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Ultrasound Stimulation for Peripheral Nerve Repair
Emily Ashbolt, Marissa Puzan, Dan Ventre, Dr. Abigail KoppesNortheastern University, Boston, MA
BMES 2016, Minneapolis, MN
2Peripheral Nerve Injuries Currently twenty million Americans with peripheral nerve
injuries [1] $150 billion a year spent on therapies nationally 51.6%: satisfactory recovery of motor function [1] 42.6%: satisfactory recovery of sensory function [1]
1. D. Grinsell and C. P. Keating, “Peripheral Nerve Reconstruction after Injury: A Review of Clinical and Experimental Therapies,” BioMed Research International, vol. 2014, Article ID 698256, 13 pages, 2014. doi:10.1155/2014/698256
3Ultrasonic Stimulation Ultrasound (US): defined as an
acoustic pressure wave
History of safe and effective use in diagnostic imaging and ablative surgeries
Recently shown to modulate central nervous system (CNS) neurons via transcranial application
Limited research into US effects on peripheral nerves, only used in in vivo models.
US guided femoral nerve block
Image credit: http://www.nysora.com/techniques/3120-ultrasound-guided-femoral-nerve-block.html/
4Ultrasound as a Non-Invasive Therapy Electrical stimulation has shown
promise to aid regeneration, but is often invasive
US can modulate neural activity transdermally or transcranially
US neuromodulation of peripheral nerve injuries could provide a non-invasive alternative to electrical stimulation
Image credit: http://www.the-scientist.com/?articles.view/articleNo/41324/title/Neuroprosthetics/
Direct peripheral nerve interfaces
5Motivation
Previous work revealed US stimulation: • Enhances rate of peripheral nerve and bone regeneration• Reversibly inhibits or excites neurons• Can disrupt neural migration in developing embryos
Despite these observations, the specific cellular mechanisms of how tissues respond to US are still poorly understood
Manlapaz, J., et al. (1964). "Effects of ultrasonic radiation in experimental focal epilepsy in the cat." Experimental neurology 10(4): 345-356.Tufail, Y., et al. (2011). "Ultrasonic neuromodulation by brain stimulation with transcranial ultrasound." nature protocols 6(9): 1453-1470.Ang, E. S., et al. (2006). "Prenatal exposure to ultrasound waves impacts neuronal migration in mice." Proceedings of the National Academy of Sciences 103(34): 12903-12910.
The purpose of this study was to determine the impact of ultrasound on peripheral neurons in vitro
6
Image sources: http://www.sprawls.org/ppmi2/USPRO/, http://www.cliparthut.com/osmosis-clipart.html, https://www.pinterest.com/pin/395050198536463573/, http://www.ninds.nih.gov/disorders/brain_basics/ninds_neuron.htm,, http://medical-dictionary.thefreedictionary.com/synapse,
How do individual neurons respond to ultrasound in vitro?
+ = ?Branching?
Probe potential for US to impact neuron morphology in order to better implement non-invasive ultrasound for PNS repair
Approach: Do peripheral neurons respond to US?
Altered neuron migration/motility?
7Approach: Neural Stimulation
Whole DRG Harvest from rat pups
DRG SensoryNeuronDissociation
Neurons seeded onto laminin coated plates
Ultrasound Stimulation –various intensities
Fix, Stain, Image neurons
Neurolucida Tracing and Analysis
6 hours
18 hours
Morphology Analysis
Stimulation
• US has been shown to modulate neuron activity does US impact peripheral neurons?
8Approach: Ultrasonic Stimulation PlatformComplex US waveform construction • Function generators and
oscilloscope: • create and visualize
waveform
• RF amplifier: • drive US transducer to
emit ultrasonic stimulus
• Water bath: • acoustic coupling agent• temperature control
Generation and Emission of US Stimulus From Immersion
Transducer
Tufail, Y., et al. (2011). "Ultrasonic neuromodulation by brain stimulation with transcranial ultrasound." Nature Protocols 6(9): 1453-1470.Tsuang, Y. H., et al. (2011). "Effects of low intensity pulsed ultrasound on rat Schwann cells metabolism." Artificial Organs 35(4): 373-383.
9Ultrasound Parameters Three Intensities of US
Stimulation Used: LOW, @ 200 mV driving voltage,
50% duty cycleMEDIUM, @ 500 mV driving
voltage, 50% duty cycleHIGH, @ 800 mV driving voltage,
75% duty cycle Core variables:
0.5 MHz frequency transducer20 Hz pulse repetition1000 cycles per pulse at 3600
total pulses delivered3 minute total duration3 cm transducer/well separation
distance Parameters designed based on
guidelines from [Tufail, 2011].
