Transcript Resident Final Research Project Laser Facial Nerve Welding in a Rabbit Model Jason D.

Resident Final Research Project
Laser Facial Nerve Welding
in a Rabbit Model
Jason D. Bloom, M.D.
University of Pennsylvania
Department of Otorhinolaryngology:
Head & Neck Surgery
June 25th, 2009
Faculty Mentors: Noam A. Cohen, M.D. &
Stephen A. Goldstein, M.D.
Special Thanks: Benjamin S. Bleier, M.D.
Otorhinolaryngology: Head and Neck Surgery at PENN
Excellence in Patient Care, Education and Research since 1870
Research Approval & Funding
This research project
has been approved by
both the U of Penn
IACUC (Protocol #
802421) & Philadelphia
VA ACORP
Awarded 2009-2010
AAO-HNSF / AAFPRS
Leslie Bernstein
Resident Research
CORE Grant
Background
Charles Gould (1957) - Introduces the term
LASER (Light Amplification by Stimulated Emission
of Radiation)
–
In his doctoral thesis on energy levels of excited thallium
Sigel & Acevado (1962) - Reported thermal
energy in tissue adhesion
–
Passing high frequency electric current through coapted
vein edges to anastomose end-to-side porto-caval shunts
in dogs (1)
Yahr & Strully (1966) – First description of laser
tissue welding in blood vessel anastomosis (2)
–
Nd-YAG laser
Almquist et al. (1984) – Argon laser in peripheral
nerve repair (3)
Fischer et al. (1985) – Published positive reports
using the CO2 laser for rat nerve repair (4)
Background
Biologic Solder
Allows for target specific laser energy absorption resulting in tissue bonds
with greater tensile strength than wounds sealed with laser energy alone (5)
The use of biologic solders has been shown to provide an added benefit in
the promotion of native wound healing mechanisms.
– Contrast to the granulomatous inflammatory response seen with suture material
– The lased solder coagulum provides a non-immunogenic scaffold (6)
– Gradually absorbed during the normal wound healing process (7,8)
Biologic solders are now being combined with wavelength specific
chromophores (9,10)
- Increased target specific energy absorption
- Decreased thermal energy leakage
- Objective basis of gauging adequacy of laser welding by providing a predictable color
change
- Examples:
Carbon Black
808nm
Fluorescein Dye
532nm
Indocyanine Green Dye
805nm
Laser Tissue Welding
Laser
Biologic Solder
ECM REMODELING
LASER WELD
Chromophore
Background
Problems associated w/ classic neurorrhaphy
– Trauma induced by handling & sutures
– Neuroma formation
– Long OR times & technically difficult surgically
– “Leakage” of axons at site of anastomosis &
in-growth of connective tissue
– Foreign body reaction from suture material
Background
Laser Nerve Welding
Eppley et al. (1989) – Laser nerve repair of the
rabbit facial nerve with CO2 laser (11)
–
–
–
neuroma formation
connective tissue invasion
axonal proliferation or extravasation outside of epineurium
– Problems:
• Histopathologic weld analysis
• Demonstrated some deleterious effects of the heat from the laser,
such as destruction of myelin and loss of axons immediately adjacent
to the anastomotic site.
• Poor anastomosis tensile strength & dehiscence
Background
Laser Nerve Welding
Trickett et al. (1997) – Albumin based solder with
indocyanine green chromophore to laser weld rat
sciatic nerves with an 800nm diode laser (12)
- Laser activated solders = stronger bonds at the anastomosis
site & less thermal damage to underlying nerve tissue
- Histology = denatured solder and underlying epineurium with
no axonal damage
Hwang et al. (2005 & 2006) – CO2 laser welding of rat
facial nerve w/o solder (13,14)
- Laser repaired nerve regeneration = microsuture repaired
nerve regeneration  immmunohistochemical nerve tracer
- Less cellular & fibroblastic response
- Less scar & neuroma formation
Hypothesis
Laser welding of the rabbit facial nerve
provides a method of creating an
atraumatic, immediate and strong repair
bond capable of allowing axonal
regeneration across the injury site without
damage to the nerve itself or the
complications associated with suture
neurorrhaphy.
Null Hypothesis: Laser nerve welding is not
an effective way to perform neurorrhapy, as
compared to suture nerve repair.
