Positron Emission Tomography in Basic ... - Wiley Online Library

Epilepsia, 48(Suppl. 4):56–64, 2007 Blackwell Publishing, Inc. C International League Against Epilepsy

Positron Emission Tomography in Basic Epilepsy Research: A View of the Epileptic Brain Stefanie Dedeurwaerdere, Bianca Jupp, and Terence J. O’Brien Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria, Australia

Summary: The neurobiological processes that result in epilepsy, known as epileptogenesis, are incompletely understood. Moreover, there is currently no therapy that effectively halts or impedes the development or progression of the condition. Positron Emission Tomography (PET) provides valuable information about the function of the brain in vivo, and is playing a central role in both clinical practice and research. This technique reliably reveals functional abnormalities in many epilepsy syndromes, particularly temporal lobe epilepsy. Unfortunately, epileptogenesis is extremely difficult to study in human patients who usually present with established epilepsy, rather than at the early stages of the process. Animal models offer the advantage of permitting the assessment of the pre-, developing, and chronic epileptic states. However, traditional techniques (e.g., histology) are only able to examine the brain at one time point during epilep-

togenesis in any one individual. Recent advances in dedicated small animal PET (saPET) allow researchers for the first time to study in vivo biomolecular changes in the brain during epileptogenesis by means of serial acquisitions in the same animal. Repeated application of in vivo imaging modalities in the same animal also decreases the effect of biological inter-individual variability and the number of animals to be used. The availability of novel PET tracers permits the investigation of a broad range of biochemical and physiological processes in the brain. Besides research on epileptogenesis, saPET can also be applied to investigate in vivo the biological effect of novel treatment strategies. saPET is widely used in many fields of pathophysiological investigation and is likely to significantly enhance epilepsy research. Key Words: Epilepsy models—Epileptogenesis— Small animal PET—Neuroimaging—Biological markers.

Epilepsy, a denominator of neurological diseases characterized by recurrent spontaneous seizures, affects approximately 3% of the population during their lifetime (Hauser and Kurland, 1975). Currently, there is no treatment available to halt or retard the development or progression of this condition. In one-third of patients, seizures are not adequately controlled by antiepileptic drugs (AEDs) (Kwan and Brodie, 2000). Seizures arise from excessive, abnormal hypersynchronized firing of a population of cortical neurons, but the precise neurobiological processes underlying epilepsy and the development of this disease are still incompletely understood. Many epilepsy syndromes, particularly temporal lobe epilepsy (TLE), are often associated with structural and functional abnormalities of the brain. Neuroimaging is increasingly taking a pivotal role in the diagnosis of epilepsy and in the decision on treatment strategies in patients, particularly those with medically refractory seizures. The use of radioisotopes to label and image molecules in-

volved in biological processes dates back to the beginning of the twentieth century see review by (Ploux and Mastrippolito, 1998). Positron emission tomography (PET) imaging was developed during the mid 1970s (Phelps et al., 1975) and applied to epilepsy research very early on (Kuhl et al., 1978). Fluorodeoxyglucose (2-deoxy2[18 F]fluoro-D-glucose, FDG), which maps glucose uptake and metabolism, has been the most common radiotracer used for PET imaging in clinical practice and research. However, PET imaging in principle provides virtually unlimited possibilities regarding the study of the function of the brain in terms of the molecules that can be labeled. The development of new probes has extended saPET applications to investigations of receptor–ligand interactions, enzyme activity, protein–protein interactions, gene expression, and cell and gene therapy (Herschman, 2003). As a noninvasive technique, PET imaging offers a number of advantages for in vivo assessment including: the acquisition of relatively fast (in the order of seconds) dynamic data; detection of pico- and femtomolar concentrations of ligand (Myers and Hume, 2002), and the potential to quantify these observations (although in practice producing semiquantitative data is often more feasible).

