ZMPCZM017000.11.03 Carey Experimentation on brain research
Ultrasound Stimulation for Peripheral Nerve Repair v7
1. Ultrasound Stimulation for
Peripheral Nerve Repair
Emily Ashbolt, Marissa Puzan, Dan Ventre, Dr. Abigail Koppes
Northeastern University, Boston, MA
BMES 2016, Minneapolis, MN
2. Peripheral 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]
2
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
3. Ultrasonic 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.
3
US guided femoral nerve block
Image credit: http://www.nysora.com/techniques/3120-ultrasound-guided-femoral-nerve-block.html/
4. Ultrasound 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
4
Image credit: http://www.the-scientist.com/?articles.view/articleNo/41324/title/Neuroprosthetics/
Direct peripheral nerve interfaces
5. Motivation
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
5
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? 6
Altered neuron migration/motility?
7. Approach: Neural Stimulation
7
Whole DRG
Harvest from
rat pups
DRG Sensory
Neuron
Dissociation
Neurons
seeded onto
laminin
coated
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?
8. Approach: Ultrasonic Stimulation
Platform
Complex 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
8
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.
9. Ultrasound Parameters
Three Intensities of US Stimulation
Used:
LOW, @ 200 mV driving voltage,
50% duty cycle
MEDIUM, @ 500 mV driving
voltage, 50% duty cycle
HIGH, @ 800 mV driving voltage,
75% duty cycle
Core variables:
0.5 MHz frequency transducer
20 Hz pulse repetition
1000 cycles per pulse at 3600
total pulses delivered
3 minute total duration
3 cm transducer/well separation
distance
Parameters designed based on
guidelines from [Tufail, 2011].
9
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
10
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 11
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
12
0.
4.
8.
12.
16.
AverageBranchQuantity
# #
Control Low Medium High
LOW, @ 200 mV driving voltage, 50% duty cycle
MEDIUM, @ 500 mV driving voltage, 50% duty cycle
HIGH, @ 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
13
0.
1125.
2250.
3375.
4500.
TotalDendriticOutgrowth(µm)
Control Low Medium High
*
#
LOW, @ 200 mV driving voltage, 50% duty cycle
MEDIUM, @ 500 mV driving voltage, 50% duty cycle
HIGH, @ 800 mV driving voltage, 75% duty cycle
(* p<0.05, # p<0.01)
14. Approach: Glial Stimulation
14
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. 15Results: 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
RFUsNormalizedtoControlWell
Low Intensity
Medium Intensity
Control
*
LOW, @ 200 mV driving voltage, 50% duty cycle
MEDIUM, @ 500 mV driving voltage, 50% duty cycle
16. Conclusions 16
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
17. Acknowledgements
Dr. Abigail Koppes
Marissa Puzan
Dan Ventre
ABNEL Lab
Northeastern College of
Science
Northeastern College of
Engineering
Thank you, Questions?
17
19. 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)
20
Tyler, W. J. (2011). "Noninvasive neuromodulation with ultrasound? A continuum mechanics hypothesis." The Neuroscientist 17(1): 25-36.
Editor's Notes
I would like to thank the conference organizers for the invitation to speak to you today about my research conducted this past year as an Undergraduate at Northeastern University regarding the effect of ultrasound stimulation on the growth of peripheral neurons
Break it down
There are currently twenty million Americans suffering from peripheral nerve injuries, many of them current or former military members
A 2005 study found that only 51.6% of those undergoing nerve repair achieve satisfactory motor recovery and even fewer (41.6%) experience satisfactory sensory recovery
Current treatments such as physical therapy and various surgical interventions have effectiveness dependent highly on the location of the injury,
nd nerve regeneration is susceptible to blocks such as scar tissue.
Ultrasound is an Acoustic Pressure wave
-Safely and effectively used in diagnostic imaging and aBLAYtive surgeries -IMAGE
-Has been shown to modulate neurons in the central nervous system transcranially
However, peripheral nerve effects are more of n unknown quantity- limited research, only in vivo models.
Why not electrical stimulation?
Has been shown to aid regeneration, but techniques are invasive- surgery, implanting elctrodes must be direct contact, not as applicable in a transcranial setting
Ultrasound can be used transdermally or transcranially and needs t
Positive and negative
Regeneration of periph nerve and bone, can reversibly inhibit or excite neurons
However, can also disrupt neural migration in developing embryos
Currently, little is known how different acoustic parameters (amplitude, frequency, intensity) impact cellular response individually. impact of ultrasonic neuromodulation on function and morphology remains unknown
Ultrasound on perph neurons in vitro
Like I said earlier, ultrasound has been shown to create reactions, such as greater outgrowth or firing, in neuronal cells in vivo but the process has not been fully explored in vitro
We attempted to explore the relationship between dorsal root ganglia being pulsed with different levels of ultrasound and two factors that could be relevant to using this technique in a therapeutic setting: total dendritic outgrowth and secondary branching.
