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)

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

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

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

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

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

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.

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.

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.

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

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

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

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

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

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

Questions?