Address correspondence and reprint requests to Stefanie Dedeurwaerdere at The University of Melbourne, Department of Medicine, Royal Melbourne Hospital, 3050 Parkville, VIC, Australia. E-mail: [email protected] doi: 10.1111/j.1528-1167.2007.01242.x

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POSITRON EMISSION TOMOGRAPHY IN BASIC EPILEPSY RESEARCH FDG-PET imaging is frequently utilized in the presurgical evaluation of TLE patients in parallel with magnetic resonance imaging (MRI) examinations to identify the epileptogenic zone. PET may localize these zones in MRI-negative patients, but does not confer specificity of etiological diagnosis (Duncan, 2002; Carne et al., 2004). While PET has been widely used in epilepsy research, clinical studies have to contend with many confounding factors such as age, course of the illness, medication regimes, and treatment response. For instance, FDG-PET can be sensitive to certain AEDs such as phenobarbital, which can cause diffuse hypometabolism (Kuzniecky, 2005). In addition, epileptogenesis is difficult to study in humans, given that in most patients, the chronic rather than the early epileptogenic stage is represented. Prospective studies, in which patients at risk are followed up until the onset of the first seizure, are time-consuming, costly and practically difficult to undertake. In vivo animal research utilizing small animal PET (saPET) to image appropriate epilepsy models potentially provides very powerful tools to further the understanding of epilepsy and to complement the information provided by clinical research. This article will discuss the different applications of saPET for epilepsy research and the relevance of this approach for clinical research and practice. IN VIVO ANIMAL RESEARCH Animal models have long been important for the investigation of the neurobiology, consequences and treatment of seizures and epilepsy. These can be divided into models of seizures and models of epilepsy. In the case of the former, the seizures are induced by the application of an acute brain insult, usually electrical or chemical, while in the latter, the seizures occur spontaneously as with human epilepsy. Generally, in these chronic models, a precipitating insult (genetic or acquired) initiates the neurobiological processes that transform a normal to an epileptic brain (i.e., epileptogenesis), inducing spontaneous recurrent seizures after a latent or silent period. The chronic epilepsy models are preferred for longitudinal in vivo studies as they allow the investigation of all stages of the disease: baseline, developing, and chronic epileptic states. Traditional neuroscience techniques (e.g., histology, immunohistochemistry, and autoradiography) are performed on postmortem tissue and are therefore limited to the assessment of one time point per animal during the epileptogenic process. When several time points are to be investigated, this approach becomes very time-consuming and requires the use of a large number of animals. This problem is exacerbated by the significant interindividual variability that is seen after an epileptogenic brain insult. In vivo imaging modalities require fewer animals given that for these studies, animals can serve as their own con-

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trols, decreasing the effects of biological variability between individuals. The development of dedicated saPET imaging offers the ability to study in vivo changes during normal development, biological processes and responses, disease initiation and progression, and the effect of therapeutic interventions using serial scans over an extended period in the same animal (Tai and Laforest, 2005). saPET imaging to date has been widely used in a number of fields of pathophysiological research such as experimental oncology, gene expression and neuroscience research and has begun to prove itself as a promising technique in epilepsy research. Technical aspects of saPET Although saPET provides many opportunities, until recently research utilizing this technique in the field of epilepsy has been lagging the applications in other fields. The relatively slow adoption of saPET for epilepsy research has been attributed to several factors, including limitations in availability of scanners (although this is rapidly changing), a lack of familiarity by preclinical researchers with PET procedures and possibilities, expensive tracer production and relatively poor spatial resolution of the older saPET detectors (usually full-width-at-halfmaximum-FWHM of 2–4 mm) (Herschman, 2003). In addition, extensive coordination is required between groups to coincide the scheduling of the epilepsy model, tracer preparation and access to the scanner. Several review articles have elaborated on limitations and technical aspects of saPET imaging, for more details on this topic please refer to (Chatziioannou, 2002; Myers and Hume, 2002; Schmidt and Smith, 2005; Tai and Laforest, 2005). Immobilizing the animal in the PET scanner is another issue that has to be taken into consideration when designing saPET studies of the brain. Although systems and procedures have been developed to use awake monkeys in PET studies (Takechi et al., 1994; Kobayashi et al., 2002), anesthesia is still the standard for ensuring immobility during in vivo imaging of small animals. A fundamental requirement of imaging is to not disturb the biological system under observation. However, it is known that anesthesia can have several effects on the brain. The anesthetic of choice needs to minimally interfere with the tracer dynamics and the research question to obtain biologically valid outcomes. For example, Matsumura et al. (2003) investigated the effect of six different anesthetic agents on FDG uptake in the rat brain. This study demonstrated significant effects of the different anesthetic agents on FDG metabolism when administered during the uptake period. Only when anesthesia was started after the initial uptake phase, i.e., 40 min after FDG injection, did the microPET images reflect glucose metabolism of the conscious state, as measured with postmortem autoradiography (Matsumura et al., 2003). However, delaying the induction of anesthesia with respect to the administration Epilepsia, Vol. 48, Suppl. 4, 2007

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of the PET ligand is of course not always possible. This limits imaging to a static, delayed imaging protocol and negates the opportunity for dynamic imaging protocols that are required for fully quantitative analysis and compartmental modeling. PET images are not always simple to interpret because of the often poor anatomical detail that they contain. Therefore, PET data analysis ideally should be performed in conjunction with MRI data or a brain atlas. A commonly used technique involves coregistration of MRI and PET images to correlate structural and functional data. The accuracy and reliability of the coregistration method critically affects the validity of the results, and therefore needs to be specifically validated. This topic is discussed in further detail by Jupp and O’Brien in this issue. At present, scanners are under development that would allow simultaneous acquisition of PET and MRI images (Catana et al., 2006; Lucas et al., 2006; Raylman et al., 2006). Combined PET/MRI imaging may greatly advance in vivo molecular imaging of the brain in the future by allowing a very precise correlation of structural (MRI) and functional changes (PET) in the living brain. The availability of anatomical MRI data can provide more than just landmarks for interpretation and coregistration of the PET data (Chatziioannou, 2002). Information obtained from MR images can also be used to improve quantification of the PET signal by providing low noise attenuation correction (Chatziioannou, 2002) and partial volume correction (see Jupp and O’Brien, this issue). MRI templates of the rat brain allow automated voxel-based or predefined volume-of-interest analysis, which may improve the accurate assessment and spatial localization of rat brain function (Casteels et al., 2006). However, this method may not be as applicable for epilepsy models like the status epilepticus (SE) model in which structural changes in e.g., hippocampus occur and intersubject variability can be large. Applications of saPET for epilepsy research Noninvasive imaging modalities, particularly PET and MRI, greatly facilitate translational research, linking basic science and clinical research. Currently, FDG-PET is routinely used during presurgical evaluation of refractory patients with partial epilepsy. However, there are many other potential applications for PET in clinical diagnosis and management. In particular, the discovery of PET biomarkers of epileptogenesis would make it possible to identify which patients are at risk for developing epilepsy after a predisposing insult. The identification of such markers would place saPET in a position to play an important role in providing a more accurate prognosis and in preclinical drug development. Potential antiepileptogenic drugs could be examined for biological effectiveness in vivo using saPET in animal models before embarking on expensive clinical trials. Furthermore, the development and optimization of methods for production of new PET raEpilepsia, Vol. 48, Suppl. 4, 2007

diotracers for use in human studies is a time consuming and expensive process (Ingvar et al., 1991). Radiotracers coupled to nonstandard isotopes with longer half-life (hours to days) are currently developed and could allow PET imaging of several time points after tracer administration (Welch et al., 2007). saPET can offer a fast and simple method for in vivo testing of putative new tracers for epilepsy applications. Finally, saPET offers the prospect to enhance our understanding of the molecular changes taking place in the brain during epileptogenesis. Imaging of seizures and epileptogenesis While saPET has great potential for in vivo investigation of the neurobiological changes that occur during epileptogenesis in animal models, its application for this purpose is still in the technical development and validation phase (Jupp et al., 2004, 2005, in press). Most studies published to date have used FDG-saPET to evaluate metabolic changes during acute seizures. Can saPET be used to image the development and progression of epileptogenesis? And if so what is the relationship of these changes to epileptogenesis and their underlying cause? The availability of a diverse range of radiotracers now enables investigation of metabolic, physiological, pharmacological and molecular processes. Tracer probes labeled with positron-emitting radionuclides with high radiochemical specific activity (ratio of the activity of the labeled compound to the mass of the unlabeled compound) are retained in tissues as a result of either binding to an appropriate receptor or conversion to a product that is metabolized and trapped in cells after uptake (Herschman, 2003). Changes in glucose metabolism FDG is the most common PET tracer that has been used in epilepsy research, including studies of animal models of epilepsy. FDG is taken up by cells in a very similar manner to glucose, but then is not further metabolized after phosphorylation by hexokinase. As a result FDG gets trapped in cells and provides a map of glucose utilization in the cell/region of interest. Kornblum et al. (2000) were the first to publish findings from in vivo FDG metabolism during acute seizures using saPET. During the seizures, FDG uptake was increased in several brain regions (Figure 1) (Kornblum et al., 2000). Rats were scanned first under baseline conditions and a week later the same rats were injected with kainic acid (KA), to induce SE, followed by FDG injection and then, after a 45 min uptake period, imaged with the UCLA microPET scanner. During SE a dramatic enhancement of glucose metabolism was demonstrated, most notably in the dorsal and ventral hippocampus, as well as the entorhinal cortex. Elevated activity was also observed in the septum, piriform and cingulate cortices. Rats exhibiting moderate to severe seizures showed 1.6- and 2.3-fold increases

