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Roles of NF-(B in Spinal Cord Injury Pathophysiology
by
Danny Salah Rafati
APPROVED BY THE SUPERVISORY COMMITTEE
________________________________
J. R. Perez-Polo, Ph.D.
________________________________
Marjorie R. Grafe, M.D., Ph.D.fillin \* mergeformat
________________________________
Norbert K. Herzog, Ph.D.
________________________________
Karin W. High, Ph.D.
________________________________
Claire E. Hulsebosch, Ph..D.
________________________________
Stuart A. Lipton, M.D., Ph. D.
________________________________
David J. McAdoo, Ph. D.
________________________________
Dean, Graduate School
Roles of NF-(B in Spinal Cord Injury Pathophysiology
by
Danny Salah Rafati, B.A.
DISSERTATION
Presented to the Faculty of the Graduate School of Biomedical Sciences at the University of Texas Medical Branch at Galveston,
in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
Approved by the Supervisory Committee
J. Regino Perez-Polo, Ph.D.
Marjorie R. Grafe, M.D. Ph. D.
Norbert K. Herzog, Ph..D.
Karin W. High, Ph.D.
Claire E. Hulsebosch, Ph.D.fillin \* mergeformat
Stuart A. Lipton, Ph.D.
David J. McAdoo, Ph.D.
September, 2005
Galveston, Texas
Key words: Decoy Oligonucleotides, Spinal Cord Injury, NF-(B
For Gregory Clark and Marianne Dauwalder
ACKNOWLEDGMENTS
I extend my sincerest and everlasting gratitude to my mentor, Dr. J. Regino Perez-Polo, for sharing his part of the universe with me. I would also like to thank members of my Supervisory Committee, Dr. Marjorie R. Grafe, Dr. Norbert K. Herzog, Dr. Karin W. High, Dr. Claire E. Hulsebosch, Dr. Stuart A. Lipton and Dr. David J. McAdoo, for sharing their minds and their laboratories.
Thanks to all my past and present friends and co-workers in Dr. Perez-Polos lab: Dr. Olivera Nesic-Taylor, Dr. Jingxin Qiu, Dr. Kasie Cole, Dr. Xiaoming Hu, Dr. Krystyn Bourne, Rachel Campbell, Diana Cittelly, Diana Ferrari, Martin Gill, Julieann Lee and Charmaine Rea. Special thanks to our lab manager, Karin Werrbach-Perez, and administrative guru, Donna Masters. Words cannot describe how all these people helped sustain me throughout the years.
Thanks are also due to Neuroscience Graduate Program Director Dr. James Blankenship and program coordinators Ms. Lonnell Simmons and Ms. Cynthia Cheatham for all the little things they do for us.
I would be remiss if I did not mention the guiding hands of Drs. Robert and Golda Leonard. They were rocks for me in my graduate career. Now they are lifelong friends.
I would especially like to thank the Rafati Family Dad, Mom, Bahige, Tarek, Dania, Zena, Halla, Mariam, Yasmene, Leenah and Noor and the Keck Family Ray, Patricia, Teresa and Lacey for their love, support, advice and encouragement.
And, finally, I would like to thank my wife, Joyce, for entering my life and promising to stay in it.
Roles of NF-(B in Spinal Cord Injury Pathophysiology
Publication No. ((((((
Danny Salah Rafati, Ph.D.
The University of Texas Medical Branch at Galveston, 2005
Supervisor: J. Regino Perez-Polo
The pathophysiology of spinal cord injury (SCI) is characterized by multiple locomotor and sensory deficits, as well as chronic pain arising partly from significant cell loss at and around the injury site. Implicated in multiple biochemical pathways affected after SCI is the transcription factor NF-(B. We used two-month-old male rats and subjected them to a contusion spinal cord injury to elucidate the differential changes in NF-(B subunits and NF-(B-dependent changes after SCI. We observed an increase in cell death 12 hours after SCI, with significant decreases in the neuronal cell marker, NeuN, at 2, 12 and 24 hours. We show significant and sustained protein level decreases in I(B family members, I(B-( and Bcl-3, suggesting early and prolonged activation of NF-(B up to 28 days after SCI. Validation of NF-(B activation was performed by immunohistochemical analyses of spinal cord cross sections and Western blot assay analyses. Immunohistochemical co-staining with NeuN and c-Rel antibodies demonstrated decreased neuronal c-Rel up to 24 hours after SCI. We also observed significant decreases in the protein levels NF-(B c-Rel up to 12 hours after SCI, signifying its inability to activate genes involved in anti-apoptotic mechanisms. NeuN co-localization with NF-(B p65 and p50 revealed neuronal increases up to 24 hours, especially in the nucleus of cells, supporting activation of NF-(B p50/p65 and p50/p65-induced apoptotic gene transcription after SCI. Western blots of p65 and p50 showed no changes in their protein levels, but whole tissue blots may dilute the immunohistochemical observations. Significant changes in NF-(B p52 and RelB were not observed, suggesting that they do not play a role in response to SCI. Genes dependent on NF-(B were also up-regulated after SCI, including Cox-2 and iNOS, important molecules in the pathophysiology of injury. We show increased protein expression of Cox-2 at 2 hours and of iNOS at 12 hours, the earliest time in which these proteins have been shown to be up-regulated after SCI. Inhibition of NF-(B p50/p65 was performed using decoy deoxyoligonucleotides containing the NF-(B consensus binding site to the Cox-2 promoter. The decoy treatment was successful in abrogating SCI-induced Cox-2 and iNOS protein increases. The decoys did not appear to significantly affect non-NF-(B-dependent gene transcription. Functional outcomes after SCI were also measured after decoy treatment. Improvements in locomotion and decreased mechanical hypersensitivity were observed. In summary, the following series of experiments were designed to test the hypothesis that NF-(B plays a central role in the pathophysiology of spinal cord injury.
