Mechanisms of Late-Delayed Radiation Induced Brain Injury

MECHANISMS OF LATE-DELAYED RADIATION INDUCED BRAIN INJURY:

INSIGHTS FROM EXTRACELLULAR MATRIX AND TRANSCRIPTIONAL

PROFILING

RACHEL N. ANDREWS, D.V.M.

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Molecular Medicine and Translational Science

April 4th 2018

Winston-Salem, North Carolina

Approved By:

J. Mark Cline, D.V.M., Ph.D., Advisor

Linda J. Metheny-Barlow, Ph.D., Chair

Gretchen Neigh, Ph.D.

Kerry O’Banion, M.D., Ph.D.

Ann M. Peiffer, Ph.D.

DEDICATION

To my father: without your guidance, love, and dogged persistence I would have become a graphic designer and presumably equally in debt.

And to my grandfather, a hopeful scientist. I hope I have made you proud.

ACKNOWLEDGEMENTS

They say it takes a village to raise a child – it apparently requires no less than four of them to develop a Ph.D. candidate. Although my academic journey has taken me many different places, I am forever grateful to those who have so generously given their time, mentorship, and support which have shaped me into the scientist I am today.

I would like to thank the Department of Pathology, Section on Comparative

Medicine for providing my research ‘home’ for the last 4 years and for their collegiality and continual emphasis on pursuing research intended to advance our understanding of human health. Above all, I must express my gratitude to my mentor, Dr. Mark Cline. His unwavering support, enthusiasm, and encouragement are what have inspired me to devote my career to translational research. You have truly been a fantastic mentor and I am deeply grateful for your time and dedication.

I would also like to extend my thanks and warmest regards to:

My committee members Dr.s Kerry O’Banion, Ann Peiffer, Gretchen Neigh and

Linda Metheny-Barlow. You have always been willing to share your knowledge, insight, and constructive criticism and I am deeply appreciative of your guidance. Thank you for your time, effort and above all, kindness. I hope this dissertation and the scientist I have become makes you proud.

My pathology mentors Dr. Nancy Kock and Dave Caudell for their infinite patience, willingness to teach, and occasional tough love when needed.

To my colleagues Kristofer Michalson, Caralyn Labriola, Hannah Atkins, Ryne

DeBo, David Hanbury, Marnie Silverstein Metzler, and Greg Dugan for your

ii camaraderie, collaborative spirit, and perpetual willingness to engage in lively intellectual debate. In the infamous words of the Joint Pathology Conference, “and a lively discussion ensued!”

Lisa O’Donnell, Cathy Mathis, Jean Gardin and Kelly Mahoney have been integral to this dissertation. None of the work contained within would have been feasible without their help and they have always been willing to lend a helping hand or a sympathetic ear.

To my collaborators from the departments of Radiation Oncology, and Integrative

Physiology and Pharmacology: thank you for your patience, tutelage, and willingness to share knowledge. Your expertise and collegiality truly embody the collaborative spirit that propels sciences forwards.

I appreciatively acknowledge the financial support of the T32 Post-doctoral

Fellowship in Laboratory Animal and Comparative Medicine Training through the

National Institute of Health and funding from the Radiation Countermeasures Center of

Research Excellence (RadCCORE) which made my education possible.

I am grateful for my undergraduate training at the University of Illinois Urbana-

Champaign and give a special, heartfelt thank you to my mentor and colleagues from

Cooke Laboratory: Paul Cooke, Gail Ekman, Melissa Cimafranca, Liz Simon, Gaurav

Tyagi, Heather Schlesser, and Juanmahel Davila. Were it not for you, I would never have discovered my passion for science. I would also like to thank the University of

Wisconsin-Madison School of Veterinary Medicine and instructors there for my doctoral training and support of my development as a physician scientist.

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And finally, I would like to convey my gratitude to the Robbins family and the Mike and Lucy Robbins Fellowship for the generous support I have received from the Mike and Lucy Robbins Graduate Scholarship Award. Much of this dissertation has grown out of his original work and would not have been possible without his foresight, effort and dedication.

“If I have seen further it is by standing on [the] shoulders of Giants." –Sir Isaac Newton

TABLE OF CONTENTS

Table of Contents ...... v List of Figures and Tables ...... vii List of Abbreviations ...... ix Abstract ...... xxi Chapter 1 ...... 1 Introduction ...... 1 References ...... 20 Chapter 2 ...... 48 Cerebrovascular remodeling and neuroinflammation is a late effect of radiation-induced brain injury in nonhuman primates (Published in the Journal of Radiation Research) .....48 Abstract ...... 49 Introduction ...... 51 Materials and Methods ...... 53 Results ...... 59 Discussion ...... 67 Acknowledgements ...... 75 References ...... 75 Chapter 3 ...... 89 Fibronectin produced by cerebral endothelial and vascular smooth muscle cells contributes to perivascular extracellular matrix in late-delayed radiation-induced brain injury. (Submitted to the Journal of Radiation Research)...... 89 Abstract ...... 90 Introduction ...... 92 Materials and Methods ...... 94 Results ...... 104 Discussion ...... 111 Acknowledgements ...... 118 References ...... 119 Chapter 4 ...... 131 Transcriptomic profiling reveals neuroinflammation, complement system involvement and impaired glutamatergic neurotransmission in white matter years post total body irradiation and months post fractionated whole brain irradiation ...... 131 Abstract ...... 132 Introduction ...... 134 Materials and Methods ...... 136 Results ...... 143 Discussion ...... 161 Acknowledgements ...... 168 References ...... 169

Chapter 5 ...... 186 References ...... 204 Appendices ...... 231 Disclaimer ...... 231 Copyright Licenses ...... 232 A. Multi-regional microvascular histomorphometry in normal and irradiated Rhesus macaques ...... 233 B. Quantification of activated microglia in normal and irradiated Rhesus macaques ...... 240 C. Brain magnetic resonance imaging (MRI) surveillance in the non-human primate radiation survivors cohort ...... 245 Curriculum Vitae ...... 254

LIST OF FIGURES AND TABLES

Chapter 1 Figures and Tables Table I ...... 11 Table II ...... 13

Chapter 2 Figures and Tables Table I ...... 57 Table II ...... 59 Figure 1 ...... 60 Table III ...... 61 Figure 2 ...... 62 Figure 3 ...... 63 Figure 4 ...... 64 Figure 5 ...... 65 Figure 6 ...... 66

Chapter 3 Figures and Tables Table I ...... 101 Figure 1 ...... 105 Figure 2 ...... 106 Figure 3 ...... 107 Figure 4 ...... 109 Figure 5 ...... 110 Figure 6 ...... 111

Chapter 4 Figures and Tables Figure 1 ...... 143 Table I ...... 143 Table II ...... 144 Table III ...... 146 Figure 2 ...... 147 Figure 3 ...... 148 Figure 4 ...... 149 Figure 5 ...... 150 Table IV ...... 151 Table V...... 153 Table VI ...... 154 Table VII ...... 155 Figure 6 ...... 157 Figure 7 ...... 159

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Figure 8 ...... 160

Chapter 5 Figures and Tables Figure 1 ...... 188 Figure 2 ...... 193

Appendix A Figures and Tables Figure 1 ...... 238

Appendix B Figures and Tables Figure 1 ...... 243

Appendix C Figures and Tables Table I ...... 246 Figure 1 ...... 249 Figure 2 ...... 250 Table II ...... 250 Figure 3 ...... 251

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ABBREVIATIONS

Micrometer μm

[18F]Fluoromisonidazole 18F-MISO

Association for the Assessment and Accreditation of Laboratory Animal Care AAALAC

ATP binding cassette subfamily B member 1 ABCB1

ATP binding cassette subfamily C member 9 ABCC9

Advanced Cell Diagnostics ACD

Angiotensin I converting enzyme ACE

Angiotensin I converting enzyme 2 ACE2

Actin, alpha 2, smooth muscle ACTA2

Alzheimer’s Disease AD

Adrenomedullin ADM

Agrin AGRN

Angiotensinogen AGT

Angiotensin II receptor type 1 AGTR1

Allograft inflammatory factor 1, IBA1 AIF-1

Amyotrophic lateral sclerosis ALS

Angiopoietin 1 ANGPT

Analysis of variance ANOVA

Amyloid precursor protein APP

Aquaporin 4 AQP4

Rho GTPase Activating Protein ARHGAP

Rho GDP Dissociation Inhibitor ARHGDI

Rho/Rac Guanine Nucleotide Exchange Factor ARHGEF

Aryl hydrocarbon receptor nuclear translocator ARNT

Acute radiation syndrome ARS

Adenosine triphosphate ATP

Brevican BCAN

1-bromo-3-chloropropane BCP

Biologically effective dose BED

BMP binding endothelial regulator BMPER

Bassoon presynaptic cytomatrix protein BSN

Cerebral amyloid angiopathy CAA

Calcium voltage-gated channel subunit alpha 1 CACNA1

Calcium voltage-gated channel auxiliary subunit beta 1 CACNB1

Calcium/calmodulin-dependent protein kinase 2 beta CAMK2B

C-C motif chemokine ligand 2 CCL2

C-C motif chemokine receptor 2 CCR2

Cluster of differentiation CD

Complement C3a receptor C3AR

Cluster of differentiation 3, T-cell receptor T3 gamma chain CD3G

Cluster of differentiation 68, Macrosialin CD68

Cell division cycle 42 CDC42

Cadherin 5 CDH5

Complementary DNA cDNA

CFB Complement factor B

Checkpoint kinase 1 CHEK1

Checkpoint kinase 2 CHEK2

Confidence interval CI

Class II Major Histocompatibility Complex Transactivator CIITA

Claudin CLDN

CAP-Gly Domain Containing Linker Protein 1 CLIP

Central nervous system CNS

Collagen type X alpha 1 chain COL

See PTGS2 COX2

Complement C3b/C4b Receptor 1 (Knops Blood Group) CR1

Cartilage acidic protein 1 CRTAC

Colony stimulating factor 1 receptor CSF1R

Chondroitin sulfate proteoglycan CSPG

Cycle threshold Ct

Computed tomography CT

Connective tissue growth factor CTGF

Catenin Beta 1 CTNNB1

C-X-C motif receptor CXCR

3,3′-Diaminobenzidine DAB

Doublecortin DCX

Differentially expressed gene DEG

Delta like canonical Notch ligand 4 DLL4

Dorsolateral prefrontal cortex (Brodmann area 46) DLPFC

Delayed-Match-to-Sample DMS

Deoxyribonucleic acid DNA

Extracellular matrix ECM

Effective dose; defined effect seen in 50% of population at this dose ED50

Ethylenediaminetetraacetic acid EDTA

Electoencephalography EEG

Epidermal growth factor EGF

Epidermal growth factor receptor EGFR

Elastin Microfibril Interfacer EMILIN

Endothelial PAS domain protein 1 EPAS1

Erythropoeitin EPO

Fibulin FBLN

False discovery rate FDR

F11 receptor F11R

Fas cell surface death receptor FAS

Fibrillin FBN

Fc Fragment Of IgE Receptor FCER

Fibroblast growth factor 1 FGF1

Vascular endothelial growth factor D FIGF

Fms related tyrosine kinase 1 FLT1

Fibronectin FN1

Fractionated whole-brain irradiation fWBI

Glial fibrillary acidic protein GFAP

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Gap junction protein alpha 1 GJA1

Gliomedin GLDN

See SLC2A1 GLUT1

Glypican GPC

Glutamate ionotropic receptor AMPA type subunit 2 GRIA2

Glutamate ionotropic receptor AMPA type subunit 5 GRIA4

Glutamate ionotropic receptor NMDA type subunit 2A GRIN2A

Glutamate ionotropic receptor NMDA type subunit 2B GRIN2B

Gray Gy

H2A Histone Family Member X H2AFX

Hyaluronan And Proteoglycan Link Protein HAPLN

Hippocampus HC

Hydrochloride HCl

Hypoxia inducible factor 1 alpha subunit HIF1A

Heme oxygenase 1 HMOX

Heparan Sulfate Proteoglycan 2, Perlecan HSPG2

Hematoxylin and Eosin H&E

Hippocampus HC

Hepatocyte growth factor HGF

HUGO Gene Nomenclature Committee HGNC

Major histocompatibility complex, class I, A HLA-A

Major histocompatibility complex, class I, F HLA-F

Major histocompatibility complex, class II, DM alpha HLA-DMA

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Major histocompatibility complex, class II, DM beta HLA-DMB

Major histocompatibility complex, class II, DO alpha HLA-DOA

Major histocompatibility complex, class II, DP alpha 1 HLA-DPA1

Major histocompatibility complex, class II, DP beta 1 HLA-DPB1

Major histocompatibility complex, class II, DQ alpha 1 HLA-DQA1

Major histocompatibility complex, class II, DR beta 5 HLA-DRB5

Hemicentin HMCN1

Human Genome Organization HUGO

Institutional Animal Care and Use Committee IACUC

See AIF1 IBA1

Intercellular adhesion molecule ICAM-1

Interferon gamma IFNG

Immunoglobulin Ig

Immunohistochemistry IHC

Interleukin IL

Ingenuity Pathway Analysis IPA

Intelligence quotient IQ

In situ hybridization ISH

Integrin subunit alpha V ITGAV

Integrin subunit beta 1 ITGB1

Junctional adhesion molecule B JAMB

Junctional adhesion molecule C JAMC

Potassium voltage-gated channel subfamily B member KCNB

xiv

Potassium voltage-gated channel subfamily J member KCNJ

Potassium two pore domain channel subfamily K member KCNK

Potassium calcium-activated channel subfamily M member KCNM

Potassium sodium-activated channel subfamily T member KCNT

Kinase insert domain receptor KDR

Kilogram kg

Laminin LAMA4

Laminin subunit alpha 5 LAMA5

Laminin subunit beta 2 LAMB2

Laminin subunit gamma 1 LAMC1

Lethal Dose; 50% population mortality LD50

Lethal Dose; 50% population mortality within 60 days LD50/60

Lactate dehydrogenase A LDHA

Linear energy transfer LET

Linear accelerator LINAC

Low Molecular Weight Protein 7, Proteasome Subunit Beta 8 LMP7

Lifespan Study, atomic bomb survivors cohort LSS

Latent transforming growth factor beta binding protein LTBP

MAS1 proto-oncogene, G protein-coupled receptor MAS1

Matrilin MATN

Midkine (neurite growth-promoting factor 2) MDK

Microfibril associated protein MFAP

Milligram mg

Major histocompatibility complex MHC

Major histocompatibility complex, class I, B MHC1B

Megahertz MHz

Micro RNA miRNA

Milliliter mL

Millimeter mm

Millisecond ms

Membrane metalloendopeptidase MME

Matrix metalloproteinase 2 MMP

Major Histocompatibility Complex, Class I-Related MR1

Magnetic resonance imaging MRI

Messenger ribonucleic acid mRNA

Membrane Spanning 4-Domains A1, see CD20 MS4A1

Megavolts MV

Nibrin NBN

Neurocan NCAN

Nuclear factor kappa B subunit 1 NFKB1

See CSPG4 NG2

Nerve Growth Factor Receptor NGFR

Non-human primate NHP

Nidogen 1 NID1

National Institute of Health NIH

NLR Family CARD Domain Containing 5 NLCR5

xvi

Nanometer nm

N-methyl-D-aspartic acid NMDA

Nitric oxide synthase 2 NOS2

Neural stem cell NSC

Neuron specific enolase NSE

No significant lesions NSL

Netrin NTN

Occludin OCLN

Oligodendrocyte progenitor cell OPC

See SERPINE1 PAI-1

Phosphorus-buffered saline PBS

Platelet derived growth factor PDGF

Platelet derived growth factor subunit B PDGFB

Platelet derived growth factor receptor PDGFR

Positron emission tomography PET

Prostaglandin E2 PGE2

Plasminogen activator, tissue type PLAT

Proteasome Subunit Beta 9 PSMB9

Prostaglandin-endoperoxide synthase 2, Cyclooxygenase-2 PTGS2

Rho family, small GTP binding protein Rac1 RAC1

Radiation Countermeasures Center of Research Excellence RadCCORE

Radiofrequency RF

Ras Homolog Family Member A RHOA

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Radiation-induced brain injury RIBI

RNA integrity number RIN

Ribonucleic acid RNA

Rho associated coiled-coil containing protein kinase ROCK

Region of interest ROI

RAR-related orphan gamma receptor RORyT

Reactive oxygen species ROS

Ribosomal ribonucleic acid rRNA

Radiation Survivor Cohort RSC

Real-time quantitative polymerase chain reaction RT-qPCR

Sphingosine-1-phosphate receptor S1PR sodium voltage-gated channel beta SCNB

Syndecan SDC

Serpin Family E Member 1, Plasminogen activator inhibitor SERPINE1

Serpin Family G Member 1 SERPING1

Solute carrier family 2 member 1, GLUT1 SLC2A1

Slit Guidance Ligand SLIT

Small nuclear ribonucleic acid snRNA

Small nucleolar ribonucleic acid snoRNA

SPARC/Osteonectin, Cwcv And Kazal Like Domains Proteoglycan SPOCK

Subcutaneous SQ

Stereotactic Radiosurgery SRS

Signal Transducer and Activator of Transcription STAT

xviii

Sievert Sv

Susceptibility weighted imaging SWI

Synaptic Ras-GTPase activating protein SYNGAP

Synaptophysin SYP

Synaptotagmin SYT

Tesla T

Type 1 diabetes mellitus T1DM

Type 2 diabetes mellitus T2DM

Typical American Diet TAD

Total body irradiation TBI

Endothelial tyrosine kinase TEK

Transmission electron microscopy TEM

Transferrin receptor TFRC

Transforming growth factor beta 1 TGFB1

Type 1 T helper cells Th1

Type 2 T helper cells Th2

Type 17 T helper cells Th17

Tissue inhibitor of metalloproteinases TIMP

Tight junction protein 1 TJP1

Tenascin C TNC

Tenascin R TNR

Tumor necrosis factor TNF

Tumor necrosis factor receptor super family member TNFRSF

xix

2,3,5-Triphenyltetrazolium chloride TTC

Triggering receptor expressed on myeloid cells 1 TREM1

Versican VCAN

Vascular endothelial growth factor VEGF

Wake Forest University School of Medicine WFUSM

Wingless-type MMTV integration site family member 1 WNT1

White matter WM

White matter hyperintensities WMH

Zona occludens 2 ZO2

ABSTRACT

Rachel N. Andrews, D.V.M.

Fractionated whole brain irradiation (fWBI), as used for the treatment of intracranial neoplasia, results in progressive cognitive impairment, and cerebrovascular and white matter injury. Although the clinical progression and morphology of late- delayed radiation-induced brain injury (RIBI) are well characterized, the pathogenesis is unclear. The long-term consequences of single high dose irradiation of the adult brain, as may occur following a nuclear accident or malicious exposure, are uncertain.

The purpose of this dissertation was to identify novel pathways and molecular effectors in a non-human primate model of RIBI and to assess if similar changes were present in long-term survivors of single high dose total body irradiation. These aims were evaluated in three separate experiments, presented in Chapters 2-4.

For Chapters 2 and 3, we evaluated brain tissue from four adult male rhesus macaques (Macaca mulatta) that developed RIBI 4-6 months post-fWBI. Changes in gene expression were assessed by RT-qPCR to screen a priori selected pathophysiologic processes. Animals with RIBI demonstrated evidence of microglial/macrophage- mediated neuroinflammation, hypoxia, vascular maturation and stabilization, extracellular matrix (ECM) deposition and remodeling and impaired glutamatergic neurotransmission.

In Chapter 3, we characterized the composition of accumulated perivascular ECM by immunohistochemistry and identified fibronectin as one of the primary components.

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By in situ hybridization, we determined that cerebral endothelial and vascular smooth muscle cells produce fibronectin in RIBI.

In Chapter 4, we expanded our analyses to evaluate five rhesus macaques which survived 12 months post-fWBI, and five which previously received high dose (6.75-8.05

Gy) TBI, 6-9 years prior to necropsy. RNA-sequencing and subsequent whole transcriptomic gene expression profiling revealed fWBI and TBI resulted in shared transcriptional patterns of CNS injury including neuroinflammation, complement fixation and impaired glutamatergic neurotransmission. fWBI animals also demonstrated increased expression of pathways associated with ECM deposition and remodeling.

In conclusion, cerebral irradiation results in persistent neuroinflammation, expression of complement factors and evidence of impaired glutamatergic neurotransmission in rhesus macaques months to years post-exposure. Perivascular fibronectin accumulation may contribute to RIBI following fWBI. These findings have led to a novel proposed mechanism of radiation-induced brain injury and the identification of several potential therapeutic targets.

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CHAPTER ONE

INTRODUCTION

Rachel N. Andrews, D.V.M.

BACKGROUND

Approximately 200,000 patients per year will receive fractionated whole-brain irradiation (fWBI) for the treatment of primary or metastatic brain cancer (1). Of those that survive long enough ( > 6 months ), 50-90% will develop late-delayed radiation- induced brain injury (RIBI) (2-4). RIBI is a progressive neurodegenerative and inflammatory condition results in cognitive decline and memory impairment that deleteriously affect quality of life for cancer patients. This loss of function is associated with progressive cerebrovascular and white matter injury (5, 6). Although the clinical progression and morphologic characteristics of RIBI are well characterized (7-12), the pathogenesis is unclear which has limited the development of effective therapeutics.

BRAIN CANCER and TREATMENT

Brain cancer is the presence of abnormally dividing cells within the cranial vault which have ceased to obey normal physiologic controls. Brain cancers may be stratified into two categories derived from site of origin: primary brain tumors and metastatic brain tumors. Primary brain tumors arise from cells located within the skull, including glia

(gliomas) and neural precursors (medulloblastoma, glioblastoma), meningeal contributors

(meningioma, melanomas), the choroid, ependyma, pituitary gland and others (13).

Primary brain tumors may be benign or malignant, depending on type and grade.

Metastatic tumors arise from organs and anatomic structures other than the brain or spinal cord, and are by definition malignant. These are the most common type of brain tumor, and occur 4-5 times more frequently than primary brain tumors (14). The most common

2 types of cancer to metastasize to the brain are: lung, breast, melanoma, colon/colorectal, and kidney/renal tumors (15-18).

The treatment of brain cancer is multimodal and the treatment regimen customized depending on a variety of patient and tumor characteristics. Tumor-dependent factors include tumor number, location, type, size and spread. Patient-dependent factors include overall patient condition and health, comorbid conditions, anticipated side-effects and previous treatment regimens. Local control of tumors by surgical excision or stereotactic radiosurgery (SRS) is constrained by tumor size, number, and anatomic location (1, 19, 20). Both modalities, due to tumor invasion and treatment margin constraints, may leave residual cancer cells within the brain. Options for chemotherapeutic treatment of brain cancer may be limited, as metastatic brain cancers may acquire resistance to chemotherapeutics used against the primary tumor (21-23).

Heterogeneity in blood-brain-barrier permeability within tumors and between patients contributes to variability in drug delivery and local concentration (24-26), complicating treatment.

Therefore, fWBI is a mainstay of treatment for intracranial neoplasia.

Radiotherapy utilizes high-energy ionizing radiation to damage cancer cells through direct damage to DNA or indirectly via free-radicals formed during ionizing events (27).

As radiation-induced DNA-damage destroys cells by inhibiting their ability to divide, rapidly proliferating cells, such as cancer cells, are more radiosensitive. As this method of cell killing is non-selective, normal, non-cancerous cells are also vulnerable to radiation- induced cellular injury and death. This normal tissue injury limits the dose of radiation that can be administered to a patient, and radiation-induced cell killing is dose-dependent.

Fractionation is the division of a large cumulative dose of radiation into several smaller doses over days to weeks. This is done to reduce normal tissue injury and enhance cancer cell killing by several mechanisms (28). First, it allows normal cells to repair, permitting the delivery of higher cumulative doses of irradiation than is possible with single doses (29). Secondly, fractionation increases the sensitivity of cancer cells to radiation by stimulating entry into vulnerable stages of the cell-cycle. Finally, fractionation aids in reoxygenation of hypoxic tumors. Molecular oxygen is a necessary co-factor in many of the chemical reactions that induce DNA-damage following exposure to ionizing radiation (30). As a result, hypoxic cells are approximately 3 times more resistant to radiation than well-oxygenated ones (31). Many solid tumors are hypoxic as their capacity for growth exceeds vascular supply and malformed tumor-associated blood vessels are prone to collapse (32,33). Fractionation results in progressive killing of well- oxygenated cells, thus reducing tumor mass and permitting migration of hypoxic cells into the aerobic compartment (34). These now re-oxygenated cancer cells are susceptible to the next fraction of radiation.

Fractionated radiotherapy may be delivered to the entire brain (whole brain irradiation) or portions (partial brain irradiation). Whole brain irradiation is used as a brain cancer treatment for aggressive, disseminating neoplasms, palliation of metastases and as prophylaxis against metastasis in a subset of high risk lung cancer patients.

However, inclusion of non-cancerous brain tissue within the field of irradiation often results in cognitive impairment, cerebral necrosis and dementia (2, 4, 35-37). Various strategies of partial brain irradiation have been proposed to ameliorate the cognitive impact of radiation therapy, supported by data demonstrating that loss of IQ in children

4 receiving cranial irradiation correlates with radiation dose to the temporal lobes (38, 39).

Approaches include SRS, discussed previously, hippocampal-avoidance, limbic circuit sparing, and neural stem cell sparing (40). Preliminary studies indicate that these sparing techniques are technologically feasible (41-45); however additional study is warranted to determine long-term outcomes including survival, cancer recurrence rates and the capacity for cognitive protection. Additional data have shown that the extent of white matter injury also correlates with extent of cognitive dysfunction (3, 5, 6), suggesting that hippocampal-avoidance alone may be insufficient to fully protect against cognitive impairment following radiotherapy.

In radiotherapy, radiation dose is measured in Gray (Gy) which conveys the amount of radiation absorbed by a given volume of tissue. It is defined as the absorption of 1 joule of radiation energy per kilogram of matter, and represents a physical quantity.

The unit Sievert (Sv) represents the health or biological effect of ionizing radiation, by combining the absorbed dose and known tissue response parameters.

RADIATION-INDUCED BRAIN INJURY

RIBI is classified into three categories based on time course of development and patient manifestations of injury: acute, early-delayed and late-delayed RIBI (46). Acute

RIBI is manifests within days to weeks of treatment and is characterized by cerebral edema and blood-brain-barrier disruption. Clinical signs related to increased intracranial pressure include headache, nausea, and mental status alteration (47). As the major risk factors for acute neurotoxicity include large doses per fraction

( > 6 Gy ) and twice-daily administration (48-50), acute RIBI is largely avoidable and

5 rare with current fWBI protocols. Early-delayed, or subacute RIBI develops 1-6 months post-irradiation and may be asymptomatic or manifest as transient demyelination and somnolence (51). The clinical signs associated with acute and early-delayed RIBI are usually mild, self-limiting and regress spontaneously.

In contrast, late-delayed RIBI is irreversible, progressive, and associated with neurologic impairment including cognitive dysfunction which negatively impacts quality of life in cancer patients (2, 37, 52). A subset of affected patients will develop severe neurologic impairment including dementia, progressive ataxia and urinary continence

(10, 35). This syndrome occurs > 6 months post-irradiation and is associated with multifocal cerebrovascular and white matter injury (3-5).

The histopathology of late-delayed RIBI has been well characterized in numerous studies in rodents, non-human primates, and human patient samples (7, 8, 10-12, 36, 53-

62). The lesions are multifocal and spatially stochastic with predilection for cerebral white matter and include demyelination, white matter necrosis, and cerebrovascular injury. Vascular changes include: blood-brain-barrier disruption with extravasation of serum products, perivascular fibrosis, and haphazard angiogenesis or telangiectasia.

Inflammation-associated histologic findings include: diffuse microglial activation, microglial aggregation at sites of necrosis, astrogliosis, and mild mononuclear perivascular cuffing. The pathogenesis of late-delayed RIBI is unclear, and there are no effective therapies.

THEORIES ON THE PATHOGENESIS OF RADIATION-INDUCED BRAIN

INJURY

Hippocampal-Mediated Cognitive Effects

Neurons were once considered radioresistant due to their post-mitotic status; however studies have demonstrated that the dentate gyrus of the hippocampus is one of two sites of active neurogenesis in the adult brain (63-65). This suggests a radiosensitive niche. As the hippocampus plays an integral role in cognition and memory (66), most preclinical studies of RIBI and radiation-induced cognitive impairment have evaluated effects on hippocampal structure and function.

Studies in rodents have demonstrated cerebral irradiation results in dose- dependent decreases in neuronal stem cells (NSC), reduced proliferation of surviving

NSC and impaired maturation into neurons (67-69). Reductions in hippocampal neurogenesis are correlated with radiation-induced cognitive impairment (70-72).

However, reduced neurogenesis does not explain the relationship between white matter necrosis and cognitive decline (4-6) and the capacity for these stem cells to differentiate into glia (including oligodendroglia) appears unimpaired (68).

Others have demonstrated radiation affects key hippocampal neuronal structures essential for synaptic plasticity and cognition, including: changes in dendritic spine morphology and density (73-76), alterations in glutamatergic ionotropic receptor subunits

(77-80), and neuronal cytoskeletal alterations (76, 81). Altered function has also been observed, including: changes in neuronal firing patterns and spike generation (82-84), impaired long-term potentiation (79, 85, 86), and impaired glutamatergic

7 neurotransmission (78, 87). Interestingly, electroencephalography in rhesus macaques given 4-8 Gy single dose total body irradiation was normal (88). Furthermore, we have shown that radiation-induced decreases in glutamatergic ionotropic receptor subunits are not hippocampal-specific in rhesus macaques (89). These data suggest that mechanisms resulting in impaired neurotransmission and signal transduction may not be restricted to the hippocampus. Thus, despite the wealth of studies evaluating the response of the hippocampus to radiation, these data should not preclude the assessment of other brain regions as contributors to radiation-induced cognitive dysfunction.

The Parenchymal Contribution to RIBI

Of the non-neuronal contributors, oligodendrocytes and their progenitors have been a focus of interest due to their potential role in demyelination and white matter necrosis. The extent of cognitive impairment is correlated with white matter injury (3, 5,

6). Rapid apoptotic depletion of oligodendrocytes occurs within 24 hours following doses of 2-30 Gy (90, 91), and oligodendrocytes are relatively radiosensitive in cell

+ + culture [d0: 1.4 Gy (92)]. Irradiation also reduces numbers of PDGFRA /NG2 oligodendroglial precursors [OPCs (93-95)], which are required to regenerate mature, myelinating oligodendrocytes (96).

