Epilepsia, 49(Suppl. 3):15–22, 2008 doi: 10.1111/j.1528-1167.2008.01506.

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SUPPLEMENT - MERRITT PUTNAM SYMPOSIUM

Molecular and diffusion tensor imaging of epileptic networks

†Aimee F. Luat and ∗ †‡Harry T. Chugani

Carman and Ann Adams Department of Pediatrics, and the Departments of †Neurology and ‡Radiology, Children’s Hospital of Michigan, Wayne State University, Detroit, Michigan, U.S.A.

SUMMARY
Several studies have shown that seizure-induced cellular and molecular changes associated with chronic epilepsy can lead to functional and structural alterations in the brain. Chronic epilepsy, when medically refractory, may be associated with an expansion of the epileptic circuitry to involve complex interactions between cortical and subcortical neuroanatomical substrates. Progress in neuroimaging has led not only to successful identification of epileptic foci for surgical resection, but also to an improved understanding of the functional and microstructural changes in long-standing epilepsy. Positron emission tomography (PET), functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI) are all promising tools that

can assist in elucidating the underlying pathophysiology in chronic epilepsy. Studies using PET scanning have demonstrated dynamic changes associated with the evolution from acute to chronic intractable epilepsy. Among these changes are data to support the existence of secondary epileptogenesis in humans. MRI with DTI is a powerful tool which has the ability to characterize microstructural abnormalities in epileptic foci, and to demonstrate the white matter fibers and tracts participating in the epileptic network. In this review, we illustrate how PET and DTI can be applied to depict the functional and microstructural alterations associated with chronic epilepsy. KEY WORDS: Epileptic networks, Secondary epileptogenesis, PET, DTI.

There has been a growing body of evidence to indicate that seizure-induced neuronal injury in chronic epilepsy can cause alterations in synaptic reorganization and connectivity (Sutula et al., 1998; Lehmann et al., 2000; Cavazos et al., 2004). It has been shown in animal models that seizures may produce long-term alterations in neuronal structures extending beyond the epileptic focus (Brener et al., 1991; Hagemann et al., 1998) so that there may be an expansion of the epileptic network with repeated seizures. For example, autoradiography studies of glucose metabolism in electrically kindled rats showed progressive recruitment of cortical and subcortical limbic structures as the stages of kindling increased (Handforth & Ackermann, 1988; 1995). Similarly, after systemic injection of kainic acid in rat, propagation of seizures in limbic and nonlimbic structures occurred (Lothman & Collins, 1981). Thus,
Address correspondence to Harry T. Chugani, M.D., Pediatric Neurology/PET Center, Children’s Hospital of Michigan, 3901 Beaubien Blvd., Detroit, MI 48201, U.S.A. E-mail: hchugani@pet.wayne.edu Blackwell Publishing, Inc. C 2008 International League Against Epilepsy

metabolic activation was noted initially in the hippocampus prior to the appearance of motor seizures. During limbic seizures, increased glucose consumption is seen in regions, such as the amygdala, entorhinal and pyriform cortices, and thalamic nuclei. With repeated seizures, metabolic activation progresses to involve many other structures, including the substantia nigra. The crucial role of subcortical structures in the propagation and behavioral manifestations of epileptic seizures has also been shown in various experimental animal models and in humans (reviewed in Norden & Blumenfeld, 2002). Taken together, these findings suggest that epileptic seizures involve widespread network interactions between cortical and subcortical structures, which contribute to the epileptic circuitry. Although advances in both structural and functional neuroimaging have led to improved localization of the epileptic focus for presurgical planning in refractory cases, novel imaging techniques can also provide a better understanding of the underlying mechanisms in epilepsy and the functional consequences of chronic epilepsy. Positron emission tomography (PET), functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI) can 15