Electronics
Well Plate
Transducer
Tufail, Y., et al. (2011). "Ultrasonic neuromodulation by brain stimulation with transcranial ultrasound." nature protocols 6(9): 1453-1470.
10
Results: Ultrasound Alters Neuron Morphology
Neurons at low, medium, and high levels of ultrasound stimulation. Scale: 100 microns. Green:
BIII tubulin
Low
High
Control
Medium
Visibly, neurite outgrowth appears greater with medium and high US compared
Neurons extend neurites in all US conditions examined
11
Approach: Neurolucida Neural Tracing
Variables Considered:
• Total Neurite Outgrowth
• Average Neurite Length
• Number of Primary Neurites Extending from Soma
• Number of Branch Points
12
Results: Higher US Stimulation,Greater Neurite Branching• No significant difference in
average tree number (primary branching)
• Significant difference in average branch number (secondary branching) # p<0.01
• Student’s t-test used to compare conditions, n=18-37
0
4
8
12
16
Ave
rage
Bra
nch
Qua
ntit
y
# #
Control
Low Medium High
LOW, @ 200 mV driving voltage, 50% duty cycleMEDIUM, @ 500 mV driving voltage, 50% duty cycleHIGH, @ 800 mV driving voltage, 75% duty cycle
13
Results: Higher Stimulation, Greater Neurite Outgrowth• Significant increase in
average total outgrowth for medium (2.83x) and high (2.25x) US
• No significant difference in average branch length
• Extended outgrowth likely comes from more branching, not longer branches
• Student’s t-test used to compare conditions, n=18-37
0
1125
2250
3375
4500
Tota
l Den
drit
ic
Out
grow
th (
µm)
Control Low Medium High
*#
LOW, @ 200 mV driving voltage, 50% duty cycleMEDIUM, @ 500 mV driving voltage, 50% duty cycleHIGH, @ 800 mV driving voltage, 75% duty cycle(* p<0.05, # p<0.01)
14
Approach: Glial Stimulation
Sciatic Nerve Harvest from rat pups
Isolate and Purify Primary Schwann cells
Schwann cells seeded onto laminin coated plates
Ultrasound Stimulation –various intensities
Alamar Blue Metabolism
6 hours
18 hours
Examine changes in Metabolism
Stimulation
• Schwann cells have been shown to enhance neurite outgrowth does US also impact peripheral glia?
15
Results: Ultrasound Increases Schwann Cell Metabolism
• Alamar Blue shows relative Schwann Cell metabolic activity After US Stimulation
• Schwann cell metabolism is increased 37% as a result of direct US with low intensity
• Student’s t-test used to compare conditions, n=4
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Low Intensity Medium Intensity Control
RFU
s N
orm
aliz
ed t
o Co
ntro
l Wel
l *
LOW, @ 200 mV driving voltage, 50% duty cycleMEDIUM, @ 500 mV driving voltage, 50% duty cycle
16Conclusions
US stimulation is capable of altering sensory neuron morphology. Ultrasound increases secondary neurite branching
Number of primary branches remains the same Ultrasound increases total neurite outgrowth
May be due to more branching, not longer neurites Investigation of the neurite branching mechanism is ongoing
Ultrasound Increases Schwann cell metabolic activity Impact of US-Schwann cell neurite extension on glia is unknown
Analysis of electrophysiological changes/ secreted factors is planned for the near future
17Acknowledgements
Dr. Abigail Koppes Marissa Puzan Dan Ventre ABNEL Lab Northeastern College of
Science Northeastern College of
Engineering
Thank you, Questions?
18
Current theories regarding potential cellular mechanisms of response to US stimulation.
BLS (BiLayer Sonophore) Theory: Oscillating acoustic pressure wave causes changes in the separation
distance between the inner and outer lipid layers of the cell membrane of the neurons. This membrane
movement allows for gas bodies to infiltrate the intervening hydrophobic space, where they can exert cavitation-
based effects. These cause shearing of the membrane and/or pore formation, increasing membrane
permeability.
Continuum Theory: Continuum theory builds off of BLS Theory, and maintains that cavitation acting upon or
near a cell membrane accounts for many observed effects, as a result of acoustic pressure waves acting on
proximal gas bodies. Fluid currents caused by the acoustic wave, such as microstreaming and microjets, alter
membrane permeability and the flux of ions across the membrane. It is believed that the resulting permeability
changes account for action potential firing or inhibition of firing, depending on acoustic parameters and
extracellular fluid composition.
19Supplemental (Theories)
20
Continuum Theory Supplemental Figure
(adapted from Tyler, 2011)
Tyler, W. J. (2011). "Noninvasive neuromodulation with ultrasound? A continuum mechanics hypothesis." The Neuroscientist 17(1): 25-36.