Data from rodent studies are partially conflicting. In mice, oligodendrocyte and

OPC populations do not recover to normal levels by 18-months post-irradiation with both single (20 Gy) and fractionated doses of irradiation (36 Gy, 6x6 Gy fractions over 2 weeks) (97, 98). Despite maintenance of myelin sheath integrity, perturbations in axonal signal transduction and nodes of Ranvier were observed. A similar lack of change in

8 myelin sheath integrity was also observed in rats 12 months post-fWBI (45 Gy, 9x5 Gy fractions, over 4.5 weeks); however oligodendrocyte number was unaffected despite demonstrated cognitive impairment (99). These data suggest that deficits in myelination are not responsible for the cognitive dysfunction observed in RIBI, however do not preclude oligodendrocyte and OPC involvement through other mechanisms. Additional study is warranted.

Astrocytes are one of the primary parenchymal components of the central nervous system [approximately 40% of all glia (100, 101)] and contribute to blood-brain-barrier integrity, nutrient transfer, neurotransmission and repair following injury (102-106).

Astrocytes are relatively radioresistant, become activated, and proliferate following single (20 Gy) and fractionated doses of radiation (40 Gy, 20x2 Gy fractions over 5 weeks) (58, 107, 108). These activated astrocytes express higher levels of glial fibrillary acidic protein (GFAP) (58, 108-111), as well many pro-inflammatory molecules including: intercellular adhesion molecule-1 (ICAM-1) cyclooxygenase-2 (COX2), interleukin-1beta (IL-1β), and interleukin-6 (IL-6) (112-115). This is presumed to facilitate leukocyte migration into the brain parenchyma in RIBI. Furthermore, both high and low-LET gamma irradiation results in decreased glutamate uptake by astrocytes, which may promote excitotoxic neuronal injury (116, 117). Although it is unlikely that selective injury to astrocytes is the primary mechanism of injury in RIBI, their reactivity to inflammation and ability to modulate glutamatergic neurotransmission (103, 104) may contribute to both vascular and parenchymal dysfunction in RIBI.

Microglia are the resident immune cells of the central nervous system and are responsible for the phagocytosis and remodeling of plaques, damaged synapses and

9 cellular debris. These cells make up approximately 12% of brain cells (118). Although microglial phagocytosis is essential to remove necrotic cellular debris, radiation induces sustained microglial activation which is thought to contribute to the progression of RIBI

(68). Single dose whole brain irradiation (6-35 Gy) induces activation-associated changes in microglia which include: proliferation, reactive oxygen species (ROS) production, and the expression of pro-inflammatory cytokines including, tumor necrosis factor alpha

(TNFα), IL-1β, IL-6, COX-2, C-C motif chemokine ligand 2 (CCL2), prostaglandin E2

(PGE2) and ICAM-1 (110, 113, 119-122). Furthermore, culture of astrocytes in microglia-conditioned media results in astrocyte activation and enhanced astrocytic expression of inflammatory mediators, demonstrating that microglial have the ability to potentiate pro-inflammatory signaling cascades in other cell types (114, 120). Additional studies suggest that this microglial-mediated neuroinflammation may contribute to RIBI by impairing neurogenesis and thus cognitive function (68, 69).

Others have shown elimination of microglia from the central nervous system

(CNS) via treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor

PLX5622 (123), and treatment with anti-inflammatories reduce microglial activation and concomitantly prevent cognitive impairment (124-126). However, other studies suggest that radiation-induced cognitive impairment is not solely microglial-dependent, as administration of the anti-inflammatory L-158,809, and implantation human embryonic stem cells can prevent or reverse cognitive impairment without affecting microglial activation or number (127-131). Thus, while microglia likely contribute to the pathogenesis of RIBI, it appears unlikely that perpetuated neuroinflammation is solely responsible for the observed cognitive dysfunction and neurologic impairment.

The Vascular Contribution to RIBI

The prevalence and spectrum of vascular injury observed in RIBI (36, 59, 60, 62,

89, 132) has led to the hypothesis that that radiation-induced vascular damage and subsequent ischemia lead to observed white matter necrosis. Supportingly, capillary loss precedes the development of cognitive decline in a rat model of RIBI (132) and several other studies have demonstrated reductions in microvascular investiture following irradiation (59, 132, 133).

However, imaging studies in humans indicate that white matter has a lower cerebral blood flow threshold for infarction, and thus, greater tolerance for reductions in perfusion than grey matter (134-136). This argues against global cerebral hypoxia as the inciting cause of RIBI but suggests that if RIBI is due to a vascular-mediated pathogenesis, that regional differences in structural vulnerability may predispose white matter to injury. Indeed, there is a reduction in both microvascular area fraction and microvessel density relative to grey matter (Table I), with fewer anastomoses and limited penetration of leptomeningeal collateral vessels (137). This transition occurs abruptly at the grey-white matter junction (138). This shift to a predominately capillary vascular bed may contribute to the susceptibility of white matter to injury.

Table I: Comparative cerebrovascular morphometrics Human Rhesus macaque Rodent (Rat) Cortical Grey Microvessel Density (profiles per mm2) 79 (139) 136* 88-165 (138) White Matter Microvessel Density (profiles per mm2) 36 (139) 52* 30 (138) Cortical Grey Microvascular % Area 1.8 (139) 2.0-2.5 (140) 2.9 (141) White Matter Microvessel % Area 1.0 (139) 0.9 (140) ND * Unpublished data, Appendix A ND: No data

Finally, white matter necrosis occurs following boron-neutron capture irradiation, a treatment modality in which the majority of the radiation dose is delivered to the vasculature (142, 143). This lends credence to the theory that damage to the radiation- induced vascular damage alone is sufficient to induce white matter necrosis.

MODELS OF RADIATION-INDUCED BRAIN INJURY

Studies investigating the mechanisms of late-delayed RIBI in humans are confounded by factors which include: socioeconomic status, genetic variation, the type and progression status of the primary cancer, concurrent and previous cancer treatment regimen, survival-interval post-irradiation and comorbid conditions such as hypertension and diabetes mellitus (144-146).

As acknowledged previously, the radiation response of cells of the central nervous system is heterogeneous, with variation in radio-sensitivity, and biological response.

Although cell-culture models are used to elucidate intricate mechanisms, these systems contain artificial constructs such as artificial media and culture reservoirs which may modulate cellular activity. Furthermore, differences in the culture microenvironment and the absence of support cell signaling may limit the translatability of findings. Even advanced three-dimensional, multicellular culture systems such as spheroids do not yet fully recapitulate the cellular and anatomic complexity of the brain, thus indicating the need for animal models.

Rodent models are commonly used in biomedical research, in part due to their small size, shorter lifespans for aging studies, reproductive efficiency, and availability of genetic manipulations (147). However, rodents possess several key genetic,

12 neuroanatomic and physiologic differences from primates which may limit the translatability of findings. While inbred rodents strains such as the C57BL/6J and

BALB/c are well-characterized and limited inter-individual genetic variability reduces confounders, this lack of genetic heterogeneity (148) may not adequately model the human spectrum of response. Inter-strain variation in radio-sensitivity (149-155) indicates that radiation responses should be evaluated across multiple strains.

Neuroanatomic differences between rodents and humans may limit translational utility in aspects of radiation research. In rodents, the hippocampus occupies a greater relative volume with an associated reduction in relative cerebral cortical volume compared to humans (Table II). With less brain volume devoted to cortical processing, this contributes to dissimilarities in the hippocampal projection pathways, innervation patterns, nuclear organization (156), and function (157, 158) between rodents and primates.

Table II: Comparative neuroanatomic volumetrics Human Rhesus macaque Rodent Cortical % Volume (cortex/total brain) 77 (162) 55 (163) 31b (162) Relative cortical volume compared to humans n/a 71% 40% White matter % Cortical volume (white/total cortex) 39 (159) 31 (159) 10a (160) 12b (161) Relative white matter volume compared to humans n/a 79% 26% 31% Hippocampus % Volume (hippocampus/total brain) 0.4 (164) 1.2 (165) 5.1a (166) Relative hippocampal volume compared to humans n/a 300% 1275% a mouse b rat

Additionally, cortical white matter in rodent brains occupies 25-30% of the relative volume of that in humans (159-161). fWBI does not result in white matter necrosis in rodents (77, 99, 129, 141), which precludes the ability to evaluate associated

13 molecular changes, disruptions in connectivity and potential therapeutic effects on this outcome. It is possible that this predominance of grey matter confers some degree of intrinsic radioprotection to the rodent brain, due to the more robust cerebrovascular structure.

Rhesus macaques (Macaca mulatta) have been used extensively in neuroscience and biomedical research. These non-human primates share a close phylogenetic relationship to humans, with evolutionary divergence 25 million years ago (167), as opposed to rodents which diverged 87 million years ago (168). As a result, there is

93.54% genomic homology (169), and 98% similarity in the coding region of the transcriptome (170) between rhesus macaques and humans. This contributes to greater physiologic, immunologic, and metabolic similarity to humans (169, 171, 172).

In addition to the aforementioned neuroanatomic similarities to humans, evolution of a larger neocortex in primates has led to the development of functional areas which do not exist in rats or mice, including dorsolateral prefrontal cortex subdivisions (173, 174).

These areas play key roles in human cognitive processes (175, 176). Furthermore, humans and non-human primates are visually oriented, unlike rodents (171, 177-179), permitting one to examine visual non-spatial and spatial cognitive processes. These and other similarities allow evaluation of higher order cognitive functions (180-187), which are difficult to model in rodents.

Finally, we and others have shown that rhesus macaques that receive fWBI develop cognitive impairment (188) and multifocal white matter necrosis and cerebrovascular injury (7, 8, 53, 54, 89), recapitulating the features of RIBI in humans.

Thus, the rhesus macaque is a more human-like alternative in which to evaluate the pathogenesis of RIBI.

THE CEREBRAL CONSEQUENCES OF SINGLE HIGH DOSE RADIATION EXPOSURE

While the histopathology and clinical manifestations of late-delayed RIBI following clinical radiotherapy are well-characterized (7-12, 53, 54), the cerebral consequences of single high dose radiation exposure on the adult brain remain unclear.

Such radiation exposures may occur following a nuclear accident or terrorism scenario, thus understanding of the long-term ramifications is essential for the risk assessment, patient care and monitoring of affected populations.

Following the terrorist attacks on September 11th 2001, the United States

Department of Homeland Security identified a large-scale nuclear exposure as one of the fifteen most likely emergency scenarios to impact the United States (189). This scenario anticipates the assembly and detonation of a 10 kiloton low-yield radiologic dispersal device (RDD), as the required materials are abundant in medicine, scientific research and in industrial applications (190, 191), generally with limited security. Such a device has the potential to expose > 100,000 people to significant doses of radiation ( > 1.5 Sv)

(192). Furthermore, increasing reliance upon nuclear power presents a growing risk for civilian radiation exposure. The United States is the world’s largest producer of commercial nuclear power, with 99 reactors in operation in 30 separate states (193).

Despite safety regulations in place to limit inadvertent radiation exposure, there have been at least 57 major nuclear accidents since the Chernobyl disaster, and 57% (56/99) of all nuclear-related accidents have occurred within the United States (194). The more

15 recent Fukushima Daiichi nuclear disaster illustrates this disastrous potential; 32 million people have been exposed to radiation due to nuclear fallout (195). As such, both accidental and malicious exposures are realistic scenarios in which high single dose total body irradiation may impact population health.

In the event of a large-scale nuclear event, thermal injury, concussive trauma and the acute radiation syndrome (ARS) will result in significant morbidity and mortality for those closest to the hypocenter. In acute injury, both the risk of cellular damage and degree of injury are proportional to the absorbed dose (196, 197). Rapidly dividing cells such as the intestinal epithelium and bone marrow are most susceptible (197). Those exposed to 2-8 Gy will require hospitalization for the management of ARS (190). There is no known treatment which will effectively prevent or reverse ARS. Management involves managing patient symptomology, including aggressive fluid therapy, blood transfusion, parenteral nutrition, neutropenic isolation precautions and prophylactic antibiotics. Administration of the bone marrow stimulating drug filgrastim (Neupogen) may improve survival for patients with the hematopoietic syndrome of ARS, but does not mitigate the gastrointestinal, cutaneous or cardiovascular manifestations (198). Those that receive greater than 8 Gy are generally administered palliative care only. Patient survival is dependent upon absorbed dose, and 50% of patients who receive 2.5 – 5 Gy are expected to perish within 60 days (199).

Those that survive the acute radiation syndrome are at risk of developing delayed effects of acute radiation exposure (DEARE). These syndromes, characterized by their late onset months to year post-irradiation are of substantial impact to public health, not only in malicious or accidental exposure scenarios, but due to their occurrence following

16 clinical irradiation in cancer therapy. DEARE may occur in virtually any tissue, and are analogous to the delayed injury that occurs following clinical radiotherapy (201, 201).

Therefore scientific advances and treatments which benefit cancer survivors may benefit those with DEARE.

Historically, the brain has been considered radioresistant, able to with standard therapeutic doses of 55-65 Gy, fractions of < 2 Gy (202) and for single doses the LD50 is approximately 15 Gy (203). Thus, the cerebral late effects of single high dose radiation exposure have been overlooked, as lethality due to the acute radiation syndrome makes survival unlikely. However, a growing body of evidence suggests that cerebral injury may occur at survivable doses of radiation (204-215). Thus there is a profound need to critically assess this compartment for evidence of injury, and identify contributing mechanisms and long-term consequences.

SUMMARY OF RESEARCH

The overall purpose of this dissertation was to elucidate novel molecular effectors and contributing mechanisms to late-delayed RIBI, which occurs in patients receiving fWBI. Our secondary objective was to assess whether similar injury-associated patterns were present in animals receiving single, high dose TBI. These aims are evaluated in three separate studies, which follow as Chapters 2-4 of this dissertation. The studies were conducted in adult, male rhesus macaques (Macaca mulatta) which had previously received fWBI (40 Gy, 8x5 Gy fractions). The biologically effective dose (BED) of this regimen is 106.7 Gy, thus comparable to a treatment of 30 fractions of 2 Gy over 6 weeks

(BED 100.2 Gy), as may be used in human brain cancer patients (132).

In Chapter Two, we screen the involvement of seven a priori selected pathophysiologic mechanisms to RIBI in four Rhesus macaques (aged: 6-11 years) developed RIBI 4-6 months post-fWBI. Patterns in gene expression were evaluated in 92 marker transcripts via RT-qPCR. The overarching functional patterns assessed were: disruption to blood-brain-barrier structural components, angiogenesis, neuroinflammation, markers of hypoxia, DNA-damage repair, neurotransmission and extracellular matrix (ECM) deposition and remodeling. We determined that RIBI is associated with gene expression patterns indicative of macrophage/microglial-mediated neuroinflammation, impaired glutamatergic neurotransmission, hypoxia, ECM deposition and remodeling, and vascular maturation and stabilization. These effects were most prominent within white matter and no significant differences in gene expression were detected in the hippocampus.

In Chapter Three, we define the composition of the abnormally-accumulated perivascular ECM in these animals by immunohistochemistry and conclude that fibronectin is one of the primary components. Through in situ hybridization, we also determine that cerebral endothelial and vascular smooth muscle cells contribute to local fibronectin production. These findings suggest that pathways modulating fibronectin deposition and matrix assembly may be potential therapeutic targets in RIBI.

In Chapter Four, we expand our analyses to evaluate animals which had developed RIBI and survived until 12-months post-fWBI (n=5, aged: 7-13 years), and others which received single high dose TBI (n=5, aged: 10-13 years, irradiation doses:

6.75-8.05 Gy) 6-9 years prior to necropsy. RNA-sequencing and subsequent whole transcriptomic gene expression profiling revealed that animals receiving TBI and animals

18 receiving fWBI demonstrate similar transcriptomic evidence of CNS injury following cerebral exposure to radiation. Shared patterns consisted of increased antigen presentation and microglial/macrophage mediated-neuroinflammation, increased expression of complement factors, and impaired glutamatergic neurotransmission.

Pathways involved in ECM deposition and remodeling were implicated in animals receiving fWBI but not TBI.

In summary, we demonstrate RIBI is associated with persistent macrophage/microglial-mediated neuroinflammation, increased complement factor expression and reduced glutamatergic neurotransmission months to years post-irradiation in rhesus macaques. We also provide evidence that perivascular fibronectin accumulation may contribute to the pathogenesis of RIBI following fWBI and reaffirm that the rhesus macaque is a reasonable translational model which recapitulates the histopathologic and neurologic deficits of late-delayed RIBI. These data ultimately have led to a novel proposed mechanism of RIBI and identification of several potential therapeutic targets.

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1950-1997. Radiation research. 2003;160(4):381-407.

215. Kharchenko VP, Zubovskii GA, Kholodova NB. [Changes in the brain of persons who participated in the cleaning-up of the Chernobyl AES accident based on the data of radiodiagnosis (single-photon emission-computed radionuclide tomography, x-ray computed tomography and magnetic resonance tomography)]. Vestn Rentgenol Radiol.

1995(1):11-4.

CHAPTER TWO

Cerebrovascular remodeling and neuroinflammation is a late effect of radiation-induced

brain injury in nonhuman primates.

Rachel N. Andrewsa, Linda J. Metheny-Barlowb,c, Ann M. Peifferb,c, David B. Hanburyd,

Janet A. Toozee, J. Daniel Bourlandb,c, Robert E. Hampsonf, Samuel A. Deadwylerf, J.

Mark Clinea

aDepartment of Pathology, Section on Comparative Medicine Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA bDepartment of Radiation Oncology, Wake Forest School of Medicine, Winston-Salem, North Carolina Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA cBrain Tumor Center of Excellence Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA dDepartment of Psychology, Averett University, Danville, Virginia Averett University, 420 West Main Street, Danville, VA 24541, USA eDepartment of Biostatistical Sciences Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA fDepartment of Physiology & Pharmacology Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA

The following manuscript was published in Radiation Research 2017, 187(5): 599-611. doi: 10.1667/RR14616.1. This chapter is reprinted with permission from Radiation

Research Managing Editor Judy E. Fye. Stylistic variations are due to the requirements of the journal.

ABSTRACT

Fractionated whole brain irradiation (fWBI) is a mainstay of treatment for patients with intracranial neoplasia; however late-delayed radiation-induced normal tissue injury remains a major adverse consequence of treatment, with deleterious effects on quality of life for affected patients. We hypothesize that cerebrovascular injury and remodeling following fWBI results in ischemic injury to dependent white matter, which contributes to the observed cognitive dysfunction.

To evaluate molecular effectors of radiation-induced brain injury (RIBI), real- time quantitative polymerase chain reaction (RT-qPCR) was performed on the dorsolateral prefrontal cortex (DLPFC, Brodmann area 46), hippocampus and temporal white matter of 4 male Rhesus macaques (age 6-10 years) which had received 40 Gray

(Gy) fWBI (8 fractions of 5 Gy each, twice per week), and 3 control comparators. All fWBI treated animals developed neurologic impairment; humane euthanasia was elected at a median of 6 months. Radiation-induced brain injury was confirmed histopathologically in all animals, characterized by white matter degeneration and necrosis, and multifocal cerebrovascular injury consisting of perivascular edema, abnormal angiogenesis and perivascular extracellular matrix deposition.

Herein we demonstrate that RIBI is associated with white matter-specific up- regulation of hypoxia-associated lactate dehydrogenase A (LDHA) and that increased gene expression of fibronectin 1 (FN1), SERPINE1 and matrix metalloprotease 2

(MMP2) may contribute to cerebrovascular remodeling in late-delayed RIBI.

Additionally, vascular stability and maturation associated tumor necrosis super family member 15 (TNFSF15) and vascular endothelial growth factor beta (VEGFB) mRNAs

49 were increased within temporal white matter. We also demonstrate that radiation-induced brain injury is associated with decreases in white matter specific expression of neurotransmitter receptors SYP, GRIN2A and GRIA4. We additionally provide evidence that macrophage/microglial mediated neuroinflammation may contribute to RIBI through increased gene expression of the macrophage chemoattractant CCL2 and macrophage/microglia associated CD68. Global patterns in cerebral gene expression varied significantly between regions examined (p < 0.0001, Friedman’s test), with effects most prominent within cerebral white matter.

INTRODUCTION

Approximately 200,000 people per year receive fractionated whole brain irradiation (fWBI) for the treatment of primary or metastatic intracranial neoplasia (1), and late-delayed normal tissue injury is a major deleterious consequence. Fifty to 70% of patients that survive long enough to develop late-delayed RIBI (> 6 months) develop higher order cognitive dysfunction that negatively affects quality of life (2, 3).

Additionally, a subset of patients will develop ataxia, urinary incontinence and profound dementia (4) characterized by memory impairment and behavioral abnormality.

Multifocal cerebrovascular and white matter injury is associated with this loss of function

(5-7), and extent of white matter injury is correlated with degree of cognitive impairment

(8-10). There is currently no effective treatment, and the pathogenesis is unclear.

Studies evaluating mechanisms of late-delayed radiation injury in humans are confounded by the concurrent presence of neoplasia, treatment with chemotherapeutics, and comorbid factors such as hypertension and diabetes mellitus (11, 12). Rodents are often used as a model for humans, but differ neuroanatomically (e.g., they possess greater capillary network density and a significantly lower white:grey matter ratio(13, 14)).

Higher order cognition is difficult to evaluate in these species. Furthermore, rodents do not develop white matter necrosis following fWBI (15, 16), as do humans. We and others (7, 17-19) have demonstrated that Rhesus macaques (Macaca mulatta) that receive fWBI (total dose of 40-80 Gy, 2-5 Gy per fraction) develop cerebrovascular and white matter lesions which are spatially, temporally and morphologically similar to those observed in humans with late-delayed RIBI (19). Furthermore, these irradiated animals developed progressive cognitive dysfunction (20), particularly at high cognitive

51 workloads, and thus serve as a more human-like alternative to rodent models. Moreover, non-human primates (NHPs) possess greater neuroanatomic and genetic similarity to humans, which may increase translatability of findings (21).

In the present study, we hypothesize that radiation-induced cognitive dysfunction is partially mediated by abnormal cerebrovascular remodeling, resulting in subsequent ischemia to dependent brain parenchyma. In order to identify potential molecular effectors which may contribute late-delayed RIBI, we conducted RT-qPCR on 92 genes associated with neuroinflammation, angiogenesis, cerebrovascular remodeling, blood- brain barrier integrity, neurotransmission and extracellular matrix deposition. We anticipated increases in mRNAs associated with inflammation, angiogenesis and extracellular matrix deposition, and decreases in those associated with blood-brain-barrier integrity, neurotransmission, and vascular maturation and stability.

MATERIALS and METHODS

Brain Tissue and Subjects

Archived frozen and formalin fixed paraffin embedded brain tissues from 7 adult, male Rhesus macaques (6-11 years old, 5.3-8.7 kg) were used in this study for molecular and histologic analyses, respectively. Whole brain irradiated animals (n=4, aged 5.6-10.7 years, 7.5-8.7 kg) received 40 Gy fWBI 4-7 months prior to necropsy as part of a previous experiment. Housing, diet, and experimental procedures are detailed in Hanbury et al. (19). Control comparators (n=3, aged 5.5-6.6 years, 5.3-8.3 kg) had been part of an unrelated study in which they had received single dose, 10 Gy thoracic irradiation, and subcutaneous injections of vehicle (1 mL SQ, sterile phosphorus-buffered saline(PBS)), twice daily for 4 months, ceasing 4 months prior to necropsy. Housing, diet, euthanasia, and tissue collection were as for the irradiated group. Both experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals and were approved by the Wake Forest University School of Medicine Institutional Animal Care and Use Committee (IACUC). The Wake Forest University School of Medicine is accredited by the Association for Assessment and Accreditation of Laboratory Animal

Care (AAALAC) and conforms to all state and federal animal welfare laws.

Irradiation

Fractionated whole brain irradiation procedures are as described in Hanbury et al.(19) and Robbins et al. (20). Briefly, NHP were sedated with ketamine HCl (15 mg/kg body weight, intramuscularly), and maintained on isoflurane gas (3% induction, 1.5% maintenance) in 100% oxygen during irradiation. A total of 40 Gy mid-plane was given

(8 fractions x 5 Gy/fraction; 2 fractions/wk x 4 wks; nominal dose rate of 4.9-5.4

Gy/min) using a clinical linear accelerator and opposed lateral fields of 6MV x rays.

Control comparators received 10 Gy, single dose thoracic irradiation delivered to the anterior-posterior thoracic mid-plane as part of a previous study. 6 MV x rays were delivered with parallel opposed anterior-posterior fields from a clinical linear accelerator at a nominal dose rate of 4 Gy/min. The field of irradiation included the heart, mediastinum and superior, lateral and inferior lung fields extending up to 4 cm caudal to the xyphoid. The maximum dose administered to the brain in thorax-only irradiated animals is estimated to be 0.1 Gy.

Clinical and Cognitive Assessment

Animals were assessed daily by trained technical staff during the study period; any animal developing impairment received veterinary neurologic evaluation, and assessment using a standardized cage-side neurologic checklist. Humane euthanasia was provided for animals with severe neurologic impairment. All whole brain irradiated animals (n=4) underwent daily cognitive assessment during the study period, using a

Delayed-Match-To-Sample task (DMS), the procedure for which has been described previously (20).

Tissue Collection, Preparation and Histopathology

Four to 7 months following irradiation, the animals were humanely euthanized in accordance with the American Veterinary Medical Association’s Guidelines on

Euthanasia (22) by deep anesthesia with pentobarbital, followed by exsanguination and perfusion of the vascular system with 2 liters of cold normal saline. The entire brain was

54 removed intact, placed in a stainless steel brain matrix with cutting guides, and sectioned coronally in 4 mm intervals. Once removed from the matrix, all slices were photographed, and then alternating sections either immediately frozen on dry ice or immersed in 4% cold paraformaldehyde for 24 hours. Fixed brain tissues were transferred to cold 70% ethanol solution until embedding.

Fixed brain tissues from 8 standard regions were then selected and trimmed in accordance with Society for Toxicologic Pathology’s Recommended Practices for

Sampling and Processing the Nervous System (23), with the addition of prefrontal cortex, and embedded in paraffin and sectioned coronally at 4 um. Sections were stained with hematoxylin and eosin and examined histologically by a board certified veterinary pathologist (JMC). Lesions were scored as described previously (19); absent (0), minimal

(1 = inflammatory or vascular changes without disruption of the neuropil or clear neuronal loss); mild (2 = focal vascular injury and inflammation with loss of neuropil or neurons and microglial activation); moderate (3 = extensive or multifocal vascular injury, hemorrhage, disruption of the neuropil, neuronal loss and microglial activation); or severe (4 = extensive or multifocal vascular injury, hemorrhage, disruption of the neuropil, neuronal loss and microglial activation, with additionally extensive zones of necrosis within neural tissue). The presence of vascular lesions in each region was recorded as follows: a: endothelial hypertrophy; b: perivascular extracellular matrix deposition, c: perivascular edema, d: disorganized vascular morphology. The assessor was blinded to the irradiation status of the animals.

Molecular assessment of cerebral gene expression

Relative gene expression was evaluated by RT-qPCR within 3 brain regions

[dorsolateral prefrontal cortex (Brodmann area 46), hippocampus and temporal white matter] from all animals. Ribonucleic acid (RNA) was isolated by homogenization of 60-

120 mg of brain tissue in TRI Reagent (Molecular Research Center, Inc.; Cincinnati, OH,

USA), followed by phase separation with 1–bromo–3–chloropropane (BCP). RNA was purified by RNeasy Mini kit (Qiagen; Valencia, CA, USA). RNA content and purity was determined at 260 nm, and with 260/280 ratios between 2.0 and 2.2, as obtained by

Nanodrop spectrophotometry (Thermo Scientific, Wilmington, DE, USA). Absence of genomic DNA contamination was confirmed during RT-qPCR by RT² qPCR Primer

Assay for Rhesus Macaque Genomic DNA (gDNA) Contamination, (Qiagen), and run concurrently with all samples. Amplification for all samples was absent or below the limit of detection (>35 Ct). cDNA was synthesized by RT2 First Strand kit (Qiagen;

Valencia, CA, USA); reaction efficiency was confirmed by Reverse Transcriptase

Control within each plate and run concurrently with all samples.

RT-qPCR was performed on an ABI 7500 Fast system (Applied Biosystems,

USA), using a Qiagen Custom RT2 Profiler Array utilizing Rhesus macaque specific primers. Plates were designed to assess 92 target genes, selected for their roles in inflammation, angiogenesis, blood-brain-barrier integrity, extracellular matrix deposition, neurotransmission, hypoxia and DNA damage repair (Table I). Standard nomenclature as designated by the HUGO Gene Nomenclature Committee (HGCN) (25) is utilized.

Table I: Classification of selected markers in RT-qPCR profiler gene expression array

Blood-Brain Angiogenesis, Neuro- Hypoxia DNA Neurons Extracellular Barrier Vascular inflammation Damage and Neuro- Matrix Structural Maturation transmission Deposition Integrity and and Stability and Transporters Remodeling

HGNC ABCB1 ANGPT1 AIF1 ADM CHEK1 DCX FN1 Symbol ABCC9 ANGPT2 CCL2 ARNT CHEK2 GRIA2 HSPG2

AGRN DLL4 CD34 EPAS1 H2AFX GRIA4 LAMA4

AQP4 FIGF CD3G EPO NBN GRIN2A LAMA5

CDH5 FLT1 CD68 FAS GRIN2B LAMB2

CLDN12 KDR IFNG HIF1A SYP LAMC1

CLDN3 MDK IL10 HMOX1 MMP10

CLDN5 NOTCH3 IL1B HMOX2 MMP12

CTNNB1 PDGFB IL6 LDHA MMP2

F11R PDGFRB MS4A1 MMP3

GFAP S1PR1 NFKB1 NID1

GJA1 S1PR2 NOS2 PLAT

JAMB S1PR3 PECAM1 SERPINE1

JAMC S1PR4 PTGS2 TGFB1

KCNJ10 S1PR5 TFRC TIMP2

KCNJ8 TEK TNF TIMP3

OCLN TNFSF15

SLC2A1 VEGFA

TJP1 VEGFB

ZO-2 VEGFC

WNT1

Calculation of Changes in Gene Expression

For each sample, the threshold cycle (Cts) for genes of interest and housekeeping genes were determined., the limit of detection was 35 Ct. Claudin-3 and S1PR4 were excluded from analyses as Ct values were below the threshold of detection for all samples. Target gene expression was normalized to the geometric mean of two control genes (β-actin and hypoxanthine-guanine phosphoribosyltransferase) in each sample; the

∆Ct [Ct (target gene)-Ct (housekeeping gene(s))] was calculated to quantitate relative gene expression. Fold change (2-∆∆Ct ) was calculated by as described by Livak (26).