16 A. F. Luat and H. T. Chugani all be utilized as powerful tools in the study of epileptic networks. behavior characterized by paroxysmal electrographic manifestations and clinical seizures” induced by seizures from a primary epileptic focus (Morrell, 1985; 1989). He described three stages in the formation of secondary epileptic foci. In the first stage, epileptiform activity in the new brain region is dependent on a trigger from a primary focus. In the second stage, epileptiform activity occurs spontaneously in the new focus. At this time, the removal or ablation of the primary focus will still lead to resolution of the epileptiform activity at the secondary site, but the recovery takes place over time. If this dependent focus is not removed, electrographic spikes and electrographic seizures begin to develop independently at the second site leading to the third stage, when the epileptiform activity in the secondary focus has become irreversible (independent secondary focus). Much of our knowledge on secondary epileptogenesis was derived from experimental animal studies. Both the kindling and kainate animal models have been utilized for studying this phenomenon (Cibula & Gilmore, 1997; Dudek & Spitz, 1997). Recently, in vitro demonstration of the formation of a secondary epileptogenic focus was described (Khalilov et al., 2003). Although the concept of secondary epileptogenesis in animals is well established, its existence in humans remains controversial. Nevertheless, there is evidence to support the notion that secondary epileptogenesis may occur in human epilepsy. For example, patients with unilateral brain lesions and epilepsy may have bilateral interictal foci (Gupta et al., 1973; Hughes, 1985; Morrell, 1985; Gilmore et al., 1994; McCarthy et al., 1997). In a long-term follow-up study on 60 patients who underwent standard anterior temporal lobe resection for lesions associated with chronic, medically intractable seizures, Eliashiv et al. (1997) observed late seizure recurrence in three patients; two had been seizurefree for 10 years and one for 15 years after surgery, before recurrence of seizures in the absence of tumor recurrence. These investigators suggested that a prolonged history of seizures prior to surgery may be associated with poor surgical outcome. Similarly, in a long-term follow-up study by Foldvary et al. (2000) on 79 patients with unilateral intractable medial temporal lobe epilepsy who underwent temporal lobectomy, 55% of the patients had at least one post-operative partial-onset seizure and 30 of them (38% of the total) experienced multiple seizures during an average of 14-years follow-up suggesting that partial seizures can be generated elsewhere in the brain of individuals who have suffered intractable mesial temporal lobe epilepsy. These findings suggest that secondary epileptogenesis at various sites distant to the lesion may develop with years of uncontrolled seizures and may contribute to the recurrence of seizures even after successful resection of the primary focus. Therefore, identification of these secondary epileptic foci during preoperative evaluation is imperative to allow

PET I MAGING IN E PILEPSY
In addition to conventional MRI, functional neuroimaging using PET and single photon emission computed tomography (SPECT) can provide complementary information to help localize the epileptic focus and often provides additional information that cannot be obtained from conventional MRI sequences. Indeed, PET scanning using various tracers has been utilized in the identification of the primary epileptic focus and dysfunctional areas outside the primary focus (Juh´ sz et al., 2000; Sood & Chugani, 2006). a The most widely available PET tracer used in epilepsy is 2-deoxy-2-[18 F]fluoro-D-glucose (FDG), which allows the rates of regional brain glucose utilization to be estimated. FDG-PET can detect focal areas of decreased glucose metabolism that are generally concordant with the epileptic cortex even in patients with normal MRI (Chugani et al., 1990; da Silva et al., 1997). In addition, there are several other tracers that have been applied in epilepsy. For example, [11 C]-flumazenil (FMZ), which binds to gamma aminobutyric acid (GABA A ) receptors (Savic et al., 1988; Henry et al., 1993), has been shown to improve localization of epileptic foci in patients with intractable epilepsy of both medial temporal and neocortical origin, including those with normal conventional MRI (Savic et al., 1988; Henry et al., 1993; Savic et al., 1993, 1995; Richardson et al., 1996; Ryvlin et al., 1998; Muzik et al., 2000; Juh´ sz et al., a 2000, 2001). [11 C] α-methyl-L-tryptophan (AMT) which is a tracer of tryptophan metabolism (Muzik et al., 1997; Chugani et al., 1998a) has been utilized in epilepsy surgery evaluation, particularly in the identification of epileptic tubers in children with intractable epilepsy and tuberous sclerosis complex (TSC) (Chugani et al., 1998b; Asano et al., 2000). In addition, AMT PET appears to be useful in the identification of the epileptic focus in children with intractable neocortical epilepsy without TSC who had malformations of cortical development with abnormal (Fedi et al., 2001) and normal MRI (Juh´ sz et al., 2003). a Other PET tracers with the potential capability of detecting epileptic brain regions include radiolabeled ligands which bind to opioid receptors (Frost et al., 1988), histamine H1 receptors (Iinuma et al., 1993), N-methyl-D-aspartate receptors (Kumlien et al., 1999) and peripheral benzodiazepine receptors (Sauvageau et al., 2002), although these tracers have not yet been validated for presurgical evaluation.

