1. E.L.E.C.T.R.O.D.E.
Evaluating Linear-Radial Electrode Conformations for Tissue Repair and Optimizing a Device for Experimentation
Team Members: Sagah Ahmed, Natalie Anzures, Zachary Bosley, Brendan Bui, Ariana Feizi, Sudabeh Jawahery, Courtney Koenig, Katherine Lakomy, Megan Lin, Poorna Natarajan, Eisha Nathan, Hiba Sayed, Eduardo Solano
Mentor: Dr. John Fisher
Poorna (1-5)

2. Introduction The Problem Groups of Interest Prevalence of Diabetes
Formation of Diabetic Ulcers
Issues with Current Treatments
Individuals with Diabetes
Medical Researchers
Healthcare Professionals
The problem:
 -Prevalence: In 2010, 1.9 million Americans over the age of 20 were diagnosed with either Type I or Type II diabetes. (1)
 -Diabetic ulcers occur in approximately 15% of patients with diabetes and are the leading cause of hospitalizations among diabetic patients. (2,3)
-Formation: Diabetic ulcers tend to develop because of diabetic neuropathy or nerve damage, which can lessen the body’s ability to feel pain and other sensations. They grow due to poor circulation and narrowing of blood vessels in the foot or leg, lack of healing/tissue repair resulting in high risks of infection and slow healing rates of ulcers6.
-Issues with current treatments: require constant application by patient, don’t directly target the wound, often have side effects such as little/too much drainage, allergic reactions, increase in necrotic tissue
People who care:
those who need treatment, those developing the treatment, those who treat

3. Wound Healing and Diabetic Ulcers
Endogenous wound healing
Angiogenesis and inflammation
Diabetic ulcers and inflammation
-Body’s natural bioelectric healing system: Human epithelial tissue maintains a natural trans-epithelial potential (TEP), which ranges anywhere between 10mV to 60mV; this naturally occurring electric potential across skin layers makes wound sites positive with respect to their surroundings25. This implies that electric fields might be responsible for the directionality of cell migration upon wounding and might play a key role in natural wound healing.
-Wound Healing and Electrical Stimulation: Electrical stimulation, when applied to wounds, can increase the production of vascular endothelial growth factor (VEGF) in the skin tissue surrounding the wound which directly correlates to wound repair29. Application of a direct-current electrical field to a wound has been found to induce cell growth and migration of cells towards the wound perimeter30. This is due to changes in membrane potentials of cells which release VEGF, which in turn cause changes in the cell’s Rho-ROCK and PI3K-Akt signal transduction pathways. INCREASE angiogenesis

4. Electrical Stimulation and Wound Healing
Deprived cell migration
Electric
Field
Body’s response to electrical stimulation
Cell migration
Proliferation
Electrical stimulation and angiogenesis
VEGF, bFGF
-Body’s natural bioelectric healing system: Human epithelial tissue maintains a natural trans-epithelial potential (TEP), which ranges anywhere between 10mV to 60mV; this naturally occurring electric potential across skin layers makes wound sites positive with respect to their surroundings25. This implies that electric fields might be responsible for the directionality of cell migration upon wounding and might play a key role in natural wound healing.
-Wound Healing and Electrical Stimulation: Electrical stimulation, when applied to wounds, can increase the production of vascular endothelial growth factor (VEGF) in the skin tissue surrounding the wound which directly correlates to wound repair29. Application of a direct-current electrical field to a wound has been found to induce cell growth and migration of cells towards the wound perimeter30. This is due to changes in membrane potentials of cells which release VEGF, which in turn cause changes in the cell’s Rho-ROCK and PI3K-Akt signal transduction pathways. INCREASE angiogenesis
Limited angiogenesis

5. Specific Aims
Specific Aim 1: To examine the effects of an optimized linearly applied electric field on cell proliferation, VEGF expression, bFGF expression and cell migration rates Specific Aim 2: To examine the effects of an optimized radially applied electric field on cell proliferation, VEGF expression, bFGF expression and cell migration rates Specific Aim 3: To examine the effects of an optimized (either radially or linearly) applied electric field on cell proliferation, VEGF expression, bFGF expression and cell migration rates in vivo
-say what’s on slide

6. Specific Aims 1 and 2: In Vitro Experiments Rationale
Verifies that electrical stimulation can promote angiogenesis
Provides preliminary data to build in vivo device
Rat aortic endothelial cells will be grown in Dulbecco’s Modified Eagle Medium with 10% Fetal Bovine Serum

7. Specific Aim 1: In Vitro Experiment Linear Electric Field Apparatus
Coat electrodes with high- purity titanium
Induces constant electric field at all points
Connected to signal generator
Sudi (6-8)
Experimental set-up of a linearly applied electric field. The anode and cathode are 1.5-in apart.

