Imaging brain inflammation in epilepsy

Accepted Manuscript Imaging brain inflammation in epilepsy Halima Amhaoul, Steven Staelens, Stefanie Dedeurwaerdere PII: DOI: Reference:

S0306-4522(14)00724-6 http://dx.doi.org/10.1016/j.neuroscience.2014.08.044 NSC 15664

To appear in:

Neuroscience

Accepted Date:

27 August 2014

Please cite this article as: H. Amhaoul, S. Staelens, S. Dedeurwaerdere, Imaging brain inflammation in epilepsy, Neuroscience (2014), doi: http://dx.doi.org/10.1016/j.neuroscience.2014.08.044

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Imaging brain inflammation in epilepsy Halima Amhaoul1, Steven Staelens2 and Stefanie Dedeurwaerdere1 1

Department of Translational Neuroscience, University of Antwerp, Universiteitsplein 1, Wilrijk,

Antwerp 2610, Belgium; [email protected], [email protected] 2

Molecular Imaging Center Antwerp, University of Antwerp, Universiteitsplein 1, Wilrijk, Antwerp

2610, Belgium; [email protected]

Corresponding author: Stefanie Dedeurwaerdere Department of Translational Neuroscience, University of Antwerp, FGEN CDE T3.07, Universiteitsplein 1, Wilrijk, Antwerp 2610, Belgium Phone number: +3232652638 [email protected] Key words: Neuroinflammation, epilepsy, non-invasive imaging, PET, MRI

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List of abbreviations AMT

α-methyl-L-tryptophan

BBB

Blood-brain barrier

CNS

Central nervous system

FLAIR

Fluid attenuated inversion recovery

GSH

Gluthatione

ICAM-1

Intercellular adhesion molecule 1

IDO

Indoleamine 2,3-dioxygenase

KASE

Kainic acid-induced status epilepticus

MAO-B

Monoamine oxidase type B

mIs

Myo-inositol

MNP

Magnetonanoparticles

MRI

Magnetic resonance imaging

MRS

Magnetic resonance spectroscopy

PET

Positron emission tomography

SPECT

Single-photon emission computed tomography

TLE

Temporal lobe epilepsy

TSPO

Translocator protein

USPIO

Ultrasmall and superparamagnetic iron oxide

VCAM-1

Vascular cell-adhesion molecule 1

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Abstract Epilepsy is a highly common chronic neurological disorder. Although symptomatic treatment is available, 30-40% of epilepsy patients still remain resistant to anti-epileptic drugs. The primary identification and extensive characterization of the pathological substrates underlying epilepsy would facilitate the development of novel treatments, including disease-modifying and antiepileptogenic therapies. A plethora of evidence points towards an undeniable role of brain inflammation in epileptogenesis. However, the exact role of this process remains unfortunately not clear. Non-invasive imaging of brain inflammation can promote our fundamental knowledge, which may lead to better insights into the role of brain inflammation in disease ontogenesis. Moreover, it will allow us to investigate whether the visualization of this process can serve as a validated biomarker for epilepsy. In turn, such can lead to major perspectives regarding the development and evaluation of anti-inflammatory treatments, and screening possibilities for patients at risk. Here, we firstly discuss the applications for imaging of the different brain inflammation constituents. Secondly, we review the available approaches for molecular imaging of brain inflammation in general and finally present the current research on the imaging of brain inflammation in patients and experimental models of epilepsy. The current imaging toolbox is limited by the range of neuroinflammatory targets, which can be visualized at present, and in addition, the often indirect approaches used. We believe that research in this field will further advance as highly specific ligands, and producible and practical imaging approaches will become available.

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Epilepsy is the denominator of a group of chronic neurological disorders characterized by the occurrence of spontaneous recurrent seizures. Approximately 65 million people worldwide suffer from epilepsy and it becomes more common as people age (Hauser et al., 1998; Brodie et al., 2009; Thurman et al., 2011). The impact of this disease on the health and the quality of life of the patient, and on society as a whole, is severe. First of all, no curative drug treatments for epilepsy exist and thus it is often acquired for life. Secondly, the current anti-epileptic drugs available, which are indeed only symptomatic, have many side-effects and are ineffective in 30-40% of the patients (Regesta and Tanganelli, 1999; Kwan and Brodie, 2000). Finally, neuropsychiatric co-morbidities, such as depression (Kanner, 2005), autism (Jensen, 2011) and schizophrenia (Stefansson et al., 1998), are common and are nowadays even seen as outcomes of the epileptogenic process (Duncan et al., 2006; Jensen, 2011; Brooks-Kayal et al., 2013). During the past decade, clinical and preclinical evidence has shown that brain inflammation is an important feature in epilepsy, even in those syndromes classically not associated with inflammation. It has been demonstrated that various inflammatory mediators are present in experimental and human epileptic tissue and that epileptogenic insults cause a rapid-onset inflammatory process in the affected brain regions (Vezzani and Granata, 2005; Friedman et al., 2009; Ravizza et al., 2011; Vezzani et al., 2011; Dedeurwaerdere et al., 2012a). Experimental studies have also shown that inflammation can cause seizures and seizures can cause inflammation (Ravizza et al., 2011; Vezzani and Ruegg, 2011) (Fig. 1). Brain inflammation is thus involved in the molecular, structural and synaptic changes, and represents a significant factor in the reorganization of a normal neuronal network into an epileptic one, a process called epileptogenesis. Addressing this inflammatory process by pharmacological manipulations in different animal models of epilepsy has been shown to efficiently decrease spontaneous recurrent seizures during the chronic phase (Maroso et al., 2010; Akin et al., 2011; Maroso et al., 2011) and may therefore lead to new anti-epileptic therapies for patients (Dedeurwaerdere et al., 2012b).

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Non-invasive imaging of brain inflammation opens new perspectives in the present highly demanding research field. We believe that it will lead to fundamental insights into the role of brain inflammation in the pathophysiology of epilepsy in vivo. Especially because longitudinal measurements in the same subject are possible, which enables the assessment of different temporal stages in disease ontogenesis (Dedeurwaerdere et al., 2007; Dedeurwaerdere et al., 2014; Shultz et al., 2014). Accordingly, these principal and essential data are needed to comprehensively assess whether brain inflammation could serve as a reliable biomarker for both epileptogenesis and chronic epilepsy.

