Use of Electrical Stimulation to Improve Nerve

Regeneration

Jan Nguyen, Claudia Wei, Jeff CoursenHieu Nguyen Group

April 2010

Hieu Data Blitz (04.29.2010)

Overview:

1.PPy conduit from TDA2.Fill with hydrogel3.Embed DRGs4.EF stimulation

Final semester project

DRG body

Final semester project

DRG bodyHydrogel

Final semester project

PPy conduitDRG

bodyHydrogel

Final semester project

PPy conduitDRG

bodyHydrogel

Electric field

• Why hydrogels? – Structural rigidity provides contact guidance for cell

adhesion & growth• Prevents conduit from collapsing

• Applications: o Allows DRGs to adhere to surface & not float in mediao Test how effective hydrogels are since goal is to have in body

• Provides 3D scaffoldo Representation of body o Allows for testing multiple layers of cells or organso Medium that serves as drug delivery device

PureCollagen

Matrigel Collagen gel with HEPES

Collagen gel with 10x DMEM

Collagen / HA hydrogel

[1] [2] [3] [4] [5]

PC12 cells in collagen hydrogel

• 1. Curt’s hydrogel– Pros:

• optimized in Schmidt’s lab to have effect on neurite extension• Can be put in final product human body

– Cons: • our success rate with this hydrogel < matrigel • Hydrogel’s current f(x) is to evaluate cell’s response to EF type doesn’t

matter

• 2. Matrigel– Pros:

• Know works in vitro b/c our success rate > other hydrogels• Preparing matrigel is not necessary

- Cons:- Can’t put into final product human body

• A. Silicone– Used as model with PC 12 cells

• B. PLGA from TDA– Used as model with PC 12 cells

• C. Ppy from TDA– Used in final project with explanted DRGs

Hydrogel

Silicone conduit

PC12 cells with NGF

• A. Cryostat Sectioning– i. fix – ii. freeze in Isopentane– iii. embed in OTC freezing medium– iv. section at -20C– v. sectioned at 40um and at 160um

http://www.bio.miami.edu/~cmallery/255/255hist/mcb5.43.microtome.jpg

http://www.chadie.de/cryotomee.jpg

Cryotome sectioning for imaging• cut thin slices to image cells inside opaque PPy

• B. Immunostaining – i. fix with 4% Paraformaldehyde– ii. Wash with PBS– iii. permeabilize with 0.02%

TritonX– iv. block with goat serum– v. stained with primary and

secondary antibodies

http://images.google.com/imgres?imgurl=http://www.abcam.com/ps/CMS/Images/immuno_staining2.jpg&imgrefurl=http://www.abcam.com/index.htmlhttp://images.google.com/imgres?imgurl=http://www.abcam.com/ps/CMS/Images/immuno_staining2.jpg&imgrefurl=http://www.abcam.com/index.htmlhttp://images.google.com/imgres?imgurl=http://www.abcam.com/ps/CMS/Images/immuno_staining2.jpg&imgrefurl=http://www.abcam.com/index.html

://www.abcam.com/index.html

• C. Imaging – Under fluorescent lighting

DRG in Ppy conduit

• Biomedical Applicationso Biosensor o Drug delivery device

• Useso Antistatic Coatingo Protective coating for photoelectrochemical cellso Battery

• Polypyrrole is a electrically conducting polymer

“Geetha, S. et al. "Biosensing and Drug Delivery by Polypyrrole." Analytica Chimica Acta (2006): 119-25. Web.

PPy was chosen for this study because it is1.Electrically conductive2.Easy to synthesize3.Flexible4.Shown to be biocompatible with mammalian cells5.Shown to promote neurite growth

Drawbacks1.NOT biodegradable2.Fragile and brittle

Huang, Yi-Cheng, and Yi-You Huang. "Biomaterials and Strategies for Nerve Regeneration." Artificial Organs 30.7 (2006): 514-22.

