A Potential Combination Therapy for Traumatic Brain Injury

A potential combination therapy for traumatic brain injury:

17beta-estradiol and memantine

Michael Robert Lamprecht

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY

2015

ABSTRACT

A potential combination therapy for traumatic brain injury: 17β-estradiol and memantine

Michael Robert Lamprecht

Every year, in the United States alone, there are 1.7 million incidences of traumatic brain

injury (TBI). Unfortunately, despite the tremendous societal and economic cost and decades of

research, current pharmacological treatments for TBI are lacking. The specific aims of this

thesis are: (1) to determine the efficacy of 17β-estradiol (E2) monotherapy treatment post-TBI,

(2) determine if a combination treatment of E2 and memantine provides statistically significant

benefits over monotherapy treatments post-TBI, and (3) to investigate the utility of an in vitro

model to recapitulate the pathobiology of an in vivo model of TBI and to assess its potential to

discover novel and clinically relevant therapeutic targets for future studies.

The neuroprotective properties of E2 have been investigated for several decades in

several different models including excitotoxicity, ischemia, and TBI. Organotypic hippocampal

slice cultures (OHSCs) were mechanically injured at specified strain and strain rates which are

relevant to TBI, and the efficacy of E2 post-TBI was investigated. Physiological concentrations

of E2 were more effective at preventing cell death than supraphysiological concentrations.

Further, GPR30, a novel G protein-coupled receptor, was not activated at physiological

concentrations. These results suggest that the classical estrogen receptors (ERs) were primarily responsible for E2-mediated neuroprotection following TBI, and that GPR30 is neither necessary

nor sufficient.

While monotherapy treatments have shown preclinical success post-TBI, none have been

successful in clinical trials. Combination therapies are a promising area of research that focuses on synergistic effects between compounds for significant increases in neuroprotection,

potentially resulting in a clinically relevant treatment. A combination treatment of E2 and

memantine was statistically more neuroprotective than either monotherapy post-TBI. Using

micro-electrode arrays (MEAs), we recorded and quantified increased evoked responses in

OHSCs after physiological concentrations of E2 and showed that memantine significantly reduces these effects. Our results suggest a potential combination treatment for TBI and a possible mechanism for its synergistic effects.

TBI is a complex injury which initiates a multitude of secondary injuries causing delayed cell death for days or beyond. The utility of in vitro models depends on their ability to

recapitulate the in vivo injury cascade after TBI. We used a genome wide approach to study

changes in gene expression after injury in both an in vitro model and an in vivo model of TBI to

compare the post-TBI pathobiology. There was a strong correlation in gene expression changes between the two models providing confidence that the in vitro model represented the in vivo

injury cascade. From these data, we searched for genes with significant changes in expression over time and identified Sorla. Sorla directs amyloid precursor protein (APP) to the recycling

pathway by direct binding and away from amyloid beta (Aβ) producing enzymes. Mutations of

Sorla have been linked to Alzheimer’s disease (AD). We confirmed the down regulation of

SORLA expression in OHSCs by immunohistochemistry (IHC) and western blotting. Together, these data suggests that the in vitro model of TBI that was tested strongly recapitulates the in vivo TBI pathobiology and is well-suited for future mechanistic or therapeutic studies. The data also suggest a novel target, Sorla, which may play a role in AD caused by TBI.

In conclusion, we discovered a potentially clinically relevant combination treatment of

E2 and memantine for post-TBI therapy. We also confirmed that our in vitro model of TBI is well representative of in vivo models, and that relevant, novel targets for future TBI studies can be elucidated with this model. A potential link between AD and TBI was suggested and warrants future study. Together, these studies address the growing public health concern of TBI.

Table of Contents

List of Figures ...... vi

List of Tables ...... vii

List of Abbreviations ...... viii

1 Introduction ...... 1

1.1 17β-estradiol and neuroprotection ...... 2

1.2 Increased excitatory post synaptic potentials induced by E2 ...... 5

1.3 Neuroprotection and memantine ...... 5

1.4 Combination therapies ...... 7

1.5 Global changes in gene expression after injury ...... 8

1.6 Significance ...... 9

2 E2 is neuroprotective after in vitro models of TBI and stroke ...... 12

2.1 Introduction ...... 12

2.2 Materials and Methods ...... 16

2.2.1 Organotypic hippocampal slice cultures ...... 16

2.2.2 In vitro model of ischemia ...... 17

2.2.3 In vitro model of TBI ...... 17

2.2.4 Cell death assessment ...... 18

2.2.5 Western blots ...... 18

2.2.6 Statistics ...... 19

2.3 Results ...... 20

2.3.1 E2 provides neuroprotection of OHSCs after OGD ...... 20

2.3.2 Therapeutic time window of E2 after OGD ...... 21

2.3.3 ERα and ERβ are necessary for neuroprotection after OGD ...... 21

2.3.4 E2 is neuroprotective after mechanical injury of OHSCs ...... 23

2.3.5 Neuroprotective concentrations of E2 do not activate GPR30 ...... 24

2.4 Discussion ...... 25

3 Combination therapy of 17β-estradiol and memantine ...... 29

3.1 Introduction ...... 29

3.2 Materials and Methods ...... 31

3.2.1 Organotypic hippocampal slice cultures ...... 31

3.2.2 In vitro model of TBI ...... 32

3.2.3 Cell death assessment ...... 32

3.2.4 Electrophysiology ...... 33

3.2.5 Statistical analysis ...... 34

3.3 Results ...... 34

3.3.1 Memantine after mechanical injury of OHSCs ...... 35

3.3.2 Combination treatments significantly decreased cell death ...... 36

3.3.3 Delayed treatment with combination therapy ...... 39

3.3.4 Memantine significantly reduced evoked responses...... 40

3.4 Discussion ...... 43

3.4.1 Estrogen as a monotherapy ...... 44

3.4.2 Memantine as a monotherapy ...... 44

3.4.3 Potential synergistic effects of E2 and memantine ...... 45

3.4.4 Combination treatment of TBI ...... 47

4 Global gene expression analysis of an in vitro and in vivo model of TBI ...... 49

4.1 Introduction ...... 49

4.2 Materials and Methods ...... 51

4.2.1 Organotypic hippocampal slice cultures ...... 51

4.2.2 In vitro model of TBI ...... 52

4.2.3 In vivo model of TBI ...... 53

4.2.4 Affymetrix gene chips...... 54

4.2.5 Microarray data analysis ...... 54

4.2.6 Immunohistochemistry ...... 55

4.2.7 Western blots ...... 55

4.3 Results ...... 56

4.3.1 Comparison of in vitro and in vivo TBI models ...... 56

4.3.2 Gene ontology (GO) analysis...... 59

4.3.3 ANCOVA analysis of in vitro TBI ...... 60

4.3.4 Protein expression of SORLA ...... 62

iii

4.4 Discussion ...... 64

4.4.1 Comparison of in vitro and in vivo TBI models ...... 65

4.4.2 GO Analysis ...... 66

4.4.3 SORLA down regulation after TBI ...... 68

5 Summary ...... 71

5.1 E2 neuroprotection after in vitro models of stroke and TBI ...... 71

5.2 Memantine neuroprotection after in vitro TBI ...... 73

5.3 17β-estradiol and memantine after in vitro TBI ...... 74

5.4 Global analysis of an in vitro and in vivo model of TBI ...... 75

5.5 Down regulation of SORLA after TBI ...... 77

5.6 Limitations ...... 78

5.7 Future Directions ...... 80

6 References ...... 85

7 Appendix A: Significant Gene Lists ...... 97

7.1 1 hour in vitro TBI ...... 97

7.2 6 hour in vitro TBI ...... 120

7.3 12 hour in vitro TBI ...... 140

7.4 24 hour in vitro TBI ...... 161

7.5 1 hour in vivo TBI ...... 170

7.6 6 hour in vivo TBI ...... 177

7.7 24 hour in vivo TBI ...... 188

8 Appendix B: Publications ...... 195

8.1 Manuscripts ...... 195

8.2 Conference proceedings ...... 196

8.3 Abstracts ...... 196

List of Figures

Figure 2.1 E2 is neuroprotective in rat OHSCs exposed to OGD...... 21

Figure 2.2 Inhibition of ERα and ERβ abrogated neuroprotection...... 22

Figure 2.3 E2 reduced cell death after TBI...... 24

Figure 2.4 High concentrations of E2 lead to phosphorylation of Akt and Erk...... 25

Figure 3.1 Memantine reduced cell death after TBI...... 36

Figure 3.2 Combination therapies were neurprotective...... 38

Figure 3.3 A combination therapy was neuroprotective 1 hour post injury...... 40

Figure 3.4 Memantine reduced E2-induced potentiation...... 42

Figure 4.1 Overview of significant changes in gene expression following TBI...... 58

Figure 4.2 Correlation of gene expression changes after TBI...... 59

Figure 4.3 GO analysis of significantly altered gene expression after TBI...... 60

Figure 4.4 SORLA expression after in vitro injury...... 63

List of Tables

Table 4.1 Enriched gene list after ANCOVA and Student's t-test analysis...... 61

vii

List of Abbreviations

Abbr. Full Name Aβ amyloid beta peptide aCSF artificial cerebrospinal fluid AD Alzheimer's disease AMPA α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate APP amyloid precursor protein CNQX 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt CNS central nervous system DIV days in vitro EPSP excitatory post synaptic potential E2 17β-estradiol FPI fluid percussion injury GFM glucose free medium IHC immunohistochemistry LTP long-term potentiation MEA microelectrode array NMDA N-methyl-D-aspartic acid OGD oxygen-glucose deprivation PDMS polydimethylsiloxane PI propidium iodide PND post natal day TBI traumatic brain injury

viii

1 Introduction

Every year, in the United States alone, there are 1.7 million incidences of traumatic brain injury (TBI) (Coronado, Xu et al. 2011). Unfortunately, despite the tremendous societal and economic cost and decades of research, current pharmacological treatments for TBI are lacking

(Narayan, Michel et al. 2002; Willis, Lybrand et al. 2004). TBI is a complex injury with many secondary injuries arising after the primary injury. Individual drugs, or monotherapies, which have been successful in experimental models, have largely failed clinically; in some instances, the benefits of treatment did not outweigh the risk of side effects (Maas, Steyerberg et al. 1999;

Narayan, Michel et al. 2002; Margulies and Hicks 2009). There are currently no FDA approved treatments targeting delayed cell death after TBI. A combination therapeutic approach targeting multiple secondary injuries simultaneously may provide clinically relevant treatment options with more success than monotherapies (Margulies and Hicks 2009). A similar approach was taken to treat HIV/AIDS, and a substantial 65% decrease in HIV/AIDS related deaths occurred from 1995-1998 (Hall, Song et al. 2008). This tremendous increase in survival was due to the introduction of highly active antiretroviral therapy (HAART) which consisted of a combination of reverse transcriptase inhibitors and protease inhibitors (Thompson, Aberg et al. 2010). This previous success motivates the investigation of combination therapies for TBI.

Primary injuries result immediately from the initial trauma of TBI and consist, in part, of contusions, damage to blood vessels, and primary axotomy. These injuries are generally considered mechanical in nature and therefore irreparable (Saatman, Duhaime et al. 2008). In addition, the primary mechanical stimulus initiates a cascade of biological events leading to what has been termed secondary injuries (Robertson 2004; Margulies and Hicks 2009). Cell death via

secondary injuries can continue for hours, days, or years after the primary injury (Sauaia, Moore

et al. 1995; Chen, Xiong et al. 2006). The ongoing nature of these injuries may suggest a potential time-frame or temporal window for therapeutic intervention to slow, stop, or repair damaged cells otherwise destined to die. Such discoveries may contribute to improved survival and decreased long-term disabilities.

In vitro models of TBI have been studied for several decades. An in vitro model of TBI was developed in our laboratory and provides a highly accurate and reproducible injury of organotypic hippocampal slice cultures (OHSCs) which is verified with high-speed video (Cater,

Gitterman et al. 2007; Morrison, Elkin et al. 2011). Our in vitro model provides several advantages to in vivo models and eliminates issues including the influence of the route of administration of drug, changes in blood flow, and partitioning effects of the blood brain barrier

(Sundstrom, Morrison et al. 2005). While these issues are important for a clinically useful treatment, they present difficult challenges to overcome in experimental models when the goal is to identify novel therapies and therapeutic targets. The presented research proposal is focused on determining the utility and biological effects of various treatments and, at this time, not their pharmacokinetics or pharmacodynamics.

1.1 17β-estradiol and neuroprotection

Estrogen is an important ovarian steroid that has also been implicated in many

physiological and psychological changes. The most potent and naturally occurring form of

estrogen, 17β-estradiol (E2), has been investigated as a neuroprotective therapy for TBI (Brown,

Suzuki et al. 2009; Zlotnik, Leibowitz et al. 2012). The neuroprotective effects of E2 have been

attributed to anti-oxidant effects (Behl, Widmann et al. 1995; Numakawa, Matsumoto et al.

2007), changes in NMDA channels (Lebesgue, Traub et al. 2010), changes in vasculature (Xia,

Yang et al. 2007) and anti-apoptotic effects (Simpson, Misso et al. 2005). An early study by

Emerson, et al, showed improved biochemical and cognitive outcomes with post TBI (FPI) injection of E2 (Emerson, Headrick et al. 1993). However, neuroprotection was dependent on gender and only male rats significantly improved post injury. Similarly, Roof, et al found that

100% of female rats compared to 75% of male rats survived an impact-acceleration closed-head injury and suggested that endogenous estrogen may have been neuroprotective (Roof and Hall

2000). In contrast, Aggarwal, et al, found that E2 had no effect on loss of acetyltransferase- positive cells post TBI (Aggarwal and Gibbs 2000). With the potential to reduce delayed cell death after TBI, further investigation of the mechanism responsible for E2 neuroprotection is a necessary and important area of research.

Estrogen induces both genomic and non-genomic responses in cells. The classical estrogen receptors ERα and ERβ are primarily responsible for the genomic effects and act as transcription factors when bound to their ligand (Emmen and Korach 2003). Some evidence supports the existence of membrane bound ERs which may be responsible for the fast, non- genomic response to E2 (Levin 2005). However, the discovery of GPR30 has muddied the issue and led many to believe that GPR30, and not membrane bound ERs, is responsible for these non- genomic effects. GPR30 is an orphan G-protein coupled receptor that is controversially implicated as an estrogen receptor (Carmeci, Thompson et al. 1997). While several groups have shown through immunofluorescence binding experiments (Funakoshi, Yanai et al. 2006) and activation of downstream pathways (Filardo, Quinn et al. 2002; Revankar, Cimino et al. 2005;

Prossnitz and Barton 2009) that estrogen does bind to and activate GPR30, the concentrations needed for these experiments (>1 nM) are well-above physiological levels (10-120 pM) (Otto,

Rohde-Schulz et al. 2008; Strom, Theodorsson et al. 2011). Other groups have shown no binding affinity of E2 for GPR30 which suggests these effects are off-target (Otto, Rohde-Schulz et al. 2008). Also, the interplay, if any, between classical estrogen receptors and GPR30 is not well understood (Lebesgue, Chevaleyre et al. 2009).

Because it is a G-protein coupled receptor and can elicit physiological responses within several seconds (Revankar, Cimino et al. 2005), GPR30 is an attractive target as the potential mediator of acute E2 neuroprotection. Evidence suggests that GPR30 activation via supra- physiological levels of E2 or its agonist G-1 leads to activation of the PI3K/Akt pathway which has well known anti-apoptotic effects (Cimarosti, Zamin et al. 2005; Gingerich, Kim et al. 2010).

Once activated, Akt exerts profound anti-apoptotic effects by phosphorylating and therefore inactivating several proteins involved in apoptosis, including GSK-3β (Cimarosti, Zamin et al.

2005). These anti-apoptotic effects support the hypothesis that GPR30 is responsible for acute

E2 mediated neuroprotection, but further research is necessary to determine if Akt activation occurs when low but still neuroprotective doses of E2 are administered, or if high doses are necessary for this increase in Akt phosphorylation and consequent neuroprotection. Because activation of GPR30 leads to potentiation of excitatory post-synaptic potentials (EPSPs) (Grassi,

Frondaroli et al. 2009), it is possible that GPR30 activation may be detrimental, and that activation of classical estrogen receptors ERα and ERβ alone may provide the greatest neuroprotection. E2 has consistently been reported in the literature to provide neuroprotection after several models of injury, but the molecular mechanism which provides neuroprotection remains elusive and requires further study (Emerson, Headrick et al. 1993; Dubal, Kashon et al.

1998; Wise and Dubal 2000; Yang, Shi et al. 2000; Yune, Kim et al. 2004; Chen, Chang et al.

2009).

1.2 Increased excitatory post synaptic potentials induced by E2

Although E2 has traditionally been linked to genomic changes caused by ERα and ERβ,

an increasing number of reports reveal rapid effects after application of E2 which are too fast to be transcriptionally mediated (Inagaki, Kaneko et al. 2012). An investigation in to the mechanism behind E2’s influence on the central nervous system (CNS) led to the discovery that

E2 causes an acute potentiation of EPSPs and enhances the magnitude of long-term potentiation

(LTP) in hippocampal slice cultures (Foy, Xu et al. 1999; Inagaki, Kaneko et al. 2012). These

increases in synaptic plasticity occurred within 3 minutes of E2 application and had a maximum

effect at 10 minutes implying a non-genomic response. In this study, the increase in EPSPs was

isolated to NMDA receptors.

In terms of pharmacological neuroprotection by E2, an enhancement of synaptic

plasticity is a double-edged sword. It is well established that TBI causes marked inhibition of

cognitive functions post injury (Moretti, Cristofori et al. 2012). An increase in synaptic

plasticity would lead to improved neuronal transmission and therefore may improve cognitive functions. However, excitotoxicity is a well known secondary injury associated with TBI. An

increase in synaptic plasticity caused by enhanced NMDA receptor transmission could lead to

increased calcium influx, exacerbating excitotoxicity. While the protective effects of E2 post

injury have been studied extensively, there is currently little research examining the negative effects of increased plasticity after E2 therapy. Mitigating negative side effects through

pharmacological means may enhance the neuroprotective effects of E2.

1.3 Neuroprotection and memantine

Under normal physiological conditions, intracellular calcium concentrations are tightly regulated. Typically the intracellular concentration is 100 nM while the extracellular concentration is about 1.8 mM, an 18,000 fold difference. However, when necessary, intracellular calcium concentrations can be increased 10-100 fold very quickly to serve as a second-messenger. In neurons, calcium increases lead to the release of neurotransmitters and are an integral part of neuronal circuit transduction. However, during excitotoxicity, sustained calcium influx can lead to activation of caspases leading to apoptosis (Matsushita, Shima et al.

2000). Increased calcium can also lead to opening of the mitochondrial permeability transition

(MPT) pore, causing swelling of the mitochondria and release of other proteins such as cytochrome c. Release of cytochrome c leads to further calcium increases in a vicious positive feedback loop inducing apoptosis (Boehning, Patterson et al. 2003). During excitotoxicity, the initial source for calcium influx is NMDA channels that have been activated due to excessive neurotransmitter release.

Memantine is an FDA-approved, uncompetitive NMDA antagonist which is prescribed primarily for Alzheimer’s patients, but is also used for Parkinson’s disease and senile dementia

(Mathys, McCarrell et al. 2013; Schneider 2013). It is unique in that it is a low affinity, use- dependent inhibitor which primarily targets extra synaptic receptors. Because it is use- dependent, its effects are greater when higher concentrations of receptor agonists are present, which may target the drug’s actions to damaged areas experiencing excitotoxicity. These properties alleviate the deleterious side effects of other NMDA antagonists such as MK-801 which produced hallucinations and psychosis in rat models (Koek, Woods et al. 1988). As an

NMDA antagonist, memantine’s primary function is to reduce calcium influx and decrease neuronal excitability, making it a potent inhibitor of excitotoxicity. The neuroprotective effects

of NMDA antagonists including MK-801 and memantine have been well documented in many

different models of CNS injury over the past 20 years (Tasker, Coyle et al. 1992; Vornov, Tasker

et al. 1994).

1.4 Combination therapies

Brain injuries occurring from TBI are complex and dynamic with multiple secondary

injury mechanisms causing delayed cell death hours, days, or years after the primary injury

(Margulies and Hicks 2009; Stoica and Faden 2010; Moretti, Cristofori et al. 2012). These

injuries can lead to progressive neurodegeneration, often accompanied by permanent loss of

sensory, motor and/or cognitive function. Individual drugs, or monotherapies, which have been

successful in experimental models, have failed clinically (Maas, Steyerberg et al. 1999; Narayan,

Michel et al. 2002; Maas, Marmarou et al. 2007; Lingsma, Roozenbeek et al. 2011). Because of

the complexity of interactions between secondary injuries, it is likely that multiple drugs,

targeting several secondary injuries, will be required to provide neuroprotection after TBI. The

clinical failure of monotherapies could be due to many factors including lack of understanding of

the underlying injury mechanisms, inadequate pre-clinical testing in multiple injury models, or

because these injuries initiate several secondary pathological cascades that a single drug can not

sufficiently reverse the negative outcome (Margulies and Hicks 2009). The goal of a

combination therapy is to combine single therapies with complementary or synergistic actions to

increase the efficacy of TBI treatment to a level which is clinically relevant (Faden 2002;

Margulies and Hicks 2009). Therefore, further investigation of the mechanisms of neuroprotection underlying monotherapies must be clear to determine the most beneficial drug

combinations.

Recent research has suggested that combination treatments may improve therapeutic outcomes following TBI. For example, the combination therapy of melatonin and dexamethasone reduced edema and brain infarctions in a mouse model of TBI (Campolo, Ahmad et al. 2013). Another successful combination included human umbilical cord blood cells and granulocyte colony stimulating factor which reduced histopathological and motor impairments in a rat model of TBI (Acosta, Tajiri et al. 2014). However, it is important to note that some drug combinations have led to worse outcomes than either monotherapy, for example, a combination of YM14673 and dextrorphan resulted in a less effective therapy after lateral FPI in rats (Faden

1993).

1.5 Global changes in gene expression after injury

Several studies have confirmed differential gene expression following injuries such as

TBI (Morrison, Eberwine et al. 2000; Morrison, Meaney et al. 2000; Poulsen, Penkowa et al.

2005; Di Pietro, Amin et al. 2010). These changes in gene expression likely coincide with the activation of different molecular pathways caused by secondary injuries such as excitotoxicity, edema, or free radical formation. With the advent of DNA/RNA microarrays, it is now possible to quantify global changes in gene expression. This powerful analysis eliminates the need to choose a specific gene of interest and to produce the necessary methods to determine changes in expression and instead allows for thousands of genes to be analyzed at once. From these data, an overall snapshot of gene expression changes can be analyzed to determine not only expression changes of a specific gene, but of an entire physiological pathway, which may lead to further insight in to the mechanisms behind injury or other interesting stimuli. An improved understanding of the pathophysiology following TBI may provide novel therapeutic targets for future drug discovery or combination therapies. Algorithms to analyze the complex datasets

produced by microarrays are constantly being developed, revised, and improved. There are several commercial and open-source academic software packages which provide tools to analyze these data such as EDGE, NetAffx, Bioconductor, and Ingenuity (Cheng, Sun et al. 2004;

Gentleman, Carey et al. 2004; Leek, Monsen et al. 2006).

Previous studies on TBI have shed light on new genes and pathways of interest but many typically focus on a single injury and a single time point after injury. Because of the complex nature of these injuries, temporal changes in gene expression may provide more insight than a single time point. For example, temporal changes in gene expression have been studied for over a decade and have been integral in understanding physiological changes during circadian rhythm cycles (Hazen, Borevitz et al. 2005; Menger, Lu et al. 2005). Instead of focusing on significant gene expression changes at a single time point, these analysis methods take in to account the temporal data and search for significant changes over time that may or may not be significant at a single time point. This analysis could provide insight to the genetic progression of a disease or pathology and could lead to novel targets for therapies which would be overlooked with analysis of a single time point. A temporal analysis of global gene expression changes in TBI would therefore provide an important dataset to validate future studies of treatment paradigms.

1.6 Significance

TBI is a growing, global public health concern, affecting nearly 10 million people

annually worldwide (Hyder, Wunderlich et al. 2007; Gean and Fischbein 2010). In the United

States alone, the incidence of TBI has risen from 1.4 million in 2002 (Langlois, Rutland-Brown

et al. 2006; Elkin, Ilankovan et al. 2011) to an estimated 1.7 million in 2006 (Faul, Xu et al.

2010), with corresponding increases in emergency department visits (1.111 million to 1.365

million), hospitalization (235,000 to 275,000), and deaths (50,000 to 52,000). Additionally, the

societal cost of TBI in the United States, including medical costs and lost productivity, is

estimated to have risen from $60 billion in 2000 to $76.5 billion in 2006 (Finkelstein, Corso et al. 2006). Despite these enormous societal costs, treatments for brain injury continue to remain

elusive. A significant contribution of this work will be to improve our understanding of the

cellular mechanisms which cause delayed cell death after TBI and to investigate a potential

combination drug therapy to ameliorate this cell death. TBI is a debilitating injury with few

treatment options post injury. Therefore, discovery of improved therapeutics is paramount to the

reduction of long-term disability and morbidity associated with these injuries. There are

currently no FDA-approved therapies specifically targeting delayed cell death for TBI. The

proposed work will focus on FDA-approved drugs which have shown experimental

neuroprotection that has not translated to clinical trials. The data provided by these experiments

will further elaborate the mechanism of neuroprotection afforded by E2 and can directly

influence the development of improved therapies for TBI.

Furthermore, combination therapies are a promising approach to complex injuries such as

TBI, and have proven successful in other diseases such as AIDS where a 65% decrease in

HIV/AIDS related deaths occurred between 1995 and 1998 with the introduction of combination

therapies (Hall, Song et al. 2008). The proposed research will test this approach with a

combination therapy of E2 and memantine, two FDA approved drugs which work through different mechanisms. Because they are FDA approved for other indications, this combination

therapy is well suited to move quickly through FDA approval for a clinical trial. Although

intense research in to therapeutics for TBI continues, no clinically relevant monotherapy

treatments have emerged which abrogate delayed cell death. Preclinical studies have provided

many potential monotherapies which show benefit after experimental TBI. One explanation for

a lack of clinical translation may be that monotherapy treatments focus primarily on one aspect

of a very complex injury (Margulies and Hicks 2009) which may be insufficient for clinical

treatment of TBI. Therefore, a combination treatment which targets more than one secondary

cascade following TBI may provide a significant benefit to patients compared to monotherapies

alone, and further research on combination therapies is necessary.

A further contribution of this work will be the discovery of potential new pathways for

therapeutic intervention. Using genome wide RNA arrays, the proposed research will follow changes in gene expression over the acute time course of TBI. These changes in gene expression correlate with the activation or deactivation of molecular pathways and will shed light on the progression of the injury. This, in turn, may provide novel pathways to target for neuroprotection after injury.

2 E2 is neuroprotective after in vitro models of TBI and

stroke1

2.1 Introduction

TBI is complex and dynamic with multiple secondary injury mechanisms causing

delayed cell death for hours, days, or years after the primary injury (Margulies and Hicks 2009;

Stoica and Faden 2010; Moretti, Cristofori et al. 2012). These secondary injuries can lead to

progressive neurodegeneration, often accompanied by permanent loss of sensory, motor and/or

cognitive function. Under circumstances of severe TBI, ischemia can occur due to the rupture of

blood vessels as well as increased intracranial pressure from swelling and edema resulting in a

loss of oxygen and glucose (Bouma, Muizelaar et al. 1991; Marion, Darby et al. 1991). Recent

studies have indicated that E2 is neuroprotective when administered after TBI and stroke.

However, the mechanism by which E2 exerts neuroprotection after injury is not fully understood.

GPR30, an orphan G-protein coupled receptor, is a recently discovered and controversial target

of E2 which has near immediate physiological response in neurons (Carmeci, Thompson et al.

1997; Foy, Xu et al. 1999). The rapid response of GPR30 to E2 has led to the speculation that

GPR30 may be responsible for the acute benefits of E2 when administered after mechanical or

ischemic injury (Lebesgue, Chevaleyre et al. 2009; Day, Floyd et al. 2013).

E2 is the most potent naturally occurring form of estrogen and exerts a multitude of

effects after binding the estrogen receptor (Shappell, Hyndman et al. 2010). After binding E2,

the classical estrogen receptors translocate to the nucleus and act as a transcription factor which

1 A modified version of this chapter previously appeared in print: Lamprecht, M.R. and Morrison, B. 3rd (2014). GPR30 activation is neither necessary nor sufficient for acute neuroprotection by 17β‐estradiol after an ischemic injury in organotypic hippocampal slice cultures. Brain Res. 1563, 131‐137.

controls the expression of many genes (Nilsson and Gustafsson 2002). E2 may also bind cell

surface receptors, causing near immediate physiological effects (Bjornstrom and Sjoberg 2004;

Zivadinovic, Gametchu et al. 2005). The neuroprotective effects of E2 have been attributed to anti-oxidant effects (Behl, Widmann et al. 1995; Numakawa, Matsumoto et al. 2007), changes in

NMDA channels (Lebesgue, Traub et al. 2010), changes in vasculature (Xia, Yang et al. 2007), and anti-apoptotic effects (Simpson, Misso et al. 2005). It is also currently FDA approved for hormone replacement therapies.

For over two decades, E2 has been investigated as a neuroprotective therapy for stroke

(Paganini-Hill, Ross et al. 1988; Paganini-Hill 1995; Simpkins and Singh 2008). In early animal studies, E2 was neuroprotective when administered before cerebral ischemia in vivo, which has led many to investigate its mechanism of prophylaxis (Simpkins, Rajakumar et al. 1997). Only two studies reported that post-ischemic administration of E2 did not reduce mortality or lesion size. In 1998, Dubal et al. compared pre-treatment with E2 administered with silastic capsules at low and high doses and acute treatment with a low dose of E2 (Dubal, Kashon et al. 1998). The authors found pre-treatment to be successful at both doses, but acute treatment with a low dose of E2 was unsuccessful. In a similar experiment, Strom et al. reported that pre-treatment with low dose silastic capsules successfully reduced infarct sizes but the same treatment had no effect when administered immediately after the injury (Strom, Theodorsson et al. 2010). Although the authors tested E2 levels after 2 days of treatment, no E2 concentration data was available immediately or shortly following the implantation of the silastic capsules. One explanation for these discrepancies is that the time frame required for low dose silastic capsules to produce a physiological concentration of E2 in vivo was beyond the therapeutic window. However, far fewer studies have investigated estrogen’s role as a post-ischemic therapy, and no consensus has

been reached on whether E2 is beneficial when administered acutely after ischemia (Yang, Shi et

al. 2000).

Less research has focused on estrogen’s neuroprotective effects on TBI, but similarly to

ischemia the results are varied. An early study by Emerson, et al, reported improved

biochemical and cognitive outcomes with post TBI (FPI) injection of E2 (Emerson, Headrick et

al. 1993). However, neuroprotection was dependent on gender and only male rats showed

significant improvement post injury. Similarly, Roof and Hall found that 100% of female rats

compared to 75% of male rats survived an impact-acceleration closed-head injury and suggested

that endogenous estrogen may have been neuroprotective (Roof and Hall 2000). In contrast,

Aggarwal, et al, found that E2 had no effect on loss of acetyltransferase-positive cells post TBI

(Aggarwal and Gibbs 2000). Although E2 has shown effectiveness as a therapeutic compound in

animal models of TBI, it has not translated clinically. There is currently a clinical phase II trial,

RESCUE-TBI, testing the safety and efficacy of Premarin IV (conjugated estrogens for

injection), which has not released results (Wigginton, Pepe et al. 2010)

Estrogen induces both genomic and non-genomic responses in cells. The classical

receptors ERα and ERβ are primarily responsible for the genomic effects and act as transcription

factors when bound to their ligand (Emmen and Korach 2003). Some evidence supports the

existence of membrane bound ERs which may be responsible for the fast, non-genomic response

to E2 (Levin 2005). However, the discovery of GPR30 has muddied the issue and led many to

believe it is GPR30, and not membrane bound ERs, which is responsible for these non-genomic

effects.

Although several groups have shown through immunofluorescence binding experiments

(Funakoshi, Yanai et al. 2006) and activation of downstream pathways (Filardo, Quinn et al.

2002; Revankar, Cimino et al. 2005; Prossnitz and Barton 2009) that estrogen does bind to and

activate GPR30, the concentrations needed for these effects (>1 nM) are far from physiological

levels (10-120 pM) (Strom, Theodorsson et al. 2010; Strom, Theodorsson et al. 2011). Other

groups have shown no binding affinity of E2 for GPR30 which suggests these effects are off-

target (Otto, Rohde-Schulz et al. 2008). Also, the interplay, if any, between classical ERs and

GPR30 is not well understood (Lebesgue, Chevaleyre et al. 2009). However, because it is a G-

protein coupled receptor and can elicit physiological responses within seconds (Revankar,

Cimino et al. 2005), GPR30 is an attractive target as the potential mediator of acute E2

neuroprotection following injury. GPR30 activation via supra-physiological levels of E2 or its

agonist G-1 activates the PI3K/Akt pathway, which has well known anti-apoptotic effects

(Cimarosti, Zamin et al. 2005; Gingerich, Kim et al. 2010). Once activated, Akt exerts profound

anti-apoptotic effects by phosphorylating and therefore inactivating several proteins involved in

apoptosis, including GSK-3β (Cimarosti, Zamin et al. 2005). In addition to the phosphorylation of Akt, GPR30 activation leads to release of heparin-bound EGF, activating EGFR and consequent phosphorylation of Erk (Filardo, Quinn et al. 2002).

In this study, we used in vitro models of TBI or stroke employing rat organotypic hippocampal slice cultures (OHSCs). Both of these models are well-established and reproducible (Sundstrom, Morrison et al. 2005), which eliminates many confounds of in vivo models including the influence of the route of administration of the drug, changes in blood flow, and partitioning effects of the blood brain barrier. Additionally, reproducibility of injury severity is improved with our in vitro model. We tested the efficacy of both physiological and supra-

physiological doses of E2 as a neuroprotective agent after mechanical injury and oxygen-glucose

deprivation (OGD). Utilizing agonists and inhibitors to classical ERs and GPR30 we begin to

define the roles of the different receptors in acute E2-mediated neuroprotection in OGD. We

also examined the phosphorylation of Akt and Erk after neuroprotective concentrations of E2.

Together, these findings provide evidence that classical ERs are responsible for acute E2-

mediated neuroprotection whereas GPR30 activation and subsequent Akt and Erk

phosphorylation are not necessary.

2.2 Materials and Methods

2.2.1 Organotypic hippocampal slice cultures

All animal procedures were approved by Columbia University's Institutional Animal

Care and Use Committee (New York, NY). OHSCs were cultured as previously described

(Stoppini, Buchs et al. 1991; Morrison, Pringle et al. 2002; Sundstrom, Morrison et al. 2005).

Briefly, Sprague Dawley rat pups (P8-P10) were rapidly decapitated, and the hippocampus excised and placed in ice-cold Gey’s balanced salt solution (Life Technologies, Grand Island,

NY) supplemented with D-glucose (4.5 mg/mL, Sigma-Aldrich, St. Louis, MO). 400 μm thick, transverse sections were cut using a McIlwain tissue chopper (Ted Pella, Redding, CA) and then plated on polydimethylsiloxane (PDMS) membranes (Speciality Manufacturing, Saginaw, MI) previously coated with laminin (80 µg/mL, Life Technologies) and poly-L-lysine (320 µg/mL,

Sigma-Aldrich) in custom-made stainless steel wells. The cultures were initially fed Neurobasal

(Life Technologies) medium supplemented with B27 (Life Technologies), GlutaMAX (2 μM,

Life Technologies), and D-glucose (4.5 mg/mL, Sigma). Cultures were maintained under standard culture conditions at 37°C and 5% CO2. After 3 days in vitro (DIV), the medium was

changed to full serum medium containing 50% MEM (Sigma), 25% heat-inactivated horse serum

(Life Technologies), 25% Hanks balanced salt solution (Sigma), GlutaMAX (2 μM, Life

Technologies), and D-glucose (4.5 mg/mL, Sigma-Aldrich). Medium was changed every 2-3

days. 17β-estradiol dipotassium salt (Sigma) was added at the stated time points and

concentrations.

2.2.2 In vitro model of ischemia

After 10 DIV, OHSCs were subjected to an ischemic injury by rinsing cultures in glucose

free medium (GFM), containing 75% DMEM, 25% Hanks balanced salt solution, and L-

glutamine (2 μM), for 5 minutes, then transferring to GFM pre-equilibrated with 95% N2 and 5%

CO2. Culture plates were immediately placed in an airtight chamber (Billups-Rothenberg, CA),

gassed with 95% N2 and 5% CO2 at 15 L/min for 10 minutes and subsequently sealed and

incubated for 30 minutes at 37°C. Control cultures were incubated for 40 minutes at 37°C in

GFM supplemented with 4.5 mg/mL glucose in 95% air and 5% CO2.