Time Table
Day
Procedure
Data Point Functional
testing
Data Point –
EMG testing
0
Rabbit Acquisition
N/A
N/A
Data Point Histology (H&E and
Masson’s Trichrome
Stain)
N/A
4
Rabbit Acclimation
N/A
N/A
N/A
5
Survival Surgery/
Functional & EMG tests
Laser Weld x 6,
Suture x 6, Neg
Control x 2
Laser Weld x 6,
Suture x 6, Neg
Control x 2
N/A
33 (wk 4)
Functional & EMG tests/
Sac-Experimental Group
#1
Laser Weld x 6,
Suture x 6, Neg
Control x 2
Laser Weld x 6,
Suture x 6, Neg
Control x 2
Laser Weld x 2, Suture
x2
61 (wk 8)
Functional & EMG testing Laser Weld x 4,
Suture x 4, Neg
Control x 2
Laser Weld x 4,
Suture x 4, Neg
Control x 2
N/A
89 (wk 12)
Functional & EMG testing Laser Weld x 4,
Suture x 4, Neg
Control x 2
Laser Weld x 4,
Suture x 4, Neg
Control x 2
N/A
Laser Weld x 4,
Suture x 4, Neg
Control x 2
Laser Weld x 4, Suture
x 4, Neg Control x 2
117 (wk 16) Functional & EMG tests/
Sac-Experimental Group
#2
Laser Weld x 4,
Suture x 4, Neg
Control x 2
Rabbit Survival Surgery
14 New Zealand White
Rabbits (3-4kg)
– Housed at the Animal
Research Facility (ARF)
of the Philadelphia
Veterans Affairs Medical
Center (PVAMC)
2 of the rabbit facial nerves
(negative control group) = facial
nerves transected with a 1cm
piece of nerve excised w/o reanastomosis
6 of the rabbits facial nerves =
suture anastomosis w/ three 9-0
monofilament nylon sutures on an
atraumatic taper needle
6 of the rabbit facial nerves =
laser welded utilizing an 810nm
diode laser and a “ribbon” of 42%
albumin-based biologic solder
coupled with an indocyanine green
dye chromophore applied with a
27 G needle
Rabbit Facial Nerve Anatomy
OR Setup
Identifying the Facial Nerve
Hand-held nerve stimulator was used * Also, used to verify that the facial
to identify the facial nerve and
nerve was cut  no longer
verify facial movement.
innervating the face
Suture Neurorrhaphy
Suture anastomosis w/
three 9-0 monofilament
polypropylene (Prolene)
sutures on an atraumatic
taper needle
* Carl Zeiss OPMI CS-1
operating microscope
Laser/Solder Platform
Biologic solder 42% albumin solution
Indocyanine green dye
Hyaluronic acid sodium
Diode laser module
- Iridex Oculight SLx
Power: 0.5W
Pulse Duration: 0.5s
Pulse Interval: 0.1s
Power Density: 15.9W/cm2
Fluency: 8.0 J/cm2
Major Wavelength Output:
810 +/- 1nm
* 600mm core diameter quartzsilica fiberoptic cable
Laser Solder Neurorrhaphy
 Microvascular clip – used to
hold nerve ends in place
 Adequacy of weld was
visually assessed by the
green colored chromophore
changing to a brown color
s/p laser energy
Surgical Complications
Surgical Complications:
Sialocele
(5/14 rabbits = 35%)
* All treated w/ repeated 20G
needle aspirations  all
completely resolved in 2wks
* No pain, distress, feeding
issues, effect on wt. gain
EMG Testing
Nicolet Viking IV EMG
- subdermal electrodes
Nerve Conduction Study
- measuring CMAPs
- amplitude
- nerve latency
Functional / Clinical Testing
Normal Facial Nerve Function
Left Facial Nerve Paralysis
Positioning of the rabbits that allows
standardized visualization and video
recording of facial movements.
Gentle nasal & forehead taps performed in an
attempt to elicit facial nerve motion.
Video tapes will then be analyzed to
objectively grade facial nerve recovery by 2
different blinded observers.
Assess the rabbit's bilateral upper lip &
whisker movement.
A scale of facial nerve movement from 0-3 will
be used to record this functional testing with
0=no movement and 3=normal facial
movement (16).
Histological Testing
6 nerve repairs from each
experimental group were harvested
and fixed in 10% buffered formalin for
subsequent H&E staining
Analysis by our histopathologist
(Eugene Einhorn, MD) for immune
response to the solder, native wound
healing progression, and extent of
collateral nerve thermal injury.
Nerve Laser Weld (H&E 4x) – 4wks
* 16 week histological analysis = ongoing
(1) Axon counts growing across the nerve repair
site (2) Organization of the axons (3) Amount
of neuroma formation (4) Amount of foreign
body/granulomatous reaction
The welds were also be prepared
and stained with Masson’s Trichrome
by our histopathologist, in order to
view the axon counts and
arrangement of nerve axons across
the repair sites.
RESULTS
Operative Time
*
* P < 0.01
N=6
N=6
Learning Curve
EMG Nerve Recovery
Clinical Nerve Recovery
Problems with the Study
• Multiple buccal branches of the
rabbit facial nerve distal to the site
of the nerve injury & repair
• Small number of rabbits in each
group
• Inability to circumferentially laser
weld the nerve
• Inconsistent EMG electrode
placement in the facial
musculature for each separate
trial
• No adequate method to
measure synkinesis in a rabbit
model
•Testing EMG vs. ENoG
Discussion /
Future Directions
Further areas of research
with this laser welding
technology:
– Peripheral nerve repair
– Microvascular arterial or
venous anastomosis
– Pharyngocutaneous
fistula repair
– Transoral robotic
surgery models
Augmentation of the
biologic solder with
growth factors and
antibiotics to further
optimize wound healing
If these welds prove
efficacious in larger
trials, this technology
would be ideally
transitioned to clinical
trials of facial nerve
injury repair.