POSITRON EMISSION TOMOGRAPHY IN BASIC EPILEPSY RESEARCH

FIG. 1. Stimulation of cerebral metabolism by KA. MicroPET scans showing FDG uptake in a control rat (A–C) and in the same rat after injection of KA (D–F). There was a marked increase in FDG uptake in limbic structures, particularly the septum (D, arrow), dorsal (E, arrow), and ventral (F) hippocampus (DH and VH, respectively). (G) Glucose utilization in the DH (grey bars) and the VH (black bars) during seizure. Rats that manifested severe seizures (grade 3, 4) exhibited enhanced hippocampal FDG uptake compared with those that showed less severe (grade 1, 2) behavioral correlates after KA injection. Results are expressed as the percentage of basal (unstimulated) FDG uptake, and regional uptake values were normalized for pontine uptake for each rat. Values are the means ± SEM of four to five rats in each group. (∗∗ p < 0.01, comparing the grade 3,4 severity group to the grade 1,2 severity group for the DH; ∗∗∗ p < 0.001, comparing the grade 3,4 severity group to the grade 1,2 severity group for the VH; a, significantly different from the DH in the grade 3,4 severity group, p < 0.05). Reproduced from Kornblum et al., 2000 with permission.

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in FDG uptake in the dorsal and ventral hippocampus, respectively (Figure 1). A recent saPET study using pilocarpine induced SE in C57Bl/6 mice confirmed that FDG uptake was most markedly increased in the hippocampus (33.2%) (Mirrione et al., 2006). Again the increase in FDG uptake correlated directly with seizure severity during the uptake period (Mirrione et al., 2006). These results agree with previous 2-deoxy-[14C]glucose (2-DG) experiments after KA (Collins et al., 1980) and pilocarpine induced seizures (Scorza et al., 2002). Also in patients, complex partial seizures result in marked increases in glucose metabolism in the seizure focus (Tatum and Stecker, 1995; Fong and Delgado-Escueta, 1999; Millan et al., 2001). The above experiments confirm saPET’s usefulness for detection of dramatically elevated neuronal activity. Based on the application of saPET for serial imaging, Kornblum et al. (2000) suggested that this technique may be particularly valuable for the in vivo evaluation of the relationship between the extent of initial hypermetabolism during SE and the degree of activation observed with spontaneous seizures as well as the extent of pathological changes following seizures. Mirrione et al. (2006) conferred that their study could provide a foundation upon which we can begin to identify genetic contributions to the metabolic signature of TLE in mice, since many transgenics are made using C57Bl/6 as the background strain. More recently, an article has been published on the investigation of a mouse model for Glut-1 haploinsufficiency including FDG saPET imaging (Wang et al., 2006). Glut-1 is the predominant glucose transporter expressed in the blood–brain barrier and responsible for glucose entry in the brain (Dick et al., 1984). GLUT1+/− mice have spontaneous epileptiform discharges, impaired motor activity, incoordination, hypoglycorrhachia, microencephaly. This mouse model displayed a diffuse hypometabolism in the brain compared with the wild-type strain measured by FDG saPET (Figure 2). Future prospects for the use of FDG saPET imaging involve the monitoring of metabolic changes occurring between the acute phase of SE and the chronic phase of spontaneous recurrent seizures. Hypometabolism in the epileptogenic zone during the interictal state is a well-described phenomenon in humans with TLE (Hong et al., 2002; Choi et al., 2003), and has been successfully used to identify regions for surgical resection in MRInegative TLE cases (Carne et al., 2004). The cause of this hypometabolism and its relation to the persistence of seizures however is still unknown. Some studies suggest that the decreased uptake may be associated with neuronal degeneration and cell loss in the region (Diehl et al., 2003; Theodore, 2004). However, a number of other studies have shown a poor correlation between the degree of hippocampal volume or cell loss and the magnitude of the hypometabolism (Henry et al., 1994; Semah et al., 1995; O’Brien et al., 1997; Knowlton et al., 2001). Epilepsia, Vol. 48, Suppl. 4, 2007

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FIG. 3. Axial FDG-PET of a kindled rat demonstrating region of decreased glucose uptake in the ipsilateral hippo campus. Left panel: FDG-PET 1 week after electrode implantation prior to any kindling stimulations. Right panel: FDG-PET 2 weeks after the cessation of kindling. Note the decreased glucose uptake in the ipsilateral hippocampus (arrow).