Probe potential for ultrasound to impact neuron morphology in order to better implement non-invasive PNS therapies.
DRGs harvested and isolated into individual cells
24 hour cycle
Seeded onto 12-well plates on laminin
Standard conditions in Neurobasal A media supplemented with 50 ng/ml nerve growth factor
Wait 6 hours, stimulate at various intensities
allow DRGs a chance to grow for 18 hours
fix, stain and image them
Do Neurolucida tracing for morphology analysis
Electronic set up blended complex waveforms and in vitro cell monolayer stimulation
Series of function generators and an oscilloscope to create and visualize waveform
RF amplifier drove the ultrasound transducer to emit the ultrasound waves
And the well plate and transducer were in a water bath, with the water being used as a coupling agent to prevent scatter, as a well as temperature control.
Here you can see an image of the set-up, as well as the parameters we used.
These variables were modified from a transcranial ultrasound study done in the Tyler lab.
We had three levels, low, medium and high, featuring increasing driving voltages and duty cycles, as well as core variables across all three levels such as our pulse repetition (20 HZ), working distance (3cm), and stimulation time (3 minutes).
After the 18 hour growth period, we stained and imaged the cells. Here you can see examples of the four levels- visually, the medium and high cells appear to have greater outgrowth.
And then this is an image from the tracing software we used, Neurolucida. It allows for the following of the processes, as well as for the marking of branch points and somas. We used it to look for the neurite lengths, both total and average per branch, as well as the number of primary and secondary branches.
Results from this study showed that higher ultrasound stimulation did indeed result in greater neurite branching.
There was no significant difference in the primary branching, or what we called the “trees” of the neuron- 2 branches off of soma, characteristic of DRGs
There was a significant increase in the number of secondary branch points, as can be seen in this graph here- nearly twice as many secondary branches.
We also saw that higher stimulation did indeed result in greater total neurite outgrowth, as can be seen in the graph here.
However, there was not a significant difference in the average branch length across the various ultrasound levels. What this tells us is that, combined with the results that higher ultrasound stimulation produced more branching, any increase in the total neurite outgrowth most likely comes from more branching from the cells, not a lengthening of existing branches.
Therapeutic effects!
Another more recent study we did that we also thought had some applicability to this realm was looking at the effects of ultrasound stimulation on glial cells as a potential method study.
Here we have the procedure for this preliminary study- it is similar to that of the DRG study in using the cells grown on well plates, the 24 hour cycle, the various intensity levels of ultrasound, but instead of looking at outgrowth or branching, we used alamar blue to look at the metabolism of the schwann cells.
Results from this small study were very interesting in that the alamar blue showed that schwann cells have significantly higher metabolic activity when pulsed with lower levels of ultrasound.
While not directly correlated with the results from the DRG study, it’s more evidence that ultrasound stimulation has significant effects on the morphology and metabolism of various neural cells.
So two main takaways from this data:
Ultrasound stimulation is capable of altering sensory neuron morphology- as I showed earlier, our studies found an increased secondary neurite branching, as well as total neurite outgrowth, in the DRGs that received a higher level of ultrasound stimulation
The glial study also showed that there is a metabolic effect of ultrasound on schwann cells, although the impact of ultrasound-schwann cell neural extension on glia is as of now unknown.
Going forward, we are hoping to do more studies and analysis on the electrophysiological changes and secreted factors of these various neural cells to understand the reasoning behind these results.
Thank you so much to everyone who made this project and my being here possible, my amazing graduate student and mentor Marissa, Dan and all the members of the ABNEL Lab, Northeastern University’s colleges of science and engineering, and Northeastern’s Office of Undergraduate Research and Fellowships.
Thank you.
Mechanism- changes in permeability of the membrane
Theories out of tyler lab
More research being done into it- including us yay
Harmful levels- only been shown to have a harmful effect in development
Again, avenue for research
Thank you so much to everyone who made this project and my being here possible, my amazing graduate student and mentor Marissa, Dan and all the members of the ABNEL Lab, Northeastern University’s colleges of science and engineering, and Northeastern’s Office of Undergraduate Research and Fellowships.
Thank you.
![Peripheral 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]
2
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](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)