•Synthesized on stainless steel•Doped with PSS (oxidative doping)•0.7 mV at 40 min•Detox methods: prestimulation vs soaking

J.M. Sansiñena, V. Olazábal, T.F. Otero, C.N. Polo da Fonseca, M-A. De Paoli, Chem. Commun., (1997) 2217

Soak•Soak for 1 day in DI water

Prestimulation•0.1 mV per cm (0.2 mV total)•2hours

Prestimulation= best method of detoxification

Endogenous Electric Fields

• Transepithelial potential

• Break in epithelium allows low resistance pathway for ions

• Resulting Electric field: 100-1000 mV cm-1

(Nuccitelli, R. 2003. Endogenous Electric Fields in Embryos During Development, Regeneration, and Wound Healing. Radiation Protection Dosimetry Vol. 106, pp. 375-383)

Role of Endogenous Electric Fields

• Development• Wound Healing• Nerve regeneration• Cell migration• Mechanisms?

Song, Bing et al. Nerve regeneration and wound healing are stimulated and directed by endogenous electric field in vivo. Journal of Cell Sciences 117 (2004):4681-4690.

Stimulation of PPy in vitro

• Electrodes: alligator clips or gold-plated mini-clips

• Stimulation: 200 mV for two hours

• PPy resistance: 10-40 kΩ

• Media (F12K) resistance: 1500 kΩ

Stimulation of Conduits in vivo

• Puck is surgically implanted near injury site

• Magnetic coil induces current in puck

• Electrodes sutured to conduit

Confidential. Virginia Technologies Inc.

Using Magnetic Fields Directly

• Why? Less invasive• Extended nerve growth

found in direction of induced current

(Macias Y. Melissa, et al. Directed and Enhanced Nuerite Growth with Pulsed Magnetic Field Stimulation. Bioelectromagnetics 21 (2000):272-286.)

Research Fall 2010

• Research Fall 2010: Stimulate rat DRGs in PPy conduit with Hydrogel.

Final semester project

DRG bodyHydrogel

PPy conduit

Electric field

Experimental setup

PPy conduit6 uL gel 8 uL gel 6 uL gel

DRG level of media

200 mV0 mV

DRG in Matrigel - stimulated

DRG

PPy conduit extended neurites onto conduit

DRG in Matrigel - control (not stimulated)

DRG

PPy conduitextended neurites into hydrogel

into Hydrogel onto PPy surface

no neurites

Collagen control II IIIII

Collagen stim

Matrigel control I IIIIIII

Matrigel stim II III IIIII

Tally for neurite extension

Columns = where the neurite extended toRows = type of hydrogel, and EF stimulation of PPy or no stimulation (control)

Quick summary

• Matrigel performed better in neurite promotion and structural support (compared to Collagen gel)

• Need more samples to draw conclusive data• Do not use cryosectioning in order to preserve

gel structure

1. Song, Bing et al. Nerve regeneration and wound healing are stimulated and directed by endogenous electric field in vivo. Journal of Cell Sciences 117 (2004):4681-4690. 2.Hou, S. "The Repair of Brain Lesion by Implantation of Hyaluronic Acid Hydrogels Modified with Laminin." Journal of Neuroscience Methods 148 (2005): 60-70. Web.3. Kotwal, A., and CE Schmidt. "Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials." Biomaterials 22 (2001): 1055-064. Print.4. Macias Y. Melissa, et al. Directed and Enhanced Nuerite Growth with Pulsed Magnetic Field Stimulation. Bioelectromagnetics 21 (2000):272-286.)5. Nuccitelli, R. Endogenous Electric Fields in Embryos During Development, Regeneration, and Wound Healing. Radiation Protection Dosimetry Vol. 106 (2003): 375-383.6. Pearson, R. et al. "Spatial Confinement of Neurite Regrowth from Dorsal Root Ganglia within Nonporous Microconduits." Phillips, J. et al.. "A Self-Organizing Collagen Guidance Conduit." Tissue Engineering 11 (2005). Web.7. Vernitskaya, T. "Polypyrrole: a Conducting Polymer; Its Synthesis, Properties and Applications." Russian Chemical Reviews 66.5 (1997): 443-57. Web.

Literature Cited

AcknowledgementsWe would like to thank:• Dr. Schmidt- for granting us the opportunity to participate in

research • Hyma- for informing us with proper lab safety techniques and

procedures• Zin, Jae, Leo, and other grad students- for their wealth of

knowledge & advice• Hieu- for taking us under your wings and providing not only

bountiful knowledge, but guidance and support in our research endeavors