2.2.3 In vitro model of TBI

After 10 DIV, OHSCs cultured on silicone membranes were subjected to a moderate

mechanical injury. The injury was induced by stretching the underlying silicone substrate to a

predetermined strain at a predetermined strain rate to produce the desired tissue injury under

motion-control. Our well-established model produces a highly accurate and reproducible injury

to OHSCs (Elkin and Morrison 2007; Morrison, Elkin et al. 2011). Tissue deformation was

verified by image analysis of high speed video (MotionPro, Redlake, Pasadena, CA) at 1000

frames per second (Elkin and Morrison 2007). Lagrangian strain of the tissue was determined by

calculating the deformation gradient tensor by locating fiduciary markers on the tissue slice

before and at maximal stretch using custom MATLAB (MathWorks, Natick, MA) scripts (Elkin and Morrison 2007).

2.2.4 Cell death assessment

Quantification of cell death in OHSC using the fluorescent vital stain, propidium iodide

(PI, Life Technologies), has been described previously (Morrison, Pringle et al. 2002; Cater,

Gitterman et al. 2007). Briefly, micrographs were taken before the induction of injury and either

1 day following OGD injury or 4 days following mechanical injury. These time points were

chosen based on previous research and represent the time after injury when maximal cell death is

observed in the respective models (Morrison, Pringle et al. 2002; Cater, Gitterman et al. 2007;

Morrison, Elkin et al. 2011). The cultures were transferred to serum free medium containing

75% MEM (Sigma-Aldrich), 25% Hanks balanced salt solution (Sigma-Aldrich), GlutaMAX (2

μM, Life Technologies), D-glucose (4.5 mg/mL, Sigma-Aldrich) and supplemented with 5

μg/mL PI for 30 minutes before imaging. Brightfield and PI images were acquired on an

Olympus IX-80 fluorescent microscope fitted with a 175 W Xenon Arc lamp (Perkin Elmer,

MA, USA), CoolSNAP ES camera (Photometrics, AZ, USA), with an excitation of 556-580 nm

and an emission of 590-630 nm (PI exposure 2 s, brightfield exposure 3 ms). Metamorph

(Molecular Devices, CA, USA) image analysis software was used to outline the CA1, CA3 and

DG regions in the brightfield image, and these regions of interest (ROI) were transferred to the

PI images taken before and after injury. Percentage cell death was determined as the area above

a threshold in the PI fluorescent image divided by the total area of the ROI (i.e. CA1 region).

The threshold was determined by the fluorescence level of uninjured, control slices imaged on

the same day as injured experimental groups. OHSCs with pre-injury cell death > 5% in the

region of interest were discarded.

2.2.5 Western blots

For each experimental condition, 9 slices were collected for protein extraction. Slices

were rinsed twice with ice-cold PBS and immediately placed in lysis buffer A (40 mM HEPES,

120 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM sodium pyrophosphate, 50 mM sodium

fluoride, 0.5 mM sodium orthovanadate, 10 mM β-glycerophosphate, Sigma). Samples were

sonicated (Sonicator 3000, Misonix, NY, USA), incubated on ice, then centrifuged to remove

cell debris. Approximately 50 µg of protein per sample were loaded in a Bis-Tris gel (Life

Technologies), and proteins were separated by electrophoresis (150 V, 1 hour). Proteins were then transferred to a nitrocellulose membrane (Life Technologies) using a semi-dry apparatus

(Thermo Fisher Scientific, Waltham, MA). After transfer, the membrane was blocked in Tris-

buffered saline (TBS, pH 7.4) with 5% bovine serum albumin (BSA) for 2 hours. Membranes

were incubated overnight at 4ºC with primary antibodies (phosphorylated Akt [Cell Signaling,

MA, USA, #9271], total Akt [Life Technologies, #44609G], phosphorylated Erk [Cell Signaling,

#4377S] , total Erk [Life Technologies, #617400], or β-tubulin [Life Technologies, #32-2600]) in

TBS-T (0.1% Tween-20, pH 7.4) and 5% BSA. Following the primary antibody incubation, membranes were washed 3x10 minutes in TBS with 0.1% Tween. For detection, the membranes were labeled with a corresponding secondary antibody (Goat-anti Rabbit Alexa Fluor 546 or

Goat-anti Mouse Alexa Fluor 647, Life Technologies). Fluorescence was detected using a CRi

Maestro 2 in vivo Imaging System (Perkin Elmer).

2.2.6 Statistics

All data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by post hoc comparisons with the Tukey-Kramer adjustment in

MATLAB (MathWorks, MA, USA); P-values less than 0.05 were deemed significant (* indicates p < 0.05).

2.3 Results

2.3.1 E2 provides neuroprotection of OHSCs after OGD

Because the CA1 region is most susceptible to injury from OGD, we focused on cell

death in this region (Pringle, Self et al. 2000; Morrison, Pringle et al. 2002; Sundstrom, Morrison

et al. 2005). OGD of OHSCs significantly increased cell death (75.1±3.3%) compared to non-

injured controls (2.6%±0.5%) in the CA1 region as shown in Figure 2.1 A. E2 was significantly

neuroprotective at 10 pM (58.0±7.4%), 100 pM (40.8±6.7%), 1 nM (60.0±7.1%) and 10 nM

(59.2±5.8%) but was not significantly protective at 100 nM (64.3±4.2%). In uninjured controls,

no significant differences in cell death in the CA1 region were observed at any E2 concentration

tested, indicating no inherent toxicity of E2 administration (data not shown). These results

indicate that estrogen has a dose-dependent therapeutic effect on ischemic injury of the OHSCs

with 100 pM being the most effective dose tested.

Figure 2.1 E2 is neuroprotective in rat OHSCs exposed to OGD. (A) PBS or E2 was administered immediately after OGD injury, and cell death was quantified 24 hours later in the CA1 region of the hippocampus using PI. * = p < 0.05, ** = p < 0.01 versus vehicle injured control. (B) Cell death was quantified 24 hours after OGD injury with vehicle or 100 pM E2 added at indicated time points after injury. * = p < 0.05, ** = p < 0.01 versus vehicle injured control. (C) Representative bright-field and PI images before injury and PI images 24 hours after injury for dose response of E2. Scale bars = 1 mm. (n >= 6) 2.3.2 Therapeutic time window of E2 after OGD

We next investigated the time window post-OGD for E2-mediated neuroprotection. E2

(100 pM) was significantly neuroprotective when administered at 1 hour (54.9±5.8%), but was not neuroprotective when administered at 2 (75.0±4.3%), 3 (80.9±2.6%), or 4 (72.8±4.9%) hours post-ischemic injury as shown in Figure 2.1 B. These results indicate that E2 neuroprotection post-OGD is time dependent and must be administered between 1 and 2 hours post injury.

2.3.3 ERα and ERβ are necessary for neuroprotection after OGD

To determine the E2 receptors necessary for neuroprotection after OGD, we used ICI

182,780 (10 µM) to inhibit ERα and ERβ and G-15 to inhibit GPR30 in combination with acute

administration of E2 (100 pM). Inhibiting the classical ERs resulted in complete abrogation of

neuroprotection of acute E2 administration, resulting in 72.3±5.6% cell death as shown in Figure

2.2. It has been suggested that GPR30 may be responsible for the acute neuroprotection afforded

by E2 after ischemia, but our data indicate no significant decrease in neuroprotection when

GPR30 was inhibited by G-15, resulting in 55.0±5.8% cell death. To further test the

involvement of GPR30 in E2 mediated neuroprotection we used the GPR30 agonist G-1.

Supporting the limited role of GPR30 in E2 neuroprotection, G-1 provided no significant

neuroprotection when administered immediately post OGD (67.0±2.7%). Furthermore,

inhibition of Akt phosphorylation (Akti-1/2, 1 µM), a downstream event of GPR30 activation, in

combination with 100 pM E2 similarly did not significantly decrease neuroprotection

(50.5±4.9%). These data suggests classical ERs are primarily responsible for acute E2 mediated

neuroprotection post-OGD in OHSCs and that GPR30 activation is not necessary or sufficient.

Figure 2.2 Inhibition of ERα and ERβ abrogated neuroprotection. PBS, 100 pM E2 and inhibitors (ICI 182,780: 10 µM, G-15: 100 nM, Akti-1/2: 1 µM), or G-1 (100 nM) were administered immediately after

OGD injury, and cell death was quantified 24 hours later in the CA1 region of the hippocampus using PI. ** = p < 0.01 versus vehicle injured control. (n >= 6) 2.3.4 E2 is neuroprotective after mechanical injury of OHSCs

For all experimental groups, a moderate injury was induced (20.1±0.1% strain,

16.8±0.2/s strain rate) that results in moderate and statistically significant cell death within the

CA1 (31.5±5.4%), CA3 (19.9±6.6%), and DG (42.8±7.0%) regions of the OHSCs compared to

uninjured controls (2.9±0.9%, 0.5±0.5%, 4.3±2.9% respectively) as measured by PI

fluorescence. Although E2 is neuroprotective in several models of TBI, it has not been

characterized in our model. Therefore, we first tested a range of concentrations of E2 after

injury. In both the CA1 and CA3 regions, E2 significantly reduced cell death after mechanical

injury compared to vehicle treated controls only at 100 pM (CA1: 13.7±4.7%, CA3: 4.1±2.4%),

but did not significantly reduce cell death at 1 nM (25.2±8.3%, 14.5±7.5%) or 10 nM

(22.9±7.0%) following TBI (Figure 2.3 A, B). E2 had no significant effect on cell death in the

DG at 100 pM, 1 nM, or 10 nM E2 (36.2±6.3%, 19.7±9.8%, 36.3±9.7%, respectively, Figure 2.3

C). We show a similar result in our OGD model and these data suggest that physiological

concentrations of E2 are more effective at reducing cell death compared to supra-physiological

concentrations after injury.

Figure 2.3 E2 reduced cell death after TBI. E2 was added at specified concentrations immediately following injury. 100 pM E2 was most neuroprotective in the (A) CA1 and (B) CA3 regions, whereas 1 nM E2 was the most neuroprotective in the (C) DG region. All n ≥ 9. 2.3.5 Neuroprotective concentrations of E2 do not activate GPR30

To investigate the involvement of GPR30 in acute E2-mediated neuroprotection, we

quantified the phosphorylation of Akt and Erk after application of E2 in OHSCs. Both Akt and

Erk are downstream targets of GPR30 and are phosphorylated when GPR30 is activated. Using

phospho-specific antibodies, we quantified the change in phosphorylation of Akt and Erk in our

OHSC model after treatment with a physiological dose of E2 (100 pM), a supra-physiological

dose of E2 (10 nM), and an agonist of GPR30 (G-1, 100 nM) by means of western blotting.

After 24 hours, we found no significant increase in Akt phosphorylation compared to vehicle

controls with the physiological concentration of E2 (97.5±14.8%) that also provided the most significant neuroprotection after both OGD and mechanical injury (Figure 2.4). However, both the supra-physiological concentration of 10 nM E2 (133.1±10.9%) and the GPR30 agonist G-1

(150.2±15.3%) produced a significant increase in Akt phosphorylation as shown in Figure 2.4 A.

As a positive control, insulin also significantly increased Akt phosphorylation (418.4±40.4%).

To further validate the activation of GPR30 at supra-physiological doses of E2, we quantified

increases in Erk phosphorylation after a 5 minute treatment with the same drugs. Again, the

physiological concentration of E2 did not increase phosphorylation of Erk compared to vehicle

24 controls (106.0±13.0%), whereas both the supra-physiological concentration of 10 nM E2

(168.4±28.5%) and G-1 (144.3±32.0%) significantly increased Erk phosphorylation as shown in

Figure 2.4 B. As a positive control, EGF also significantly increased Erk phosphorylation

(273.1±46.4%).

Figure 2.4 High concentrations of E2 lead to phosphorylation of Akt and Erk. (A) Representative western blot of phosphorylated Akt, total Akt, and associated β-tubulin expression. Quantitative analysis of western blots versus PBS (n=6/group). The expression of phosphorylated Akt was normalized to β- tubulin expression, and experimental groups were normalized to PBS-treated groups. * = p < 0.05 versus vehicle control. (B) Representative western blot of phosphorylated Erk 1/2, total Erk 1/2, and associated β-tubulin expression. Quantitative analysis of western blots versus PBS-treated group (n=5/group). The expression of phosphorylated Erk was normalized to β-tubulin expression, and experimental groups were normalized to the PBS-treated group. * = p < 0.05 versus vehicle control. 2.4 Discussion

Our findings demonstrate that a physiological level of E2 is neuroprotective after both an ischemic or mechanical injury. The difference between our study and those which have found no protection from E2 after ischemic or mechanical injury is the immediate application of a physiological dose of E2 to the affected brain tissue at a given time with no dependence on drug

delivery methods (Lebesgue, LeBold et al. 2006; Bruce-Keller, Dimayuga et al. 2007; Strom,

Theodorsson et al. 2010). Because of the short therapeutic window, E2 must be administered

quickly to provide neuroprotection. This hypothesis is supported by multiple studies which have

reported that bolus injections of E2 after an injury were neuroprotective (Simpkins, Rajakumar et

al. 1997; Yang, Shi et al. 2000; Saleh, Cribb et al. 2001; Liu, Wang et al. 2007; Day, Floyd et al.

2013).

The classical mechanism of action for estrogens occurs through ERs located within the

cytoplasm. E2 easily penetrates lipid bi-layers to reach its canonical target receptors, ERα and

ERβ (MacGregor and Jordan 1998). However, it has recently been shown that E2 can also bind a G-protein receptor, GPR30, albeit at high concentrations (Filardo, Quinn et al. 2000). The binding constant of E2 to GPR30 is approximately 6.6 nM (Revankar, Cimino et al. 2005).

Interestingly, the physiological concentrations of E2 found in the rat range from 10-400 pM, far below the binding constant of E2 to GPR30 (Herbison 2009). E2 has a wide range of effects at low, physiological concentrations which differ from the effects at saturating concentrations above 1 nM (Coser, Chesnes et al. 2003). We suggest that at least some of the differing effects of low versus high concentrations of E2 can be attributed to GPR30 activation and may be responsible for the E2 dose dependent neuroprotection shown in our study.

We found that a low, physiological concentration of 100 pM E2 was the most neuroprotective after OGD and TBI. To investigate the effect of GPR30 activation at this concentration, we used inhibitors specific to the classical receptors ERα and ERβ or GPR30 in our model of OGD. Inhibition of the classical receptors with ICI 182,780 abrogated the neuroprotective effects of E2 whereas inhibition of GPR30 with the antagonist G-15 had a non- significant effect. Direct activation of GPR30 with the agonist G-1 provided no significant

neuroprotection against OGD. Taken together, these data suggest that GPR30 was not involved

in E2-mediated neuroprotection after OGD at this concentration. This conclusion conflicts with

in vivo studies indicating that the GPR30 agonist G-1 was similarly as neuroprotective as E2 post

ischemic or mechanical injury (Lebesgue, Traub et al. 2010; Day, Floyd et al. 2013). However,

these studies cannot be directly compared to ours because we are focusing on parenchymal brain

tissue whereas the effects of GPR30 activation may extend to vasculature or other currently

unknown targets not present in our OHSCs.

The activation of the PI3K pathway and subsequent Akt phosphorylation is a well-

documented anti-apoptotic event and a downstream event of GPR30 activation (Song, Ouyang et

al. 2005). Because of its anti-apoptotic properties, increases in Akt phosphorylation have been a

speculative cause of decreased neuronal cell death after treatment with E2 or G-1 (Honda,

Sawada et al. 2000; Cimarosti, Zamin et al. 2005; Gingerich, Kim et al. 2010; Day, Floyd et al.

2013), however, our data suggests that inhibition of Akt phosphorylation has only a minor effect

on neuroprotection. Furthermore, our most neuroprotective concentration of E2 did not

significantly increase phosphorylated Akt. Previous studies have not tested the effect of direct

inhibition of Akt as opposed to inhibition through ERs to validate the requirement of Akt

phosphorylation for neuroprotection after treatment with E2 or G-1 (Honda, Sawada et al. 2000;

Cimarosti, Zamin et al. 2005; Gingerich, Kim et al. 2010; Day, Floyd et al. 2013). We also

investigated the phosphorylation of Erk, another established downstream effect of GPR30

activation (Filardo, Quinn et al. 2000; Dennis, Field et al. 2011). Physiological E2

concentrations produced no significant increase of phosphorylated Erk, further substantiating our hypothesis that GPR30 activation occurs only at supra-physiological concentrations of E2 and that GPR30 activation is not necessary for E2-mediated neuroprotection.

It is important to note that our results are based on in vitro models that lack effects due to

blood circulation, perfusion, and the blood brain barrier among others in vivo. These additional

factors may alter the therapeutic effect and time window of an in vivo E2 treatment after injury.

For example, activation of GPR30 has previously been shown to cause vasodilation, an effect

which alone may lead to better outcomes after injury (Lindsey, Carver et al. 2011). It is possible

that non-parenchymal effects of GPR30 activation may be as neuroprotective in vivo as the direct

effects of E2 on parenchymal tissue.

Several studies have suggested that GPR30 is responsible for the acute neuroprotective

effects of E2. However, our data suggests GPR30 has no direct neuroprotective effects on

parenchymal tissue. Our observations support the hypothesis that E2 is therapeutic at

physiological levels post injury and that these effects are not dependent on GPR30. However, further study is needed to elucidate the mechanism of action. A greater understanding of the therapeutic pathways activated by E2 may lead to more specific therapeutic treatments for stroke and TBI and warrants considerable attention.

3 Combination therapy of 17β-estradiol and memantine2

3.1 Introduction

The pathology of TBI is complex and dynamic with multiple secondary injury

mechanisms causing delayed cell death for hours, days, or years after the primary injury

(Margulies and Hicks 2009; Stoica and Faden 2010; Moretti, Cristofori et al. 2012). These

secondary injuries can lead to progressive neuro-degeneration, often accompanied by permanent

loss of sensory, motor and/or cognitive function. Individual drugs, or monotherapies, which

have been successful in experimental models, have failed clinically (Maas, Steyerberg et al.

1999; Narayan, Michel et al. 2002; Maas, Marmarou et al. 2007; Lingsma, Roozenbeek et al.

2011). Because of the complexity of interactions between secondary injury mechanisms, it is

likely that multiple drugs targeting several secondary injury cascades will be required to provide

neuroprotection after TBI (Margulies and Hicks 2009).

Although many monotherapies have demonstrated efficacy in animal models of TBI,

none have been clinically effective, i.e. providing a benefit to patients compared to those receiving a placebo (Narayan, Michel et al. 2002). These failures could be due to many factors including lack of understanding of the underlying injury mechanisms, inadequate pre-clinical testing in multiple injury models, or because TBI initiates several secondary pathological cascades that a single drug can not sufficiently reverse the negative outcome (Margulies and

Hicks 2009). The goal of a combination therapy is to combine two or more therapeutics with

2 A modified version of this chapter is currently submitted for print: Lamprecht, M.R. and Morrison, B. 3rd (Submitted). A combination therapy of 17β‐estradiol and memantine is more neuroprotective than monotherapies in an organotypic brain slice culture model of traumatic brain injury.

complementary or synergistic actions to increase the efficacy of TBI treatment to a level that is

clinically relevant (Faden 2002; Margulies and Hicks 2009; Stoica and Faden 2010).

E2 is the most potent naturally occurring form of estrogen and exerts a multitude of

effects after binding the estrogen receptor. After binding E2, the classical estrogen receptors

translocate to the nucleus and act as transcription factors which control the expression of many genes (Nilsson and Gustafsson 2002). E2 may also bind cell surface receptors, causing near immediate physiological effects (Bjornstrom and Sjoberg 2004; Zivadinovic, Gametchu et al.

2005). Reduced oxidative stress, anti-inflammation, improved vascular function, decreased apoptosis and attenuated excitotoxicity are all benefits associated with E2 therapy after injury

(Behl, Widmann et al. 1995; Simpson, Misso et al. 2005; Numakawa, Matsumoto et al. 2007;

Xia, Yang et al. 2007; Liu and Zhao 2013). It is also currently FDA-approved for hormone replacement therapies. Although E2 has shown efficacy as a therapeutic compound in animal models of TBI, it has not translated clinically. There is currently a clinical phase II trial,

RESCUE-TBI, testing the safety and efficacy of Premarin IV (conjugated estrogens for injection), which has not released results (Wigginton, Pepe et al. 2010).

Memantine is a non-competitive, use-dependent NMDA channel antagonist. It is FDA- approved to treat Alzheimer’s disease and is generally well tolerated. Excitotoxicity is a major secondary injury mechanism following TBI, and memantine significantly reduces cell death in models of excitotoxicity (Lipton 2006). Although memantine, like E2, has shown promise as a treatment for TBI in animal models, it has not translated clinically.

In this study, we hypothesized that a combination treatment of E2 and memantine would work synergistically to provide neuroprotection after a mechanical injury of organotypic

hippocampal slice cultures (OHSCs). Our in vitro model of TBI is well established as an

alternative to in vivo experiments and was an appropriate system to test a combination therapy,

which requires several experimental groups (Morrison, Elkin et al. 2011). A combination

treatment of E2 and memantine significantly decreased cell death compared to vehicle controls

and monotherapies in the CA1 region of the hippocampus after TBI. E2 increases NMDA

channel conductance, and we hypothesized that memantine may reduce this effect and

potentially improve neuroprotection by decreasing excitotoxic effects (Foy, Xu et al. 1999). We

show increased Schaffer-collateral evoked responses in the CA1 region after the addition of E2

and that memantine significantly reduced this potentiation. These data suggests that the

combination therapy of E2 and memantine warrants more detailed testing in vivo.

3.2 Materials and Methods

3.2.1 Organotypic hippocampal slice cultures

All animal procedures were approved by Columbia University's Institutional Animal

Care and Use Committee (New York, NY). OHSCs were cultured as previously described

(Stoppini, Buchs et al. 1991; Morrison, Pringle et al. 2002; Sundstrom, Morrison et al. 2005).

Briefly, Sprague Dawley rat pups (P8-P10) were rapidly decapitated, and the hippocampus excised and placed in ice-cold Gey’s balanced salt solution (Life Technologies, Grand Island,

NY) supplemented with D-glucose (4.5 mg/mL, Sigma-Aldrich, St. Louis, MO). 400 μm thick, transverse sections were cut using a McIlwain tissue chopper (Ted Pella, Redding, CA) and then plated on PDMS membranes (Speciality Manufacturing, Saginaw, MI) previously coated with laminin (80 µg/mL, Life Technologies) and poly-L-lysine (320 µg/mL, Sigma-Aldrich) in custom-made stainless steel wells. The cultures were initially fed Neurobasal (Life

Technologies) medium supplemented with B27 (Life Technologies), GlutaMAX (2 μM, Life

Technologies), and D-glucose (4.5 mg/mL, Sigma). Cultures were maintained under standard

culture conditions at 37°C and 5% CO2. After 3 DIV, the medium was changed to full serum

medium containing 50% MEM (Sigma), 25% heat-inactivated horse serum (Life Technologies),

25% Hanks balanced salt solution (Sigma), GlutaMAX (2 μM, Life Technologies), and D-

glucose (4.5 mg/mL, Sigma-Aldrich). Medium was changed every 2-3 days. 17β-estradiol

dipotassium salt and memantine hydrochloride (Sigma) were added at the stated time points and

concentrations.

3.2.2 In vitro model of TBI

After 10 DIV, OHSCs cultured on silicone membranes were subjected to a moderate

mechanical injury. The injury was induced by stretching the underlying silicone substrate to a

predetermined strain at a predetermined strain rate to produce the desired tissue injury under

motion-control. Our well-established model produces a highly accurate and reproducible injury

to OHSCs (Morrison, Eberwine et al. 2000; Morrison, Meaney et al. 2000; Sundstrom, Morrison

et al. 2005; Cater, Gitterman et al. 2007; Elkin and Morrison 2007; Morrison, Elkin et al. 2011).

Tissue deformation was verified by image analysis of high speed video (MotionPro, Redlake,

Pasadena, CA) at 1000 frames per second (Elkin and Morrison 2007). Lagrangian strain of the

tissue was determined by calculating the deformation gradient tensor by locating fiduciary

markers on the tissue slice before and at maximal stretch using custom MATLAB (MathWorks,

Natick, MA) scripts (Elkin and Morrison 2007).

3.2.3 Cell death assessment

Quantification of cell death in OHSC using the fluorescent vital stain, PI (Life

Technologies), has been described previously (Morrison, Pringle et al. 2002; Cater, Gitterman et

al. 2007). Briefly, micrographs were taken before the induction of injury and 4 days following

injury. The cultures were transferred to serum free medium containing 75% MEM (Sigma-

Aldrich), 25% Hanks balanced salt solution (Sigma-Aldrich), GlutaMAX (2 μM, Life

Technologies), D-glucose (4.5 mg/mL, Sigma-Aldrich) and supplemented with 5 μg/mL PI for

30 minutes before imaging. Brightfield and PI images were acquired on an Olympus IX-80

fluorescent microscope fitted with a 175 W Xenon Arc lamp (Perkin Elmer, MA, USA),

CoolSNAP ES camera (Photometrics, AZ, USA), with an excitation of 556-580 nm and an

emission of 590-630 nm (PI exposure 2 s, brightfield exposure 3 ms). Metamorph (Molecular

Devices, CA, USA) image analysis software was used to outline the CA1, CA3 and DG regions

in the brightfield image, and these regions of interest (ROI) were transferred to the PI images

taken before and after injury. Percentage cell death was determined as the area above a threshold

in the PI fluorescent image divided by the total area of the ROI (i.e. CA1 region). The threshold

was determined by the fluorescence level of uninjured, control slices imaged on the same day as

injured experimental groups. OHSCs with pre-injury cell death > 5% in the region of interest

were discarded.

3.2.4 Electrophysiology

For electrophysiological recordings, an MEA60 system (Multichannel Systems,

Reutlingen, Germany) was used. Micro-electrode arrays (MEAs) were first made hydrophilic

with gas plasma treatment and treated with nitrocellulose (Thermo Fisher Scientific, Waltham,

MA) for slice culture adhesion. Slices were transferred from Biopore CM membranes (EMD

Millipore, Billerica, MA) to the MEA and perfused with artificial cerebral spinal fluid (aCSF,

125 mM NaCl, 3.5 mM KCl, 26 mM NaHCO3, 1.2 mM KH2PO4, 1.3 mM MgCl2, 2.4 mM

CaCl2, 10 mM D-glucose, pH = 7.4, Sigma-Aldrich) at 37°C, and aerated with 95% O2/5% CO2, as previously described (Yu and Morrison 2010). Evoked responses were generated with

bipolar, biphasic stimuli (a positive phase for 100 µs followed by a negative phase for 100 µs)

applied to the Schaffer collateral axons using a programmable stimulator (STG2004,

Multichannel Systems). A stimulus-response curve was generated for each slice culture, as

previously described (Yu and Morrison 2010). The I50 stimulation current, the current which

produced a half-maximal response from the tissue, for each slice was determined with a custom

MATLAB script, and this current was used to stimulate the OHSC at 1 minute intervals. Only

recordings from electrodes in the CA1 region that exhibited a steady baseline were used for

analysis (Heuschkel, Fejtl et al. 2002). After 20 minutes of stable baseline evoked responses,

slices were perfused with aCSF containing 100 pM E2 for 20 minutes followed by 20 minutes of

aCSF containing 100 pM E2 and 10 µM memantine. Data was analyzed with custom MATLAB scripts to determine the peak-to-peak values of the evoked response. Evoked responses were normalized to the average evoked response of the final 10 minutes of baseline recordings; the normalized average of the final 10 minutes of E2 alone versus with E2 and memantine were used for statistical analysis.

3.2.5 Statistical analysis

All data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by post hoc comparisons with the Tukey-Kramer adjustment in

MATLAB (MathWorks, MA, USA); P-values less than 0.05 were deemed significant (* indicates p < 0.05).

3.3 Results

For all experimental groups, a moderate injury was induced (20.1±0.1% strain,

16.8±0.2/s strain rate) that results in moderate and statistically significant cell death within the

CA1 (31.5±5.4%), CA3 (19.9±6.6%), and DG (42.8±7.0%) regions of the OHSCs compared to

uninjured controls (2.9±0.9%, 0.5±0.5%, 4.3±2.9% respectively) as measured by PI

fluorescence. Although E2 and memantine are neuroprotective in several models of TBI, neither

has been characterized in our model. In section 2.1, we showed that E2 was significantly

neuroprotective at a concentration of 100 pM in our model of TBI. Here, we tested memantine

at 3 different concentrations post-injury.

3.3.1 Memantine after mechanical injury of OHSCs

Memantine monotherapy significantly reduced cell death when administered at 10 µM

(CA1: 8.2±1.9%, CA3: 0.2±0.1%, DG: 0.7±0.3%, Figure 3.1), the highest concentration tested,

compared to vehicle-treated controls in all regions. In the CA3 and DG region, 1 µM memantine

significantly reduced cell death (4.3±1.7%, 18.1±4.3%, respectively), but did not in the CA1

(21.0±4.3%). Memantine did not significantly reduce cell death when administered at 100 nM

(CA1: 22.±1.9%, CA3: 22.±1.9%, DG: 22.±1.9%) in any region.

Figure 3.1 Memantine reduced cell death after TBI. Compounds were added at specified concentrations immediately following injury. 10 µM memantine was the most neuroprotective concentration in all regions. All n ≥ 9. 3.3.2 Combination treatments significantly decreased cell death

In the CA1, 8 of the 9 concentration combinations significantly reduced cell death

compared to vehicle-treated, injured controls (Figure 3.2 A). Cell death was significantly

reduced after treatment with a combination of 100 nM memantine + 100 pM E2 (13.6±4.8%), 1

uM memantine + 100 pM E2 (6.9±2.6%), 10 uM memantine + 100 pM E2 (2.6±1.7%), 100 nM

memantine + 1 nM E2 (13.3±4.0%), 1 uM memantine + 1 nM E2 (6.4±3.0%), 10 uM memantine

+ 1 nM E2 (9.8±5.4%), 1 uM memantine + 10 nM E2 (10.5±4.0%), and 10 uM memantine + 10

nM E2 (8.2±2.5%). Only the combination of 100 nM memantine + 10 nM E2 (17.5±3.3%) did

not significantly reduce cell death compared to injured controls. The combination of 10 uM

memantine + 100 pM E2 was also significantly more neuroprotective than treatment at any

concentration with either E2 or memantine alone.

In the CA3, only 3 of the 9 combinations significantly reduced cell death compared to vehicle controls (Figure 3.2 B). These combinations were 10 uM memantine + 100 pM E2

(1.9±1.8%), 1 uM memantine + 10 nM E2 (0.34±0.3%), and 10 uM memantine + 10 nM E2

(0.1±0.1%). The combinations of 100 nM memantine + 100 pM E2 (2.1±1.8%), 1 uM memantine + 100 pM E2 (1.3±0.9%), 100 nM memantine + 1 nM E2 (3.3±1.3%), 100 nM memantine + 10 nM E2 (3.6±1.6%), 1 uM memantine + 1 nM E2 (3.2±3.0%), 10 uM memantine

+ 1 nM E2 (0.8±0.6%) did not significantly reduce cell death.

All 9 drug combinations significantly reduced cell death compared to vehicle controls in the DG (Figure 3.2 C). Cell death was significantly reduced after treatment with a combination of 100 nM memantine + 100 pM E2 (11.3±7.9%), 1 uM memantine + 100 pM E2 (8.1±5.3%), 10 uM memantine + 100 pM E2 (3.8±3.6%), 100 nM memantine + 1 nM E2 (6.8±3.9%), 1 uM memantine + 1 nM E2 (2.5±1.2%), 10 uM memantine + 1 nM E2 (1.8±0.9%), 100 nM memantine + 10 nM E2 (5.9±1.7%), 1 uM memantine + 10 nM E2 (5.1±3.3%), and 10 uM memantine + 10 nM E2 (0.2±0.1%).

Figure 3.2 Combination therapies were neurprotective. Several of the combination therapies were significantly more neuroprotective compared to vehicle treatment. The combination of 100 pM E2 and 10 µM memantine was significantly more neuroprotective than either monotherapy for the CA1 region. All n ≥ 8. # = p < 0.05 versus 100 pM E2 monotherapy. & = p < 0.05 versus 10 µM memantine monotherapy. The solid line represents the vehicle mean cell death for all regions, large dashes represent the 100 pM E2

38 mean cell death for CA1, CA3 and 1 nM E2 for DG, and the small dashes represent the 10 µM memantine mean cell death for all regions. 3.3.3 Delayed treatment with combination therapy

The most effective combination treatment (100 pM E2 + 10 µM memantine) was tested

for its efficacy when administered at 1, 2, or 3-hours post injury. Because full media changes

were used to administer the combination therapy at each time-point, time-matched vehicle

controls were also quantified. There was no significant change in cell death between vehicle

treated controls within a given region (CA1: 37.0±4.5%, CA3: 12.7±2.9%, DG: 26.0±4.5%), and

they were combined for statistical analysis. At 1 hour post injury, the combination treatment

significantly reduced cell death (CA1: 18.6±4.5%, CA3: 0.3±0.1%, DG: 2.9±1.2%), whereas

treatment at 2 or 3 hours post injury did not significantly reduce cell death (CA1: 38.5±5.9%,

CA3: 10.1±5.1%, DG: 24.9±9.6% and CA1: 51.6±9.8%, CA3: 23.8±12.4%, DG: 41.2±10.7%

respectively, Figure 3.3 A-C) in all regions.

Figure 3.3 A combination therapy was neuroprotective 1 hour post injury. Vehicle-treated injured cultures for all timepoints were not significantly different and were combined on a per-region basis. All n ≥ 6. 3.3.4 Memantine significantly reduced evoked responses

After 20 minutes of perfusion with 100 pM E2, the amplitude of evoked responses in the

CA1 region increased by 29.4±5.5% (Figure 3.4 A) above the baseline amplitude. After

perfusion of E2 alone, the addition of memantine to the aCSF in the continued presence of E2

significantly decreased the evoked responses to 7.6±2.9% above the baseline amplitude (Figure

3.4 B, C).

Figure 3.4 Memantine reduced E2-induced potentiation. (A) An OHSC on an MEA. The oval indicates CA1 electrodes which were included in analysis. White stars represent Schaffer collateral stimulating electrodes. (B) A representative trace from a single electrode after (1) 20 minutes of 100 pM E2 potentiation and (2) 20 minutes of 100 pM E2 + 10 µM memantine (offset by 50 ms for clarity). (C) Memantine significantly reduced E2 induced potentiation of evoked response amplitude.

3.4 Discussion

After TBI, the primary mechanical stimulus initiates a cascade of biological events

leading to secondary injuries (Robertson 2004; Margulies and Hicks 2009). Cell death via

secondary injuries can continue for hours, days, or years after the primary injury (Margulies and

Hicks 2009; Stoica and Faden 2010; Moretti, Cristofori et al. 2012). These ongoing cascades

leading to delayed cell death provide a potential temporal window for therapeutic intervention to

slow, stop, or repair damaged cells otherwise destined to die and may contribute to improved

survival and decreased long-term disabilities.

Although intense research in to TBI therapeutics continues, no clinically relevant

monotherapy treatments have emerged to abrogate delayed cell death and improve outcome

(Margulies and Hicks 2009). Preclinical studies have provided many potential monotherapies

that have shown benefit after TBI in experimental models. One explanation for a lack of clinical

translation may be that monotherapy treatments focus primarily on one aspect of a very complex

injury and may be too limited to show clinical efficacy. For example, some of the secondary

injuries which occur after TBI include ischemia, hypoxia, edema, excitotoxicity, inflammation,

and free radical formation, among others (Haddad and Arabi 2012).

Combination treatments are a promising alternative to monotherapies for the treatment of

TBI (Margulies and Hicks 2009). A similar approach has proven successful in other diseases

such as AIDS for which a 65% decrease in HIV/AIDS related deaths occurred between 1995 and

1998 with the introduction of combination therapies (Hall, Song et al. 2008). In 2008, a

workshop organized by the National Institute of Neurological Disorders and Stroke brought

together clinicians and scientists from a variety of disciplines to identify promising combination

treatments for TBI (Margulies and Hicks 2009). It was recommended that potential combination

therapies should “combine agents with complementary targets and effects (e.g., mechanisms and

time-points), rather than focusing on a single target with multiple agents.” To that end, we chose

to test two FDA-approved drugs with very different physiological effects, but that may have

complementary mechanisms of action to produce a synergistic effect.

3.4.1 Estrogen as a monotherapy

Estrogen is an important steroid hormone that has also been implicated in many

physiological and psychological changes. The most potent and naturally occurring form of

estrogen, E2, has been investigated as a neuroprotective therapy for TBI (Brown, Suzuki et al.

2009; Zlotnik, Leibowitz et al. 2012; Day, Floyd et al. 2013). An early study by Emerson, et al,

reported improved biochemical and cognitive outcomes with post-TBI (FPI) injection of E2

(Emerson, Headrick et al. 1993). Neuroprotection was dependent on gender, and only male rats

significantly improved post injury. Similarly, Roof and Hall found that 100% of female rats

compared to 75% of male rats survived an impact-acceleration closed-head injury and suggested

that endogenous estrogen may have been neuroprotective (Roof and Hall 2000). In addition to

TBI, E2 is well known to be neuroprotective in models of ischemia (Simpkins, Rajakumar et al.

1997; Cimarosti, Zamin et al. 2005; Liu, Wang et al. 2007; Simpkins and Singh 2008; Lamprecht

and Morrison 2014). In our hands, E2 was neuroprotective only at a low, physiological dose of

100 pM and only in the CA1 and CA3 regions as show in section 2.1. There was no reduction in

cell death in the DG with E2 alone.