S ECONDARY E PILEPTIC F OCI
Morrell coined the word secondary epileptic foci as “trans-synaptic and long-lasting alterations in nerve cell
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17 Molecular and Diffusion Tensor Imaging of Epileptic Networks modification of surgical treatment in order to achieve a good surgical outcome. correlated to clinical seizure variables. It was noted that the change in seizure frequency between the two PET scans correlated positively with the change in the extent of the cortical glucose hypometabolism. Most patients with persistent or increased seizure frequency showed enlargement of the cortical areas of glucose hypometabolism (Fig. 1A and B). On the other hand, the extent of glucose hypometabolism remained stable or even decreased if seizures came under control. This observation suggests that the extent of glucose metabolism alterations correlates with major changes in clinical seizure frequency suggesting that clinical progression or persistence of severe, intractable epilepsy can lead to involvement of progressively larger cortical areas of neuronal dysfunction, as reflected by the size of the cortical glucose hypometabolism. Conversely, at least some of the cortical hypometabolism seen on PET scans may represent reversible changes in neuronal function. These findings are consistent with the observations of other investigators who have shown that some foci of glucose hypometabolism disappear after seizure control achieved either medically (Matheja et al., 2000) or with surgery (Akimura et al., 1999; Spanaki et al., 2000; Joo et al., 2005). All together, these observations support the notion that intractable epilepsy in children is a progressive condition, and that the areas of focal glucose hypometabolism undergo dynamic changes related to seizure activity. Persistent epilepsy in children may recruit progressively larger areas of brain into the seizure network. Whether this process is linked etiologically with the development of an epileptic encephalopathy in some children remains to be determined.

PET E VIDENCE OF THE E XISTENCE OF S ECONDARY E PILEPTOGENIC F OCI IN H UMAN E PILEPSY
It is well-known that the extent of brain glucose hypometabolism shown on PET scans of patients with epilepsy is not static but undergoes dynamic changes depending upon the chronicity and intractability of epilepsy. For example, PET scans of glucose metabolism in patients with new onset partial epilepsy rarely show focal abnormalities (Matheja et al., 2001; Gaillard et al., 2002). Recently, Gaillard et al. (2007) were not able to demonstrate progression of hypometabolism in a short longitudinal study over 2–3 years. On the other hand, patients with chronic partial epilepsy often exhibit areas of glucose hypometabolism not only in the primary epileptic focus, but also in remote yet interconnected cortical areas (da Silva et al., 1997; Jokeit et al., 1997; Juh´ sz et al., 2000; Takaya a et al., 2006). Longitudinal changes in the extent of glucose hypometabolism using sequential PET scans in children with partial epilepsy have been demonstrated by our group (Benedek et al., 2006). In this longitudinal study of 15 children with partial epilepsy and normal MRI scans, two FDG-PET scans were performed 7–44 months apart and the extent of hypometabolic cortex on the side of electroencephalography (EEG)-verified epileptic focus and its changes between the two PET scans were measured and