FIG. 2. FDG-PET scans in GLUT-1+/+ and GLUT-1+/− mice. Representative PET images of a pair of GLUT-1+/+ and GLUT1+/− mice are shown. The upper and lower panels represent the comparable horizontal and axial sections, respectively. Reproduced from Wang et al., 2006 with permission.

Furthermore it is well established that patients can have marked hypometabolism of FDG-PET in the epileptogenic temporal lobe in the absence of any obvious cell loss or any other pathological or imaging abnormality (O’Brien et al., 2001; Carne et al., 2004). Our group is currently using in vivo imaging with saPET to investigate the relationship between the development of hypometabolism and epileptogenesis using the post-KA SE and electrical amygdala kindling models, both chronic epilepsy models of TLE in the rat. In the post-KA SE model whole brain FDG uptake was found to be dramatically reduced as early as 24 h after the KA treatment. This decreased uptake was found to persist during the 6week follow-up period and was not affected by the onset of spontaneous recurrent seizures (present in all animals) or any observed reduction in volume of cerebral structures on T2 -weighted MRI (Jupp et al., in press). These findings suggest that decreased FDG uptake develops before and independently from the hippocampal volume loss and the onset of spontaneous recurrent seizures. This is likely to reflect a decrease in glucose utilization (i.e., metabolism) rather than transport, as previous studies have shown an upregulation of glucose transporters in the brain after KAinduced SE (Gronlund et al., 1996). Given the time course demonstrated in this study, these metabolic changes may reflect some of the earliest neurobiological changes occurring during limbic epileptogenesis. However, at present we have insufficient data to conclude whether FDG hypometabolism is a biomarker for development of spontaneous epilepsy. Epilepsia, Vol. 48, Suppl. 4, 2007

saPET studies in the amygdala kindling model, demonstrated decreased glucose uptake in the ipsilateral compared with the contralateral hippocampus (Figure 3), independent of any change in hippocampal volume. This reduced uptake appeared after 4 weeks of kindling stimulations (twice per day for 6 days per week) and persisted when assessed again 2 weeks after kindling had ceased. This indicates that the hypometabolism was not due to the acute effect of electrical stimulation or seizures. Similar to what is observed after SE; it may reflect a change in metabolism in this structure in response to the development of epileptogenic changes (Jupp et al., in press). Changes in receptor expression The utilization of saPET to image receptor function and tracer kinetics in vivo in experimental animals continues to attract the interest of both the pharmaceutical industry and basic scientists. The receptor that has most commonly been imaged using PET in human epilepsy studies has been the central benzodiazepine (cBZ) receptor using radiolabeled flumazenil (FMZ). Changes in expression and function of the GABAA /cBZ receptor complex are well described in human focal epilepsy. Many studies have shown that FMZ-PET can detect abnormalities (mostly decreased binding) in the epileptogenic zone of patients with both TLE and extratemporal epilepsy appearing to be better defined than FDG-PET studies (Koepp et al., 2000). Currently, the possibility of using FMZ, and other receptor ligands, to study epilepsy models with saPET has begun to gain attention. Receptor PET imaging is more complex than FDG imaging because of the need to use modeling to calculate receptor density (Bmax ) and affinity (Kd ) (rather than just brain uptake as is done with FDG-PET). Receptor studies have generally required arterial blood sampling to calculate an input function (Ingvar et al., 1991). This may significantly hamper serial in vivo saPET receptor imaging studies over weeks or months in longitudinal studies during epileptogenesis. It would be beneficial to develop noninvasive techniques deriving the input function from a reference tissue devoid of receptors or from images of the heart (Wu et al., 1996; Laforest et al., 2005). Also by

POSITRON EMISSION TOMOGRAPHY IN BASIC EPILEPSY RESEARCH using an (arterial) probe to monitor radioactivity the need for blood sampling could be reduced (Pain et al., 2004). The last two options are only feasible when the radiotracer does not undergo significant metabolism (Sossi and Ruth, 2005) or when a standard curve describing the metabolism is usable. Liefaard et al. (2005) developed a population pharmacokinetic model to quantify both Bmax and Kd of the receptor complex from a single PET study using a single bolus injection. This model is based on the injection of a range of doses, all sufficient to saturate the receptor, in different individuals. Although the method requires blood sampling, it is potentially readily applicable to human studies (Liefaard et al., 2005). Recently, the same group used this approach to investigate changes in GABAA /cBZR complex in the kindling model of TLE (Liefaard et al., 2006). Fully kindled animals were implanted with a jugular vein catheter for administration of [11 C]FMZ and a femoral artery cannula for blood sampling. This study found a 36% decrease in the receptor density, but no changes in receptor affinity after amygdala kindling in rats. [11 C]FMZ has the disadvantage of a short-half life. As a result multiple syntheses are required if tracer doses are to be achieved for each individual or multiple subjects require study at any given time point. For this reason, our group has begun to investigate the use of [18 F] labeled FMZ. An additional advantage of this isotope is that protons emitted by 18 F do not diffuse as far in tissue before annihilation, resulting in better image resolution compared with 11 C. We have explored

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the potential of 2 -[18 F]fluoroflumazenil ([18 F]FFMZ) to study the GABAA /cBZR complex in rats utilizing saPET (Figure 4) (Dedeurwaerdere et al., 2006). In the hippocampus (region with high GABAA /cBZR density), signal intensity was higher (10%) than in the whole brain during the first 10 min of uptake. In the pons (low GABAA /cBZR density), the signal intensity was approximately 20% lower than in the whole brain during 25–30 min of the scan. Presaturation and displacement studies showed a high nonspecific component in the measured signal, which may be due to recirculation of [18 F]fluoroethanol, a metabolite of [18 F]FFMZ. The significant nonspecific activity of this tracer in the brain limits the ability to detect changes in GABAA /cBZR density during epileptogenesis and therefore alternative ligands need to be investigated (Dedeurwaerdere et al., 2006). Currently, we are testing an [18 F] labeled analogue of FMZ (Figure 4) and our preliminary results show that with this compound nonspecific binding is low and therefore the best candidate for imaging and quantification of cBZ receptors in the rat brain using serial saPET. Another receptor radiotracer with great potential for small animal epilepsy models with saPET is 2 -methoxyphenyl-(N-2 -pyridinyl)-p-18 F-fluoro18 benzamidoethylpiperazine ([ F]MPPF), which labels 5-HT1A receptors. A human PET study using [18 F]MPPF demonstrated that the in vivo availability of 5-HT1A receptors is decreased in the epileptogenic zone of patients with focal epilepsy compared with healthy subjects (Merlet et al., 2004). This decrease was highly

FIG. 4. Distribution of FMZ tracers in the rat brain. Axial, horizontal, and sagittal planes are taken through the right hippocampus. Template MRI (T2 -weighted) of the rat brain is showed in the left panel. Middle and right panel demonstrate the distribution of [18 F]FFMZ and an alternatively labeled FMZ tracer, respectively in the rat brain. The latter shows much more specificity for brain binding and a better brain to background contrast.

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correlated to the degree of epileptogenicity of cortical areas as determined by intracerebral recordings, and was suggested to reflect more than pathological changes or neuronal loss in the epileptic focus (Merlet et al., 2004). A preclinical study has used [18 F]MPPF autoradiography in a rat model of limbic epilepsy after injection of KA (Van Bogaert et al., 2001), but the tracer has not yet been applied for in vivo saPET imaging. PET studies utilizing [11 C]carfentanil (µ-opiate agonist), [11 C]methylnatrindol (δ-receptor-opiate antagonist), [11 C]alpha-methyl-L-tryptophan (analogue of Ltryptophan, the precursor for 5-HT) have also demonstrated their application for use in human epileptic patients. To the authors’ knowledge none of the above receptor tracers has yet been utilized in saPET for epilepsy research.

Changes in reporter gene expression Perhaps the fastest growing application of saPET technology has been the development of reporter gene systems to monitor gene expression, cell transplantation and distribution of gene delivery vehicles (Herschman, 2003; Jacobs et al., 2003). To date, these techniques have not been used in epilepsy research, but potentially have powerful applications for the in vivo monitoring of gene expression during epileptogenesis.