3.4.2 Memantine as a monotherapy

Memantine is an FDA-approved, uncompetitive NMDA antagonist, which is prescribed

primarily for Alzheimer’s disease, but is also used to treat Parkinson’s disease and senile

dementia (Mathys, McCarrell et al. 2013; Schneider 2013). It is a low affinity, use-dependent inhibitor that does not affect learning and memory formation through LTP as other, more potent

NMDA inhibitors can (Frankiewicz, Potier et al. 1996; Parsons, Danysz et al. 1999). Because its

antagonism is use-dependent, its effects are greater when higher concentrations of receptor

agonists are present, which may target the drug’s actions to damaged areas experiencing

excitotoxicity. These properties alleviate the deleterious side effects of other NMDA antagonists

such as MK-801, which produce hallucinations and psychosis (Koek, Woods et al. 1988). As an

NMDA antagonist, memantine’s primary function is to reduce calcium influx and decrease

neuronal excitability, making it a potent inhibitor of excitotoxicity (Volbracht, van Beek et al.

2006). The neuroprotective effects of NMDA antagonists including MK-801 and memantine

have been well documented in many different models of CNS injury over the past 20 years

(Tasker, Coyle et al. 1992; Vornov, Tasker et al. 1994). We found memantine was

neuroprotective at the highest dose tested (10 µM) in the CA1, CA3 and DG.

3.4.3 Potential synergistic effects of E2 and memantine

We found that many of the combination treatments of E2 and memantine resulted in a

significant reduction in cell death compared to vehicle controls. Of particular interest, some

combinations of drug concentrations that were not neuroprotective by themselves became

significantly neuroprotective when combined (e.g. 100 nM memantine + 10 nM E2 in all

regions). One combination significantly reduced cell death in the CA1 region more than either

of the most effective monotherapies (100 pM E2 + 10 µM memantine). These data suggest a

potentially synergistic effect between the two drugs that does not occur when either is used as a

monotherapy. We suggest that memantine may reduce potentially negative effects of E2.

E2 induces numerous downstream events. While many studies have confirmed that E2 is

neuroprotective, a few have also noted potentially negative side effects of treatment. Some of these potentially negative side effects may include increased oxidative stress, increased inflammation, and increased excitotoxicity (Strom, Theodorsson et al. 2011). Short term treatment with E2 has been shown to increase EPSPs and enhance LTP through NMDA receptors (Foy, Xu et al. 1999). It is possible that increased potentiation due to E2 may exacerbate injury through the well-established excitotoxicity cascades that are a major secondary injury mechanisms after TBI.

In particular, the conductance of NR2B containing NMDA receptors was increased after

E2 exposure, and this effect was due to the recruitment of NR2B subunits to synapses (Snyder,

Cooke et al. 2011). Although previous studies examined these effects after 72 hours of E2 exposure in vivo, Foy et al have shown near immediate (<10 minutes) potentiation of both EPSPs and LTP after E2 exposure, with increases of 180% and 177%, respectively (Foy, Xu et al.

1999). These studies were conducted in experimental conditions of low Mg2+ and DNQX to

isolate NMDA receptors as much as possible.

NR2B subunit-specific inhibitors tend to have a better therapeutic profile in many animal

models when compared to traditional NMDA antagonists such as MK-801 (Parsons, Danysz et

al. 1998). In particular, previous studies of ifenprodil and eliprodil, both NR2B specific

inhibitors, significantly reduced NMDA-induced release of acetylcholine. Memantine had a similar effect whereas the more potent NMDAR inhibitor, MK-801, was less effective. This data suggests that memantine may be more selective at inhibiting NR2B-containing compared to

NR2A-containing NMDARs (Parsons, Danysz et al. 1999).

Because of the direct link between increased NR2B conductances after E2 exposure, we

chose memantine for a combination therapy for its potential to reduce potentiation caused by E2

after injury, as well as its previous success in animal models of TBI. We found that memantine

significantly reduced E2 induced potentiation, supporting our suggested mechanism. However,

further testing is necessary to confirm if E2 does, in fact, increase excitotoxicity.

3.4.4 Combination treatment of TBI

Our study is the first to show that the combination of E2 and memantine significantly

reduced cell death in a model of TBI. We have also shown that this neuroprotection is

significantly more effective than either E2 or memantine alone and have proposed and tested a

potential mechanism for this synergy. This is a promising result for a potentially clinically

relevant therapy for TBI. It is important to note that both E2 and memantine are currently FDA

approved drugs, which makes the combination treatment a candidate to be fast tracked for

clinical trial approval.

Although the combination of E2 and memantine reduced cell death in our model of TBI,

it has yet to be tested in vivo and faces many challenges before clinical testing. The most

neuroprotective combination of E2 and memantine significantly reduced cell death up to 1 hour

post injury, which may limit its usefulness in the clinic. Because of the time necessary for

transport to the hospital, triage wait times, and informed consent issues, it is difficult to enter

patients in to clinical trials before 6 hours (Faden 2002). Our in vitro model of TBI lacks effects

due to blood circulation, perfusion, and the blood brain barrier among others in vivo. Testing the

combination in higher order animals of different age and sex as well as further understanding the

mechanistic link between E2 and memantine neuroprotection are also needed. The

pharmacological consequences of combining drugs are an important and difficult hurdle that

requires detailed pharmacokinetic studies. These studies should optimize the dose, route of

administration, timing, and evaluate the mechanism of action. Nevertheless, our study suggests a

potential combination therapy for the acute treatment of TBI.

4 Global gene expression analysis of an in vitro and in vivo

model of TBI3

4.1 Introduction

Every year, in the United States alone, there are approximately 1.7 million traumatic

brain injuries (TBI) responsible for an estimated $76.5 billion in losses due to direct medical

costs and lost productivity (Coronado, Xu et al. 2011; Roger, Go et al. 2011). Unfortunately,

despite the tremendous societal and economic cost and decades of research, current

pharmacological treatments for TBI are lacking, and TBI remains a leading cause of long term

disability (Narayan, Michel et al. 2002; Willis, Lybrand et al. 2004; Taussky, Tawk et al. 2011;

Kelley and Martin-Schild 2012). It is estimated that 75% of TBIs are due to concussions or other

forms of mild TBI, and a growing body of research on chronic traumatic encephalopathy has revealed serious and long term detrimental effects of mild brain injuries (Gavett, Stern et al.

2011; Baugh, Robbins et al. 2014).

The primary mechanical event sets in motion a myriad of secondary injury cascades that cause delayed cell death that may continue for days or longer (Stoica and Faden 2010; Moretti,

Cristofori et al. 2012). Effective clinical treatments for TBI have been elusive, perhaps because

of this complex pathophysiology. A better understanding of the secondary mechanisms leading to delayed cell death and dysfunction after TBI may support the discovery and development of

novel and clinically viable treatments. To this end, several in vivo and in vitro models have been

developed to investigate TBI (Morrison, Elkin et al. 2011; Gold, Su et al. 2013). Both in vitro

3 A modified version of this chapter is currently being prepared for print: Lamprecht M.R., Elkin B.S., Kesavabhotla K., Crary J.F., Raghupathi R., Morrison B. 3rd. Correlation of genome‐wide expression after traumatic brain injury in vitro and in vivo implicates a role for SORLA.

and in vivo models have respective strengths and weaknesses, but ultimately the utility of in vitro

models depends on their ability to reproduce the in vivo TBI cascade (Morrison, Elkin et al.

2011).

The primary motivation of this study was to determine the degree of transcriptomic similarity between an in vivo and an in vitro model of TBI. Global gene expression changes

likely coincide with the activation of different molecular pathways as the secondary injury

process unfolds. Therefore, a high degree of correlation between the models would support the

utility of in vitro models as research tools to complement in vivo models.

With the advent of DNA/RNA microarrays, it is now possible to quantify global changes

in gene expression (Fodor, Rava et al. 1993). This powerful method eliminates the need to

choose a specific gene of interest and to produce the necessary tools (e.g. primers) for its study

but instead allows for thousands of genes to be analyzed simultaneously. From these data, entire

physiological pathways can be studied, which may lead to further insight in to injury

mechanisms.

Previous studies of TBI have shed light on new genes and pathways of interest but many

of these studies focused on a single injury model and a single time point after injury (Morrison,

Eberwine et al. 2000; Poulsen, Penkowa et al. 2005; Di Pietro, Amin et al. 2010). Because of the

complex nature of the post-traumatic pathology, temporal changes in gene expression may

provide more insight than from a single time point. For example, temporal changes in gene

expression have been studied for over a decade and have been integral in understanding

physiological changes during circadian rhythm cycles (Hazen, Borevitz et al. 2005; Menger, Lu

et al. 2005; Leek, Monsen et al. 2006; Aryee, Gutierrez-Pabello et al. 2009). A temporal analysis

could provide insight to the progression of a disease or pathology and could identify novel

targets for therapies which would be overlooked with analysis at a single time point.

Our in vitro model, which has previously been characterized, utilized OHSCs that were

subjected to an equi-biaxial deformation, which was verified by high-speed video analysis

(Morrison, Meaney et al. 2000; Yu, Tsay et al. 2006; Cater, Gitterman et al. 2007; Elkin and

Morrison 2007; Morrison, Elkin et al. 2011). To serve as a comparison to the in vitro data, a

group of animals was subjected to a closed-head injury using a pneumatically driven piston

(Raghupathi and Huh 2007; Huh, Widing et al. 2008; DiLeonardi, Huh et al. 2009). Changes in

gene expression between the two models were highly correlated at similar time points after

injury. From these data, we identified a transmembrane protein SORLA (sorting protein-related

receptor with A-type repeats) that was significantly reduced after TBI, and confirmed its reduced

expression with both Western blotting and immunohistochemistry in our in vitro model.

Interestingly, SORLA has been implicated in Alzheimer’s disease (AD), and mutations that

reduce SORLA’s ability to function properly may lead to late onset AD by decreasing amyloid

beta (Aβ) clearance possibly linking TBI as a risk factor for AD (Buggia-Prevot and Thinakaran

2014; Caglayan, Takagi-Niidome et al. 2014). In conclusion, our study confirms a strong

correlation of the pathobiology between our in vitro model and that of an in vivo model, further

supporting its utility as a research tool for the discovery of potential therapeutic targets after TBI.

4.2 Materials and Methods

4.2.1 Organotypic hippocampal slice cultures

Animal procedures to generate OHSCs were approved by Columbia University's IACUC.

OHSCs were cultured as previously described (Stoppini, Buchs et al. 1991; Morrison, Pringle et

al. 2002; Sundstrom, Morrison et al. 2005). Briefly, Sprague Dawley rat pups (post natal day

[PND] 8-10) were rapidly decapitated, and the hippocampus excised and placed in ice-cold

Gey’s balanced salt solution (Life Technologies, Grand Island, NY) supplemented with D-

glucose (4.5 mg/mL, Sigma-Aldrich, St. Louis, MO). 400 μm thick, transverse sections were cut

using a McIlwain tissue chopper (Ted Pella, Redding, CA) and then plated on

polydimethylsiloxane membranes (Speciality Manufacturing, Saginaw, MI) previously coated

with laminin (80 µg/mL, Life Technologies) and poly-L-lysine (320 µg/mL, Sigma-Aldrich) in

custom-made stainless steel wells. The cultures were initially fed Neurobasal (Life

Technologies) medium supplemented with B27 (Life Technologies), GlutaMAX (2 μM, Life

Technologies), and D-glucose (4.5 mg/mL, Sigma-Aldrich). Cultures were maintained under standard culture conditions at 37°C and 5% CO2. After 3 DIV, the medium was changed to full

serum medium containing 50% MEM (Sigma-Aldrich), 25% heat-inactivated horse serum (Life

Technologies), 25% Hanks balanced salt solution (Sigma-Aldrich), GlutaMAX (2 μM, Life

Technologies), and D-glucose (4.5 mg/mL, Sigma-Aldrich). Medium was changed every 2-3

days.

4.2.2 In vitro model of TBI

After 10 DIV, OHSCs cultured on silicone membranes were subjected to a moderate

mechanical injury. The injury was induced by stretching the underlying silicone substrate to a

predetermined strain at a predetermined strain rate to produce the desired tissue injury under

motion-control. Our well-established model produces a highly accurate and reproducible injury

to OHSCs (Sundstrom, Morrison et al. 2005; Cater, Gitterman et al. 2007; Morrison, Elkin et al.

2011). Tissue deformation was verified by image analysis of high speed video (MotionPro,

Redlake, Pasadena, CA) at 1000 frames per second (Elkin and Morrison 2007). Lagrangian

strain of the tissue was determined by calculating the deformation gradient tensor by locating

fiduciary markers on the tissue slice before and at maximal stretch using custom MATLAB

(MathWorks, Natick, MA) scripts (Elkin and Morrison 2007).

4.2.3 In vivo model of TBI

Animal procedures for closed-head TBI were approved by Drexel University’s IACUC

and were in compliance with the Guide for the Care and Use of Animals. Brain injuries were

induced using a pneumatically driven controlled cortical impact device (AmScien Incorporated,

Richmond, VA) as described by Dixon et al. Briefly, rat pups (PND11) were anesthetized with

isofluorane (2%) using a nose cone, and once a loss of a tail-pinch reflex was observed, a

midline incision was made to expose the skull. The periosteum was reflected, the animal was

placed in a restrainer, and the head was supported by a soft foam pad to make it level with the

body. The restrainer was positioned under the cortical impact device, the nose cone was

removed, and the zero-point for the indenter was made on the skull over the left parietal cortex,

midway between the lambda and bregma sutures. The exposed skull was subjected to a 5 mm

diameter piston impact of 2.0 mm depth, 5 m/s velocity, and 100 ms duration for moderate

injury. Sham-injured animals underwent the same procedures, except the piston was not

activated. The total time from initiation of anesthesia to removal of the nose cone prior to

zeroing the impactor tip was typically 5–6 min. Following recovery from injury/anesthesia, the

animals were re-anesthetized with isofluorane for the scalp to be sutured, and the pups were

returned to the dam. Animals were placed on a heating pad maintained at 37°C to maintain body

temperature throughout the procedures and recovery.

4.2.4 Affymetrix gene chips

OHSC samples, including time-matched controls, were collected after the in vitro model

of TBI at 1, 6, 12, and 24 hours post injury. Whole hippocampal samples, including time-

matched controls, were collected after the in vivo model of TBI at 1, 6, and 24 hour post injury.

Tissue was collected, flash frozen, and stored at -80C until total RNA was isolated using an

RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocols. The quality of

RNA was assessed using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA). Purified

RNA was used to synthesize cDNA using the HT One-Cycle cDNA Synthesis Kit (Affymetrix,

Santa Clara, CA) following the manufacturer’s protocols. Amplified cRNA was produced from the cDNA template using the 3’ IVT Labeling Kit (Affymetrix). The cRNA was then hybridized

to the Affymetrix Rat 230 2.0 chips, washed, and scanned at the Columbia Genome Center

according to the manufacturer’s protocols.

4.2.5 Microarray data analysis

Using custom MATLAB scripts, each array was normalized for variations in background

using the robust Multi-array Average (RMA) procedure.(Irizarry, Hobbs et al. 2003). After

performing a Student’s t-test between experimental groups on an individual gene basis, the p-

values were adjusted for false discovery rate (FDR) using the Storey-Tibshirani method to improve confidence in the p-values (Storey and Tibshirani 2003). An FDR corrected p-value <

0.05 and a two-fold biological change in expression were chosen as criteria for identifying genes whose expression was altered both statistically and biologically. Analysis of covariance

(ANCOVA) was used to determine whether gene expression over time was significantly different between experimentally injured groups and non-injured controls. An ANCOVA p- value < 0.05 was considered significant. To enrich for the most significantly changed genes in

our dataset for further analysis, we focused on genes which were significant in both the Student’s

t-test and ANCOVA.

To determine the correlation between the two injury models, the Pearson correlation

coefficient was determined for the 323 genes found to be significant in both the in vitro and in

vivo models by the Student’s t-test.

Using DAVID Bioinformatics Resources provided by the NIH, each time point was

analyzed for the most enriched gene ontology (GO) terms. We chose to focus on biological

process terms and have excluded cellular component and molecular function GO terms from

analysis. Specifically, GO FAT biological process terms as described by DAVID were used for

analysis. The GO FAT subset excludes the broadest GO terms so they do not overshadow more

specific terms.

4.2.6 Immunohistochemistry

All steps were performed with a Ventana Benchmark Ultra automated staining instrument

(Ventana Medical Systems, Tucson, AZ). Formalin-fixed, paraffin-embedded tissue samples were cut at 6 um. Using the automated Ventana equipment, paraffin was removed at 72°C, and antigen retrieval was achieved by incubation with CC1 reagent (Ventana Medical Systems) at

95°C. Following antigen-retrieval, slides were incubated with anti-SORL1 (Sigma, S9200,

1:1000) for 32 minutes before being washed. Tissue nuclei were counter-stained using hematoxylin. Slides were then dehydrated using gradient alcohols and coverslipped.

4.2.7 Western blots

Additional samples were collected to confirm the transcriptional results at the protein level. For in vitro injured and uninjured controls, 9 slices were collected for protein extraction.

Slices were rinsed twice with ice-cold PBS and immediately placed in lysis buffer A (40 mM

HEPES, 120 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM sodium pyrophosphate, 50 mM

sodium fluoride, 0.5 mM sodium orthovanadate, 10 mM β-glycerophosphate, Sigma). Samples

were sonicated (Sonicator 3000, Misonix, NY, USA), incubated on ice, and then centrifuged to

remove cell debris. Approximately 50 µg of protein per sample was loaded in a 4-12% Bis-Tris

gel (Life Technologies), and proteins were separated by electrophoresis (150 V, 1 hour).

Proteins were then transferred to a nitrocellulose membrane (Life Technologies) using a semi-

dry apparatus (Thermo Fisher Scientific). After transfer, the membrane was blocked in Tris-

buffered saline (TBS, pH 7.4) with 5% bovine serum albumin (BSA) for 2 hours. Membranes

were incubated overnight at 4ºC with primary antibodies (Anti-SORL1 [Sigma, S9200], Anti-β-

tubulin to serve as an internal control [Life Technologies, 32-2600]) in TBS-T (0.1% Tween-20,

pH 7.4) and 5% BSA. Following the primary antibody incubation, membranes were washed

3x10 minutes in TBS-T. For detection, the membranes were labeled with a corresponding

secondary antibody (goat-anti rabbit Alexa Fluor 546 or goat-anti mouse Alexa Fluor 647, Life

Technologies). Fluorescence was detected using a CRi Maestro 2 in vivo Imaging System

(Perkin Elmer, Akron, OH). Grayscale images were analyzed in ImageJ using the gel analysis

function. SORLA band intensities were normalized by the intensity of the β-tubulin band.

4.3 Results

4.3.1 Comparison of in vitro and in vivo TBI models

Across all time points, a total of 2073 unique genes were differentially expressed in at least 1 time point after in vitro TBI, and a total of 877 genes were differentially expressed in at least 1 time point after in vivo TBI compared to uninjured controls. After in vitro TBI, 988, 866,

861, and 374 genes were significantly differentially expressed at 1, 6, 12, and 24 hours after

injury, respectively, compared to uninjured controls. For the in vivo TBI model, 312, 445, and

308 genes were significantly differentially expressed at 1, 6, and 24 hours after injury,

respectively, compared to uninjured controls. There were a large number of differentially

expressed genes found at multiple time points for both models of injury, and this overlap is

shown in Figure 4.1 A-B. Comparing the overlap between the two models, 323 genes were

differentially expressed in both models of injury as shown in Figure 4.1 C. In total, the in vitro

model replicated about 37% of the changes in gene expression of the in vivo model of TBI,

although almost three times as many genes were significantly regulated in the in vitro model.

We next investigated how correlated gene expression was between the two models of

TBI for the 323 differentially expressed genes common to both the in vivo and in vitro models.

The comparison of fold changes is graphed in Figure 4.2. There was a strong correlation in gene

expression between the in vitro and in vivo models at matching time points (1 hour: r = 0.54, 6

hour: r = 0.64, 24 hour: r = 0.59). Within the in vitro injury group, there were very strong

correlations between the 6 and 12 hour time points (r = 0.89), and strong correlations between

the 6 and 24 hour time points (r = 0.67) and the 12 and 24 hour time points (r = 0.62). This trend

was similar for the in vivo injury between the 6 and 24 hour time points (r = 0.85). A moderately negative correlation between the 1 hour time point and all later time points within a given model

(Figure 4.2) suggests an abundance of genes were initially differentially expressed post injury

and returned to baseline over time.

For an overall comparison of our in vitro to in vivo TBI models, we combined the gene

expression fold changes for the 37% of genes which were significantly regulated in both models

at identical time points after injury: 1 hour in vitro vs. 1 hour in vivo, 6 hour in vitro vs. 6 hour in

vivo, and 24 hour in vitro vs. 24 hour in vivo. Gene expression fold changes for the time-point

57 matched analysis had a combined Pearson correlation r = 0.69. This strong correlation gives us confidence that our in vitro model is representative of in vivo TBI.

Figure 4.1 Overview of significant changes in gene expression following TBI. Venn diagrams show the number of overlapping genes whose expression was significantly altered compared to time-matched controls for (A) in vitro TBI, (B) in vivo TBI, and (C) in vitro vs. in vivo TBI.

Figure 4.2 Correlation of gene expression changes after TBI. Scatter plots of log2 fold changes of the 323 genes that were significantly and differentially expressed in both models. Pearson correlation coefficients (r) are located in the top left corner. Axes represent log2 fold change from -5 to 5. Dashed line encloses in vitro versus in vivo comparisons. Both models were strongly correlated, particularly at matching time points.

4.3.2 Gene ontology (GO) analysis

After analysis for the most significantly enriched GO terms in our dataset, TBI-relevant

GO terms were chosen, and the number of genes within a GO term for in vitro and in vivo injuries were graphed (Figure 4.3). Several TBI relevant GO terms contain significantly differentially expressed genes after both injury models, and furthermore, many of the genes in these relevant groups were identical as indicated in Figure 4.3.

Figure 4.3 GO analysis of significantly altered gene expression after TBI. The number of significantly altered genes from each model within the specified GO groups is graphed. The vertical black bar represents the number of identical genes which were significantly regulated in both models within the specified GO group. TBI-relevant GO groups were remarkably similar between the in vitro and in vivo models, and many identical genes were altered in both models.

4.3.3 ANCOVA analysis of in vitro TBI

The comparison of significant genes between the ANCOVA analysis and Student’s t-test

yielded 32 genes representing changes of particular interest (Table 4.1). These genes are listed

in Table 1. One of these genes, SORLA, is predominately expressed in the central nervous

system and is predictive of late onset AD, and was further validated with western blot and

immunohistochemistry (Buggia-Prevot and Thinakaran 2014; Caglayan, Takagi-Niidome et al.

2014).

Table 4.1 Enriched gene list after ANCOVA and Student's t-test analysis.

Affymetrix ID Gene Name ANCOVA p‐value solute carrier organic anion 1380783_at transporter family, member 5A1 0.01050155 1368261_at neurexin 3 0.036895826 sortilin‐related receptor, LDLR class A 1393933_at repeats‐containing 0.012764344 1368290_at cysteine‐rich, angiogenic inducer, 61 0.038822208 1394095_at gypsy retrotransposon integrase 1 0.028431777 acyltransferase like 1B; similar to H43E16.1; BTB (POZ) domain 1373179_at containing 8 0.042848911 1376574_at CD93 molecule 0.016903661 1384144_at nucleoredoxin 0.047020786 ganglioside‐induced differentiation‐ 1394286_at associated protein 1‐like 1 0.038374163 nuclear receptor subfamily 4, group 1369217_at A, member 3 0.003272821 small glutamine‐rich tetratricopeptide repeat (TPR)‐ 1377920_at containing, beta 0.022492303 1385036_at synuclein, alpha interacting protein 0.011087624 1391146_at cadherin 11 0.010498705 ras‐related C3 botulinum toxin 1388332_at substrate 1 0.022143913 solute carrier family 30 (zinc 1383632_at transporter), member 1 0.006240913 isopentenyl‐diphosphate delta 1388872_at isomerase 1 0.04378311 1389280_at tubby like protein 1 0.014260733 BTB (POZ) domain containing 10; similar to K+ channel tetramerization 1396485_at protein 0.03527607 DEAH (Asp‐Glu‐Ala‐His) box 1379101_at polypeptide 36 0.019789344 guanine nucleotide binding protein (G protein), beta polypeptide 1; guanine nucleotide binding protein 1371071_at (G protein), beta polypeptide 4 0.029507691 family with sequence similarity 84, 1390176_at member B 0.002971343 1391807_at transcription factor CP2 0.039669544 1393725_at desmoglein 1 beta 0.035248779 1378205_at 0.028691264 1379531_at 0.020121219

1386059_at 0.032877844 1376458_at 0.038650084 1374022_at 0.041078106 1396050_at 0.047522184 1390240_at 0.012810421 1374713_at 0.012079601 1390425_at 0.02633515

4.3.4 Protein expression of SORLA

Although SORLA is widely expressed in the brain, it is enriched in the neurons of the

hippocampus (Motoi, Aizawa et al. 1999). Cytoplasmic puncta were observed in the CA1

pyramidal neurons of uninjured controls (Figure 4.4 A-B). Interestingly, after mechanical injury of the OHSC, these puncta were reduced or completely missing from the cytoplasm of pyramidal neurons (Figure 4.4 C-D). Injured regions also exhibited a vacuolar appearance, suggesting cell death. These data further support our gene chip data, corroborating reduced SORLA expression after injury. To further confirm this claim, we measured protein expression of SORLA via

Western blotting after TBI. We found that SORLA expression was significantly reduced by 44% compared to uninjured control OHSCs (p < 0.05, Figure 4.4 E-F).

Figure 4.4 SORLA expression after in vitro injury. Micrographs of SORLA staining in uninjured (A: 4X, B: 40X) and moderately injured (C: 4X, D: 40X) OHSCs. An overall decrease in SORLA staining was observed after injury, and distinct SORLA staining puncta were missing from the apical dendrites. (E) Normalized SORLA protein expression after in vitro injury of OHSCs was significantly decreased after injury. (F) A representative Western blot of SORLA and β-tubulin.

4.4 Discussion

TBI is a major public health concern which affects approximately 1.7 million American’s

every year (Coronado, Xu et al. 2011). The dearth of treatment options for TBI patients is a

point of concern, and further research is necessary to address this issue. In vitro models of TBI

have the advantage of being higher throughput than in vivo methods, but their utility depends on

their ability to replicate the in vivo injury cascade(s). To that end, this study compared the global

changes in gene expression after TBI in both an in vitro and an in vivo model. Our in vitro

model of TBI utilized organotypic brain slice cultures (OHSCs) derived from PND 8-10 animals

that maintain the cellular diversity and structure of the hippocampal structure in vivo (Stoppini,

Buchs et al. 1991; Gahwiler, Capogna et al. 1997). Our custom built injury device delivered a specified equi-biaxial strain to the tissue, similar to what would occur in vivo during a diffuse closed-head TBI (Morrison, Meaney et al. 2000; Cater, Gitterman et al. 2007; Morrison, Elkin et al. 2011). Therefore, we chose a well-established closed-head injury model using PND 11 rat pups for comparison (Raghupathi and Huh 2007; DiLeonardi, Huh et al. 2009). Time points were chosen to investigate the near immediate cellular response (1 hour) which may lead to downstream cascades at later time points (6, 12 hours) and potential stabilization and recovery

(24 hours).

Some of the significant gene expression changes in our current study have been identified previously either in genone-wide or single-gene studies (Morrison, Eberwine et al. 2000;

Matzilevich, Rall et al. 2002; Poulsen, Penkowa et al. 2005; Ishikawa, Uchino et al. 2006; Di

Pietro, Amin et al. 2010; Barr, Alexander et al. 2011; Di Pietro, Amorini et al. 2013; White, Ford et al. 2013). Di Pietro et al., using the same in vitro model as ours, investigated mild (10%) and severe (50%) injuries in OHSCs at 24 hours post injury and found significant changes in

expression of inflammatory and apoptotic genes such as Cox-2, Cxcl2, and Mmp2 (Di Pietro,

Amin et al. 2010). Interestingly, they found more significant gene expression changes in mild

TBI than severe at the 24 hour time point and suggest this may explain neuronal dysfunction after mild TBI. However, we found 603 significant changes in gene expression at 1 hour which were not significantly altered at any other time point after injury. A 24 hour time point provides a single snapshot of gene expression changes, and our data suggests that this time point may miss many gene expression changes after injury. It is possible that, in their model, the severe injury may induce significant gene expression changes at an earlier time point than mild TBI, which would be overlooked by focusing on a single time point. Both Crack et al. and Poulsen et al. used an in vivo TBI injury and analyzed gene expression changes at multiple time points after injury (Poulsen, Penkowa et al. 2005; Crack, Gould et al. 2009). However, no temporal analysis method was employed; instead analysis focused on gene expression changes at individual time points. In both studies, similar inflammatory genes were found to be differentially expressed. A re-analysis of their data sets, taking into account the temporal aspects could reveal interesting and statistically significant changes that would otherwise be missed. White et al. analyzed gene

expression changes 24 hours after TBI in rats (White, Ford et al. 2013). They compared

ipsilateral to contralateral tissue and found significant changes in inflammatory genes. The

authors of this study acknowledged the temporal limitation of their study, and suggested that a

larger study investigating many time points would be necessary to track changes in gene

expression. To our knowledge, ours is the first study of global gene expression after TBI which

takes advantage of multiple time points to further enrich our conclusions and provide novel

targets for future study.

4.4.1 Comparison of in vitro and in vivo TBI models

Gene expression between the two models was strongly and positively correlated 1 hour

after injury. Interestingly, these initial changes in gene expression were moderately negatively

correlated with later changes in gene expression suggesting a transient burst of expression

followed by a return to baseline reminiscent of post-traumatic immediate early gene expression

(Robertson 1992; Dutcher, Underwood et al. 1999). This relationship was apparent in both the in

vitro and in vivo models. The strongest correlations within a model were between the in vitro 6

and 12 hour time points and the in vivo 6 and 24 hour time points. This trend extended to the

cross-model comparison at the later time points (6, 12, and 24 hour) as well. Biological fold

changes between the in vitro and in vivo model at matched time-points (1 hour in vitro vs. 1 hour in vivo, 6 hour in vitro vs. 6 hour in vivo, and 24 hour in vitro vs. 24 hour in vivo) had a strong correlation (r = 0.69). This data suggests that the post injury changes in gene expression follow a similar trend and therefore the in vitro model is well representative of the in vivo model.

4.4.2 GO Analysis

Several TBI-related GO terms were enriched after injury in both models. Not surprisingly, several GO terms related to inflammation were enriched including response to wounding (GO:0009611), response to extracellular stimulus (GO:0009991), defense response

(GO:0006952), cellular response to stress (GO:0033554), and inflammatory response

(GO:0006954). These GO terms contain genes activated by damage to the organism. Genes from both models that fall within these GO categories included Cd14, Cox-2, Ptges, Cxcl2, and

Ccl2. Ccl2 has been implicated in recruitment of macrophages after TBI, and elevated levels can be measured for 10 days after trauma in severe TBI patients (Semple, Bye et al. 2010). Cox-2 is one of the most widely studied enzymes responsible for inflammation and pain and is the target of most NSAID drugs such as ibuprofen (Mitchell, Akarasereenont et al. 1993; Engelhardt

1996). Cox-2 has previously been targeted as a potential therapy for TBI and has shown promise in preclinical studies (Strauss 2008).

Secondary injuries after TBI can lead to delayed cell death for hours, days, or even years

(Sauaia, Moore et al. 1995; Chen, Xiong et al. 2006). Genes within the GO category positive regulation of apoptosis (GO:0043065) were significantly altered in both injury models and could prove to be targets for therapeutic intervention. For instance, Wwox is an essential mediator of

TNF- induced apoptosis and is upregulated after injury in our models of TBI (Yang and Zhang

2008). Mmp2, which was significantly increased after closed-head TBI and is implicated in synapse loss, was also found to be upregulated in our study immediately after injury (Ding,

Kreipke et al. 2009). Jnk2 plays an important role in neuronal cell death as well as astrocytes activation, and inhibitors to Jnk2 have shown promising preclinical results (Ries, Silva et al.

2008; Kang, Simon et al. 2011; Fernandes, Harder et al. 2012; Tran, Sanchez et al. 2012).

The GO term transcription (GO:0006350) included several differentially expressed genes in our study that could have wide-ranging effects on the cellular response after injury. Some of

these genes are well known markers of injury, such as Atf3, and others are part of the

transcription machinery such as Polr2a, which forms the largest subunit of RNA polymerase II

(Greer, McGinn et al. 2011). Perhaps more interestingly, some of the identified genes are

directly associated with development and neurogenesis, including Notch-2 and Adnp, respectively. It has previously been reported that peptides based on Adnp can have direct, neuroprotective effects after closed head injury in mice (Beni-Adani, Gozes et al. 2001). Adnp has also been shown to be vital to embryogenesis and neurogenesis, and could have direct implications in neural repair after injury (Mandel, Rechavi et al. 2007).

4.4.3 SORLA down regulation after TBI

By combining different statistical approaches (single time point t-tests and ANCOVA),

32 genes were identified as being of particular interest. From this enriched subset of genes,

Sorla, which plays a key role in recycling amyloid precursor protein (APP) from the cell surface via the endocytic pathway, was identified. Specifically, SORLA directs trafficking of APP into recycling pathways by direct binding. Common mutations of SORLA, which are predictive of late-onset AD, reduce the binding affinity of SORLA for APP, leaving APP to enter Aβ-

generating compartments and accumulate (Buggia-Prevot and Thinakaran 2014; Caglayan,

Takagi-Niidome et al. 2014). It is speculated that SORLA, in this manner, is mechanistically involved in late-onset AD. Accumulating evidence from epidemiological studies implicate TBI as a possible risk factor in AD development (Van Den Heuvel, Thornton et al. 2007). Although the link between TBI and AD is controversial, extensive research continues to explore the relationship, if any, between injury and disease. At the forefront of this research is an

apolipoprotein E (APOE) genotype (ε4) which results in higher Aβ deposition following head

injury. However, the link between the APOE ε4 genotype and TBI is inconclusive and no other

biological explanation has been well substantiated. In our model, Sorla was down regulated

following TBI, which may lead to a disruption in APP recycling leading to the accumulation of

Aβ, suggesting a possible link to late-onset AD. Therefore, the identification of decreased Sorla

expression after injury was an interesting and potentially clinically relevant discovery from our in vitro model of TBI.

Immunohistochemical (IHC) staining of SORLA in uninjured OHSCs was similar to previous reports in both rat and human tissue (Motoi, Aizawa et al. 1999). Punctate staining was

specific to neurons located at the base of the apical dendrites within structures hypothesized to be

botrysomes (Figure 4.4 A-B), suggesting that SORLA expression was not altered during the

process of culturing the OHSCs. Further confirming our microarray analysis, SORLA

expression was considerably lower, and puncta were almost non-existent after in vitro TBI

(Figure 4.4 C-D). Western blotting analysis of SORLA expression confirmed that SORLA was

significantly down regulated after mechanical injury in our in vitro model. These results suggest

a potential mechanism for Aβ accumulation after injury and that SORLA may be a potential

therapeutic target for post-TBI treatment, as an abundance of Aβ can lead to neuronal cell death

(Reifert, Hartung-Cranston et al. 2011). Further research to determine the injury threshold for

SORLA reduction and the temporal response are necessary to better understand the connection

between TBI and SORLA changes. These data suggest the potential therapeutic benefit of

targeting SORLA after injury, and may unveil links between TBI and neurodegenerative

diseases.

Although several genes were highly correlated in both models of TBI, there were many

genes which did not overlap between the two models. It is possible that the severity of injury

was not equal in both models, although moderate levels were specifically chosen. Further work

will be required to determine the importance of these non-overlapping genes. Although many

inflammatory genes were upregulated in our in vitro model, the OHSCs are missing the systemic

component of the inflammatory response, which could have major effects on the efficacy of any

therapy tested in vitro. Other limitations of the in vitro model include a lack of a blood brain

barrier and circulatory system. We also opted to include more time points in our study at the

expense of more arrays per experimental condition. Although the high correlation of genes is a

promising result, a more extensive study with additional arrays would provide higher statistical

confidence.

Utilizing microarrays, we compared our in vitro model of TBI to an in vivo model. We

found many overlapping genes that were categorized in similar GO groups. The correlation of

gene expression between models was high; of the 12 Pearson correlations made between the two

models, 7 had a strong positive correlation (r > 0.50). A remaining 2 had moderately negative

correlations (r < -0.40) and were comparisons of the in vivo 1 hour to the in vitro 6 and 12 hour

time points. These negative correlations could potentially represent immediate early genes

responding to the initial mechanical injury, which then normalize at later time points, but this

speculation would require further study to confirm. These data, taken together and with the

previously mentioned limitations, provide confidence in using the in vitro model to represent the

in vivo TBI cascade. This important conclusion supports the use of in vitro models such as ours

for drug discovery and mechanistic studies which may reduce the number of animals necessary for TBI research. Furthermore, our study identified significant changes in Sorla expression, which previously has not been implicated in TBI research, and may provide a new connection between TBI and AD, as well as an avenue for therapeutic intervention.