Statistics

Statistical analyses were performed under the guidance of a professional statistician (JAT) and in Statistica 13 (StataCorp, College Station, TX: StataCorp LP) unless noted. ∆Ct values were used for all comparisons. The effect of brain region on relative gene expression was evaluated by Friedman’s test in GraphPad Prism (version

6.07 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com) followed by paired T-tests or Wilcoxon Signed Rank for parametrically and non- parametrically distributed data, respectively. Gene expression with respect to irradiation status was stratified by region and evaluated as follows: Normality and equality of variances were assessed by Shapiro-Wilk and Levene’s tests respectively. Non- parametric data were compared by Mann Whitney U. Parametric data were compared by

Student’s T and Welch’s tests for data with equal and unequal variances, respectively.

Fold changes ≥ ±2 and p values < 0.01 were considered significant to adjust for the small sample size. As these data were considered preliminary analyses, no adjustments for multiple comparisons were made.

RESULTS

Clinical Parameters

All four whole brain irradiated animals were euthanized during the study period

(4.3-6.6 months post-irradiation, mean: 5.6 months) due to the development of severe neurologic signs (Table II). Concurrent unilateral inner ear infection precluded interpretation of the clinical signs in NHP1, as impaired vestibular function secondary to otitis media/interna could not be clearly distinguished from a central nervous system lesion affecting the vestibular system. Neurologic deficits in the remaining three animals were attributed to late-delayed radiation-induced brain injury, as no other inciting cause or evidence of local or systemic disease was identified clinically or histopathologically.

Table II: Summary of clinical neurological deficits in irradiated NHP

Age Onset Survival interval Subject (years) (post-irradiation) Neurologic Signs (post-irradiation) NHP1 10.7 4.4 months Ataxia, dysphagia 5.3 months Concurrent mastoiditis and otitis media/interna NHP2 5.7 4.0 months Visual impairment (incomplete menace 4.3 months response and impaired object tracking) Progressive ataxia and lethargy

NHP3 6.8 5.0 months Ataxia, weakness and intermittent head- 6.0 months pressing NHP4 5.6 5.5months Decreased dexterity, progressive ataxia 6.6 months

As control comparators had received single, high dose thoracic irradiation as part of a previous study, clinical data regarding cardiac and respiratory function were reviewed prior to use of archived tissues. Neurologically these animals were normal.

Respiratory rate, heart rate, pulse oximetry and ejection fraction as determined by echocardiography were within normal limits until euthanasia.

Histopathology

All fWBI treated animals demonstrated histopathologic lesions consistent with late-delayed RIBI, including white matter degeneration, necrosis and cerebrovascular injury consisting of perivascular edema with or without protein deposition, perivascular fibrosis, endothelial hypertrophy and disorganized angiogenesis (Fig 1). Individual animals differed in lesion severity and distribution of vascular morphologic abnormality

(Table III), although forebrain white matter was affected in all animals examined.

Figure 1: Cerebrovascular pathology in late-delayed radiation-induced brain injury.

Histopathology illustrating the spectrum of vascular injury in animals with late-delayed radiation-induced brain injury. A) Normal arteriole from an non-irradiated animal. B) Perivascular accumulation of brightly eosinophilic protein and fibrin. C) Disorganized angiogenesis characterized by haphazardly arranged, tortuous small caliber vessels, often lacking distinct lumina. Endothelial nuclei are enlarged with prominent nucleoli. D) Accumulation of fibrous extracellular matrix surrounding arterioles. Reactive (gemistocytic) astrocytes are numerous within the surrounding parenchyma. E) Pericapillary fibrous extracellular matrix, similar to that in D.

Table III: Assessment of white matter necrosis and cerebrovascular remodeling in late- delayed radiation induced brain injury

ANIMAL ID NHP1 NHP2 NHP3 NHP4

Lesion Type Necrosis Vascular Necrosis Vascular Necrosis Vascular Necrosis Vascular

Region/Structure

Forebrain: +++ a,b,d ++ a,b ++++ a,b,d ++ b,c White matter

Forebrain: ++ a,d + a,b,d ++ a,d - - Cortical grey matter

Prefrontal cortex - - - - ++ a,d - -

Basal ganglia/striatum + ------

Hippocampus - - + a,b,d - - - -

Thalamus na na ++++ a,b,c,d ++++ a,b,d - -

Midbrain ++++ b ++ a,d ++ a,b,d - -

Cerebellum + - - - + - - -

Brainstem +++ - ++ a,b +++ b na na

Spinal cord na na + b + - - - a endothelial hypertrophy b perivascular extracellular matrix deposition c perivascular edema d disorganized vascular morphology na: not assessed, tissue unavailable for analysis

Regional Effects of Irradiation on Gene Expression

The effect of irradiation on relative gene expression differed with respect to region (p < 0.0001, Friedman’s test); further analyses were stratified by region. No significant change in hippocampal gene expression (p > 0.01 for all genes examined) was

61 associated with late-delayed RIBI. Mean fold changes were of the greatest magnitude within temporal white matter (-19 to 49 fold).

Deposition of Extracellular Matrix

RIBI was associated with increased expression of fibronectin 1 (FN1: DLPFC: 3.2 fold up-regulated, p < 0.01; WM: 4.6 fold up-regulated, p < 0.0001), Serpin E1

(SERPINE1: WM: 20.8 fold up-regulated, p < 0.0006) and matrix metallopeptidase 2

(MMP2: DLPFC: 6.5 fold up-regulated, p < 0.003; WM: 34.6 fold up-regulated, p <

0.001), factors associated with extracellular matrix deposition and remodeling (Fig. 2).

No significant changes were observed in expression of TGFB1, nidogen 1, HSPG2, tissue plasminogen activator, or any of the laminins, tissue inhibitors of matrix metalloproteinases, and other matrix metalloproteinases evaluated.

Figure 2: Gene expression changes associated with extracellular matrix deposition. )

t # p < 0 .0 0 0 4 * p < 0 .0 0 0 6

C # p < 0 .0 0 5

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Late-delayed radiation-induced brain injury was associated with up-regulated expression of MMP2 (35 fold), SERPINE1 (21 fold) and fibronectin1 (3 fold) within white matter and MMP2 (6 fold) and fibronectin1 (3 fold) within dorsolateral prefrontal cortex. Bars denote mean fold change, individual points represent gene expression values for each animal. Dashed line indicates 2 fold-expression change. DLPFC: Dorsolateral prefrontal cortex, HC: Hippocampus, WM: Temporal white matter. * p-value indicates with the effect of radiation on relative-gene expression; # p-value signifies differences in regional gene expression with respect to irradiation status.

Angiogenesis, Vascular Maturation and Stabilization

The relative expression of factors associated with vascular stability and survival

(tumor necrosis factor super family member 15 (TNFSF15: 44.4 fold up-regulated, p <

0.003) and vascular endothelial cell growth factor beta (VEGFB: 2.3 fold up-regulated, p

< 0.002)) was increased within white matter in late-delayed RIBI (Fig. 3).

Figure 3: Increased expression of vascular maturation and stability-assosciated factors

# p < 0 .0 0 5 # p < 0 .0 0 2

# p < 0 .0 0 4 ) t

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Expression of TNFSF15 and VEGFB mRNAs was up-regulated within temporal WM in late-delayed RIBI, 44 and 2 fold respectively. Bars denote mean fold change, individual points represent gene expression values for each animal. Dashed line indicates 2 fold-expression change. DLPFC: Dorsolateral prefrontal cortex, HC: Hippocampus, WM: Temporal white matter. *p-value indicates with the effect of radiation on relative-gene expression; # p-value signifies differences in regional gene expression with respect to irradiation status.

Irradiation had no significant effect on other members of the vascular endothelial cell growth factor (VEGF) family (VEGFA, VEGFC, VEGFD or associated receptors

KDR and FLT1). No significant changes were seen in other molecules associated with angiogenesis and vascular maturation and stabilization [angiopoietin1 or 2 (ANGPT1,

ANGPT2), DLL4, FIGF, midkine (MDK), NOTCH3, PDGFB, PDGFRB, sphingosine-1- phosphate receptors 1-5, TEK, or WNT1].

Neuroinflammation

Relative expression of macrophage/microglia associated CD68 and CCL2 was up- regulated within both DLPFC (CCL2: 30.5 fold up-regulated, p < 0.002) and temporal

WM (CCL2: 49.0 fold up-regulated, p < 0.003; CD68: 27.8 fold up-regulated, p < 0.005)

(Fig 4B). Endothelial associated PECAM1 and lymphocyte associated CD3G expression was also increased within temporal WM (PECAM1: 4.8 fold up-regulated, p < 0.0005;

CD3G: 23.2 fold up-regulated, p < 0.0003) (Fig. 5A-B). fWBI had no significant effect on the relative gene expression of inflammatory mediators IL6, AIF1, IFNG, TNF,

COX2, NOS2, IL10, NFKB1, transferrin (TFRC) or B-cell associated MS4A1.

Figure 4: Evidence of macrophage/microglial mediated neuroinflammation.

A) Focal white matter necrosis with axonal degeneration, mineralization and infiltration by macrophages/microglia. B) Late-delayed RIBI was associated with increased gene expression of CD68 (28 fold) and CCL2 (49 fold) within temporal white matter. Expression of CCL2 was also increased within dorsolateral prefrontal cortex CCL2 (31 fold up-regulated). Bars denote mean fold change, individual points represent gene expression values for each animal. Dashed line indicates 2 fold-expression change. DLPFC: Dorsolateral prefrontal cortex, HC: Hippocampus, WM: Temporal white matter. p-value indicates with the effect of radiation on relative-gene expression; # p-value signifies differences in regional gene expression with respect to irradiation status'

Hypoxia

Expression of LDHA (2.9 fold up-regulated, p < 0.004) and FAS (12.7 fold- upregulated, p < 0.0004) was increased within WM in irradiated animals (Fig. 5C-D). No significant changes were observed in the relative expression of hypoxia-inducible factor family members (HIF1a, EPAS1, ARNT), erythropoietin (EPO), or heme oxygenase 1 or

2 (HMOX1, HMOX2).

Figure 5: Additional changes in gene expression.

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A) Gene expression of endothelial inflammation associated PECAM1 was 4.8 fold increased within temporal WM. B) Expression of T-lymphocyte associated CD3G was 23.2 increased within WM. C) Hypoxia associated LDHA expression was 2.9 fold increased within temporal WM. D) Late-delayed RIBI was associated with a 12.7 fold increase in gene expression of cell surface death receptor FAS in temporal WM. Bars denote mean fold change, individual points represent gene expression values for each animal. Dashed line indicates 2 fold-expression change. DLPFC: Dorsolateral prefrontal cortex, HC: Hippocampus, WM: Temporal white matter. *p-value indicates with the effect of radiation on relative-gene expression; # p-value signifies differences in regional gene expression with respect to irradiation status.

Neurotransmission

RIBI was associated with decreased expression of neurotransmission associated synaptophysin (SYP: 3.4 fold down-regulated, p < 0.004), glutamate receptor, ionotropic,

AMPA4 (GRIA4: 6.7 fold down-regulated, p < 0.007) and glutamate ionotropic receptor

NMDA type subunit 2A (GRIN2A: 6.7 fold down-regulated, p < 0.01) in white matter

(Fig. 6). There was no significant effect on the expression of GRIA2 and GRIN2B.

Figure 6: Decreases in gene expression associated with neurotransmission.

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Late-delayed radiation-induced brain injury was associated with decreased expression of neurotransmission associated synaptophysin (-3.4 fold) , and glutamatergic neurotransmitter receptor mRNAs for GRIA4 (-6.7 fold) and GRIN2A (-6.7 fold) within white matter. Bars denote mean fold change, individual points represent gene expression values for each animal. Dashed line indicates 2 fold-expression change. DLPFC: Dorsolateral prefrontal cortex, HC: Hippocampus, WM: Temporal white matter.*p-value indicates with the effect of radiation on relative-gene expression; # p-value signifies differences in regional gene expression with respect to irradiation status

Blood Brain Barrier Structural Integrity

Late-delayed RIBI did not affect the relative gene expression of astrocyte- associated GFAP, AQP4, Connexin 43 or Agrin. No significant effect was observed on relative gene expression of endothelial cell-cell junction associated VE-Cadherin, B-

Catenin, zona occludens 1 and 2, claudins 5 and 12, occludin, and junctional adhesion

66 molecules 1, 2 and 3. Expression of claudin 3 was below the level of detection in all samples. fWBI did not affect the relative gene expression of channel associated KCNJ10,

KCNJ8, ABCB1 or ABCC9.

DNA Damage

No significant changes were observed in any of the markers examined associated with DNA damage and repair [CHEK1, CHEK2, Nibrin (NBN), and H2AFX].

DISCUSSION

These data reaffirm that non-human primates develop multifocal cerebrovascular and white matter lesions in late-delayed RIBI that are consistent with those observed in human patients. As the primary clinical manifestation of late-delayed RIBI in patients is progressive cognitive decline (2, 3), the parent study for all whole brain irradiated animals (n=4) included daily cognitive assessment as used previously (20). As neurological decline necessitated humane euthanasia prior to experimental endpoint, cognitive data were incomplete and full statistical analysis of cognition for the animals in the present cohort was not possible. Task measured cognitive decline preceded ataxia observations; however, as the ataxia developed, animals stopped working the task.

Previous data have demonstrated working memory impairment beginning four months post-irradiation (20).

Performance on this DMS task is reliant on processing by both the prefrontal cortex (rule based) and hippocampus (working and/or short-term); thus the impaired cognitive function cannot be localized to either brain region solely on the basis of DMS task outcome. Rather, impaired performance on this task necessitates error analysis of the behavioral task and/or further experimental manipulations to determine the mechanism of impaired performance. In other studies, extra-hippocampal injury (prefrontal cortical lesioning) alone is sufficient to impair performance on the DMS task (27), and electrophysiology demonstrates that inter-laminar processing by the DLPFC is required for successful execution of the task (28). These injuries suggest that NHPs err when following the wrong task rule, as directed by the DLPFC, rather than incorrectly remembering due to hippocampal processing. This, in conjunction with our demonstrated

68 regional patterns in gene expression, challenges the postulation that hippocampal-sparing irradiation alone will be sufficient to spare cognitive function after fWBI (29, 30).

Our gene expression data reveal that whole brain irradiation does not result in significant changes in hippocampal gene expression for our selected targets. Rather, fWBI results in patterns of gene expression indicating that cerebrovascular remodeling, neuroinflammation and alterations in neurotransmission contribute to late-delayed RIBI, and that these changes most prominently affect white matter.

Our molecular data indicate that fibronectin 1, MMP2 and SERPINE1 may contribute to vascular remodeling in RIBI and influence the deposition of perivascular extracellular matrix. Fibronectin, exuded in soluble form from the vascular compartment as a component of blood plasma or secreted by endothelial cells, is a major component of extracellular matrix following cell-mediated assembly, and its expression is up-regulated in a number of fibrotic diseases (31, 32). Additionally, fibronectin triggers the activation of microglia in vitro (33), and thus may stimulate macrophage/microglial activation in

RIBI. Fibronectin is also increased in the cortex(34) and plasma(35) of humans with clinical and subclinical Alzheimer’s disease. The cortical deposition of fibronectin may play a role in the pathology of cognitive dysfunction.

Both fibronectin and SERPINE1 expression may be influenced by Rho\ROCK signaling (36, 37), suggesting a common pathway of induction. Increased SERPINE1 expression in late-delayed radiation-induced brain injury is known to contribute to perivascular fibrosis through the inhibition of fibrinolysis (38, 39) and stimulation of fibronectin matrix assembly through interaction with αvβ5 and α5β1 integrins (40).

Furthermore, endothelial cell death in radiation-induced intestinal injury is dependent on

SERPINE1 expression (41), which serves as a potential explanation for the decreases in microvascular density observed following fWBI in rat studies (16, 42).

Increased expression of the gelatinase MMP2 is associated with blood-brain barrier disruption in rodent models of focal transient cerebral ischemia (43), leukemia

(44), and pneumococcal meningitis (45), and in human brain tissue affected by cerebral amyloid angiopathy-associated hemorrhagic stroke (46). Additionally, MMP2 expression and activity is increased in rodents receiving high, single dose whole-brain irradiation (10

Gy) and fWBI (40 Gy, 8 fractions x 5 Gy/fraction) and associated with the degradation of type IV collagen (47), which may contribute to blood-brain barrier breakdown and perivascular edema in RIBI. In addition to the potential of MMP2 to degrade extracellular matrix, increased expression and activation contributes to cardiac, hepatic and renal fibrosis (48-50). We postulate that MMP2 contributes to perivascular fibrosis in RIBI by remodeling the deposited substrate.

The perivascular deposition of extracellular matrix components, including fibronectin, may create a diffusion barrier and induces ischemic injury to the dependent brain parenchyma. We propose that cerebral white matter is most vulnerable to this ischemic injury, as the cerebrovascular architecture transitions from larger bore penetrating arterioles and venules to a predominately capillary network within white matter (51) and capillary density is decreased relative to grey matter(52). Our hypothesis is supported by the white matter-specific increased relative expression of LDHA, an enzyme which catalyzes the conversion of L-lactate to pyruvate in the final step of anaerobic glycolysis. While there were no significant changes in relative gene expression

70 amongst members of the hypoxia-inducible factor family (HIF1A, EPAS, ARNT), regulation of these factors most commonly occurs at the protein level (53, 54), which may explain lack of change at the transcriptomic level. Additionally, hypoxia induces the expression of both SERPINE1 (55) and fibronectin (56, 57), and thus hypoxia may potentiate perivascular fibrosis in radiation-induced brain injury. Hypoxia is known to induce the expression of cell-surface death receptor Fas (58), however the white matter- specific up-regulation of Fas is difficult to interpret as it is involved in many physiologic processes necessitating apoptosis.

As histologic evaluation of tissue revealed disorganized vasculature with reactive nuclei, reminiscent of endothelial proliferation, we anticipated increased expression of markers associated with the induction of angiogenesis. On the contrary, we found no increase in markers associated with new blood vessel formation, but increased expression of VEGFB and TNFSF15, factors involved in vascular quiescence and stability. VEGFB, unlike other members of the vascular endothelial growth factor (VEGF) family, does not induce the formation of new blood vessels, but is critical for the survival and maintenance of newly formed and pre-existing vasculature (59). Additional evidence from rodent studies suggests that VEGFB participates in vascular repair following brain injury (60). Circulating TNFSF15 also increased following brain injury, and greater blood levels of TNFSF15 as compared to VEGFA was associated with higher rates of recovery in patients (61). Furthermore, rodent studies demonstrated that administration of

TNFSF15 to mice in an experimental model of traumatic brain injury resulted in decreased neuroinflammation, and increased blood-brain barrier integrity with decreased permeability (62). Further characterization of these tissues including assessment of

71 microvascular area fraction is required to meaningfully interpret the alterations in angiogenic gene expression; however, we postulate that increased expression of VEGFB and TNFSF15 may represent an adaptive response to ongoing cerebrovascular injury.

Neurotransmission-associated synaptophysin, GRIA4 and GRIN2A are most prominently decreased within irradiated white matter. This finding contradicted our initial hypothesis that decreases in markers associated with synapses and neurotransmission would be most prominent within the cortex and hippocampus, where the neuron soma and synapses themselves reside. We propose these decreases in expression reflect a loss of deep white matter synapses, sites in which axons communicate directly to NG2+ oligodendrocyte progenitor cells (OPCs) via vesicular release of the neurotransmitter glutamate (63-65). Whether the observed reduction in

GRIN2A and GRIA4 mRNAs is a reflection of axonal loss or a reduction in number of

NG2+ precursor cells requires further investigation, particularly in non-human primates.

Rodent data are conflicting. Studies completed in rats demonstrate that following initial depletion, NG2+ OPC populations in irradiated spinal cord begin proliferating by 2 weeks following high single dose irradiation (66), with recovery to near normal numbers by 6 weeks (66, 67) to 6 months (68). In contrast, studies in mice demonstrate that OPC populations are less severely affected following irradiation, but lack the capacity to repopulate the irradiated area (69). While both astrocytes and NG2+ precursors express

GRIA4 (70, 71), the observed decreased relative expression is not presumed to be associated with decreases in total astrocyte number, as GFAP, AQP4 and AGRN were not significantly altered. We additionally infer from these data that disruption of the

72 blood-brain barrier is less likely to be due to transcriptionally mediated alterations of astrocyte associated integrity.

Instead, the increased expression of CCL2 and CD68, in conjunction with the aggregates of macrophages/microglia observed on histopathology and infiltration of parenchyma by IBA-1 positive cells in our previous work (19), indicates that RIBI occurs in an environment of macrophage/microglial mediated neuroinflammation. Increased expression of CCL2 directly induces the proliferation and activation of microglia (72,

73), and further drives neuroinflammation by stimulating the recruitment of circulating monocytes into the CNS parenchyma (73). CD68 expression, in addition to its historical use as a marker of macrophages and microglia, is also associated with the phagocytic capacity of a cell (74). Accordingly, the white matter specific increase in CD68 expression may correlate to the histologically observed necrosis, a process necessitating phagocytosis and debris removal. Furthermore, enhanced phagocytic capacity is associated with microglial activation (75) that in turn produces a number of pro- inflammatory cytokines (76-78), which may contribute to a sustained pro-inflammatory milieu.

In addition to macrophage/microglial activation and monocyte recruitment, CCL2 increases the expression and surface localization of PECAM1 within endothelial cells

(79). Further studies in cell culture and mouse models demonstrate that PECAM1 facilitates the endothelial transmigration of leukocytes, including CD3+ T-lymphocytes

(80-82). Thus, increased expression of PECAM1 may contribute to the lymphocytic perivascular aggregates observed histologically in RIBI (83, 84), which we believe correspond to the increased expression of CD3G observed in this study. We do not

73 believe that the concurrent otitis media/interna in one animal affected neuroinflammatory gene expression, as the infection did not extend within the cranial vault and no outliers were detected during statistical analysis. Further characterization of the neuroinflammatory contributors, including localization, quantification and immunophenotyping is pending and will be the focus of our future studies.

While these data provide insight into transcriptional changes in radiation-induced brain injury, further characterization of these factors at both the transcriptional and protein level is required to determine cell of origin and tissue compartment distribution.

Additionally, we acknowledge that these analyses are correlative and do not necessarily indicate causation, but indicate potential molecular effectors and provide insight on possible pathogenic mechanism. Further study is required to elucidate the complex role of these molecules in radiation-induced brain injury; however the factors presented may represent potential targets for therapeutic intervention.

We also acknowledge that the tissues in the present study were acquired from young adult NHP and do not fully recapitulate the aged patient population (approximate age: 60 years) (85-87) receiving fWBI. Retrospective analysis of patient populations indicate that aged patients receiving fWBI have poorer survival than younger patient subpopulations (88, 89), although these results are confounded by tumor progression, treatment regimen, concurrent medical illness, and neurologic comorbidity. Data regarding the neuroinflammatory effects of fWBI on the aging brain are conflicting (15,

90-92), and warrant additional study.

Finally, although gene targets were categorized according to function, we recognize that the selected targets do not behave independently and may contribute to several physiologic processes. Nonetheless, these are the first reported cerebral gene expression changes from NHP receiving a clinically-relevant course of fractionated whole brain irradiation, and set the groundwork for future studies into the molecular mechanisms underlying RIBI and associated cognitive impairment.

ACKNOWLEDGEMENTS

The authors would like to thank Lisa O’Donnell for her invaluable guidance in mRNA extraction and gene expression analysis. This was supported by 1R01CA155293-

01A1 (Robbins/Deadwyler). Dr. Andrews was supported by NIH grant T32 OD010957.

Drs. Bourland, Hanbury, Peiffer, and Cline were also supported by NIH grant U19-

AI67798 awarded to Nelson Chao, Duke University (JMC: primate core leader, Wake

Forest School of Medicine). Dr. Tooze is supported by the Biostatistics and

Bioinformatics Shared Resource, NCI Cancer Center Support Grant P30 CA012197.

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16.

CHAPTER THREE

Fibronectin produced by cerebral endothelial and vascular smooth muscle cells

contributes to perivascular extracellular matrix in late-delayed radiation-induced brain

injury.

Rachel N. Andrewsa, David L. Caudella, Linda J. Metheny-Barlowb,c, Ann M. Peifferb,c,

Janet A. Toozed, J. Daniel Bourlandb,c, Robert E. Hampsone, Samuel A. Deadwylere, J.

Mark Clineb,c

aDepartment of Pathology, Section on Comparative Medicine Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA bDepartment of Radiation Oncology, Wake Forest School of Medicine, Winston-Salem, North Carolina Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA cBrain Tumor Center of Excellence Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA dDepartment of Biostatistical Sciences Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA eDepartment of Physiology & Pharmacology Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA

This manuscript has been submitted to Radiation Research and is under revision. Stylistic variations are due to the requirements of the journal.

ABSTRACT

Late-delayed radiation-induced brain injury (RIBI) is a major adverse effect of fractionated whole-brain irradiation (fWBI). Characterized by progressive cognitive dysfunction, and associated cerebrovascular and white matter injury, RIBI deleteriously impacts quality of life for cancer patients. Despite extensive morphological characterization of the injury, the pathogenesis is unclear, thus limiting the development of effective therapeutics.

We previously reported that RIBI is associated with increased gene expression of the extracellular matrix (ECM) protein fibronectin (FN1). We hypothesized fibronectin contributes to perivascular ECM, which may impair diffusion to the dependent parenchyma, thus contributing to the observed cognitive decline. Herein, we sought to determine the localization of fibronectin in RIBI and further characterize the composition of perivascular ECM, as well as identify the cell of origin for FN1 by in situ hybridization. Briefly, fibronectin localized to the vascular basement membrane of morphologically normal blood vessels from control comparators and animals receiving fWBI, and to the perivascular space of edematous and fibrotic vascular phenotypes of animals receiving fWBI. Additional mild diffuse parenchymal staining in areas of vascular injury suggested blood-brain-barrier disruption and plasma fibronectin extravasation. Perivascular ECM lacked amyloid and contained lesser amounts of collagens I and IV, which localized to the basement membrane. These changes occurred in the absence of alterations in microvascular area fraction or microvessel density.

Fibronectin transcripts were rarely expressed in control comparators, and were most

90 strongly induced within cerebrovascular endothelial and vascular smooth muscle cells following fWBI.

Our results demonstrate that fibronectin is produced by cerebrovascular endothelial and smooth muscle cells in late-delayed RIBI and contributes to perivascular

ECM, which we postulate may contribute to diffusion barrier formation. We propose that pathways that antagonize fibronectin deposition and matrix assembly or enhance degradation may serve as potential therapeutic targets in RIBI.

INTRODUCTION

Approximately 200,000 patients per year receive fractionated whole-brain irradiation (fWBI) for the treatment of intracranial neoplasia (1). Of patients that survive long enough to develop late-delayed radiation-induced brain injury [RIBI, (> six months)], 50-90% will demonstrate higher order cognitive dysfunction which adversely affects patient day-to-day function and quality of life (2-4). This loss of function is associated with progressive cerebrovascular and white matter injury (5, 6).

Cerebrovascular manifestations of injury include perivascular edema suggestive of ongoing injury, as well as perivascular extracellular matrix (ECM) accumulation

(fibrosis) indicative of chronicity. Despite numerous studies characterizing the morphology of RIBI (7-12), the pathogenesis and contributing mechanisms remain unclear, thus limiting the development of effective therapies for affected patients.

Studies investigating the pathogenesis of RIBI in humans are confounded by the concurrent presence of neoplasia or other comorbid conditions such as diabetes mellitus and hypertension (13, 14). We and others have shown that rhesus macaques (Macaca mulatta) that receive fWBI (total dose of 40-80 Gy, 2-5 Gy per fraction) recapitulate the histologic features and progressive cognitive impairment (7, 8, 15-17) observed in humans with RIBI. While rodent models have been used to investigate the pathogenesis and mitigation of RIBI, rodents differ in neuroanatomic (18) and cerebrovascular structure (19) compared to primates. Moreover, rodents do not develop white matter necrosis following fWBI (20, 21), which may limit translatability of findings. Therefore, the NHP model is a reasonable alternative in which to investigate the pathogenesis of

RIBI.

One of the hallmark histopathologic lesions of RIBI is multifocal, spatially stochastic, perivascular ECM accumulation with predilection for white matter (9, 22-24).

Similar perivascular fibrosis is associated with impaired blood flow in heart disease (25) and contributes to impaired gas exchange in interstitial lung disease (26-28). Thus, we postulate that perivascular ECM accumulation may be a key event in the pathogenesis of

RIBI, resulting in diffusion barrier formation and subsequent ischemic injury to dependent white matter. Necrosis and disruption of white matter neurotransmission negatively impacts cognition.

ECM is a heterogeneous amalgamation of proteins, proteoglycans, glycoproteins and other molecules. Therefore, our previous gene expression study included preliminary screening of a several ECM contributors in a non-human primate model of RIBI (NHP)

(29) as mechanisms of degradation, accumulation, and remodeling vary by target ECM molecule and implicate different signaling pathways for future analysis. Of the ECM components evaluated, which included several laminins, proteoglycans, and nidogen, only fibronectin (FN1) was differentially regulated in RIBI (29). Thus we hypothesized that fibronectin is a key contributor to ECM in RIBI. While the majority of fibronectin in the body is produced in the liver and circulates as a component of blood plasma (30), detection of FN1 mRNAs suggested a component of local production. We further hypothesized that the cell of origin would be the cerebral endothelial cell. In the present study we sought to localize fibronectin and further characterize the composition of perivascular ECM and to identify the cell of origin for FN1 transcripts. Additionally, we examined the gene expression of several factors which contribute to fibronectin matrix deposition and assembly in an effort to identify potential molecular mechanisms of RIBI.

