The Effects of Ultrasound on Planarian Photoreceptor RegenerationANDREW TAGLE | ROSE-ANNE NICOLAS | JASMINE REYES | BEZA NEGUSSIE

Why this topic?◦Personal interests:

◦Diagnostic imaging

◦Ultrasound and skincare (Hissong, et. al)

◦ Application of ultrasound has shown:

◦ Accelerated muscle healing in rats (Montali et. al)

◦ Enhanced blood flow, increased membrane permeability, altered connective tissue permeability and nerve conduction in living tissue (Binder et. al)

◦ Faster bone regeneration in rabbits (Takebe et. al)

◦ Treatment of Alzheimer's in rats (Hynynen et. al)

Planaria

◦ Capable of regenerating lost body parts

◦ Pieces up to 1/279th of the organism can still regenerate a head

◦ Contain tissues and organs homologous to humans

◦ Model organism for investigating regeneration

Background

Morphological and Functional Recovery of the Planarian Photosensing System during Head Regeneration-Takeshi Inoue, et. Al

◦ Planaria exhibit negative phototaxis (light avoidance behavior)

◦ Intact planaria able to detect direction of light

◦ Decapitated planaria unable to recognize direction of light

Planaria Exhibit Negative Phototaxis by Day 5

Morphological and Functional Recovery of the Planarian Photosensing System during Head Regeneration-Takeshi Inoue, et. Al

QuestionWhat effect does ultrasound have on planaria

photoreceptor regeneration?

Previous Ultrasound Experiment◦ Applied ultrasound different days after cut◦ Concluded that applying ultrasound one day after decapitation was most beneficial

Ultrasound’s effects on planarian photoreceptor regeneration-Chan et., al

◦ Challenges: Budding

How can we stop budding?

◦Planaria will bud (fission) and split apart from each other

◦ Ran experiments to reduce budding

◦ Cut in two different areas

◦ Placed planaria in 100 mL of water or 200 mL of water

Tail Pieces Reduce Budding

Voltage Test

◦Find voltage to use for ultrasound application

◦3 Groups that receive 60 seconds of ultrasound◦200 mV (n=10)◦300 mV (n=10)◦400 mV (n=10)

◦ Timed how long it takes for each planaria to escape inner ring

Voltage Test Results

Voltage Test Results

Physical Differences

Ultrasound Application

◦ Planaria cut near tail to reduce budding 24 hours . . .◦ Planaria were treated with 200 mV of

ultrasound ~24 hours after being cut

◦ 3 groups (n=36)

◦ Control: No treatment (n=12)

◦ 1 treatment (n=12)

◦ 2 treatments (n=12)

◦ Proceed with phototaxis assay

How to Test Photoreceptor Function

◦ Planaria exhibit negative phototaxis ◦ Shadow box

◦ Acclimate planaria to dark ~15 minutes prior to testing

◦ Conducted in a dark room

◦ Test for 90 seconds

◦ Conduct assay on Day 0 and Day 3-Day 7 and on day 9

◦ Hoped to see difference between treated planaria and control.

Control Results

Shadow Box Results

*** ******

**

NS

***

= p < 0.001

= p < 0.01

NS = Non Significant

*****

Control vs. 1 US Treatment Results

Shadow Box Results

NS

NS

*** ***** *** ****** *** ***

*

**

**

***

= p < 0.001

= p < 0.01

NS = Non Significant

**

Control vs. 2 US Treatment Results

Shadow Box Results

NS NSNS

*** ****** ***

***

*** ***

****

***

= p < 0.001

= p < 0.01

NS = Non Significant

**

Summary ◦Using tail piece of planaria help reduce budding

◦Applying 2 doses of 200 mV of ultrasound at 30 minutes aparts improves photoreceptor regeneration

◦Takes ~7-9 days for tail piece to reach day 0 performance in phototaxis assay

Future Directions

◦More than 2 treatment groups

◦Run assay for a longer period (>90 seconds)

Acknowledgements

◦ Bryan White

◦ Pierre Mourad

◦ Cameron Whalen

◦ Ivan Owen

◦ Engineering program for sharing their lab with us

Questions?