8. Specific Aim 2: In Vitro Experiment Radial Electric Field Apparatus
Outer electrode made from polyethylene
Inner electrode made with commercial titanium rod
Non-constant electric field
Analyze effects on angiogenic factors in different areas
Experimental set-up of a radially applied electric field. The outer hoop is positively charged, whereas the inner cylindrical electrode is negatively charged.

9. Specific Aims 1 and 2: In Vitro Experiments Experimental Design
Pulsed monophasic current at (ideally) 0.1 V, 15 µA and 50 Hz
Applied for 30 minute periods, once per day for 2 days
Five control cultures will not receive any electrical stimulation
Pulsed monophasic current.

10. Specific Aims 1 and 2: In Vitro Experiments Cell Analysis
Cell Proliferation
Counting using hemocytometer
Measure growth factor expression (VEGF, bFGF)
ELISA
qPCR
Immunohistochemistry
Western Blotting
Cell Migration
Microscopy

11. Specific Aims 1 and 2: In Vitro Experiments Success Criterion
Significant increase in cell migration
Increased migration is associated with increased healing rates
Significant increase in cell proliferation
Increased proliferation is associated with increased healing rates
Increase in angiogenic growth factors and expression
Increased growth factors are associated with increased reparative cell activity

12. Specific Aim 3: In Vivo Experiments Diabetic Rat Model
Induce wounds in diabetic rats
Use best electrode setup determined from in vitro trials to treat wounds
Set-up will be tested on 4- 8 week old Sprague- Dawley rats
-diabetic rats
-how to induce ulcer
-4 groups
-the experimental control group will be treated with parameters used in a device that uses a multi-electrode assay for the treatment of pressure ulcers
-The electrical stimulation will be applied for 30 minutes, five days a week for a period of two weeks. Before each trial, the electrodes will be placed on sterile pads cleaned with saline solution. The electrodes will then be strapped tightly onto the subject in such a way as to both prevent electrode displacement and to position the anode directly above the wound with the cathode placed towards the neuraxis7.
Arbitrary waveform generator
Provides flexibility in our design without rebuilding multiple times
Manipulates voltage, amplitude, frequency, wave type

13. Prototype Design Model off of in vivo device
Build cheap and small prototype for a more marketable device
Battery/Power Cable
Sudi (13-15)
-main goal: build a prototype with the best parameters determined from in vivo trials!
- We believe our planned electrical prototype will successfully administer electrical impulses to the diabetic ulcer sites according to the optimal voltage and placement of electrodes determined in the experimental trials. This device will hopefully be a model for a more marketable device that allows for convenient electrical stimulation for diabetic ulcer patients.
Electrodes

14. Present at senior thesis conference
Projected Timeline
Spring 2012
Lab training
Finish proposal
HHMI grant
In vitro devices
Fall 2012
In vitro testing
In vivo device
Junior Colloquia
IACUC approval
NSF grant
Spring 2013
In vivo testing
Begin prototype
ACCIAC grant
Fall 2013
Start thesis
Finish prototype
Spring 2014
Present at senior thesis conference

15. Housing and Care for Rats
Projected Budget
Categories
Expense (dollars)
Cell Culture
800
Electrode Materials 25
Circuitry
180
In vivo Model
9420
Housing and Care for Rats
200
Cell Assay Supplies
2000
TOTAL
12,625
-diabetic rats
-how to induce ulcer
-4 groups
-the experimental control group will be treated with parameters used in a device that uses a multi-electrode assay for the treatment of pressure ulcers
-The electrical stimulation will be applied for 30 minutes, five days a week for a period of two weeks. Before each trial, the electrodes will be placed on sterile pads cleaned with saline solution. The electrodes will then be strapped tightly onto the subject in such a way as to both prevent electrode displacement and to position the anode directly above the wound with the cathode placed towards the neuraxis7.
Arbitrary waveform generator
Provides flexibility in our design without rebuilding multiple times
Manipulates voltage, amplitude, frequency, wave type

16. Questions?