Quest for surrogate markers of epileptogenesis Only a limited number of patients will develop epilepsy after a brain insult such as a head injury or stroke. The “Holy Grail” of in vivo imaging research is to find a biological marker that could predict the development of epilepsy after such an initiating insult. This would open a window for treatment opportunities with neuroprotective agents during the latent period to prevent the progression and development of the condition. PET would become a very powerful tool to monitor in vivo the effectiveness of potential neuroprotective treatments. Currently, no biological marker predictive for seizure outcome has been identified. Our group has found that FDG, the most common radiotracer used in epilepsy PET studies, may not have such predictive value. However, glucose metabolism is very nonspecific, being affected by a large variety of processes. Possibly, other tracers that label particular neurobiological processes or substances may prove to have greater predictive value for seizure outcome. In addition, biological markers may be sought to identify other related processes like apoptosis, necrosis and neurogenesis. The identification of such biological markers will most likely occur in parallel with in vitro and postmortem molecular research. Potential surrogate markers can then be labeled and tested using saPET.


saPET in the development and evaluation of new treatment strategies Many pharmaceutical companies are actively pursuing molecular imaging as part of their drug discovery program (Chatziioannou, 2002). PET imaging can be used to investigate the biodistribution, metabolism, location of action and brain kinetics of labeled conventional and novel AEDs. While labeling the drug of interest directly may present some challenges, through this process it will be possible to estimate the degree to which this drug penetrates the blood–brain barrier (Sossi and Ruth, 2005). Further, the mechanism of action of a particular treatment regime can be studied through the use of other PET tracers (e.g., FDG) to evaluate the effects and efficacy of treatments on biological processes (e.g., glucose uptake referring to neuronal activity) in the brain. While there are currently few published studies implementing this approach with saPET, the proof of concept was demonstrated in a study in which the mechanism of action of vagus nerve stimulation (VNS) was investigated using FDG in rats (Dedeurwaerdere et al., 2005). Although VNS is well established as an effective treatment in a proportion of patients with medically refractory epilepsy, its mechanism of action remains largely unknown. The study aimed to use saPET to identify the brain structures affected by VNS, and investigate whether this changed over a prolonged period of stimulation. A baseline, an acute (after first activation of the VNS electrode) and a chronic (after 1 week of continuous VNS) FDG-PET scan was performed. During acute VNS, glucose metabolism was significantly decreased in the left hippocampus, while increases were found in both olfactory bulbs. During chronic VNS, a significant decrease in left/right uptake ratio in the striatum was observed. These results indicate that acute and chronic VNS interact with regions important for seizure control. This pilot study demonstrated the value of saPET as an explorative tool for studying rat brain metabolism and responses during several stages of VNS therapy. In the future, the imaging of cerebral activation in long-term studies in vivo in rats may be applied to assess other therapeutic interventions like drug treatments and experimental therapies like deep brain stimulation, transcranial magnetic stimulation, ketogenic diet, etc. CONCLUSIONS In vivo imaging techniques such as saPET, which provide the possibility to perform serial follow-up studies to investigate the development of functional changes in the brain over time, will become important tools for epilepsy research. saPET can be used to investigate a wide range of physiological changes during the course of epileptogenesis in vivo and provide the ability to correlate such

POSITRON EMISSION TOMOGRAPHY IN BASIC EPILEPSY RESEARCH changes with seizure outcome. So far, findings utilizing this method suggest that epileptogenesis is a progressive process that continues following the onset of spontaneous recurrent seizures. In the future, saPET may also play a role in the development and evaluation of new treatments for epilepsy. An important advantage of saPET is that the same molecular radiotracers can be used for both animal research and clinical applications facilitating a more rapid progression from preclinical research to clinical practice. Acknowledgment: The authors would like to acknowledge Rodney Hicks (for the contribution to the FDG and FMZ imaging work and critically reading the manuscript), Damian Myers (for the contribution to the FMZ imaging work and for critically reading the manuscript), David Binns (for the contribution to the FDG and FMZ imaging work), and Peter Roselt, MarieClaude Gregoire and Lucy Vivash for their involvement in the FMZ work. This work was supported by the CRC for Biomedical Imaging Development.

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