TABLE OF CONTENTS
Page
LIST OF TABLES.IX
LIST OF FIGURESx
ABBREVIATIONSXII TOC \t "Heading 1,2,Chapter Title,1"
Chapter 1: INTRODUCTION
Models of SCI 1
Biochemistry of SCI 4
NF-(B Family of Proteins - General Overview 6
I(B Family of Proteins - General Overview 9
Decoy Deoxyoligonucleotides 11
Cyclooxygenase-2 and Inducible Nitrous Oxide Synthase 13
Hypothesis 18
CHAPTER 2: METHODS
Animals 19
Injury 19
Tissue Extraction 20
"Decoy" Deoxyoligonucleotide Preparation 20
Decoy Treatment 21
Immunocytochemistry 22
Protein Extraction 23
Protein Concentration Determination 24
RNA Isolation 24
DNA Microarray 25
Cell Death Detection ELISA Assay 27
Western Blot Assay Analysis 28
Electrophorectic Mobility Shift Assays (EMSA) 30
Locomotor Function Testing 31
Mechanical Stimuli Testing 31
Thermal Stimuli Testing 32
Statistical Analyses 33
CHAPTER 3: RESULTS
Spinal Cord Injury Causes Cell Death 34
SCI Causes Decreases In Cytoplasmic I( B - a n d B c l - 3 3 7
S C I T r i g g e r s D e c r e a s e d E x p r e s s i o n o f N F - ( B c - R e l 4 0
S C I T r i g g e r s I n c r e a s e d E x p r e s s i o n o f N F - ( B p 6 5 4 3
S C I T r i g g e r s I n c r e a s e d E x p r e s s i o n o f N F - ( B p 5 0 4 6
S C I D o e s N o t A l t e r E x p r e s s i o n o f N F - ( B p 5 2 o r R e l - B 4 9
S C I S t i m u l a t e s N F - ( B p 5 0 / p 6 5 - D e p endent Inflammatory Gene Expression 52
NF-(B Decoys Penetrate Into Injured Spinal Cord 54
Cox-2 Promoter NF-(B Decoys Bind Specifically to p50/p65 56
Cox-2 Promoter NF-(B Decoy Treatment After SCI Abrogates Cox-2 Protein Expression 59
Cox-2 Promoter NF-(B Decoy Treatment After SCI Affects Other Known NF-(B-Dependent Targets 60
Cox-2 Promoter NF-(B Decoy Treatment Improves Functional Measures After SCI 62
CHAPTER 4: DISCUSSION 68
REFERENCE liST .93
VITA111
LIST OF TABLES
PAGE
TABLE 1. Immunohistochemical Assessment of NF-(B Changes after SCI...79
LIST OF FIGURES
PAGE
TOC \h \z \t "Heading 8" \c HYPERLINK \l "_Toc106598341" Figure 1. The NF-(B and I(B family of Proteins. 9
HYPERLINK \l "_Toc106598342" Figure 2. Formation of Reactive Oxygen Species via Reduction of Iron (Fe2+) or Oxidation of Nitric Oxide 14
HYPERLINK \l "_Toc106598343" Figure 3. Schematic Pathway for NF-(B Mediated Effects after SCI 17
Figure 4. Spinal Cord Cell Death Levels a f t e r C o n t u s i o n I n j u r y i n R a t & & & & & & & . . . 3 5
H Y P E R L I N K \ l " _ T o c 1 0 6 5 9 8 3 4 5 " F i g u r e 5 . N e u N S t a i n i n g o f S p i n a l C o r d N e u r o n s a n d P r o t e i n L e v e l s a f t e r S C I . 3 6
H Y P E R L I N K \ l " _ T o c 1 0 6 5 9 8 3 4 6 " F i g u r e 6 . S p i n a l C o r d C y t o p l a s m i c I ( B - P r o t e i n L e v e l s a f t e r S C I . 38
HYPERLINK \l "_Toc106598347" Figure 7. Spinal Cord Cytoplasmic Bcl-3 Protein Levels after SCI. 39
HYPERLINK \l "_Toc106598348" Figure 8. Double Immunofluorescent Staining of NF-(B c-Rel and NeuN after SCI. 41
HYPERLINK \l "_Toc106598349" Figure 9. Spinal Cord Nuclear NF-(B c-Rel Protein Levels after SCI. 42
HYPERLINK \l "_Toc106598350" Figure 10. Spinal Cord Nuclear and Cytoplasmic NF-(B c-Rel Protein Levels after SCI 43
HYPERLINK \l "_Toc106598351" Figure 11. Double Immunofluorescent Staining of NF-(B p65 and NeuN after SCI 45
HYPERLINK \l "_Toc106598352" Figure 12. Spinal Cord Nuclear and Cytoplasmic NF-(B p65 Protein Levels after SCI. 46
HYPERLINK \l "_Toc106598353" Figure 13. Double Immunofluorescent Staining of NF-(B p50 and NeuN after SCI 48
HYPERLINK \l "_Toc106598354" Figure 14. Spinal Cord Nuclear and Cytplasmic NF-(B p50 Protein Levels after SCI. 49
HYPERLINK \l "_Toc106598355" Figure 15. Double Immunofluorescent Staining of NF-(B p52 and NeuN after SCI. 50
HYPERLINK \l "_Toc106598356" Figure 16. Double Immunofluorescent Staining of NF-(B RelB and NeuN after SCI 51
HYPERLINK \l "_Toc106598357" Figure 17. Cox-2 mRNA Expression after SCI 52
HYPERLINK \l "_Toc106598358" Figure 18. Cox-2 Protein Expression after SCI 53
HYPERLINK \l "_Toc106598359" Figure 19. iNOS Protein Expression after SCI. 55
HYPERLINK \l "_Toc106598360" Figure 20. Distribution of Decoy Deoxyoligonucleotides after Injection into Injured Spinal Cord 56
HYPERLINK \l "_Toc106598361" Figure 21. Effects of Cox-2 Decoy on NF-(B Activation after SCI. 57
HYPERLINK \l "_Toc106598362" Figure 22. Binding Specificity of Cox-2 Decoys to NF-(B p50/p65. 59
HYPERLINK \l "_Toc106598363" Figure 23. Cox-2 Decoy effects on Cox-2 Protein Expression after SCI 59
HYPERLINK \l "_Toc106598364" Figure 24. Cox-2 Decoy effects on iNOS Protein Expression after SCI 60
HYPERLINK \l "_Toc106598363" Figure 25. Cox-2 Decoy effects on I( B - P r o t e i n E x p r e s s i o n a f t e r S C I 6 2
H Y P E R L I N K \ l " _ T o c 1 0 6 5 9 8 3 6 3 " F i g u r e 2 6 . C o x - 2 D e c o y e f f e c t s o n I ( B - P r o t e i n E x p r e s s i o n a f t e r S C I 6 3
H Y P E R L I N K \ l " _ T o c 1 0 6 5 9 8 3 6 4 " F i g u r e 2 7 . L o c o m o t o r R e c o v e r y a f t e r 1 5 0 k D y n e S C I i n C o x - 2 D e c o y - T r e a t e d R a t s 64
HYPERLINK \l "_Toc106598363" Figure 28. Locomotor Recovery after 200 kDyne SCI in Cox-2 Decoy-Treated Rats 65
HYPERLINK \l "_Toc106598364" Figure 29. Effects of Decoys on Mechanical Sensitization after SCI 66
HYPERLINK \l "_Toc106598364" Figure 30. Schematic pathway for NF-(B medeiated effects after SCI. 92
ABBREVIATIONS
ANOVA Analysis of variance
BBB Basso, Beattie and Bresnahan
BCA Bicinchronic acid
CNS Central nervous system
Cox Cyclooxygenase
d Day
DTT Dithiothreitol
ECL Enhanced chemiluminescence lighting system
EMSA Electromobility shift assay
eNOS Endothelial nitric oxide synthase
FDR False discovery rate
h Hour
IH Infinite Horizon
iNOS Inducible nitric oxide synthase
min Minute
MnSOD Manganese superoxide dismutase
NF-(B Nuclear Factor-(B
nNOS Neuronal nitric oxide synthase
NO Nitric oxide
NOS Nitric oxide synthase
NYU New York University
OD Optical density
PMSF Phenylmethylsulfonylfluoride
PNS Peripheral nervous system
RNS Reactive nitrogen species
ROS Reactive oxygen species
SAM Statistical analysis of microarrays
SEM Standard error of the mean
SCI Spinal cord injury
Chapter 1: INTRODUCTION
Spinal cord injury (SCI) is caused by any one of a myriad of destructive forces to the spinal cord, resulting in a pathophysiology characterized by multiple locomotor and sensory deficits, in addition to altered nociception and hyperalgesia. The Spinal Cord Injury Information Network ADDIN REFMGR.CITE 2005167Spinal Cord Injury Information NetworkInternet Communication167Spinal Cord Injury Information Network2005Spinal CordSpinal Cord InjuriesinjuriesNot in FileUniversity of Alabama at Birmingham.Facts and figures at a glance.June, 2005http://www.spinalcord.uab.edu/show.asp?durki=21446University of Alabama at Birmingham.Facts and figures at a glance.June, 200535(2005) reports an estimated 11,000 new cases per year in the United States, with a predicted total patient population of 250,000. Medical and technological advances over the past few decades have greatly improved both the survival rate and the survival time of SCI patients. Moreover, quality of life has also improved as a result of these advances, allowing for life-spans approaching those of the non-injured population. Despite the promising progress, there remains a pressing need for more effective treatment strategies. Since the highest percentage of injuries manifest in individuals between the ages of 16-30, victims of an SCI must deal with a plethora of injury related problems early in life and for many years after.
Models of SCI
Rats are both cost effective and have established paradigms for use in SCI studies. Several injury models exist for the systematic study of SCI, including contusions, compressions, ischemia, crush injuries, transections and hemisections ADDIN REFMGR.CITE Mautes2000197Vascular events after spinal cord injury: contribution to secondary pathogenesisJournal197Vascular events after spinal cord injury: contribution to secondary pathogenesisMautes,A.E.Weinzierl,M.R.Donovan,F.Noble,L.J.2000/7anatomy & histologyArteriesblood supplycomplicationsenzymologyetiologyEvolutionHemorrhageHumansInflammationinjuriesmetabolismMetalloendopeptidasesphysiopathologyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesNot in File673687Phys.Ther.807Neurosurgical Research Laboratory, Saarland University Medical School, Homburg, GermanyPM:10869130Phys.Ther.1(Mautes et al., 2000). Each attempts to mimic certain aspects of injury and the specific biochemical and behavioral outcomes seen after. Two widely used paradigms are the hemisection and contusion models employed on rats.
The hemisection model allows for precise, consistent and reproducible severing of the spinal cord. It permits the study of a number of events after SCI, including loss of specific functions and, especially, central chronic pain ADDIN REFMGR.CITE Hains200138Reduction of pathological and behavioral deficits following spinal cord contusion injury with the selective cyclooxygenase-2 inhibitor NS-398Journal38Reduction of pathological and behavioral deficits following spinal cord contusion injury with the selective cyclooxygenase-2 inhibitor NS-398Hains,B.C.Yucra,J.A.Hulsebosch,C.E.2001/4Amino AcidsanalysisAnimalsantagonists & inhibitorsBehavior,AnimalcomplicationsContusionsCyclooxygenase InhibitorsDinoprostonedrug effectsetiologyExcitatory Amino AcidsFree RadicalsHeatHindlimbHyperalgesiaHypesthesiaImmunohistochemistryInflammationIsoenzymesLipid PeroxidationMalemetabolismMotor ActivityNitrobenzenesPainpathologypharmacologyPhysical StimulationphysiopathologyProstaglandin-Endoperoxide SynthasepsychologyRatsRats,Sprague-DawleyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesSulfonamidesNot in File409423J.Neurotrauma184Department of Anatomy and Neurosciences and Marine Biomedical Institute, University of Texas Medical Branch, Galveston 77555-1069, USAPM:11336442J.Neurotrauma1Hulsebosch2000104Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentinJournal104Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentinHulsebosch,C.E.Xu,G.Y.Perez-Polo,J.R.Westlund,K.N.Taylor,C.P.McAdoo,D.J.2000/12Acetic AcidsAminesAnalgesicsAnimalsBehavior,AnimalChronic DiseaseContusionsCyclohexanecarboxylic Acidsdrug effectsdrug therapygamma-Aminobutyric AcidHeatMaleMotor ActivityPainPain MeasurementPain ThresholdPhysical StimulationphysiopathologypsychologyRatsRats,Sprague-DawleyReaction TimeResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord Injuriestherapeutic useTime FactorsNot in File12051217J.Neurotrauma1712Marine Biomedical Institute, University of Texas Medical Branch, Galveston 77555-1069, USA. cehulseb@utmb.eduPM:11186233J.Neurotrauma1Mills2000149AIDA reduces glutamate release and attenuates mechanical allodynia after spinal cord injuryJournal149AIDA reduces glutamate release and attenuates mechanical allodynia after spinal cord injuryMills,C.D.Xu,G.Y.Johnson,K.M.McAdoo,D.J.Hulsebosch,C.E.2000/9/28Animalsantagonists & inhibitorscomplicationsdrug effectsdrug therapyetiologyExcitatory Amino Acid AntagonistsGlutamic AcidHyperalgesiaIndansinjuriesMaleMechanoreceptorsmetabolismMicrodialysisNeuronspathologypharmacologyPhysical StimulationphysiopathologyRatsRats,Sprague-DawleyReceptors,Metabotropic GlutamateResearch Support,Non-U.S.Gov'tSpinal CordSpinal Cord InjuriesNot in File30673070Neuroreport1114The Department of Anatomy and Neurosciences, The University of Texas Medical Branch at Galveston, 77555-1069, USAPM:11043525Neuroreport1Mills2001148Strain and model differences in behavioral outcomes after spinal cord injury in ratJournal148Strain and model differences in behavioral outcomes after spinal cord injury in ratMills,C.D.Hains,B.C.Johnson,K.M.Hulsebosch,C.E.2001/8AnimalsBehavior,AnimalComparative StudyDisease Models,AnimalHeatHyperalgesiainjuriesMaleMotor ActivityPainPhysical StimulationphysiologyphysiopathologyRatsRats,Long-EvansRats,Sprague-DawleyRats,WistarResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Species SpecificitySpinal CordSpinal Cord InjuriesNot in File743756J.Neurotrauma188Department of Anatomy and Neurosciences. University of Texas Medical Branch at Galveston, 77555-1043, USAPM:11526981J.Neurotrauma1(Hains et al., 2001; Hulsebosch et al., 2000; Mills et al., 2001; Mills et al., 2000). Indeed, the consistency of pain formation appears to be greatest in this model of SCI ADDIN REFMGR.CITE Mills2001148Strain and model differences in behavioral outcomes after spinal cord injury in ratJournal148Strain and model differences in behavioral outcomes after spinal cord injury in ratMills,C.D.Hains,B.C.Johnson,K.M.Hulsebosch,C.E.2001/8AnimalsBehavior,AnimalComparative StudyDisease Models,AnimalHeatHyperalgesiainjuriesMaleMotor ActivityPainPhysical StimulationphysiologyphysiopathologyRatsRats,Long-EvansRats,Sprague-DawleyRats,WistarResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Species SpecificitySpinal CordSpinal Cord InjuriesNot in File743756J.Neurotrauma188Department of Anatomy and Neurosciences. University of Texas Medical Branch at Galveston, 77555-1043, USAPM:11526981J.Neurotrauma1(Mills et al., 2001). It is an injury similar to that caused by stab wounds, gunshots and other penetrating injuries to the spinal cord. Aside from its consistency in producing central chronic pain, hemisection has several distinct advantages over the contusion model. There is no need for complex surgical equipment such as a contusion device, providing for procedural ease. Also with hemisection there is no need for manual bladder expression or prolonged antibiotic treatment. Despite these advantages, the hemisection model is limited because penetrating injuries to the spinal cord represent a minority of total SCI cases and are therefore less clinically relevant.
