293 on 27.02.2017 by CS (TIS)The following work describes the development and application of a neurological system to definitively profile the auditory responses of aquatic animals, presented as audiograms showing hearing threshold verses sound frequency. The accuracy of such information is essential for the optimisation of bio-technical devices such as the Acoustic Fish Deterrent (AFD) barrier deployed in the Illinois River to prevent the migration of non-indigenous Asian carp into Lake Michigan, and in the impact assessment of anthropogenic underwater sounds on the hearing of cetaceans and other marine animals. The ensuing Auditory Brainstem Response (ABR) electrophysiological recording technique developed at the University of Plymouth and described in this thesis is classified by the UK Home Office as being non-invasive, yielding high quality data from vertebrates in the absence of anaesthetics or implanted electrodes. The ABR technique was further refined to allow for the recording of evoked potentials in response to either the sound pressure or particle motion component of an acoustic signal, from animals stationed both at and below the water surface and ranging in size from a few millimetres to nearly a meter in length. The electrophysiological studies have resulted in the publication of three peer reviewed manuscripts, one of which is the first to define hearing for any animal from the order Acipenseriform (sturgeons and paddlefish). In addition to the development of the electrophysiology system and protocols, the inner ear morphology of the animals tested in this work were studied at the ultrastructural level, along with detailed descriptions of the afferent nerve pathway from the ear to the brain. Current literature shows a paucity of information on consistent and meticulous removal of inner ear parts necessary to identify damage to the ultrastructure that is symptomatic of hearing loss. In order for the acquisition of concise and reliable data, the dissection and preparation technique for Scanning Electron Microscopy (SEM) was refined for each species investigated and has resulted in the publication of a further three peer reviewed manuscripts on inner ear morphology

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin