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Purpose: The development of nonradioactive and targeted magnetonanoparticles (MNP) capable of crossing the blood–brain barrier (BBB) and of concentrating in the epileptogenic tissues of acute and chronic animal models of temporal lobe epilepsy to render these tissues visible on magnetic resonance imaging (MRI).
Methods: Nonradioactive alpha methyl tryptophan (AMT) was covalently attached to MNP composed of iron oxide and dextran. A rodent model of temporal lobe epilepsy was prepared by injecting kainic acid into the right hippocampus. AMT-MNP or plain MNP was injected in the tail-vein of two animals during the acute stage 3 days after status epilepticus, and AMT-MNP in five animals during the chronic stage. MRIs were obtained before and after particle injection in all animals. Intracranial EEGs were obtained in all chronic animals after completion of MRI studies.
Results: AMT-MNP crossed the BBB and intraparenchymal uptake was visible on MRI. In the acute condition, AMT-MNP appeared to localize to both hippocampi, whereas plain MNP only identified unilateral, presumably inflammatory, changes. In the chronic condition, AMT-MNP uptake correlated with the occurrence of spontaneous seizures, and the location of uptake appeared to agree with bilateral or unilateral epileptogenicity confirmed by subsequent intracranial EEG.
Discussion: Nonradioactive AMT-MNP can cross the BBB and may accurately localize epileptogenic cerebral regions. The MNP-MRI approach is potentially applicable to the use of any bioactive molecules as ligands for imaging normal and abnormal localized cerebral functions, accurately, safely, and inexpensively.
The American Epilepsy Society and the National Institute of Neurological Disorders and Stroke have coauthored a list of benchmarks to be attained in the search for a cure for epilepsy (Jacobs et al., 2001), the first of which is the identification of a reliable surrogate marker of epileptogenesis and epileptogenicity. A reliable surrogate marker that can be noninvasively measured could (1) provide an inexpensive means to localize the epileptogenic region for surgical resection in patients with pharmacoresistant epilepsy who are candidates for surgery; (2) predict which patients are likely to develop epilepsy following a potentially epileptogenic brain injury, in order to institute antiepileptogenic treatment; and (3) determine which of the many therapeutic approaches, including a large number of antiepileptic drugs, vagus nerve stimulation, and deep brain stimulation, are likely to be effective in individual patients without the need to wait for another seizure to occur. One putative surrogate marker that has received recent attention is alpha methyl tryptophan (AMT), which has been used as a positron emission tomography (PET) ligand to identify epileptogenic tissues in several epilepsy conditions (Fedi et al., 2001; Duchowny, 2003; Juhasz & Chugani, 2003; Juhasz et al., 2003; Natsume et al., 2003; Juhasz et al., 2004; Kagawa et al., 2005).
This proof of principle study demonstrates the use of nanotechnology to covalently attach AMT to magnetonanoparticles (MNP) visible on magnetic resonance imaging (MRI). These particles cross the blood–brain barrier (BBB) and concentrate in epileptogenic tissues during the acute and chronic stages of an animal model of temporal lobe epilepsy, permitting their localization with standard MRI. Beyond its immediate application in epilepsy, this represents the first demonstration of a novel approach to utilize standard MRI for ligand-based functional neuroimaging, which would have the following advantages: (1) there would be no radiation risk, and thus no limit on the number of times the study could be repeated; (2) the technique could be employed in any MRI facility without need for modification; (3) tracers are relatively easy to synthesize in a short period of time; (4) they could be purchased relatively inexpensively and could be kept for prolonged periods of time without special storage requirements. This MNP-MRI approach is potentially applicable to the use of any bioactive molecules as ligands for imaging normal and abnormal cerebral function, accurately, safely, and inexpensively.
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The results of this study provide important proof of principle for several aspects of the hypothesis that conjugated MNP, which are not radioactive, could be used to delineate specific cerebral functions on MRI. Most importantly, the MRI patterns observed before and after conjugated MNP injection provide evidence that these magnetized particles cross the BBB in this epilepsy model (Oby & Janigro, 2006), to be taken up by brain parenchyma, and that the negative enhancement observed in epileptogenic tissues after MNP injection was not due to blood products. Whether MNP uptake across the blood–brain barrier also occurs in normal nonepileptogenic rat brain remains to be demonstrated.
The fact that AMT-MNP concentration does not merely indicate nonspecific inflammatory changes associated with KA injection (Vezzani et al., 2008) is suggested by the difference between the MRI pattern seen with AMT-MNP and that with plain MNP during the acute phase. The latter, which are known to be taken up in areas of inflammation (Dousset et al., 1999a, 1999b), concentrated only unilaterally on the side of the kainate injection, while the former were concentrated bilaterally. Epileptic status in unilateral intrahippocampal kainate-injected animals, however, involves hippocampal and parahippocampal structures bilaterally, and the subsequent epileptogenic process can be bilateral or unilateral (Bragin et al., 1999). Therefore, this suggestion that plain MNPs only indicate inflammation, and not epileptogenicity, needs to be substantiated by further studies; given that only one rat was investigated with each nanoparticle, it is possible that the rat injected with plain MNP had a strictly unilateral epileptogenic process and that these particles could also identify areas of epileptic activity.