Most SCIs are caused by vertebral subluxation; vertebrae and discs shift, crushing the spine. Most often the vertebrae realign after injury, although their position is not stable. The resulting injury is akin to a bruise of the spinal cord. The contusion model more closely reproduces this injury. It has been in use since it was originally described in 1911 ADDIN REFMGR.CITE Allen1911198Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal cloumn: preliminary reportJournal198Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal cloumn: preliminary reportAllen,A.R.1911Spinal CordinjuriesNot in File878JAMA57JAMA1Allen1914199Remarks in histopathological changes in spinal cord due to impact: an experimental studyJournal199Remarks in histopathological changes in spinal cord due to impact: an experimental studyAllen,A.R.1914Not in File141147J.Nerv Ment Dis41J.Nerv Ment Dis1(Allen, 1911; Allen, 1914). This weight drop method, with certain modifications to produce a controlled, reproducible injury, continues to be used today ADDIN REFMGR.CITE Dohrmann1972200Experimental spinal cord trauma. A historical reviewJournal200Experimental spinal cord trauma. A historical reviewDohrmann,G.J.1972/12AnimalsCatsDisease Models,AnimalDogsHaplorhinihistoryHistory,19th CenturyHistory,20th CenturypathologyRabbitsRatsSpinal CordSpinal Cord InjuriesUnited StatesNot in File468473Arch.Neurol.276PM:4628439Arch.Neurol.1Dohrmann1972201Research in experimental spinal cord trauma: past and present--a brief reviewJournal201Research in experimental spinal cord trauma: past and present--a brief reviewDohrmann,G.J.Wick,K.M.1972/12pathologyResearchSpinal CordSpinal Cord InjuriesNot in File115124J.Neurosurg.Nurs.42PM:4485981J.Neurosurg.Nurs.1Lifshutz2004202A brief history of therapy for traumatic spinal cord injuryJournal202A brief history of therapy for traumatic spinal cord injuryLifshutz,J.Colohan,A.2004/1/15Anesthesia,GeneralAnimalsEgyptGreecehistoryHistory,15th CenturyHistory,16th CenturyHistory,17th CenturyHistory,18th CenturyHistory,19th CenturyHistory,20th CenturyHistory,21st CenturyHistory,AncientHistory,MedievalHumansinjuriesMilitary MedicineNeurologyNeurosurgeryNeurosurgical ProceduresPortraitsPrognosisRabbitsResearch Support,Non-U.S.Gov'tSpinal CordSpinal Cord InjuriestherapyTractionNot in FileE5Neurosurg.Focus.161Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. jlifshutz@neuroscience.mcw.eduPM:15264783Neurosurg.Focus.1(Dohrmann, 1972; Dohrmann and Wick, 1972; Lifshutz and Colohan, 2004). Animals injured with such devices exhibit various degrees of bilateral paralysis and sensation loss, as well as changes in nociceptive and hyperalgesic states.
One of the more recent well-characterized contusion devices, the New York University (NYU) impactor, has aided in providing a standard model for developing optimal therapies for treating SCI ADDIN REFMGR.CITE Constantini199495The effects of methylprednisolone and the ganglioside GM1 on acute spinal cord injury in ratsJournal95The effects of methylprednisolone and the ganglioside GM1 on acute spinal cord injury in ratsConstantini,S.Young,W.1994/1AnimalsAnnexinsantagonists & inhibitorsBody WaterBody WeightCentral Nervous Systemchemistrydrug effectsDrug Synergismdrug therapyG(M1) GangliosideHematocritHyponatremiaLaminectomyMalemetabolismMethylprednisolonepathologypharmacologyphysiopathologyPotassiumprevention & controlRatsReference ValuesResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,Non-P.H.S.Research Support,U.S.Gov't,P.H.S.SodiumSpinal CordSpinal Cord InjuriesNot in File97111J.Neurosurg.801Department of Neurosurgery, New York University Medical Center, New YorkPM:8271028J.Neurosurg.1Gruner199237A monitored contusion model of spinal cord injury in the ratJournal37A monitored contusion model of spinal cord injury in the ratGruner,J.A.1992AnimalsContusionsDisease Models,AnimalpathologyRatsSpinal CordSpinal Cord InjuriesNot in File123126J.Neurotrauma92Department of Neurosurgery, New York University Medical Center, New YorkPM:1404425J.Neurotrauma1(Constantini and Young, 1994; Gruner, 1992). It led to the first methodical analysis of locomotor, histological and anatomical outcomes after reproducible SCI ADDIN REFMGR.CITE Basso1996150Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transectionJournal150Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transectionBasso,D.M.Beattie,M.S.Bresnahan,J.C.1996/6AnimalsBehavior,AnimalContusionsDisease Models,AnimalFemaleHindlimbHistocytochemistryinjuriesLocomotionMalepathologyphysiologyphysiopathologyRatsResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesNot in File244256Exp.Neurol.1392Ohio State University, Columbus, Ohio 43210, USAPM:8654527Exp.Neurol.1(Basso et al., 1996), along with various biochemical and interventional studies ADDIN REFMGR.CITE Bennett199984NGF levels decrease in the spinal cord and dorsal root ganglion after spinal hemisectionJournal84NGF levels decrease in the spinal cord and dorsal root ganglion after spinal hemisectionBennett,A.D.Taglialatela,G.Perez-Polo,R.Hulsebosch,C.E.1999/3/17AnimalsbloodCordotomyEnzyme-Linked Immunosorbent AssayGanglia,SpinalLateralityMalemetabolismNerve Growth