The identification of such a non-invasive biomarker will give rise to an extended list of possibilities for clinical research and care. Validated markers for brain inflammation would enable new approaches for the development of novel therapies. The non-invasive character of these biomarkers makes it possible to evaluate anti-inflammatory treatment in patients. Furthermore, the evaluation of other treatment strategies could also benefit from this marker by investigating the indirect effect of the particular therapy on brain inflammation, representing a measure of brain damage. In addition, the development of markers for specific inflammatory processes could also guide the development of drugs which could inhibit epileptogenesis. Secondly, imaging markers of brain inflammation would be extremely helpful in selecting those patients who should enrol in a clinical trial to evaluate antiinflammatory treatments in patients with established epilepsy. Finally, in drug-resistant epilepsy, these markers could provide an additional and objective readout for treatment efficacy besides the routine clinical examination and determination of seizure frequency. Previously, it has been suggested that hippocampal sclerosis, which is highly associated with brain inflammation (gliosis), is a risk factor for drug refractoriness. This means that brain inflammation might serve as a biomarker for identifying pharmacoresistant patients. In line, a recent study showed that imaging of brain inflammation could serve as a biomarker for anti-epileptic drug resistance (Bogdanovic et al., 2014).

7 This in turn may lead to better treatment strategies and to an improvement in the quality of healthcare. Currently, resective surgery of the epileptic focus is the first (non-pharmacological) treatment option in line for patients with intractable focal epilepsy. There are cases of epilepsy that are cured surgically, and many which go into spontaneous remission. However, in some patients the ictal onset zone cannot be clearly delineated, which makes them less suitable for surgery. Moreover, some patients do not show any structural alterations on images acquired by magnetic resonance imaging (MRI). 20 to 30%, which is probably still an underestimation, of patients with chronic focal epilepsy syndromes are MRI negative (Cascino et al., 1991; Li et al., 1995; Wiebe et al., 2001; Tellez-Zenteno et al., 2010). This raises the necessity of using additional tools, not only for more accurate delineation, but also for the identification of the epileptic focus in MRI negative patients. Whether brain inflammation could assist in more punctual confinement of the ictal onset zone is doubtful as it is known that excitotoxicity can cause inflammation. Thus, the spread of epileptic activity beyond the epileptic focus will probably cause inflammation in the surrounding brain areas (Juhasz et al., 2013). Therefore, it needs to be demonstrated whether it can be useful in the pre-surgical evaluation of the relevant anatomical site in those patients without any typical pathological hallmarks (Kumar et al., 2008).

We would also like to emphasize an important goal in epilepsy research, namely altering epileptogenesis. If future research were to confirm that brain inflammation is associated with causal factors in the development of epilepsy, it may become conceivable that we would be able to identify patients at risk for developing epilepsy from a brain inflammation scan. For instance, patients who experienced traumatic brain injury, febrile seizures or stroke could be systematically examined and followed-up by non-invasive imaging of brain inflammation. It is plausible that a peripheral or cerebrospinal fluid biomarker profile would be determined in parallel as it is reported that combining fluid biomarkers with imaging improves diagnostic accuracy (Schoonenboom et al., 2008; Zhang et

8 al., 2008; Shaffer et al., 2013). This would, eventually, lead to better guiding of the patient during disease ontogenesis and the immediate application of disease modifying or anti-epileptogenic therapies, if available. Furthermore, a reduction in healthcare costs and economic burden will follow concomitantly.

1. Different players in brain inflammation The central nervous system (CNS) lacks a conventional lymphatic system and is tightly surrounded by the blood-brain barrier (BBB) formed by specialized endothelial cells. The BBB is the brain’s intrinsic defence mechanism against influences from outside the CNS and the traffic of peripheral immune cells is limited. The brain, however, is not an immune privileged site (Fig. 1). Firstly, peripheral immune-competent cells can infiltrate into the brain when the BBB is disrupted, which has been frequently reported in epilepsy (Mihaly and Bozoky, 1984; van Vliet et al., 2007; Marchi et al., 2012). Secondly, the brain has its own innate immune system, which is believed to be formed primarily by microglia and astrocytes. The first key player, namely the microglia, continuously senses the environment in search for detrimental agents. Even subtle deviations from the normal intra-cerebral milieu can alert microglia. In turn, they become activated and as a result undergo morphological, molecular and functional changes. Under these circumstances they mediate defensive immune responses by producing different inflammatory mediators, becoming antigen presenting cells and exerting phagocytic actions. Microglia can present different activation patterns depending on the inflammatory mediators they produce (Miron et al., 2013). Typecasting into static phenotypes (M1 vs. M2) is likely an oversimplification as various intermediate polarization states of microglia seem apparent. The second player of the brain’s innate immune system, namely the astrocytes, has important supportive functions, such as their role in regulation of blood flow and in synapse function. When activated, they also have a role in the protection of CNS cells and tissue, and a role in regulation of brain inflammation (Sofroniew, 2009). However a recent hypothesis also suggests a gain

9 of bad or a loss of good functions upon activation (Bush et al., 1999; Brambilla et al., 2005; Takano et al., 2005).

Microglia, astrocytes, endothelial cells of the BBB, infiltrated peripheral immune-competent cells and neurons can all produce inflammatory mediators, including interleukins, chemokines and neurotrophic factors. Several cytokines can lead to glutamate release, inhibition of glutamate reuptake or even changes in the subunit composition of the glutamate receptor, which all can contribute to neuronal hyperexcitability (Fig. 1). Interleukine 1β (IL-1β) can exert pro-convulsive activity through its receptor mediated phosphorylation of the NMDA receptor (Fig. 1A), thus, leading to increased neuronal calcium influx (Balosso et al., 2008). An abnormal increase of the intracellular calcium concentration does not only lead to hyperexcitability, but also to the opening of the mitochondrial permeability transition pore, resulting in mitochondrial swelling and the production of reactive oxygen species (Fig. 1B)(Lemasters et al., 2009). In turn, this can cause neuronal cell death. Glial cells can also produce inducible nitric-oxide synthases, which are capable of producing large amounts of nitric-oxide, leading to apoptosis (Brown and Bal-Price, 2003). Next to neuronal hyperexcitability and cell death, cytokines can also promote extravasation of peripheral immunecompetent cells into the brain by the upregulation of the expression of leukocyte adhesion molecules, such as vascular cell-adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which is the case in epilepsy (Duffy et al., 2012). The infiltrated cells can subsequently contribute to the inflammatory cascades going on.

2. Imaging brain inflammation Mapping of brain inflammation in vivo is made possible by the development and the continuous improvement of valuable neuroimaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). During the past decade, many advances have been made

10 regarding data acquisition and analysis, leading to better spatial resolution. At present, the main principle for visualizing brain inflammation directly is based both on targeting specific molecules expressed on (e.g. translocator protein) or taken up (e.g. exogenous magnetic particles) by immunecompetent cells. Another successful approach is the labelling of immune-competent cells outside the body after isolation. A more indirect approach, for instance, comprises visualizing BBB dysfunction and the expression of adhesion molecules. The non-invasive and specific monitoring of the inflammatory processes has already found its implementation in both experimental and clinical studies. This review summarizes in the first part the common approaches currently available for imaging the different constituents of brain inflammation (Fig. 1) and then focuses in the second part on those applied for epilepsy, both in experimental animal models and in patients.

2.1 Imaging microglial activation A prominent hallmark of activated microglia is the increased expression of the translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor. Within the brain parenchyma, this protein is located in the outer mitochondrial membrane, where it is part of the mitochondrial permeability transition pore and has a role in the translocation of cholesterol from the outer to the inner mitochondrial membrane (Casellas et al., 2002; Papadopoulos et al., 2006; Rupprecht et al., 2010). Expression of TSPO in healthy brain tissue is very low, but the tremendous increase in the TSPO levels specifically under inflammatory conditions renders it the main target for imaging microglial activation. The non-benzodiazepine ligand 11C-PK11195 (Le Fur et al., 1983a; Le Fur et al., 1983b) was one of the first tracers used to visualize this process by mapping TSPO expression based on a selective ligand-receptor interaction. In fact, the vast majority of PET imaging studies on TSPO in humans has been performed with this tracer. Nevertheless, several limitations can be attributed to PK11195 including low binding potential, high non-specific binding (Petit-Taboue et al., 1991; Shah et al., 1994) and therefore an unfavourable signal to noise ratio, and problematic quantification or modelling of receptor density (Petit-Taboue et al., 1991; Venneti et al., 2006). Many new TSPO PET

11 ligands have been developed in an attempt to overcome these shortcomings and to improve the quantification of TSPO expression. Indeed, new PET and SPECT radioligands that outperform PK11195 are made available after extended research, including 18F-DPA-714 (Chauveau et al., 2009), 18F-GE180 (Wadsworth et al., 2012) and 18F-PBR111 (Schweitzer et al., 2010; Van Camp et al., 2010; Wadsworth et al., 2012). Many experimental and clinical studies, investigating various CNS disorders with TSPO PET imaging, have already been published and are reviewed by Schweitzer et al. (2010). Although increased TSPO expression is primarily confined to activated microglia (Scarf and Kassiou, 2011), a number of recent published studies, both experimental (Chen et al., 2004; Ji et al., 2008; Lavisse et al., 2012; Dickens et al., 2014) and clinical (Cosenza-Nashat et al., 2009), have shown an increased TSPO binding in astrocytes. Cosenza-Nashat et al. (2009) demonstrated elevated TSPO levels in microglia, macrophages and some hypertrophic astrocytes with a distribution varying depending on the disease, disease stage and proximity to the lesion or relation to infection. Additional research is still necessary to characterize and further validate the TSPO expression in astrocytes. The above studies indicate that the cellular localization of TSPO is probably disease dependent, and even within a certain disease different cellular localizations may be assigned to this protein, probably time and brain region dependent. Such a disease-specific expression may relate to the different functional roles TSPO might have induced by conditions specific for the disease. For interpreting TSPO imaging results, TSPO should be seen as a marker for the evaluation of reactive gliosis. However, when the research question enquires knowledge about the cellular localization, histological co-localization studies are recommendable to determine cell-specificity if possible.

Upon activation, microglia not only show increased TSPO expression, but other molecules are also upregulated. Such molecules are potential targets for the development of imaging markers to visualize microglial activation and could form a promising alternative or complementary approach to TSPO tracers. For example, the cannabinoid receptor type 2 (CB2) (Fig.1), which is primarily known for its role in antinociception, is proposed to have anti-inflammatory properties (Tolon et al., 2009).

12 The CB2 receptor is a promising target as it has already been shown that it is upregulated in microglia in different neurological disorders, including Alzheimer’s disease, Huntington’s disease and multiple sclerosis (Benito et al., 2003; Benito et al., 2007; Sagredo et al., 2009). Whether the CB2 receptor is exclusively located in microglia has to be studied further. In addition, some limitations can be ascribed to this receptor. Firstly, the cannabinoid receptor type 1 (CB1), involved in psychoactive effects of cannabinoids, is abundantly present in the CNS. Therefore the CB2 receptor ligands should be highly selective over CB1 to be able to detect neuroinflammatory changes. Secondly, species differences in binding affinity, also between human and rodents, have been demonstrated (Rhee et al., 1997; Iwamura et al., 2001; Khanolkar et al., 2007; Ashton et al., 2008; Verbist et al., 2008). However, no polymorphisms of the CB2 receptor are known in contrast to TSPO (cfr. infra). Several potential candidates for PET imaging of the CB2 receptor are under investigation. These newly synthesized PET probes have been evaluated in vitro and in vivo over the past few years and include 18

F-triazine derivatives (Hortala et al., 2014), 11C-KD2 (Mu et al., 2013), 18F-FE-PEO (Riss et al., 2013),

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F-oxoquinoline derivatives (Turkman et al., 2012),18F-FE-GW405833 (Evens et al., 2011) and 11C-

NE40 (Evens et al., 2012). Further research is still necessary to fully characterize the kinetics and quantification capacities of these tracers, and validation studies are essential for widespread and productive use in pre- and clinical studies. TSPO ligands, in contrast, are better characterized and are thoroughly validated with histological techniques. Therefore they remain, for now, the preferred tool for determining microglial activation.

Finally, (micro)glial activation might also be monitored by quantification of the methionine uptake (Fig. 1). Methionine is an essential amino acid and an important intermediate in the biosynthesis of many phospholipids. The uptake of this amino acid is mainly visualized by the PET tracer 11Cmethionine, which is actually an important tracer for the evaluation of brain tumours (Bergstrom et al., 1983; Derlon et al., 1989). However, it is speculated that higher methionine uptake might be related to (micro)glial activation and proliferation (Madakasira et al., 2002; Stober et al., 2006).

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2.2 Imaging astroglial activation The best known hallmark of activated astrocytes is the upregulation of intermediate filament proteins, in particular the glial fibrillary acidic proteins. To date no in vivo imaging probes exist for the visualization of these or analogue proteins. The expression of many other proteins, including monoamine oxidase type B (MAO-B), is also altered (Eddleston and Mucke, 1993). This enzyme is abundantly present in astrocytes and is upregulated specifically upon activation (Ekblom et al., 1993; Ekblom et al., 1994; Saura et al., 1994; Gulyas et al., 2011). At present, MAO-B is actually the main target available for in vivo visualization of astroglial activation. The principal tool for neuroimaging MAO-B activity in the majority of clinical studies has been PET imaging. Over the past decades, different radioligands have been developed, including N,N-11C-dimethylphenethylamine, 18Ffluorodeprenyl,11C-L-deuterium deprenyl and 11C-L-deprenyl (Kersemans et al., 2013). The latter is, to date, most used in experimental and human studies (Santillo et al., 2011; Carter et al., 2012). Some limitations characterize 11C-L-deprenyl; these include the short half-life of carbon-11 (20.4 min) and the entrance of its main radiometabolite11C-L-methamphetamine into the brain. In addition, regulatory aspects related to the medicinal chemistry of the precursor requiring amphetamine as an intermediate step can hamper its use. However, this can be avoided by synthesizing 18Ffluorodeprenyl (Nag et al 2012). Other PET radioligands, such as18F-fluororasagiline, are under investigation too (Nag et al., 2012; Nag et al., 2013).

Myo-inositol (mIs) and gluthatione (GSH) are two brain metabolites which are also associated with glial activation, especially with astroglial activation (Raps et al., 1989; Brand et al., 1993; LopezVillegas et al., 1995; Dringen et al., 1999; Filibian et al., 2012). GSH, which is an antioxidant with beneficial effects on neurons, is mainly produced by astrocytes (Raps et al., 1989; Dringen et al., 1999), whereas mIs is particularly located in astrocytes (Brand et al., 1993). By using MRS, these metabolites can be identified and quantified.

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2.3 Imaging blood-brain barrier dysfunction and peripheral immunecompetent cell infiltration The main principle for imaging BBB dysfunction is based on the injection of radiolabeled agents or compounds with magnetic characteristics that do not cross the BBB under physiological conditions. Disruption of this barrier allows these agents to leak into the brain parenchyma, where they emit positrons or photons detected by PET or SPECT, respectively, or cause alterations in the MR relaxation time (T1). A widely used non-isotopic agent in clinical routine is Gd-DTPA, which is visualized by MRI. Usually, MR contrast agents are administered intravenously as a bolus followed by MRI at a specific point in time post-injection (Dijkhuizen, 2011). Recent discussions in the field point towards insufficient detection of subtle BBB leakage when using a bolus injection as it fails to provide adequate and steady state concentrations in the blood (Nagaraja et al., 2007; Knight et al., 2009; van Vliet et al., 2014). It would seem that prolonged infusion of the contrast agent improves the sensitivity of BBB permeability assessments (Nagaraja et al., 2007; Knight et al., 2009). PET and SPECT candidates, for example68Ga-EDTA (PET) and 99mTc-DTPA (SPECT), are also available and have been reviewed by Wunder et al. (2009).

Extravasation of peripheral immune cells, such as macrophages and T-lymphocytes, is promoted under inflammatory conditions because of both an increase in vascular permeability and a higher expression of cell-adhesion molecules located on endothelial cells, such as VCAM-1 and ICAM-1 (Oude Engberink et al., 2008). Different approaches exist to image leukocytes and their circulation in vivo. A first possible method is to reinject radiolabeled leukocytes into a subject, after isolation and labelling them with 99mTc-HMPAO or 111In-oxinate. This enables trafficking by imaging with SPECT (Akopov et al., 1996; Murphy et al., 2000; Spinelli et al., 2000; Liberatore et al., 2003; Ballinger and Gnanasegaran, 2005; Wunder et al., 2009). A second available method is the injection of ultrasmall and superparamagnetic iron oxide (USPIO) particles into the blood of a subject where they are

15 subsequently taken up by phagocytic white blood cells (Jander et al., 2007; Nighoghossian et al., 2007; Stoll and Bendszus, 2009). These agents cause a hypointense signal on T2-weighted MRI images (Weinstein et al., 2010). The higher expression of adhesion molecules on endothelial cells provides a third and indirect target to assess the infiltration of peripheral immune cells. Antibodies targeting these molecules have been developed over the past ten years and studied in different models of inflammation (McAteer et al., 2007; Rossin et al., 2008; Hoyte et al., 2010; Shao et al., 2011).

2.4 Imaging inflammatory mediators and tryptophan metabolism Direct imaging of inflammatory mediators would be a relevant angle for mapping the effectors causing the inflammatory changes associated with a particular neurological disorder. The knowledge about the context-specific production of inflammatory mediators leading to a pro- or antiinflammatory environment could guide the researcher or clinician to more specific interventions. However, this is still not an optimized or successful approach so far, though, studies have been performed using radiolabeled inhibitors of nitric oxide synthases (Zhang et al., 1996; Zhang et al., 1997; Pomper et al., 2000; de Vries et al., 2004) or cyclo-oxygenases (McCarthy et al., 2002; de Vries et al., 2003; Majo et al., 2005; Wust et al., 2005; Prabhakaran et al., 2007). Some researchers report a prominent association between inflammatory mediators and specific imaging techniques, including fluid-attenuated inversion recovery (FLAIR)-MRI (Varella et al., 2011). The latter imaging modality nulls fluid and thus suppresses signals from the cerebrospinal fluid. Consequently, it enhances the visualization of lesions. Of course, this approach can obviously be designated as an indirect approach for the visualization of inflammatory mediators. Likewise, in his review, Obenaus (2013) pointed towards possible associations between the activation of glial cells and different MRI techniques, including structural MRI, diffusion-weighted imaging and diffusion tensor imaging.

16 Alterations in the tryptophan metabolism have also been associated with inflammatory processes and are primarily visualized by 11C-α-methyl-L-tryptophan (AMT) PET. Increased AMT uptake is associated with an increase in serotonin synthesis; however, under inflammatory conditions tryptophan may be metabolized by indoleamine 2,3-dioxygenase (IDO) in the tryptophan catabolism pathway (Yamazaki et al., 1985; Haber et al., 1993; Chugani and Muzik, 2000; Chugani and Chugani, 2005). This pathway leads to the production of kynurenine, which is subsequently transformed into quinolinic acid, which is potentially neurotoxic, or into kynurenic acid, which is potentially neuroprotective (Zunszain et al., 2012). Different inflammatory cytokines, including interferon γ and IL-1β, can induce the production of IDO, thus, making the association between alterations in tryptophan metabolism and brain inflammation (Taylor and Feng, 1991). Recently, Akhtari et al. (2008) developed a new tool for the visualization of tryptophan uptake by conjugating AMT with magnetonanoparticles (MNP). These particles can cross the BBB and can be visualized by MRI. Although successfully applied, no other studies using this modality have been performed yet.

3. Imaging brain inflammation in epilepsy 3.1 Imaging microglial activation in epilepsy In vitro binding assays with TSPO ligand PK11195, executed on post-surgical tissue of patients with drug-resistant epilepsy, demonstrated a higher binding of PK11195 in specific regions important for seizure generation, including the temporal lobe (Johnson et al., 1992; Kumlien et al., 1992). In addition, Sauvageau et al. (2002) showed a significant increase in TSPO gene expression only in patients with hippocampal sclerosis compared to controls and patients without the former pathological hallmark. Furthermore, they demonstrated a histological correlation between the higher TSPO binding and the higher density of glial cells, strengthening even more the use of TSPO as a neuroimaging biomarker for brain inflammation. Likewise, comparable results could be demonstrated in an experimental model of epilepsy, namely the kainic acid-induced status epilepticus (KASE) model. Harhausen et al. (2013) demonstrated a higher uptake of the TSPO tracer

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F-DPA-714 in regions associated with epileptic activity and correlated this with

microglial/macrophage activation using histological techniques. These in vitro studies forecasted the application of TSPO PET ligands for assessing gliosis in vivo.

Dedeurwaedere et al. (2012a) demonstrated a higher uptake of this tracer one week after status epilepticus (early epileptogenesis) in the KASE rat model (Fig. 2). The uptake was significantly higher in different epileptogenic regions, including the hippocampus and the amygdala. The increased in vivo binding was validated by post-mortem autoradiography with the tracer 125I-CLINDE, a specific TSPO SPECT ligand, and Ox42 immunohistochemistry for visualizing microglia. This study demonstrated promising results for the feasibility of PBR111 for imaging brain inflammation during epileptogenesis. Recently, Bogdanovic et al. (2014) measured TSPO binding using 11C-PK11195 PET in the self-sustained status epilepticus rat model. They showed a higher uptake in the parietal and occipital lobe of epilepsy rats compared to controls. Interestingly, they succeeded in finding a higher uptake of the tracer in drug-resistant animals compared to the animals which did respond to the drugs. In the past decade, the implementation of TSPO as a marker for brain inflammation in epilepsy patients was limited to small-sized studies or case reports with different rationale. These provided additional information about the involved cells located in the lesion detected by MRI (Goerres et al., 2001), identified the epileptogenic zone based on focally increased inflammation in order to guide epilepsy surgery more accurately (Kumar et al., 2008; Butler et al., 2013). Microglial activation was not only observed in temporal lobe epilepsy (TLE), but also in other epilepsy syndromes, such as Rasmussen’s encephalitis and focal cortical dysplasia (Banati et al., 1999; Butler et al., 2013). Recently, Hirvonen et al. (2012) published for the first time a more extensive study using TSPO PET imaging in 16 TLE patients with the PET ligand 18F-PBR028. An increased uptake in the hippocampal regions near the epileptogenic focus was demonstrated, which suggests the involvement of brain

18 inflammation by the activation of glial cells. The question still remains whether increased TSPO has a functional role or is solely an epiphenomenon of epileptogenesis.

Zurolo et al. (2010) studied histologically the expression of the CB2 receptor in focal epileptogenic lesions in surgical specimen of patients with focal cortical dysplasia and cortical tubers. They showed upregulation of the CB2 receptor both in balloon and giant cells in which the CB2 expression colocalized with GFAP, and in microglia and macrophages. Although the clear relationship between the CB2 receptor and glial activation, no in vivo preclinical or clinical studies have been executed yet in epilepsy.

