Neuroinflammatory Alterations via CD-36 in Traumatic Brain Injury

134 Pages·2015·2.85 MB·English

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USF Tampa Graduate Theses and Dissertations USF Graduate Theses and Dissertations
January 2015
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Diana G. Hernandez-Ontiveros
University of South Florida, [email protected]
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Neuroinflammatory Alterations via CD-36 in Traumatic Brain Injury

by

Diana G. Hernandez-Ontiveros

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy of Medical Sciences
Department of Molecular Pharmacology and Physiology
with a Concentration in Neurosciences
Morsani College of Medicine
University of South Florida

Major Professor: Paula C. Bickford, Ph.D.
Andreas G. Seyfang, Ph.D.
Dominic P. D’Agostino, Ph.D.
Daniel Kay-Pong Yip, Ph.D.

Date of Approval:
July 8, 2015

Keywords: Fatty acid translocase, inflammation, oxidized low density lipoprotein, macrophages,
soluble receptor of advanced end glycation
Copyright © 2015, Diana G. Hernandez-Ontiveros
DEDICATION
I would like to dedicate this work to my parents and family members who have been a
propelling force and anchor throughout my entire professional education in the USA. In addition
to my parents, Francisco and Carmen, I would like to also give recognition to my grandparents,
Paca and Rogelio, for their love, and for playing an active role in my education, and
encouragement to pursue my academic goals. At the same time, to my brother Cesar for his
friendship, constant protection and optimism since we left our hometown. I am deeply thankful
to my husband Anthony, and the “Russo-Scaglione clan” for their love, affection, help, and
motivation to continue moving forward with my life aspirations. I am very grateful and fortunate
for being able to find friends who have helped me along the road: “Muchísimas gracias Coqui
P., el Deland G., el Kentucky G., Amanda G., Drashti, and M. Li.” My profound admiration and
appreciation to all these significant people in my life, who in one way or another have
contributed to my professional education.
ACKNOWLEDGMENTS
I would like to state my sincere gratitude to my mentor Dr. Paula C. Bickford, for allowing
me to learn under her mentorship, for her continuous advice and patience to mentor me
throughout my graduate studies in order to piece together this dissertation and doctorate
degree. This academic endeavor was possible through a common effort from many
collaborators. Thus, I would like to acknowledge every lab member with whom I have teamed up
over the past five years to acquire research experience in the field of neuroscience. Their
contribution has been very valuable in all activities related to this dissertation project. I would
like to thank: faculty, post-docs, graduate and undergraduate students, USF lab technicians and
staff, and all volunteers who have dedicated their time and effort to help complete my
dissertation, in particular Arum Yoo, Lyanne M. Suarez, Jenny Kim, Christian Cerecedo, Daniela
Aguirre Raigoza, Diego Lozano, and Stephanny Reyes. My special thanks to Dr. Mibel Pabon,
Dr. Jared Ehrhart, Dr. Josh Morganti, Dr. Naoki Tajiri, Dr. Byeong Cha, and Amanda Garces for
being there throughout my graduate school, for cheering and encouraging me to improve myself
in my studies and scientific skills. Finally, I particularly would like to thank all faculty members of
my committee: Dr. Dominic P. D’Agostino, Dr. Andreas G. Seyfang, Dr. Daniel K.P. Yip, and Dr.
Stanley M. Stevens Jr., who kindly steered me in the right direction with their useful criticism in
order to improve my knowledge in this field and the quality of this dissertation. My special
thanks to Dr. Eric S. Bennett and Dr. Christopher C. Combie for their support and concern on
the final stages of my dissertation. All of these individuals inspire me to continue expanding my
scientific knowledge and my research abilities in the medical and neurosciences fields.
i

TABLE OF CONTENTS

List of Figures ............................................................................................................................ iv

Abstract...................................................................................................................................... vi

Chapter 1: Introduction ................................................................................................................ 1
1.1 Traumatic Brain Injury (TBI) ....................................................................................... 1
1.1.1 Societal Burden Costs ............................................................................... 10
1.1.2 Gaps in Basic Science Knowledge of TBI ................................................. 10
1.1.3 Gaps in Translational and Clinical Knowledge in TBI ................................ 11
1.2 Cell Death Mechanisms Associated with Secondary Damage in TBI ...................... 13
1.2.1 Neuroinflammation .................................................................................... 17
1.3 CD-36, Fatty Acid Translocase ................................................................................ 21
1.4 Role of CD-36 in Atherosclerosis ............................................................................ 22
1.5 Role of CD-36 in Stroke .......................................................................................... 24
1.6 Role of CD-36 in TBI ............................................................................................... 27
1.7 Role of CD-36 in Other Neurodegenerative Diseases ............................................. 29
1.7.1 Alzheimer’s Disease .................................................................................. 29
1.7.2 Cerebral Amyloid Angiopathy .................................................................... 31
1.7.3 Parkinson’s Disease ............................................................................................ 32