MATERIALS AND METHODS

Tissue and Subjects

Quantitative immunohistochemical and gene expression analyses were performed on the same animals as in Andrews et al. (29). Whole-brain irradiated animals (n=4, aged

5.6-10.7 years, 7.5-8.7 kg) received 40 Gy fWBI 4-7 months prior to necropsy as part of a previous experiment. All four whole-brain irradiated animals were euthanized during the study period (4.3-6.6 months post-irradiation, mean: 5.6 months) due to the development of severe neurologic signs consisting of progressive ataxia, visual impairment, and lethargy or weakness. Late-delayed RIBI was verified by histopathology

(29).

Control comparators (n=3, aged 5.5-6.6 years, 5.3-8.3 kg) had been part of an unrelated study in which they had received single dose, 10 Gy thoracic irradiation 8 months prior to necropsy, and subcutaneous injections of vehicle (1 mL SQ, sterile phosphorus-buffered saline(PBS)), twice daily for 4 months, ceasing 4 months prior to necropsy. Neurologically these animals were normal. Respiratory rate, heart rate, pulse oximetry and ejection fraction were within normal limits until euthanasia.

Animal husbandry and experimental procedures are detailed in Hanbury et al. (7).

All procedures were approved by the Wake Forest University School of Medicine

Institutional Animal Care and Use Committee (IACUC) and were in accordance with the Guide for Care and Use of Laboratory Animals. The Wake Forest University School of Medicine is accredited by the Association for Assessment and Accreditation of

Laboratory Animal Care (AAALAC) and adheres to all state and federal animal welfare laws.

Irradiation

Irradiation procedures for both groups of animals are described in Andrews et al.(29). Animals receiving fWBI were administered a total of 40 Gy mid-plane (8 fractions x 5 Gy/fraction; 2 fractions/wk x 4 wks; nominal dose rate of 4.9-5.4 Gy/min) using a clinical linear accelerator and opposed lateral fields of 6MV x rays. These fields were designed to match human whole-brain radiation fields, enclosing the cranial contents (nominal field size of 11 x 7 cm^2, field edge tangential to the base of skull) with the central axis of each lateral beam placed at the respective outer canthus. The eyes and olfactory region were shielded from radiation by cylindrical eye blocks. fWBI is further described in Hanbury et al.(7) and Robbins et al. (15). NHP receiving fWBI were sedated with ketamine HCl (15 mg/kg body weight, intramuscularly), and maintained on isoflurane gas (3% induction, 1.5% maintenance) in 100% oxygen during irradiation.

Control comparators received 10 Gy single dose thoracic irradiation delivered to the anterior-posterior thoracic mid-plane as part of a previous study. The maximum dose administered to the brain is estimated to be less than 0.1 Gy ( < 1%). 6 MV x rays were delivered with parallel opposed anterior-posterior fields from a clinical linear accelerator at a nominal dose rate of 4 Gy/min. The field of irradiation included the heart, mediastinum and superior, lateral and inferior lung fields extending 4 cm below the xyphoid.

Tissue Collection and Histopathology

Tissue collection, processing and archival procedures were the same for all subjects. At the time of necropsy, the animals were humanely euthanized in accordance with the American Veterinary Medical Association’s Guidelines on Euthanasia (31) by deep anesthesia with pentobarbital followed by exsanguination and perfusion of the vascular system with 2 liters of cold normal saline. The brain was removed intact, and sectioned coronally in 4 mm intervals using a stainless steel brain matrix with cutting guides. Once removed from the matrix, all slices were photographed. Alternating sections were either immediately frozen on dry ice or immersed in 4% cold paraformaldehyde for

24 hours.

Fixed tissues were embedded in paraffin and sectioned coronally at 4 μm, then stained with hematoxylin and eosin. All animals were assessed by a board certified veterinary pathologist (JMC) who was blinded to the irradiation status of all animals.

Each animal received a full histopathologic assessment in accordance with the Society for

Toxicologic Pathology’s Recommended Practices for Sampling and Processing the

Nervous System (32) with the addition of a section of prefrontal cortex containing

Brodmann area 46. A minimum of 8 tissue sections were reviewed, structures included but were not limited to: cerebral cortex, subcortical and deep white matter structures, striatum, hippocampus, thalamus, midbrain, cerebellum and brainstem. The presence of necrosis, inflammatory changes and vascular lesions was tabulated and scored.

Histopathologic scoring is detailed in Andrews et al. 2017 (29).

Special Stains, Immunohistochemistry (IHC) and RNAScope ®

Formalin fixed, paraffin embedded brain tissues containing deep white matter

(centrum semiovale) were sectioned coronally at 4 μm. IHC and RNAScope® in situ hybridization assays were performed on a Leica Bond RX (Leica Biosystems, Buffalo

Grove, IL) automated stainer. Sections from animals receiving fWBI and control comparators were stained concurrently.

The composition of perivascular ECM was evaluated by IHC for fibronectin

(rabbit polyclonal; 1:100; catalog #ab652; Abcam), collagens I (rabbit monoclonal;

1:1000; catalog #ab138492; Abcam) and IV (rabbit polyclonal; 1:300; catalog #ab6586;

Abcam). Slides were air-dried overnight and loaded onto the Leica Bond RX. All sections were dewaxed, rinsed, and underwent heat-induced epitope retrieval for 20 minutes at 99°C in the following solutions: fibronectin, citrate buffer pH 6.0; collagen I and IV, EDTA buffer pH 8.0). Endogenous peroxidase activity was blocked by 10 minute incubation with IHC/ISH Super Blocking Novocastra solution (catalog #PV6122, Leica

Biosystems). All slides were incubated with the primary antibody for 15 minutes and then rinsed. Secondary antibody and chromagen application were as according to manufacturer’s specifications (Bond Polymer Refine Red Detection Kit, catalog #

DS9390, Leica Biosystems). Slides were then removed from the instrument, dehydrated, cleared with xylene and coverslipped.

Amyloid was detected with Putchler’s modification of Bennhold’s Congo Red stain. Paraffin embedded, formalin fixed brain tissues were sectioned coronally at 10 μm.

Positive identification of amyloid was defined as green birefringence under polarized light.

Fibronectin transcripts were visualized by in situ hybridization RNAScope 2.5

Assay, with concurrent visualization of the cerebral microvasculature by anti-GLUT1

IHC. In situ hybridization was performed according to manufacturer’s specifications, using a RNAScope 2.5 LS Reagent Kit – Brown (Advanced Cell Diagnostics (ACD), catalog #: 322100) and standard Leica reagents as follows. Slides were loaded onto the

Leica Bond RX, then dewaxed, followed by heat-induced epitope retrieval in EDTA buffer pH 8.0 at 95°C for 15 minutes. Enzyme-induced epitope retrieval was completed in ACD protease III (catalog #322340) for 15 minutes at 40°C, followed by quenching of endogenous peroxidase by 10 minute incubation in hydrogen peroxide at room temperature. A custom-made probe optimized for the rhesus macaque FN1 sequence

(1289-2251 of XM_015110901.1) was applied for 2 hours at 42°C followed by an amplification series at 42°C and subsequent probe labelling at room temperature. Slides were stained with Bond Polymer Refine Detection (ACD, catalog #: DS9800) for 20 minutes then rinsed. Slides were counterstained with hematoxylin for 5 minutes and rinsed, followed by ACD Bluing for 2 minutes.

Antibodies were stabilized in preparation for IHC by 10 minute incubation with

IHC/ISH Super Blocking (Leica Biosystems, cat# PV6122). Slides were then incubated for 15 minutes with a rabbit polyclonal anti-glucose transporter GLUT1 antibody (rabbit polyclonal, 1:1000; Abcam, catalog #ab652) and rinsed. The secondary antibody and chromogen were visualized with the Bond Polymer Refine Red Detection Kit or when without concurrent in situ hybridization, Bond Polymer Refine Detection and applied

98 according to manufacturer specifications. Slides were then removed from the instrument, dehydrated, cleared with Xylene and coverslipped.

Histomorphometry

Digital images were captured using an Olympus BX61VS virtual slide microscope using VS120 software at 20x magnification. Microvessel density, cross- sectional microvascular area fraction, and in situ hybridization probe density and probe area were quantified on the same tissue section. All parameters were quantified from a single tissue section within deep white matter (centrum ovale) within a rectangular region of interest (ROI) no less than 15 mm2 (equivalent to a minimum of 60 40x high power fields). Microvessel density and cross-sectional microvascular area fraction were computed in VisioPharm (version 6.5.0.2303; Broomfield, CO, USA) using the following modifications to APP #10022 (“CD31, Tumor Neovascularization”) and an unbiased counting frame. The microvasculature was identified using an AEC_DAB-DAB-input channel refined to exclude brown objects and the recognition algorithm was thresholded to exclude all background objects. Microvessel density was defined as the number of microvascular profiles per mm2. Cross-sectional microvascular area fraction was defined as the total area occupied by blood vessels including lumina divided by the total area

(area within blood vessels/total area of the region of interest) and expressed as percent area (microvascular area fraction * 100). The cross sectional area for each microvessel profile was also recorded, analyses are detailed in the statistical methods.

Microvascular profiles generated by the previous analysis were converted to a single region of interest (vascular) with the remaining surrounding area defined as

99 parenchyma. Fibronectin in situ hybridization probe foci were identified using a Fast

Red_DAB-DAB-input channel and the recognition algorithm was thresholded to exclude all non-brown objects. Probe density was calculated as the number of probe foci per mm2.

Cumulative probe area was defined as (cumulative probe area / anatomic subcompartment ROI area) * 100) μm2. The cross sectional area for each probe aggregate was also recorded. The assessor was blinded to the irradiation status of the animals.

Transmission Electron Microscopy (TEM)

The cerebral microvasculature was visualized in archived, formalin fixed, 4-mm coronal sections of brain tissue by trans-illumination on a dissecting microscope. The surrounding 3 mm3 were sub-dissected then post-fixed in 2% osmium tetroxide in phosphate buffer for one hour, followed by dehydration in EtOH. Samples were prepared for resin infiltration by incubation in propylene oxide then gradually infiltrated with 1:1,

1:2, and pure solutions of Spurr’s resin, and cured 70° C oven overnight. 90 nm sections were obtained with a Reichert-Jung Ultracut E ultramicrotome (Reichert-Jung; Vienna,

Austria), stained with lead citrate and uranyl acetate and viewed with a FEI Tecnai Spirit

TEM (FEI Company; Hillsboro, OR, USA) operating at 80 kV. Images were acquired with an AMT 2Vu CCD camera (Advanced Microscopy Techniques; Woburn, MA,

USA).

Gene Expression Analyses

Relative gene expression was evaluated by RT-qPCR within 3 brain regions

[dorsolateral prefrontal cortex (Brodmann area 46), hippocampus, and deep temporal white matter (centrum semiovale)] from all animals and conducted on the same samples

100 as in Andrews et al. (29). Additional factors were selected for their involvement in ECM deposition and remodeling, and RAAS or Rho/ROCK signaling (Table 1). Gene names are reported in accordance with the recommendations of the HUGO Gene Nomenclature

Committee (HGNC).

Table I: Classification of markers in RT-qPCR profiler gene expression array

Renin-Angiotensin Extracellular Matrix Rho/ROCK System Deposition and Remodeling HGNC ACE ROCK1 MMP9

Symbol ACE2 ROCK2 CTGF AGT RHOA ITGAV AGTR1 CDC42 ITGB1 MME RAC1 ACTA2 MAS1 COL1A1

RNA was extracted via a modified Qiagen RNeasy Mini kit, purification, quality control and RT-qPCR procedures are detailed in Andrews et al. (29). RT-qPCR was performed on an ABI 7500 Fast system (Applied Biosystems, USA) using a Qiagen

Custom RT2 Profiler Array utilizing Rhesus macaque specific primers. Cycle thresholds

(Ct) were acquired for all target and housekeeping genes per sample; the limit of detection was set to 35 Ct. Relative gene expression (∆Ct) for each target gene was determined by normalization of the target gene Ct to the geometric mean of two housekeeping genes (β-actin and hypoxanthine-guanine phosphoribosyltransferase).

Relative expression was calculated as 2-(Target gene – geometric mean of housekeeping genes). Calculation of fold change (2-∆∆Ct) was as described by Livak (33).

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Statistical Analyses

Statistical analyses were performed under the guidance of a statistician (JAT) and in GraphPad v. 7.03 (StataCorp, College Station, TX: StataCorp LP) unless noted.

Normality and equality of variance were assessed by Shapiro-Wilk and Levene’s tests, respectively.

The effect of fWBI on cumulative microvascular area fraction and microvessel density was compared by Student’s T-test.

Two separate, 2x2 repeated-measures ANOVA were conducted to compare the main effects of irradiation status and anatomic location (vascular and parenchymal) and the interaction effect between irradiation status and anatomic location on in situ hybridization probe focus density and cumulative probe focus area, respectively. Samples were matched by subject with respect to anatomic location (e.g.; vascular and parenchymal compartments were measured within the same animal). Post-hoc comparisons were performed to assess the effect of irradiation within regions using

Bonferroni’s procedure. Multiplicity adjusted p-values are reported, and p < 0.05 was considered significant.

As values for individual subjects were similarly distributed within irradiation groups, individual probe focus areas and individual microvascular profile areas were pooled within groups with respect to irradiation status. Differences in distribution between groups were confirmed by Kolmogorov-Smirnov test. The effect of radiation was compared between groups by Mann-Whitney U due to right-skew. Individual probe focus area data were stratified by region. P values < 0.05 were considered significant.

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Individual probe focus areas and individual microvascular profiles are reported as median with 95% confidence intervals (CI). Microvascular area fraction, microvessel density and cumulative probe focus area are reported as mean with 95% CI. Probe focus density data were square-root transformed to achieve normality, then back-transformed.

Geometric mean and 95% CI are reported.

Gene expression analyses with respect to irradiation status were stratified by region and were evaluated as follows: Non-parametric data were compared by Mann

Whitney U. Normally distributed data were compared by Student’s T and Welch’s tests for data with equal and unequal variances, respectively. Significance determined at p <

0.01.

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RESULTS

Histopathology

All animals receiving fWBI developed multifocal cerebrovascular and white matter injury consistent with late-delayed RIBI. Notable lesions included perivascular edema with serum protein extravasation, perivascular ECM aggregation and white matter necrosis. The cerebrovascular lesions were multifocal, spatially-stochastic and occurred primarily within white matter. Multifocal accumulation of perivascular ECM was observed in all animals receiving fWBI. These lesions were noted in the white matter (4/4 animals), cortical grey matter (1/4 animals), hippocampus (1/4 animals), thalamus (2/3 animals), midbrain (2/4 animals), brainstem (2/3 animals) and spinal cord (1/3 animals); a denominator less than 4 indicates the tissue section was not available for analysis. Full histopathologic assessment is detailed in Andrews et al (29). No significant lesions were present in control comparators.

Characterization of Perivascular ECM

Fibronectin preferentially localized to the pia mater, arachnoid mater, and basement membrane zone of morphologically normal blood vessels in both groups

(Figures 1A-B and 2D). Additional irregular, regionally-extensive parenchymal fibronectin immunoreactivity was observed in areas of vascular injury in animals receiving fWBI (Figure 1B), and interpreted as vascular leakage. Fibronectin was a major contributor to the perivascular material in edematous (Figure 2E) and fibrotic vascular morphologies (Figure 2F). In edematous vascular morphologies, the material within the

104 perivascular space was also immunoreactive for collagen-I (Figure 2H), and collagen-IV

(Figure 2K).

Figure 1: Distribution of perivascular ECM contributors in radiation-induced brain injury

(low magnification).

A) Fibronectin IHC; control: Frontal cortex and subtending white matter. Fibronectin (red) localized to the vascular basement membrane zone, pia and arachnoid. B) Fibronectin IHC; fWBI: Frontal cortex and subtending white matter. The distribution of fibronectin (red) was similar to that in the control animals (A), with additional regionally extensive parenchymal staining adjacent to foci of necrosis and associated abnormal vasculature (black arrows). C) Collagen-I IHC; control: Subcortical white matter, temporal cortex: The microvasculature lacked collagen-I (red). D) Collagen-I IHC; fWBI: Subcortical white matter, temporal cortex. Collagen-I (red) localized to the basement membrane and wall of occasional small caliber arterioles and capillaries (black arrows), and was absent in others (white arrows). E) Collagen-IV IHC; control and F) Collagen-IV IHC; fWBI: Subcortical white matter, temporal cortex. Collagen-IV (red) localized to the basement membrane zone of the cerebral microvasculature, the distribution was comparable between control and WBI groups. All slides were counterstained with Hematoxylin.

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Figure 2: Distribution of perivascular ECM contributors in vascular pathomorphologies in radiation-induced brain injury (high magnification)

Column 1: Morphologically normal microvessels, control. Column 2: Edematous vascular pathomorphology, fWBI. Material within the perivascular space was immunoreactive for fibronectin (E), collagen-I (H) and collagen-IV (K). Column 3: Perivascular fibrosis, fWBI. Row 1: Morphology of vascular injury in RIBI; hematoxylin and eosin. A) Minimal expansion of the perivascular space (black arrows, glia limitans). B) Prominent expansion of the perivascular space (black arrows, glia limitans) by eosinophilic material interpreted as plasma extravasation. The adjacent neuropil is edematous and contains several reactive (gemistocytic) astrocytes (asterix). C) Prominent expansion of the perivascular space (black arrows, glia limitans) by loose, fibrillar extracellular matrix. Row 2: Fibronectin immunohistochemistry. Fibronectin (red) localized to the basement membrane zone in morphologically normal microvessels (D) and was present throughout the perivascular space in edematous (E) and fibrotic vascular phenotypes (F) in animals receiving fWBI. Row 3: Collagen-I immunohistochemistry. Collagen-I (red) localized adjacent to the basement membrane; it was scant to absent in control comparators (G) and diffuse in animals receiving fWBI (I). Row 4: Collagen-IV immunohistochemistry. Collagen-IV localized to the vascular wall and basement membrane in all vascular phenotypes (J-L); distribution was comparable between fWBI animals and control comparators.

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Collagen-I localized adjacent to the basement membrane zone and was scant to absent in the subcortical white matter of control animals (Figures 1C and 2G). Diffuse, basement membrane zone staining for collagen-I was present in scattered, otherwise normal appearing small caliber arterioles and capillaries (Figure 1D), and fibrotic vascular phenotypes (Figure 2I) of animals receiving fWBI.

Figure 3: Expansion of Virchow-Robin space by perivascular ECM.

A) Transmission electron micrograph indicating abundant fibrillar extracellular material surrounding a blood vessel (*). This material lacks the regular periodicity of type I collagen. Virchow-Robbin’s space also contains a few lymphocytes and macrophages containing phagocytosed debris consistent with hemosiderin (M). B) Hematoxylin and Eosin stain, corresponding histopathology indicating aggregated perivascular ECM, as well as perivascular inflammatory cells, as in (A). Black arrows indicate the glia limitans.

Collagen-IV localized to the microvascular wall and basement membrane zone of the cerebral vasculature in both groups (Figures 1E-F and 2J,L). The distribution was

107 comparable between both groups. TEM of affected vessels demonstrated medium electron dense, fibrillar material without regular periodicity within Virchow-Robbin’s space (Figure 3). This material did not stain for amyloid.

Localization of Fibronectin mRNAs by In Situ Hybridization

fWBI resulted in a prominent induction of FN1 expression in the vascular compartment (endothelial and vascular smooth muscle cells; Figure 4B-C) with a 1259- fold increase in anatomic sub-compartment fractional area ( p < 0.006), a 206-fold increase in mean probe focus density ( p < 0.005 ) and a 2-fold individual probe aggregate area (p < 0.002; Figure 4D ). FN1 expression was also increased within in the parenchyma (glia) following fWBI, albeit to a lesser extent, with increases in mean probe focus density ( 18-fold increase, p < 0.03; Figure 4D ) and median probe aggregate area

( 1.3-fold increase, p < 0.0001; Figure 4D), but no difference in cumulative anatomic sub- compartment fractional area. There was a significant interaction effect between irradiation status and anatomic sub-region for both probe focus number ( p < 0.03 ) and cumulative percent area ( p < 0.04 ).

Transcript aggregates greater than 40 μm2 were present only in animals receiving fWBI and largest within the vascular compartment (median: 1.901 μm2, range: 0.2 -

321.1 μm2; p < 0.0001). Fibronectin transcripts were rarely expressed in non-irradiated cerebrovascular cells (geometric mean: 0.4 foci per mm2; 95% CI 0.0-1.2) and glia

(gemometric mean: 2.4 foci per mm2; 95% CI: 1.3-3.8).

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Figure 4: fWBI was associated with prominent induction of FN1 expression with predilection for the vascular compartment.

A) Control comparator: normal microvessels. Fibronectin transcripts were rare within control animals. B) fWBI-receiving animal: Fibronectin transcripts primarily localized to the vascular compartment. C) fWBI receiving animal: Fibronectin was expressed within endothelial (white arrow) and vascular smooth muscle cells (black arrow). D) Within the vascular compartment, fWBI was associated with a 1259-fold increase in fibronectin transcript fractional area due to an 18-fold increase in probe density and 1.3-fold increase in median individual probe focus area. Expression following fWBI was lesser within the parenchymal compartment (glia), with an 18-fold increase in mean probe focus density and 1.3-fold increase in median individual probe focus area. Chromogen, Vector Red: Endothelial cells, anti-GLUT1 IHC. Chromogen, DAB: Fibronectin transcripts, in-situ hybridization probe foci

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Figure 5: fWBI was associated with an increase in median vessel diameter.

fWBI resulted in a shift in microvessel cross-sectional area distribution (A); histologically manifested an increase in dilated small caliber vessels in animals receiving fWBI (C), as compared to control animals (B). Correspondingly, there was a 57.4% increase in median cross-sectional microvessel area per vascular profile, cumulative microvascular area fraction and microvessel density did not differ between groups (D). Due to marked right skew, panel A was truncated at 360 um2 (range: 0.2 – 7284.6 um2). Bars indicate the mean for microvascular area fraction and microvessel density, and median for microvessel profile area. Error bars: 95% CI; KS: Kolmogorov-Smirnov test

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Microvascular Area Quantification

RIBI was associated with a shift in microvessel cross-sectional area distribution ( p < 0.0001; Kolmogorov-Smirnov test, Figure 5A), resulting in a 57.4% increase in median microvessel cross-sectional profile area (p < 0.0001, Figure 5D). This corresponded to dilated small caliber blood vessels on histopathology (Figure 5B-C).

There was no significant different in cumulative microvascular area fraction or microvessel density between groups.

Figure 6: Late-delayed RIBI was associated with an 88-fold increase in MMP9 expression within temporal white matter (centrum semiovale).

The center line denotes the median relative expression value; bar limits denote 25th and 75th percentiles.

Error bars represent minimum and maximum values. Relative expression was defined as 2-[(MMP9 – Geometric

Mean of Housekeeping Genes (ACTB and HPRT1)] . * p-value indicates with the effect of radiation on relative-gene expression; # p-value signifies differences in regional gene expression with respect to irradiation status

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Gene Expression Assessment of Contributing Molecular Pathways

Late-delayed RIBI was associated with an 88-fold increase in MMP9 expression within temporal white matter ( p < 0.002 , Figure 6 ). There were no differences in gene expression in members of the Rho/ROCK (ROCK1, ROCK2, RHOA, CDC42 or RAC1) or RAAS signaling pathways (ACE, ACE2, AGT, AGTR1, MME or MAS1) or other

ECM-associated molecules (CTGF, ITGAV, ITGB1, ACTA2 or COL1A1) within any region examined.

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DISCUSSION

Herein, we have demonstrated that fibronectin is a significant contributor to perivascular ECM in late-delayed RIBI and following fWBI, expression of the encoding gene FN1 is strongly induced within cerebrovascular endothelial and smooth muscle cells following irradiation.

Fibronectin is an approximately 440 kiloDalton glycoprotein and major component of ECM. The majority of fibronectin is produced by the liver and circulates in the plasma as dimeric, soluble fibronectin. Fibronectin may also be secreted locally by endothelial cells, fibroblasts, chondrocytes, synovial cells and myocytes (34, 35).

Assembly into an insoluble fibrous matrix is a cell-mediated process, facilitated by binding of fibronectin to integrin α5β1 with subsequent induction of cytoskeletal contraction (36, 37). Resultant clustering of integrins on the cell-surface brings multiple fibronectin dimers into close proximity, promoting fibronectin-fibronectin interactions, self-association, fibril elongation and matrix assembly. The assembled fibronectin matrix may then further promote fibrosis through association with collagen fibrils, other ECM contributors, and resultant cell-ECM interactions (38). Spontaneous folding and rearrangement of secondary elements within assembled fibronectin matrix intermittently exposes additional binding sites (39, 40), thus providing opportunity to modify deposited

ECM and represents an appealing target for interventional therapies.

Fibronectin transcripts were identified with cerebrovascular endothelial cells, vascular smooth muscle, and glia. A significant interaction effect between irradiation status and anatomic sub-compartment was detected for both fibronectin probe density and

113 fractional area. As the magnitude of change in expression was greatest within the vascular sub-compartment, we infer that up-regulation of fibronectin occurs with predilection for the cerebral microvasculature (within endothelial cells and vascular smooth muscle cells). This is supportive of a differing radiobiology between the two anatomic sub-compartments. Cellular morphology and proximity to areas of necrosis suggest that the parenchymal (glial) contributor to fibronectin expression is the activated microglia/macrophage. Definitive identification of the cell of origin is the focus of future studies. Others have demonstrated that activated macrophages produce fibronectin

(41, 42) and that fibronectin induces microglial activation (43, 44). We theorize that fibronectin-induced macrophage/microglial activation may contribute to the persistent neuroinflammation in RIBI via an autocrine signaling loop.

In the present study, we establish that fibronectin is a major contributor to perivascular fibrous ECM with lesser amounts of perivascular collagen-I and IV, as evidenced by immunohistochemistry and supported by TEM. The differential expression of perivascular collagen-I and collagen-IV immunoreactivity between edematous and fibrotic vascular phenotypes suggest transient accumulation of pre-fibrillar collagen precursors during lesion progression. Clearance of these contributors is presumed partially mediated by radiation-induced matrix metalloprotease expression, particularly

MMP2 and MMP9 (29, 45-48), a matrix metalloprotease which is induced by fibronectin

(49, 50), and functions to degrade collagen IV (51).

Previous studies in rodent and cell culture models indicate MMP9 expression is increased at four hours to one week following single doses of irradiation (45-48).We reaffirm that radiation exposure induces the expression of MMP9, and furthermore,

114 translate these findings to a clinically relevant, non-human primate model of fractionated whole-brain radiotherapy.

Kyrkanides et. al.,(47) have shown that COX2-selective inhibition prior to and following irradiation reduces MMP9 expression. Therefore, persistent neuroinflammation may contribute to ECM remodeling through MMP9-mediated effects. We also hypothesize that MMP9-mediated basement membrane degradation facilitates extravasation of soluble fibronectin, providing additional substrate for matrix assembly.

Blood-brain-barrier insufficiency as a consequence of fWBI has been demonstrated previously (52-55). While the proportion of extravasated soluble fibronectin to locally produced cellular fibronectin remains undetermined, both are presumed to contribute to perivascular fibronectin accumulation and thus play a role in the pathogenesis of RIBI.

We also affirm that perivascular ECM deposition occurs without corresponding increase in white matter microvascular density, indicating increased accumulation of matrix, rather than a reflection of enhanced microvascular investiture. Interestingly, while overall microvascular area fraction did not differ between irradiation and control comparators, there was a shift in individual microvascular profile distribution, resulting in a greater median individual microvessel profile area. This was unexpected as fibronectin matrix assembly is typically associated with cytoskeletal contracture rather than dilation, as contraction is necessary for integrin clustering, which facilitates fibronectin-fibronectin interactions and subsequent assembly. Artefactual vasodilatation due to perfusion of the vascular system with saline at the time of necropsy is unlikely, as the necropsy procedure for animals receiving fWBI and control comparators was identical. We postulate that the difference in vessel size may reflect defects in

115 cytoskeletal coordination or contractile ability within endothelial cells or their adjacent mural contributors (vascular smooth muscle, pericytes), or a vasodilatory compensatory response to a hypoxic environment. Selective capillary loss may also contribute to the shift in microvessel profile area.

Additional irregular, regionally extensive background staining for fibronectin in irradiated animals was suggestive of blood-brain barrier insufficiency and exudation of plasma containing fibronectin into the adjacent parenchyma, as mentioned previously. In addition to MMP9-mediated basement membrane degradation, other studies have indicated that radiation-induced cytoskeletal rearrangement and concomitant contraction of the cell membrane results in disruption of junctional apposition (56-58), a mechanical process which may not be reflected at the transcriptional level. Previous studies suggest that radiation-induced cytoskeletal contracture may be mediated by activation of the

Rho/ROCK pathway as part of the acute endothelial radiation response (57, 59-62).

Radiation-modulated endothelial-fibronectin interactions occur in a RhoA/ROCK- dependent manner (63). While our gene expression analyses did not detect changes in the

Rho/ROCK family member mRNAs, activation of RhoA is primarily regulated at the protein level by phosphorylation. Thus functional alterations may not have been detectable by our transcriptional analyses. A recent study demonstrated decreased phosphorylated RhoGDI in irradiated endothelial cells (0.5 Gy, 1-7 days post-irradiation)

(59). RhoGDI functions to sequester RhoA and prevent its activation, and may be a promising target for future mechanistic analyses.

The role of fibronectin in RIBI may be particularly relevant in the tumor bearing brain. Increased fibronectin extravasation due to the permeability of tumor-associated

116 vasculature (64-68), and local production by glioma cells (69-71) may lead to increased fibronectin substrate available for matrix assembly. This could potentially exacerbate

RIBI. Data regarding the influence of fibronectin on glioma cell migration are conflicting

(71-75), however short hairpin RNA (shRNA)-mediated fibronectin knockdown results in delayed tumor growth in a mouse glioma model (76). These data suggest that signaling mechanisms which modulate fibronectin deposition and matrix assembly may be relevant targets for therapeutic intervention.