Although the use of 11C-methionine PET in epilepsy is still in its infancy, it has already been demonstrated that in drug-resistant patients with focal cortical dysplasia (Sasaki et al., 1998; Madakasira et al., 2002) and with Rasmussen’s encephalitis (Maeda et al., 2003) a higher uptake of methionine in the epileptic foci is present. Interpretation of the higher uptake in patients with epilepsy is not straightforward. A possible hypothesis is that reactive glial cells cause the higher uptake (Madakasira et al., 2002) as it is considered that uptake reflects carrier-mediated transport over the cell membrane or the proliferative activity of cells (Herholz et al., 1998; Chugani and Muzik, 2000; Langen et al., 2000; Chugani and Chugani, 2005).

3.2 Imaging astroglial activation in epilepsy As early as 20 years ago, supportive data were generated for the use of deprenyl as an in vivo imaging tool for brain inflammation in epilepsy by visualizing MAO-B binding (Kumlien et al., 1992). Post-surgical tissue specimens of patients with TLE were analyzed by 3H-L-deprenyl autoradiography binding assays and these demonstrated a higher MAO-B binding associated with gliosis. So far, studies applying deprenyl for imaging purposes in animal models of epilepsy are lacking. However, different studies in epilepsy patients have been successfully executed. A higher binding in

19 epileptogenic regions (identified by MRI and EEG), including the hippocampus and the amygdala, was demonstrated in TLE patients by means of 11C-deuteriumdeprenyl PET supporting the presence of reactive astrocytes (Kumlien et al., 1995; Kumlien et al., 2001). In accordance with these findings, Buck et al. (1998) had already reported a higher binding in the temporal lobe using the MAO-B SPECT ligand 123I-Ro43-0463 in patients with TLE. Besides its use for identifying inflammation, some studies investigated whether the visualization of MAO-B binding is a more sensitive diagnostic tool for delineating the epileptic focus for epilepsy surgery (Kumlien et al., 2001). A comparison with 18FDGPET resulted in no added value for 11C-deuterium deprenyl alone, but rather designated the latter as a complementary method in doubtful cases.

As mentioned earlier, mIs and GSH levels can also be measured to assess astroglial activation. In a preclinical setting, Filibian et al. (2012) demonstrated with MRS a progressive increase of mIs and GSH in the hippocampus during epileptogenesis, which then stabilized during the chronic period when the animals were epileptic. Of interest was the positive correlation between reactive astrocytes by immunohistochemistry and the levels of both metabolites by MRS (Fig. 3), supporting the use of these metabolites to visualize brain inflammation in vivo. Various clinical studies have been executed to assess reactive astrogliosis in epilepsy by means of MRS (Mizuno et al., 2000; Turkdogan-Sozuer et al., 2000; Hammen et al., 2008). An increase of mIs in epileptogenic tubers of patients with tuberous sclerosis (Mizuno et al., 2000), in the hippocampus of patients with TLE (Hammen et al., 2008) and in the cortex of a patient with Rasmussen’s encephalitis (TurkdoganSozuer et al., 2000) has been demonstrated. The increase was consistently associated with (astro)gliosis. However, other studies in patients with TLE demonstrated similar or decreased mIs values compared to healthy controls (Simister et al., 2002; Mueller et al., 2003; Riederer et al., 2006; Briellmann et al., 2007; Doelken et al., 2008). Filibian et al. (2012) speculated that these differences could be due to treatment or to heterogeneity in the underlying pathology.

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3.3 Imaging blood-brain barrier dysfunction and peripheral immunecompetent cell infiltration in epilepsy In different experimental epilepsy models BBB permeability was visualized by gadolinium contrastenhanced MRI, both acute (Roch et al., 2002; Danjo et al., 2013) and longitudinal (Bouilleret et al., 2000) post-precipitating insult. The main finding was the detection of BBB disruption acutely after the insult but not chronically. This is inconsistent with the findings achieved by conventional ex vivo techniques. Van Vliet et al. (2014) succeeded in the in vivo demonstration of longitudinal BBB disruption in a chronic epilepsy model. They used contrast-enhanced MRI with prolonged step-down infusion of gadobutrol, which is a gadolinium based contrast agent, instead of bolus injection. Quantification of BBB permeability at two points in time during epileptogenesis (acute and chronic phase) demonstrated non-uniform leakage in the limbic regions throughout epileptogenesis, with a decrease over time. These findings were confirmed by post-mortem histology studies. Gilad et al. (2012) performed an imaging study using 99mTc-DTPA SPECT on patients suffering from post-stroke seizures. About 85.7% of the patients studied displayed BBB disruption.

To date no in vivo studies visualizing peripheral immune cells, either experimental or clinical, have been performed in the field of epilepsy research, though, a recent study was published in which seizure-induced endothelial activation in a chronic epilepsy model was assessed by means of antibodies targeting VCAM-1 (Duffy et al., 2012). These antibodies were conjugated with micronsized particles of iron oxide (MPIO) and were visualized by MRI. The spatial expression profile of VCAM-1 was congruent with brain vessels in regions important for epileptic activity, such as the hippocampus and was in agreement with previous post-mortem studies (Librizzi et al., 2007; Fabene et al., 2008).

3.4 Imaging inflammatory mediators and tryptophan metabolism in epilepsy

21 Up to now, there have been no in vivo studies which have visualized inflammatory mediators directly. Varella et al. (2011) demonstrated an association between FLAIR-MRI and particular inflammatory mediators in patients with mesial temporal sclerosis. More specifically, the main finding was a positive correlation between the pathological substrates identified as hyperintense signals on FLAIRMRI and the inflammatory mediators’ nitric oxide and IL-1β determined in post-surgical specimens. Although indirectly demonstrated, these findings suggest that the hyperintense signals could be the result of increased expression of the inflammatory mediators associated with increased excitotoxicity in TLE. One could argue whether this is the most precise method for visualizing inflammatory mediators; however, a direct tool targeting these molecules themselves is still lacking, probably out of complexity.

