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.