Figure 1. (A) Transaxial 2-deoxy-2[18 F]fluoro-D-glucose positron emission tomography (FDG PET) image of a 15-year-old child, whose seizure frequency increased between the two scans from one per day to more than 10 seizures per day. Note the expansion of the areas of the left temporal and frontal lobe hypometabolism (arrows). (B) Three-dimensional surface rendering of the objectively marked FDG PET abnormalities showing the hemispheric expansion of cortical glucose hypometabolism. Epilepsia C ILAE

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18 A. F. Luat and H. T. Chugani et al., 1993; Muzik et al., 2000; Juh´ sz et al., 2001). a Furthermore, cortical regions remote from the presumed epileptic focus (as indicated by scalp EEG), detected as areas of decreased FMZ binding are often identified (Juh´ sz & Chugani, 2003). In the study of Juh´ sz et al. a a (2001) on patients with intractable localization-related epilepsy with MRI-verified brain lesions, remote areas of FMZ-PET abnormalities were noted in areas having direct cortico–cortical connections with the primary lesional region suggesting that well-established cortico–cortical pathways (e.g., superior longitudinal fasciculus, inferior longitudinal fasciculus, and arcuate fasciculus) may be involved in propagation of epileptic discharges to cause alterations in remote areas. On the other hand, some of the FMZ-binding abnormalities outside the primary focus may disappear following surgical removal of the primary epileptic focus (Savic et al., 1998). These observations suggest that such FMZ abnormalities beyond the primary epileptic focus may represent areas of secondary epileptic foci, and that some of these changes may be potentially reversible, as predicted by Morrell (1985, 1989).

Figure 2. 2-deoxy-2-[18 F]fluoro-D-glucose positron emission tomography (FDG PET) (A) showed areas of glucose hypometabolism in the right frontal and parietal cortex (arrows). Flumazenil (FMZ) PET (B) disclosed a smaller area of decreased FMZ binding involving the right frontal cortex. Electrocorticography captured seizures of right frontal onset overlapping with the area of decreased FMZ binding. Epilepsia C ILAE

T HE U SE OF D IFFUSION T ENSOR I MAGING IN E PILEPSY
DTI is a new MRI technique, which is based upon the ability of MRI to assess the direction and magnitude of water diffusion in tissues in vivo by utilizing the principle of anisotropic diffusion of water molecules in the white matter tracts of the brain (Le Bihan et al., 1986). DTI measurements reflect the random thermal displacement of water molecules and the technique is more sensitive than conventional MRI in detecting microstructural changes in the brain. Two important parameters: the apparent diffusion coefficient (ADC), which measures the overall magnitude of diffusion, and the fractional anisotropy (FA), which measures the directional preference of the diffusion motion, can be calculated using DTI. By detecting these changes, DTI provides tissue information about microscopic barriers, which can be affected by various disease processes. Furthermore, it has the capability of tracking the white matter fibers by assessing the connectivity of the main fiber direction. The use of DTI in epilepsy has great potential value. Studies on experimentally induced status epilepticus showed reductions in ADC in various brain regions involving both limbic and extralimbic structures including the medial thalamus, suggesting that sustained epileptic activity is associated with complex interactions involving both cortical and subcortical structures (Zhong et al., 1993; Nakasu et al., 1995; Fabene et al., 2003). The decreased ADC was attributed to the presence of cytotoxic edema which may be due to a shift of extracellular water into the intracellular space, resulting in a reduction of free