5 Summary

5.1 E2 neuroprotection after in vitro models of stroke and TBI

E2 is the most potent, naturally occurring form of estrogen which exerts a multitude of

effects on cells. Several decades ago, a study revealed that women statistically survive stroke

more often than men (Paganini-Hill, Ross et al. 1988). Since then, many studies have

investigated the neuroprotective effects of E2 (Paganini-Hill 1995; Simpkins, Rajakumar et al.

1997; Yang, Shi et al. 2000; Liu, Wang et al. 2007; Simpkins and Singh 2008; Sarkaki, Khaksari

et al. 2011; Day, Floyd et al. 2013). Reduced oxidative stress, anti-inflammation, improved

vascular function, decreased apoptosis and attenuated excitotoxicity are all benefits associated

with E2 therapy after injury (Behl, Widmann et al. 1995; Simpson, Misso et al. 2005;

Numakawa, Matsumoto et al. 2007; Xia, Yang et al. 2007; Liu and Zhao 2013).

In Chapter 2, we hypothesized that E2 would be neuroprotective in both an in vitro model

of stroke and an in vitro model of TBI. In Chapter 4, we confirmed that expression of

inflammatory- and apoptotic-related genes were significantly altered after injury in our model of

TBI, which further supports utility for E2 after injury. Additionally, delayed mechanisms of

injury in both TBI and stroke may include reduced blood flow due to either primary damage to

blood vessels or due to increased intracranial pressure from edema. Furthermore, our genome

wide analysis of gene expression after in vitro TBI in Chapter 4 revealed no significant changes

in estrogen receptor expression, suggesting the E2 pathway is likely intact following injury and

can exert its neuroprotective effects. We therefore first tested a range of E2 concentrations after

a mechanical injury or OGD.

We assessed concentrations of E2 which ranged from physiological levels (10 pM – 120

pM) to supraphysiological levels greater than 1 nM in both models of injury (Strom,

Theodorsson et al. 2010). Previous research has shown divergent effects of E2 depending on

concentration, and these differences may be responsible for conflicting results in studies of E2-

mediated neurorprotection after injury (Strom, Theodorsson et al. 2010; Strom, Theodorsson et

al. 2011). To our surprise, in both our stroke and TBI models, a lower, physiological

concentration of E2 was more neuroprotective than supraphysiological concentrations. To better

understand why lower concentrations of E2 were more effective, we investigated the effects of

different E2 receptors on neuroprotection after OGD in OHSCs.

Classical ERs, ERα and ERβ, are found in the cytoplasm, and after binding E2,

translocate to the nucleus to act as transcription factors that control the expression of many genes

(Nilsson and Gustafsson 2002). E2 may also bind cell surface receptors, causing near immediate

physiological effects (Foy, Xu et al. 1999; Bjornstrom and Sjoberg 2004; Zivadinovic, Gametchu

et al. 2005). More recently, GPR30, an orphan G-protein coupled receptor, has been shown to

respond quickly to E2 (Filardo 2002; Filardo, Quinn et al. 2002; Revankar, Cimino et al. 2005;

Prossnitz and Barton 2009; Liu, Zhang et al. 2012). It is important to note that binding constant

of GPR30 for E2 is approximately 6.6 nM, well above its physiological range (Revankar, Cimino

et al. 2005). Using inhibitors to the classical ERs or GPR30, we found that only the classical

ERs had a significant effect on neuroprotection after OGD, in agreement with many previous

studies of E2-mediated neuroprotection after stroke (Simpkins, Rajakumar et al. 1997; Yang, Shi

et al. 2000; Liu, Wang et al. 2007; Simpkins and Singh 2008). This data suggested that GPR30

played only a small role in neuroprotection and that its activation at higher concentrations of E2

may inhibit neuroprotection, as indicated by increased cell death at higher E2 concentrations.

To further support our hypothesis that GPR30 is not activated at physiological

concentrations of E2, we quantified the phosphorylation of the downstream targets of GPR30,

AKT and ERK. Using phospho-specific antibodies, significant increases in phosphorylation only occurred at the high, supraphysiological concentration tested (10nM). To our knowledge, this is the first study to directly compare the divergent effects of E2 concentrations on E2- mediated neuroprotection and the involvement of classical ERs and GPR30. This important result could explain the divergent effects previously reported (Strom, Theodorsson et al. 2011).

5.2 Memantine neuroprotection after in vitro TBI

Excitotoxicity is a well known secondary injury cascade activated after TBI, which is

caused by an excessive release of excitatory amino acids such as glutamate that eventually leads

to neuronal cell death (Nilsson, Hillered et al. 1990; Baker, Moulton et al. 1993; Palmer, Marion

et al. 1993). Excitatory amino acid inhibitors have shown success after experimental models of

excitotoxicity and TBI (Willis, Lybrand et al. 2004; Volbracht, van Beek et al. 2006). We chose

memantine, a non-competitive, use-dependent NMDA inhibitor, which is already FDA approved

for use in AD, because it is well tolerated clinically and has previously shown success mitigating

excitotoxicity (Parsons, Danysz et al. 1998; Parsons, Danysz et al. 1999; Volbracht, van Beek et

al. 2006; Schneider 2013). Our genome wide analysis of gene expression in Chapter 4 revealed

significant changes in NMDA subunits following injury, suggesting a possible change in NMDA

homeostasis and its potential as a therapeutic target. Therefore, in Chapter 3, we investigated

memantine as a monotherapy and tested 3 concentrations after mechanical injury of OHSCs. In

our model of TBI, memantine was significantly neuroprotective at 10 µM compared to vehicle

controls, while 100 nM and 1 µM were not. This concentration of memantine is consistent with

results from other models of injury (Parsons, Danysz et al. 1999). These data suggested

memantine may be a good candidate for a combination treatment after TBI along with E2.

5.3 17β-estradiol and memantine after in vitro TBI

In Chapter 3, we investigated a combination therapy of E2 and memantine as a treatment

after TBI. Combination therapies are a promising area of TBI research because they aim to

combine individual compounds with complementary or synergistic actions to increase the

efficacy of monotherapy treatment to a level that is clinically relevant (Faden 2002; Margulies

and Hicks 2009). In Chapter 2, we assessed E2 as a monotherapy in our model of TBI.

Although E2 proved to be effective at the 100 pM concentration after injury, we also showed that

E2 at high doses is not as effective as low doses. To that end, we decided to investigate a

combination therapy of E2 with a drug that may target some of the potentially negative side

effects of E2 (Strom, Theodorsson et al. 2011).

E2 enhances EPSPs and LTP (Foy, Xu et al. 1999). We hypothesized that an

enhancement of evoked responses may lead to increased excitotoxicity and that memantine may

reduce or eliminate this effect. We were able to quantify the potentiation effect of E2 on evoked

responses in our OHSCs using an MEA. We stimulated OHSCs at the Schaffer collaterals and

analyzed responses in the CA1 region. Indeed, after potentiation by E2, memantine statistically

reduced evoked responses to near baseline values. For these reasons, we proposed that

memantine would be an ideal candidate to work synergistically with E2 as a combination

treatment for TBI.

In agreement with previous literature, both E2 and memantine were neuroprotective after

TBI (Parsons, Danysz et al. 1999; Rao, Dogan et al. 2001; Day, Floyd et al. 2013). We then

tested all 9 combinations of the concentrations evaluated as monotherapies after TBI. We found

that many of the combinations were neuroprotective after TBI compared to vehicle controls,

even for concentrations that were not previously neuroprotective individually. For instance,

neither 1 nM E2 nor 1 µM memantine statistically reduced cell death in the CA1 region after

TBI, but the combination was neuroprotective compared to vehicle controls. Further, the

combination of 100 pM E2 and 10 µM memantine was significantly more neuroprotective than

either monotherapy in the CA1 region. We are the first to show that the combination of E2 and memantine were significantly more neuroprotective after TBI than either monotherapy, and this is a promising result for future combination therapy studies.

5.4 Global analysis of an in vitro and in vivo model of TBI

After TBI, the primary mechanical event sets in motion a myriad of secondary injury cascades that cause delayed cell death that may continue for days or longer (Stoica and Faden

2010; Moretti, Cristofori et al. 2012). A better understanding of the secondary mechanisms leading to delayed cell death and dysfunction after TBI may support the discovery and development of novel and clinically viable treatments. To this end, several in vivo and in vitro models have been developed to investigate TBI (Raghupathi and Huh 2007; Morrison, Elkin et al. 2011; Gold, Su et al. 2013). Both in vitro and in vivo models have respective strengths and weaknesses, but ultimately the utility of in vitro models depends on their ability to reproduce the

in vivo TBI cascade (Morrison, Elkin et al. 2011).

In Chapter 4, we investigated the transcriptomic similarity between an in vitro model and

an in vivo model of TBI. Our aim was to quantify the degree of similarity and determine how

well the in vitro model of injury represented the in vivo pathophysiology. We chose an in vitro

model of TBI that is well established and reproducible that was also used for studies in Chapters

2 and 3 (Morrison, Meaney et al. 2000; Sundstrom, Morrison et al. 2005; Cater, Gitterman et al.

2007; Morrison, Elkin et al. 2011). Because this model requires culturing tissue from young animals (P8-10) and simulates a diffuse TBI, we chose to compare it to a closed-head impact model of in vivo injury using P11 rat pups (Raghupathi and Huh 2007; DiLeonardi, Huh et al.

2009). To quantify gene expression changes, we hybridized cRNA constructed from injured and uninjured tissue collected at several time points from both models to Affymetrix Rat 230 2.0 gene chips. These gene chips contain over thirty thousand probes to different genes.

After quality control and background intensity adjustment, using a Student’s t-test with a false discovery rate corrected p < 0.05, and a biological fold change > 2 or < 0.5, we determined significant gene expression changes at 1, 6, 12, and 24 hours after our in vitro model of TBI and

1, 6, and 24 hours after our in vivo model of TBI. We identified significant changes in 2073 and

877 unique genes after in vitro or in vivo injury, respectively and found that 36.8% of the

differentially regulated genes were identical between the two models.

To better understand how well the in vitro model represented the in vivo model, we used

Pearson’s correlation of the fold changes for the 323 genes that were significant in both models.

There was a strong correlation in gene expression between the in vitro and in vivo models at

matching time points. We also found moderately negative correlations between the 1 hour time

points and any later time points, suggesting an early immediate response to injury which returned

to baseline during the next 6-24 hours.

We then categorized significant gene expression changes by gene ontology (GO) to

identify the most enriched GO groups after injury. We found many overlapping GO groups

between the in vitro and in vivo model, further confirming that a similar molecular post injury

environment exists in both models. Our data is in agreement with several other gene expression

studies which show an increase in inflammatory cascades and genes, such as Cox-2, Cxcl2, and

Mmp (Di Pietro, Amin et al. 2010; White, Ford et al. 2013). Some studies have investigated

genomic changes at different time points, but did not include any temporal analysis and instead

investigated changes at distinct time points. We are the first, to our knowledge, to investigate

early global gene expression changes and include a temporal analysis as discussed in Chapter 4.

5.5 Down regulation of SORLA after TBI

In Chapter 4, to identify significant temporal changes in gene expression, we used

analysis of covariance (ANCOVA) which combines aspects of regression and analysis of variance (ANOVA). We then looked for genes which were significant in both the Student’s t- test and ANCOVA and found 32 genes which met these criteria. From this enriched set of genes, a particular gene of interest, SORLA, which has been linked to late-onset AD was identified. An increasing body of evidence implicates TBI as a risk factor in some instances of

AD (Van Den Heuvel, Thornton et al. 2007; Moretti, Cristofori et al. 2012), but direct evidence is lacking, and the leading theory, that the APOE genotype ε4 is responsible, is controversial

(Van Den Heuvel, Thornton et al. 2007). Therefore, the identification of decreased SORLA expression after injury was an interesting and potentially clinically relevant discovery from our in vitro model of TBI that could link TBI to AD.

Although SORLA is widely expressed in the brain, it is predominately expressed in the neurons of the hippocampus (Motoi, Aizawa et al. 1999). Cytoplasmic puncta of SORLA staining were observed in the CA1 pyramidal neurons of uninjured controls. Interestingly, after

mechanical injury, these puncta were reduced or completely absent in otherwise healthy pyramidal neurons. SORLA protein expression was significantly decreased 43.8% after injury as

confirmed by western blotting. These data provide a compelling alternative theory linking TBI

directly to Aβ accumulation which could lead to AD.

The genes identified in Chapter 4, such as SORLA, may have a significant impact on future TBI research. The mechanisms behind E2 and memantine neuroprotection after TBI are not well understood and identifying potential downstream pathways that are affected by these compounds could be fruitful, leading to improved therapeutics. Also, further analysis of the pathways activated after TBI could lead to novel therapeutic targets that could be investigated as monotherapies or included in a combination therapy as discussed in Chapter 3.

5.6 Limitations

An overarching limitation of the studies presented is the use of OHSCs. Although previous studies have shown OHSCs maintain the cellular diversity and structure of the hippocampal structure in vivo (Stoppini, Buchs et al. 1991; Gahwiler, Capogna et al. 1997), they lack effects due to blood circulation, perfusion, and the blood brain barrier, among others, in

vivo. Despite these concerns, in Chapter 4 we compared our in vitro model of TBI to an in vivo

model and found a high degree of similarity, suggesting they are generally reliable models of

hippocampal tissue. Also, a comprehensive comparison of the translation between in vitro and in

vivo models of TBI found them to be reliable predictors of drug efficacy (Morrison, Elkin et al.

2011). However, any potential treatments inferred from these studies would need to be

confirmed in vivo.

Because we are using OHSCs, drug delivery is simplified for the reasons previously

discussed, speeding the search for novel therapies. However, these additional factors may alter

the therapeutic effect and time window of an in vivo treatment after injury. The presented

windows of therapeutic efficacy (~1 hour) for E2 monotherapy or the combination therapy of E2

and memantine may be too short for a clinically relevant treatment. Typically several hours after

an injury are required before any treatment can be initiated due to the time it takes to travel to the

hospital, triage and diagnosis, and family consent for clinical trials (Faden 2002). However, in

vivo E2 treatments in rats have shown efficacy up to 6 hours after ischemic injuries. The

progression of injury may take longer in vivo, and therefore delayed treatment in vivo remains an

important future study.

There are several limitations of our mechanical injury model. It takes advantage of

equibiaxial stretch, but some investigators have postulated that shear strain is a better model of

TBI-induced deformation in vivo. However, if we assume the OHSCs are incompressible and

that they are 3 dimensional, if they are stretched in-plane they will be compressed in the

orthogonal direction. Because the cultures remain in the same plane of focus during injury, high

speed camera analysis of the deformation can verify the induced stretch. Furthermore, we are using rat tissue and human tissue may respond differently to similar injuries. Comparison of our

results with human tissue would alleviate these concerns. Also, it is unclear which cell types are

dying and being stained by PI. Because of their location within the pyramidal cell layer, we

assume they are primarily neurons, but double staining with PI and cell type markers would

confirm these assumptions.

Our OGD model also has associated limitations. We took several precautions to

eliminate oxygen and glucose from the experimental chamber, however, it is likely that there was

residual glucose in the OHSCs and that oxygen was not completely eliminated. Using oxygen

sensors and glucose assays could verify their concentrations. Also, we chose a specific amount

of time for OGD. During stroke, arteries can be blocked for seconds to hours or longer,

requiring surgical intervention to open them. Therefore, our data is relevant to a very specific

duration or severity of injury. Also, our model does not reproduce the effects of a penumbra, as

the entire tissue is subjected to the same severity of OGD, making it difficult to directly correlate

with in vivo studies, which have a significant penumbra.

In Chapter 4, we discovered thousands of differentially expressed genes after TBI.

Although several genes were highly correlated in both models of TBI, there were many genes

that were not regulated in the same manner between the two models. It is possible that the

severity of injury was not equal in both models, although moderate levels were chosen. Further

work is necessary to determine the importance of these non-overlapping genes. Also, although

many inflammatory genes were upregulated in our in vitro model, the in vivo inflammatory

response is missing from our in vitro model, and these responses could have major effects on the

efficacy of any therapy tested in vitro. For the experimental design, we opted to include more

time points in our study at the expense of more arrays per experimental condition. Although the

high correlation of genes is a promising result, a follow-up study with more arrays could provide

more statistically significant results.

5.7 Future Directions

E2 was neuroprotective in both in vitro models of TBI and stroke, but questions still

remain. We found that E2 was neuroprotective at a physiological dose immediately following

either mechanical or ischemic injury and up to 1 hour post OGD. Confirming this delayed

treatment response for E2 monotherapy in mechanical injury would be important for any future

TBI studies as it could have clinical significance. Similarly, the rate at which E2 is administered

may be crucial to neuroprotection. In our model, we directly apply E2 to the OHSCs following

injury. Although many studies have shown efficacy with a bolus injection of E2 following

injury, other methods of delivery, such as with time-release silastic capsules, have not been

successful (Strom, Theodorsson et al. 2010). However, bolus injections of E2 lead to immediately supraphysiological levels of E2 in the rat. We hypothesize that the most effective dosing regimen may be one in which the concentration of E2 is brought rapidly up to a physiological concentration (e.g. 100 pM) and then stably maintained at that concentration over time. The mechanism of E2 neuroprotection could also be explored in more depth. Although we investigated the receptors which may be involved, we did not investigate the role of E2 in free radical scavenging or antioxidant effects, or its ability to reduce the inflammatory response.

GPR30 is a controversial target of E2, and more research is necessary to determine its

role in E2-mediated neuroprotection. In vivo studies have shown success using G-1, a GPR30

agonist, after models of stroke and TBI in rat (Day, Floyd et al. 2013). It is possible that GPR30

plays an important role in the systemic inflammatory response or in blood flow as the basis for

neuroprotection in vivo, effects which could not be tested in our in vitro model. Also, GPR30 is well known to phosphorylate AKT and ERK which we explored in Chapter 2. Both of these proteins, when phosphorylated, are capable of inhibiting apoptosis, which could be an alternative mechanism of neuroprotection. However, this anti-apoptotic mechanism is not support by our data that suggested GPR30 activation had little effect on E2-mediated neuroprotection. We show higher concentrations of E2 are less neuroprotective than concentrations which do not

phosphorylate these proteins, so further research is necessary to explain this disparity in the

effect of GPR30 activation in vivo vs. in vitro.

There are several methods to achieve more confidence in our conclusion that GPR30 is

not an important factor in E2-mediated neuroprotection. Using RNAi against or using OHSCs

from knockout rats of either classical ERs or GPR30 would eliminate completely the individual

receptor’s component of neuroprotection. This would further bolster any of our results which

were obtained with chemical inhibitors. Also, confirming well known downstream effects of E2

such as up-regulation of NGF, BDNF, or IGF-1 would further support our claim that classical

ERs are responsible for neuroprotection conferred by E2, whereas GPR30 is not.

In Chapter 3 we investigated a combination treatment of E2 and memantine which has

similar but more extensive future directions than Chapter 2. Again, delayed treatment studies

would need to be performed in vivo to determine the efficacy of treatment after injury. However,

because it is a combination of two drugs, many more experiments will be necessary to determine

the pharmacology associated with a combination treatment. Previous studies have shown

successful monotherapies that have negative consequences as a combination therapy (Faden

1993), and therefore there may be an adverse reaction to a combination treatment of E2 and

memantine in vivo. The correct dosing of both drugs would require extensive research as we

tested only 9 combinations. The lowest concentration of each drug necessary for maximal neuroprotection would be desirable for future clinical trials. In our study, combinations of low

doses provided superior and significant neuroprotection compared to higher concentrations of

monotherapies.

The biological mechanism that provides neuroprotection from the combination treatment also requires extensive research. We provided a potential mechanism in which memantine reduced an undesireable side-effect of E2, specifically electrophysiological potentiation, but

these results are not conclusive. To test this particular hypothesis, Ca2+ influx could be imaged and quantified after exposure of OHSCs to E2. If increased potentiation in OHSCs after E2 exposure is due to increased NMDA potentiation as proposed, Ca2+ should increase accordingly,

and memantine should block it.

In Chapter 2, we investigated the phosphorylation of AKT and ERK after exposure to

different concentration of E2. In Chapter 3, we found that 1 nM E2 combined with 1 uM memantine provided significant neuroprotection in the CA1 region, where neither monotherapy

was effective. It is possible that 1 nM E2 may activate GPR30 and cause phosphorylation of

AKT and ERK, although we did not test this concentration for its effects on AKT and ERK

phosphorylation. We showed in Chapter 2 that GPR30 activation did not contribute to overall

neuroprotection after OGD, but inhibiting GPR30 did cause a slight, although non-significant

decrease in cell death in the CA1 region. Because it may play some role in neuroprotection, we

could further test if the combination treatment of E2 and memantine successfully inhibits the

negative side effects of E2 while still activating GPR30 and phosphorylating AKT, a well-known

anti-apoptotic molecular event. Further understanding the downstream events of GPR30 could

also be fruitful, revealing a more direct path to AKT phosphorylation that may provide additional

neuroprotection without negative side effects.

In Chapter 4, we analyzed and compared the transcriptomic changes after an in vitro and in vivo TBI. Although we focused primarily on the similarities between these two models, we could learn a great deal by looking at their differences. We have already discussed the

limitations of our OHSC model including the lack of an immune system or blood circulation.

Analysis of the in vivo specific changes in gene expression may reveal the downstream cellular

events which do not take place in our in vitro model. These important differences may benefit

future studies of potential therapeutics in our in vitro model of TBI. Also, different injury

severities result in a multitude of gene expression changes (Di Pietro, Amin et al. 2010).

Another potential explanation of the differences observed in our study may be injury severity. A

future study looking at several different injury severities in both models would be ideal.

There was a distinct negative correlation between gene expression at the 1 hour time

points and all other time points. We hypothesize that these genes were initially altered

immediately post injury and were returning to baseline over time. Genes which are altered soon

after injury are likely to initiate or contribute to many of the secondary injury cascades of TBI

such as excitotoxicity and free radical formation. Therefore, further analysis of this group of

genes may provide future therapeutic targets.

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7 Appendix A: Significant Gene Lists

7.1 1 hour in vitro TBI

Affymetrix ID Gene Symbol p‐value 1367568_a_at Mgp 0.025882125 1367602_at Cited2 0.00038588 1367652_at Igfbp3 0.022127569 1367658_at Shank3 0.000155628 1367731_at Gnb1 0.00150013 1367749_at Lum 0.00023669 1367753_at Sec31a 1.90E‐05 1367799_at Eef1a2 0.000288069 1367802_at Sgk1 2.09E‐05 1367836_at Cpt1a 0.000606656 1367847_at Nupr1 0.022769153 1367880_at Lamb2 9.54E‐06 1367896_at Car3 0.004195809 1367948_a_at Kdr 0.004053175 1367973_at Ccl2 0.007642388 1367984_at Scaf1 0.000186145 1367998_at Slpi 0.000507057 1368014_at Ptges 0.005615473 1368015_at Ptges 0.001905203 1368025_at Ddit4 0.000311017 1368030_at Gnai3 0.008378923 1368046_at Slc31a1 0.010679126 1368091_at Oplah 0.014998436 1368146_at Dusp1 0.000188231 1368147_at Dusp1 0.00195384 1368171_at Lox 0.034335256 1368223_at Adamts1 0.000563264 1368224_at Serpina3n 0.018382072 1368248_at Cds1 0.001427352 1368249_at Klf15 1.17E‐05 1368255_at Ntm 0.000487089 1368261_at Nrxn3 0.000202537 1368279_at Mllt3 3.10E‐06 1368280_at Ctsc 0.000815511 1368281_at Dpep1 0.000243664 1368306_at Grin2c 0.000549793

1368321_at Egr1 0.000171065 1368370_at Adcy4 0.000222147 1368384_at Klk6 0.000145853 1368440_at Slc3a1 0.020868421 1368462_at Itpka 0.002254129 1368505_at Rgs4 0.041743457 1368509_at Bbs2 0.00050056 1368527_at Ptgs2 0.000185728 1368572_a_at Grin1 0.000380039 1368612_at Itgb4 0.000179589 1368646_at Mapk9 0.005829692 1368675_at Chn2 0.001070857 1368676_at Dync2h1 1.20E‐05 1368692_a_at Chka 4.05E‐06 1368696_at Fxyd7 0.001855075 1368719_at Pfkfb4 0.002148747 1368760_at Cxcl2 0.005834341 1368782_at Sstr2 0.003137291 1368826_at Comt 0.001181603 1368929_at Nploc4 0.000985503 1368948_at Msn 0.003073573 1368958_at Pacsin1 0.000479579 1368982_at Pkia 0.005817592 1368984_at 2‐Sep 0.004025936 1369002_at Soat1 0.005708456 1369003_at Dedd 0.00012219 1369007_at Nr4a2 0.031589687 1369035_a_at Kcnj6 0.002606213 1369077_at Asah1 0.003803372 1369089_at Prkcg 0.001402974 1369099_at Slc30a1 0.000607848 1369128_at Grik5 9.76E‐05 1369132_at Slc18a2 0.000265777 1369191_at Il6 0.003740251 1369202_at Mx2 0.032601893 1369248_a_at Xiap 0.010366559 1369251_a_at Syn1 0.003569603 1369268_at Atf3 0.001820207 1369294_at Bst1 0.002250075 1369312_a_at Csnk1a1 0.002303839 1369344_at Wdr7 0.000814676 1369378_at Slc23a2 0.000660419 1369400_a_at Pfkfb2 3.01E‐05

1369409_at Nab1 0.001142323 1369452_a_at Picalm 0.001689792 1369504_at Tgfbr1 0.004141212 1369505_at Cds2 0.000384092 1369511_at Ednra 4.76E‐05 1369514_at Insrr 6.12E‐05 1369518_at Pik3r3 0.001259327 1369560_at Gpd1 0.001369774 1369571_at Golph3 0.017420471 1369590_a_at Ddit3 0.000125825 1369610_at Lin7c 0.004262805 1369647_at Calcrl 0.000556886 1369648_at Calcrl 0.000329494 1369650_at Pak2 0.00666368 1369662_at Scn2a1 0.007183015 1369666_at Gpd2 0.000155985 1369721_at Strn 0.000622392 1369736_at Emp1 0.018052101 1369758_at Gpam 0.006197333 1369785_at Ppat 0.00109899 1369791_at Clcn4‐2 0.001885653 1369814_at Ccl20 0.022789776 1369868_at Magt1 0.003163815 1369871_at Areg 0.000327468 1369903_at Gabrb3 0.024389982 1369912_at Crk 0.001788378 1369920_at Hist1h1t 5.08E‐05 1370016_at Nell2 0.010626793 1370114_a_at Pik3r1 0.000640213 1370131_at Cav1 0.017817974 1370132_at Fkbp1b 0.006995857 Cav2 /// 1370135_at LOC100362824 0.020905852 1370137_at Agps 7.76E‐05 1370146_at Glrb 0.005321264 1370155_at Col1a2 0.000205278 1370177_at PVR 0.023783684 1370259_a_at Pth1r 1.30E‐05 1370301_at Mmp2 0.034857512 1370307_at Agrn 8.88E‐05 Ceacam1 /// 1370371_a_at Ceacam10 0.014458358 1370372_at Rasd2 0.001570463

1370391_at Crabp2 0.012019753 1370432_at Pou3f1 0.002047598 1370438_at Nos1ap 0.000402868 1370463_x_at RT1‐CE16 0.000104308 Lilrb3l /// LOC683463 1370493_a_at /// LOC690948 0.000128865 1370542_a_at Elf1 0.000354767 1370553_at Stx2 0.000342965 1370562_at Calcb 5.41E‐05 1370570_at Nrp1 0.015182018 1370623_at Fgl2 0.000255227 1370630_a_at Fgfr2 0.002593935 1370648_a_at Wipf3 5.72E‐06 1370682_at Lilrb3l 0.000292897 1370717_at Synrg 0.001787603 1370728_at Il13ra1 0.029477477 1370746_at Prkacb 0.003177643 1370770_s_at Kitlg 8.06E‐05 1370775_a_at Calca 8.67E‐05 1370780_at Rab31 0.001216173 1370823_at Bambi 0.000380158 1370857_at Acta2 3.34E‐06 1370864_at Col1a1 0.000570595 1370913_at Rsad2 0.001897872 1370917_at Hsf1 1.07E‐06 1370922_at Ctxn1 0.000641644 1370954_at P4ha1 1.44E‐05 1370959_at Col3a1 0.001488388 1370989_at Ret 7.86E‐05 1371015_at Mx1 0.006644905 1371019_at Trib1 0.002072632 1371059_at Prkar2a 0.000507593 1371060_at Trim23 0.007638097 1371069_at Slc22a23 0.003404856 1371071_at Gnb4 0 1371103_at Rab6a 0.024200082 1371131_a_at Txnip 4.72E‐05 1371139_at Pls3 0.011235476 1371173_a_at Cast 0.001474381 1371179_a_at Fgfr2 0.000736952 1371184_x_at Tpm3 2.44E‐05 1371239_s_at Tpm3 0.00656426 1371472_at [] 0.001365662

100

1371479_at Mettl7a 2.15E‐06 1371500_at Ltbp4 0.001588941 1371527_at Emp1 0.000270605 1371583_at Rbm3 7.39E‐05 1371687_at Canx 0.011890769 1371731_at RGD1566215 0.000188172 1371924_at Olfml3 0.000999689 1371970_at Fam111a 0.032940209 1372048_at Podxl2 6.51E‐05 1372063_at [] 7.09E‐05 1372208_at Ppp1r1b 1.87E‐05 1372219_at Tpm2 0.00816685 1372345_at [] 0.001791775 1372389_at Ier2 7.62E‐05 1372426_at Adamtsl4 0.001266956 1372492_at Cldn10 0.000444531 1372599_at Mgst2 0.001830936 1372617_at Btbd2 9.54E‐07 1372646_at RGD1305645 0.000284255 1372699_at Znf775 1.67E‐05 1372823_at [] 0.033850789 1372958_at Otub1 4.23E‐05 1372967_at Geft 1.57E‐05 1372977_at Map4k5 0.005027771 1373118_at LOC689074 0.000243664 1373191_at [] 1.54E‐05 1373218_at [] 0.003015459 1373260_at LOC686779 1.39E‐05 1373278_at [] 1.48E‐05 1373358_at Usp8 0.000682652 1373403_at LOC684871 0.007912934 1373410_at Mef2c 0.018950224 1373508_at Selt 0.001933694 1373534_at Sfrs18 0.000211 1373628_at [] 0.005314708 1373648_at LOC681849 0.002308071 1373651_at [] 0.003246248 1373656_at Ppp1r3d 0.003213227 1373663_at Dmkn 0.001956463 1373685_at Ankrd37 2.60E‐05 1373740_at [] 0.006410956 1373750_at Leprel2 8.78E‐05 1373751_at [] 0.000101566

101

1373757_at Trafd1 1.04E‐05 1373759_at [] 0.000127673 1373777_at [] 9.55E‐05 1373807_at Vegfa 1.92E‐05 1373814_at R3hdm2 0.000115454 1373839_at [] 6.82E‐05 1373886_at Reep1 0.001805663 1373911_at Postn 0.003864884 1373931_at RGD1304952 1.91E‐05 1373975_at Inmt 0.032182217 1373992_at MGC108823 0.005870283 1374022_at [] 3.34E‐06 1374062_x_at Mapre3 0.000256181 1374115_at Mzf1 0.001273274 1374159_at Als2cl 1.72E‐05 1374171_at LOC100360403 0.015707731 1374207_at Angpt2 0.009431005 1374211_at RGD1566084 0.002491474 1374263_at [] 0.0179739 1374303_at Alkbh2 0.001718402 1374370_at [] 6.28E‐05 1374419_at LOC100362008 0.002934992 1374425_at Tle1 0.001050293 1374444_at [] 0.000327349 1374529_at [] 0.001911223 1374655_at [] 0.003648937 1374659_at Arpp‐21 0.005784988 1374684_at [] 0.000432372 1374713_at [] 4.77E‐05 1374746_at [] 0.000935137 1374778_at [] 0.002159834 1374785_at Clec2l 0.01348722 1374786_at [] 0.001700759 1374803_at [] 0.015105367 1374868_at [] 0.039569795 1374874_at RGD1561042 0.000456095 1374957_at [] 0.000334978 1375005_at [] 0.004131079 1375043_at Fos 0.000132203 1375053_at [] 4.03E‐05 1375129_at Fgfr2 0.000211239 1375176_at Fam172a 0.001157045 1375223_at [] 6.47E‐05

102

1375271_at Nek9 0.000924408 1375319_at [] 0.020536304 1375331_at [] 0.010800719 1375374_at [] 0.016723156 1375449_at [] 0.020859838 1375495_at [] 0.000624359 1375535_at [] 0.000617325 1375596_at Trpm1 7.43E‐05 1375625_at Paqr8 0.000115395 1375664_at Tnrc6a 0.00015986 1375696_at Ifnar1 0.001706481 1375703_at Mll5 6.58E‐05 1375707_at [] 7.51E‐06 1375751_at [] 0.000540376 1375754_at Impact 0.015224099 1375793_at [] 0.001215279 1375815_at [] 0.003131628 1375829_at Fam45a 0.003720999 1375868_at [] 0.00833869 1376028_at Msl1 0.000330329 1376130_a_at Dtnb 2.06E‐05 1376137_at Plekhb2 0.011893749 1376175_at Gbas 0.000329137 1376200_at MGC72974 3.30E‐05 1376208_at [] 0.000491858 1376224_at Elfn2 0.000905871 1376233_at Dlgap3 1.05E‐05 1376240_at [] 0.000178933 1376251_at [] 0.001868188 1376311_at Ntng1 0.000824451 1376388_at [] 0.01025033 1376397_at [] 2.85E‐05 1376425_at Tgfb2 0.000517368 1376434_at [] 0.007049918 1376438_at [] 0.01070255 1376460_at Hrnbp3 0.000155449 1376558_at Shisa7 5.28E‐05 1376562_at Tnik 0.001748502 1376707_at C1qtnf4 0.011658907 1376747_at [] 4.27E‐05 1376780_at Fam103a1 0.019756675 1376810_at Mphosph8 /// Parp4 0.000512958 1376872_at [] 0.000406325

103

1377133_at LOC680687 5.57E‐05 1377174_at Sbf1 0.000791013 1377193_at [] 0.009493589 1377208_at [] 0.007888794 1377234_at [] 1.91E‐05 1377243_at [] 1.91E‐05 1377340_at Tfpi2 0.025631905 1377353_a_at Tnfsf13 0.001063347 1377403_at [] 0.014602304 1377451_at RGD1305704 0.007651568 1377457_a_at Sorl1 0.000577569 1377461_at [] 0.000352442 1377472_at Extl1 1.21E‐05 1377508_at [] 0.000846863 1377532_at Phf20 2.09E‐05 1377534_at Stk32c 0.000593305 1377554_at Tnfsf9 0.0021469 1377570_at [] 0.000154853 1377583_at [] 7.75E‐06 1377614_at Ino80e 0.002293706 1377635_at Fmo2 0.000457823 1377644_at RGD1308706 9.55E‐05 1377656_at Mak16 0.002829433 1377663_at Rnd3 0.013658643 1377694_at Luc7l3 0.008072734 1377784_at [] 0.005250216 1377808_at Dcaf6 0.024695218 1377822_at Ccdc64 0.001346827 1377883_at [] 6.12E‐05 1377902_a_at Rad52 0.010839105 1377920_at Sgtb 1.48E‐05 1377985_at RGD1309198 0.001395524 1378030_at [] 4.86E‐05 1378051_at [] 0.002110601 1378124_at Ppm1b 0.01031357 1378238_at Anapc1 0.018964887 1378279_at Gabarap 0.000363469 1378412_at [] 0.002127647 1378460_at [] 0.005964875 1378483_at Prl7b1 0.005137563 1378506_at Pik3c2a 6.34E‐05 1378563_at RGD1308722 0.00098598 1378566_at [] 0.009260297

104

1378582_at Trabd 0.014848709 1378613_at [] 0.001189828 1378616_at [] 0.000145912 1378631_at [] 0.000723362 1378636_at LOC100359642 0.000266612 1378639_at Snupn 0.034193635 1378659_at [] 1.56E‐05 1378700_at [] 4.22E‐05 1378705_at Setd5 2.80E‐05 1378736_at [] 0.001942992 1378797_at [] 0.006850958 1378831_at [] 8.89E‐05 1378840_at [] 0.000911474 1378844_at [] 0.015907645 1378862_at [] 0.006836116 1378873_at Hils1 0.000417173 1378877_at [] 4.63E‐05 1378887_at [] 0.000858247 1378945_at [] 0.000268519 1378947_at Tns4 0.000321269 1378951_at [] 0.004303813 1378954_at [] 0.010851622 1378977_at [] 0.002135336 1378990_at [] 7.19E‐05 1379003_at [] 0.001324713 1379011_at [] 0.001581311 1379029_at [] 0.002751172 1379038_at [] 0.026954532 1379060_at [] 4.74E‐05 1379070_at [] 0.001099229 1379073_at Slc25a23 0.002912939 1379078_at [] 0.000747323 1379119_at [] 0.000262141 1379141_at [] 0.009811044 1379170_at [] 4.62E‐05 1379187_at [] 0.014445126 1379201_at Dhx8 0.038195252 1379202_at Ddi2 7.87E‐06 1379212_at [] 0.006088376 1379219_at RGD1308626 0.007044792 1379226_at Aim1l 0.001215696 1379233_at [] 0.002737463 1379234_a_at Cdc45l 0.010520041

