Bioeffective Ultrasound at Very Low Doses: Reversible Manipulation of Neuronal Cell Morphology and Function in Vitro

Abstract

Direct and safe manipulation of neurons by external means is an increasingly studied therapeutic modality with the potential to treat many neurological diseases. Anticipating such future applications, we investigated reversible bioeffects of very low dose focused ultrasound on neuronal cell morphology and function in vitro. To test morphological changes, undifferentiated PC12 cells were serum-cultured. The culture plates were placed on an inverted optical microscope. An f/1.1 ultrasound transducer with a water-filled coupling cone was focused on the culture and excited with 30-ms 4.67-MHz 100-kPa pulses. To test functional changes, rat hippocampal slices were cultured and individually transferred to the well of a 60-channel multi electrode array. An f/2.1 ultrasound transducer with a water-filled coupling cone was focused on a culture and excited with 100-Ps 4.04-MHz 77-kPa pulses. The culture was stimulated before and after the ultrasonic stimulus with a 100-Ps 100-PA biphasic electrical stimulus. Optical microscopy of PC12 cultures under insonification revealed that cells that were clustered near the ultrasound focal region elongated by approximately 2 Pm during insonification and returned to approximately their original shapes following insonification. We conclude that the acoustic radiation force is capable of reversibly deforming cultured cells. In the rat hippocampal cultures, the ultrasonically and electrically evoked responses exhibited similar biphasic waveforms. In addition, robust electrically evoked responses following insonification indicated that the insonified cultures remained viable. We conclude that low-dose ultrasound can stimulate neurons; the mechanism is currently under investigation.

Hippocampal culture stimulus with 4-megahertz ultrasound

“…Optical microscopy of insonified cultured cells (PC12 cells from the rat adrenal gland) confirms this and demonstrates reversible distortion [2]. These observations are consistent with the early work of Wood [3] and more recent studies by Haake [4] and Lee [5] and their coworkers.…”

“…Full experimental details were presented previously [2,10] and are summarized here. Hippocampal slices were obtained from 8-day old Sprague Dawley rats and cultured for 6 days [2]. The in vitro cultures were exposed to multiple 100-ms 4.04-MHz ultrasound pulses from a 42-mm diameter, 90-mm spherical cap transducer.…”

“…The rat hippocampal culture in combination with a multi-channel electrode array offers a precise means of spatially and temporally tracking responses to ultrasonic stimuli. To investigate how different hippocampal regions responded to the same stimulus, additional data from a previously reported experiment [2,10] were analyzed.…”

“…Responses in the form of electrical potentials from a sixty channel electrode array (Multi Channel Systems GmbH, Reutlingen Germany) were digitized and recorded [2]. The ultrasonically-evoked field potentials were averaged over three sweeps and two hippocampal regions determined with optical microscopy: CA 1 (Cornu Ammonis, mostly pyramidal cells) and DG (Dentate Gyrus, mostly granular cells).…”

Towards multifocal ultrasonic neural stimulation: pattern generation algorithms

“…In the context of hyperthermia treatments, for example, it has been suggested that multi-focal ultrasound distributions would result in a more uniform temperature field, which could be more finely controlled and quicker to create [1][2][3]. In ultrasonic neuro-modulation, an emerging application of FUS [4][5][6][7][8][9], multi-focal ultrasonic distributions may also be used for patterned simultaneous stimulation, inhibition or modulation of neural structures and populations, much as multi-focal light distributions are already being applied for parallel photostimulation [10][11][12][13]. In contrast to visible light, FUS is not heavily scattered or absorbed when penetrating biological tissues and may be applied completely non-invasively to various neural structures including cortical areas and even deep-seated nuclei [9].…”