FactorsphysiologyRatsRats,Sprague-DawleyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesNot in File889893Neuroreport104Marine Biomedical Institute and Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77555-1069, USAPM:10208566Neuroreport1Chu200215Delayed cell death signaling in traumatized central nervous system: hypoxiaJournal15Delayed cell death signaling in traumatized central nervous system: hypoxiaChu,D.Qiu,J.Grafe,M.Fabian,R.Kent,T.A.Rassin,D.Nesic,O.Werrbach-Perez,K.Perez-Polo,R.2002/2AnimalsAnoxiaApoptosisblood supplyCell DeathCentral Nervous SystemCerebrovascular AccidentDeathgeneticsIschemiaNecrosisNeuronsphysiologyphysiopathologyResearch Support,Non-U.S.Gov'tSignal TransductionSpinal CordSpinal Cord InjuriesTime FactorsIn File97106Neurochem.Res.271-2Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, USAPM:11926281Neurochem.Res.1Hulsebosch2000104Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentinJournal104Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentinHulsebosch,C.E.Xu,G.Y.Perez-Polo,J.R.Westlund,K.N.Taylor,C.P.McAdoo,D.J.2000/12Acetic AcidsAminesAnalgesicsAnimalsBehavior,AnimalChronic DiseaseContusionsCyclohexanecarboxylic Acidsdrug effectsdrug therapygamma-Aminobutyric AcidHeatMaleMotor ActivityPainPain MeasurementPain ThresholdPhysical StimulationphysiopathologypsychologyRatsRats,Sprague-DawleyReaction TimeResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord Injuriestherapeutic useTime FactorsNot in File12051217J.Neurotrauma1712Marine Biomedical Institute, University of Texas Medical Branch, Galveston 77555-1069, USA. cehulseb@utmb.eduPM:11186233J.Neurotrauma1Nesic200214DNA microarray analysis of the contused spinal cord: effect of NMDA receptor inhibitionJournal14DNA microarray analysis of the contused spinal cord: effect of NMDA receptor inhibitionNesic,O.Svrakic,N.M.Xu,G.Y.McAdoo,D.Westlund,K.N.Hulsebosch,C.E.Ye,Z.Galante,A.Soteropoulos,P.Tolias,P.Young,W.Hart,R.P.Perez-Polo,J.R.2002/5/15administration & dosageanalysisAnimalsantagonists & inhibitorsCluster AnalysisContusionsDizocilpine MaleateExcitatory Amino Acid AntagonistsFluorescent Antibody TechniqueGene Expression ProfilinggeneticsInflammationInjections,SpinalMalemetabolismNeuroprotective AgentsOligonucleotide Array Sequence AnalysispharmacologyRatsRats,Sprague-DawleyReceptors,N-Methyl-D-AspartateResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.RNA,MessengerSpinal CordSpinal Cord InjuriesIn File406423J.Neurosci.Res.684Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0652, USA. Olnesic@utmb.eduPM:11992467J.Neurosci.Res.1Qiu200116Bcl-xL expression after contusion to the rat spinal cordJournal16Bcl-xL expression after contusion to the rat spinal cordQiu,J.Nesic,O.Ye,Z.Rea,H.Westlund,K.N.Xu,G.Y.McAdoo,D.Hulsebosch,C.E.Perez-Polo,J.R.2001/11AnimalsApoptosisbiosynthesisBlotting,WesterncomplicationsContusionsDnaDNA FragmentationetiologyFluorescent Antibody Technique,DirectgeneticsHistonesLocomotionMalemetabolismMicroscopy,ConfocalNecrosisNeuronsPainpathologyphysiologyphysiopathologyProteinsProto-Oncogene ProteinsProto-Oncogene Proteins c-bcl-2RatsRats,Sprague-DawleyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesSpinothalamic TractsIn File12671278J.Neurotrauma1811Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, USAPM:11721745J.Neurotrauma1(Bennett et al., 1999; Chu et al., 2002; Hulsebosch et al., 2000; Nesic et al., 2002; Qiu et al., 2001b). However the NYU device can cause confounding secondary injury due to impactor rebound. This secondary injury can be avoided with the Infinite Horizon (IH) impactor. The IH impactor also allows the user to control such parameters as the level of force administered and the dwell time of the impactor probe. Altogether, the IH system provides for a single unit with the capacity for a dynamic range of injuries ADDIN REFMGR.CITE Scheff2003125Experimental modeling of spinal cord injury: characterization of a force-defined injury deviceJournal125Experimental modeling of spinal cord injury: characterization of a force-defined injury deviceScheff,S.W.Rabchevsky,A.G.Fugaccia,I.Main,J.A.Lumpp,J.E.,Jr.2003/2AdultAginganalysisAnimalsDisease Models,AnimalEquipment DesignetiologyFemaleinstrumentationMotor ActivityNeurologypathologyphysiopathologyRatsRats,Sprague-DawleyResearch Support,Non-U.S.Gov'tSpinal CordSpinal Cord InjuriesNot in File179193J.Neurotrauma202The Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40536-0230, USA. ssheff@uky.eduPM:12675971J.Neurotrauma1(Scheff et al., 2003).
Regardless of contusion injury model, the rats require a large amount of post-operative care, including, but not limited to, twice daily manual bladder expression and prolonged antibiotic treatment at least until bladder function returns. Prior to receiving an SCI the rats must undergo a dorsal laminectomy at the desired level. This is in contrast to most human spinal injuries, in which significant cord compression via swelling may result in the confines of the vertebral canal. Despite these demands and limitations, we use a contusion injury with the IH impactor model in all experiments in order to characterize most accurately the pathophysiology of SCI.