105

1379242_at LOC684996 0.00091368 1379309_at Tbl1xr1 0.002925634 1379331_at [] 0.000646114 1379360_at Gatc 3.36E‐05 1379383_at [] 0.003441215 1379426_at [] 5.81E‐05 1379551_at Dus4l 9.99E‐05 1379636_at Fam82a1 0.029916763 1379661_at [] 0.001467109 1379673_at Uap1l1 0.000967205 1379733_at [] 0.037709951 1379742_at [] 0.00013268 1379754_at Stau2 3.52E‐06 1379776_at [] 0.030202091 1379988_a_at LOC305633 0.000252485 1379995_at [] 0.000118911 1380004_at [] 8.94E‐07 1380024_at [] 0.007780075 1380066_at Tfr2 0.000426769 1380108_at Wbp1 0.000323832 1380111_at [] 1.38E‐05 1380159_at Peo1 0.006614149 1380161_at [] 0.001198351 1380168_at Etv4 0.004055679 1380233_x_at [] 0.000262201 1380350_at [] 0.000235319 1380357_a_at [] 0.002420545 1380523_at Fbxo15 0.000243664 1380540_at [] 0.002506137 1380557_at Bckdha 0.001227856 1380606_at [] 0.000127673 1380631_at LOC100366273 0.001024306 1380656_at [] 4.77E‐05 1380658_at [] 0.032899857 1380672_at [] 0.004336476 1380676_at [] 0.000103354 1380677_at [] 0.001866221 1380727_at [] 0.013796389 1380783_at Slco5a1 0.016353309 1380786_at [] 0.003045619 1380850_at [] 2.03E‐06 1380974_at [] 0.001347721 1381038_at [] 2.93E‐05

106

1381041_at [] 2.26E‐06 1381060_at [] 6.44E‐05 1381110_at [] 0.006851912 1381115_at [] 0.015377402 1381204_at [] 8.53E‐05 1381213_at Nfix 1.55E‐05 1381216_at [] 0.000464857 1381261_at [] 7.27E‐06 1381298_at [] 0.001008153 1381306_at [] 0.020146132 1381320_at [] 0.005947471 1381331_at Cmtm2a 5.65E‐05 1381346_at [] 0.000273347 1381375_at [] 0.000333488 1381377_at [] 9.86E‐05 1381386_at Pop5 0.003485441 1381463_at [] 0.000537515 1381509_at Nbr1 0.00906831 1381546_at [] 0.003173828 1381557_at Gna14 0.000127137 1381571_at [] 0.000645697 1381609_at Zfp335 0.001945734 1381621_at [] 0.017572761 1381642_at [] 0.001307964 1381684_at [] 0.00543195 1381724_at [] 0.004915595 1381751_at [] 2.69E‐05 1381786_at [] 1.19E‐06 1381866_at [] 3.46E‐06 1381887_x_at RGD1311595 0.00027895 1381892_at Nav2 0.015456617 1381932_at Fam46a 0.00352931 1381950_at [] 0.001852751 1382090_at [] 0.002672553 1382135_at RGD1307882 2.49E‐05 1382156_at [] 0.001630545 1382203_at Gdf1 /// Lass1 0.019314706 1382282_at Lix1l 4.45E‐05 1382295_at [] 0.00285542 1382315_at Tsc22d4 0.008074939 1382334_at [] 0.001797199 1382357_at [] 0.000122786 1382478_at Btbd3 0.000780106

107

1382537_at Rragc 0.005790114 1382540_at [] 2.97E‐05 1382543_at [] 0.012291312 1382566_at Il7r 5.60E‐06 1382594_at [] 0.000160336 1382669_at Bin2a 0.008529246 1382679_at Wdr43 0.000315249 1382700_at [] 0.000427842 1382717_at Fam168a 0.000567913 1382777_at [] 0.002416492 1382814_at Odz3 7.21E‐05 1382838_at [] 0.000938535 1383001_at [] 0.001464248 1383200_at Ccdc81 0.001349926 1383212_at [] 0.000635445 1383252_at Sars2 0.000268519 1383355_at Abca1 0.004937649 1383422_at Bend5 0.005155504 1383448_at Irf9 0.007772982 1383461_at Wdyhv1 4.29E‐06 1383497_at [] 0.029549301 1383513_at [] 0.001433253 1383526_at [] 0.000582695 1383566_at [] 0.049737394 1383632_at [] 0.001765072 1383654_a_at Fn3k 0.001655221 1383682_at [] 0.004135847 1383684_at Asf1b 0.000118971 1383691_at [] 0.001779079 1383694_at Rnf208 0.000604093 1383695_at Vipr1 0.017265201 1383743_at Lrrc16a 0.001343012 1383749_at Phospho1 0.001793265 1383780_at Hectd1 0.02083385 1383836_at [] 0.000112474 1383872_at [] 0.012879312 1383922_a_at Ard1a 0.000676751 1384055_at [] 0.012700081 1384101_at Wasl 0.000643671 1384144_at Nxn 1.88E‐05 1384182_at Fermt2 4.21E‐05 1384207_at Nif3l1 0.000326574 1384212_at Rbms3 0.000658393

108

1384271_at [] 0.000329912 1384305_at Slc45a1 0.010135472 1384346_at [] 0.000412464 1384371_at Tead3 0.000492692 1384392_at Cyp26b1 2.52E‐05 1384479_at Galnt3 0.000928402 1384516_at Mtf2 0.006149769 1384533_at [] 0.001347423 1384545_at [] 0.001278758 1384573_at Papola 0.001897335 1384601_at Lrba 0.002698541 1384631_at [] 0.000205278 1384646_at [] 0.003360272 1384674_at [] 0.001241863 1384693_at [] 0.018446088 1384743_at [] 0.000937343 1384747_at [] 0.007034302 1384779_at Cplx3 0.005471826 1384780_at Cpne4 0.000312328 1384800_at Zic3 0.007701874 1384826_a_at [] 0.000532866 1384836_at Calml3 0.001959682 1384850_at [] 0.000972629 1384861_at [] 0.008178592 1384884_at RGD1307595 9.70E‐05 1384992_at Hdac10 0.001499116 1385107_at Slc6a14 9.86E‐05 1385110_at [] 1.56E‐05 1385116_at Pcdhb21 0.002268791 1385132_at [] 0.001474619 1385193_at [] 0.0115695 1385222_at [] 0.001991868 1385260_at [] 0.000358582 LOC100364003 /// 1385338_at Zfp758 0.003324091 1385346_at Wbp4 7.39E‐06 1385347_at [] 0.001188874 1385349_at Cetn4 0.004502296 1385376_at [] 0.004599035 1385418_at LOC688717 0.000185132 1385431_at [] 0.008312225 1385433_at Mtss1 0.000135481 1385450_at Ucn2 0.01133585

109

1385469_at Igf2bp1 0.012128055 Adora3 /// 1385515_at LOC100363178 7.94E‐05 1385526_at Atg5 0.000984728 1385544_at Nrl 0.022671819 1385579_at P4ha3 0.002755582 1385631_at [] 0.001290798 1385633_at [] 5.79E‐05 1385643_at [] 0.001455545 1385651_at [] 0.00013864 1385691_at [] 0.018096328 1385766_at Als2cr11 0.00153935 1385780_at [] 0.034848571 1385791_at Vill 0.024381697 1385813_at RGD1310251 6.48E‐05 1385909_at [] 0.000403285 1385950_at Fam131b 0.001610458 1385973_at [] 1.86E‐05 1386003_at Morf4l1 7.99E‐06 1386016_at Hspb11 0.001666904 1386047_at Galnt13 0.003939211 1386059_at Hist2h2be 0.000895023 1386234_at [] 0.004823804 1386295_at [] 0.007879973 1386321_s_at Trib3 0.000300229 1386361_at Teddm1 0.02061367 1386494_at RGD1311624 0.005886197 1386573_a_at [] 0.017657995 1386594_at [] 0.021955907 1386660_at [] 0.017605007 1386679_at [] 0.003757715 1386705_at [] 0.000448525 1386726_at [] 0.001594126 1386777_at Pot1a 0.004483104 1386822_s_at RGD1311823 0.008241892 1386884_at Htra1 0.012599349 1386896_at Khdrbs1 7.39E‐06 1386962_at Plcb4 0.006244183 1386986_at Ogfr 2.54E‐05 1387002_at Bud31 0.006035686 1387093_at Slco1a4 0.000168264 1387149_at Erap1 0.006569028 1387160_at Kcne3 0.009765506

110

1387161_at Slc1a6 0.00207907 1387190_at Dgka 0.002856612 1387194_at Adap1 0.003534436 1387198_at Inpp5d 0.000134349 1387450_at Tgfa 0.000469565 1387456_at Stau2 0.000668645 1387471_at Cela2a 0.004109502 1387570_at Manea 0.024653554 1387578_a_at P2rx2 0.001899958 1387580_at Srd5a2 0.00100255 1387615_at St8sia2 0.002450407 1387630_at Elovl5 0.012954414 1387644_at Btc 0.001464963 1387648_at Cxcl6 0.010458887 1387707_at Slc2a3 0.005368471 1387725_at Gulo 0.000132084 Acaa1 /// 1387783_a_at RGD1562373 0.011249602 1387868_at Lbp 0.004450202 1387914_at Cyp27a1 0.000895381 1387921_at Zc3h14 0.002496123 1387968_at Slc6a15 5.37E‐05 1387985_a_at Obp3 0.000767291 1388001_at Lrit1 0.002308309 1388018_at Sele 0.006263852 1388043_at Atp4a 0.000716925 1388065_at Insrr 0.010191679 1388159_at CYTB 0.00469631 1388241_at Insl3 0.005792916 1388274_at Bmyc 0.002853274 1388386_at Chmp1a 6.74E‐05 1388410_at Ugp2 0.005664945 1388448_at Cdc42se2 0.000157118 1388461_at Shd 0.009503007 1388487_at Add1 0.000936031 1388500_at [] 0.0001508 1388560_at Wdr77 0.005260468 1388592_at [] 0.00032115 1388627_at Ctsq /// RGD1564827 0.006246746 1388632_at Rab6b 0.001303554 1388654_at Mrpl52 0.00207895 1388667_at Man2a1 0.005660772 1388725_at Leprot 5.21E‐05

111

1388751_at [] 0.001185298 1388786_at [] 0.014825821 1388788_at Gcdh 0.000462234 1388840_at Plekhg2 0.010778069 1388873_at Trip12 0.004524469 1388877_at Mrps5 0.013055325 1388910_at Urg4 0.007937789 1388939_at Col15a1 9.47E‐05 1388982_at [] 5.97E‐05 1388983_at RGD1305045 0.016877651 1389067_at Slco4a1 0.006515503 1389134_at [] 0.001017213 1389163_at Trim32 9.98E‐05 1389186_at [] 0.000418782 1389214_at [] 0.018341184 1389238_at Pes1 1.66E‐05 1389239_at [] 0.000724912 1389242_at [] 0.001088738 1389245_at Psmd7 0.000471592 1389280_at [] 2.26E‐06 1389292_at Rab18 0.032694638 1389326_at Rfc3 0.000113845 1389327_at Mrpl32 0.009566903 1389329_at Lgals8 1.07E‐06 1389331_at [] 0.000640631 1389350_at Apoh 0.004798412 1389371_at [] 1.12E‐05 1389476_at RGD1307100 0.000607908 1389483_at Dpy19l1 0.000953496 1389492_at [] 0.000126004 1389557_at Tex261 7.41E‐05 1389565_at [] 0.001595557 1389570_at Inpp5k 0.005451977 1389583_at [] 0.00183928 1389606_at Anapc1 0.002537489 1389670_at Hoxa10 0.001198709 1389675_at Ier5l 0.009344697 1389701_at [] 1.76E‐05 1389706_at Dclk1 6.11E‐05 1389743_at [] 3.10E‐05 1389746_at RGD1564228 0.001052558 1389749_at [] 0.000226617 1389796_at [] 0.005071342

112

1389797_at [] 0.033822894 1389804_at Relt 0.00649935 1389855_at Ppp2r2c 0.002452314 1389906_at Fdft1 0.011850059 1389922_at Hadh 0.001999795 1389925_at [] 0.004334331 1390032_at Rbms2 7.54E‐05 1390056_at Nck2 0.014854968 1390085_at [] 0.001319945 1390099_at Ube3a 0.011896253 1390122_at [] 0.002463043 1390201_at Rap1a 0.001343548 1390202_at [] 0.000211239 1390299_at [] 0.004194558 1390358_at Cacna2d3 5.65E‐05 1390400_a_at [] 0.016268253 1390425_at [] 0.000338197 1390437_at Sema5a 0.000668168 1390476_at Zbtb39 0.000515044 1390516_at [] 0.000222206 1390528_at [] 0.001057863 1390603_at [] 0.00119406 1390627_a_at Tbx3 0.000232041 1390642_at [] 0.007649303 1390719_at [] 0.006211281 1390781_at Abcb10 0.015699267 1390831_at [] 0.003133416 1390864_at [] 0.001589894 1390908_at [] 6.44E‐05 1390975_at [] 0.006449282 1390985_at [] 0.002865195 1390995_at Fkbpl 4.49E‐05 1391029_at [] 0.000421047 1391047_at [] 0.000741065 1391062_at RGD1566052 0.000261307 1391095_at Mmp19 0.004005551 1391138_at [] 5.58E‐05 1391142_at [] 0.000166297 1391153_at [] 0.011681676 1391166_at [] 0.000391245 1391192_at [] 0.004420877 1391209_at [] 0.000245214 1391214_at Fam26f 0.021750033

113

1391232_at [] 0.001631439 1391329_at [] 0.003165066 1391359_at [] 0.006554604 1391362_at [] 0.004386306 1391572_at Cars 0.01211524 1391595_at Larp6 0.002341509 1391601_at Lrrn2 0.003259718 1391629_at [] 0.001521111 1391635_at Ctdspl 0.00116086 1391644_at LOC687565 0.000999093 1391663_at [] 0.000101924 1391665_at [] 0.000220716 1391695_at [] 0.000377655 1391718_at [] 0.002744913 1391719_at [] 0.004792333 1391792_at [] 0.000185847 1391798_at [] 0.000230789 1391802_at [] 4.51E‐05 1391807_at Tcfcp2 2.80E‐06 1391843_at [] 0.000285149 1391918_at [] 0.001711726 1391935_at Eif4e3 0.004966795 1391945_at [] 0.008803487 1391973_at [] 1.76E‐05 1392002_at Pcdhb22 0.001251638 1392056_at [] 0.000507116 1392058_at Txnl1 0.000403643 1392143_at [] 0.00886184 1392163_at [] 0.000154376 1392180_at Sp1 0.00077045 1392266_at Rnf190 0.000654578 1392304_at [] 0.000696599 1392397_at [] 0.003254592 1392551_at LOC503192 0.002048612 1392637_at [] 0.000120163 1392687_at Pigq 0.015001416 1392698_a_at Gtf3c4 0.000693083 1392722_at Dapp1 4.30E‐05 1392787_at [] 0.00296694 1392788_at [] 0.002775312 1392827_at Tmem126b 0.003266215 1392862_at [] 0.000301898 1392895_at [] 0.009676039

114

1393033_at Yars2 0.002790153 1393043_at Utp18 0.012721419 1393082_at Ppp1r14c 0.0115273 1393105_at Zfp259 4.52E‐05 1393128_at Gpatch4 0.008391321 1393156_at [] 0.003538728 1393202_a_at Igf2bp3 3.32E‐05 1393216_at Slc33a1 0.034875751 1393222_at Vps18 0.001880288 1393224_at [] 0.004449129 1393240_at Efemp2 0.000163794 1393265_at Syncrip 0.003863096 1393273_at [] 0.00500536 1393392_at [] 0.000434637 1393434_at [] 0.006038368 1393473_at RGD1563866 0.001015008 1393484_at [] 0.000828147 1393524_at [] 0.005334497 1393537_at [] 0.000713646 1393539_at [] 0.000108421 1393553_at [] 0.010441959 1393561_at [] 0.000218153 1393604_at Gpn3 0.00681603 1393608_at [] 0.000961244 1393652_at Zbtb1 0.002379537 1393655_at Rad54l 4.71E‐05 1393658_at [] 0.008906961 1393664_at [] 0.000770092 1393687_at [] 5.81E‐05 1393703_at Hivep3 0.007114589 1393721_at Zc3h11a 1.04E‐05 1393722_at Fem1c 0.013945341 1393818_s_at Fam71e1 8.94E‐06 1393876_at Hoxc5 5.30E‐06 1393896_at Lamc3 8.23E‐05 1393937_at [] 0.0134691 1393967_at [] 0.003774941 1393970_at Kdm4d 0.004053235 1393983_at Xpot 0.006651759 1393996_at Dnajb14 0.026067138 1394010_at Rsf1 6.47E‐05 1394095_at Gin1 0.000514627 1394130_at Trim44 0.034262538

115

1394134_at Dock8 0.001782239 1394212_at RGD1306487 0.000742018 1394241_at Clip1 0.047787189 1394286_at [] 0.002502263 1394361_a_at Wnt2 0.000899434 1394464_at LOC690830 0.003205597 1394466_at [] 9.44E‐05 1394491_at [] 0.000738263 1394519_at [] 4.34E‐05 1394535_at Kif16b 0.006794453 1394550_at LOC691221 0.002514839 1394556_at [] 0.003661037 1394627_at Snx19 0.000386596 1394674_at [] 0.000551641 Ercc4 /// Ercc4l1 /// LOC100365348 /// 1394686_at LOC688631 0.001551449 1394787_at RGD1309138 0.001985014 1394820_at [] 0.005373776 1394835_at [] 0.000229478 1394843_at [] 0.002751887 1394915_at [] 0.010046244 1394957_at Nisch 0.011309385 1394970_at Cars 0.002482593 1394972_at Trove2 0.015680909 1395007_at Znf23 2.26E‐06 1395022_at [] 0.002438784 1395035_at [] 0.008091033 1395037_at Fhad1 0.000351191 1395064_at RGD1564943 0.002959132 1395092_at [] 0.018346012 1395132_at Utrn 0.000876486 1395139_at Kif16b 0.000999808 1395217_at [] 0.000293016 1395233_at [] 0.000237465 1395240_at [] 0.000203311 1395247_at [] 0.015544534 1395254_at [] 0.002938151 1395289_at Vps26b 0.00011456 1395368_at [] 0.00434947 1395383_at RGD1311066 0.026441336 1395398_at [] 0.003228068 1395425_at Cpsf6 0.005938411

116

1395429_at Chrna7 0.009826481 1395452_at [] 0.003713369 1395455_at Hnrnph3 0.006790042 1395550_at RGD1565629 0.000645757 1395642_at Nol9 0.000715554 1395689_at Fam169a 0.000119209 1395705_at [] 0.009849191 1395729_at [] 0.001860738 1395733_at [] 0.000157058 1395737_at Cd276 0.000416636 1395740_at [] 0.002391756 1395761_at [] 0.04085964 1395769_at [] 0.000845551 1395770_at [] 0.012834668 1395777_at [] 0.000913858 1395790_at Zbtb33 0.01985085 1395799_at Itsn2 0.008963585 1395826_at Ppp2r5e 0.007959068 1395842_at [] 0.01517725 1395876_at [] 0.004579306 1395915_at [] 0.000968933 1395918_at [] 0.014375687 1395956_at [] 0.013106227 1395979_at [] 8.26E‐05 1396007_at [] 0.002331734 1396008_at [] 0.046251833 1396009_at [] 0.006680131 1396012_at [] 0.009266257 1396021_at Zfp68 0.000142455 1396041_at [] 0.006415486 1396050_at [] 9.29E‐05 1396065_at [] 2.49E‐05 1396081_at Spire1 0.00782913 1396105_at LOC688133 0.027156293 1396116_at Sdccag1 0.000427663 1396194_at [] 9.71E‐05 1396211_at [] 0.013161063 1396226_at [] 0.000450194 1396255_at Kif16b 0.002487063 1396271_at [] 0.001774192 1396283_at [] 0.002377749 1396314_at [] 0.00162077 1396380_at [] 0.001671076

117

1396413_at [] 0.024018764 1396441_at [] 0.008190811 1396444_at Mrpl12 0.002015948 1396445_at Ribc2 0.006281853 1396474_at [] 0.001509786 1396486_x_at RGD1564162 1.30E‐05 1396490_at [] 0.003066719 1396505_at [] 0.00079155 1396509_at Tmem132e 0.012963533 1396524_at [] 0.000692844 1396559_at [] 0.000204384 1396563_at [] 0.009714723 1396589_at [] 7.63E‐06 1396610_at [] 0.000276208 1396611_at [] 0.004573882 1396693_at [] 0.000570536 1396709_at [] 0.001190066 1396717_at [] 0.002042174 1396771_at [] 0.0084939 1396817_at [] 0.013184667 1396823_at [] 0.00664717 1396856_at [] 0.000171661 1396858_at [] 7.75E‐05 1396859_at Zfp423 0.000138164 1396876_at [] 0.015997171 1396883_at [] 0.000233591 1396921_at [] 0.025526524 1396934_at [] 0.001007915 1396936_at [] 0.015004814 1397008_at [] 0.031565309 1397017_at [] 0.000271857 1397029_at [] 0.00067991 1397089_at [] 0.002055168 1397134_at [] 0.000127256 1397181_at [] 6.22E‐05 1397198_at [] 0.028091788 1397211_at Grb10 0.009609759 1397234_at Gpatch1 0.000942528 1397328_at [] 0.020958185 1397370_at [] 0.006444454 1397382_at [] 0.020362735 1397403_at [] 0.010566235 1397411_at Fgd2 0.001539469

118

1397415_at Atl1 0.005794585 1397428_at [] 0.00849247 1397438_at Magi1 0.021267414 1397444_at [] 0.008049786 1397553_s_at Prdm2 0.008084774 1397621_at [] 0.001487732 1397626_at [] 0.009112597 1397628_at RGD1305592 0.009815395 1397699_at Dak 0.005506396 1397793_at [] 0.008848369 1397884_at Sel1l3 0.034209907 1397885_at [] 7.09E‐05 1397953_at LOC687696 0.006825507 1397955_at Rtel1 0.005620062 1397980_at [] 0.008610845 1398031_at Pyroxd2 0.002680183 1398047_at [] 0.009965003 1398053_at [] 0.008037031 1398074_at [] 3.89E‐05 1398120_at [] 0.001009822 1398179_at [] 0.025968194 1398187_at [] 0.008674145 1398203_at [] 0.001014411 1398271_at Pclo 0.001160324 1398285_at Ak3l1 0.004443348 1398287_at Plau 0.000665069 1398337_at LOC290341 0.007866204 1398341_at Cisd3 0.001227737 1398360_at Elovl1 0.015466332 1398372_at Rmnd5b 0.012441516 1398386_at Zcchc6 0.000175416 1398388_at [] 0.00065589 1398416_at Rnasen 0.001057506 1398433_at [] 4.18E‐05 1398482_at Bcl3 0.020232022 1398534_at [] 7.93E‐05 1398582_at Rps6ka5 0.000285208 1398623_at Chrnb4 7.11E‐05 1398626_s_at Actr2 4.03E‐05 1398635_at [] 0.002076864 1398655_at Myod1 0.002256632 1398772_at Nsfl1c 0.000209749 1398808_at Impa1 0.005128741

119

1398809_at Nde1 0.010137081 1398907_at Ormdl2 0.006978154 1398928_at Cuta 0.000249684 1398976_at Ncor1 0.016197681 1398984_at Tm2d2 0.007125139 1399002_at Mrps17 0.002340913 1399008_at Ankrd17 0.002304971 1399010_at Cops7a 0.00224936 1399011_at Cops6 0.002686262 1399036_at Usp47 0.021030128 1399059_at Ezh1 0.000744462 1399097_at [] 0.000252545 1399110_at Zbtb22 0.006830692 1399114_at Gtf2e2 0.002591252

7.2 6 hour in vitro TBI

1367731_at Gnb1 0.00246805 1367806_at Gls 0.001096487 1367817_at Hdgf 9.15E‐05 1367836_at Cpt1a 0.005653262 1367882_at Map1a 0.001004994 1367910_at Smad4 0.00145179 1367970_at Pfn2 0.005036235 1367975_at Anxa3 0.003546894 1367984_at Scaf1 0.000554442 1368030_at Gnai3 0.001154482 1368046_at Slc31a1 0.006314755 1368066_at Bak1 0.001150012 1368290_at Cyr61 0.015582442 1368319_a_at Homer1 0.008225203 1368332_at Gbp2 0.000814557 1368347_at Col5a3 0.000465989 1368398_at Cacna1h 0.003961325 1368401_at Gria2 0.000154972 1368411_a_at Map2 4.69E‐05 1368439_at Sox10 0.000454068 1368506_at Rgs4 0.001070976 1368519_at Serpine1 0.045165539 1368646_at Mapk9 0.002088964 1368703_at Pdlim5 0.000639856

120

1368814_at Aldh6a1 0.000561714 1368853_at Vsnl1 0.005664349 1368864_at Synpr 0.001809061 1368866_at Eif2c2 0.000972033 1368867_at Eif2c2 0.000121295 1368883_at Nov 0.000231147 1368912_at Trh 0.025995255 1368930_at Kcnn4 5.07E‐05 1368943_at Rnase4 0.004762888 1368944_at Dlg1 0.019716442 1368948_at Msn 0.012722194 1368976_at Cd38 0.001794457 1368981_at Aqp4 0.00103569 1368982_at Pkia 0.004415035 1368984_at 2‐Sep 0.000498652 1369002_at Soat1 0.002978981 1369007_at Nr4a2 0.003471136 1369026_at Arfip1 0.003020704 1369041_at Nlgn1 0.002638578 1369067_at Nr4a3 0.001074851 1369077_at Asah1 0.004702926 1369078_at Mapk1 0.000858963 1369083_at Cirbp 0.000824928 1369099_at Slc30a1 0.004986644 1369110_x_at RT1‐EC2 0.010033667 1369129_at Rasgrp1 0.001083076 1369130_at Rasgrp1 0.000706136 1369152_at Ppp3r1 0.010794461 1369172_at Pde1a 0.002352238 1369182_at F3 0.000621498 1369202_at Mx2 0.000833392 1369220_at Dnm1l 0.002357006 1369227_at Chm 0.000833988 1369248_a_at Xiap 0.005027473 1369265_at Senp2 0.013683677 1369269_at Galnt1 0.00106597 1369290_at Ccr5 0.00234592 1369297_at Ppp2r2c 0.021406531 1369311_at Scn2b 0.001557827 1369312_a_at Csnk1a1 0.001440466 1369323_at Leprot 0.002955318 1369332_a_at Rims1 0.025503993 1369344_at Wdr7 0.00131017

121

1369378_at Slc23a2 0.009588718 1369402_at Adnp 0.00335747 1369409_at Nab1 0.005582452 1369415_at Bhlhe40 0.020168662 1369417_a_at Opcml 0.007524192 1369421_at Top1 0.000873148 1369427_at Mpeg1 0.030987978 1369452_a_at Picalm 0.008292913 1369462_at Gad2 0.001048148 1369472_a_at Atf2 0.001380503 1369501_at Zfp260 0.001423121 1369526_at Acadsb 0.002730131 1369541_at Tmod2 0.000474095 1369571_at Golph3 0.001337707 1369610_at Lin7c 0.003299177 1369614_at Rap2b 0.008665085 1369627_at Sv2b 0.003956795 1369640_at Gja1 0.00194031 1369648_at Calcrl 0.004167795 1369650_at Pak2 0.000636697 1369689_at Nsf 0.010241508 1369718_at Ssr3 0.001331806 1369721_at Strn 0.004890144 1369733_at Ctnnb1 0.001850784 1369756_a_at Slc4a4 0.00063777 1369758_at Gpam 0.004957855 1369791_at Clcn4‐2 0.001223564 1369794_a_at Pfkfb3 0.006181717 1369857_a_at Slc14a1 0.002209902 1369868_at Magt1 0.001318395 1369903_at Gabrb3 0.001836598 1369905_at Gabra4 0.001483083 1369912_at Crk 0.001530886 1369942_at Actn4 2.96E‐05 1370114_a_at Pik3r1 0.002401948 1370131_at Cav1 0.003698707 Cav2 /// 1370135_at LOC100362824 0.004582405 1370146_at Glrb 0.002712607 1370166_at Sdc2 0.001796067 1370214_at Pvalb 0.001666546 1370430_at RGD1561357 0.002439618 1370501_at Ube2g1 0.00274837

122

1370513_at Tpm1 0.001366437 1370529_a_at Pld1 0.001511931 1370541_at Nr1d2 0.002319098 1370543_at Ogt 0.003043056 1370595_a_at Kcnip4 0.00413239 1370630_a_at Fgfr2 0.012322307 1370656_a_at Homer1 0.006320775 1370668_a_at Cnksr2 0.000222206 1370688_at Gclc 0.006313264 1370690_at Hspa9 0.017690241 1370728_at Il13ra1 0.000654817 1370746_at Prkacb 0.000918031 1370760_a_at Gad1 0.00765717 1370780_at Rab31 0.002408683 1370913_at Rsad2 0.006830812 1370950_at Ppap2b 0.001744688 1370955_at Adam10 0.000420988 1370956_at Dcn 0.008432686 1371007_at Epha5 0.000725091 1371015_at Mx1 0.029924929 1371019_at Trib1 0.015397131 1371024_at Cux1 0.000438273 1371028_at Tgoln1 0.012764633 1371033_at RT1‐Bb 0.017652154 1371038_at Cebpg 0.002098084 1371042_at Map4k3 0.002105951 1371055_at Rab12 0.003426969 1371059_at Prkar2a 0.000922143 1371060_at Trim23 0.00228411 1371063_at Sh3gl2 0.003380179 1371103_at Rab6a 0.001139343 1371113_a_at Tfrc 0.012003243 1371139_at Pls3 0.003772557 1371173_a_at Cast 0.001100063 1371177_a_at Cask 0.002511263 1371179_a_at Fgfr2 0.006586194 1371186_at Itga6 0.000988424 1371209_at RT1‐CE5 0.011189938 1371263_a_at Camk2d 0.000690341 1371442_at Hyou1 0.002251685 1371687_at Canx 0.001048207 1371703_at Ahnak 0.000542045 1371859_at Poldip3 0.000924349

123

1371910_at [] 0.001042604 1372336_at [] 0.000651956 1372427_at Raver1 0.000753343 1372481_at [] 0.004383147 1372935_at Tmem119 0.000775695 1372977_at Map4k5 0.002611101 1373054_at Slc44a1 0.00044477 1373257_at Arpp‐21 0.00480628 1373303_at Sfrs2ip 0.000118256 1373321_at RGD1306622 0.000880718 1373358_at Usp8 0.009169161 1373401_at Tnc 0.000716746 1373508_at Selt 0.001742125 1373514_at Rnf213 0.002614617 1373601_at [] 0.002237678 1373717_at [] 0.000928879 1373811_at Rap1gds1 0.005216539 1374263_at [] 0.038180172 1374283_at Falz 0.001317501 1374337_at Rnf213 0.002162516 1374367_at Grxcr1 0.026899695 1374655_at [] 0.01083523 1374684_at [] 0.000422597 1374786_at [] 0.017619491 1375120_at Id4 0.001989782 1375130_at LOC679612 0.001450777 1375176_at Fam172a 0.000398457 1375193_at Lrp11 0.008747339 1375194_at [] 0.006468713 1375254_at Slc35a1 0.001641214 1375267_at Ppic 0.0024122 1375271_at Nek9 0.006128728 1375294_at [] 0.000489473 1375319_at [] 0.001223862 1375331_at [] 0.00521189 1375342_at Nfic 0.000142872 1375343_at [] 0.002547979 1375374_at [] 0.00311321 1375382_at Mdc1 0.000219047 1375388_at [] 0.001051784 1375402_at [] 0.001321912 1375421_a_at Pja2 0.003268123 1375449_at [] 0.042800069

124

1375459_at Srpk2 0.000291228 1375532_at [] 0.011156559 1375535_at [] 0.000758767 1375538_at [] 0.003908813 1375696_at Ifnar1 0.003446281 1375754_at Impact 0.001317263 1375765_at [] 0.000948727 1375777_at [] 0.007701874 1375783_at [] 0.008316159 1375793_at [] 0.000259876 1375829_at Fam45a 0.004190028 1375862_at Pxdn 4.20E‐05 1376009_at [] 0.00175488 1376137_at Plekhb2 0.013720512 1376157_at Uba6 0.002737284 1376224_at Elfn2 0.000524044 1376251_at [] 0.00714004 1376344_at [] 0.01860851 1376388_at [] 0.002753258 1376463_at [] 0.003204525 1376490_at [] 0.000246346 1376531_at [] 0.012204766 1376568_at [] 0.000609159 1376727_at Yipf4 0.006161451 1376755_at Rarb 0.002023578 1376780_at Fam103a1 0.007688522 1376867_at Hspc159 0.004255593 1376910_at Znf536 6.63E‐05 1376938_at [] 0.000222206 1377029_at [] 2.71E‐05 1377133_at LOC680687 1.67E‐02 1377151_at [] 0.009653568 1377193_at [] 0.018769264 1377208_at [] 0.001266718 1377305_at [] 0.002763152 1377352_at [] 0.002590895 1377403_at [] 0.007172108 1377451_at RGD1305704 0.026445448 1377614_at Ino80e 0.001885653 1377633_a_at Stt3a 0.001146078 1377694_at Luc7l3 0.009319186 1377784_at [] 0.007615984 1377871_at [] 0.000149131

125

1377928_at Tm4sf20 0.006832004 1377947_at [] 0.002335548 1377985_at RGD1309198 0.017834961 1377992_at [] 0.044265389 1378009_at [] 0.002855897 1378189_at [] 0.045068204 1378232_at Clcn3 0.032679498 1378238_at Anapc1 0.014977336 1378279_at Gabarap 0.019740403 1378412_at [] 0.000852764 1378460_at [] 0.013381481 1378566_at [] 0.007578135 1378639_at Snupn 0.010471702 1378656_at Bbs5 0.015154064 1378678_at [] 0.00148952 1378688_at [] 0.000644445 1378701_at [] 0.000134885 1378702_at [] 0.00247097 1378705_at Setd5 1.82E‐02 1378717_at [] 7.39E‐05 1378754_at [] 0.003576815 1378832_at [] 0.012443781 1378877_at [] 3.39E‐02 1378937_at [] 0.001161575 1378945_at [] 0.001306295 1378959_at [] 0.044058919 1378968_at Bcl2l14 0.004151225 1378969_at Mast4 0.000489712 1379011_at [] 0.003844082 1379029_at [] 0.01202637 1379066_at Slc5a3 0.001668096 1379073_at Slc25a23 0.001615942 1379076_at [] 0.003777862 1379078_at [] 0.004204691 1379083_at Adat3 0.002278686 1379141_at [] 0.005336702 1379187_at [] 0.000288129 1379201_at Dhx8 0.002658725 1379210_at [] 0.001448035 1379212_at [] 0.005104303 1379226_at Aim1l 0.000205159 1379233_at [] 0.000349998 1379234_a_at Cdc45l 0.002022386

126

1379235_x_at Cdc45l 0.02199173 1379262_at Acot9 0.000974178 1379278_at Ints9 0.007938504 1379279_at Stim2 0.009366989 1379309_at Tbl1xr1 0.011333525 1379327_at Smarca5 0.002035141 1379329_at LOC685729 0.002625346 1379357_at [] 0.000554323 1379358_at Samd4a 0.00017041 1379371_at [] 0.041908145 1379411_at [] 0.015308321 1379413_at Nmnat1 0.000592649 1379422_at Alg13 0.008084536 1379451_at [] 0.005768716 1379501_at [] 0.003655136 1379570_at Fbll1 0.000832677 1379572_at Mab21l1 7.03E‐05 1379594_at [] 0.001208127 1379602_at [] 0.000851691 1379633_a_at Uba7 0.000523806 1379636_at Fam82a1 0.000585079 1379661_at [] 0.003935933 1379676_a_at Dnase1l1 0.000631869 1379697_at [] 0.012558222 1379729_at Cpsf6 0.002943635 1379733_at [] 0.009916782 1379761_at [] 0.000921845 1379823_at [] 0.001776636 1379848_at Dfnb59 0.00665313 1379871_at Rfx7 0.001178503 1380068_at [] 0.001484096 1380345_at [] 0.013439178 1380398_at [] 0.000604808 1380422_at RGD1560686 0.000188708 1380614_at [] 0.001265764 1380677_at [] 0.011164308 1380704_a_at [] 0.001507878 1381306_at [] 0.007155776 1381396_s_at Klf15 0.012948275 1381446_at Fam75a4 0.003644407 1381499_at [] 0.000179768 1381546_at [] 0.000886321 1381561_at [] 0.04157871