Chapter 2: Materials and Methods ............................................................................................ 34
2.1 Animals ................................................................................................................... 35
2.2 Surgical Procedures ............................................................................................... 35
2.3 Histology.................................................................................................................. 36
2.4 Tissue Collection for Protein Analysis ...................................................................... 37
2.5 Immunohistochemistry ............................................................................................. 38
2.5.1 CD-36/MCP-1 immunohistochemistry in Brain .......................................... 38
2.5.2 CD-36/Iba-1 immunohistochemistry in Brain ............................................. 38
2.5.3 CD-36/NeuN immunohistochemistry in Brain ............................................ 39
2.5.4 CD-36/GFAP immunohistochemistry in Brain ............................................ 39
2.5.5 CD-36 immunohistochemistry in Spleen .................................................... 40
2.6 Laser Scanning Confocal Microscopy ..................................................................... 40
2.7 Fluorescencent Microscopy ..................................................................................... 40
2.8 Statistical Analysis on Indirect Immunofluorescence in Brain .................................. 41
2.9 Statistical Analysis on Fluorescence Intensity in Spleen .......................................... 41
2.10 Tissue Processing ................................................................................................ 41
2.11 Western Blot ......................................................................................................... 42
2.12 Statistical Analysis on Western Blot ...................................................................... 42
2.13 Cell Culture ........................................................................................................... 43
2.13.1 Measurement of Cell Viability: Calcein-AM Fluorescence Dye ................ 43
2.13.2 Measurement of Mitochondrial Activity: MTT Assay ................................ 44
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2.14 Preliminary in vitro Experiments............................................................................. 44
2.14.1 In Vitro study CD-36 and sRAGE expression .......................................... 44
2.14.2 CD-36, Spleen and TBI ........................................................................... 44
2.14.3 TBI and CD-36 Immune Response in Neonatal Rats .............................. 45
2.14.4 sRAGE Modulates CD-36 Expression in Neonatal Spleen and
Brain after TBI ........................................................................................ 45

Chapter 3: Inflammatory Role of CD-36 in an Animal Model of TBI .......................................... 46
3.1 Introduction ........................................................................................................... 46
3.1.1 Why CD-36 as a Biomarker of Inflammation? ........................................... 46
3.1.2 Inflammatory Pathways Related to CD-36 ................................................. 47
3.1.3 Neuroinflammation in TBI Accompanied by CD-36 Expression in
Brain and Spleen ...................................................................................... 48
3.2 Specific Materials and Methods ............................................................................. 49
3.2.1 Surgical Procedures ................................................................................. 49
3.2.2 Immunohistochemistry of Brain ................................................................ 50
3.2.3 Immunohistochemistry of Spleen ............................................................. 51
3.2.4 Statistical Analysis on Indirect Immunofluorescence in Brain .................... 52
3.2.5 Statistical Analysis of Fluorescence Intensity in Spleen ............................ 53
3.2.6 Western Blot ............................................................................................. 53
3.2.7 Statistical Analysis on Western Blot .......................................................... 54
3.3 Results .................................................................................................................. 55
3.3.1 CD-36 Expression in the TBI Brain ........................................................... 55
3.3.2 CD-36 in the TBI Spleen .......................................................................... 61
3.3.3 Western Blot ............................................................................................ 67
3.3.3.1 Protein Detection in the Brain Cortex ......................................... 67
3.3.3.2 Protein Detection in the Spleen ................................................. 67
3.4 Discussion ............................................................................................................. 72

Chapter 4: Pharmacological Interventions Designed to Abrogate TBI Related Inflammation
May Improve Functional Outcome ................................................................................. 78
4.1 Introduction ............................................................................................................ 78
4.2 Specific Materials and Methods .............................................................................. 79
4.2.1 Measurement of Cell Viability: Calcein-AM fluorescence dye .................... 80
4.2.2 Measurement of mitochondrial activity: MTT assay .................................. 80
4.2.3 Data Analysis ........................................................................................... 81
4.3 Results .................................................................................................................. 81
4.4 Discussion ............................................................................................................. 82

Chapter 5: Stem Cell Therapy in Combination with sRAGE May Ameliorate
Neuroinflammation in an In-Vitro Cell Model of TBI ............................................................. 88
5.1 Introduction ........................................................................................................... 88
5.2 Specific Materials and Methods .............................................................................. 90
5.2.1 Measurement of Cell Viability: Calcein-AM Fluorescence Dye .................. 91
5.2.2 Measurement of Mitochondrial Activity: MTT Assay ................................. 91
5.2.3 Data Analysis ............................................................................................ 92
5.3 Results .................................................................................................................. 92
5.4 Discussion ............................................................................................................. 93