Although control comparators received 10 Gy thoracic irradiation 8 months prior to necropsy as part of a separate study, the estimated dose to the brain is far lower ( < 0.1

Gy ). There is little information regarding the long-term consequences of single, low dose irradiation in the adult brain. Studies in rodents suggest that doses of < 0.1 Gy do not significantly impair spatial learning or memory (77) and that cerebrovascular permeability returns to normal by 1 month post-irradiation (78). Retrospective analyses of the Chernobyl and atomic bomb survivor cohorts suggest that increased risk of cerebrovascular injury occurs at doses > 0.15 – 0.5 Sieverts (79-82). The cerebral consequences of radiation-induced cardiopulmonary injury in these animals are presumed minimal. Clinical data regarding cardiac and respiratory function prior to death were carefully reviewed prior to use of archived tissue. Respiratory rate, heart rate, pulse oximetry and ejection fraction were within normal limits until euthanasia.

The young adult NHP used in this study may not fully recapitulate the aged patient population (approximate age: 60 years) (83-85) receiving fWBI. Survival is poorer in aged patient subpopulations than younger comparators; however these findings are confounded by comorbid neurologic and systemic health conditions, tumor

117 progression and treatment regimen. Data regarding the role of fibronectin in the aging brain are conflicting (86-88) and warrant additional study in the context of neurodegenerative diseases generally.

In conclusion, we have demonstrated the fibronectin is preferentially produced by cerebrovascular endothelial and smooth muscle cells, and is a major component of perivascular fibrous ECM in late-delayed RIBI. The dynamic nature of the fibronectin extracellular scaffold (39, 40) suggests that even chronically established ECM containing fibronectin may be susceptible to remodeling. Thus, we propose that altering the cellular and molecular dynamics of fibronectin matrix assembly to favor degradation may represent a potential therapeutic target for late-delayed RIBI and other fibrotic conditions in which fibronectin is a contributor.

ACKNOWLEDGEMENTS

The authors would like to thank Lisa O’Donnell and Cathy Mathis for their indispensable enthusiasm, histologic expertise and unending support. This work was supported in part by the NCI Cancer Center Support Grant (P30CA012197) to the

Comprehensive Cancer Center of Wake Forest Baptist Medical Center, and the North

Carolina Biotechnology Center (2015-IDG-1006). We also acknowledge the support of the Comparative Pathology Laboratory Shared Resources of the Comprehensive Cancer

Center of Wake Forest Baptist Medical Center. TEM was obtained with the assistance of the WFUSM Cellular Imaging Shared Resource. This work was supported by

1R01CA155293-01A1 (Robbins/Deadwyler). Dr. Andrews was supported by NIH grant

T32 OD010957.

118

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130

CHAPTER FOUR

Transcriptomic profiling reveals neuroinflammation, complement system involvement,

and impaired glutamatergic neurotransmission in white matter years post total body

irradiation and months post fractionated whole brain irradiation

Rachel N. Andrews, D.V.M.

131

ABSTRACT

Fractionated whole-brain irradiation, as used for the treatment of intracranial neoplasia, is associated with progressive neurodegeneration and neuroinflammation however the long-term consequences of high dose cerebral irradiation, as may occur during a nuclear accident or malicious exposure, are unknown.

In order to assess the long-term consequences of single high dose cerebral irradiation, we have compared RNA sequencing obtained transcriptomic profiles from

Rhesus macaques (Macaca mulatta) receiving single dose total body irradiation (TBI: n=5, 6.75 - 8.05 Gy, 6-9 years prior to necropsy) to those which had received a prior course of fractionated whole brain radiotherapy with a biologically effective dose (BED) similar to that used in human brain cancer treatment (fWBI: n=5, 40 Gy, 8x5 Gy fractions; 12 months prior to necropsy) and control comparators (n=5). Gene expression profiles from the dorsolateral prefrontal cortex (DLPFC), hippocampus (HC), and deep white matter (centrum semiovale, WM) were compared.

Stratified analyses by treatment and region revealed radiation-induced transcriptomic alterations were most prominent in fWBI animals, and within treatment groups, primarily affected white matter. Canonical pathway, ontologic molecular function, and supervised analyses revealed that TBI and fWBI animals demonstrated shared patterns of injury including white matter neuroinflammation, increased expression of complement factors, and evidence of T-cell activation. Both irradiated groups also demonstrated evidence of impaired glutamatergic neurotransmission and signal transduction within white matter, but not within the DLPFC or HC. Signaling pathways

132 involved in extracellular matrix deposition and remodeling, and changes in ECM components were noted within the WM of fWBI animals, but not TBI animals.

Thus it appears that animals receiving TBI are susceptible to neurological injury similar to that observed following fWBI, and these changes persist years post-irradiation.

Transcriptomic profiling reaffirmed that macrophage/microglial-mediated neuroinflammation is present in RIBI and our data provides novel evidence that the complement system may contribute to the pathogenesis of RIBI. Finally, these data also challenge the assumption that the hippocampus is the predilection site of injury in RIBI, and present evidence that alteration in glutamatergic neurotransmission and neuronal signal transduction is a manifestation of white matter injury.

133

INTRODUCTION

There have been 57 major nuclear accidents since the Chernobyl disaster (1), despite safety regulations to limit radiation exposure, and occupational, medical and malicious exposure poses a significant threat to public health and safety. Those that survive the acute radiation syndrome are at risk of developing late-delayed sequelae which may not appear until several years post-irradiation; including pulmonary and cardiac fibrosis, cataracts, muscle wasting, and cancer (2-6). While the brain is considered relatively radioresistant [ED50 12-14 Gray (Gy)] (7); recent studies in non- human primate long-term radiation survivors indicate that late-delayed radiation injury occurs in other organs at doses lower than anticipated (4, 8). Clear understanding of the molecular mechanisms of radiation injury is necessary to mitigate the health risks associated with radiation exposure, however little is known regarding the long-term consequences of single, high dose, cerebral radiation exposure.

A progressive, neuroinflammatory and neurodegenerative brain disease occurs in patients receiving fractionated whole brain irradiation (fWBI) for the treatment of intracranial neoplasia. Characterized by multifocal cerebrovascular injury and white matter necrosis (9-24), late-delayed radiation-induced brain injury (RIBI) results in cognitive dysfunction and memory impairment which negatively impacts patient quality of life (25-27). A subset of patients will develop profound dementia, incontinence and ataxia (28).

Studies investigating the long-term consequences of single, high dose irradiation in humans are confounded by variations in radiation source, dose rate, shielding and the

134 effects of concomitant thermal and mechanical trauma (5, 29). Additionally, large scale tissue repositories of these cohorts have not been established. While rodent models have been used to investigate the late-effects of radiation exposure, the radiation biology and dose response of rodents differs from humans. Further dissimilarities in cerebrovascular structure and white matter investiture (e.g.; rodents have a lower white:grey matter ratio)

(30) may limit the translatability of findings. Finally, rodents do not develop white matter necrosis as a component of RIBI following fWBI (31-34); thus lacking a key histologic feature of the disease. Alternatively, non-human primates possess greater genomic homology to humans; and recapitulate the histopathologic manifestations of late-delayed

RIBI following fWBI (9, 11, 12). Therefore, non-human primates are a more human-like alternative in which it is appropriate to study RIBI.

We hypothesized that non-human primates receiving single, high dose total body irradiation (TBI) develop similar patterns of brain injury as seen in fWBI, albeit of lesser severity and may take longer to develop. Herein, we investigate patterns of change in the cerebral transcriptome from animals receiving single, high dose TBI; and compare them to animals with histologically confirmed radiation-induced brain injury, as well as thorax- only irradiated control comparators. As our previous targeted gene expression analysis

(35) indicated evidence of macrophage/microglial-mediated neuroinflammation, extracellular matrix (ECM) deposition, evidence of hypoxia, impaired glutamatergic neurotransmission within white matter, and factors associated with vascular maturation and increased stability, we anticipated that RNA sequencing would reveal similar patterns of injury.

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METHODS

Animals

Archived frozen and formalin fixed paraffin embedded brain tissues from three groups of young adult, post-pubertal, male Rhesus macaques (n=5 per group) were compared to evaluate changes in the cerebral transcriptome following radiation exposure.

Five animals (aged: 10-13 years; 6.9-19.7 kg) received high dose TBI (median:

7.2 Gy; range: 6.75-8.05 Gy) a median of 7.8 years prior to necropsy (6.2 - 8.8 years). 3-4 months post-irradiation, these animals were enrolled into a cohort of long-term irradiation survivors. These animals were monitored closely for sequelae associated with prior irradiation. Procedures included: cerebral and cardiac magnetic resonance imaging

(MRI), annual thoracic CT, bronchoalveolar lavage and cytology, bone marrow aspiration, annual echocardiographic and electrocardiographic examination.

Whole brain irradiated animals (n=5; aged: 7-13 years; 8.3-9.5 kg) received 40 Gy fWBI, 12-15 months prior to necropsy as part of a separate experiment. As the parent study included cognitive assessment for which the award for performance was sip of juice, these animals were water regulated and hydration status was monitored daily.

Additional experimental procedures are described in detail in Robbins et. al., 2011 (36) and Hanbury et. al, 2015 (12).

Control comparators (n=5; aged: 5-7 years, 5.5-8.3 kg) received 10 Gy thoracic- only irradiation as part of previous experiments.

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All animals received daily clinical observations by trained veterinary personnel.

All procedures were performed in accordance with the Guide for Care and Use of

Laboratory Animals, and approved by the Wake Forest University School of Medicine

(WFUSM) Institutional Animal Care and Use Committee (IACUC). The Wake Forest

University School of Medicine is accredited by the Association for Assessment and

Accreditation of Laboratory Animal Care (AAALAC) and adheres to all state and federal animal welfare laws.

Diet

TBI animals and control comparators were fed a Typical American Primate diet

[(TAD); Diet #5L0P; LabDiet, Land O’Lakes Inc.; St. Louis, MO], which is designed to approximate the macronutrients of a western dietary profile. Animals T2 and T5 were maintained on TAD for 3 and 2 years respectively, and then transitioned back to commercially available monkey chow (diet #5038, LabDiet) for the management of Type

2 diabetes mellitus [(T2DM), hemoglobin A1C > 6.5%]. Additional detail is provided in

DeBo et. al., 2016 (4). fWBI animals were fed diet #5038.

Irradiation

Irradiation procedures for control comparators and fWBI animals are described in

Andrews et. al. (35), fWBI is further detailed in Hanbury et. al. (12) and Robbins et. al.

(36). The irradiation procedure for animals receiving TBI is described in Yu et. al (37).

Briefly, animals receiving fWBI were given a total of 40 Gy mid-plane (8 fractions x 5 Gy/fraction, 2 fractions/wk x 4 wks) of 6 MV x rays at a nominal dose rate of 4.9-5.4 Gy/min from a clinical linear accelerator (LINAC) at the WFUSM. The

137 biologically effective dose (BED) of this regimen is 106.7 Gy, and thus comparable to a brain tumor treatment of 30 fractions of 2 Gy in 6 weeks (BED: 100.2) (38). The opposed lateral fields were designed to match human whole-brain irradiation fields, enclosing the cranial contents (nominal field size: 11 x 7 cm2, with the field edge tangential to the base of the skull). The central axis of each lateral beam was placed at the respective outer canthus, and the eyes and olfactory region were shielded from radiation by cylindrical eye blocks. NHP receiving fWBI were sedated with ketamine HCl (15 mg/kg body weight, intramuscularly), and maintained on isoflurane gas (3% induction, 1.5% maintenance) in 100% oxygen during irradiation.

TBI animals received 6.75-8.05 Gy (median: 7.2 Gy; range: 6.75-8.05 Gy) total body irradiation administered via a 6 MV LINAC a nominal dose rate of 0.8 +/- .025

Gy/min. 50% of the dose was delivered from the anterior-posterior direction, and the remaining dose was delivered from the posterior-anterior direction.

Control comparators received 10 Gy thoracic irradiation to the anterior-posterior thoracic mid-plane. 6 MV x rays were delivered with parallel opposed anterior-posterior fields from a clinical linear accelerator at a nominal dose rate of 4 Gy/min. The field of irradiation included the heart, mediastinum and lung fields extending up to 4 cm caudal to the xyphoid. The maximum dose to the brain is estimated to be 0.1 Gy.

Tissue Collection and Histopathology

Tissue collection, processing, archival and histopathology procedures were the same for all subjects. At the time of necropsy, the animals were humanely euthanized in accordance with the American Veterinary Medical Association’s Guidelines on

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Euthanasia (39) by deep anesthesia with pentobarbital, followed by exsanguination and perfusion of the vascular system with 2 liters of cold normal saline. The brain was removed intact, and sectioned coronally in 4 mm intervals using a stainless steel brain matrix with cutting guides. Once removed from the matrix, all slices were photographed.

Alternating sections either immediately frozen on dry ice or immersed in 4% cold paraformaldehyde for 24 hours.

Fixed tissues were embedded in paraffin and sectioned coronally at 4 μm; then stained with hematoxylin and eosin. All animals were assessed by a board certified veterinary pathologist (JMC) and received a full histopathologic assessment in accordance with the Society for Toxicologic Pathology’s Recommended Practices for

Sampling and Processing the Nervous System (40), with the addition of a section of prefrontal cortex containing Brodmann area 46. Lesions were scored as described previously (12, 35); absent (0), minimal (1 = inflammatory or vascular changes without disruption of the neuropil or clear neuronal loss); mild (2 = focal vascular injury and inflammation with loss of neuropil or neurons and microglial activation); moderate (3 = extensive or multifocal vascular injury, hemorrhage, disruption of the neuropil, neuronal loss and microglial activation); or severe (4 = extensive or multifocal vascular injury, hemorrhage, disruption of the neuropil, neuronal loss and microglial activation, with additionally extensive zones of necrosis within neural tissue). The presence of vascular lesions in each region was recorded as follows: a: endothelial hypertrophy; b: perivascular ECM deposition, c: perivascular edema, d: disorganized vascular morphology. The assessor was blinded to the irradiation status of the animals.

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RNA Extraction Methods

Total mRNA was extracted from 100 mg of brain tissue from three brain regions

[(Dorsolateral prefrontal cortex (Brodmann area 46), hippocampus (HC) and deep white matter (centrum semiovale)] for all animals. Tissue was placed into a 1.4 mm ceramic bead tube with 1 ml QIAzol lysis reagent, and homogenized using a Bead Ruptor 24

(Omni International). The tissue sample tube was processed on the Bead Ruptor for 1 cycle at a speed of 4.7 m/s for 20 sec; and repeated up to three times until sample was completely homogenized. Aliquots of homogenized lysates equivalent to 40 mg tissue were extracted for total RNA using the RNeasy Microarray Tissue Mini kit (Qiagen).

Extracted RNA was DNase-treated and purified using the RNA Clean and Concentrator-5 kit (Zymo Research), then assessed for RNA quality using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies). cDNA Library Preparation and Sequencing Methods

Total RNA from 45 samples was used to prepare cDNA libraries using the

Illumina® TruSeq Stranded Total RNA with Ribo-Zero Gold Preparation kit (Illumina

Inc.) and the SciClone NGS Work Station (Perkin Elmer). RIN values for the RNA samples ranged from 7.1 to 9.3. Briefly, 750 ng of total RNA was rRNA depleted followed by enzymatic fragmentation, reverse-transcription and double-stranded cDNA purification using AMPure XP magnetic beads. The cDNA was end repaired, 3′ adenylated, with Illumina sequencing adaptors ligated onto the fragment ends, and the stranded libraries were pre-amplified with PCR. The library size distribution was validated and quality inspected on a Bioanalyzer DNA 1000 chip (Agilent Technologies,

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USA). The quantity of each cDNA library was measured using the Qubit 3.0 (Thermo

Fisher, USA). Three library pools were formed, each containing 15 libraries. Library pools were sequenced to a target read depth of 28M reads per library using single-end 76 cycle sequencing with the High Output 75-cycle kit (Illumina Inc.) on the Illumina

NextSeq 500.

Data Analysis

Raw read quality was assessed by FASTQC analysis (Babraham Bioinformatics).

Uniquely mapped reads ranged from 21M-36M reads per sample. Reads with >Q20 quality score were aligned to the Ensembl Macaca mulatta genome build Mmul_1 using the STAR aligner (41) and gene counts determined by featureCounts software (42).

Differentially expressed genes (DEGs) were assessed by DESeq2 (43). Significant DEGs were conservatively defined as log2fold change ratios ≥ ±1 and p < 0.05 after adjustment for false discovery (Benjamini-Hochberg). Genes identities were preliminarily mapped in

Ingenuity Pathway Analysis (IPA)(44) using Ensembl identification numbers for the

Rhesus macaque and assigned the corresponding HGNC identifier. Genes which did not map in IPA were identified by secondary screening in PANTHER (45, 46) and UniProt

(47), then backmapped to the encoding gene by the corresponding HGNC identifier.

Ensembl ID which did not correspond to an identified gene were recorded, and excluded from pathway analysis.

DEGs were analyzed for significant enrichment of biological pathways and signaling networks using IPA. Gene ontologies were evaluated using the GO Enrichment

Analysis tool powered by PANTHER (45, 46) using a Fisher’s Exact test with False

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Discovery Rate (FDR) correction for multiple comparisons. Biological process and molecular function analyses were hierarchically clustered and the most specific subclass is reported. Hierarchies were sorted by fold-enrichment value, and the 10 greatest fold- enriched hierarchies are reported. FDR corrected p values < 0.05 were considered significant.

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RESULTS

Demographics

Control comparators were younger than fWBI ( mean difference: 4.18 years; p <

0.002 ) and TBI groups ( mean difference: 6.06 years; p < 0.0001 ) (Figure 1A). The ages of animals receiving fWBI and TBI were comparable ( p = 0.18 ). There was no difference in animal weight between groups (Figure 1B), the TBI animals weighing > 15 kg were diagnosed with T2DM (Figure 1B, white triangles). Differences in radiation dose

(Figure 1C) and survival interval between groups (Figure 1D) were due to differences in experimental design.

Figure 1, Table I: Subject Demographics

Group Age (years) Weight (kg) Dose to Brain (Gy) Survival Interval (years) Control 5.5 ± 0.7 6.66 ± 1.24 0.1 0.7 TBI 11.5 ± 1 11.84 ± 5.47 7.19 ± 0.53 7.8 ± 1.1 fWBI 9.7 ± 2.2 8.6 ± 0.80 40 1.2 ± 0.1 Mean ± standard deviation, ∆:Animal with type 2 diabetes

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Clinical Findings

Causes of death and comorbid conditions are summarized in Table 2.

Neurologically, all animals (n=15) were normal. Four out of five (4/5) TBI animals developed neoplasia during the study period. Three out of four (3/4) tumors were of

Table II: Subject Causes of Death and Comorbid Illness Group Animal ID Cause of Death/Comorbid Illness Control C1 Experimental euthanasia C2 Experimental euthanasia C3 Experimental euthanasia C4 Experimental euthanasia C5 Experimental euthanasia fWBI F1 Experimental euthanasia F2 Experimental euthanasia F3 Experimental euthanasia F4 Experimental euthanasia F5 Experimental euthanasia TBI T1 Clinical euthanasia Pulmonary bullous emphysema; with rupture and pneumothorax Neoplasia, hemangiosarcoma, pulmonary

T2 Clinical euthanasia Fatal fasting syndrome of obese macaques T2DM

T3 Clinical euthanasia Pulmonary bullous emphysema; with rupture and pneumothorax Neoplasia, seminal vesicle, poorly-differentiated neuroendocrine tumor

T4 Clinical euthanasia Neoplasia, renal tubular carcinoma with polycythemia Neoplasia, subcutis, chrondrolipoma Neoplasia, subcutis, poorly-differentiated neuroendocrine tumor

T5 Clinical euthanasia T2DM Bacterial pneumonia Chronic renal disease Hypertension Neoplasia, neuroendocrine tumor, heart base

144 neuroendocrine origin, and the remaining neoplasm was a hemangiosarcoma (n=1); there was no evidence of intracranial metastasis for any animal. Pulmonary bullous emphysema, with bulla rupture and resultant pneumothorax necessitated the euthanasia of two out of five (2/5) TBI animals. Two out of five (2/5) TBI animals developed T2DM, 4 years post-irradiation.

As control comparators had received high dose thoracic irradiation as part of a previous study, clinical data regarding respiratory and cardiac function were reviewed prior to selection as control comparators. Respiratory rate, heart rate, and pulse oximetry were within normal limits until euthanasia.

Histopathology

All animals receiving fWBI (5/5) developed multifocal white matter necrosis and cerebrovascular injury (Table III).

There were no significant lesions within control comparators. There was no evidence of white matter necrosis in animals receiving TBI. In Animal T002, perivascular spaces surrounding penetrating arterioles in the rostral frontal cortex contained multifocal aggregates of hemosiderin. A few arterioles within the caudal caudate nucleus were surrounded by homogeneous eosinophilic ECM.

Animal T004 was humanely euthanized due to multiple neoplasia (Table 2), 8 years post-irradiation. Histopathologic evaluation revealed a focal vascular malformation consistent with a cavernous hemangioma within the subcortical white matter of the right inferior occipital lobe (Appendix C, Figure 3).

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Table III: Histopathologic grading of RIBI in animals receiving fWBI

WHITE MATTER NECROSIS F1 F2 F3 F4 F5 Forebrain: White matter + ++++ ++ +++ +++ Forebrain: Cortical grey matter – +++ + + ++ Prefrontal cortex – – – + ++ Basal ganglia/striatum – +++ – – – Hippocampus – – – – – Thalamus – ++ – ++ ++ Midbrain – ++ – + +++ Cerebellum – + – ++ ++ Brainstem – ++ – + – Spinal cord – ++ na + –

VASCULAR F1 F2 F3 F4 F5

Forebrain: White matter a, c a,b,d - a, b, d b,d Forebrain: Cortical grey matter - a, d d a, d b Prefrontal cortex - - - - - Basal ganglia/striatum - - a - - Hippocampus c - - - - Thalamus - - - - - Midbrain - a, d - - b Cerebellum - - - - b Brainstem - a,d - - - Spinal cord - - - - - a endothelial hypertrophy b perivascular extracellular matrix deposition c perivascular edema d disorganized vascular morphology

Transcriptomic Profiling

DEGs were stratified by region and treatment group, and quantified (Figure 2). In any region examined, the number of DEGs was greatest in fWBI animals. White matter

146 contained the greatest number of DEGs within treatment groups, followed by the DLPFC, and then HC. 133 DEGs were expressed within the white matter of both fWBI and TBI groups. 2 DEGs (MEIS3, SCEL) were expressed within the DLPFC of both fWBI and

TBI groups (Figure 3).

Figure 2: Venn-diagram of regional differences in differential gene expression by region, across irradiation groups.

fWBI TBI

1815 39 55 172 0 2 WHITE DLPFC WHITE DLPFC MATTER 16 MATTER 0 23 21 0 0 Shared 32 (fWBI x TBI) 0 HIPPOCAMPUS HIPPOCAMPUS

133 0 2 WHITE DLPFC MATTER 0 0 0 0 HIPPOCAMPUS

Both fWBI and TBI treatment groups are normalized to the expression values of the control group. A greater number of genes were differentially regulated in all regions in animals receiving fWBI, than comparable regions in TBI animals. White matter contained the highest number of DEGs within both treatment groups.

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Figure 3: MEIS3 and SCEL are differentially expressed within the DLPFC of both fWBI

and TBI animals.

) l

r 0 .0

t MEIS3 was down-regulated within the C /

x -0 .5 DLPFC (log2fold-change: -1.02) and

(

e WM (log2fold-change: -1.67) of fWBI

g -1 .0 n animals, and the DLPFC (log fold-

a 2 h

C -1 .5 * change: -1.12) of TBI animals.

d * l

o fW B I

F -2 .0 2

g T B I SCEL was up-regulated within the o

L -2 .5 * D L P F C H C W M DLPFC in animals receiving fWBI (log2fold-change: 1.01) and animals

M E IS 3 )

l receiving TBI (log2fold-change: 1.01). r 1 .5 t

C /

x * * T

( Bars denote mean log2fold-change,

e 1 .0

g error bars are set to log2fold standard

n a

h error. Dashed line indicates log2fold- C

d 0 .5

l change of ±1.0.

o F

2 g

o DLPFC: Dorsolateral prefrontal L 0 .0 D L P F C H C W M cortex, HC: Hippocampus, WM: S C E L Temporal white matter. *FDR adjusted p-value < 0.05 Canonical Pathway Analysis

DEGs were imported into Ingenuity Pathway Analysis for canonical pathway identification; the top 10 enriched canonical pathways are reported in order of decreasing statistical significance, all pathways p < 0.05 (Figure 4). Shared canonical pathways were detected between regions and treatments (Figure 5A-B). As overall patterns suggested shared processes involving inflammation, ECM remodeling, and neurotransmission/signal transduction; for further analysis the contributing genes were grouped by broader function (Table IV).

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Figure 4: Top 10 Enriched Canonical Pathways in Ingenuity Pathway Analysis.

P e r c e n ta g e o f G e n e s in R e fe r e n c e S e t Percentage of Genes in Reference Set 0 1 0 2 0 3 0 4 0 5 0 0 10 20 30 40 50 fW B I D L P F C fWBI Hippocampus A n tig e n P re se n ta tio n P a th w a y Antigen Presentation Pathway H e p a tic F ib ro sis/H e p a tic S te lla te C e ll A c tiv a tio n Allograft Rejection Signaling A th e ro sc le ro sis S ig n a lin g OX40 Signaling Pathway A llo g ra ft R e je c tio n S ig n a lin g Th1 Pathway O X 4 0 S ig n a lin g Th2 Pathway

C D C 4 2 S ig n a lin g Cdc42 Signaling

D e n d ritic C e ll M a tu ra tio n Th1 and Th2 Activation Pathway

T h 1 P a th w a y B Cell Development

B C e ll D e v e lo p m e n t Atherosclerosis Signaling

T h 2 P a th w a y Autoimmune Thyroid Disease Signaling

fW B I W h ite M a tter 0 1 0 2 0 3 0 4 0 5 0 T B I W h ite M a tte r 0 1 0 2 0 3 0 4 0 5 0 N e u ro in fla m m a tio n S ig n a lin g P a th w a y C o m p le m e n t S y ste m H e p a tic F ib ro sis / H e p a tic S te lla te C e ll A c tiv a tio n T H e lp e r C e ll D iffe re n tia tio n G ra n u lo c y te A d h e sio n a n d D ia p e d e sis C a lc iu m S ig n a lin g A m y o tro p h ic L a te ra l S c le ro sis S ig n a lin g N e u ro in fla m m a tio n S ig n a lin g P a th w a y G lu ta m a te R e c e p to r S ig n a lin g A m y o tro p h ic L a te ra l S c le ro sis S ig n a lin g C o m p le m e n t S y ste m G ra n u lo c y te A d h e sio n a n d D ia p e d e sis T R E M 1 S ig n a lin g T R E M 1 S ig n a lin g

A n tig e n P re se n ta tio n P a th w a y A n tig e n P re se n ta tio n P a th w a y

T h 1 a n d T h 2 A c tiv a tio n P a th w a y A llo g ra ft R e je c tio n S ig n a lin g H y p e rc y to k in e m ia /h y p e rc h e m o k in e m ia T y p e I D ia b e te s M e llitu s S ig n a lin g in th e P a th o g e n e sis o f In flu e n z a All comparisons are normalized to the expression values of the matching region from the control group. Pathways are listed in descending order of decreasing statistical significance, all FDR adjusted p-values were < 0.05. The stacked bars indicate the percentage of up-regulated (red) or down- regulated (blue) DEGs out of a reference gene set curated by IPA. Data are not shown for DLPFC or HC comparisons as zero (0) enriched canonical pathways were detected.

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Figure 5: Overlapping canonical pathways across brain regions and irradiation groups.

A B

fWBI DLPFCfWBI HCfWBI WMTBI WM fWBI fWBI Calcium signaling DLPFC HC Hypercytokinemia/Hyperchemokinemia 1 1 T-helper cell differentiation 6 ALS signaling 1 0 Complement system 2 3 Granulocyte adhesion and diapedesis 0 1 Neuroinflammation TREM1 signaling

1 Glutamate receptor signaling 1 0 0 0 T1DM Hepatic fibrosis / Hepatic stellate cell activation 5 fWBI TBI Dendritic cell maturation Atherosclerosis signaling WM WM B cell development CDC42 signaling OX40 signaling pathway Th1 pathway Th2 pathway Autoimmune thyroid disease signaling Th1 and Th2 activation Allograft rejection signaling Antigen presentation

Patterns in canonical pathway expression were shared across regions, and across treatments. A) Shared and exclusive pathways are quantitated, and displayed by region and treatment. B) Boxes are color coded to the corresponding Venn-diagram sector. White indicates that the following canonical pathway was not within the top 10 enriched pathways for that region and treatment.

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Table IV: Functional grouping of canonical pathways Neurotransmission & Inflammation ECM Signal Transduction Neuroinflammation CDC42 signaling Calcium signaling Granulocyte adhesion and diapedesis Atherosclerosis signaling ALS signaling Hepatic fibrosis / hepatic stellate cell Hepatic fibrosis / hepatic Glutamate receptor activation stellate cell activation signaling Complement system TREM1 signaling Antigen presentation pathway Th1 and Th2 activation pathway Type I diabetes mellitus signaling Allograft rejection signaling OX40 signaling Th1 pathway Th2 pathway B Cell development Dendritic cell maturation Autoimmune thyroid disease signaling T-Helper cell differentiation Role of hypercytokinemia / hyperchemokinemia in the pathogenesis of influenza

Canonical pathway analysis indicated enrichment of inflammation and immunologic signaling patterns within all regions in fWBI animals and within the WM of

TBI animals. ECM-associated pathways were enriched within fWBI animals only.

Neurotransmission and signal transduction-associated pathways were enriched within the

WM of fWBI and TBI animals. Comparisons with the greatest numbers of shared pathways were between the DLPFC and HC of fWBI animals (significantly enriched pathways n=6), and the WM of fWBI and TBI animals (significantly enriched pathways n=5), suggesting that the radiation response in RIBI is differentially regulated by region.

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19 genes were implicated in the top 10 canonical pathways within fWBI DLPFC,

10 in fWBI HC, and 192 in fWBI WM. 31 contributing genes were identified in the TBI

WM, and there were no enriched canonical pathways detected within the TBI DLPFC or

HC.

Gene Ontology

Genes which contributed to the top 10 canonical pathways were evaluated for contributing molecular functions in Gene Ontology. As there were no enriched canonical pathways detected within the DLPFC or HC of TBI animals, these regions were omitted from ontologic analysis.