127

1381619_at [] 0.00104773 1381621_at [] 0.006377399 1381622_at [] 0.001768291 1381724_at [] 0.003165722 1381843_at [] 0.003296971 1381874_at Sox2 0.004992306 1381887_x_at RGD1311595 0.00194788 1381892_at Nav2 0.004678071 1382024_at Dnajb6 0.005176902 1382203_at Gdf1 /// Lass1 0.013535976 1382217_at [] 0.003674746 1382295_at [] 0.003784239 1382298_at [] 0.000549614 1382375_at [] 0.012692213 1382401_at [] 0.005726278 1382413_at Nup155 0.001482368 1382434_at Entpd5 0.002728462 1382452_at Sdpr 0.000141621 1382478_at Btbd3 0.002165675 1382525_at Lypd6b 0.006294489 1382543_at [] 0.008589387 1382546_at Phf11 0.002799213 1382586_at [] 0.00096339 1382724_at Pigo 0.002238035 1382865_at Tsga14 0.00154525 1382974_at [] 0.012460172 1382976_at [] 0.013792753 1383212_at [] 0.000495672 1383215_at LOC304558 0.001473904 1383355_at Abca1 0.006722808 1383426_at Pstpip1 0.001521766 1383448_at Irf9 0.001349986 1383497_at [] 0.001424789 1383513_at [] 0.001830995 1383526_at [] 0.032216012 1383634_at Zfp653 0.000195622 1383682_at [] 0.005048037 1383691_at [] 0.001553178 1383706_at RGD1305733 0.003028274 1383775_at [] 0.006060302 1383780_at Hectd1 0.031061172 1383844_at Arhgef15 0.007946909 1383870_at Hoxb6 0.0149979

128

1383872_at [] 0.006247759 1383881_at [] 0.000349104 1383930_at [] 0.004101932 1383957_at Nudt3 0.005106568 1384134_at [] 0.007217467 1384146_at [] 0.002421796 1384213_at Pdcd6ip 0.000259757 1384250_a_at [] 0.000906348 1384315_at Pdk3 0.017660975 1384345_at Ptgr2 0.000790834 1384364_at Fbxo4 0.001028121 1384485_at Ptpru 0.000726938 1384657_at Mfhas1 0.001526475 1384674_at [] 0.004185915 1384693_at [] 0.000556469 1384747_at [] 0.000460505 1384776_x_at RGD1564722 0.002004266 1384792_at Prpf40a 0.01374197 1384800_at Zic3 0.001212716 1384836_at Calml3 0.002155066 1384843_at Spaca3 0.001906395 1384861_at [] 0.006977737 1384949_at [] 0.002102435 1385027_at [] 0.00669837 1385055_at RGD1563458 0.000114381 1385108_at [] 0.001254857 1385117_at [] 0.00047493 1385132_at [] 0.000630498 1385193_at [] 0.005895555 1385194_at Rb1cc1 0.001926899 1385201_at Tmem182 0.002968848 1385249_at [] 2.22E‐05 1385316_at Pvrl3 0.003542364 1385333_at Aatk 0.000581563 LOC100364003 /// 1385338_at Zfp758 0.006553888 1385412_at LOC100364488 0.016429305 1385431_at [] 0.001754403 1385438_at ND4 0.001080155 1385450_at Ucn2 0.011505604 1385458_a_at RGD1306959 0.003160954 1385469_at Igf2bp1 0.016461909 1385474_at Rnf160 0.001573563

129

1385485_x_at [] 0.004520237 1385506_at [] 0.00223726 1385525_at Clec1b 0.000877142 1385578_at [] 0.000827551 1385603_at [] 0.003115892 1385605_at Gpr84 0.000435114 1385631_at [] 0.003724694 1385665_at Adam19 0.0004673 1385667_x_at [] 0.000527263 1385673_at Rpain 0.011158347 1385724_at Ccdc74a 0.002753854 1385725_at LOC684444 0.004138112 1385770_at Ulk4 0.000722408 1385788_at Ephb3 0.000841498 1385817_at Nek4 0.010620713 1385990_at RGD1565545 0.002320707 1385999_at Yme1l1 0.000290751 1386013_at LOC100359753 0.002952218 1386016_at Hspb11 0.003007531 1386031_at Prkd3 0.006000757 1386047_at Galnt13 0.00364852 1386215_at Ggps1 0.000324845 1386295_at [] 0.018062055 1386497_at [] 0.001592696 1386543_at Fbxl8 0.00394845 1386573_a_at [] 0.002579093 1386592_at [] 0.00385344 1386610_at Rcbtb2 7.33E‐05 1386679_at [] 0.007199884 1386777_at Pot1a 0.002335787 1386822_s_at RGD1311823 0.001497209 1386837_x_at Znf574 0.002477527 1386884_at Htra1 0.002179325 1386907_at Eno3 0.000129223 1386935_at Nr4a1 0.000204742 1386940_at Timp2 0.000452578 1386962_at Plcb4 0.043771565 1386970_at Eif2b4 0.00589776 1386989_at S1pr2 0.000959277 1387002_at Bud31 0.001284957 1387043_at Lypd3 0.0207057 1387087_at Cebpb 0.001573086 1387115_at Ikbkap 0.000586271

130

1387149_at Erap1 0.007631421 1387167_at Slc7a1 0.000259101 1387190_at Dgka 0.003305912 1387198_at Inpp5d 0.000724792 1387219_at Adm 0.003922343 1387230_at Slc12a3 0.00159061 1387239_a_at Padi4 0.023764968 1387264_at Kcnk6 0.00222677 1387423_at Lhcgr 0.001139641 1387428_at Cacnb1 0.003533244 1387450_at Tgfa 0.001897931 1387482_at Grid2 0.016992211 1387538_at Acaca 0.001977503 1387570_at Manea 0.005882621 1387602_a_at Htr3b 0.001029968 1387615_at St8sia2 0.028636456 1387630_at Elovl5 0.018081844 1387639_at Hpcal4 0.001258373 1387644_at Btc 0.001574755 1387648_at Cxcl6 0.006537139 1387658_at Eef2k 0.002906382 1387661_a_at Oprl1 0.000332952 1387670_at Gpd2 0.001165211 1387671_at Sctr 0.000270486 1387674_at Cnr1 0.008828521 1387707_at Slc2a3 0.006997407 1387743_at Gpr20 0.012178957 Acaa1 /// 1387783_a_at RGD1562373 0.001607537 1388001_at Lrit1 0.001169682 1388018_at Sele 0.003862381 1388038_at Atrn 9.27E‐05 Eef1a1 /// LOC100360150 /// LOC100360413 /// 1388110_at RGD1566344 0.000929534 1388159_at CYTB 0.011088014 1388210_at Acot2 0.000625551 1388241_at Insl3 0.002915263 1388243_at Gpr176 0.031183124 1388253_at Scd 0.000374198 1388274_at Bmyc 0.012889147 1388289_s_at Fshb 0.009332538

131

1388410_at Ugp2 0.000832796 1388461_at Shd 0.002742589 1388463_at Tex264 0.000147104 1388472_at [] 0.000534058 1388560_at Wdr77 0.004122734 1388631_at Azi2 0.002677739 1388654_at Mrpl52 0.000924885 1388722_at Dnajb1 0.000623584 1388723_at Bre 0.000398576 1388751_at [] 0.005981326 1388757_at Adrbk1 0.006065011 1388840_at Plekhg2 0.008408427 1388872_at [] 0.000411928 1388873_at Trip12 0.002379119 1388877_at Mrps5 0.00213176 1388894_at [] 0.002665877 1388910_at Urg4 0.006782234 1388950_at Col9a2 0.004215717 1388955_at Ehd2 0.000175416 1388967_at Tcfe3 0.010777056 1388968_at Mpped1 0.000347793 1389079_at Dhrs7c 0.001491666 1389112_at Nkd1 0.001976371 1389245_at Psmd7 0.007332802 1389267_at [] 0.000179768 1389292_at Rab18 0.002646804 1389311_at [] 0.000122786 1389350_at Apoh 0.001583457 1389388_at [] 0.000670373 1389487_at Dnajb12 0.00236845 1389493_at Abtb1 0.0006091 1389545_at Tbl1xr1 0.008044124 1389620_at Suv420h2 0.000444531 1389675_at Ier5l 0.005604684 1389763_at [] 0.001223743 1389770_at Ttll9 0.003178477 1389804_at Relt 0.001036048 1389820_at Cox7c 0.023055553 1389828_at Cebpd 0.005403101 1389881_at [] 0.000362933 1389884_at Ccdc88b 0.000477493 1389906_at Fdft1 0.002826572 1389922_at Hadh 0.023051202

132

1389924_at Cpd 0.000679314 1389987_at Trib1 0.002177537 1390008_at Cited4 0.000534773 1390022_at Arpc5 6.90E‐05 1390069_at [] 0.004964352 1390082_at Hip1 0.001213074 1390099_at Ube3a 0.005551398 1390111_at Fam91a1 0.000277042 1390158_at LOC304903 0.001955986 1390296_at [] 0.000273705 1390299_at [] 0.003086329 1390322_at [] 0.000489473 1390345_at [] 0.000808001 1390373_at Smad5 0.003250837 1390400_a_at [] 0.000882745 1390454_at Nipsnap1 0.002878487 1390528_at [] 0.003312588 1390576_at LOC684233 0.002835155 1390591_at Slc17a3 0.002243757 1390603_at [] 0.000722826 1390615_at Kpna1 0.00319165 1390642_at [] 0.001121163 1390661_at [] 0.000284553 1390709_at [] 0.001650751 1390719_at [] 0.001236796 1390729_x_at Cpsf7 0.001101375 1390731_at [] 0.003113747 1390766_at Tuba8 0.00138092 1390831_at [] 0.00639075 1390864_at [] 0.000963986 1390957_at RGD1562018 0.00026083 1390975_at [] 0.011325479 1390990_at [] 0.000174165 1391011_at [] 0.00042069 1391028_at [] 0.000481963 1391040_at Rbbp8 0.001318216 1391072_at Fcer2 0.004585385 1391153_at [] 0.009251237 1391187_at Ppl 0.000703216 1391192_at [] 0.013457417 1391201_at Wdhd1 0.001398325 1391273_at Naglt1 0.002446175 1391362_at [] 0.001623035

133

1391416_at Ankrd17 0.002305985 1391476_at [] 0.000524044 1391479_at [] 0.002319813 1391506_at Phf17 0.001583695 1391523_at [] 0.009593487 1391531_at LOC691729 0.0033741 1391572_at Cars 0.002704322 1391629_at [] 0.003809691 1391635_at Ctdspl 0.007574678 1391666_at Aak1 0.003976226 1391695_at [] 0.000527322 1391718_at [] 0.002457142 1391719_at [] 0.002392054 1391743_at Elavl1 0.000320137 LOC100362394 /// 1391756_at LOC100365991 0.004148066 1391805_at Klhl32 0.000647664 1391904_at [] 0.002361298 1391920_at RGD1564722 0.004056096 1392134_x_at Srgap2 0.000853419 1392143_at [] 0.006617785 1392210_at RGD1307443 0.01272738 1392247_at [] 0.000770807 1392262_at [] 0.01938349 1392493_at [] 0.024593592 1392528_at Csf2ra 0.040073335 1392534_at Pmepa1 0.020190835 1392551_at LOC503192 0.001194 1392618_at [] 0.004115105 1392663_at RGD1305500 0.005250752 1392687_at Pigq 0.003253937 1392698_a_at Gtf3c4 0.011839867 1392788_at [] 0.003029466 1392794_at [] 0.001738071 1392822_at Nf1 0.000821054 1392861_at [] 0.005192936 1392870_at [] 0.000261486 1392904_at Pwwp2a 0.000926495 1392915_at Col11a1 0.001690686 1392932_at Leng8 0.000794411 1392940_at [] 0.004979909 1392967_at MGC116121 0.002656758 1392968_at [] 0.000292897

134

1393033_at Yars2 0.001669169 1393043_at Utp18 0.003242254 1393087_at Recql5 0.007552683 1393128_at Gpatch4 0.001920104 1393155_at Plk3 0.000864923 1393156_at [] 0.002102494 1393216_at Slc33a1 0.002085805 1393224_at [] 0.002878845 1393273_at [] 0.006405354 1393295_at LOC498662 0.000253797 1393340_at Mlxip 0.003794193 1393378_at [] 0.003964603 1393435_at Plch2 0.002374172 1393462_at Lrrc18 0.005373478 1393473_at RGD1563866 0.010747552 1393484_at [] 0.002281725 1393524_at [] 0.005380154 1393537_at [] 0.016501129 1393553_at [] 0.005039156 1393583_at Etaa1 0.000685871 1393604_at Gpn3 0.013916492 1393614_at Necab3 0.001241863 1393627_at Osap 0.001356483 1393653_at [] 0.002973616 1393658_at [] 0.002131462 1393703_at Hivep3 0.001272023 1393723_at Plk5 0.000451922 1393738_s_at Mfhas1 0.023844659 1393784_at [] 0.002991498 1393819_x_at [] 0.009070516 1393901_at Runx1t1 0.00243336 1393909_at RGD1305110 0.006526411 1393937_at [] 0.009726584 1393976_at Slbp 0.000136912 1393977_at [] 0.002077222 1393996_at Dnajb14 0.000604987 1394006_at Psme1 0.000181675 1394027_at Nup62cl 0.000419021 1394095_at Gin1 0.001541197 1394130_at Trim44 0.008618295 1394241_at Clip1 0.002959251 1394315_at LOC683788 0.006003022 1394367_at Dll4 0.000768721

135

1394406_at [] 0.000993848 1394524_at LOC688495 0.000568926 1394530_at [] 0.000311315 1394531_at [] 0.012309909 1394535_at Kif16b 0.002529025 1394542_at [] 0.000182271 1394544_at [] 0.006045342 1394556_at [] 0.001169205 1394635_at [] 0.001334071 1394660_at [] 0.000119567 1394717_at Tbc1d5 0.001716197 1394761_at Arhgap42 0.00158298 1394764_at Lmtk2 0.000977755 1394795_at [] 0.012460113 1394797_at [] 0.001222313 1394820_at [] 0.012293577 1394843_at [] 0.001113594 1394861_at [] 0.004932523 1394957_at Nisch 0.005465508 1394986_at Fam120b 0.000901222 1395035_at [] 0.001814961 1395037_at Fhad1 0.000521183 1395092_at [] 0.001641512 1395096_at [] 0.011619449 1395139_at Kif16b 0.002336741 1395231_at Cdca4 0.002495527 1395238_at [] 0.007912517 1395254_at [] 0.014603496 1395265_at Gns 0.000558138 1395293_at Sulf2 0.003034592 1395376_at Ddx11 0.001517057 1395383_at RGD1311066 0.027804077 1395393_at [] 0.003053188 1395398_at [] 0.000336945 1395400_at RGD1305508 0.000779331 1395410_at Ppp2r3a 0.002297342 1395415_at [] 0.000448644 1395425_at Cpsf6 0.03507024 1395452_at [] 0.000843108 1395506_at [] 0.001437843 1395560_at [] 0.000859022 1395594_at Wnt11 0.002316058 1395600_at Ccdc117 0.024347425

136

1395608_at [] 0.001265049 1395634_at [] 0.001060784 1395637_at Asphd2 0.001867473 LOC316460 /// 1395641_at LOC689991 0.002288759 1395656_at [] 0.001151264 1395679_at LOC681924 0.001558781 1395692_at [] 0.008696139 1395727_at Rabggta 0.0018242 1395740_at [] 0.005884349 1395761_at [] 0.007399797 1395769_at [] 0.000372171 1395770_at [] 0.001440406 1395777_at [] 0.003185391 1395790_at Zbtb33 0.018612385 1395799_at Itsn2 0.010279536 1395817_at [] 0.001425862 1395826_at Ppp2r5e 0.007553935 1395842_at [] 0.001067519 1395874_at [] 0.00525558 1395905_at [] 0.003527403 1395915_at [] 0.004657388 1395918_at [] 0.002749145 1395942_at [] 0.008385301 1395956_at [] 0.021211207 1395988_at Zdhhc22 0.000649929 1396004_at [] 0.030150592 1396007_at [] 0.002125084 1396008_at [] 0.002887726 1396009_at [] 0.002548933 1396012_at [] 0.002378345 1396026_at [] 0.006263673 1396034_at Ces7 0.005840004 1396039_at Slc22a12 0.002008915 1396046_at [] 0.001526177 1396081_at Spire1 0.002970696 1396188_at Rhoj 0.001259267 1396211_at [] 0.005579114 1396217_at [] 0.004874349 1396222_at [] 0.002190054 1396255_at Kif16b 0.003731191 1396259_at Frem3 0.020855963 1396299_at [] 0.000971437

137

1396309_at Tbc1d19 0.006566286 1396380_at [] 0.009898484 1396389_at LOC687758 0.007517338 1396413_at [] 0.001773357 1396414_at [] 0.001772761 1396433_at [] 0.000396848 1396438_at [] 0.002518058 1396441_at [] 0.014632881 1396475_at [] 0.007220149 1396478_at Tmem164 0.003559291 1396490_at [] 0.001338959 1396509_at Tmem132e 0.014152884 1396524_at [] 0.008339047 1396555_at [] 0.009562075 1396563_at [] 0.001291156 1396567_at [] 0.006053627 1396609_at [] 0.004618645 1396761_at [] 0.002054095 1396769_at [] 0.000106096 1396771_at [] 0.014846087 1396786_at [] 0.011684597 1396793_at Ppfia2 0.00121522 1396797_at Nova2 0.003308475 1396817_at [] 0.005081177 1396823_at [] 0.000756621 1396826_at [] 0.00608933 1396842_at Atg9a 0.003022552 1396876_at [] 0.001571953 1396902_at [] 0.004127443 1396923_at Ophn1 0.002758384 1396926_at Vac14 0.0017488 1396931_at [] 0.001413107 1396936_at [] 0.003038764 1396948_at [] 0.004407346 1397047_at [] 0.006257653 1397198_at [] 0.001421154 1397199_at [] 0.000195622 1397279_at Itgb5 0.008472741 1397299_at Frmpd1 0.000147104 1397328_at [] 0.006850541 1397329_at [] 0.016378522 1397370_at [] 0.020618439 1397382_at [] 0.036411881

138

1397403_at [] 0.001940489 1397411_at Fgd2 0.005738616 1397413_at [] 0.000125587 1397415_at Atl1 0.011216283 1397428_at [] 0.004910588 1397438_at Magi1 0.003728092 1397440_at Megf10 0.004477739 1397442_at LOC100359695 0.003978908 1397444_at [] 0.00586611 1397546_at Spats2 0.000947356 1397553_s_at Prdm2 0.012437344 1397621_at [] 0.026028156 1397626_at [] 0.020248234 1397628_at RGD1305592 0.003575444 1397634_at Anks1a 0.00893265 1397673_at [] 0.00231719 1397675_at Eif4h 0.00109911 1397714_at RGD1307235 0.004891574 1397793_at [] 0.012711942 1397831_at [] 0.006685615 1397864_at [] 0.004288912 1397884_at Sel1l3 0.005305827 1397953_at LOC687696 0.014431119 1397955_at Rtel1 0.00024873 1397974_at [] 0.00336057 1397980_at [] 0.00319469 1397993_at [] 0.008232534 1398015_at [] 0.003632247 1398031_at Pyroxd2 0.018066168 1398039_at [] 0.000938416 1398053_at [] 0.001412451 1398065_at Sobpl 0.00542438 1398110_at [] 0.008492112 1398135_at [] 0.004204214 1398151_at [] 0.001554966 1398156_at [] 0.000408828 1398170_at [] 0.00104779 1398179_at [] 0.000318289 1398183_at [] 0.011043787 1398187_at [] 0.005777955 1398190_at [] 0.016579151 1398224_at [] 0.002299368 1398236_at [] 0.012034774

139

1398271_at Pclo 0.00054431 1398275_at Mmp9 0.001136303 1398281_at Ocln 0.002150297 1398291_at Slc26a5 5.98E‐05 1398336_at Rnf25 0.000729263 1398337_at LOC290341 0.006356418 1398341_at Cisd3 0.004728138 1398355_at Trpm7 0.001615346 1398360_at Elovl1 0.001463413 1398372_at Rmnd5b 0.004276812 1398482_at Bcl3 0.000832796 1398491_at Ildr1 0.009285212 1398510_at [] 0.002142668 1398574_a_at Cdc2l5 0.001389802 1398627_at [] 0.000749052 1398655_at Myod1 0.003774405 1398710_at Cyp2u1 0.00015372 1398808_at Impa1 0.006492257 1398809_at Nde1 0.008769572 1398867_at Prpf19 0.010698915 1398903_at Esd 0.000462294 1398907_at Ormdl2 0.002084374 1398935_at [] 0.002426088 1398976_at Ncor1 0.000250578 1398984_at Tm2d2 0.00153029 1399010_at Cops7a 0.005475164 1399011_at Cops6 0.013940036 1399013_at Tmem85 0.005586922 1399024_at Scyl1 0.008061826 1399035_at Polr3h 0.004209042 1399036_at Usp47 0.005510747 1399051_at Patl1 0.00202924 1399098_at Glo1 0.022911012

7.3 12 hour in vitro TBI

Affymetrix ID Gene Symbol p‐value 1367581_a_at Spp1 0.007123828 1367619_at Pgrmc1 0.004737973 1367633_at Glul 0.000162363 1367657_at Btg1 0.00603652

140

1367749_at Lum 0.010421872 1367795_at Ifrd1 0.001832843 1367806_at Gls 0.001307428 1367814_at Atp1b1 0.024078488 1367910_at Smad4 0.006399751 1367942_at Acp5 0.001831353 1367970_at Pfn2 0.016461015 1367977_at Snca 0.001164317 1368030_at Gnai3 0.009614468 1368044_at Scg2 0.002584815 1368059_at Crym 0.001216888 1368082_at Slc4a2 0.001713395 1368114_at Fgf13 0.004582822 1368124_at Dusp5 0.004668474 1368144_at Rgs2 0.000179172 1368145_at Pcp4 0.004475772 1368151_at Matr3 0.026235104 1368240_a_at Prkcb 0.0043571 1368247_at Hspa1a /// Hspa1b 0.003062248 1368256_at Serpini1 0.041519165 1368271_a_at Fabp4 0.034645379 1368290_at Cyr61 0.02367717 1368373_at Rgs7 0.00159663 1368398_at Cacna1h 0.001359105 1368411_a_at Map2 0.00011003 1368439_at Sox10 0.006716609 1368450_at Myo5a 0.000118852 1368505_at Rgs4 0.009220481 1368506_at Rgs4 0.000393152 1368513_at Enpep 0.002339125 1368527_at Ptgs2 0.001429319 1368564_at Slc17a6 0.001636088 1368565_at Slc1a3 0.033590674 1368632_at Foxg1 0.029254794 1368646_at Mapk9 0.038814068 1368677_at Bdnf 0.000517011 1368716_at Ppp1r14c 0.000613928 1368799_at Birc5 0.014424264 1368814_at Aldh6a1 0.002367497 1368853_at Vsnl1 0.000625193 1368854_at Vsnl1 0.01695919 1368856_at Jak2 0.000538945 1368858_at Ugt8 0.000366628

141

1368864_at Synpr 0.000386477 1368883_at Nov 0.000216722 1368908_at Anxa4 0.001591563 1368912_at Trh 0.000974119 1368925_a_at Arhgef7 0.000445724 1368943_at Rnase4 0.005817592 1368944_at Dlg1 0.002138138 1368956_at Pcdh8 0.000751913 1368964_at Lrrn3 0.004324198 1368975_at Cd38 0.001405001 1368981_at Aqp4 0.003760219 1368982_at Pkia 0.002013803 1368984_at 2‐Sep 0.030240536 1369002_at Soat1 0.006664276 1369007_at Nr4a2 0.000657082 1369008_a_at Olfm1 0.002504468 1369026_at Arfip1 0.000631273 1369041_at Nlgn1 0.000914216 1369067_at Nr4a3 0.003473103 1369077_at Asah1 0.001810789 1369078_at Mapk1 0.006386399 1369079_at Nxph1 0.000655532 1369083_at Cirbp 0.000445426 1369085_s_at Snrpn /// Snurf 0.000126302 1369099_at Slc30a1 0.005123615 1369117_at Calca 0.000202477 1369129_at Rasgrp1 0.000707686 1369130_at Rasgrp1 0.001315057 1369151_at Dlk1 0.001437783 1369152_at Ppp3r1 0.016082048 1369220_at Dnm1l 0.004269958 1369227_at Chm 0.003925026 1369248_a_at Xiap 0.003846884 1369265_at Senp2 0.002793074 1369269_at Galnt1 0.019899607 1369285_at Pggt1b 0.000807464 1369312_a_at Csnk1a1 0.002754211 1369344_at Wdr7 0.002423048 1369378_at Slc23a2 0.004887343 1369402_at Adnp 0.047737062 1369409_at Nab1 0.01407361 1369415_at Bhlhe40 0.001144528 1369417_a_at Opcml 0.049217701

142

1369421_at Top1 0.023566723 1369472_a_at Atf2 0.001477122 1369482_a_at Syn2 0.000134707 1369501_at Zfp260 0.005567133 1369526_at Acadsb 0.016268671 1369541_at Tmod2 0.015894175 1369571_at Golph3 0.003426909 1369610_at Lin7c 0.004633606 1369627_at Sv2b 0.009275913 1369640_at Gja1 0.011856914 1369651_at Thy1 0.00022459 1369677_at Cnr1 0.00600636 1369686_at Dclk1 0.005055785 1369689_at Nsf 0.002023399 1369690_at Nsf 0.000103891 1369718_at Ssr3 0.019362807 1369731_at Cnksr2 0.020800889 1369756_a_at Slc4a4 0.001074433 1369770_at Sstr1 0.000611663 1369781_at Grm7 0.001290679 1369791_at Clcn4‐2 0.001993775 1369813_at Dnajc5 0.001052916 1369822_at Kit 0.000787497 1369897_s_at Gnas 0.043980539 1369903_at Gabrb3 0.004409134 1369905_at Gabra4 0.001167536 1369912_at Crk 0.026158571 1369977_at Uchl1 0.006204367 1370024_at Fabp7 0.036496818 1370041_at Stmn2 0.007994056 1370042_at Stmn2 0.000324845 1370058_at Nefl 0.003121316 1370059_at Nefl 0.000523329 1370081_a_at Vegfa 0.000329137 Eef1a1 /// LOC100360150 /// 1370109_s_at LOC100360413 0.036296427 1370114_a_at Pik3r1 0.001980841 1370116_at 3‐Sep 0.000207961 1370125_at Hapln1 0.000545621 1370131_at Cav1 0.009452999 Cav2 /// 1370135_at LOC100362824 0.005283713

143

1370141_at [] 0.008593857 1370146_at Glrb 0.000604808 1370150_a_at Thrsp 0.001282811 1370166_at Sdc2 0.001758099 1370167_at Sdc2 0.002463102 1370168_at Ywhaq 0.000838995 1370205_at Slco1c1 0.012069106 1370229_at Ndrg4 0.006786048 1370310_at Hmgcs2 0.000739753 1370455_a_at Olfm3 0.00097245 1370463_x_at RT1‐CE16 0.001793385 1370501_at Ube2g1 0.00404489 1370503_s_at Epb4.1l3 0.005624712 1370518_a_at Stxbp1 0.000636816 1370541_at Nr1d2 0.01025027 1370543_at Ogt 0.001378834 1370562_at Calcb 3.41E‐03 1370575_a_at Azin1 0.002333343 1370595_a_at Kcnip4 0.006962061 1370630_a_at Fgfr2 0.036445737 1370656_a_at Homer1 0.00086534 1370680_at Stau2 0.007294297 1370688_at Gclc 0.005661488 1370690_at Hspa9 0.005655766 1370705_at Znf597 0.000690222 1370728_at Il13ra1 0.001500607 1370746_at Prkacb 0.002230823 1370760_a_at Gad1 0.002045393 1370773_a_at Kcnip2 0.000989854 1370775_a_at Calca 5.33E‐04 1370803_at Zwint 0.042611301 1370830_at Egfr 0.00037986 1370891_at Cd48 0.004127383 1370950_at Ppap2b 0.000139833 1370951_at Ppap2b 0.000628233 1370955_at Adam10 0.006066024 1370956_at Dcn 0.000160813 1371038_at Cebpg 0.000715852 1371042_at Map4k3 0.003676295 1371055_at Rab12 0.008292019 1371057_at Gabra5 0.011396348 1371060_at Trim23 0.0022192 1371063_at Sh3gl2 0.005900085

144

1371103_at Rab6a 0.003654599 1371113_a_at Tfrc 0.001330853 1371114_at Kcnj4 0.004744887 1371122_at Tank 0.001145899 1371139_at Pls3 0.018686295 1371173_a_at Cast 0.005441606 1371177_a_at Cask 0.00116384 1371179_a_at Fgfr2 0.024456501 1371298_at H19 0.015760899 1371319_at Itm2b 0.043416083 1371320_at Itm2b 0.046549737 1371485_at [] 0.002573013 1371642_at Eif4a2 0.00391978 1371840_at S1pr1 0.016377211 1371922_at Car12 0.017202377 1371951_at Fhl2 0.001750469 1371956_at LOC683077 0.004528582 1372183_at Kpna1 0.00036782 1372186_a_at Top2a 0.001935184 1372190_at Aqp4 0.046690822 1372437_at Skp1 0.024682462 1372440_at Serpine2 0.008915365 1372511_at Dazap2 0.001686454 1372513_at Rac1 0.000832796 1372631_at Tk1 0.000141621 1372702_at Prp2l1 0.001188397 1372771_at [] 0.001237989 1373016_at Dld 0.000429034 1373058_at Tmem30a 0.003319144 1373067_at Ctnnb1 0.015561342 1373093_at Errfi1 0.002131164 1373257_at Arpp‐21 0.015393257 1373321_at RGD1306622 0.007128596 1373326_at [] 0.001390457 1373403_at LOC684871 0.003930509 1373508_at Selt 0.000841379 1373514_at Rnf213 0.000268996 1373601_at [] 0.001401305 1373661_a_at Cxcr4 0.021410823 1373718_at Tubb2a 0.019094229 1373773_at Gpm6a 0.015162766 1373819_at [] 0.000280082 1373865_at Snap91 0.005136013

145

1374004_at Prepl 0.000264168 1374263_at [] 0.028572023 1374529_at [] 0.025909066 1374659_at Arpp‐21 0.012451351 1374684_at [] 0.020079374 1374775_at Mki67 0.002768397 1374787_at [] 9.58E‐05 1374794_at Kif15 0.008438885 1375120_at Id4 0.00286901 1375130_at LOC679612 0.003569722 1375137_at Arpc2 0.00051719 1375140_at Mbnl2 0.020727932 1375182_at [] 0.000440419 1375184_at [] 0.034408391 1375193_at Lrp11 0.001258314 1375194_at [] 0.000525713 1375254_at Slc35a1 0.002151728 1375267_at Ppic 0.000318825 1375319_at [] 0.002389193 1375335_at Hsp90ab1 0.000293851 1375343_at [] 0.000225186 1375422_at [] 0.000525236 1375449_at [] 0.033704698 1375532_at [] 0.000432909 1375535_at [] 0.011135459 1375538_at [] 0.00085783 1375627_at Akirin2 0.012269974 1375676_at [] 0.000192702 1375696_at Ifnar1 0.010450363 1375705_at [] 9.02E‐05 1375754_at Impact 0.001901865 1375762_at Dnajb5 0.003994703 1375765_at [] 0.001987338 1375777_at [] 0.011552572 1375829_at Fam45a 0.001543343 1375881_at Dstn 0.000346899 1375882_at [] 0.016538978 1375903_a_at Yaf2 0.00432241 1376009_at [] 0.001768887 1376072_at Fam174a 0.01082027 1376128_at [] 0.000318587 1376157_at Uba6 0.002521396 1376186_at [] 0.000368655

146

1376193_at [] 0.005821109 1376321_at Fam38a 0.003981471 1376344_at [] 0.009395599 1376388_at [] 0.00257647 1376443_at [] 0.041891456 1376463_at [] 0.00163585 1376658_at Raph1 0.008805394 1376727_at Yipf4 0.000644445 1376816_at [] 0.00039196 1376856_at RGD1310414 0.007354975 1376867_at Hspc159 0.001820207 1376893_at Nrsn1 0.00271523 1376980_at Htr2c 0.011594832 1377013_at [] 0.002082467 1377133_at LOC680687 9.82E‐03 1377151_at [] 0.021992087 1377190_at [] 0.003196776 1377208_at [] 0.01337409 1377296_at [] 0.010096431 1377305_at [] 0.008995533 1377352_at [] 0.000306964 1377403_at [] 0.001686454 1377451_at RGD1305704 0.002730131 1377508_at [] 0.002246499 1377513_at [] 4.32E‐05 1377518_at Camk1g 0.00664413 1377531_at [] 0.002267778 1377554_at Tnfsf9 0.002318382 1377569_at Sav1 0.020223498 1377656_at Mak16 0.002753735 1377688_at [] 0.000223041 1377689_at Kntc1 0.001934588 1377708_at LOC499339 0.000274181 1377729_at Elovl4 0.001157999 1377784_at [] 0.010989249 1377838_at RGD1562218 0.003140569 1377928_at Tm4sf20 0.003429055 1377947_at [] 0.003022909 1377985_at RGD1309198 0.013012826 1378007_at RGD1306148 0.000147522 1378009_at [] 0.017265081 1378013_at [] 0.010911822 1378102_at Pcnxl2 8.17E‐05

147

1378116_at Zbtb45 0.005993486 1378227_at Rbpms2 0.002323449 1378238_at Anapc1 0.002992988 1378419_at Rnase2 0.002154946 1378436_at LOC100364311 0.042497933 1378471_at Raver2 0.001071215 1378472_at [] 0.04145056 1378492_at [] 0.045007825 1378494_at [] 8.61E‐05 1378508_at [] 0.035241127 1378566_at [] 0.001265049 1378587_at Ankrd40 0.023182809 1378688_at [] 0.002303362 1378701_at [] 0.000149429 1378702_at [] 0.000509977 1378717_at [] 0.001273394 1378754_at [] 0.002791047 1378832_at [] 0.000217795 1378859_at Alx3 0.011724114 1378929_at [] 0.003828347 1378968_at Bcl2l14 0.000789285 1378969_at Mast4 0.001838446 1378978_a_at Prr12 0.027112246 1379001_at [] 0.000336766 1379006_at LOC691396 0.000203252 1379007_at [] 0.000586212 1379018_at [] 0.000893474 1379029_at [] 0.028456092 1379066_at Slc5a3 0.003813088 1379070_at [] 0.001472414 1379073_at Slc25a23 0.001644731 1379076_at [] 0.003864527 1379083_at Adat3 0.000964284 1379141_at [] 0.002712488 1379187_at [] 0.010691702 1379210_at [] 0.00525856 1379212_at [] 0.006622791 1379226_at Aim1l 0.00195384 1379233_at [] 0.042348802 1379234_a_at Cdc45l 0.008935332 1379235_x_at Cdc45l 0.012087941 1379246_at RGD1308742 0.000535905 1379268_at LOC100362155 0.006671429

148

1379278_at Ints9 0.00119561 1379279_at Stim2 0.00214529 1379309_at Tbl1xr1 0.020940304 1379327_at Smarca5 0.001177967 1379329_at LOC685729 0.00222528 1379346_at Cdc73 0.009633184 1379357_at [] 0.024571657 1379358_at Samd4a 0.000850558 1379412_at [] 0.004839301 1379413_at Nmnat1 0.001559615 1379420_at RGD1565002 0.034861565 1379422_at Alg13 0.000312746 1379451_at [] 0.014905632 1379501_at [] 0.00043869 1379572_at Mab21l1 0.0033288 1379594_at [] 0.000170946 1379599_at [] 0.004562736 1379633_a_at Uba7 0.000835657 1379636_at Fam82a1 0.002317786 1379661_at [] 0.016193867 1379676_a_at Dnase1l1 0.003818035 1379697_at [] 0.00984782 1379717_at LOC360997 0.000750542 1379733_at [] 0.018612146 1379768_at [] 0.000176787 1379778_at RGD1307983 0.000585079 1379871_at Rfx7 0.004544377 1379879_at [] 0.010508418 1380110_at Jak2 0.024053335 1380185_at RGD1564541 0.000130892 1380222_at [] 0.001399159 1380320_at Lin54 0.008131385 1380381_at Alg2 9.23E‐05 1380422_at RGD1560686 0.000714064 1380440_at [] 0.010494232 1380567_at [] 0.020546198 1380586_at [] 0.001237035 1380672_at [] 0.006330371 1380673_at [] 0.021187305 1380677_at [] 0.002141953 1380936_at Tbata 0.000254929 1381172_at [] 0.000746071 1381260_at [] 0.000648439

149

1381430_at [] 0.000177681 1381509_at Nbr1 0.024640083 1381546_at [] 8.83E‐05 1381568_at Nek4 0.002125502 1381621_at [] 0.028431118 1381700_at [] 0.000873208 1381724_at [] 0.003256917 1381892_at Nav2 0.018167675 1382030_at Gtf2h2 0.000836492 1382065_at [] 0.011410296 1382098_at [] 0.029322267 1382203_at Gdf1 /// Lass1 0.014190257 1382217_at [] 0.000470877 1382254_at [] 0.014518499 1382294_at [] 0.000382185 1382375_at [] 0.001104713 1382413_at Nup155 0.031616688 1382457_at Khdrbs3 0.004569173 1382514_at [] 0.001703024 1382525_at Lypd6b 0.004034758 1382543_at [] 0.040830553 1382586_at [] 0.004098475 1382658_at LOC100364718 0.027629077 1382690_at Cgm4 0.000304759 1382865_at Tsga14 0.001370966 1382974_at [] 0.010875463 1382976_at [] 0.03692317 1382988_at [] 0.046330154 1382993_at Bbc3 0.018384457 1383001_at [] 0.000238895 1383032_at [] 0.033064008 1383040_a_at LOC100360384 0.042565942 1383045_at [] 0.011344671 1383195_at [] 0.002987146 1383242_a_at [] 0.000290036 1383312_at [] 0.000652075 1383332_at [] 0.000415981 1383355_at Abca1 0.004913688 1383394_at [] 0.000692248 1383419_at Lhfpl3 0.019617796 1383426_at Pstpip1 0.005038679 1383448_at Irf9 0.001496315 1383497_at [] 0.000752032