Biochemistry of Spinal Cord Injury
The primary trauma of SCI compresses neural tissue, damages blood vessels, disrupts axons, and ruptures cell membranes. Within minutes the spinal cord swells, secondary ischemia ensues, autoregulation of blood flow ceases, and spinal neurogenic shock leads to systemic hypotension ADDIN REFMGR.CITE McDonald200212Spinal-cord injuryJournal12Spinal-cord injuryMcDonald,J.W.Sadowsky,C.2002/2/2AdolescentAdultElectric StimulationetiologyFemaleHumansMaleNerve RegenerationphysiologyphysiopathologyrehabilitationResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesIn File417425Lancet3599304Department of Neurology, Spinal Cord Injury Neuro-Rehabilitation Section, and Restorative Treatment and Research Program, Washington University School of Medicine, St Louis, MO 63110, USA. mcdonald@neuro.wustl.eduPM:11844532Lancet1McDonald200212Spinal-cord injuryJournal12Spinal-cord injuryMcDonald,J.W.Sadowsky,C.2002/2/2AdolescentAdultElectric StimulationetiologyFemaleHumansMaleNerve RegenerationphysiologyphysiopathologyrehabilitationResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesIn File417425Lancet3599304Department of Neurology, Spinal Cord Injury Neuro-Rehabilitation Section, and Restorative Treatment and Research Program, Washington University School of Medicine, St Louis, MO 63110, USA. mcdonald@neuro.wustl.eduPM:11844532Lancet1(McDonald and Sadowsky, 2002). Damage to, or killing of, neighboring cells ensues due to events triggered by the early responses, which includes ischemia (and hypoxia), release of neurotransmitters in toxic amounts (e.g. glutamate) from cells, and electrolyte imbalances, notably Na+ and Ca2+ ADDIN REFMGR.CITE McDonald200212Spinal-cord injuryJournal12Spinal-cord injuryMcDonald,J.W.Sadowsky,C.2002/2/2AdolescentAdultElectric StimulationetiologyFemaleHumansMaleNerve RegenerationphysiologyphysiopathologyrehabilitationResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesIn File417425Lancet3599304Department of Neurology, Spinal Cord Injury Neuro-Rehabilitation Section, and Restorative Treatment and Research Program, Washington University School of Medicine, St Louis, MO 63110, USA. mcdonald@neuro.wustl.eduPM:11844532Lancet1(McDonald and Sadowsky, 2002).
A distinctive feature of SCI is a wave of secondary injury that lasts up to four weeks after the initial insult and spreads to four or more spinal cord segments away from the lesion locus ADDIN REFMGR.CITE Basso1996150Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transectionJournal150Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transectionBasso,D.M.Beattie,M.S.Bresnahan,J.C.1996/6AnimalsBehavior,AnimalContusionsDisease Models,AnimalFemaleHindlimbHistocytochemistryinjuriesLocomotionMalepathologyphysiologyphysiopathologyRatsResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesNot in File244256Exp.Neurol.1392Ohio State University, Columbus, Ohio 43210, USAPM:8654527Exp.Neurol.1Beattie200081Review of current evidence for apoptosis after spinal cord injuryJournal81Review of current evidence for apoptosis after spinal cord injuryBeattie,M.S.Farooqui,A.A.Bresnahan,J.C.2000/10AnimalsApoptosisAstrocytesBrainBrain InjuriesCell DeathDeathHumansIschemiametabolismMicrogliaNecrosisNerve DegenerationNeuronspathologyphysiologyphysiopathologySignal TransductionSpinal CordSpinal Cord InjuriesNot in File915925J.Neurotrauma1710Department of Neuroscience, Ohio State University Medical Center, Columbus, USA. beattie.2@osu.eduPM:11063057J.Neurotrauma1(Basso et al., 1996; Beattie et al., 2000). There is a continued, delayed cell death that augments dysfunction and interferes with recovery due to injury-triggered inflammatory responses. The cell death seen displays both necrotic and apoptotic phenotypes. While necrotic cell death typifies cellular fates at the injury locus, a significant amount of apoptotic cell death occurs in the peri-lesion areas. Necrotic cell death is caused by massive energy failure and induction of inflammation. In contrast, apoptosis is an energy-requiring, controlled cell death with typical morphological hallmarks, including cell shrinkage, membrane blebbing, nuclear condensation and DNA fragmentation ADDIN REFMGR.CITE Chu200215Delayed cell death signaling in traumatized central nervous system: hypoxiaJournal15Delayed cell death signaling in traumatized central nervous system: hypoxiaChu,D.Qiu,J.Grafe,M.Fabian,R.Kent,T.A.Rassin,D.Nesic,O.Werrbach-Perez,K.Perez-Polo,R.2002/2AnimalsAnoxiaApoptosisblood supplyCell DeathCentral Nervous SystemCerebrovascular AccidentDeathgeneticsIschemiaNecrosisNeuronsphysiologyphysiopathologyResearch Support,Non-U.S.Gov'tSignal TransductionSpinal CordSpinal Cord InjuriesTime FactorsIn File97106Neurochem.Res.271-2Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, USAPM:11926281Neurochem.Res.1(Chu et al., 2002). These dual cell death phenomena are analogous to that seen in hypoxia/ischemia in the central nervous system (CNS) ADDIN REFMGR.CITE Chu200215Delayed cell death signaling in traumatized central nervous system: hypoxiaJournal15Delayed cell death signaling in traumatized central nervous system: hypoxiaChu,D.Qiu,J.Grafe,M.Fabian,R.Kent,T.A.Rassin,D.Nesic,O.Werrbach-Perez,K.Perez-Polo,R.2002/2AnimalsAnoxiaApoptosisblood supplyCell DeathCentral Nervous SystemCerebrovascular AccidentDeathgeneticsIschemiaNecrosisNeuronsphysiologyphysiopathologyResearch Support,Non-U.S.Gov'tSignal TransductionSpinal CordSpinal Cord InjuriesTime FactorsIn File97106Neurochem.Res.271-2Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, USAPM:11926281Neurochem.Res.1(Chu et al., 2002).
