DTIC ADA421543: 810 NM Light Treatment of Acute Spinal Cord Injury Alters the Immune Response and Improves Axonal Regeneration and Functional Recovery

154 Pages·1.5 MB·English

by  Defense Technical Information Center

Report Documentation Page Form Approved
OMB No. 0704-0188
Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and
maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,
including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington
VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it

does not display a currently valid OMB control number.
1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
2003 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
810 NM LIGHT TREATMENT OF ACUTE SPINAL CORD INJURY
5b. GRANT NUMBER
ALTERS THE IMMUNE RESPONSE AND IMPROVES AXONAL
REGENERATION AND FUNCTIONAL RECOVERY 5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Byrnes /Kimberly R.
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Uniformed Service University of the Health Sciences REPORT NUMBER
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
Spinal cord injury (SCI) results in substantial and often permanent impairment of function due to the lack
of regeneration of damaged axons. Despite vigorous research, no cure for SCI has been found. Light
therapy (LT), through the absorption of light by target tissue, improves healing in a number of injury
models. However, no study to date has assessed the ability of LT to facilitate the regeneration of specific
spinal cord tracts. Our hypothesis was that transcutaneous application of 810 nm light promotes axonal
regeneration and functional reinnervation following transection of the corticospinal tract (CST) by
changing the extracellular milieu of the spinal cord. Three studies were implemented to investigate this
hypothesis. First, anterograde and retrograde tract tracing techniques were used to investigate axonal
regrowth after SCI and LT. LT (810 nm) was applied at the site of acute injury to the CST of adult rats.
Anterograde tract tracing demonstrated that LT improved axonal regrowth after injury, with significant
increases in axon number (199 +/- 12) and distance of regrowth (8.7 +/- 0.8 mm) as compared to controls
(p<0.01). Double label retrograde tract tracing revealed that transected axons regrew and reinnervated
motor neurons in the lumbar spinal cord in the light treated group only (p<0.05). Functional analyses
revealed that this regeneration was coupled with significant improvement in 2 tests of CST performance,
angle of rotation and ladder beam cross time (p<0.05). Second, to explore the effect of LT on the spinal
cord cellular environment, we investigated the inflammatory response after SCI, using quantitative
immunohistochemistry techniques. This study revealed that LT suppressed the invasion/activation of
macrophages, microglia and T lymphocytes after SCI (p<0.001) and delayed the activation of astrocytes.
The third study explored gene expression after SCI and LT. A number of cytokines and chemokines were
assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of interleukin 6,
monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS) was suppressed
at 6 hours post-injury by LT (p<0.01). These results demonstrate that LT has an anti-inflammatory effect
on the spinal cord after injury and significantly improves axonal regeneration and functional recovery.
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON

a. REPORT b. ABSTRACT c. THIS PAGE SAR 153
unclassified unclassified unclassified
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
COPYRIGHT STATEMENT

The author hereby certifies that the use of any copyrighted material in the dissertation
manuscript entitled:

“810 nm Light Treatment of Acute Spinal Cord Injury Alters the Immune Response and
Improves Axonal Regeneration and Functional Recovery”

is appropriately acknowledged and, beyond brief excerpts, is with the permission of the
copyright owner.

Kimberly R. Byrnes
Neuroscience Program
Uniformed Services University of the Health Sciences
ii
ABSTRACT

810 NM LIGHT TREATMENT OF ACUTE SPINAL CORD INJURY ALTERS THE
IMMUNE RESPONSE AND IMPROVES AXONAL REGENERATION AND
FUNCTIONAL RECOVERY

Kimberly R. Byrnes
Directed by Juanita J. Anders, Ph.D., Associate Professor of Anatomy, Physiology, and
Genetics, and Neuroscience