PET U SING [11 C] F LUMAZENIL (FMZ)
Based on the observation of altered gammaaminobutyric acid (GABA) inhibitory mechanisms in the epileptic focus demonstrated in experimental and human epilepsy (Craig & Colasanti, 1986; Lloyd et al., 1986; Pitkanen et al., 1987), several investigators have explored the use of PET scanning with FMZ in the identification of the epileptic focus. FMZ is a benzodiazepine receptor antagonist that binds to the alpha subunit of the GABA A receptors. PET scanning with [11 C] FMZ is sensitive in detecting mesial temporal sclerosis (Savic et al., 1988; Henry et al., 1993), but has also been used in the identification of epileptogenic cortex in extratemporal lobe epilepsy (Savic et al., 1995; Richardson et al., 1996; Ryvlin et al., 1998; Muzik et al., 2000). Compared with FDG-PET, the cortical area showing decreased FMZ binding is usually smaller than the cortical region of glucose hypometabolism (Fig. 2) and has been found to be a better indicator of the seizure focus and areas of frequent spiking on electrocorticography (Fig. 3) (Savic
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19 Molecular and Diffusion Tensor Imaging of Epileptic Networks

Figure 3. Flumazenil-positron emission tomography (FMZ PET) of a 7.2-year-old girl with intractable epilepsy projected on a 3-dimensional brain surface. Areas of >10% decreased FMZ binding are seen in black. Seizure onset was noted in the right inferior temporal cortex (yellow diamond) and areas of frequent (>10/min) interictal spiking (orange circle) were noted in the right temporal and frontal cortex. Both the seizure onset zone and the area of rapid seizure spread (circle with cross) were overlapping and/or adjacent to the areas of decreased FMZ binding. Scalp ictal EEG showed an anterior temporal focus but did not disclose epileptiform activity in the frontal region. Epilepsia C ILAE diffusion. A similar observation was noted in patients with new onset prolonged seizure and acute symptomatic seizures (Farina et al., 2004; Parmar et al., 2006) where decreased ADC in the hippocampus corresponded to the side of the EEG focus. Interictal DTI has been utilized to further characterize the microstructural abnormalities of epileptic foci (Wieshmann et al., 1999; Rugg-Gunn et al., 2001, 2002; Assaf et al., 2003; Thivard et al., 2005). Increases in diffusivity (ADC) and reductions in anisotropy (FA) likely related to neuronal loss, gliosis and structural disorganization were noted not only in patients with acquired partial epilepsies but also in subjects with cryptogenic partial epilepsies, thus indicating a higher sensitivity of this modality to detect epilepsy-related changes as compared with conventional MRI (Rugg-Gunn et al., 2001). Since subcortical brain structures have been implicated in the propagation of seizure spread through cortico– subcortical epileptic circuitries (Morillo et al., 1982; Gale, 1992; Chugani et al., 1994), we evaluated DTI changes in the hippocampus and subcortical brain structures in 14 children with temporal lobe epilepsy: seven with and seven without secondary generalization (Kimiwada et al., 2006). Five patients had MRI signs of hippocampal sclerosis. None of the subjects showed any structural or signal changes on conventional MRI in the thalamus or basal ganglia. Decreased FA (p < 0.001) and increased ADC (p = 0.003) values were found in the hippocampi ipsilateral to the seizure focus. Significant decreases of FA (p = 0.002) were also seen in the contralateral hippocampi, despite unilateral seizure onset and excellent surgical outcome in patients who underwent surgery. FA and ADC values of patients with generalized versus partial seizures did not show significant differences in this preliminary study involving relatively few patients. However, there was a weak trend for increased FA in the ipsilateral thalami of children with generalized seizures (p = 0.12). In addition, a trend for increased ADC was also found in the ipsilateral thalami of children with generalized seizures (p = 0.09) but not in those with partial epilepsy only (p = 0.46). Our observations added evidence for the existence of microstructural changes of the hippocampus and perhaps, the ipsilateral thalamus in children with temporal lobe epilepsy suggesting that further, detailed characterization of the structural
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20 A. F. Luat and H. T. Chugani