Another aspect of secondary cell injury involves the release of excessive amounts of glutamate from dying neurons ADDIN REFMGR.CITE Panter1990151Alteration in extracellular amino acids after traumatic spinal cord injuryJournal151Alteration in extracellular amino acids after traumatic spinal cord injuryPanter,S.S.Yum,S.W.Faden,A.I.1990/1Amino AcidsanalysisAnimalsAspartic AcidCentral Nervous SystemExcitatory Amino Acidsgamma-Aminobutyric AcidGlutamatesGlycineinjuriesMalemetabolismMicrodialysisNeurologyRabbitsResearch Support,U.S.Gov't,Non-P.H.S.Research Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesNot in File9699Ann.Neurol.271Department of Neurology, University of California, San FranciscoPM:2301932Ann.Neurol.1(Panter et al., 1990), resulting in excitatory amino acid toxicity ADDIN REFMGR.CITE Nicotera1999156Excitotoxins in neuronal apoptosis and necrosisJournal156Excitotoxins in neuronal apoptosis and necrosisNicotera,P.Lipton,S.A.1999/6Amino AcidsAnimalsApoptosisCell DeathDeathdrug effectsExcitatory Amino AcidsHumansinjuriesNecrosisNeurodegenerative DiseasesNeuronspathologyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.toxicityNot in File583591J.Cereb.Blood Flow Metab196Department of Molecular Toxicology, Faculty of Biology, University of Konstanz, GermanyPM:10366188J.Cereb.Blood Flow Metab1Nicotera1997157Neuronal necrosis and apoptosis: two distinct events induced by exposure to glutamate or oxidative stressJournal157Neuronal necrosis and apoptosis: two distinct events induced by exposure to glutamate or oxidative stressNicotera,P.Ankarcrona,M.Bonfoco,E.Orrenius,S.Lipton,S.A.1997AnimalsApoptosisdrug effectsGlutamic AcidHumansMitochondriaNecrosisNeuronsNitric OxideOxidative StressphysiologyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.StresstoxicityultrastructureNot in File95101Adv.Neurol.72Institute of Environmental Medicine, Karolinska Institute, Stockholm, SwedenPM:8993688Adv.Neurol.1Gillessen2002153Excitatory amino acid neurotoxicityJournal153Excitatory amino acid neurotoxicityGillessen,T.Budd,S.L.Lipton,S.A.2002AnimalsAspartic AcidBrainBrain InjuriesBrain IschemiaCalciumchemistrycytologyEpilepsygeneticsGlutamic AcidHumansmetabolismMitochondriaNervous System DiseasesNeurodegenerative DiseasespathologypharmacologyReactive Nitrogen SpeciesReactive Oxygen SpeciesReceptors,GlutamateSignal TransductiontoxicityNot in File340Adv.Exp.Med.Biol.513Institut fuer Pharmakologie und Toxikologie, Bereich Studien und Wissenachaft, Neuherbergstrasse 11, 80937 Muenchen, GermanyPM:12575816Adv.Exp.Med.Biol.1(Gillessen et al., 2002; Nicotera et al., 1997; Nicotera and Lipton, 1999). A positive feedback loop of evoked Na+ and Ca2+ depolarization ensues, and further glutamate is released ADDIN REFMGR.CITE Regan199118Glutamate neurotoxicity in spinal cord cell cultureJournal18Glutamate neurotoxicity in spinal cord cell cultureRegan,R.F.Choi,D.W.1991Animalsantagonists & inhibitorsCell DeathCells,CulturedcytologyDeathDextromethorphanDextrorphandrug effectsGlutamatesGlutamic AcidGlycineN-MethylaspartateNeuronspharmacologyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesIn File585591Neuroscience432-3Department of Neurology, Stanford University School of Medicine, CA 94305PM:1681469Neuroscience1Choi199219Excitotoxic cell deathJournal19Excitotoxic cell deathChoi,D.W.1992/11Amino AcidsAnimalsBrainCalciumCell DeathcytologyDeathGlutamatesGlutamic AcidHumansModels,NeurologicalNeuronsphysiologyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriesIn File12611276J.Neurobiol.239Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110PM:1361523J.Neurobiol.1Liu199920Neurotoxicity of glutamate at the concentration released upon spinal cord injuryJournal20Neurotoxicity of glutamate at the concentration released upon spinal cord injuryLiu,D.Xu,G.Y.Pan,E.McAdoo,D.J.1999Amino AcidsAnimalsAspartic Acidblood supplychemically inducedContusionsGlutamic AcidMalemetabolismMicrodialysisNerve DegenerationNeuronsNeurotoxinspathologyRatsRats,Sprague-DawleyResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Spinal CordSpinal Cord InjuriestoxicityIn File13831389Neuroscience934Department of Neurology, University of Texas Medical Branch, Galveston 77555, USAPM:10501463Neuroscience1(Choi, 1992; Liu et al., 1999; Regan and Choi, 1991). Inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF- ) a l s o p l a y s i g n i f i c a n t r o l e s i n n e u r o d e g e n e r a t i o n a f t e r S C I A D D I N R E F M G R . C I T E <