Inflammation-associated canonical pathways were identified in all brain regions from fWBI animals and the WM of TBI animals. The contributing DEGs (fWBI DLPFC:

15 DEGs, fWBI HC: 6 DEGs, fWBI WM: 172 DEGs and TBI WM: 22 DEGs) were evaluated for patterns in molecular function (Table V). Evidence of complement system activity was identified in the WM of both irradiated groups. Functions common to major histocompatibility complex (MHC) class II-mediated peptide antigen presentation were noted in the fWBI DLPFC, fWBI HC and TBI WM. Ontologic patterns suggesting pro- inflammatory chemokine signaling were present in the WM of both irradiated groups.

Functions consistent with modulation of neurotransmission noted in the fWBI WM

(glutamate-gated calcium ion channel activity, NMDA glutamate receptor activity and calcium-dependent protein kinase C activity) illustrate the cross-talk between neuroinflammatory signaling pathways and neurotransmission.

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Table V: Molecular function analysis of neuroinflammatory genes in irradiated brain.

Complement MHC Class II, peptide antigen fWBI, DLPFC Fold Enrichment p value MHC class II receptor activity > 100.00 1.50E-04 platelet-derived growth factor binding > 100.00 1.50E-04 MHC class II protein complex binding > 100.00 2.66E-04 peptide antigen binding > 100.00 5.29E-05 integrin binding 34.91 3.63E-02

fWBI, Hippocampus Fold Enrichment p value MHC class II receptor activity > 100.00 1.26E-05 peptide antigen binding > 100.00 1.03E-06 MHC class II protein complex binding > 100.00 1.11E-02

fWBI, White Matter Fold Enrichment p value opsonin receptor activity 89.16 1.20E-03 tumor necrosis factor binding 79.25 2.78E-02 ICAM-3 receptor activity 79.25 2.75E-02 glutamate-gated calcium ion channel activity 71.33 1.76E-03 NMDA glutamate receptor activity 67.93 1.42E-04 calcium-dependent protein kinase C activity 59.44 3.81E-02 complement component C3b binding 59.44 3.78E-02 benzodiazepine receptor activity 59.44 2.39E-03 lipoteichoic acid binding 59.44 3.75E-02 CCR2 chemokine receptor binding 59.44 3.72E-02

TBI, White Matter Fold Enrichment p value MHC class II receptor activity > 100.00 2.78E-02 platelet-derived growth factor receptor binding > 100.00 4.06E-02 CXCR chemokine receptor binding > 100.00 4.13E-02 chemokine activity 53.68 1.06E-02 cytokine binding 34.38 5.49E-03 cytokine receptor activity 28.28 3.99E-02 serine-type endopeptidase activity 16.99 7.86E-03 peptide binding 13.13 4.87E-02 Analyses completed in Gene Ontology enrichment analysis tool. Maximum fold-enrichment values are truncated at 100. The top 10 enriched ontologies are reported in order of decreasing fold-enrichment and statistical significance, multiplicity adjusted p < 0.05 were considered significant. Pathways in orange are associated with complement; pathways in blue are associated with MHC class II peptide antigen presentation.

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ECM-associated canonical pathways were detected in all regions of fWBI animals and none from TBI animals. The contributing genes (fWBI DLPFC: 12 DEGs, fWBI HC:

4 DEGs, fWBI WM: 43) were evaluated or patterns in molecular function (Table VI).

Enriched ECM-pertinent molecular functions were not detected in the HC of fWBI animals. Platelet derived growth factor signaling was implicated in both regions, and functions which contribute to ECM deposition and remodeling were detected in the fWBI

WM. Notably, PDGF signaling was partially indicated due to differential regulation of platelet derived growth factor receptor alpha (PDGFRA) expression (DLPFC: log2fold change: -1.743, p value: 8.01E-28; WM: log2fold change: -5.88, p value: 9.79E-78 ). This is most likely associated with oligodendrocyte progenitor depletion, however

Table VI: Molecular function analysis of extracellular matrix and remodeling-associated genes in fWBI brain. ECM remodeling PDGF

fWBI, DLPFC Fold Enrichment p value platelet-derived growth factor binding > 100.00 2.96E-04

fWBI, White Matter Fold Enrichment p value tumor necrosis factor binding > 100.00 5.93E-03 platelet-derived growth factor binding > 100.00 6.18E-06 interleukin-1 receptor activity > 100.00 1.80E-02 tumor necrosis factor-activated receptor activity 99.63 1.50E-06 platelet-derived growth factor receptor binding 95.65 1.16E-03 phosphatidylinositol-4,5-bisphosphate 3-kinase activity 43.48 2.51E-06 extracellular matrix structural constituent 42.92 5.27E-07 integrin binding 29.62 1.85E-06 protease binding 24.98 3.89E-06 collagen binding 22.77 3.80E-02 Analyses completed in Gene Ontology enrichment analysis tool. Maximum fold-enrichment values are truncated at 100. The top 10 enriched ontologies are reported in order of decreasing fold-enrichment and statistical significance, multiplicity adjusted p < 0.05 were considered significant. Pathways in orange are associated with extracellular matrix remodeling; pathways in blue are associated with platelet derived growth factor signaling.

154 expression of collagen 1 and 3 subunits (COL3A1, COL1A1) in the DLPFC and WM, and epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), fibroblast growth factor 1 (FGF1), platelet derived growth factor beta (PDGFB) and hepatocyte growth factor (HGF) in WM implicate a role for this pathway in ECM deposition.

Alteration of canonical pathways involved in neurotransmission and signal transduction were detected within the white matter of both irradiated groups.

Table VII: Molecular function analysis of neurotransmission and signal transduction associated genes in white matter. Ca++ Glutamate

fWBI, White Matter Fold Enrichment p value neurotransmitter receptor activity involved in regulation of > 100.00 2.64E-03 postsynaptic cytosolic calcium ion concentration glutamate-gated calcium ion channel activity > 100.00 4.11E-05 NMDA glutamate receptor activity > 100.00 2.13E-06 adenylate cyclase inhibiting G-protein coupled glutamate receptor > 100.00 4.97E-03 activity glutamate binding > 100.00 2.05E-04 extracellularly glutamate-gated ion channel activity > 100.00 7.71E-07 high voltage-gated calcium channel activity > 100.00 2.34E-04 glycine binding > 100.00 3.94E-04 L-glutamate transmembrane transporter activity 88.41 2.30E-02 calcium channel regulator activity 53.05 3.19E-03

TBI, White Matter Fold Enrichment p value ionotropic glutamate receptor activity > 100.00 6.99E-03 extracellularly glutamate-gated ion channel activity > 100.00 6.75E-03 ligand-gated calcium channel activity > 100.00 1.01E-02 actinin binding 97.87 2.87E-02 neurotransmitter binding 80.93 4.01E-02

Analyses completed in Gene Ontology enrichment analysis tool. Maximum fold-enrichment values are truncated at 100. The top 10 enriched ontologies are reported in order of decreasing fold-enrichment and statistical significance, multiplicity adjusted p < 0.05 were considered significant. Pathways in orange are associated with calcium ion regulation; pathways in blue are associated with glutamatergic neurotransmission and glutamate regulation.

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Analysis of the contributing genes to neurotransmission and signal transduction- associated canonical pathways (32 fWBI DEGs, 10 TBI DEGs) primarily demonstrated alteration in mechanisms modulating glutamatergic neurotransmission and calcium channel activity (Table VII).

Supervised Analyses

Neuroinflammatory analyses indicated that MHC class II peptide antigen presentation and the complement system play a role in RIBI. As antigen presentation may lead to T-cell activation, and the adaptive immune system may regulate complement activation, T-cell activation and differentiation gene were also assessed.

Gene expression associated with MHC class II antigen presenting molecules

(HLA-DPB1, HLA-DQA1, HLA-DMA) was increased within all brain regions in fWBI and WM in TBI (Figure 6A). HLA-DPA1 and HLA-DRB5 were also expressed within all brain regions in fWBI animals, and HLA-DMB within the DLPFC and WM of fWBI animals.

Complement factor associated gene expression was increased within the WM of fWBI and TBI animals (Figure 6B). CFB and C7 were also expressed within the fWBI

DLPFC, and C3AR within the fWBI HC. Complement regulatory proteins CD59, C4BP,

SERPING1, and CR1 were differentially regulated in fWBI, but not in TBI.

C4+ T-cell activation is a consequence of antigen presentation by MHC class II molecules on antigen presenting cells (microglia), thus pathways associated with T-cell differentiation were assessed (Figure 6C).

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Figure 6: Differentially regulated neuroinflammatory genes in RIBI

A B C

A n tig e n P re s e n ta tio n C o m p le m e n t T -C e ll A c tiv a tio n

L C

D H W

I I I

B B B

W W W

f f f T

f T

f f f T

H L A -D P B 1 C F B H L A -D Q A 1

H L A -D Q A 1 C 1 Q C H L A -D M A

H L A -D M A C 1 Q A H L A -D R B 5

H L A -D P A 1 C 2 H L A -D M B 3

C L IP C 7 R O R y t

H L A -D R B 5 C 1 r H L A -D O A

H L A -D M B C R 1 T N F -a

L H L A -D O A C 3 A R 2 F C E R 1 G o

g C IIT A C 1 s N G F R 2

M H C I-ß IT G B 2 IL -1 0 F

M R 1 IF IL 1 0 R A d

1 N L R C 5 C 5 A R S T A T 6 C

H L A -F S E R P IN G 1 H L A -A a

H L A -A C D 5 9 IL 1 0 R B g

e P S M B 9 D F T N F R S F 1 B 0 L M P 7 C 4 B P B T G F B R 2

IL 2 R G

T N F R S F 1 A

C D 8 6 -1 C D 4 0

IL 2 1 R

IL 1 2 R B 1

Differentially regulated gene contributors to canonically-enriched pathways of neuroinflammation: antigen presentation, the complement system and T-cell activation. Gene lists were curated by Ingenuity Pathway

Analysis. Gene IDs are HGCN identifiers. Red: Up-regulation, Green: Down-regulation. White boxes containing ‘x’ indicate the gene was not differentially regulated within that region. All log2fold changes were < ±1 and multiplicity adjusted p values (Benjamini-Hochberg) < 0.05.

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Gene expression related to T-cell activation and differentiation was most prominent within the WM of fWBI animals. CD3D and CD3E expression was increased within the WM of fWBI animals (data not shown). RAR-related orphan gamma receptor

(RORyt), a molecule involved in chronic inflammatory signaling through Th17 cells, was up-regulated within the WM of fWBI and TBI animals. STAT6, involved in Th2 differentiation, was up-regulated in the WM of fWBI animals. Co-stimulatory molecules essential for the activation of antigen-presenting cells (CD86), T-cell (CD86) and B-cells

(CD40) were up-regulated within fWBI WM. These data provide preliminary evidence that MHC class II antigen presentation may induce T-cell activation in RIBI.

Ontologic evaluation suggested that ECM deposition and remodeling in the white matter may contribute to RIBI in fWBI animals. A comprehensive list of ECM contributors was compiled by literature review (47-55). Of several classes of ECM molecules, differential gene regulation was noted in basement membrane contributors, proteoglycans, fibrous proteins, glycoproteins and ECM molecules generally enriched in

CNS, bone, cartilage and teeth (Figure 7). No changes were noted in any of the keratan sulfate proteoglycans, vascular-associated fibrinogen chains, vitronectin or von

Willebrand factor, small interstitial proteoglycans (decorin, biglycan, asporin), laminins, agrin, aggrecan or nidogen.

As previous analyses suggested that radiation exposure alters neurotransmission and signal transduction pathways in white matter, we completed a supervised review of

DEGs in the WM of TBI and fWBI animals for contributing molecular functions. The oligodendrocyte precursor marker platelet derived growth factor alpha (PDGFRA) was

158

Figure 7: Differentially regulated ECM transcripts in white matter following fWBI

f f

C O L 4 A 3 2 M F A P 2 C O L 4 A 1 C O L 3 A 1 B a s e m e n t H S P G 2 F ib r o u s P r o te in s M e m b ra n e C O L 8 A 2 C O L 4 A 4 C O L 2 4 A 1 1 F B L N 2 C O L 1 9 A 1

S D C 1 0 M A T N 3 S P O C K 2 G L D N H A P L N 3 T N C G P C 5 F N 1 V C A N -1 P ro te o g ly c a n s M A T N 2 C S P G 4 G ly c o p ro te in s H M C N 1 G P C 6 E M IL IN 3 H A P L N 4 -2 F B N 2 B C A N F B N 3 C S P G 5 T N R N C A N -3

H A P L N 1 L T B P 2 E n r ic h e d in B o n e s , N T N G 1 C R T A C E n ric h e d in C N S -4 C a rtila g e a n d T e e th S L IT 3 B M P E R

Supervised analysis of differentially expressed extracellular matrix genes in WM in RIBI post-fWBI. Gene

IDs are HGCN identifiers. Red: Up-regulation, Green: Down-regulation. All log2fold changes were < ±1 and multiplicity adjusted p values (Benjamini-Hochberg) < 0.05.

decreased in all regions following fWBI but not TBI (Figure 8; DLPFC log2fold change:

-1.74, p value: 8.01E-28; HC log2fold change: -1.75, p value: 1.80E-21; WM log2fold change: -5.88, p value: 9.79E-78). Glutamatergic NMDA receptor subunit GRIN2B

159

(fWBI log2fold change: -1.74, p value: 0.002; TBI log2fold change: -1.22, p value: 0.04) and AMPA receptor subunit GRIA3 (fWBI log2fold change: -2.52, p value: 1.50E-08;

TBI: log2fold change: -1.24, p value: 0.02) were decreased in WM but not DLPFC or HC in both irradiated groups

Figure 8: Decreases in glutamatergic neurotransmitter receptor subunits in RIBI occurs

independently of PDGFRA expression

)

l r t 2

C 0 0

/ x

T 0 (

-1 e

g -2 -1 n

a * * -2

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-4

C d l fW B I * -3 o -6 -2 F T B I * 2 *

g -8 * -4 o

L D L P F C H C W M D L P F C H C W M D L P F C H C W M

P D G F R A G R IN 2 B G R IA 3 Supervised analysis of oligodendrocyte precursor marker, platelet derived growth factor alpha (PDGFRA) and glutamatergic neurotransmitter receptor subunits GRIN2B and GRIA3. PDGFRA was decreased in DLPFC, HC and WM 12 months post-fWBI but not 6-9 years post-TBI. Glutamatergic NMDA receptor subunit GRIN2B and AMPA receptor subunit GRIA3 expression were decreased in white matter in both fWBI and TBI groups. Bar: Mean log2fold change. Error bars: log fold change standard error. Log2 fold changes < ±1 and multiplicity adjusted p values (Benjamini-Hochberg) < 0.05 were considered significant.

Additional down-regulated neurotransmission-associated genes detected in WM of both irradiated groups were bassoon presynaptic cytomatrix protein (BSN), calcium voltage-gate channel subunit alpha 1 G (CACNA1G), CACNA1I, calcium voltage-gated channel auxillary subunit beta 1 (CACNB1), calcium/calmodulin-dependent protein kinase 2 beta (CAMK2B), potassium voltage-gated channel subfamily B member 1

(KCNB1), potassium two pore domain channel subfamily K member (KCNK5), potassium calcium-activated channel subfamily M alpha 1 (KCNMA1), potassium sodium-activated channel subfamily T member 1 (KCNT1), synaptotagamin 2 (SYT2),

160

SYT5 and SYT6. CACNA1D, KCNK12, synaptic Ras-GTPase activating protein 1

(SYNGAP1), sodium voltage-gated channel beta subunit 2 (SCN2B) were differentially regulated in TBI WM. The gene encoding glutamate transporter excitatory amino acid transporter 2 (EAAT2; gene: SLC1A2) was down-regulated in the WM of fWBI animals.

DISCUSSION

Herein, we have characterized the transcriptomic signature of radiation-induced brain injury in the Rhesus macaque and demonstrate that fWBI and TBI manifest shared patterns of injury months and years post-irradiation, respectively. Our analyses reaffirm that RIBI is associated with microglial/macrophage mediated-neuroinflammation, and present novel evidence that the complement system may contribute to injury. We also demonstrate that ECM deposition and remodeling appear to play a role in RIBI following fWBI but not TBI, and characterize radiation-induced changes in the matrixome.

Furthermore, we also present data which challenge the existing hypothesis that radiation- induced brain injury is primarily due to perturbation of hippocampal function and structure.

In the present study, regional stratification of DEGs demonstrates a relative paucity of gene expression changes within the hippocampus as compared to white matter

(55 HC DEGs vs. 1815 WM DEGs in fWBI, 2 HC DEGs vs. 172 WM DEGs in TBI).

These findings are consistent with our previous gene expression analyses (35). Given the integral role of the hippocampus in memory and cognition, and numerous preceding studies which demonstrate radiation-induced effects on hippocampal structure and function (32, 57-76), hippocampal gene expression patterns data were carefully reviewed

161 for the involvement of neurotransmission, signal transduction and stem cell signaling pathways. The involved signaling pathways were consistent with inflammatory processes and we did not detect alteration in neurogenic or synapse markers. These data refute the claim that radiation-induced changes in neurotransmission are solely due to altered hippocampal function. Our data are transcriptional and structural and proteomic studies are needed to confirm this hypothesis, however these data suggest that hippocampal- mediated dysfunction may contribute less to RIBI in primate than in rodents. These data also suggest that hippocampal-sparing irradiation strategies may prove insufficient to prevent radiation-induced cognitive dysfunction in patients receiving fWBI.

Hippocampal protection is not feasible in malicious or accidental exposure scenarios.

Thus, there is a need to develop mitigation strategies of radiation-induced brain injury, which do not rely solely on hippocampal protection.

The present study also provides evidence that the brain is vulnerable to injury from single doses of total body irradiation (6.05-8.5 Gy), which are much lower than the previously assumed threshold for CNS injury (7). This suggests that even patients that do not develop the acute radiation CNS syndrome (77) following an accidental or malicious exposure may still be at risk of developing more subtle neurologic injury. The functional ramifications of this injury are currently unknown, but preliminary cognitive test in this same cohort of animals suggests that animals receiving high dose TBI may demonstrate less cognitive flexibility (78). Thus, ARS survivors may benefit from more aggressive surveillance and monitoring of cognitive function.

Contrary to our initial assumption, modulation of ECM deposition and remodeling-associated pathways were noted in fWBI WM but not in TBI WM. This

162 suggests an inherent difference in the radiation response to fractionated vs single dose irradiation. In an effort to further understanding of radiation-induced alterations in the matrixome, we have provided an unbiased assessment of all differentially regulated ECM contributors to fWBI WM (Figure 7). Notably, fWBI is associated with cognitive impairment and in some patients, dementia, progressive neurodegeneration and neurological impairment; while the animals in our study receiving TBI are neurologically normal. Thus, we postulate that the deposition and remodeling of ECM may play a role in the enhanced pathogenicity of RIBI following fWBI. These data are preliminary and additional study is warranted.

As the rhesus gene identifiers were transcribed to human identifiers for analysis, the MHC class II molecules referred to herein as HLA-transcripts should be referred to as

MAMU-transcripts. Our data reaffirm that irradiation induces microglial activation (75,

79-84), a process which leads to up-regulation MHC class II antigen presentation molecules (85-88). These molecules are responsible for the presentation of extracellularly-derived peptide antigens derived from the phagocytosis and processing of cellular debris. The ultimate purpose of this process is to present antigens to helper T- cells, which results in their activation (89). In the absence of exogenous sources of foreign antigens, we presume that MHC class II molecules must be presenting endogenously derived antigens which we hypothesize are acquired from the phagocytosis of necrotic debris (exposed intracellular antigens) or proteins altered by radiation (90).

Therefore, we propose the novel hypothesis that sustained microglial activation following irradiation may induce auto-sensitization and self-reactivity leading to long-term perpetuation of injury.

163

Furthermore, antibody-mediated immunity, as results from auto-sensitization, may induce complement activation via the classical pathway (C1 complex-associated).

As our data demonstrate increased expression of C1 complement factors, we hypothesize that a portion of complement activation in RIBI be due to auto-antibody production.

While complement is known to mediate radiation-induced cell-killing (91-93), we are the first to demonstrate that complement activation may play a role in the pathogenesis of

RIBI.

Complement activation may also to synaptic degeneration and loss. Complement- mediated synaptic pruning is responsible for the development of efficient neuronal circuitry during development (94). In adults, complement may play a role in the synaptic loss associated with neurodegenerative diseases. In transgenic mouse models of

Alzheimer’s disease, complement factor C1q is required for the induction of amyloid- beta oligomer-mediated synaptic loss (95, 96). C3 knockout mice are protected against age-related synaptic loss and cognitive decline (97) and demonstrate enhanced spatial learning (98). These findings suggest that neuroinflammation may modulate impairment in neurotransmission and signal transduction in RIBI.

Canonical pathway and ontologic analysis demonstrated differential expression of glutamatergic neurotransmission and signal transduction-associated mRNAs in the WM of fWBI and TBI animals. This finding was unexpected, as alteration of neurotransmitter-associated, stem cell, or signal transduction-associated transcripts was noticed in the hippocampus of fWBI or TBI animals, as mentioned previously.

164

Within the white matter, both glia and axons possess the cellular machinery required for synaptic vesicular neurotransmission, including NMDA, AMPA and kainate receptors, and glutamate transporters. Unmyelinated axons communicate with adjacent

NG2+ oligodendroglial progenitor cells via action potential-induced vesicular glutamate release, which may facilitate myelination (99-102). We propose the novel hypothesis that the reductions in neurotransmission-associated transcripts within white matter are due to radiation-induced loss or dysfunction of these deep white matter synapses. Radiation- induced oligodendrocyte progenitor suppression (103-106) or ischemic injury (99-102) may both contribute to loss of PDGFRA+ oligodendroglial precursors, thus reducing the number of deep white matter synapses. However, in TBI animals we observed reduction in glutamatergic neurotransmitter receptors (GRIN2B and GRIA3) in the face of normal levels of PDGFRA, suggesting that deep white matter synaptic impairment may occur independently of oligodendrocyte progenitor cell loss.

Additionally, the scavenging of extracellular glutamate by astrocytes and oligodendrocytes via glutamate transporters is essential to maintaining normal glutamate homeostasis (107). Impaired scavenging may contribute to glutamate excess, which may thus result in excitotoxic injury to oligodendrocytes via glutamatergic receptor binding induced calcium influx (108-110). Indeed, inhibition of glutamate scavenging by administration of dihydrokainate results in oligodendroglial loss, massive demyelination and severe axonal damage in optic nerves of rats (111). As our data demonstrate decreased expression of the encoding gene for EAAT2, SLC1A2, in the WM of fWBI animals, it is possible that excitotoxic injury may contribute to cognitive dysfunction in

RIBI by contributing to demyelination and white matter injury. Sustained

165 neuroinflammation may exacerbate these effects, as the pro-inflammatory cytokine TNF- a reduces glutamate transporter expression in oligodendrocytes (112), and enhances glutamate synthesis in microglia (113). The significance of differential regulation of

MEIS3 and SCEL in the DLPFC of fWBI and TBI animals is unclear. MEIS3 encodes a homeobox protein presumed to play a role in transcriptional regulation (114, 115). SCEL is a component of the cornified envelope of keratinocytes (116-120).

While we acknowledge that differing diet, age and comorbidities between groups may have affected cerebral gene expression, we nonetheless report the first cerebral transcriptomic data from NHP years following single high dose TBI. In the event of a large scale nuclear accident or malicious exposure, anatomic sites of radiation exposure and extent of shielding within the exposed population will be heterogeneous, often with multiple organ systems affected as demonstrated by studies of the Hiroshima and

Chernobyl survivors, and the NHP RSC (4, 8). Therefore, radiation-associated comorbidities are anticipated in survivor populations, and related alterations in cerebral gene expression are relevant to consider for long-term radiation survivors.

The Typical American Diet (TAD) is pro-inflammatory, atherogenic and obesogenic and models the dietary contributions to chronic disease present in western populations. As control comparators were also maintained on TAD, comparisons between groups are considered valid and may underestimate the magnitude of change in animals receiving maintained on typical monkey chow.

Despite statistical differences in age, all groups of animals were within similar life stages (young adult, post-pubertal), and biologically comparable. As previous studies

166 have demonstrated age and development-associated changes in hippocampal gene expression in rodents (121-123) and rhesus macaques (124) the agreement in hippocampal gene expression between TBI and control animals (0 DEGs, mean age difference 6.01 years) supports that these animals were in similar life stages.

In summary, we are the first to report whole transcriptomic profiling from a non- human primate model of RIBI following fWBI, and to assess transcriptomic alterations in non-human primate survivors of single high dose total body irradiation (6.75-8.05 Gy).

We present novel evidence that these animals manifest evidence of CNS injury years post-irradiation, with similarities to the more characterized RIBI following fWBI. These analyses reaffirm the involvement of macrophage/microglial-mediated neuroinflammation in RIBI, and suggest that long-term impairment in glutamatergic neurotransmission and neuronal signal transduction may mediate RIBI following fWBI and TBI. Furthermore, due to unbiased survey of the entire transcriptome, these data provide insight into the pathogenesis of RIBI, and we have identified novel signaling pathways, including involvement of complement and the acquired-immune system which may contribute to injury months and years post-irradiation. Finally, these data challenge the assumption that alterations in hippocampal structure and function are primarily responsible for cognitive impairment in RIBI, and provide evidence that alterations in neurotransmission pathways may be more prominently affected in white matter.

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ACKNOWLEDGEMENTS

This work was supported in part by the Cancer Genomics Shared Resource supported by the Wake Forest Baptist Comprehensive Cancer Center’s NCI Cancer

Center Support Grant P30CA012197, P30 CA012197-39 (Cancer Center grant) and AN

3932506 (instrumentation grant). Drs. Andrews and Cline were supported by NIH grant

U19-AI67798 awarded to Nelson Chao, Duke University (JMC: primate core leader,

Wake Forest School of Medicine).

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71.

185

CHAPTER FIVE

Impact, Conclusions and Future Directions

Rachel N. Andrews, D.V.M.

186

DISCUSSION

Summary

In summary, we have further characterized the molecular manifestations of RIBI in a non-human primate model, the Rhesus macaque, and reaffirm data acquired from rodent studies that macrophage/microglial-mediated neuroinflammation may contribute to the pathogenesis of RIBI (1-7). We have also identified preliminary evidence of potential novel mechanisms of RIBI including complement-mediated injury and self- reactivity, deep-white matter synaptic dysfunction, and Rho\ROCK-mediated perivascular fibronectin accumulation. Additional study is warranted to assess the contribution of these pathways to RIBI. Finally, we reaffirm that the Rhesus macaque is a reasonable model in which to study the pathogenesis of RIBI as it recapitulates the histopathologic (8-12) and functional features (13) of RIBI.

Proposed Mechanism

From these findings, we propose a novel mechanism of radiation-induced brain injury which accounts for the perpetuation of injury months post-irradiation. Others have shown that exposure to ionizing radiation results in immediate activation of RhoA/ROCK signaling (14-17) in endothelial cells, cytoskeletal contraction and stress fiber formation

(14, 18, 19). This abrupt endothelial cell contracture leads to mechanical rupture of inter- endothelial cell junctions (14, 18-20). Resultant disruption of blood-brain-barrier integrity permits the extravasation of plasma containing fibronectin into the perivascular space and adjacent parenchyma (21). Fibronectin binding to cell surface α5β1 integrins sustains endothelial cell contraction (22-24), and a combination of stress fiber formation

187 and reduction in cell surface area facilitates integrin clustering. This clustering brings multiple fibronectin dimers into proximity and promotes fibronectin-fibronectin interactions, self-association, fibril elongation and matrix assembly.

Figure 1: Proposed novel mechanisms of radiation-induced brain injury

Perivascular fibronectin matrix accumulation increases the diffusion distance, and the resulting diffusion barrier results in ischemia and subsequent necrosis. White matter is susceptible to this injury (25) due to its neurovascular architecture (lesser microvascular investiture, fewer anastomoses and relative lack of penetrating arterioles compared to grey matter)(Chapter 1, Table I; (26). Ischemic tissues are then bound by self-reactive IgM molecules, which leads to classical complement activation and fixation,

188 and further necrosis (27-29). Although a single IgM molecule is capable of activating complement efficiently, several IgG molecules may also activate complement (30) which suggests the capacity for a component of self-reactivity in RIBI.

Irradiation also induces microglial activation (1-7), which leads to up-regulation

MHC class II antigen presentation molecules (31-34). Microglial phagocytosis of necrotic cellular debris leads to the processing of exposed intracellular antigens or proteins altered by radiation (35). These microglia may then present processed antigens to T-cells, leading to effector T-cell induction (36). Stimulated effector T-cells may then activate B-cells which lead to the production of auto-antibodies in RIBI, thus leading to sustained complement activation and long-term perpetuation of injury.

Finally, due to radiation-induced oligodendrocyte progenitor suppression (37-40) or ischemic injury (41, 42), radiation results in persistent loss or reduced function of deep white matter synapses. These unique structures are pathways of communication between oligodendrocyte precursors and unmyelinated axons, characterized by vesicular glutamatergic neurotransmission (41-44), may play an integral role in coordinating myelination. Furthermore, persistent neuroinflammation may potentiate further cerebrovascular and white matter injury as microglial activation and impaired astrocytic glutamate uptake result in an excess of extracellular glutamate, leading to excitotoxic cellular injury (45-49). Ultimately, disruption of white matter tract integrity, whether by ischemic injury, immune-modulated mechanisms, or impaired excitatory neurotransmission, may negatively impact cognition via slowed impulse transmission due to demyelination and axonal damage (50, 51). Others have demonstrated that white matter injury results in decreased coherence in patterns detectable by

189 electroencephalography (EEG), and is associated with cognitive impairment (52, 53).

Several neurologic disorders characterized by prominent white matter injury also result in cognitive impairment, including toluene leukoencephalopathy, Binswanger’s disease, chronic traumatic encephalopathy, multiple sclerosis and metachromic leukodystrophy

(51, 54, 55). This suggests that white matter injury, as occurs in RIBI, may be sufficient to negatively impact cognitive function.

Thus, we present a novel mechanism of RIBI which accounts for multiple interactions between glial, neural and vascular contributors, and provides direction for further research.

IMPACT

Previous research has emphasized the importance of alterations in hippocampal structure and function in RIBI. The regional gene expression data presented in chapters 2 and 4 challenge the hypothesis that the hippocampus is the primary site of injury in RIBI.