The potential for AMT-MNP to be a specific surrogate marker of brain tissue involved in spontaneous seizures was supported by the chronic studies, which demonstrated concentration of this tracer in the four rats that exhibited spontaneous seizures, but not in the rat with no spontaneous seizures. The epileptogenicity of brain tissue demonstrating AMT-MNP uptake was confirmed by the electrophysiological studies showing that these areas were capable of generating both interictal and ictal discharges, while no such epileptic abnormalities were seen in the rat that did not concentrate AMT-MNP or demonstrate spontaneous behavioral seizures.
The correlation between bilateral versus unilateral AMT-MNP uptake and bilateral versus unilateral electrophysiological evidence of epileptogenicity is suggestive that this tracer could be a reliable surrogate marker to localize the epileptogenic region; however, the results are by no means definitive. The number of rats is small and the pattern of negative enhancement on MRI could not be quantitatively measured by densitometry. Many more animals will be needed to confirm these visual MRI analyses with quantitative data that can be analyzed statistically. The electrophysiological evidence of lateralization is more convincing, but also subject to limitations.
The two rats with bilateral AMT-MNP concentration had bilateral independent IIS and pHFOs, indicating bilateral epileptogenicity. The seizures appeared to begin bilaterally as well; however, this diffuse onset pattern usually indicates that the site of ictal generation is actually some distance from the electrode contacts. It is not possible, therefore, to determine whether seizures began independently from both sides, or consistently from one side, with rapid propagation. In the two rats with unilateral AMT-MNP concentration, IIS predominated on that side, and pHFOs, including FR in one rat, were limited to that side. In these rats, seizures consistently began on the side of apparent AMT-MNP concentration, with subsequent propagation to the contralateral side.
These electrophysiological studies remain restricted, because it was not possible to sample all areas with and without AMT-MNP uptake. Consequently, a clear delineation of the spatial relationship between areas of uptake and epileptogenesis cannot be demonstrated. Further studies with a much larger sample size are necessary to define how well AMT-MNP might not only localize, but also define the extent of, the epileptogenic region.
It is curious that none of the rats in this study demonstrated the unilateral hypersynchronous ictal onsets commonly encountered in our previous studies of intrahippocampal kainate-induced epilepsy (Bragin, et al., 1999). Lewis rats were used for these experiments because they had been used for previous MNP studies, whereas all of our previous electrophysiological investigations of intrahippocampal kainate-induced epilepsy were carried out with Wistar rats. It is possible that a strain difference could have accounted for our inability to record clearly focal hypersynchronous ictal onsets. The absence of fast ripples in three of the four epileptic rats, on the other hand, is not surprising in view of the fact that only fixed electrodes were used. Because FR are generated from small, discrete clusters of neurons, they are difficult to find without moveable electrodes (Bragin, et al., 2002). The ripple frequency pHFOs recorded here, however, have the same significance as fast ripples, because ripple frequency oscillations do not occur in normal dentate gyrus (Bragin et al., 2004).
This proof-of-principle study involves only a few animals, and much more research is necessary to demonstrate that AMT-MNP, or MNPs conjugated with other ligands, could eventually be used to image localized cerebral function with MRI in humans. The pharmacokinetics of these particles, their metabolism and elimination, will need to be defined. In particular, the fate of maghemite in the brain needs to be determined, particularly if this technique is to be used in people with epilepsy, given that iron is known to be epileptogenic (Wilmore et al., 1978). Although the particles could still be visualized on MRI 24 h after injection, they were not seen with Perl's iron stain on tissue samples obtained weeks after the injection. This could mean that the particles were completely eliminated by this time, but they could be too small to be detected with routine histological methods. Electron microscopic studies are in progress to answer this question.
Reliable surrogate markers of epileptogenesis (the development of an epileptic abnormality) and epileptogenicity (the presence and severity of an epileptogenic abnormality) would greatly enhance our ability to diagnose, treat, and prevent epilepsy, and this is an area of active research. AMT is only one of a number of putative tracers that could eventually be useful in this regard. Although AMT PET has demonstrated that this compound is concentrated in epileptogenic tissue in a few forms of human epilepsy (Fedi et al., 2001; Duchowny, 2003; Juhasz et al., 2003; Natsume et al., 2003; Juhasz, 2004; Kagawa et al., 2005), its value as a reliable surrogate marker remains controversial, and very few clinical centers have access to this positron tracer. Our results are preliminary; however, if they lead to the ability to perform similar investigations with MRI, it could greatly facilitate this research in centers that may not have PET or the AMT tracer, and greatly reduce the cost of the extensive clinical investigations that would be necessary to fully examine the potential value of AMT or any of the other putative tracers, as reliable surrogate markers for epilepsy. More importantly, the proof of principle with respect to the ability of conjugated MNPs to cross the BBB, and their potential for identifying localized cerebral biochemical processes in a manner similar to that of PET, and to a certain extent SPECT, means that this functional MRI approach could be available not only to study other potential surrogate markers for epilepsy, but theoretically, any bioactive molecules that might be tracers for imaging normal and abnormal localized cerebral functions with high structural resolution MRI.