Figure 4. (A) 2-deoxy-2-[18 F]fluoro-D-glucose positron emission tomography (FDG PET) image of a child with tuberous sclerosis complex (TSC) showing multiple areas of cortical glucose hypometabolism representing multiple cortical tubers (thin arrows). (B) Alpha [11 C] methyl-L-tryptophan positron emission tomography (AMT PET) of the same child showing a single tuber which showed increased AMT uptake (thick arrow). The patient underwent left temporoparietal resection, including the tuber with increased AMT uptake. He became seizure-free for 15 months. After 4 years of follow-up, he achieved Engel class IIA outcome. Epilepsia C ILAE and functional changes in brain regions beyond the epileptic temporal lobe can be obtained by DTI. ADC was much higher in the epileptogenic tubers. The high ADC values in cortical tubers are reflective of the loss of the structural barrier in water motion, perhaps due to looser integrity of the tissues, presence of hypomyelination, gliosis, or loss of neurons. Since DTI-fiber tractography can noninvasively evaluate the tissue microenvironment of cerebral white matter tracts, it can be applied to evaluate the integrity and connectivity of the white matter tracts connecting cortical and subcortical brain structures. With the growing body of evidence suggesting the involvement of subcortical structures in the epileptic network, DTI-fiber tractography can potentially be used to detect and define the epileptic circuitry as it evolves with chronicity and increasing severity of epilepsy. We have explored the use of DTI-fiber tractography in identifying epileptogenic tubers. Based on our preliminary data, some of the epileptogenic tubers (defined as ictal onset zone by electrocorticography) showed thalamic connectivity with significantly higher FA values when compared with the contralateral homotopic side suggesting that this may represent an aberrant connectivity to the thalamus. Whether this observation is related to the involvement of the thalamus in the epileptic circuitry or to the phenomenon of thalamo-cortical retargetting secondary to reorganization following early brain injury as has been observed in experimentally induced cerebrocortical microgyria (Rosen et al., 2000) remains to be elucidated.

T HE U SE OF DTI IN THE I DENTIFICATION OF E PILEPTOGENIC T UBERS IN TSC
Whereas, FDG-PET is not able to distinguish between epileptogenic and nonepileptogenic tubers, AMT-PET scanning has proven to be a useful tool in the identification of epileptogenic tubers and has improved the outcome of epilepsy surgery in TSC (Kagawa et al., 2005). Cortical tubers on FDG PET are typically seen as areas of glucose hypometabolism. AMT-PET, on the other hand, is able to highlight epileptogenic tubers as areas of increased AMT uptake interictally while nonepileptogenic tubers show decreased uptake of AMT (Fig. 4) (Chugani et al., 1998; Asano et al., 2000). With recognition of the potential role of DTI in the localization of epileptogenic cortex in partial epilepsy, its potential use in the identification of the epileptogenic tuber has also been explored. Jansen et al. (2003), in a study of four patients, found increased ADC values in epileptogenic tubers, based on interictal spiking (derived from high-resolution magnetoencephalography and scalp EEG). They noted that while all tubers showed high ADC values compared to the surrounding cortex, the increase in
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21 Molecular and Diffusion Tensor Imaging of Epileptic Networks

C ONCLUSION
Provocative evidence exists to suggest that epileptic networks may undergo dynamic changes as a result of persistent seizures, particularly in the immature brain, thus recruiting more structures into the epileptic network. In children, expansion of an epileptic network may be related to the development of an epileptic encephalopathy or the establishment of secondary epileptic foci that, if independent, also must be identified and resected in epilepsy surgery. Currently, we do not have adequate methods of distinguishing “dependent” from “independent” secondary epileptic foci. However, advances in neuroimaging using PET scanning, DTI, and functional MRI have the potential capability of delineating and defining the epileptic network thus contributing to improved outcomes in epilepsy surgery.

ACKNOWLEDGMENTS
This work was supported by NIH grant RO1 NS/RR38324 (to H.C.). We are grateful to the staff of the PET Center at Children’s Hospital of Michigan, Wayne State University for the collaboration and assistance in performing the studies described above. Conflict of interest: The contributing authors to this article have declared no conflicts of interest.

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Epilepsia, 49(Suppl. 3):15–22, 2008 doi: 10.1111/j.1528-1167.2008.01506.x