LNW Conclusions
Clinical / Functional:
― Improved facial nerve functional recovery on video analysis with the laser weld
group
Electrophysiological:
― Improved facial nerve electrophysiological/EMG nerve recovery at all time points
in the laser weld group, especially at early (1 month) & late (4 month) time points
Histological:
― H&E staining demonstrating negligible collateral thermal nerve or tissue damage
― Biologic solder supporting the progression of the native wound healing response
― Ongoing histopath: Utilizing Masson’s Trichrome stain  (1) Axon counts
growing across the nerve repair site (2) Organization of the axons (3) Amount of
neuroma formation (4) Amount of foreign body/granulomatous reaction
Mechanical:
― No learning curve or operating microscope necessary with the laser weld group
― Significantly reduced neurorrhaphy time with the laser weld group
References
1. Sigel B, Acevado FJ, “Vein anastomosis by electrocoaptive union,” Surg Forum. 1962;13:233-5.
2. Yahr WZ, Strully KJ, “Blood vessel anastamosis by laser and other biomedical applications,” J
Assoc Adv Med Instrum. 1966;(1):28-31.
3. Almquist EE, Nachemson A, Auth D, Almquist B, Hall S. “Evaluation of the use of the argon
laser in repairing rat and primate nerves,” J Hand Surg Am. 1984;9:792-299.
4. Fischer DW, Beggs JL, Kenshalo DJ, Shetter AG. “Comparative study of microepineurial
anastomoses with the use of CO2 laser and suture techniques in rat sciatic nerves: Part 1.
Surgical technique, nerve action potentials, and morphological studies,” Neurosurgery.
1985;17:300-308.
5. Gil Z, Shaham A, Vasilyev T, Brosh T, Forer B, Katzir A, Fliss DM. “Novel laser tissuesoldering technique for dural reconstruction,” J Neurosurg. 2005 Jul;103(1):87-91.
6. Kirsch AJ, Miller MI, Hensle TW, Chang DT, Shabsigh R, Olsson CA, Connor JP, “Laser tissue
soldering in urinary tract reconstruction: first human experience,” Urology. 1995 Aug;46(2):261-6.
7. Lauto A, Trickett R, Malik R, Dawes JM, Owen ER, “Laser-activated solid protein bands for
peripheral nerve repair: an vivo study,” Lasers Surg Med. 1997;21(2):134-41.
8. Lauto A, Kerman I, Ohebshalon M, Felsen D, Poppas DP, “Two-layer film as a laser soldering
biomaterial,” Lasers Surg Med. 1999;25(3):250-6.
9. Talmor M, Bleustein CB, Poppas DP, “Laser tissue welding: a biotechnological advance for the
future,” Arch Facial Plast Surg. 2001 Jul-Sep;3(3):207-13.
10. Oz MC, Johnson JP, Parangi S, Chuck RS, Marboe CC, Bass LS, Nowygrod R, Treat MR,
“Tissue soldering by use of indocyanine green dye-enhanced fibrinogen with the near infrared
diode laser,” J Vasc Surg. 1990 May;11(5):718-25.
11. Eppley BL, Kalenderian E, Winkelmann T, Delfino JJ. “Facial nerve graft repair: suture versus
laser-assisted anastomosis,” Int J Oral Maxillofacial Surg. 1989;18:50-54.
References
12. Trickett I, Dawes JM, Knowles DS, Lanzetta M, Owen ER. “In vitro Laser Nerve Repair:
Protein Solder Strip Irradiation or Irradiation Alone?,” Int Surg. 1997;82:38-41.
13. Hwang K, Kim SG, Kim DJ, Lee CH. “Laser Welding of Rat’s Facial Nerve,” Journal of
Craniofacial Surgery. 2005;16(6):1102-1106.
14. Hwang K, Kim SG, K DJ. “Facial-Hypoglossal Nerve Anastomosis Using Laser Nerve
Welding,” Journal of Craniofacial Surgery. 2006;17(4):687-691.
15. Yian CH, Paniello RC, Spector JG. “Inhibition of Motor Nerve Regeneration in a Rabbit Facial
Nerve Model.” Laryngoscope. 2001;111:786-791.
16. Bleier BS, Palmer JN, Sparano AM, et al. “Laser-assisted cerebrospinal fluid leak repair: an
animal model to test feasibility.” Otolaryngol Head Neck Surg. 2007;137(5):810-4.
17. Bleier BS, Palmer JN, Gratton MA, Cohen NA. “In Vivo Laser Tissue Welding in the Rabbit
Maxillary Sinus.” American Journal of Rhinology. 2008;22:625-628.
18. Bleier BS, Gratton MA, Leibowitz JM, Palmer JN, Newman JG, Cohen NA. “Laser-Welded
Endoscopic Endoluminal Repair of Iatrogenic Esophageal Perforation: An Animal Model.”
Otolaryngology – Head and Neck Surgery. 2008;139:713-717.
THANK YOU!
Special Thanks To:
- My Faculty Mentors
- Dr. O’Malley
- Dr. Cohen, Dr. Palmer, Dr. Bleier &
The Penn Rhinology Lab
- Perry DeRitis & VA ARF Staff
- VA Neurology Service