150

1383513_at [] 0.01603663 1383529_at [] 0.002299786 1383634_at Zfp653 0.003805161 1383691_at [] 0.000710964 1383706_at RGD1305733 0.001393676 1383763_at [] 0.004183888 1383775_at [] 0.002531052 1383789_at [] 0.003456354 1383792_at Sytl1 0.0060004 1383851_at Ccdc96 0.000840485 1383870_at Hoxb6 0.006034732 1383872_at [] 0.031670332 1383930_at [] 0.002789974 1383957_at Nudt3 0.009486795 1384032_at [] 0.034054995 1384054_at [] 0.002959669 1384062_at [] 0.011883259 1384067_at [] 0.000154555 1384069_at Lrp11 0.000280023 1384134_at [] 0.01058048 1384213_at Pdcd6ip 0.003488481 1384214_a_at [] 0.001623333 1384224_at [] 0.001115322 1384273_at Shpk 0.011481285 1384281_at [] 0.025092483 1384322_at [] 0.000283003 1384341_at Faah 0.002877533 1384356_at Fam151b 0.005861044 1384657_at Mfhas1 0.001764894 1384674_at [] 0.03006649 1384675_at Apeg3 0.001254559 1384693_at [] 0.015923321 1384747_at [] 0.003864408 1384792_at Prpf40a 0.015765607 1384800_at Zic3 0.006774783 1384836_at Calml3 0.011071146 1384932_at Pftk1 0.003852129 1384949_at [] 0.012783527 1384960_at Cftr 0.00091207 1385035_at Usp12 0.016136646 1385132_at [] 0.004455924 1385193_at [] 0.008361757 1385201_at Tmem182 0.001361191

151

1385255_at Itga9 0.042857766 1385316_at Pvrl3 0.004884124 1385319_at Arhgap27 0.03941679 1385333_at Aatk 0.004528642 1385355_at [] 0.002659321 1385412_at LOC100364488 0.013864636 1385431_at [] 0.001801491 1385438_at ND4 0.010981739 1385450_at Ucn2 0.000858426 1385458_a_at RGD1306959 0.017276883 1385485_x_at [] 0.001492381 1385506_at [] 0.007996321 1385549_at LOC685964 0.007283926 1385578_at [] 0.001158178 1385603_at [] 0.0013026 1385631_at [] 0.015363872 1385724_at Ccdc74a 0.006554484 1385770_at Ulk4 0.002872348 1385793_at [] 0.006530643 1385999_at Yme1l1 0.002586067 1386013_at LOC100359753 0.041400433 1386031_at Prkd3 0.006152391 1386069_at Sp2 0.019757867 1386295_at [] 0.011449039 1386497_at [] 0.002047181 1386573_a_at [] 0.001839697 1386660_at [] 0.013427079 1386822_s_at RGD1311823 0.002619386 1386823_at Irx5 0.014384449 1386837_x_at Znf574 0.00181073 1386855_at [] 0.002280355 1386884_at Htra1 0.019316614 1386925_at Arpc1b 0.002010465 1386962_at Plcb4 0.011391401 1386970_at Eif2b4 0.000525236 1387002_at Bud31 0.004603744 1387043_at Lypd3 0.006606638 1387115_at Ikbkap 0.000551701 1387148_at Gprasp1 0.00037986 1387149_at Erap1 0.012075067 1387220_at Mcpt9 0.014288962 1387264_at Kcnk6 0.000598669 1387326_at Spam1 0.00050652

152

1387428_at Cacnb1 0.002262235 1387482_at Grid2 0.002937079 1387501_at Synpo 0.001762211 1387570_at Manea 0.005796611 1387639_at Hpcal4 0.001464009 1387707_at Slc2a3 0.025426447 1387739_at Cd8b 0.001249909 1387743_at Gpr20 0.000564873 Acaa1 /// 1387783_a_at RGD1562373 0.00695461 1387810_at Keap1 0.000579 1387929_at Pmf31 0.006239295 1387995_a_at Ifitm3 0.002901316 1388001_at Lrit1 0.023873985 1388005_at Prpf18 0.003988266 1388018_at Sele 0.013680518 1388138_at Thbs4 0.004585624 1388165_at Porf‐2 0.000573993 1388210_at Acot2 0.000498772 1388241_at Insl3 0.025216341 1388243_at Gpr176 0.010276914 1388253_at Scd 0.000617802 1388278_at Hspa1l 0.001278698 1388289_s_at Fshb 0.004888177 1388329_at Krt13 0.010360301 1388364_at Ndufs3 0.006511092 1388410_at Ugp2 0.006857336 1388417_at Anapc5 0.000725269 1388471_at Tcp11l2 0.003172696 1388472_at [] 0.001547158 1388513_at [] 0.00067842 1388560_at Wdr77 0.002538919 1388631_at Azi2 0.001081407 1388632_at Rab6b 0.002062559 1388654_at Mrpl52 0.001195669 1388722_at Dnajb1 0.002862394 1388751_at [] 0.03311801 1388757_at Adrbk1 0.003378868 1388768_at Sgsm2 0.000488818 1388873_at Trip12 0.00878644 1388877_at Mrps5 0.009770036 1388894_at [] 0.00334394 1388910_at Urg4 0.002447248

153

1388955_at Ehd2 0.000355005 1388967_at Tcfe3 0.001595616 1388968_at Mpped1 0.000686586 1388983_at RGD1305045 0.01761508 1389058_at Trim29 0.010178208 1389067_at Slco4a1 0.029242754 1389112_at Nkd1 0.01222527 1389282_at Itga3 7.35E‐05 1389292_at Rab18 0.045689344 1389298_at [] 0.009449065 1389350_at Apoh 0.000766039 1389360_at Fxyd3 5.43E‐05 1389361_at Cluap1 0.006139875 1389393_at Fam108c1 0.016045451 1389464_at Lnx1 0.003962398 1389487_at Dnajb12 0.000838518 1389530_at Slc35e1 0.000558972 1389575_at RGD1311703 0.000218034 1389673_at [] 0.034449935 1389675_at Ier5l 0.043081701 1389753_at Brp16 0.005844593 1389770_at Ttll9 0.002829671 1389772_at [] 0.000407577 1389804_at Relt 0.002062202 1389820_at Cox7c 0.032328844 1389828_at Cebpd 0.007686973 1389884_at Ccdc88b 0.004223704 1389897_at Cops7a 0.011413157 1389906_at Fdft1 0.00220269 1389987_at Trib1 0.001218736 1390011_at [] 0.001290381 1390056_at Nck2 0.022175729 1390069_at [] 0.004285574 1390082_at Hip1 0.002125084 1390088_at Nog 0.000427127 1390099_at Ube3a 0.006471634 1390109_at [] 0.012210727 1390296_at [] 0.015956283 1390299_at [] 0.002420247 1390364_at [] 0.015254319 1390373_at Smad5 0.000314295 1390400_a_at [] 0.001720071 1390454_at Nipsnap1 0.00764209

154

1390492_a_at Usp40 0.024701238 1390536_at [] 0.000914931 1390603_at [] 0.001293659 1390615_at Kpna1 0.000521183 1390620_at RGD1306746 0.002363443 1390642_at [] 0.010915697 1390679_at [] 0.000909746 1390719_at [] 0.001390994 1390731_at [] 0.002125144 1390824_at RGD1304963 0.010765135 1390831_at [] 0.013776779 1390864_at [] 0.004415274 1390957_at RGD1562018 0.004187107 1390975_at [] 0.012078047 1391028_at [] 0.003153682 1391040_at Rbbp8 0.031531334 1391065_at [] 0.014941096 1391072_at Fcer2 0.0019871 1391089_at [] 0.028466225 1391095_at Mmp19 0.025073409 1391106_at [] 0.003062129 1391151_at Arsg 0.00076139 1391157_at [] 0.000531316 1391192_at [] 0.001570165 1391273_at Naglt1 0.000977874 1391354_at [] 0.0004583 1391416_at Ankrd17 0.001926661 1391444_at [] 0.001290917 1391461_at RGD1306576 0.002872646 1391473_at [] 0.00086534 1391496_at Polr3f 0.001252115 1391523_at [] 0.001583219 1391534_at Elovl2 0.000380635 1391601_at Lrrn2 0.00555867 1391629_at [] 0.023993492 1391635_at Ctdspl 0.003430724 1391660_at [] 0.00589627 1391666_at Aak1 0.000985086 1391670_at [] 0.001190066 1391695_at [] 0.037502885 1391714_at Plag1 0.000295401 1391718_at [] 0.002064943 1391719_at [] 0.002172649

155

1391721_at [] 0.000774384 1391747_at Rbm12 0.007031679 1391904_at [] 0.017354488 1391984_at [] 0.022276342 1391985_at Nrip3 0.00334686 1392047_at [] 0.004433811 1392056_at [] 8.48E‐05 1392092_at Rab33a 0.009063363 1392143_at [] 0.011264741 1392175_at Ncor1 0.008869588 1392210_at RGD1307443 0.000573039 1392217_at [] 0.003618121 1392262_at [] 0.010712743 1392493_at [] 0.035011649 1392528_at Csf2ra 0.034610152 1392551_at LOC503192 0.002647579 1392671_at Thoc3 5.67E‐05 1392788_at [] 0.019187927 1392822_at Nf1 0.000174642 1392839_at Bri3bp 0.014150321 1392861_at [] 0.019934714 1392904_at Pwwp2a 0.002113104 1392967_at MGC116121 0.006991327 1393043_at Utp18 0.009179592 1393128_at Gpatch4 0.005672157 1393153_at [] 0.000278831 1393155_at Plk3 0.006599665 1393156_at [] 0.002435863 1393166_at LOC100359435 0.000729442 1393216_at Slc33a1 0.006687284 1393273_at [] 0.02217263 1393310_at [] 0.002103686 1393340_at Mlxip 0.005075812 1393378_at [] 0.017334342 1393435_at Plch2 0.00908196 1393489_at Rsb66 0.002738535 1393530_at [] 0.011701226 1393553_at [] 0.037149191 1393583_at Etaa1 0.001569629 1393588_at Cldn14 0.00603199 1393609_at [] 0.015623808 1393636_at [] 8.05E‐05 1393653_at [] 0.000866294

156

1393655_at Rad54l 1.93E‐02 1393663_at Slc36a2 0.000956357 1393703_at Hivep3 0.01494801 1393722_at Fem1c 0.001887858 1393819_x_at [] 0.001164436 1393901_at Runx1t1 0.000613213 1393909_at RGD1305110 0.001379252 1393946_at Fusip1 0.001986384 1393975_a_at Fam132a 0.002356589 1393977_at [] 0.049494445 1393996_at Dnajb14 0.000811458 1394019_at [] 0.003354967 1394057_at Ndp 0.000802159 1394095_at Gin1 0.015621305 1394241_at Clip1 0.005873203 1394286_at [] 0.025609732 1394290_at [] 0.0017277 1394315_at LOC683788 0.000991404 1394406_at [] 0.034724355 1394433_at Katnal1 0.003433824 1394531_at [] 0.000886202 1394535_at Kif16b 0.03904283 1394547_at [] 0.017190099 1394549_at [] 0.000437617 1394556_at [] 0.003399193 1394618_at [] 0.000166774 1394619_at [] 0.019239545 1394687_at [] 0.000528455 1394761_at Arhgap42 0.001243234 1394792_at [] 0.000535607 1394820_at [] 0.017475009 1394837_at [] 0.001526356 1394843_at [] 0.00108707 1394957_at Nisch 0.004903913 1394958_at [] 0.007288814 1395008_at Itsn1 0.004551768 1395035_at [] 0.001611948 1395139_at Kif16b 0.004998803 1395238_at [] 0.003729105 1395254_at [] 0.021234632 1395265_at Gns 0.000998497 1395383_at RGD1311066 0.047166586 1395393_at [] 0.003435254

157

1395398_at [] 0.020319283 1395400_at RGD1305508 0.00206542 1395410_at Ppp2r3a 0.026433706 1395452_at [] 0.006045938 1395487_at [] 0.001731992 1395560_at [] 0.00225246 1395600_at Ccdc117 0.004338503 1395610_at Tspan33 0.013709664 1395637_at Asphd2 0.001111209 LOC316460 /// 1395641_at LOC689991 0.004707694 1395644_at Abca8 0.000484586 1395656_at [] 0.000839114 1395679_at LOC681924 0.00740689 1395727_at Rabggta 0.000922918 1395740_at [] 0.001718998 1395750_at [] 0.0024243 1395751_at [] 0.001980901 1395769_at [] 0.00167948 1395777_at [] 0.012862563 1395799_at Itsn2 0.016281068 1395842_at [] 0.005746782 1395876_at [] 0.017517209 1395905_at [] 0.002029479 1395915_at [] 0.009586573 1395942_at [] 0.026252925 1395962_at Chchd5 0.000699997 1396007_at [] 0.041268229 1396008_at [] 0.005074024 1396009_at [] 0.011244535 1396012_at [] 0.00183785 1396034_at Ces7 0.00190264 1396039_at Slc22a12 0.002468467 1396048_at Cachd1 0.004578054 1396081_at Spire1 0.035808921 1396134_at [] 0.000611067 1396188_at Rhoj 0.000817537 1396211_at [] 0.002214253 1396217_at [] 0.006516099 1396221_at B3galt6 0.000618696 1396222_at [] 0.00710839 1396241_at [] 0.006724834 1396309_at Tbc1d19 0.003089547

158

1396375_at [] 0.001584888 1396389_at LOC687758 0.041710556 1396390_at LOC100270669 0.015334487 1396394_at [] 0.000465572 1396395_at [] 0.000462234 1396399_at [] 0.01172626 1396413_at [] 0.002159476 1396414_at [] 0.008579969 1396438_at [] 0.019092679 1396490_at [] 0.002196312 1396509_at Tmem132e 0.00102514 1396563_at [] 0.006442428 1396746_at [] 0.001358509 1396761_at [] 0.003769219 1396786_at [] 0.004734755 1396804_at [] 0.004195035 1396817_at [] 0.004593968 1396823_at [] 0.007183909 1396840_at [] 0.001996696 1396842_at Atg9a 0.003821909 1396876_at [] 0.003383815 1396948_at [] 0.002947331 1397047_at [] 0.016571462 1397078_at Itfg2 0.001055121 1397094_at [] 0.003486574 1397198_at [] 0.005621076 1397279_at Itgb5 0.001383007 1397288_at [] 0.002820194 1397328_at [] 0.018741369 1397382_at [] 0.007810414 1397411_at Fgd2 0.005122423 1397413_at [] 0.03376013 1397428_at [] 0.008037269 1397444_at [] 0.023497999 1397546_at Spats2 0.001934648 1397553_s_at Prdm2 0.001796186 1397626_at [] 0.013040662 1397628_at RGD1305592 0.005528688 1397714_at RGD1307235 0.013007402 1397793_at [] 0.004623175 1397861_at Vstm2b 0.000276566 1397884_at Sel1l3 0.029862046 1397894_at [] 0.008181155

159

1397950_at Smg7 0.002114415 1397955_at Rtel1 0.000891209 1397974_at [] 0.00310272 1397980_at [] 0.002915621 1398015_at [] 0.018493354 1398039_at [] 0.005850434 1398053_at [] 0.000496805 1398065_at Sobpl 0.002787113 1398110_at [] 0.014960349 1398135_at [] 0.014585555 1398151_at [] 0.001762807 1398170_at [] 0.004674494 1398179_at [] 0.003285289 1398180_at [] 0.00251168 1398183_at [] 0.001570582 1398187_at [] 0.044130325 1398204_at [] 0.000474095 1398224_at [] 0.000973821 1398236_at [] 0.000698805 1398275_at Mmp9 0.006764889 1398281_at Ocln 0.004191756 1398285_at Ak3l1 0.002711535 1398336_at Rnf25 0.00064683 1398337_at LOC290341 0.001706123 1398355_at Trpm7 0.010942876 1398360_at Elovl1 0.031752229 1398362_at Notch2 0.00138247 1398364_at RGD1359529 0.002275229 1398372_at Rmnd5b 0.011233211 1398482_at Bcl3 0.010770202 1398491_at Ildr1 0.032970786 1398510_at [] 0.002292931 1398559_at [] 0.000207067 1398627_at [] 0.003907919 1398655_at Myod1 0.000804663 1398808_at Impa1 0.004694104 1398809_at Nde1 0.021074712 1398842_at Cltc 0.009082794 1398907_at Ormdl2 0.005645275 1398976_at Ncor1 0.000672221 1398984_at Tm2d2 0.003343642 1399010_at Cops7a 0.035020769 1399011_at Cops6 0.017137289

160

1399035_at Polr3h 0.018409252 1399088_at Tlk2 0.000261724

7.4 24 hour in vitro TBI

Affymetrix ID Gene Symbol p‐value 1367581_a_at Spp1 0.000921845 1367973_at Ccl2 0.020125985 1368014_at Ptges 0.014620185 1368202_a_at Dab2 0.00541079 1368247_at Hspa1a /// Hspa1b 0.01038301 1368270_at Apobec1 0.000800967 1368271_a_at Fabp4 0.013383389 1368420_at Cp 0.005216122 1368490_at Cd14 0.00545013 1368505_at Rgs4 0.001005292 1368527_at Ptgs2 0.001695037 1368530_at Mmp12 0.015347004 1368677_at Bdnf 0.001875222 1368814_at Aldh6a1 0.049631655 1368887_at [] 0.001721025 1368912_at Trh 0.011233807 1369007_at Nr4a2 0.000731468 1369047_at Sult1d1 0.015029073 1369099_at Slc30a1 0.016663551 1369151_at Dlk1 0.008509934 1369194_a_at Cdkn2a 0.026872933 1369217_at Nr4a3 0.001001477 1369248_a_at Xiap 0.009986281 1369285_at Pggt1b 0.012669802 1369497_at LOC24906 0.016137362 1369560_at Gpd1 0.000519753 1369610_at Lin7c 0.011376023 1369728_at LOC680097 0.002506197 1369756_a_at Slc4a4 0.008895457 1369791_at Clcn4‐2 0.011112809 1369814_at Ccl20 0.023094773 1370113_at Birc3 0.0104478 Cav2 /// 1370135_at LOC100362824 0.007932544 1370146_at Glrb 0.047491789

161

1370307_at Agrn 5.33E‐04 Ceacam1 /// 1370371_a_at Ceacam10 0.009245932 1370454_at Homer1 0.001297653 1370570_at Nrp1 0.001248241 1370572_at Gpr149 0.000701189 1370623_at Fgl2 0.016048431 1370630_a_at Fgfr2 0.019536197 1370652_at Ntrk2 0.004488707 1370830_at Egfr 0.014704227 1370902_at Akr1b8 0.004598975 1370955_at Adam10 0.014996946 1370997_at Homer1 0.000765562 1371028_at Tgoln1 0.036183715 1371033_at RT1‐Bb 0.012739003 1371179_a_at Fgfr2 0.0140962 1371194_at Tnfaip6 0.002862454 1371250_at Pf4 0.013760209 1371298_at H19 0.026463747 1371369_at Col6a2 0.014612079 1371447_at Plac8 0.017492831 1371771_at LOC313641 0.002730131 1372031_at [] 0.003182173 1372235_at [] 0.001133263 1372326_at Slc2a3 0.000240505 1372345_at [] 0.000617802 1372372_at Cmbl 0.010462105 1372439_at Col4a1 0.001063466 1372492_at Cldn10 0.014599681 1372760_at [] 0.004374981 1372823_at [] 0.002740383 1372935_at Tmem119 0.000719607 LOC100360189 /// LOC363363 /// LOC501476 /// LOC680682 /// LOC685369 /// LOC689362 /// 1373177_x_at LOC691793 0.004921436 1373179_at [] 3.83E‐05 1373245_at Col4a1 0.000762701 1373403_at LOC684871 0.00095129 1373759_at [] 9.04E‐05 1373932_at [] 0.017570853

162

1374247_at LOC100363145 0.043381095 1374263_at [] 0.005357802 1374283_at Falz 0.016104698 1374367_at Grxcr1 0.022385955 1374655_at [] 0.002708376 1374684_at [] 0.002152801 1374713_at [] 6.12E‐04 1374775_at Mki67 0.001424372 1374778_at [] 0.007904291 1374786_at [] 0.036382675 1375120_at Id4 0.00227505 1375139_at [] 0.000840068 1375305_at Snhg11 0.004368424 1375331_at [] 0.005479217 1375449_at [] 0.007847667 1375535_at [] 0.001332402 1375573_at [] 0.000319481 1375693_at [] 1.92E‐05 1375740_at [] 0.000398993 1375777_at [] 0.030973911 1376157_at Uba6 0.007802248 1376321_at Fam38a 0.005667567 1376344_at [] 0.007597685 1376458_at [] 0.00016737 1376477_at [] 0.003050506 1376574_at [] 0.001882613 1376688_a_at LOC685020 0.041410148 1376727_at Yipf4 0.034690022 1376729_at RGD1309388 0.005899787 1376755_at Rarb 0.015995204 1376911_at [] 0.002344012 1376967_at [] 0.000694871 1377008_at RGD1566401 0.000553012 1377193_at [] 0.022454977 1377208_at [] 0.002428174 1377451_at RGD1305704 0.008831561 1377516_at [] 0.000423551 1377602_at Nudt2 0.003692269 1377694_at Luc7l3 0.002078295 1377720_x_at [] 0.000271201 1377784_at [] 0.024291575 1377808_at Dcaf6 0.003866255 1378205_at [] 0.000862181

163

1378452_at Prss36 0.041548014 1378472_at [] 0.002189636 1378506_at Pik3c2a 2.67E‐03 1378536_at Hook1 0.013779223 1378582_at Trabd 0.0158723 1378593_at [] 0.007956028 1378639_at Snupn 0.003615856 1378658_at Clca4 0.002342343 1378693_at Shc2 0.000460029 1378754_at [] 0.038160801 1378768_at [] 0.007733882 1378794_at [] 0.01408112 1378859_at Alx3 0.0307886 1378949_at Ece2 0.011819124 1378981_at [] 0.0019629 1379013_at RGD1306404 0.003660917 1379101_at Dhx36 3.10E‐06 1379327_at Smarca5 0.019098163 1379360_at Gatc 1.04E‐04 1379371_at [] 0.004262328 1379383_at [] 0.014191031 1379531_at [] 0.001024067 1379541_at [] 0.002058625 1379881_at [] 0.003476918 1379929_at [] 0.001597166 1380032_at Slain2 0.000608206 1380066_at Tfr2 0.000935316 1380141_at Nsun4 0.001134157 1380245_at [] 0.00050807 1380412_at Elac1 0.008455515 1380606_at [] 0.004228532 1380695_at [] 0.00059855 1380783_at Slco5a1 4.57E‐05 1381042_at Anapc10 0.007811904 1381306_at [] 0.036192894 1381309_at LOC100361360 0.001144528 1381386_at Pop5 0.00093019 1381538_at [] 0.000710785 1381724_at [] 0.006862521 1381892_at Nav2 0.02212894 1381936_at Camk2g 0.036054194 1382030_at Gtf2h2 0.002855718 1382203_at Gdf1 /// Lass1 0.024725974

164

1382298_at [] 0.001310706 1382315_at Tsc22d4 0.006646752 1382478_at Btbd3 0.042427421 1382514_at [] 0.036875665 1382525_at Lypd6b 0.026168704 1382586_at [] 0.024783015 1382865_at Tsga14 0.016125023 1383355_at Abca1 0.014558494 1383395_at Agmat 0.002133369 1383434_at Pycr1 0.000634611 1383513_at [] 0.046378076 1383691_at [] 0.000527143 1383777_at [] 0.014788151 1383780_at Hectd1 0.001207352 1383870_at Hoxb6 0.018831253 1383954_at [] 0.002638936 1384055_at [] 0.005163431 1384293_at Ncor1 0.001098692 1384315_at Pdk3 0.000549436 1384448_at RGD1565844 0.001161397 1384496_at [] 0.000267565 1384508_at [] 0.012697637 1384545_at [] 0.003084898 1384693_at [] 0.019115329 1384831_at Slc7a13 0.000667572 1385011_at Smarca4 0.018941462 1385036_at Sncaip 0.000162005 1385058_at Cldn8 0.002028227 1385193_at [] 0.021595597 1385207_at RGD1310384 0.003649712 1385308_at Mogat1 0.004824817 LOC100364003 /// 1385338_at Zfp758 0.013514161 1385355_at [] 0.011059344 1385450_at Ucn2 0.000437617 1385544_at Nrl 0.017222047 1385583_at Tyr 0.001053929 1385631_at [] 0.01615113 1385638_at [] 0.012540877 1385691_at [] 0.004189491 1385724_at Ccdc74a 0.039415598 1385766_at Als2cr11 0.043621182 1385791_at Vill 0.00579679

165

1385794_at MGC94891 0.000528753 1385817_at Nek4 0.005498886 1386295_at [] 0.040407658 1386573_a_at [] 0.027232111 1386594_at [] 0.002683878 1386602_at Rab40b 0.036403358 1386679_at [] 0.011698663 1386797_at Slc39a6 0.029210627 1386884_at Htra1 0.008193076 1386903_at S100b 0.001951039 1386919_at Prl8a4 0.008599639 1386962_at Plcb4 0.005042017 1386991_a_at Bad 0.004821539 1387149_at Erap1 0.019988775 1387160_at Kcne3 0.033412576 1387190_at Dgka 0.039808929 1387222_at Cript 0.012482285 1387230_at Slc12a3 0.004436672 1387270_at Hhex 0.007745266 1387354_at Stat1 /// Stat4 0.005998492 1387433_a_at Slc25a27 0.016026914 1387456_at Stau2 0.005414784 1387482_at Grid2 0.021519184 1387548_at Has2 0.006710291 1387615_at St8sia2 0.005790532 1387618_at Bcl2l10 0.009646952 1387630_at Elovl5 0.048136234 1387647_at Tff1 0.000213265 1387648_at Cxcl6 0.014961541 1387791_at Ace 0.000338972 1387852_at Thrsp 0.034711719 1388001_at Lrit1 0.011739969 1388061_a_at Epha7 0.010510325 1388241_at Insl3 0.003424525 1388297_at Eef1g 0.004665792 1388332_at [] 0.0062356 1388410_at Ugp2 0.037484169 1388438_at Nap1l4 0.001295805 1388500_at [] 0.003826499 1388598_at LOC691995 0.006436825 1388613_at Isca1 0.002213478 1388667_at Man2a1 0.012343884 1388673_at Lsp1 0.000276029

166

1388691_at Ubqln4 0.049887776 1388786_at [] 0.002150595 1388840_at Plekhg2 0.005281806 1388873_at Trip12 0.012777805 1388902_at Loxl1 0.017528415 1388916_at [] 0.003731906 1388939_at Col15a1 2.34E‐04 1389134_at [] 0.00339222 1389245_at Psmd7 0.011112392 1389298_at [] 0.000150502 1389639_at RGD1565350 6.68E‐05 1389770_at Ttll9 0.025901496 1389804_at Relt 0.039160013 1389922_at Hadh 0.009996295 1390058_at LOC361990 0.00289613 1390099_at Ube3a 0.020056307 1390141_at Mthfd1l 0.02010715 1390176_at Fam84b 0.015791953 1390240_at [] 0.030937374 1390642_at [] 0.049546421 1390709_at [] 0.001955688 1390727_at [] 0.000262618 1390831_at [] 0.013696313 1391020_at Btbd17 0.004090905 1391109_x_at [] 0.011463046 1391146_at Cdh11 0.001315475 1391192_at [] 0.04236573 1391227_at [] 0.004898608 1391325_at [] 0.031640708 1391359_at [] 0.004282951 1391362_at [] 0.030025244 1391532_at Rap2ip 0.025122821 1391635_at Ctdspl 0.023361444 1391657_at Aldh5a1 0.012170076 1391695_at [] 0.015676796 1391718_at [] 0.001060426 1391719_at [] 0.002245128 1391731_at [] 0.010566175 1391904_at [] 0.04993546 1392072_at [] 0.046425521 1392143_at [] 0.013217926 1392254_at Map4 0.000355542 1392262_at [] 0.004441679

167

1392281_at [] 0.046224475 1392549_at [] 0.005725503 1392698_a_at Gtf3c4 0.023797274 1392967_at MGC116121 0.031354368 1393019_at [] 0.001757383 1393043_at Utp18 0.004632115 1393080_at Zfr 0.004021704 1393216_at Slc33a1 0.02156204 1393224_at [] 0.018924415 1393273_at [] 0.00826323 1393286_at Nr5a1 0.000870824 1393310_at [] 0.006277561 1393414_at [] 0.008309722 1393535_at [] 0.003686309 1393537_at [] 0.015476882 1393582_at [] 0.010478556 1393604_at Gpn3 0.027834356 1393652_at Zbtb1 0.00506556 1393682_at Gpr34 0.002348781 1393725_at Dsg1b 0.001981914 1393738_s_at Mfhas1 0.00868988 1393933_at Sorl1 0.002033353 1393937_at [] 0.004669666 1394095_at Gin1 0.01825273 1394130_at Trim44 0.00261122 1394241_at Clip1 0.033414423 1394470_at Fam49a 0.014609694 1394554_at [] 0.002924919 1394621_at Smox 0.006618381 1394660_at [] 0.005604982 1394795_at [] 0.048377395 1394820_at [] 0.022356629 1394915_at [] 0.00258261 1394934_at Zfp709l2 0.00089854 1394966_at Lppr5 0.009815395 1395057_at [] 0.020931005 1395092_at [] 0.013063073 1395238_at [] 0.002060056 1395254_at [] 0.01159507 1395383_at RGD1311066 0.002284467 1395452_at [] 0.030818939 1395462_at Nkx3‐1 0.001343608 1395641_at LOC316460 /// 0.003363848

168

LOC689991 1395705_at [] 0.008906126 1395740_at [] 0.004569173 1395751_at [] 0.024167895 1395799_at Itsn2 0.042034149 1395918_at [] 0.002596498 1395921_at Usp35 0.006904244 1396004_at [] 0.001627088 1396007_at [] 0.00896287 1396039_at Slc22a12 0.029569387 1396048_at Cachd1 0.005129635 1396116_at Sdccag1 0.001389742 1396211_at [] 0.006674528 1396222_at [] 0.01593256 1396271_at [] 0.005334139 1396380_at [] 0.006604552 1396485_at Btbd10 0.013652623 1396490_at [] 0.033290029 1396771_at [] 0.014473021 1396786_at [] 0.006532788 1396921_at [] 0.002310634 1397328_at [] 0.011317372 1397329_at [] 0.018464446 1397403_at [] 0.014065027 1397492_at Lmo7 0.000779629 1397508_at Ddx18 0.001079977 1397519_at Adipor2 0.002975464 1397560_at [] 0.000543356 1397621_at [] 0.010215104 1397626_at [] 0.009913504 1397699_at Dak 0.006543756 1397714_at RGD1307235 0.026901603 1397793_at [] 0.011223376 1397884_at Sel1l3 0.006520629 1397953_at LOC687696 0.008707702 1398110_at [] 0.014164448 1398120_at [] 0.00683254 1398340_at Ccndbp1 0.00744319 1398372_at Rmnd5b 0.002327561 1398635_at [] 0.002005696 1398809_at Nde1 0.025250137 1398867_at Prpf19 0.002167702 1398931_at LOC686289 /// 0.000282347

169

Tmem223 1398976_at Ncor1 0.028516889 1399030_at Wdr45 0.017173886 1399035_at Polr3h 0.012114882 1399110_at Zbtb22 0.015205145

7.5 1 hour in vivo TBI

Affymetrix ID Gene Symbol p‐value 1367712_at Timp1 0.007043898 1367823_at Timp2 0.01631844 1367973_at Ccl2 0.000365138 1368030_at Gnai3 0.012280583 1368046_at Slc31a1 0.00905478 1368073_at Irf1 0.00184536 1368124_at Dusp5 0.007717252 1368144_at Rgs2 0.00372982 1368146_at Dusp1 0.005832136 1368223_at Adamts1 0.01347512 1368321_at Egr1 0.000344515 1368332_at Gbp2 0.012187719 1368490_at Cd14 0.000500739 1368527_at Ptgs2 0.011708915 1368606_at Slco1a5 0.040345073 1368646_at Mapk9 0.021656394 1368683_at Olr1 0.000857711 1368753_at Camkk2 0.01303792 1368760_at Cxcl2 0.000305295 1368813_at Cebpd 0.017987549 1368826_at Comt 9.25E‐05 1368864_at Synpr 0.031046212 1368944_at Dlg1 0.012233078 1368956_at Pcdh8 0.007720232 1368982_at Pkia 0.010287642 1369007_at Nr4a2 0.016750634 1369041_at Nlgn1 0.019780755 1369067_at Nr4a3 0.001632452 1369078_at Mapk1 0.025300205 1369129_at Rasgrp1 0.046765327 1369130_at Rasgrp1 0.018464983 1369265_at Senp2 0.024576724

170

1369268_at Atf3 0.004998624 1369269_at Galnt1 0.028373837 1369325_at Lyst 0.031488061 1369390_a_at Dpp6 0.035152197 1369452_a_at Picalm 0.03595984 1369501_at Zfp260 0.04520309 1369625_at Aqp1 0.024404109 1369626_at Ide 0.025756121 1369720_at Myo1b 0.013465405 1369781_at Grm7 0.02059269 1369815_at Ccl3 0.003364921 1369839_at Amph 0.043672919 1369885_at [] 0.043318212 1370141_at [] 0.002037883 1370146_at Glrb 0.048024893 1370174_at Ppp1r15a 0.005174637 1370177_at PVR 0.003083706 1370224_at Stat3 0.017954588 1370301_at Mmp2 0.025683761 1370454_at Homer1 0.007695794 1370501_at Ube2g1 0.046162188 1370527_a_at Csnk1d 0.03183037 1370543_at Ogt 0.022532344 1370652_at Ntrk2 0.026889086 1370668_a_at Cnksr2 0.044540286 1370760_a_at Gad1 0.011365116 1370773_a_at Kcnip2 0.049376488 1370792_at Mapre1 0.019705296 1370832_at Ccl4 1.46E‐03 1370997_at Homer1 0.006793737 1371028_at Tgoln1 0.01722008 1371060_at Trim23 0.022884548 1371103_at Rab6a 0.040697694 1371108_a_at Atp1a1 0.033971429 1371113_a_at Tfrc 0.017957687 1371139_at Pls3 0.043222904 1371170_a_at Il1a 0.000135243 1371527_at Emp1 0.022539139 1371595_at [] 0.004278779 1372016_at Gadd45b 5.88E‐05 1372213_at LOC500300 0.000372708 1372389_at Ier2 4.68E‐03 1372520_at Mcl1 0.0003075

171

1373053_at [] 0.000553191 1373157_at Usp47 0.035593152 1373759_at [] 3.08E‐05 1373866_at Coq10b 0.001181841 1374404_at Jun 0.007377446 1374446_at Tiparp 0.002572775 1374529_at [] 0.007142544 1374752_at Mdfic 0.044167638 1375043_at Fos 0.000441551 1375140_at Mbnl2 0.038462698 1375538_at [] 0.003468037 1375595_at Arih1 0.008691728 1375829_at Fam45a 0.027850866 1375973_at Arih1 0.002059937 1376011_at [] 0.040485382 1376129_at [] 0.037811875 1376263_at Metrn 0.049253345 1376569_at Klf2 0.023199618 1376925_at Palmd 0.002028704 1377100_at Pds5b 0.036866069 1377208_at [] 0.003696561 1377340_at Tfpi2 5.28E‐05 1377684_at Rars2 1.09E‐02 1377960_at [] 0.034855604 1377962_at Mybl2 0.005138576 1378040_at Chst14 0.009355307 1378079_at Golga3 0.009463728 1378227_at Rbpms2 0.010744154 1378238_at Anapc1 0.013091803 1378251_at Treml1 0.020904303 1378388_at [] 0.017346084 1378506_at Pik3c2a 1.49E‐03 1378565_at [] 0.016721427 1378566_at [] 0.009743929 1378645_at [] 0.007295728 1378656_at Bbs5 0.025942862 1378773_at [] 0.000917494 1378818_at [] 0.042111635 1378831_at [] 1.40E‐02 1378877_at [] 1.35E‐02 1378887_at [] 3.11E‐05 1378917_at Rexo1 0.008305073 1378959_at [] 0.042725205