Increases in tryptophan metabolism, typically associated with tuberous sclerosis, have also been reported in non-tuberous epileptic foci (Chugani et al., 2011; Kumar et al., 2011; Chugani et al., 2013). Akhtari et al. (2008) showed increased uptake of AMT-MNP in regions associated with epileptic activity, including the hippocampus, during the acute and chronic phase of epileptogenesis in the KASE model. In a case report of a patient with new-onset drug-resistant status epilepticus, AMT-PET facilitated diagnosis and assisted in treatment success (Juhasz et al., 2013). AMT-PET demonstrated an increased uptake within and adjacent to the epileptogenic zone. In addition, an increased expression of IL-1β, its receptor and IDO was exclusively shown in the AMT upregulated brain region post-mortem (Fig. 4). Therefore, it can be stated that AMT-PET may serve as a noninvasive imaging biomarker of brain inflammation in epilepsy (Chugani, 2011; Juhasz et al., 2013).

4. Concluding remarks A multitude of very suitable and highly relevant imaging tools for the in vivo visualization of brain inflammation in epilepsy are available and are already being applied in patients and animal models of epilepsy. However, it remains necessary for new approaches to be developed, as there is, first of all,

22 a tremendous imbalance between markers of brain inflammation and the present available imaging tools. Secondly, as discussed earlier, some of these imaging markers cannot be specifically assigned to a particular inflammatory component. For instance, TSPO and MAO-B are not exclusively present in microglia and astrocytes respectively and do not per se provide information about the inflammatory mediators produced by these glial cells. Furthermore, indirect approaches are regularly used to identify brain inflammation. This could be an obstacle especially in experimental studies where, for example, the aim is either to define the key players in more detail so that a context- and disorder-specific profile can be revealed, or to evaluate interventions with a specific target by pharmacological manipulation.

It should be noted that, despite their availability, it seems that in vivo imaging tools for brain inflammation are not commonly used in epilepsy research. For instance, different post-mortem and post-surgical data support the use of TSPO as a biomarker for gliosis in epilepsy while not many in vivo animal studies have been executed yet. Studies in patients have been done to a greater extent but mostly on a single patient base. This could be for different reasons, including the absence of an imaging unit on site, the unfamiliarity with these techniques or the complexity of PET. The partial volume effect (activity spillin or -out) in PET, which is limited by its spatial resolution, may dilute the signal. PET is also a signal integrated over time and this period of recording allows for downstream effects to occur. Often, when evaluating new biomarkers, dynamic acquisitions with continuous blood sampling and metabolite analysis at discrete time points is required, rendering the image acquisition laborious and data post processing challenging. Finally, recent in vitro and in vivo studies have shown an irrefutable heterogeneity in binding potential of different TSPO ligands, including PBR028, PBR111 and PBR06 (Kreisl et al., 2010; Owen et al., 2011; Hirvonen et al., 2012; Owen et al., 2012). Apparently, a common inter-subject variability in affinity for these ligands exists, distinguishing three types of binders, namely high, low and mixed affinity binders. As a consequence, it complicates interpretation of quantified PET images as these no longer solely reflect receptor

23 density but also receptor affinity. Thus, it requires further genetic testing for determination of the TSPO binding class to correct the acquired PET data (Owen et al., 2012). In clinical settings, it is also of the utmost importance that the techniques are non-invasive and of high comfort for the patient. The practical ease of executing the technique is also important for the clinician so that its use can become more widespread and productive. A recent review signified that MRI techniques for the quantification of BBB disruption are not routinely used in the clinic, owing to complex and exhausting scanning protocols (Chassidim et al., 2013). The capacity of the technique to quantify brain inflammation on an individual base is also a prerequisite for its use in clinical settings. The use of in vivo techniques for the visualization of brain inflammation is recommended whenever possible and relevant, so that the motivation of scientists to develop new and improve the existing imaging tools remains.

In conclusion, although it is already used in the clinic, imaging of brain inflammation in epilepsy is still in its infancy. Many targets for brain inflammation have been identified and these are potential candidates for the development of novel sensitive and specific non-invasive imaging methods for the detection and quantification of brain inflammation. A vast effort, but which could possibly lead to various imaging biomarkers of brain inflammation and thus possibly providing improved research, diagnostic, prognostic and evaluation tools.

5. Acknowledgements We would like to thank JD Linnegar for copy-editing this manuscript and B Dewilde for creating the basic template of figure 1. Halima Amhaoul and Stefanie Dedeurwaerdere are supported by the BijzonderOnderzoeksFonds (BOF) of the University of Antwerp, the Research Foundation Flanders (FWO) funding 1.5.110.14N, 1.5.144.12N and ERA-NET NEURON G.A009.13N, and finally by the Queen Elisabeth Medical Foundation (Q.E.M.F.) for Neurosciences. Steven Staelens is supported by the BijzonderOnderzoeksFonds (BOF) of the University of Antwerp.

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6. Authors’ contributions The first author, Halima Amhaoul, conducted the literature research necessary for the successful writing of this review and prepared the first draft and changes to the final manuscript. The second and last author, Steven Staelens and Stefanie Dedeurwaerdere respectively, edited the manuscript and gave rise to new ideas and insights.

7. Conflicts of interest We declare no conflicts of interest.

8. Role of the funding source There was no role for any funding source in the preparation or the decision to submit this review for publication.