While we acknowledge that the absence of transcriptional alteration does not preclude structural or functional change in neurons or neurogenic precursors, our data suggest that the assumption that radiation-induced impairment in neurotransmission is restricted to the hippocampus is erroneous. Therefore we presume that hippocampal-sparing irradiation techniques will be insufficient to result in full neuroprotection. Thus there is a need to identify additional therapies and mitigation strategies.

As mentioned briefly, our work in Chapters 2 and 4 unexpectedly demonstrates that reductions in neurotransmission and signal transduction-associated transcripts occur most prominently within white matter. We propose the novel hypothesis that this is due

190 to radiation-induced loss or dysfunction in deep-white matter synapses, structures essential for communication between oligodendrocyte precursors and unmyelinated axons (41, 42). This represents a new research direction into the pathogenesis of RIBI and may further elucidate the role of oligodendrocyte progenitors in radiation-induced brain injury. Although it is premature to suggest modulation of deep white matter synaptic transmission is a viable therapy for RIBI, it is a new area of study to identify therapeutic targets. Furthermore, identification of the role of deep-white matter synapses in impaired neurotransmission and the development of assays to detect changes in deep white matter synapses may translate to other models of neurodegenerative disease.

Another impact of this work is our identification of another potential mechanism of RIBI: complement activation and self-reactivity. Although others have previously demonstrated the contribution of macrophage/microglial-mediated neuroinflammation to radiation-induced brain injury, the precise mechanism by which microglial potentiate

RIBI is unclear. In the absence of neoplasia and exogenous sources of foreign antigens

(infection) recruitment of the complement system suggests self-reactivity. This mechanism, outlined briefly in the previous section, suggests a novel role for microglia/macrophages in RIBI as antigen-presenting cells which drive autosensitization.

The suggestion that inhibition of antigen presentation or complement antagonism are viable treatments for RIBI is premature; however assessment of the role of these pathways and related self-reactivity is a promising new direction for future research.

Potential Radioprotectants, Mitigators and Treatments of Radiation-Induced Brain

Injury

191

Based on the pathways indicated by our molecular analyses, we propose that the following compounds are promising potential interventions for RIBI. Evaluation of these compounds in translation models of RIBI will provide mechanistic insight into the pathogenesis, and potentially, develop novel therapies for the treatment of RIBI. Efficacy and safety testing of these compounds in translational models will be essential prior to assessment in human patients.

Fasudil hydrochloride is a potent, selective RhoA/Rho-kinase (ROCK) inhibitor approved for the treatment of cerebral vasospasm and ischemic stroke in China and

Japan. It has also been used in rodent studies to improve memory and learning in aged rats (56), reduce infarct area following middle cerebral artery occlusion (57), and provide neuroprotection in models of Alzheimer’s (58), amyotrophic lateral sclerosis (ALS) (59), and Parkinson’s disease (60). Similar compounds protect hippocampal neurons from excitotoxicity-induced death (61).

Supervised analysis of our transcriptomic data from Chapter 4 indicates several regulators of Rho activity (62) are differentially regulated following fWBI (Table I).

Alterations in the gene expression of Rho-assosciated coiled-coil-containing protein kinases (ROCK1 and ROCK2) and the majority of Rho-family small GTPases were not detected by differential gene expression analysis. However, as these molecules are regulated by phosphorylation and protein level interactions (63-65), differential expression at the transcript level is presumed unlikely. Proteomic analysis is necessary to confirm phosphorylation of Rho, and will be the focus of future study. Interestingly, Rho family GTPase 1 (Rnd1), a protein which decreases stress fiber formation and focal adhesions, was down-regulated (log2fold change: -1.567, p value: 0.01). Up-regulation of

192

Rac2 (log2fold change: 3.216, p = 8.45 E-13) another Rho-GTPase, may be related to phagocytosis and reactive oxygen species (66) production in RIBI.

Figure 2: Differentially Expressed Regulators of Rho-family Small GTPases in fWBI

White Matter e e g g n n a a h h C C d l d o e l e o F lu l u 2 a F g 2 a o v g v L p o p L

A R H G A P 2 4 0 .0 3 5 Supervised analysis revealed differential A R H G A P 1 5 expression of several Rho-family small 1 .5 A R H G A P 2 5 GTPase regulators in white matter 0 .0 3 0 following fWBI. Functions are as A R H G A P 1 8 follows: Rho GTPase Activating Proteins A R H G A P 3 6 1 .0 0 .0 2 5 (ARHGAPs): Dephosphorylation A R H G A P 3 0 (inhibition) of Rho; Rho Guanine A R H G A P 2 7 0 .5 Nucleotide Exchange Factors 0 .0 2 0 A R H G A P 4 (ARHGEFs): Phosporylation (activation) A R H G A P 2 0 of Rho; Rho Dissosication Inhibitors 0 0 .0 1 5 (ARHGDIs): Sequestration (inhibition of A R H G A P 4 4 Rho). Data were stratified by function, A R H G A P 2 8 and sorted from highest to lowest -0 .5 0 .0 1 0 A R H G E F 3 8 log2fold changes. Log2Fold Changes > A R H G E F 2 5 ±2 and p < 0.05 were considered -1 .0 0 .0 0 5 significant. A R H G D IB

A R H G D IG

Due to variation in directionality of log2fold changes, directionality of effect on

Rho-family activation cannot be inferred; however these data suggest a role for modulation of Rho signaling pathways in RIBI. Others have demonstrated radiation- induced Rho-GTPase activation (14), decreased levels of the RhoA inhibitor p-Rho GDI

(15), and differential gene expression of several Rho family small GTPases in human

193 tissues with radiation induced intestinal fibrosis (67). Thus Rho\ROCK inhibition appears to be a reasonable therapeutic target in RIBI.

One potential limitation to assessing Fasudil as a treatment in RIBI is route of administration. The recommended dosage for the treatment of cerebral vasospasm and ischemic stroke in humans is 30 mg delivered via intravenous infusion over 30 minutes 2-

3 times daily. However, once daily subcutaneous injection in aged rats (56) was effective in improving learning and working memory, and oral administration in drinking water in mice attains therapeutic levels and was well tolerated when administered long-term (4-5 weeks) (59, 68). These data suggest that alternative routes of administration may still be effective. The potential for long-term, oral administration increases the likelihood that

Fasudil could be incorporated as a treatment option in RIBI. We hypothesize that Fasudil will be most effective as a radioprotectant by counteracting radiation-induced endothelial cell contracture (14, 16, 17, 69), however it may also be effective as a mitigator or treatment. We hypothesize that at later time periods, Fasudil may improve cognition by preventing or reversing perivascular fibrosis by inhibiting RhoA/ROCK dependent endothelial-fibronectin interactions (70).

Tiplasinin is a first-generation, orally active, plasminogen-activator inhibitor

(PAI-1) antagonist. PAI-1 is a pro-thrombotic, pro-inflammatory and pro-fibrotic compound which contributes to a variety of fibrotic conditions in the lung (71-74), gastrointestinal tract (75), vasculature (76, 77), and kidney (78-81). We and others have demonstrated that irradiation induces increased expression of PAI-1 and its encoding gene, SERPINE1 (75, 79, 80, 82-85). Increased expression of PAI-1 is also associated with poor prognosis, increased metastasis, and radioresistance in cancer (86-90), thus

194 inhibition of PAI-1 may be beneficial in the treatment of both cancer and RIBI.

Tiplasinin reduces airway fibrosis in a mouse model of asthma (71) and aortic modeling in a rat model of hypertension (77). We hypothesize that it will be effective in reducing perivascular extracellular matrix deposition in RIBI by reducing fibronectin matrix assembly (91).

As genetic PAI-1 deficiency in humans is associated with mild to moderate hemorrhagic diathesis (92), careful dose titration will be required with the goal of reducing PAI-1 excess without completely eradicating PAI-1 activity. Although a Phase-I clinical trial evaluating Tiplasinin for the treatment of thrombosis in humans was halted due to similar concerns, safety testing in rodents showed no toxic effects at up to 2000 mg/kg per day, 1500 times the efficacious dose (93). Dogs administered 10 mg/kg and rats administered up to 100 mg/kg had no abnormalities in bleeding time, activated partial thromboplastin time, prothrombin time or platelet aggregation (94, 95). Although preclinical studies seem to indicate a wide safety margin, due to increased risk of hemorrhage in the acute and late phases of the cerebral radiation response (96-99),

Tiplasinin would likely be most suitable as a mitigator in RIBI. Assessment of safety in preclinical trials is essential.

FUTURE DIRECTIONS

The majority of the work contained within this dissertation has used gene expression analysis for the screening of potential novel pathways and molecular effects of

RIBI. Subsequent structural and functional assessment of the identified targets and related structures will be essential to confirm the contribution of these pathways to the

195 pathogenesis in RIBI. In particular, studies are warranted investigating the role of white matter ischemia, complement activation and deep-white matter synapses in RIBI. We also propose that evaluation of the effects of low dose total body irradiation will be beneficial for public health and risk assessment, particularly for large scale accidental or malicious exposure scenarios.

We and others have developed the working hypothesis that radiation-induced cerebrovascular injury, and subsequent vascular remodeling and extracellular matrix deposition lead to diffusion barrier formation and resultant ischemia. This assumption is pivotal, as different mitigation strategies are indicated for vascular protection versus other mechanisms of injury. Despite its importance, the assumption that cerebrovascular injury and hypoxia precede the formation of white matter necrosis has not been definitively proven. Prospective studies interrogating the temporal and spatial relationship between hypoxia and necrosis in RIBI would be indispensable in evaluating mechanisms of RIBI.

2,3,5-Triphenyltetrazolium chloride (TTC) or pimonidazole hydrochloride assays permit post-mortem evaluation of tissue ischemia and hypoxia respectively, however are limited to a single time point and require sacrifice of the animal.

Alternatively, longitudinal assessment of in vivo hypoxia markers such as the positron emission tomography ligand [18F]fluoromisonidazole (18F-MISO), with time matched susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) to assess necrosis and hemorrhage may provide insight into this relationship. Unfortunately, in vivo assessment of such markers and the cerebral vasculature are limited by various technological constraints including the resolution of the imager, relative scarcity of extremely high resolution imagers, and the size of the animal that a given imager can

196 accommodate. Computer tomography angiography would provide structural assessment of the vasculature, however would not provide sufficient resolution to assess microvascular injury and structural perturbation. As the initial injury likely occurs at the microvascular level, readily accessible imaging technologies may not be capable of dissecting the intricate temporospatial relationship between vascular injury, ischemia and necrosis.

Our transcriptomic analyses demonstrate that increased gene expression of complement factors is associated with previous cerebral irradiation. The functional consequences of this up-regulation are unknown. The majority of complement factors in the body are produced by the liver and circulate in plasma as inactive precursors. Our detection of transcripts suggests a component of local cerebral complement production. A number of studies investigating the role of complement in RIBI need to be performed.

Although the up-regulation of complement factors in Chapter 4 suggests that complement activation may contribute to RIBI, complement factors are inactive until proteolytically cleaved. Thus, determination of whether complement activation and fixation occur in brain tissue in RIBI is essential and logical next steps.

Immunohistochemical characterization complement proteins and activation products will provide assessment of sub-pathway involvement [classical (antibody-mediated), alternative or lectin], activation status, and identification of fixation targets (100, 101).

C4d immunohistochemistry is used as a marker of allograft rejection in humans (102-

104), as C4d covalently binds to the fixation target, thus persisting in tissue longer than other complement markers. Interestingly, Chernobyl liquidators diagnosed with postradiation encephalopathy (105) produced autoantibodies against neuron specific enolase

197

(NSE), further supporting our hypothesis that self-reactivity may play a role in the pathogenesis of RIBI.

If complement activation is determined to occur in RIBI, identification of the inciting antigens is essential. This will identify which cell types and structures to protect against self-reactive injury. Early intervention will be necessary to prevent epitope- spreading. This phenomenon occurs in many autoimmune diseases, in which persistent tissue destruction and immune activation against a single dominant target antigen results in furthered antigen presentation and sensitization against secondary targets (106, 107).

Should shared dominant antigens be recognized across the majority of patients with RIBI, design of antigen-specific treatments and implementation early in the course of disease may more effectively mitigate injury.

However, given the complex role of the immune system in cancer, careful consideration should be given to immunomodulatory therapies in tumor-bearing patients as to avoid impairment of the anti-tumoral response. Assessment of complement inhibiting and antigen-presentation inhibiting drugs in tumor-bearing translational models will be of particular importance.

Complement activation may also contribute to RIBI by mediating synaptic degeneration and loss. During development, complement-mediated synaptic elimination is required to refine projection pathways and form efficient neuronal circuitry (108). In adults, complement may mediate synaptic loss and cognitive dysfunction, as C3 knockout mice demonstrate enhanced spatial learning (109) and are protected against age-related synaptic loss and cognitive decline (110). As complement fixation may mediate synaptic

198 loss in RIBI, further characterization of this neuroimmune response is a prospective target for future study.

Assessment of sex differences in the response to RIBI was not possible in these retrospective studies, as tissues from male animals only were available for analyses. Data regarding the effect of sex in radiation response are conflicting. Although estrogen is radioprotective in mice (111-113), cerebral irradiation in young mice results in greater impairment of white matter development in females than in males (114). In humans, the overall lethality risk for total body irradiation in women is approximately 35% greater than that for men (115). Although it may seem reasonable to hypothesize that women are at a greater risk of developing radiation-induced brain injury, this does not appear to be supported by clinical data in patients with RIBI.

Retrospective analyses indicate that sex had no effect on the incidence of cerebral radiation necrosis, cognitive function, clinical outcome, and self-reported anxiety and depression in patients treated for nasopharyngeal carcinoma with intensity modulated radiotherapy in which the temporal lobe was irradiated (116-118). Sex had no effect on the incidence of late cerebrovascular complications in survivors of childhood treatment for primary central nervous system cancer (119, 120). Interestingly, males treated with whole-brain irradiation and motexafin gadolinium had shorter survival intervals than female counterparts (121). However, it is important to consider that studies investigating sex-differences in RIBI sensitivity and progression in humans are confounded by differences in primary tumor origin [(<1% of breast cancer cases occur in males (122)], the incidence of metastatic disease [males are more likely than female to develop brain metastases for any given tumor type with the exception of sex-specific cancers (123,124)]

199 and concurrent treatment regimens [subsequent chemotherapy with certain agents increases the risk of radiation necrosis (125, 126)]. Although sex does not appear to play a role in the development of RIBI, the non-human primate model would provide the opportunity to definitively assess sex differences in the cerebral radiation response in the absence of concurrent neoplasia or confounding chemotherapeutic regimens.

The Cerebral Effects of Low Single Dose Total Body Irradiation

While we have explored the long-term consequences of single, high dose radiation exposure (6.75 - 8.05 Gy), the long-term ramifications of lower doses requires evaluation.

Following a nuclear detonation, concussive forces and conflagration result in significant mortality near the hypocenter (127). Thus, persons near enough to receive an acute lethal dose of radiation ( > 4 Gy) (128) are likely to have died in the initial explosion (129), or succumb to combined injury (130-132). This relationship strengthens with increasing device size, as the conflagration radius scales more rapidly than the lethal ionizing radiation dose radius (133-135). For devices > 10 kilotons, the thermal injury radius exceeds that of the radius of lethal ionizing radiation exposure (129).

Accordingly, evaluation of the Life Span Study cohort (LSS)(136) reveals that the dose distribution to survivors of the Hiroshima and Nagasaki atomic bombs is highly right skewed, with a mean dose of 0.2 Gy. Analyses exclude survivors who received doses < 0.005 Gy. Less than 0.6% (564 / 105,427) of LSS enrollees received >2 Gy

(137). The average absorbed doses in Chernobyl survivors are lower, with 96% of those exposed (evacuees, contaminated area residents) receiving 0.01 - 0.05 Sv (138). Less than

0.005% of exposed persons received doses of 1 – 16 Sv (139). Despite receiving

200 relatively low doses of radiation, retrospective studies have demonstrated survivors receiving > 0.15 - 0.5 Sv may be at risk of developing cerebrovascular pathology including stroke and dyscirculatory encephalopathy (140-143). Chernobyl liquidators receiving > 0.3 Sv have demonstrated EEG abnormalities and seizure activity (144-150), cognitive impairment (151-153), PET, CT and MRI evidence of cerebral atrophy (154) and autoimmunity to NSE (105). These data suggest that single low doses of radiation ( 0

– 2 Gy ) may have deleterious effects on the central nervous system, and additional study is warranted.

Radiation-Induced Brain Injury: A Potential Model of Microvascular Dementia and

Synaptic Dysfunction in Neurodegenerative Disease

Approximately 50 million people world-wide currently have dementia (155), an abnormal decrease in memory and cognitive ability of severity sufficient to impair quality of life. Although increasing age is associated with increased risk, dementia is not a normal process of aging, but due to various neurodegenerative diseases or conditions.

Dementia health care related costs totaled 818 billion dollars in 2015 (155), and the aging population is expected to nearly triple by 2050 (156). In most cases, there are no effective treatments to or stop disease progression. There are no current animal models which fully recapitulate all features of dementias (157, 158). Thus, there is a profound need to develop translational models of AD and other dementias to facilitate the development of therapies.

60-80% of dementia cases are due to Alzheimer’s disease (AD) (159). This progressive and incurable neurodegenerative condition is characterized by accumulation

201 of proteins with the brain: amyloid-beta plaques in the parenchyma (160), and abnormally phosphorylated tau within neurons which form neurofibrillary tangles (161,

162). The pathogenesis remains unclear, which has hampered the development of effective therapies.

Others have shown that exposure to ionizing radiation increases tau-phosporylation in cultured neurons (163), as well as parenchymal and vascular amyloid-beta deposition

[cerebral amyloid angiopathy (CAA)] in human patients receiving fWBI (164).

Furthermore, synaptic loss and white matter injury both are noted in early AD and correlate with rapidity and extent of cognitive decline (165-173). In particular, white matter hyperintensities (WMH) or leukoariosis, are thought to modulate disease progression in AD (165, 166, 170, 173-178). These magnetic resonance findings are generally accepted to represent microvascular injury and secondary ischemic damage to white matter (173). Therefore, RIBI in non-human primates and AD in humans may share common features.

Finally, complement activation and up-regulation of C1q are observed in the human brain in Alzheimer’s disease (AD) (179, 180). In mouse models of Alzheimer’s disease

[amyloid precursor protein (APP) over expressing, and APP/mutant presenilin], C1q- deficiency decreased synaptic loss despite comparable levels of amyloid-beta to C1q- competent mice (181). Furthermore, injection of amyloid-beta oligomers into wild type mice induced C1q localization at synapses, and synaptic loss was prevented when these mice were administered an antibody against C1q (182). Although further characterization of complement-mediated synaptic loss in RIBI is required, RIBI may

202 serve as an inducible model in which it is feasible to evaluate early contributing events to

AD including microvascular and white matter injury and neuroinflammatory contributors.

Vascular dementia alone accounts for approximately 10% dementia cases, but may be comorbid in up to 50% of Alzheimer’s disease patients (183). As mentioned briefly, microvascular injury is associated with white matter hyperintensities (WMH) on

MRI. A 2010 metanalysis of 46 studies in human patients revealed that risk of dementia is significantly associated with WMH (184). Furthermore, WMH were significantly associated with incident vascular dementia with WMH (185-187). Thus, as fWBI reliably induces multifocal cerebral microvascular injury and associated white matter necrosis, we and other propose (188) that RIBI be a viable translational model of vascular dementia.

Conclusion

In conclusion, we have reaffirmed that the rhesus macaque is an anatomically and physiologically translational model in which it is appropriate to study the effects of RIBI.

We also have provided insight into the pathogenesis of RIBI, which is presumed multifactorial and likely involves interaction between vascular, glial, neuronal and immune system contributors. In part, the perivascular deposition of fibronectin likely plays a role in cerebrovascular dysfunction following fWBI. We have also identified novel potential contributors to RIBI which warrant additional study, including assessment of deep white matter synaptic impairment, and complement activation and self-reactivity following fWBI. We have also identified several novel prospective treatments for RIBI which require future preclinical assessment.

203

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APPENDICES

DISCLAIMER: Permission was granted by Radiation Research to reprint Chapter 2 as part of this dissertation.

231

Dear Rachel,

Radiation Research grants to Requester, Rachel N. Andrews, the permission to re- publish: Andrews RN, Metheny-Barlow LJ, Peiffer AM, Hanbury DB, Tooze JA,

Bourland JD, Hampson RE, Deadwyler SA, Cline JM. “Cerebrovascular Remodeling and

Neuroinflammation is a Late Effect of Radiation-Induced Brain Injury in Non-Human

Primates.” Radiat Res. 2017 May;187(5):599-611. doi: 10.1667/RR14616.1. Epub 2017

Mar 6. PubMed PMID: 28398880; PubMed Central PMCID: PMC5508216.

Please ensure that full acknowledgment of previous publication is cited.

Congratulations on making it to this final step.

All the best, Judy

Judy E. Fye

Managing Editor

Radiation Research

Indiana University School of Medicine

Department of Radiation Oncology

535 Barnhill Drive, RT 041

Indianapolis, IN 46202 e-mail: [email protected]

(317) 944 1308

232

APPENDIX A

Multi-regional microvascular histomorphometry in normal and irradiated Rhesus

macaques

Rachel. N. Andrews, D.V.M.

233

METHODS

Animals and Tissue

The animals were the same as those assessed in Chapters 2 and 3. Briefly, archived, paraffin embedded brain tissues containing the dorsolateral prefrontal cortex

(Brodmann area 46), hippocampus, and deep white matter (centrum semiovale) from 7 adult, male Rhesus macaques (Macaca mulatta) (6-11 years old, 5.3-8.7 kg) were evaluated for changes in microvascular investiture related to prior fractionated whole brain irradiation (fWBI). 4, adult male rhesus macaques (aged 5.6-10.7 years, 7.5-8.7 kg) received 40 Gy fWBI 4-7 months prior to necropsy as part of a previous experiment.

Control comparators (n=3, aged 5.5-6.6 years, 5.3-8.3 kg) were part of an unrelated study in which they were given single dose 10 Gy thoracic irradiation, and subcutaneous injections of vehicle (1 mL SQ, sterile phosphorus-buffered saline(PBS)), twice daily for 4 months, ceasing 4 months prior to necropsy. Housing, diet, euthanasia, and tissue collection are as detailed in Hanbury et al. (1), and the same for both groups.

Both studies were performed in accordance with the Guide for Care and Use of

Laboratory Animals and all procedures approved by the Wake Forest University School of Medicine Institutional Animal Care and Use Committee (IACUC). The Wake Forest

University School of Medicine conforms to all state and federal animal welfare laws and is accredited by the Association for Assessment and Accreditation of Laboratory Animal

Care (AAALAC).

Irradiation

Fractionated whole brain irradiation procedures are as described in Hanbury et al.(1) and Robbins et al. (2). NHP were sedated with ketamine HCl (15 mg/kg body

234 weight, intramuscularly), and maintained on isoflurane gas (3% induction, 1.5% maintenance) in 100% oxygen during irradiation. Animals were irradiated using a clinical linear accelerator, and a total of 40 Gy mid-plane was given (8 fractions x 5 Gy/fraction;

2 fractions/wk x 4 wks; nominal dose rate of 4.9-5.4 Gy/min) in opposed lateral fields of

6MV x rays.

Control comparators were given 10 Gy, single dose thoracic irradiation to the anterior-posterior thoracic mid-plane in parallel opposed anterior-posterior fields of 6

MV x rays via a clinical linear accelerator. The nominal dose rate was 4 Gy/min. The field of irradiation included the heart, mediastinum and superior, lateral and inferior lung fields extending up to 4 cm caudal to the xyphoid. The total dose to the brain in thoracic irradiated animals is estimated to be < 0.1 Gy.

Histology and Immunohistochemistry (IHC)

Formalin fixed, paraffin embedded brain tissues containing 4 mm coronal slabs of dorsolateral prefrontal cortex, hippocampus and deep white matter (centrum semiovale) were sectioned coronally at 4 μm. IHC was performed on a Leica Bond RX (Leica

Biosystems, Buffalo Grove, IL) automated stainer. Sections from animals receiving fWBI and control comparators were stained concurrently. The cerebral vasculature was visualized with a rabbit polyclonal anti-glucose transporter GLUT1 antibody (rabbit polyclonal, 1:2000; Abcam, catalog #ab652). Slides were air-dried overnight and loaded onto the Leica Bond RX. All sections were dewaxed, rinsed, and underwent heat-induced epitope retrieval for 20 minutes at 99°C in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked by 10 minute incubation with IHC/ISH Super Blocking Novocastra solution (catalog #PV6122, Leica Biosystems). All slides were incubated with the

235 primary antibody for 15 minutes and then rinsed. Secondary antibody and chromagen application were as according to manufacturer’s specifications (Bond Polymer Refine

Detection, ACD, catalog #: DS9800). Slides were then removed from the instrument, dehydrated, cleared with xylene and coverslipped.

Histomorphometry

Digital images were captured using an Olympus BX61VS virtual slide microscope using VS120 software at 20x magnification. Regions of interest were delineated as follows: the dorsal segment of Brodmann area 46 was manually delineated using a brain atlas for the rhesus macaque (3), rectangular ROIs no less than 0.6 mm2 and

5.5 mm2 were placed within the boundaries of the dentate gyrus and centrum semiovale, respectively. ROIs were equivalent to minimum of 21, 4 and 34 40x high power fields for the DLPFC, HC and WM respectively.

Microvessel density and cross-sectional microvascular area fraction were measured in VisioPharm (version 6.5.0.2303; Broomfield, CO, USA) using the following modifications to APP #10022 (“CD31, Tumor Neovascularization”) and an unbiased counting frame. Imported images were pre-processed with a Red-Blue -> Mean value filter; images were thresholded to exclude all background objects. Microvessel density was defined as the number of microvascular profiles per mm2. Cross-sectional microvascular area fraction was defined as the total area occupied by blood vessels including lumina divided by the total area (area within blood vessels/total area of the region of interest) and expressed as percent area (microvascular area fraction * 100).

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Statistical Analyses

Two seperate 2x3 analyses of variance (ANOVAs) were conducted to compare the main effects of irradiation status and region, and the interaction effects between radiation status and region on microvessel density and microvascular area fraction. Post- hoc comparisons were performed to assess the effect of irradiation within regions using

Bonferroni’s procedure. Multiplicity adjusted p-values are reported, and p < 0.05 was considered significant.

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RESULTS

Microvessel density, but not microvascular area fraction differed between regions evaluated (p < 0.0001). Microvessel density and microvascular area fraction were not affected by irradiation. There was no interaction effect between irradiation status and region. The increased variance in hippocampal microvessel area fraction is presumed due to measurement of a venule in cross-section within the dentate gyrus in one animal.

Figure 1: Microvascular histomorphometric in irradiated and normal Rhesus macaques

Representative micrograph of GLUT-1 immunohistochemistry (endothelial cells) in deep temporal white matter. Microvessel density, but not microvascular area fraction differed between regions ( p < 0.0001). Irradiation did not affect microvessel density or microvascular area fraction. Bars: Mean, Error bars: standard deviation.

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REFERENCES

1. Hanbury DB, Robbins ME, Bourland JD, Wheeler KT, Peiffer AM, Mitchell EL, et al. Pathology of Fractionated Whole-Brain Irradiation in Rhesus Monkeys ( Macaca mulatta ). Radiation research. 2015.

2. Robbins ME, Bourland JD, Cline JM, Wheeler KT, Deadwyler SA. A model for assessing cognitive impairment after fractionated whole-brain irradiation in nonhuman primates. Radiation research. 2011;175(4):519-25.

3. Paxinos G. The Rhesus Monkey Brain in Stereotaxic Coordinates:

Elsevier/Academic Press; 2009.

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APPENDIX B

Quantification of activated microglia in normal and irradiated Rhesus macaques

Rachel N. Andrews, D.V.M.

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METHODS

Animals and Tissue

The animals were the same as those assessed in Chapters 2 and 3.

Histology and Immunohistochemistry

Separate, formalin fixed, paraffin embedded brain tissues containing 4 mm coronal slabs of dorsolateral prefrontal cortex, hippocampus and deep white matter

(centrum semiovale) were sectioned coronally at 4 μm. IHC was performed on a Leica

Bond RX (Leica Biosystems, Buffalo Grove, IL) automated stainer. Sections from animals receiving fWBI and control comparators were stained concurrently. Activated microglia were visualized with a mouse monoclonal anti-IBA antibody (1:2000;

Millipore, catalog #MABN92). Slides were air-dried overnight and loaded onto the Leica

Bond RX. Sections were dewaxed, rinsed, and underwent heat-induced epitope retrieval for 20 minutes at 99°C in EDTA buffer pH 9.0. Endogenous peroxidase activity was blocked by 10 minute incubation with IHC/ISH Super Blocking Novocastra solution

(catalog #PV6122, Leica Biosystems). Slides were incubated with the primary antibody for 15 minutes and then rinsed. Secondary antibody and chromagen application were as according to manufacturer’s specifications (Bond Polymer Refine Red Detection Kit, catalog # DS9390, Leica Biosystems). Slides were then removed from the instrument, dehydrated, cleared with xylene and coverslipped.

Histomorphometry

Digital images were captured using an Olympus BX61VS virtual slide microscope at 20x magnification using VS120 software. Regions of interest were selected

241 as follows: the dorsal segment of Brodmann area 46 was manually delineated using a brain atlas for the rhesus macaque (1), rectangular ROIs no less than 0.7mm2 and 13.3 mm2 were placed within the boundaries of the dentate gyrus and centrum semiovale, respectively. ROIs were equivalent to minimum of 12, 2 and 8 40x high power fields for the DLPFC, HC and WM respectively.

Microglial percent area was quantified as [(area red staining / total ROI area)*100] and measured in VisioPharm (version 6.5.0.2303; Broomfield, CO, USA) using a custom, automated image processing algorithm. Imported images were pre- processed with a H&E - Eosin filter; and thresholded to exclude all background (non-red) objects.

Gene Expression Analyses

RNA extraction, real-time quantitative polymerase chain reaction and gene expression analyses were completed as described in Chapters 2 and 3.

Statistical Analyses

All statistical analyses were completed in GraphPad Prism version 7.0 (GraphPad

Software, La Jolla California USA, www.graphpad.com). A 2x3 analysis of variance

(ANOVA) was conducted to compare the main effects of irradiation status and region, and the interaction effects between radiation status and region on microglial activation.