Chapter 6: Discussion ............................................................................................................... 98
6.1 Relevance of CD-36 ................................................................................................ 98
iii
6.2 Inflammation and TBI............................................................................................... 98
6.3 Effects of sRAGE in an in Vitro Cell Model of Inflammation .................................. 102
6.4 Therapeutic Effects of Combination Stem Cell Therapy and sRAGE ..................... 103

References ............................................................................................................................ 107

Appendix A: Publications ........................................................................................................ 120

Appendix B: Publisher’s Permission to use Figure 1 ............................................................... 122

About the Author ....................................................................................................... END PAGE
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LIST OF FIGURES

Figure 1: Mechanisms associated with secondary damage in TBI ......................................... 15

Figure 2: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact
at 24 hours post-TBI ................................................................................................ 56

Figure 3: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact
at 48 hours post-TBI ................................................................................................ 57

Figure 4: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact
at 7 days post-TBI.................................................................................................... 58

Figure 5: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact
at 60 days post-TBI .................................................................................................. 59

Figure 6: A-D Co-localization of CD-36/NeuN at 24 hours after TBI ....................................... 60

Figure 7: A-D Minimal co-localization of CD-36/ GFAP at 24 hours after TBI ....................... 62

Figure 8: Spleen CD-36 immunodetection at 24 hours post TBI ............................................ 63

Figure 9: Spleen CD-36 immunodetection at 48 hours post TBI ............................................ 64

Figure 10: Spleen CD-36 immunodetection at 7 days post TBI. ............................................... 65

Figure 11: Spleen CD-36 immunodetection at 60 days post TBI .............................................. 66

Figure 12: CD-36 brain cortex protein expression at 24 hours post-TBI ................................... 68

Figure 13: CD-36 brain protein expression 48 hours post-TBI .................................................. 69

Figure 14: CD-36 protein expression in spleen 24 hours post-TBI ........................................... 70

Figure 15: CD-36 protein expression in spleen of 48 hours post-TBI ........................................ 71

Figure 16: CD-36 protein expression in spleen of 7 days post-TBI. .......................................... 72

Figure 17: In Vitro hNP1 cells relative cell viability Calcein assay using sRAGE. ..................... 83

Figure 18: In Vitro hNP1 cells relative metabolic activity MTT assay using sRAGE. ................. 84

Figure 19: In Vitro hNP1 cells relative cell viability Calcein assay using stem cells. ................. 94
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Figure 20: In Vitro hNP1 cells relative metabolic activity MTT assay using stem cells .............. 95
vi

ABSTRACT

Traumatic brain injury (TBI) has become an increasingly unmet clinical need due to
intense military conflicts worldwide. Directly impacted brain cells suffer massive death, with
neighboring cells succumbing to progressive neurodegeneration accompanied by inflammatory
and other secondary cell death events. Subsequent neurodegenerative events may extend to
normal areas beyond the core of injury, thereby exacerbating the central nervous system’s
inflammatory response to TBI. Recently CD-36 (cluster of differentiation 36/fatty acid
translocase (FAT), a class B scavenger receptor of modified low-density lipoproteins (mLDLs) in
macrophages, has been implicated in lipid metabolism, atherosclerosis, oxidative stress, and
tissue injury in cerebral ischemia, and in certain neurodegenerative diseases.
Accordingly, we proposed that CD-36 has a pivotal role in the neuroinflammatory
cascade that further contributes to the pathology of TBI. First, we explored the
neuroinflammatory role of CD-36 after acute and chronic stages of TBI. Second, we employed a
neuroinflammatory model to test the therapeutic effect of the soluble receptor of advanced end-
glycation product (sRAGE); previously shown to abrogate increased CD-36 expression in
stroke. Third, we further examined ameliorating TBI related inflammation as a therapeutic
pathway by combination of stem cell therapy and sRAGE. At acute stages of TBI, we observed
brain co-localization of CD-36, monocyte chemoattractant protein 1 (MCP-1) and ionized
calcium-binding adapter molecule 1 (Iba-1) on impacted cortical areas, significant increases of
CD-36 and MCP-1 positive cells in the ipsilateral vs. contralateral hemispheres of TBI animals in
acute, but no significant increases of Iba-1 expressing cells over time. In early acute stages of
TBI immunoblotting showed overexpression of CD-36 in brain cortex when comparing ipsilateral