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Figure legends Figure 1. Activation of the innate-like immune system of the brain in epilepsy highlighting potential targets for non-invasive in vivo imaging of brain inflammation. A. Cascades of inflammation causing hyperexcitability. B. Hyperexcitability and seizures causing inflammation and cell death. The primary goal of brain inflammation is to defend the brain against all harmful compounds and to restore the strictly maintained cerebral milieu. However, under certain circumstances, still not unravelled completely, it might promote CNS pathologies. This figure demonstrates a cascade of negative events, including higher neuronal excitability (NMDA receptor phosphorylation), oxidative stress (ROS production), glutamate excitotoxicity and, eventually, neuronal cell death, as a result of the production and release of pro-inflammatory cytokines such as IL-1β. These events are counteracted by, for instance, beneficial effects of microglia producing neurotrophic factors, exerting phagocytic actions to remove debris and toxic components or inhibiting the effect of pro-inflammatory cytokines via the CB2 receptor. The antioxidant GSH could also exert beneficial effects on the neuron. IDO can lead to protective as well as detrimental effects on the neuron via the tryptophan catabolism pathway. In addition, infiltration of peripheral immune-competent cells through ICAM-1 and VCAM-1 signaling is shown, which also contributes to the production of inflammatory mediators. GSH, mIs, methionine, ICAM-1, VCAM-1, TSPO, CB2 receptor and MAO-B could in principle serve as targets for the in vivo visualization of brain inflammation in epilepsy. Abbreviations: BBB= blood-brain barrier, CB2= cannabinoid receptor type 2, Ca= calcium, GSH= gluthatione, ICAM-1= intercellular adhesion molecule 1, IDO= indoleamine 2,3-dioxygenase, IL-1β= interleukine 1β, MAO-B= monoamine oxidase type B, mGlu= metabotropic glutamate receptor, mIs= myo-inositol, NMDA= N-methyl-D-aspartate, ROS= reactive oxygen species, TSPO= translocator protein, VCAM-1= vascular cell-adhesion molecule. Adapted from Devinsky et al. (2013), Jacobs and Tavitian (2012) and Vezzani et al. (2011). Figure 2. In the KASE model of TLE, Dedeurwaerdere et al. (2012) evaluated brain inflammation by the use of in vivo 18F-PBR111 PET imaging. Corresponding post-mortem autoradiography and immunohistochemistry on the coronal sections in the representative control and KASE rats, 7 days post-status epilepticus induction, are shown. Arrows indicate the different brain regions named on the ipsilateral hemisphere, in which higher tracer uptake is demonstrated in line with the immunohistochemistry results (microglial marker OX-42) and the autoradiographic results (TSPO). HC, hippocampus; THAL, thalamus; AMYG, amygdala; PIR, piriform cortex. (Dedeurwaerdere et al., 2012) Figure 3.In a preclinical study of Filibian et al. (2012) longitudinal 1H-MRS measurements were done in the pilocarpine rat model of epilepsy to study metabolite content in the hippocampus that could reflect the extent and duration of glial activation. In this figure, correlation analysis between immunohistochemical signal (GFAP, S100β) and 1H-MRS metabolite level (mIn, GSH) in the hippocampus of epilepsy rats is shown. Scatter plot in panels A, B, and C show positive and significant correlation between GFAP signal and mIn/Cr (A) or GSH/Cr (B) levels, and between the number of S100β immunoreactive astrocytes (a subpopulation of GFAP-positive glia) and mIn/Cr levels (C). (Filibian et al., 2012) Figure 4. Juhasz et al. (2013) described a case report of a patient with new-onset drug-resistant status epilepticus in which AMT-PET facilitated diagnosis and assisted in treatment success. In this

36 figure, immunostaining findings of resected epileptogenic tissue is shown. A. Curvilinear 3D reconstruction of the brain (5 mm under the cortical surface to emphasize gyri and sulci) using the preoperative volumetric MRI with overlay of intracranial subdural electrodes. The arrow indicates the MRI-defined lesion in the subcortical area, whereas the outlined region in red corresponds to the highest AMT uptake in the superior temporal gyrus. Electrodes in yellow represent seizure onset, and those in blue showed rapid seizure propagation. Electrodes number 30 (superior temporal gyrus) and number 4 (middle temporal gyrus) are labeled because the cortex under these electrodes was sampled for immunohistochemistry. B. GFAP immunohistochemistry shows extensive reactive astrocytes in the image-guided biopsy which is obtained from the non-enhancing, T2weighted/FLAIR hyperintense lesion prior to implantation of the intracranial electrodes. C. Strong IDO (red) and IL-1β (green) coexpression is shown in tissue obtained from electrode number 30 which corresponds to an AMT-positive region involved in seizure onset in the superior temporal gyrus. D. In contrast, sparse IDO and IL-1β coexpression is shown in the tissue obtained from electrode number 4 which corresponds to an AMT-negative region involved in seizure onset in the anterolateral temporal cortex. Nuclei are stained in blue by DAPI counterstaining. E and F. Immunostaining for IL-1 receptor, type 1 (IL-1R1) is shown in the same regions again demonstrating higher expression in the AMT-positive tissue (electrode number 30; E) compared with the AMT-negative tissue (electrode number 4; F). (Juhasz et al., 2013)

Figure 1 hh Ca2+ Pro-inflammatory cytokines

A. Inflammation causes increased excitability

NMDA

IL-1β

mGlu

Activated astrocyte VCAM-1

ICAM-1

BBB dysfunction ICAM-1

Glutamate excitotoxicity

hh Ca2+

MAO-B mIs

GSH

Pro-inflammatory cytokines

-

IL-1β

VCAM-1

Pro-inflammatory cytokines

CB2

Microglia

ROS TSPO

Neuron

Methionine uptake

Cell death

IDO

cytokines

Inflammatory infiltration

Surveying microglia

Phagocytosis

Pro-inflammatory cytokines

Primed microglia

Neurotrophic factors

TSPO

CB2

Microglia Methionine uptake

CB2

B. Increased excitability or seizures cause inflammation

Figure 2

Figure 3

Figure 4

hh Ca2+ Pro-inflammatory cytokines

A. Inflammation causes increased excitability

NMDA

IL-1β

mGlu

Activated astrocyte VCAM-1

ICAM-1

BBB dysfunction ICAM-1

Glutamate excitotoxicity

hh Ca2+

MAO-B mIs

GSH

Pro-inflammatory cytokines

-

IL-1β

VCAM-1

Pro-inflammatory cytokines

CB2

Microglia

ROS TSPO

Neuron

Methionine uptake

Cell death

IDO

cytokines

Inflammatory infiltration

Surveying microglia

Phagocytosis

Pro-inflammatory cytokines

Primed microglia

Neurotrophic factors

TSPO

CB2

Microglia Methionine uptake

CB2

B. Increased excitability or seizures cause inflammation

37

• • • •

Brain inflammation important in epileptogenesis, but exact role not known. Non-invasive imaging of this process can promote our knowledge and insights. Approaches for molecular imaging of brain inflammation are available. However, this modality still resides in its infancy in epilepsy research and care.