Post-hoc comparisons were performed to assess the effect of irradiation within regions using Bonferroni’s procedure. Multiplicity adjusted p-values are reported, and p < 0.05 was considered significant.

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RESULTS

Irradiation (main effect: p < 0.001) and brain region ( main effect: p < 0.0012) affect the percent area occupied by activated microglia. The presence of a significant radiation status x region interaction ( p < 0.001) implies that microglial activation is differentially regulated by region. Post-hoc analysis reveals that this effect occurs within white matter ( p < 0.0001 ), but not dorsolateral prefrontal cortex or hippocampus.

Figure 1: Irradiation resulted in increased microglial activation within white matter

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A) IBA-1 immunohistochemistry, control animal: Low magnification assessment demonstrates a relative lack of activated microglia in cortex and white matter. B) IBA-1 immunohistochemistry, fWBI animal: Irradiation resulted in a prominent increase in activated microglia, most prominently within white matter. C) Microglial activation following irradiation is differentially regulated by region (# main effect of region: p < 0.0012 ) and occurs within white matter ( *p < 0.001, Bonferroni post-hoc ). D) Gene expression of AIF1, the IBA-1 encoding gene is also up-regulated within white matter ( p < 0.05 ) and significantly different than expression levels in the DLPFC. Bars: mean, Error bars: standard deviation.

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Gene expression of AIF (allograft inflammatory factor), the gene which is transcribed into IBA-1, was increased within white matter ( p < 0.05 ), but not prefrontal cortex or hippocampus.

REFERENCES

1. Paxinos G. The Rhesus Monkey Brain in Stereotaxic Coordinates:

Elsevier/Academic Press; 2009.

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APPENDIX C

Brain Magnetic Resonance Imaging Surveillance in the Non-Human Primate Radiation

Survivors Cohort

Rachel N. Andrews, D.V.M

METHODS

Subjects

Archived magnetic resonance imaging (MRI) sequences were evaluated from 170

Rhesus macaques, aged 3-18 years (median age: 6 years). 141 were males, and were 29 females. 53 animals (median age: 6.2 years; range: 4-18) had no prior history of irradiation, and were either part of unrelated studies or adopted as control comparators and enrolled within the non-human primate Radiation Survivor Cohort (RSC). 117 animals (median age: 6.5 years; range: 3-18 years) were given single dose total body irradiation (1.14 - 8.5 Gy) as part of separate studies at other institutions and then adopted into the RSC for long-term surveillance for the sequelae of radiation exposure. Tabulated demographic data include age, radiation dose, and age at irradiation (Table I).

Table I: Subject Demographics

Irradiation Age at Age at Age at to Diagnosis Lesion Assessment Irradiation Dose Interval Diagnosis n (months) (months) (Gy) (months) (months)

Non- Irradiated 53 74 [42-220] - - - -

Irradiated NSL 101 71 [34-198] 50 [28-183] 6.6 [1.14-8.5]* - -

Irradiated SWI 16 86 [51-118] 45.5 [36-80] 7.4 [4-8.4]* 12.5 [7-136] 86 [51-176]

NSL: No significant lesions SWI: Susceptibility weighted imaging (brain lesion) * p < 0.05 All data are presented as median [range]

Animals were housed in stainless steel cages, in temperature and humidity controlled rooms with a 12 hour light/dark cycle. Diet was contingent upon the

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experimental study in which the animal was enrolled; animals were either fed commercially available monkey chow (Purina, St. Louis, MO) or Typical American

Primate diet [(TAD); Diet #5L0P; LabDiet, Land O’Lakes Inc.; St. Louis, MO] designed to approximate the macronutrients of a western dietary profile. All animals received daily clinical assessment by trained veterinary personnel including cage-side neurologic evaluation when indicated; all animals were neurologically normal. All procedures were approved by the Wake Forest University School of Medicine (WFUSM) Institutional

Animal Care and Use Committee (IACUC), and performed in accordance with all state and federal animal welfare laws. WFUSM is accredited by the Association for

Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Magnetic Resonance Imaging

All animals were sedated with ketamine HCl (15 mg/kg body weight, IM), and maintained on inhaled isofluorane (3% induction, 1.5% maintenance) in 100% oxygen anesthesia for the duration of the MRI procedure. Images were acquired on a 3.0 Tesla

Siemens Skyra clinical magnetic resonance scanner, operating on D13 platform with a maximum gradient field strength = 45 mT/m. Sequences were optimized for non-human primates and acquired using a dedicated, 8 channel, non-human primate RF coil (Rapid

MR International, Columbus, OH) tuned to 127.7 MHz.

T1-weighted anatomic images were obtained using a 3D volumetric MPRAGE sequence (TR=2700 msec; TE=3.39 msec; TI=880 msec; FA=8 degrees; 160 slices, voxel dimension=0.5 x0.5 x 0.5 mm). Brain lesions consistent with hemorrhage and necrosis were identified by Susceptibility Weighted Image (SWI) sequence (TR=28ms, TE=20ms,

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Flip angle=15 degrees, NEX=2, with voxel size = 0.5 x 0.5 x 0.5 mm ). SWI and T1 sequences were co-registered, and the anatomic location of SWI-hypointense foci was determined from the corresponding T1 structural image. Lesion dimensions were measured on T1 structural images in three perpendicularly oriented planes.

Statistical Analyses

All statistical analyses were completed in GraphPad Prism version 7.0 (GraphPad

Software, La Jolla California USA, www.graphpad.com). Animals were stratified by irradiation and SWI lesion status. Normality was assessed by Shapiro-Wilk test. Non- parametric data (age at irradiation, age at lesion diagnosis, dose, and irradiation to diagnosis interval) were compared by Mann Whitney U. The age at time of assessment between non-irradiated, irradiated NSL and irradiated SWI groups was compared by

Kruskal-Wallis one-way analysis of variance (ANOVA). The association between prior radiation exposure and brain lesions on MRI was assessed by Fisher’s exact test, and the odds ratio calculated by Woolf-logit method. Significance for all analyses was set to p <

0.05.

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RESULTS

Susceptibility Weighted Imaging (SWI) Hypointense Brain Lesions on MRI

Focal and multifocal T1 hypointense, SWI hypointense brain lesions consistent with necrosis and chronic hemorrhage were noted in 16 irradiated animals, and zero non- irradiated comparators (Figure 1). No significant proprioceptive, locomotor or cranial nerve deficits were noted on cage-side neurologic evaluation.

Figure 1: Animals receiving TBI developed hypointense lesions on susceptibility weighted imaging comparable to those which occur in RIBI following fWBI.

fWBI TBI

Left: Multifocal hemorrhage and necrosis (white arrows) in an animal that developed RIBI after receiving 40 Gy fWBI (8x5 Gy fractions, 2x per week) as part of a separate study. Scan taken at 12 months post- irradiation. Right: Comparable lesions in an animal which had received 8.0 Gy TBI, 1 year prior to MRI.

Animals receiving previous total body irradiation were more likely than non- irradiated comparators to have SWI hypointense brain lesions on MRI (14% vs 0% respectively, p = 0.003, Fisher’s exact test, Figure 2A). Lesions occurred with a predilection for white matter (14/16). Animals that developed brain lesions received

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higher doses of TBI (Figure 2B). 3 out of 16 lesions developed during the surveillance period and are termed ‘incident’ lesions; the remaining 13 were present at the time of first scan (‘prevalent’ lesions). Incident lesions took longer to develop ( p = 0.002 ) and animals that developed incident lesions were older ( p = 0.04 ) than those with prevalent lesions. There was no difference in the dose of irradiation, or age at irradiation between animals developing incident and prevalent SWI lesions.

Figure 2: TBI is associated with MRI detectable brain lesions, which occur at higher doses of radiation

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Table II: Incident and Prevalent SWI Lesions

Age at Age at Median Irradiation to n Assessment Irradiation Median Dose Diagnosis Interval (months) (months) (Gy) (months)

Incident SWI 3 108 [96-118]* 40 [36-52] 7.55 [7.55-7.85] 82 [82-136]*

Prevalent SWI 13 68 [51-111]* 27 [39-80] 7 [4-8.5] 11 [7-75]*

SWI: Susceptibility weight imaging (brain lesion) * p < 0.05 All data are presented as median [range]

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Correlative Gross and Histopathology

Of the sixteen animals diagnosed with SWI brain lesions, one animal (age: 11 years; dose: 7.2 Gy; irradiation-death interval: 8 years) was humanely euthanized due to illness unassociated with neurologic dysfunction (multiple neoplasms: chondrolipoma, renal carcinoma and poorly-differentiated neuroendocrine neoplasia). There was no gross or histologic evidence of metastatic disease.

Figure 3: Gross and histopathologic evaluation of a prevalent SWI lesion

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Focal brain (prevalent) lesion was first noted six years post-irradiation, at the time the first MRI scan was completed . A) T1 MRI indicating focal parenchymal loss with central T1 isointense heterogeneity. B) SWI MRI indicating the presence of iron or calcium (blood or necrosis, respectively). C) Gross necropsy demonstrating focal brain lesion containing hemorrhage. D) Corresponding histopathology (hematoxylin and eosin): Subcortical white matter contained a focal, well demarcated vascular vascular malformation (cavernous hemangioma).

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6 year-post irradiation, a 1.9 x 1.9 x 1.5 mm, heterogeneously T1 hypointense,

SWI hypointense focus, consistent with focal necrosis and/or hemorrhage was noted within the subcortical white matter of the right inferior occipital lobe. Two years later, the lesion had increased to 2.3 x 2.8 x 2.6 mm. At necropsy, the occipital white matter contained a 2.7 x 2.1 mm, firm, well-demarcated, irregular, dark red-brown focus.

Histopathologic evaluation revealed a well demarcated vascular malformation composed of irregularly dilated and haphazardly arranged vascular channels with walls variably thickened by dense, homogeneous extracellular matrix, consistent with a cavernous hemangioma. The adjacent parenchyma was multifocally edematous, contained scattered gemistocytic (reactive) astrocytes, and macrophages containing hemosiderin (evidence chronic hemorrhage). Cavernous hemangiomas are sequelae of cerebral irradiation in humans (1-3).

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REFERENCES

1. Cutsforth-Gregory JK, Lanzino G, Link MJ, Jr. RDB, Flemming KD.

Characterization of radiation-induced cavernous malformations and comparison with a nonradiation cavernous malformation cohort. Journal of neurosurgery. 2015;122(5):1214-

22.

2. Jain R, Robertson PL, Gandhi D, Gujar SK, Muraszko KM, Gebarski S.

Radiation-Induced Cavernomas of the Brain. American Journal of Neuroradiology.

2005;26(5):1158-62.

3. Heckl S, Aschoff A, Kunze S. Radiation-induced cavernous hemangiomas of the brain: a late effect predominantly in children. Cancer. 2002;94(12):3285-91.

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WAKE FOREST SCHOOL OF MEDICINE

Curriculum Vitae

NAME: Rachel N. Andrews, D.V.M

CURRENT ACADEMIC TITLE: Post-doctoral Fellow MMTS Ph.D Candidate

ADDRESS: Department of Comparative Medicine Wake Forest School of Medicine Medical Center Boulevard Winston-Salem, NC 27157-1010 (336) 716-1561 (office) (630) 329-1229 (cell) e-mail: [email protected]

EDUCATION (in chronologic order):

2010 University of Illinois: Urbana-Champaign Champaign-Urbana, Illinois Bachelors of Science: Animal Sciences

2014 University of Wisconsin: Madison Madison, Wisconsin Doctor of Veterinary Medicine

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POSTDOCTORAL TRAINING:

Present Molecular Medicine and Translational Sciences Wake Forest University Winston-Salem, North Carolina Ph.D. Candidate

Research Advisor: J. Mark Cline, D.V.M, Ph.D Thesis: Mechanisms of Late-Delayed Radiation- Induced Brain Injury: Insights from Extracellular Matrix and Transcriptional Profiling

PROFESSIONAL LICENSURE

2014 - present Veterinary Medicine, State of Wisconsin, #7148-50

EMPLOYMENT:

Academic Appointments (in chronologic order):

Wake Forest of Medicine, Wake Forest University:

2014 - Present Postdoctoral Fellow Department of Comparative Medicine

Professional Experience

2007 - 2010 Research Assistant, Cooke Laboratory University of Illinois, Champaign-Urbana, Illinois.

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2012 - 2014 Research Assistant, MacAnulty Laboratory University of Wisconsin, Madison, Wisconsin.

2014 Extern, Veterinary Medicine Rotations Exotics Care Marathon Veterinary Clinic, Marathon, Florida

Diagnostic Pathology Colorado State University, Fort Collins, Colorado Texas A&M University, College Station, Texas

Lab Animal Medicine and Toxicologic Pathology Abbvie, Waukegan, Illinois

Zoo Pathology University of Illinois, Chicago, Illinois

EXTRAMURAL APPOINTMENTS AND SERVICE

Other: 2016 Chairperson of the Organizational Committee Wake Forest University 2nd Annual Comparative Medicine Research Symposium 26 July 2016 Winston-Salem, NC

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2017 Correspondence Co-Chair Wake Forest University 3rd Annual Comparative Medicine Research Symposium 27th July 2017 Winston-Salem, NC

OTHER PROFESSIONAL APPOINTMENTS AND ACTIVITIES

Tissue Collection and Protocol Development

2014 – 2017 Vervet Research Colony Standard Operating Procedure: Collection the Central Nervous System for Histopathologic Assessment and Molecular Analyses

2015 Alzheimer-type Change and Atherosclerosis Pilot Study

2015 Alzheimer Disease Core Center Collection of the Central Nervous System for Neuropathologic Assessment of Alzheimer-Type Change in the Non- human Primate

2015 – present Cline Laboratory, Collection the Central Nervous System for Histopathologic Assessment and Molecular Analyses in the Long-term Radiation Survivors Cohort

2016 Ko Laboratory, Collection of the trigeminal ganglion and nerve branches

Animal Resources Program

2014 – present Diagnostic necropsy and histopathology support for the Wake Forest Animal Resources Program, ~100 cases.

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PROFESSIONAL MEMBERSHIPS

2017 – present Wake Forest Alzheimer’s Disease Core Center Member

2016 – present American Association for the Advancement of Science Member

2015 - present Radiation Research Society Scholar in Training, Post-doctoral

HONORS AND AWARDS

2016 - 2017 Mike and Lucy Robbins Graduate Scholarship

2016 Travel Award, Scholar in Training Radiation Research Society

2015 Travel Award, Scholar in Training Radiation Research Society

2014 ACVP Excellence in Veterinary Pathology

INVITED PRESENTATIONS AND SEMINARS

1. Normal Tissue Injury Following Whole Brain Irradiation. Brain Tumor Center of Excellence, Wake Forest University School of Medicine. October 2014.

2. Radiation encephalopathy. National Primate Centers Virtual Pathology Conference. October 2014.

3. Standardization of the assessment of neuropathology associated with Alzheimer’s type change in the non-human primate. Investigative Pathology Grand Rounds. Wake Forest University School of Medicine. November 2014.

4. Radiation Induced Cerebrovascular Pathology; A Model for Delayed-Onset Radiation Induced Cognitive Dysfunction. Non-Human Primate Models, Cancer Center Retreat, in collaboration with D. Hanbury. Wake Forest University School of Medicine. December 2014.

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5. Late-Delayed Radiation Induced Cerebrovascular Disease. Molecular Medicine and Translational Sciences Graduate Seminar. Wake Forest University School of Medicine. March 2015.

6. Corticomedullary tubular hypoplasia. National Primate Centers Virtual Pathology Conference. June 2015.

7. Late-Delayed Radiation Induced Cerebrovascular Injury. 1st Annual Comparative Medicine Research Symposium. Wake Forest Biotech Place. July 2015.

8. Gene Expression Changes in Late-Delayed Radiation Induced Brain Injury. Molecular Medicine and Translational Sciences Graduate Seminar. Wake Forest University School of Medicine. February 2016.

9. Radiation Induced Cognitive Dysfunction: Gene Expression and Mitigation. Brain Tumor Center of Excellence, Wake Forest University School of Medicine. April 2016.

10. Neuroinflammation and Cerebrovascular Remodeling in Late-Delayed Radiation Induced Brain Injury. 2nd Annual Comparative Medicine Research Symposium. Wake Forest Biotech Place. July 2016.

11. Cerebrovascular Remodeling and Neuroinflammation in Late-Delayed Radiation Induced Brain Injury, speaker. Molecular Medicine and Translational Sciences Graduate Seminar. Wake Forest University School of Medicine. January 2017.

12. Radiation-Induced Neurovascular Injury, speaker. Research Review, Department of Pathology, Section on Comparative Medicine. Wake Forest University School of Medicine, Clarkson Campus. May 2017.

13. Fibronectin contributes to perivascular ECM in radiation-induced brain injury. 3rd Annual Comparative Medicine Research Symposium. Wake Forest University School of Medicine, Bowman Gray Center for Medical Education. July 2017.

14. Neurovascular Remodeling in Radiation-Induced Brain Injury. Research Review, Department of Pathology, Section on Comparative Medicine. Wake Forest University School of Medicine, Clarkson Campus. September 2017.

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15. The Role of Fibronectin in Radiation-Induced Brain Injury. Research Review, Department of Pathology, Section on Comparative Medicine. Wake Forest University School of Medicine, Clarkson Campus. September 2017.

16. The Role of Fibronectin in Radiation-Induced Brain Injury. Molecular Medicine and Translational Sciences Seminar. Wake Forest University School of Medicine, Bowman-Gray Campus. October 2017.

17. Microvascular Contribution to Alzheimer’s Disease Progression and Resilience. 2017-2018 Dean’s Research Symposia Series, Bowman Gray Center for Medical Education. December 2017.

18. The RIBIseq Pilot. Transcriptomic Insights into the Pathogenesis of Late-Delayed Radiation-Induced Brain Injury. Radiation Biology Section Meeting, Wake Forest University School of Medicine. February 2018.

GRANTS:

CURRENT

Department of Pathology Pilot Grant (Andrews RN, Co-P.I., Cline JM, Co-P.I., no paid effort) 2017-2018 The Late Effects of High Dose, Single Exposure Total Body Irradiation on the Cerebral Transcriptome $25,000 /year direct cost This pilot project will acquire preliminary data on changes in the cerebral transcriptome induced in the years following exposure to ionizing radiation; in both the contexts of cancer treatment (fractionated irradiation) and accidental or malicious exposures (single high dose irradiation).

Collaborators: Gregory A. Hawkins

U19 AI677988 (Chao N, P.I.; Andrews RN, Post-doctoral fellow, 100% effort) 08/01/15 - 07/31/20 Center for Medical Countermeasures Against Radiation $1,313,387 /year direct cost This research consortium, serves to collectively and collaboratively develop possible agents to detect, mitigate and treat those people exposed to deterministic doses of radiation

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PENDING

P50 CA234092-01 (Debinski, PI; Andrews RN, Co-I) 04/01/2019 - 03/31/2024

Novel Therapies for Glioblastoma This Specialized Program of Research Excellence (SPORE) application addresses an unmet need in medicine, the management of glioblastoma (GBM). It has three projects and four cores in addition to Developmental Research and Career Enhancement Programs. The application is highly translational as one project includes a clinical trial and the other two are seeking IND approvals to begin clinical examination of novel therapies. Based on inter- and trans-disciplinary neuro-oncology research and strong inter-institutional collaborations, we propose to overcome the most significant barriers to effective treatment of GBM and to introduce novel biotherapeutic and immunotherapy approaches. The projects are supported by Administrative, Biospecimen, Biostatistics and Large Animal Shared Resources.

OD024623 (Copolla G, P.I.; Andrews RN, Co-I, 20% effort) 07/01/2018 - 06/30/2022 Genetic Control of Gene Expression in the Developing Primate Brain $203,288 /year direct cost The identification of genetic variants controlling gene expression constitutes a major goal toward a better understanding of brain physiology and susceptibility to disease. Through whole-genome sequencing, genomic characterization of multiple brain regions across development, and high-throughput functional validation of resulting variants, we seek to elucidate the impact of genetic variation on gene expression in the developing primate brain. These studies will advance our understanding of the genetic contribution to human brain in health and disease. In this project, I served as a neuropathology consult, coordinated necropsies, and conducted brain sample collections.

PAST GRANT HISTORY (in chronological order):

NIH 2T32OD010957 (Cline JM, PD; Andrews RN, Trainee) 04/01/13-03/31/18 Laboratory Animal & Comparative Medicine Training $309,920 /year direct cost

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BIBLIOGRAPHY:

Peer-Reviewed Journal Articles:

Snow WW, Cline JM, Bourland JD, Michalson KT, Andrews RN, Sempowski GD, Register TC, and Caudell DL. Transcriptional Profiling of Nonhuman Primate Lymphoid Organs Following Exposure to Total Body Irradiation. In preparation.

Latimer CS, Shively CA, Keene CD, Jorgensen MJ, Andrews RN, Register TC, Montine TJ, Wilson AM, Neth BJ, Mintz A, Maldjian JA, Whitlow CT, Kaplan JR, and Craft S. A Non-Human Primate Model of Early Alzheimer’s Disease Pathologic Change: Implications for Disease Pathogenesis. In submission, Alzheimer’s and Dementia, 2018.

Andrews RN, Caudell DL, Metheny-Barlow LJ, Peiffer AM, Tooze JA, Bourland JD, Hampson RE, Deadwyler SA, Cline JM. Fibronectin produced by cerebral endothelial and vascular smooth muscle cells contributes to perivascular extracellular matrix in late-delayed radiation-induced brain injury. Radiat Res. In revision, Radiation Research 2018.

Tyrrell D, Bharadwaj M, Jorgensen M, Register TC, Shively CA, Andrews RN, Neth B, Dirk Keene CD, Mintz A, Craft S, Molina A. Blood-Based Bioenergetic Profiling Reflects Differences in Brain Bioenergetics and Metabolism. Oxidative Metabolism and Cellular Longevity. 2017. doi: 10.1155/2017/7317251. Epub: 2017 Oct 2.

Andrews RN, Metheny-Barlow LJ, Peiffer AM, Hanbury DB, Tooze JA, Bourland JD, Hampson RE, Deadwyler SA, Cline JM. Cerebrovascular Remodeling and Neuroinflammation is a Late Effect of Radiation-Induced Brain Injury in Non- Human Primates. Radiat Res. 2017 May;187(5):599-611. doi: 10.1667/RR14616.1. Epub 2017 Mar 6. PubMed PMID: 28398880.

Postupna N, Latimer CS, Larson EB, Sherfield E, Paladin J, Shively CA, Jorgensen MJ, Andrews RN, Kaplan JR, Crane PK, Montine KS, Craft S, Keene CD, Montine TJ. Human Striatal Dopaminergic and Regional Serotonergic Synaptic Degeneration with Lewy Body Disease and Inheritance of APOE ε4. Am J Pathol. 2017 Feb 14. pii: S0002-9440(17)30060-3. doi: 10.1016/j.ajpath.2016.12.010. [Epub ahead of print] PubMed PMID: 28212814.

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Hanbury DB, Peiffer AM, Dugan G, Andrews RN, Cline JM. Long-Term Cognitive Functioning in Single-Dose Total-Body Gamma-Irradiated Rhesus Monkeys ( Macaca mulatta ). Radiat Res. 2016 Oct 14. [Epub ahead of print] PubMed PMID: 27740889.

Cimafranca MA, Davila J, Ekman GC, Andrews RN, Neese SL, Peretz J, Woodling KA, Helferich WG, Sarkar J, Flaws JA, Schantz SL, Doerge DR, Cooke PS. Acute and chronic effects of oral genistein administration in neonatal mice. Biol Reprod. 2010 Jul;83(1):114-21. doi:10.1095/biolreprod.109.080549. Epub 2010 Mar 31. PubMed PMID: 20357267; PubMed Central PMCID: PMC2888966.

Abstracts/Scientific exhibits/Presentations at national meetings:

Latimer CS, Shively CA, Keene CD, Jorgenseon MJ, Andrews RN, Register TC, Montine TJ, Wilson AM, Neth B, Mintz A, Maldjian J, Whitlow CT, Kaplan JR, Craft S. A Non-Human Primate Model of Early Alzheimer’s Disease Pathologic Change. Poster to be presented at the 2018 Alzheimer's Association International Conference; Chicago, Illinois.

Andrews RN, Caudell DL, Metheny-Barlow LJ, Peiffer AM, Bourland JD, Hampson RE, Deadwyler SA, Cline JM. Cerebrovascular endothelial cells produce fibronectin in late-delayed radiation-induced brain injury in non-human primates. Poster to be presented at 63rd Annual Meeting of the Radiation Research Society, 2017; Cancun, Mexico.

Caudell DL, Dugan G, Andrews RN, Kock N, Chao N, Cline JM. Neoplastic outcomes following high dose total body irradiation in rhesus macaques. Poster to be presented at the 63rd Annual Meeting of the Radiation Research Society, 2017; Cancun, Mexico.

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DeBo RJ, Michalson KT, Dugan GO, Lees CJ, Hanbury DB, Caudell DL, Andrews RN, Vujaskovic Z, Batinic-Haberle, Bourland JD, Cline JM, Register TC. Evaluation of a superoxide dismutase mimetic as a potential mitigator of acute and delayed responses of the lung and heart to thoracic gamma irradiation. Poster to be presented at 63rd Annual Meeting of the Radiation Research Society, 2017; Cancun, Mexico.

Labriola CL, Andrews RN, Balamayooran G, Hutchison AR, Caudell DL, Jorgensen MJ, Cline JM and ND Kock. Placenta accreta in three African green monkeys (Chlorocebus aethiops sabaeus). Poster to be presented at the 2017 American College of Veterinary Pathologist and American Society for Veterinary Clinical Pathology Concurrent Meeting; Vancouver, British Columbia, Canada.

Andrews RN, Metheny-Barlow LJ, Hanbury DB, Peiffer AM, Bourland DJ, Hampson RE, Deadwyler SA, Tooze JA, Cline JM. Cerebrovascular remodeling and neuroinflammatory gene expression is up-regulated in late-delayed radiation induced brain injury. Poster presented at 62nd Annual Meeting of the Radiation Research Society, 2016; Waikoloa, HI.

Cline JM, Dugan G, Andrews RN, Kavanagh K, Bourland DJ, Hanbury D, Peiffer AM, Register TC, Caudell D, Chao NJ. Multisystemic Late Effects of Radiation Exposure in Nonhuman Primates. 62nd Annual meeting of the Radiation Research Society, 2016; Waikoloa, HI.

Andrews RN, Hampson RE, Metheny-Barlow LJ, Hanbury DB, Bourland JD, Peiffer AM, Cline JM, Deadwyler SA. Altered Gene Expression Following Whole Brain Irradiation in Nonhuman Primates. Poster presented at Society for Neuroscience 2016; San Diego, CA.

Neth BJ, Mintz A, Sai K, Gage HD, Shively C, Register TC, Jorgensen MJ, Andrews RN, Atkins HM, Uberseder B, Cline JM, Cunnane S, Castellano CA, Keene CD, Montine TJ, Maldjian J, Wagner B, Hughes TM, Craft S. Dual-Tracer Acetoacetate and Glucose Metabolism are Associated with Neuropathologic Amyloid Burden and Alzheimer Biomarkers in CSF. Alzheimer's & Dementia, Volume 12, Issue 7, Supplement, July 2016, Page P519, ISSN 1552-5260, http://dx.doi.org/10.1016/j.jalz.2016.06.1021.

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Wilson QN, LeGrande A, Hutchison R, Balamayooran G, Andrews RN, Atkins H, Michalson KT, Gee MK, Kock ND and Caudell D. The Clinical Management of an Outbreak of Yersinia enterocolitca in a Colony of African Green Monkeys (Chlorocebus aethiops sabeus). Poster presented at Association of Primate Veterinarians Workshop, 2016; Charlotte, NC.

Latimer C, Kaplan J, Shively C, Cline JM, Andrews RN, Matt Jorgensen M, Craft S, Montine TJ, Keene CD. The Aging Vervet as a Model of Alzheimer’s Disease. Poster presented at the 92nd Annual Meeting of the American Association of Neuropathologists; 2016.

Andrews RN, Hanbury DH, Peiffer AM, Dugan G, Cline JM. Single dose total body irradiation (TBI) results in magnetic resonance imaging (MRI) detectable brain lesions in non-human primates. Poster presented at: 61st Annual Meeting of the Radiation Research Society; 2015; Weston, FL.

Deadwyler SA, Peiffer A, Sexton CA, Daunais JB, Hanbury DB, Mitchell EL, Wheeler KT, Bourland JD, Cline JM, Andrews RN, Hampson, RE. Effects of whole-brain- irradiation (WBI) on primate brain and related cognitive function. Poster presented at: Neuroscience 2015; Chicago, IL.

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WAKE FOREST SCHOOL OF MEDICINE

Teaching Portfolio

TEACHING RESPONSIBILITIES – INTRAMURAL

2014 COMD 703: Diseases of Laboratory Animals, Incidental and Age-Related Lesions in Non-Human Primates

2014 – present COMD 712: Comparative Pathology Conference, Clinical Pathology. Recurring lecture, approximately once per every two months.

2014 – present COMD 712: Comparative Pathology Conference, General Pathology

2014: Functional Neuroanatomy

2015: Angiogenesis

2015: Angiogenesis in Neoplasia 2016: Apoptosis

2016: Response of the Vascular Wall to Injury

2017: The Molecular Basis of Neoplasia: Proto-oncogenes

2017: The Molecular Basis of Neoplasia: Tumor Suppressors

2017: The Molecular Basis of Neoplasia: Metabolic Alterations and Cancer Cell Survival

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2015 - present COMD 706: Animal Models in Biomedical Research

2015, 2017: Animal Models of Neurodegenerative Disease

2017: Bioinformatics in Translational Research

TEACHING RESPONSIBILITIES – EXTRAMURAL

2014 – present North Carolina State University, Primate Pathology Conference, veterinary students 2014: Radiation Encephalopathy 2015: Retroperitoneal Fibromatosis 2016: Cervical Intraepithelial Neoplasia 2016: Placenta Accreta 2017: Prostatic Melioidosis

2015 Winston Salem State University, DPTS 6206: Pharmacology for Physical Therapy Doctoral Students Pharmacology of Antiviral Drugs Pharmacology of Antifungal Drugs Anti-Cancer Chemotherapeutics Immunosuppressive Therapies

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