Spinal cord injury (SCI) results in substantial and often permanent impairment of
function due to the lack of regeneration of damaged axons. Despite vigorous research, no
cure for SCI has been found. Light therapy (LT), through the absorption of light by
target tissue, improves healing in a number of injury models. However, no study to date
has assessed the ability of LT to facilitate the regeneration of specific spinal cord tracts.
Our hypothesis was that transcutaneous application of 810 nm light promotes axonal
regeneration and functional reinnervation following transection of the corticospinal tract
(CST) by changing the extracellular milieu of the spinal cord. Three studies were
implemented to investigate this hypothesis. First, anterograde and retrograde tract tracing
techniques were used to investigate axonal regrowth after SCI and LT. LT (810 nm) was
applied at the site of acute injury to the CST of adult rats. Anterograde tract tracing
demonstrated that LT improved axonal regrowth after injury, with significant increases in
axon number (199 +/- 12) and distance of regrowth (8.7 +/- 0.8 mm) as compared to
iii
controls (p<0.01). Double label retrograde tract tracing revealed that transected axons
regrew and reinnervated motor neurons in the lumbar spinal cord in the light treated
group only (p<0.05). Functional analyses revealed that this regeneration was coupled
with significant improvement in 2 tests of CST performance, angle of rotation and ladder
beam cross time (p<0.05). Second, to explore the effect of LT on the spinal cord cellular
environment, we investigated the inflammatory response after SCI, using quantitative
immunohistochemistry techniques. This study revealed that LT suppressed the
invasion/activation of macrophages, microglia and T lymphocytes after SCI (p<0.001)
and delayed the activation of astrocytes. The third study explored gene expression after
SCI and LT. A number of cytokines and chemokines were assessed using reverse
transcriptase-polymerase chain reaction (RT-PCR). Expression of interleukin 6,
monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS)
was suppressed at 6 hours post-injury by LT (p<0.01). These results demonstrate that LT
has an anti-inflammatory effect on the spinal cord after injury and significantly improves
axonal regeneration and functional recovery.
iv
810 NM LIGHT TREATMENT OF ACUTE SPINAL CORD INJURY ALTERS
THE IMMUNE RESPONSE AND IMPROVES AXONAL REGENERATION AND
FUNCTIONAL RECOVERY

By
Kimberly R. Byrnes

Dissertation submitted to the faculty of the
Program in Neuroscience of the
Uniformed Service University of the Health Sciences
In partial fulfillment of the requirements for the degree of
Doctor of Philosophy 2003
v
ACKNOWLEDGEMENTS

I would first and foremost like to thank Dr. Juanita J. Anders, my advisor and
mentor over these past 6 years. Without her help, guidance, and friendship, this thesis
would not have been possible. I’ve learned an immeasurable amount about science,
academics and research. Thank you.

I would also like to thank all of the members of my committee, Drs. Rosemary
Borke, Sonia Doi, Howard Bryant, Leslie McKinney and Ronald Waynant. Without their
advice, time and commitment, I would not have been able to see this project through to
its conclusion. Their effort in helping me to complete this journey has been inspiring.

I owe an enormous thank you to the many people over the years that helped me
with the actual workload of this project. For his patience, countless answers to my
countless questions, and the amount of laser information that I never knew I’d need to
learn, I thank Dr. Ilko Ilev. To Xingjia Wu, Kimberly Smith, Lauren Barna, Reed
Heckert, Amy Van Horn, Heather Gerst, and Dr. Holly Nash, thank you for your hours
upon hours of hard work for caring about my project.

I would like to acknowledge the faculty and staff of the Neuroscience Program
and the Graduate Education Office, specifically Drs. Cinda Helke, Michael Sheridan and
Regina Armstrong and Ms. Janet Anastasi, for encouraging me to become involved in the
vi
graduate education process on more than one level and helping me to learn more than I
ever thought I would in graduate school.

I would also like to acknowledge the advice and support of several faculty and
staff of USUHS, including Drs. Diane Borst, Michelle Chenault, Cathy Joswick, Joe
McCabe, Harvey Pollard and Linda Porter. And I’d like to thank those outside of
USUHS who also supported my journey, Drs. David Baxter, Laure Haak, Leonardo
Longo and Jan Tunér.

I am extremely grateful to the friends that I have made along the way. Without
their support, guidance and advice, I would not be where I am today. I would be remiss
if I did not specifically acknowledge Ashley Poindexter, Tara Romanczyk, Alisa
Schaefer and Sharon Dominick for their love and encouragement.

Finally, I thank my parents, Rose and Randolph Byrnes, for their never ending
support and love. Thank you for giving me a chance to make you proud. I still owe you
a beach house, but hopefully this will hold you till then.
vii
TABLE OF CONTENTS

APPROVAL SHEET i
COPYRIGHT STATEMENT ii
ABSTRACT iii
TITLE PAGE v
ACKNOWLEDGMENTS vi
TABLE OF CONTENTS viii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS xiii
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LIGHT PROMOTES REGENERATION AND FUNCTIONAL
RECOVERY AFTER SPINAL CORD INJURY
Title Page 24
Introduction 24
Results/Conclusions 25
Methods 31
Figures 35

CHAPTER 3: LIGHT ALTERS CELLULAR INVASION AND ACTIVATION
FOLLOWING SPINAL CORD INJURY
Title Page 40
viii
Acknowledgements 41
Abstract 42
Introduction 43
Methods 46
Results 50
Discussion 56
Tables/Figures 64
CHAPTER 4: LIGHT SUPPRESSES CYTOKINE AND CHEMOKINE GENE
EXPRESSION AFTER SPINAL CORD INJURY
Title Page 70
Abstract 72
Introduction 74
Material and Methods 77
Results 81
Discussion 84
Acknowledgements 90
Tables/Figures 91
CHAPTER 5: DISCUSSION 96
REFERENCES 106
APPENDIX 132
ix