172

1378964_at [] 0.024548709 1378969_at Mast4 0.02751714 1378981_at [] 0.006360292 1378986_at [] 0.017259836 1379013_at RGD1306404 0.005759656 1379029_at [] 0.015653253 1379066_at Slc5a3 0.00957489 1379076_at [] 0.039880753 1379201_at Dhx8 0.027593434 1379212_at [] 0.046736479 1379217_at Acvr1 0.027999997 1379226_at Aim1l 0.024975181 1379309_at Tbl1xr1 0.047521174 1379329_at LOC685729 0.01386404 1379360_at Gatc 3.16E‐03 1379368_at Bcl6 0.015167415 1379413_at Nmnat1 0.010885119 1379420_at RGD1565002 0.022287071 1379442_at [] 0.000439525 1379461_at Zfp445 0.016039968 1379541_at [] 0.000968695 1379594_at [] 0.014280081 1379661_at [] 0.016066253 1380159_at Peo1 0.000134945 1380364_at RGD1561115 0.009497762 1380558_at Dlx3 0.037947059 1380974_at [] 0.000680804 1381039_at Dnah1 0.002468646 1381100_at [] 0.008076072 1381110_at [] 0.015649557 1381572_at RGD1565787 0.022408009 1381612_at [] 0.022097111 1381621_at [] 0.042540908 1381672_at Ldlrap1 0.020113111 1382101_at Hs2st1 0.000258982 1382217_at [] 0.044180036 1382259_a_at LOC474147 0.000133097 1382363_at Mpp5 0.000213861 1382459_at Ska3 0.016447306 1382489_at RGD1564964 0.013526678 1382669_at Bin2a 0.000208437 1382690_at Cgm4 0.026033998 1383312_at [] 0.017504334

173

1383395_at Agmat 0.021377206 1383448_at Irf9 0.004774749 1383497_at [] 0.03090328 1383706_at RGD1305733 0.041660488 1384228_at [] 0.004029036 1384496_at [] 0.017001688 1384626_at [] 0.01220578 1384831_at Slc7a13 0.000133395 1385005_at [] 0.006969929 1385011_at Smarca4 0.033480048 1385025_a_at Gpr137b 0.001683891 1385059_at Znf655 0.007507145 1385130_at Fezf2 0.004029393 1385156_at [] 0.039637387 1385157_at Cbx1 0.014437199 1385333_at Aatk 0.007659435 1385355_at [] 0.015037179 1385412_at LOC100364488 0.017939925 1385561_at Pex1 0.026732683 1385656_at Tmem174 0.049502909 1386573_a_at [] 0.036872685 1386660_at [] 0.004458726 1386823_at Irx5 0.035176158 1386997_at Eif4e 0.040054798 1387043_at Lypd3 0.033596456 1387055_at Nae1 0.019386828 1387219_at Adm 0.026387215 1387507_at Il1rap 0.006977081 1387896_at Podn /// Scp2 0.023095369 1388138_at Thbs4 0.036829352 1388243_at Gpr176 0.028414845 1388410_at Ugp2 0.005657196 1388547_at Cldn4 0.006391048 1388598_at LOC691995 0.015008807 1388631_at Azi2 0.01477766 1388673_at Lsp1 0.011460662 1388779_at Zfp180 0.000462055 1388967_at Tcfe3 0.030099392 1388968_at Mpped1 0.03882587 1389112_at Nkd1 0.036953509 1389177_at Perp 0.004307866 1389278_at Tspyl2 0.009747148 1389343_at LOC100361944 /// 0.035963655

174

RGD1560187 1389489_at [] 0.046428204 1389493_at Abtb1 0.029504538 1389545_at Tbl1xr1 0.049213469 1389675_at Ier5l 0.036685824 1389767_at RGD1304924 0.027487934 1389804_at Relt 0.010307014 1389820_at Cox7c 0.028701186 1389884_at Ccdc88b 0.022152543 1389906_at Fdft1 0.015186191 1390082_at Hip1 0.006033003 1390135_at RGD1311595 0.020884752 1390299_at [] 0.023540139 1390364_at [] 0.025146782 1390400_a_at [] 0.024034977 1390454_at Nipsnap1 0.031311452 1390719_at [] 0.002916217 1390746_at [] 0.003460228 1391028_at [] 0.006626129 1391165_at Sost 0.02073729 1391201_at Wdhd1 0.028686106 1391232_at [] 0.018012524 1391273_at Naglt1 0.022013187 1391416_at Ankrd17 0.043210745 1391514_at [] 0.039381921 1391629_at [] 0.031844139 1391635_at Ctdspl 0.001109958 1391721_at [] 0.011626959 LOC100359473 /// 1391818_at Tmco4 0.007068455 1391863_at [] 0.041280866 1392210_at RGD1307443 0.030101418 1392560_at Pip5k1a 0.0107162 1392575_at Znf654 0.000716925 1392589_at Rfc5 0.043930709 1392663_at RGD1305500 0.005897641 1393019_at [] 0.02635181 1393784_at [] 0.003728271 1393977_at [] 0.028490663 1393996_at Dnajb14 0.016292453 1394163_at [] 0.023370087 1394174_at Defa‐rs1 0.044591248 1394252_at Spock3 0.002351224

175

1394404_at Strn4 0.01791513 1394406_at [] 0.023625195 1394516_at [] 0.000864923 1394535_at Kif16b 0.043934405 1394957_at Nisch 0.009102345 1395013_at RGD1559610 0.010125339 1395035_at [] 0.010381103 1395051_at [] 0.004320324 1395057_at [] 0.039391041 1395265_at Gns 0.0122087 LOC100361684 /// 1395285_at LOC100365086 0.040613413 1395393_at [] 0.022533715 1395410_at Ppp2r3a 0.032533765 LOC100362414 /// 1395454_at Ostm1 0.016057611 1395600_at Ccdc117 0.011948168 1395637_at Asphd2 0.007451236 1395644_at Abca8 0.015342355 1395679_at LOC681924 0.035481751 1395740_at [] 0.017671049 1395777_at [] 0.001204968 1395824_at Ccno 0.037067294 1395942_at [] 0.041059494 1396008_at [] 0.0391289 1396021_at Zfp68 0.011401296 1396023_at [] 0.042974114 1396081_at Spire1 0.035806298 1396216_at Lrrc8a 0.048598886 1396226_at [] 0.002062917 1396255_at Kif16b 0.028511286 1396362_at [] 0.022450447 1396414_at [] 0.002962709 1396438_at [] 0.035907626 1396471_at [] 0.024268031 1396555_at [] 0.032535851 1396563_at [] 0.015425384 1396613_at [] 0.003563941 1396746_at [] 0.012530327 1396761_at [] 0.031444848 1396797_at Nova2 0.030216455 1396840_at [] 0.008641005 1396936_at [] 0.004955292

176

1396948_at [] 0.035668731 1397208_at [] 0.009197474 1397489_at [] 0.021100998 1397508_at Ddx18 0.002989769 1397799_at Ccdc40 0.007120371 1397852_at Tada1l 0.005611777 1397896_at RGD1566400 0.021760702 1397980_at [] 0.029423714 1398037_at [] 0.004379273 1398179_at [] 0.035217822 1398187_at [] 0.043907583 1398196_at [] 0.022430122 1398236_at [] 0.041887283 1398263_at Syne1 0.034734607 1398337_at LOC290341 0.018951416 1398362_at Notch2 0.011482179 1398364_at RGD1359529 0.009594917 1398479_at Ryr3 0.023394823 1398482_at Bcl3 0.02428472 1398490_at [] 0.028951049 1398510_at [] 0.001419008 1398655_at Myod1 0.045342088 1398976_at Ncor1 0.019892573 1399032_at Ercc1 0.022825956 1399051_at Patl1 0.006324053 1399098_at Glo1 0.025505662

7.6 6 hour in vivo TBI

Affymetrix ID Gene Symbol p‐value 1367577_at Hspb1 0.000579 1367581_a_at Spp1 0.000822 1367614_at Anxa1 0.006489 1367661_at S100a6 0.001604 1367669_a_at Map1lc3b 0.020777 1367679_at Cd74 0.021474 1367712_at Timp1 5.60E‐05 1367823_at Timp2 0.034574 1367846_at S100a4 0.001368 Fcgr2a /// LOC100362543 /// 1367850_at LOC498276 0.012035

177

1367973_at Ccl2 1.55E‐06 1367981_at Rabep1 0.000818 1367998_at Slpi 0.000572 1368000_at C3 0.019722 1368014_at Ptges 0.000172 1368187_at Gpnmb 0.005749 1368223_at Adamts1 0.007439 1368322_at Sod3 3.69E‐05 1368332_at Gbp2 0.00197 1368382_at S100a3 0.004811 1368419_at Cp 0.020964 1368420_at Cp 0.000154 1368440_at Slc3a1 0.043102 1368489_at Fosl1 0.000257 1368490_at Cd14 0.000244 1368494_at S100a8 0.000569 1368527_at Ptgs2 0.045341 1368537_at Dctn4 0.019611 1368704_a_at Cspg5 0.031546 1368728_at P2ry12 0.007619 1368778_at Slc6a6 0.037617 1368820_at Nfyc 0.036572 1368840_at Tmem176b 0.00855 1368858_at Ugt8 0.008397 1368894_at Cap2 0.027719 1368914_at Runx1 0.007229 1368921_a_at Cd44 0.002941 1368943_at Rnase4 0.003321 1368986_at Slc17a7 0.036349 1369022_at Svop 0.04117 1369041_at Nlgn1 0.002459 1369059_at Trpm7 0.011417 1369066_at Madd 0.028984 1369077_at Asah1 0.027504 1369078_at Mapk1 0.033754 1369079_at Nxph1 0.000504 1369081_at Neu1 0.025735 1369084_a_at Bok 0.005203 1369130_at Rasgrp1 0.016748 1369220_at Dnm1l 0.017022 1369249_at Ankh 0.046404 1369265_at Senp2 0.017702 1369268_at Atf3 6.65E‐05

178

1369355_at Grm5 0.044391 1369402_at Adnp 0.016038 1369417_a_at Opcml 0.006784 1369487_a_at Kcnj4 0.044045 1369609_at Cldn11 0.006506 1369617_at Ube2n 0.041282 1369621_s_at Fkbp1a 0.038372 1369629_at Adk 0.003837 1369688_s_at Ptk2b 0.030623 1369714_at Dnajc14 0.011814 1369733_at Ctnnb1 0.025353 1369755_at B3gat2 0.039978 1369811_at Cplx1 0.028269 1369815_at Ccl3 0.003696 1369847_at Kcnab1 0.027531 1369885_at [] 0.032404 1369943_at Tgm2 0.000679 1369973_at Xdh 0.007038 1370011_at Ak1 0.040635 1370059_at Nefl 0.032926 1370080_at Hmox1 0.001082 1370130_at Rhoa 0.043746 1370134_at Slc33a1 0.006749 1370146_at Glrb 0.009945 1370150_a_at Thrsp 0.024203 1370177_at PVR 0.004314 1370186_at Psmb9 0.001862 1370205_at Slco1c1 0.023137 1370249_at Tspo 0.002717 1370445_at Pla1a 0.000237 1370501_at Ube2g1 0.042276 1370503_s_at Epb4.1l3 0.041828 1370609_a_at Slc16a7 0.012005 1370651_a_at Inppl1 0.017545 1370669_a_at Pde10a 0.03594 1370680_at Stau2 0.020079 1370690_at Hspa9 0.034678 1370693_a_at Cnp 0.043937 1370757_at Cacng3 0.047777 1370792_at Mapre1 0.044135 1370832_at Ccl4 0.018082 1370883_at RT1‐Da 0.006554 1370951_at Ppap2b 0.035898

179

1371026_at Ppfia4 0.011157 1371051_at Grinl1a 0.022748 1371055_at Rab12 0.039825 1371060_at Trim23 0.027695 1371063_at Sh3gl2 0.03115 1371079_at Fcgr2a /// Fcgr2b 2.74E‐06 1371101_at Ryk 0.037326 1371113_a_at Tfrc 0.010774 1371139_at Pls3 0.028843 1371237_a_at Mt1a 0.002091 1371298_at H19 0.000114 1371447_at Plac8 0.002462 1371527_at Emp1 0.037475 1371575_at [] 0.005211 1371785_at Tnfrsf12a 0.00398 1372064_at Cxcl16 0.000366 1372180_at Sdc3 0.024911 1372930_at Sp110 0.025301 1373403_at LOC684871 0.001074 1373759_at [] 0.022464 1373777_at [] 0.025875 1373814_at R3hdm2 0.036209 1374085_at Mxd4 0.034507 1374113_at Baz1a 0.011975 1374129_at LOC690789 /// Oaz2 0.04404 1374207_at Angpt2 0.023997 1374284_at Rassf4 0.004247 1374610_at [] 0.007566 1374857_at Gar1 0.002722 1375043_at Fos 0.003277 1375084_at Serinc2 0.000568 1375137_at Arpc2 0.027254 1375140_at Mbnl2 0.026202 1375153_at Sox2 0.039692 1375170_at S100a11 0.007876 1375268_at [] 0.004616 1375388_at [] 0.012099 1375468_at Abcc5 0.037293 1375630_at Nhp2l1 0.020572 1375714_at RGD1562952 0.005365 1375829_at Fam45a 0.034237 1375870_a_at Rbms1 0.035233 1375876_at RGD1308049 0.035339

180

1375903_a_at Yaf2 0.014653 1376100_at Tubb6 9.68E‐05 1376102_at Tmbim1 0.048627 1376150_at S1pr3 0.000901 1376197_at [] 0.042128 1376481_at Adamts9 0.001174 1376579_at Lap3 0.023128 1376727_at Yipf4 0.002483 1376952_at [] 0.026521 1376996_at [] 0.021261 1377889_at Urb1 0.039208 1377894_at [] 0.013951 1377896_at Rab22a 0.030583 1377925_at [] 0.020079 1377947_at [] 0.006476 1378037_at Rnf215 0.00025 1378122_a_at [] 0.034817 1378151_at [] 0.019128 1378189_at [] 0.026783 1378238_at Anapc1 0.019278 1378388_at [] 0.028664 1378450_at [] 0.000308 1378477_a_at Fhod1 0.049453 1378484_at Rasl12 0.002712 1378532_at [] 0.031601 1378546_at [] 0.02869 1378582_at Trabd 0.026843 1378696_at [] 0.000633 1378705_at Setd5 0.006939 1378724_at Nfkbil1 0.001246 1378751_at Mapk4 0.009155 1378754_at [] 0.016505 1378781_at [] 0.047593 1378832_at [] 0.031048 1378847_x_at H2afx 0.000515 1378887_at [] 0.000821 1378914_a_at LOC308990 0.000626 1378927_at [] 0.016703 1378956_at [] 0.007157 1378976_x_at [] 0.047418 1379007_at [] 0.044765 1379018_at [] 0.034482 1379027_at Wwc1 0.01985

181

1379029_at [] 0.02393 1379066_at Slc5a3 0.007496 1379076_at [] 0.020821 1379102_at [] 0.033885 1379137_at [] 0.003609 1379212_at [] 0.048532 1379329_at LOC685729 0.01285 1379341_at Usp6nl 0.007654 1379357_at [] 0.049115 1379420_at RGD1565002 0.032287 1379442_at [] 0.018774 1379446_at [] 0.006348 1379450_at Cttnbp2nl 0.007672 1379465_at LOC311134 0.012396 1379498_at [] 0.032239 1379567_at Med13 0.005366 1379590_at Zrsr1 0.010072 1379594_at [] 0.0157 1379599_at [] 0.041861 1379760_at Tnfaip8l2 0.009351 1379767_at [] 0.044741 1379768_at [] 0.011212 1379809_at Plekhf2 0.015034 1379843_at Vps36 0.014982 1379852_at Znf512b 0.038858 1379912_at [] 0.03703 1379965_at [] 0.009047 1380014_at [] 0.019549 1380068_at [] 0.000499 1380283_at Tshz1 0.007162 1380320_at Lin54 0.013624 1380358_at Caprin1 0.003529 1380695_at [] 0.003283 1380923_at [] 0.011752 1381042_at Anapc10 0.000587 1381125_at [] 0.005833 1381469_a_at Gigyf1 0.032655 1381490_at [] 0.04814 1381834_at [] 0.047458 1381899_at [] 0.027987 1381951_at [] 0.016752 1382217_at [] 0.029426 1382231_at [] 0.00047

182

1382270_at [] 0.027665 1382375_at [] 0.022428 1382426_at RGD1563798 0.016596 1382430_at [] 0.021897 1382459_at Ska3 0.034675 1382470_at [] 0.026704 1382564_at [] 0.037844 1382612_at [] 0.011115 1382669_at Bin2a 0.020707 1382717_at Fam168a 0.008635 1382803_at Map4k1 0.000393 1382969_at [] 0.023035 1382976_at [] 0.025961 1382977_at [] 0.040878 1382990_at [] 0.028182 1383307_at Tmem186 0.020999 1383312_at [] 0.011704 1383349_at Rfx5 0.017304 1383363_at [] 0.004048 1383434_at Pycr1 0.006463 1383484_at [] 0.001397 1383540_at [] 0.014197 1383706_at RGD1305733 0.014773 1383997_at [] 0.020176 1384150_at Mid1 0.033734 1384235_at [] 0.002871 1384329_at Prss22 0.002081 1384435_at Tmem68 0.028044 1384437_at Smarca1 0.00461 1384544_at Pon3 0.043775 1384545_at [] 0.008551 1384718_at [] 0.035809 1384724_at [] 0.004759 1384747_at [] 0.00209 1384765_at Rhbdf2 0.015178 1384829_at [] 0.028235 1384989_at Plekhn1 0.032897 1385006_at [] 0.004329 1385027_at [] 0.000725 1385201_at Tmem182 0.041566 1385280_at [] 0.000611 1385319_at Arhgap27 0.047255 1385355_at [] 0.010367

183

1385420_at Dvl3 0.031519 1385474_at Rnf160 0.025098 1385506_at [] 0.02515 1385544_at Nrl 0.005176 1385561_at Pex1 0.030165 1385626_at MGC95152 0.041396 1385876_at LOC691984 0.044008 1386031_at Prkd3 0.035782 1386073_at Psmd14 0.000192 1386172_at [] 0.002648 1386359_x_at [] 0.037262 1386884_at Htra1 0.00145 1386970_at Eif2b4 0.022007 1387319_at Ccl11 0.001412 1387421_at Kcnip3 0.022124 1387450_at Tgfa 0.037145 1387524_at Cysltr2 0.008781 1387551_at Kcnh7 0.013176 1387584_at Shh 0.003527 1387599_a_at Nqo1 0.037936 1387613_at Npy2r 0.043866 1387661_a_at Oprl1 0.043849 1387698_at Kcnj11 0.005546 1387791_at Ace 0.008762 1387871_at [] 0.025352 1388134_at Eef1d 0.009828 1388210_at Acot2 0.047076 1388243_at Gpr176 0.011075 LOC300024 /// Ly6al 1388273_at /// Ly6c 0.014353 1388289_s_at Fshb 0.046695 1388471_at Tcp11l2 0.022926 1388472_at [] 0.046182 1388474_at Ube2i 0.032766 1388534_at [] 6.01E‐05 1388574_at Wars 0.044046 1388810_at Abce1 0.042077 1388846_at Bcl2l12 0.008064 1388966_at [] 0.027505 1388967_at Tcfe3 0.013919 1388974_at Srp19 0.005961 1389067_at Slco4a1 0.040088 1389112_at Nkd1 0.037084

184

1389177_at Perp 0.016337 1389268_at Poll 0.01243 1389298_at [] 0.014972 1389326_at Rfc3 0.002251 LOC100361944 /// 1389343_at RGD1560187 0.008422 1389364_at Ndfip2 0.015723 1389388_at [] 0.031369 1389402_at Csrnp1 0.030299 1389449_at Fitm1 0.007936 1389466_at Ccdc6 0.006405 1389486_at [] 0.041614 1389487_at Dnajb12 0.009386 1389570_at Inpp5k 0.000757 1389596_at Cox18 0.022157 1389617_at Elk3 0.048496 1389670_at Hoxa10 0.002593 1389820_at Cox7c 0.044566 1389828_at Cebpd 0.031334 1389906_at Fdft1 0.027588 1389986_at [] 0.026037 1389987_at Trib1 0.046386 1390015_at [] 0.042552 1390129_at [] 0.012594 1390135_at RGD1311595 0.023008 1390155_at Abhd6 0.048924 1390158_at LOC304903 0.02215 1390364_at [] 0.033373 1390425_at [] 0.03433 1390438_at Hdac5 0.014045 1390454_at Nipsnap1 0.0249 1390489_at [] 0.003659 1390810_at [] 0.034654 1390831_at [] 0.022046 1391020_at Btbd17 0.011802 1391029_at [] 0.029932 1391273_at Naglt1 0.042327 1391331_at [] 0.031264 1391378_at [] 0.024827 1391442_at Ehd3 0.00293 1391514_at [] 0.039126 1391597_at Gpr75 0.017633 1391670_at [] 0.02846

185

1391681_at Ccne2 0.014344 1391682_at [] 0.044461 1391729_at [] 0.024656 1391746_at [] 0.026329 LOC100362394 /// 1391756_at LOC100365991 0.011077 1391851_at [] 1.01E‐05 1391863_at [] 0.043397 1392072_at [] 0.000498 1392493_at [] 0.024467 1392536_at [] 0.011449 1392768_at Asf1a 0.002339 1393018_at [] 0.005586 1393181_at Nfasc 0.01385 1393254_at Stk36 0.033905 1393279_at [] 0.00525 1393356_at [] 0.024305 1393510_at Golsyn 0.017591 1393524_at [] 0.00136 1393632_at C1qtnf7 0.035093 1393715_s_at T2 0.025308 1393747_at Spink4 0.01379 1393909_at RGD1305110 0.043037 1393937_at [] 0.000205 1394059_s_at Yme1l1 0.010398 1394163_at [] 0.045435 1394180_at [] 0.042128 1394388_at LOC100125371 0.000469 1394389_at [] 0.043674 1394441_at [] 0.036058 1394590_at Nkiras1 0.030165 1394634_at Dnajc10 0.008599 1394670_at Obfc1 0.008496 Ercc4 /// Ercc4l1 /// LOC100365348 /// 1394686_at LOC688631 0.002278 1394735_at Catsper2 0.017754 1394766_at [] 0.035514 1394795_at [] 0.047633 1394812_at Als2cl 0.016464 1394820_at [] 0.034407 1394902_at [] 0.012266 1394959_at [] 0.02712

186

1394987_at [] 0.005357 1395096_at [] 0.027312 1395107_at [] 0.028524 1395205_at [] 0.030109 1395265_at Gns 0.039803 1395487_at [] 0.003208 1395502_at Ppp2r5d 8.74E‐05 1395773_at [] 0.006154 1395934_at LOC684934 0.03212 1396000_at [] 0.024375 1396003_at [] 0.012386 1396008_at [] 0.043117 1396021_at Zfp68 0.034111 1396026_at [] 0.04527 1396033_at [] 0.033131 1396133_at Itgb5 0.024947 1396178_at [] 0.00439 1396181_at F8 0.012033 1396195_at Ankrd13a 0.016953 1396217_at [] 0.001982 1396220_at [] 0.02393 1396221_at B3galt6 0.002806 1396299_at [] 0.03607 1396322_at LOC100364796 0.002346 1396329_at [] 0.006933 1396405_at [] 0.013197 1396506_at [] 0.002347 1396592_at [] 0.020669 1396635_at [] 0.004013 1396728_at [] 0.011092 1396745_at [] 0.027623 1396746_at [] 0.037209 1396761_at [] 0.011409 1396806_at [] 0.047263 1396814_at [] 0.010555 1396908_at [] 0.006684 1396926_at Vac14 0.027553 1396948_at [] 0.025886 1397440_at Megf10 0.020864 1397722_at [] 0.025151 1397980_at [] 0.039888 1398107_at Ggct 0.00203 1398175_at [] 0.007988

187

1398178_at [] 0.029242 1398187_at [] 0.016238 1398204_at [] 0.025292 1398263_at Syne1 0.01047 1398355_at Trpm7 0.008506 1398678_at [] 0.03077 1398685_at [] 0.006304 1398793_at Cdc5l 0.011104 1399088_at Tlk2 0.022788 1399098_at Glo1 0.019257

7.7 24 hour in vivo TBI

Affymetrix ID Gene Symbol p‐value 1367555_at Alb 0.003605 1367568_a_at Mgp 0.015707 1367570_at Tagln 0.000257 1367574_at Vim 6.35E‐05 1367577_at Hspb1 0.000457 1367581_a_at Spp1 0.005778 1367584_at Anxa2 0.000255 1367612_at Mgst1 0.000937 1367614_at Anxa1 0.000943 1367628_at Lgals1 0.000419 1367631_at Ctgf 0.039667 1367661_at S100a6 0.000448 1367676_at Hmgb2 0.008063 1367679_at Cd74 0.001918 1367705_at Glrx1 0.020252 1367712_at Timp1 8.72E‐05 1367776_at Cdk1 0.000882 1367794_at A2m 0.002448 1367823_at Timp2 0.031009 1367846_at S100a4 0.00012 1367847_at Nupr1 0.011208 1367849_at Sdc1 0.000944 Fcgr2a /// LOC100362543 /// 1367850_at LOC498276 0.004617 1367925_at Mvp 0.005405 1367973_at Ccl2 0.001129 1367974_at Anxa3 0.000789

188

1367975_at Anxa3 0.004399 1367998_at Slpi 0.001355 1368000_at C3 0.001381 1368010_at Ptpn6 0.000332 1368187_at Gpnmb 9.89E‐05 1368270_at Apobec1 0.000748 1368332_at Gbp2 0.000178 1368353_at Gfap 2.90E‐05 1368356_a_at Erap1 0.040718 1368382_at S100a3 0.001936 1368384_at Klk6 0.003149 1368420_at Cp 0.000143 1368490_at Cd14 1.19E‐06 1368494_at S100a8 0.001601 1368537_at Dctn4 0.030962 1368558_s_at Aif1 0.010509 1368666_a_at Lphn3 0.045952 1368704_a_at Cspg5 0.045432 1368742_at C5ar1 0.000923 1368770_at Gcnt1 0.013771 1368778_at Slc6a6 0.017349 1368820_at Nfyc 0.049773 1368859_at Ppm1a 0.034997 1368879_a_at Gnao1 0.037991 1368914_at Runx1 0.002591 1368921_a_at Cd44 0.003274 1368957_at Gng7 0.036682 1369022_at Svop 0.031869 1369079_at Nxph1 0.001255 1369130_at Rasgrp1 0.023891 1369265_at Senp2 0.041351 1369326_at Akap6 0.048471 1369402_at Adnp 0.04446 1369421_at Top1 0.034349 1369714_at Dnajc14 0.031104 1369733_at Ctnnb1 0.042947 1369885_at [] 0.0288 1369973_at Xdh 0.003453 1369979_at Skap2 0.003695 1370080_at Hmox1 0.001967 1370090_at Lcp2 0.002131 1370154_at Lyz2 8.92E‐05 1370186_at Psmb9 7.11E‐05

189

1370228_at [] 0.01606 1370249_at Tspo 0.00031 1370355_at Scd1 0.003855 1370488_a_at Ptprs 0.049489 Ugt1a1 /// Ugt1a2 /// Ugt1a3 /// Ugt1a5 /// Ugt1a6 /// Ugt1a7c /// 1370613_s_at Ugt1a8 /// Ugt1a9 0.005953 1370669_a_at Pde10a 0.04905 1370690_at Hspa9 0.032404 1370883_at RT1‐Da 0.001979 1370885_at Ctsz 8.64E‐05 1370973_at Scn7a 0.001354 1371037_at Pros1 0.046441 1371074_a_at Mcm6 0.000208 1371079_at Fcgr2a /// Fcgr2b 1.72E‐05 1371140_a_at Accn1 0.041149 1371298_at H19 0.000949 1371400_at Thrsp 0.000419 1371447_at Plac8 0.007866 1371575_at [] 0.000381 1371643_at Ccnd1 0.004369 1371862_at Rrm1 0.006659 1371913_at Tgfbi 0.0068 1372013_at Ifitm1 0.004394 1372070_at Ifi30 0.000536 1372097_at [] 0.000591 1372254_at Serping1 0.000785 1372404_at Rac2 0.00209 1372406_at Mcm3 0.001876 1372585_at [] 0.002711 1372886_at Tacc3 0.001078 1372930_at Sp110 0.011436 1373025_at C1qc 0.000111 1373161_at Tmem98 0.040697 1373490_at Gmfg 0.000558 1373575_at Fcer1g 5.51E‐05 1373658_at Racgap1 0.006831 1373722_at Kif20a 0.005782 1373785_at Pld4 0.000521 1373823_at Cks2 0.00028 1373889_at Igsf7 /// LOC363715 0.001034

190

/// RGD1561778 1373911_at Postn 0.004108 1373932_at [] 0.030359 1374036_at Mcm2 1.07E‐06 1374284_at Rassf4 0.00406 1374367_at Grxcr1 0.015551 1374449_at Cdca3 0.004145 1374540_at Cdca7 0.001979 1374730_at Tyrobp 0.000648 1374775_at Mki67 0.001148 1374778_at [] 0.027516 1374794_at Kif15 0.002226 1375010_at Cd68 0.002674 1375170_at S100a11 0.000937 1375183_at Id4 0.016231 1375404_at [] 0.032019 1375542_at Rdx 0.041214 1375633_at Clic1 0.000413 1375716_at Ifngr2 0.027076 1376055_at Mcm5 0.002943 1376100_at Tubb6 0.000264 1376102_at Tmbim1 0.011459 1376137_at Plekhb2 0.036614 1376138_at [] 0.003739 1376150_at S1pr3 0.000295 1376185_at Kifc1 0.011269 1376579_at Lap3 0.023275 1376952_at [] 0.03707 1376967_at [] 0.048715 1377092_at [] 0.00201 1377119_at Pura 0.039756 1377350_at [] 0.003801 1377710_at [] 0.016139 1377925_at [] 0.038996 1378052_at [] 0.047083 1378399_at LOC498145 0.018223 1378450_at [] 9.86E‐05 1378461_at [] 0.001991 1378484_at Rasl12 0.000176 1378529_at [] 0.049519 1378582_at Trabd 0.012842 1378696_at [] 0.004601 1378705_at Setd5 0.032173

191

1378847_x_at H2afx 0.000833 1378927_at [] 0.046902 1378959_at [] 0.04296 1379137_at [] 0.001991 1379420_at RGD1565002 0.03025 1379465_at LOC311134 0.011979 1379498_at [] 0.016475 1379524_at Wdr34 0.008743 1379531_at [] 0.000366 1379567_at Med13 0.001286 1379643_at LOC100359906 0.010726 1379768_at [] 0.010278 1379809_at Plekhf2 0.024924 1379812_at Nnmt 0.025238 1379843_at Vps36 0.000318 1379870_at Chrnb1 0.003611 1379912_at [] 0.00233 1379965_at [] 0.000737 1380032_at Slain2 0.000197 1380056_at [] 0.003413 1380068_at [] 0.002763 1380222_at [] 0.001129 1380283_at Tshz1 0.009398 1380335_at [] 0.001016 1380459_at Nacc2 0.010723 1380527_at [] 0.001189 1380578_at [] 0.015864 1380695_at [] 0.002305 1380782_at [] 0.000463 1380877_at [] 0.038498 1380923_at [] 0.015664 1380980_at RGD1309414 0.000429 1381042_at Anapc10 0.002115 1381125_at [] 0.000814 1381127_at [] 0.007508 1381138_at [] 0.005779 1381189_at Fzr1 0.021976 1381327_a_at Cr2 0.0044 1381328_at Cr2 0.02444 1381445_at [] 0.002673 1381538_at [] 0.001589 1381688_a_at [] 0.02537 1381746_at [] 0.007739

192

1381920_at [] 0.000963 1381955_at Fam183b 0.005155 1381971_at Sox18 0.020146 1381987_at [] 0.001816 1382082_at Nfyb 5.14E‐05 1382217_at [] 0.026394 1382231_at [] 0.001379 1382259_a_at LOC474147 0.003078 1382305_at [] 0.013308 1382310_at RGD1563296 0.013287 1382325_at Gcat 0.004394 1382426_at RGD1563798 0.019027 1382430_at [] 0.0209 1382507_at Cd96 0.000402 1382803_at Map4k1 0.003056 1382908_at Epha4 0.03694 1383307_at Tmem186 0.027998 1383408_at [] 0.009496 1383434_at Pycr1 0.005492 1383477_at Uchl5 0.007367 1383687_at [] 0.010842 1383944_at [] 0.043593 1384122_at [] 0.04844 1384131_at Atl2 0.017969 1384146_at [] 0.007062 1384537_at [] 0.022529 1384545_at [] 0.007254 1384718_at [] 0.024453 1385090_at Rad17 0.003032 1385188_at RGD1309188 0.011404 1385220_at Zwilch 0.003958 1385506_at [] 0.025784 1385544_at Nrl 0.018497 1385561_at Pex1 0.036819 1385565_at Rad50 0.002089 1385578_at [] 0.043737 1385600_at [] 0.002174 1385628_at [] 0.015918 1386073_at Psmd14 0.000422 1386359_x_at [] 0.029681 1386586_at Pak1ip1 0.015971 1386822_s_at RGD1311823 0.011626 1387239_a_at Padi4 0.018342

193

1387319_at Ccl11 0.000537 1387674_at Cnr1 0.011996 1387698_at Kcnj11 0.013232 1388021_at Sfrp4 0.004696 1388148_a_at Lrpap1 0.005486 1388159_at CYTB 0.006088 LOC300024 /// Ly6al 1388273_at /// Ly6c 0.019482 1388289_s_at Fshb 0.039441 1388367_at Pole4 0.002217 1388435_at Crygs 0.008148 1388534_at [] 0.000122 1388722_at Dnajb1 0.006688 1389067_at Slco4a1 0.035381 1389268_at Poll 0.04572 1389326_at Rfc3 0.023556 1389381_at Sqstm1 0.005035 1389499_at [] 0.013979 1389533_at Fbln2 0.002542 1389570_at Inpp5k 0.0027 1389749_at [] 0.030915 1389767_at RGD1304924 0.045969 1389827_at Fdx1 0.000689 1389966_at [] 0.015549 1390011_at [] 0.002413 1390489_at [] 0.045913 1390515_at [] 0.019794 1390744_at [] 0.017271 1390779_at [] 0.035662 1390810_at [] 0.034301 1390939_at [] 0.04603 1391201_at Wdhd1 0.000105 1391212_at Tceal1 0.040282 1391670_at [] 0.048977 1391794_at [] 0.007402 1391851_at [] 0.000305 1391863_at [] 0.026259 1391874_at [] 0.004499 1392537_at LOC684811 0.029943 1393181_at Nfasc 0.029304 1393185_at LOC691918 0.035995 1393473_at RGD1563866 0.040905 1393510_at Golsyn 0.032312

194

1393535_at [] 0.001642 1393652_at Zbtb1 0.029382 1393747_at Spink4 0.000207 1393872_at Sel1l3 0.004734 1393937_at [] 0.008248 1394114_at [] 0.037664 1394284_at [] 0.000309 1394429_at [] 0.010191 1394605_at RGD1566149 0.000209 1394987_at [] 0.028053 1395034_at [] 0.044356 1395088_at [] 0.002305 1395096_at [] 0.048622 1395234_at [] 0.036875 1395289_at Vps26b 0.040767 1395430_at [] 0.029619 1395750_at [] 0.035169 1395773_at [] 0.016392 1395853_at Clca3 0.00504 1396003_at [] 0.044534 1396025_at Gpr61 0.045701 1396194_at [] 0.018488 1396217_at [] 0.018871 1396220_at [] 0.029091 1396313_at [] 0.019283 1396745_at [] 0.040865 1396942_at [] 0.00098 1397524_at [] 0.018342 1398475_at Dars2 0.022184 1398678_at [] 0.044741

8 Appendix B: Publications

8.1 Manuscripts

Lamprecht M.R., Elkin B.S., Kesavabhotla K., Crary J.F., Raghupathi R., Morrison B. 3rd (In preparation). Correlation of genome-wide expression after traumatic brain injury in vitro and in vivo implicates a role for SORLA.

195

Lamprecht, M.R. and Morrison, B. 3rd (Submitted). A combination therapy of 17β-estradiol and memantine is more neuroprotective than monotherapies in an organotypic brain slice culture model of traumatic brain injury.

Lamprecht, M.R. and Morrison, B. 3rd (2014). GPR30 activation is neither necessary nor

sufficient for acute neuroprotection by 17β-estradiol after an ischemic injury in organotypic

hippocampal slice cultures. Brain Res. 1563, 131-137.

Dixon S.J., Lemberg K.M., Lamprecht MR, Skouta R., Zaitsev E.M., Gleason C.E., Patel D.N.,

Bauer A.J., Cantley A.M., Yang W.S., Morrison B. 3rd, Stockwell B.R. (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 149(5), 1060-1072.

8.2 Conference proceedings

Lamprecht M.R., McKnight T.E., Ericson M.N., Morrison B. 3rd (2010). VACNF arrays for

recording dopamine concentrations in the brain. The 36th Annual Northeast Bioengineering

Conference at Columbia University. New York, NY

Muratore R., Szulman E., Kalisz A., Xu N., Lamprecht M.R., Simon M.J., Yu Z., Morrison B.

3rd (2008). Bioeffective ultrasound at very low doses: reversible manipulation of neuronal cell

morphology and function in vitro. 8th International Symposium on Therapeutic Ultrasound,

Minneapolis, MN

8.3 Abstracts

Lamprecht M.R. and Morrison B 3rd (2011). Memantine and Estradiol Combination Therapy as

Treatment for Traumatic Brain Injury. BMES Annual Meeting, Hartford, Connecticut

196

Lamprecht M.R., Elkin B.S. and Morrison B 3rd (2011). Combinational drug therapies as

treatment for traumatic brain injury. National Neurotrauma Society Symposium, Ft. Lauderdale,

Florida.

197