Magnetic Resonance Imaging of Functional ... - Wiley Online Library

Epilepsia, 48(Suppl. 4):11–17, 2007 Blackwell Publishing, Inc. C International League Against Epilepsy

Magnetic Resonance Imaging of Functional Anatomy: Use for Small Animal Epilepsy Models ∗ Andre Obenaus and †Russell E. Jacobs ∗ Non-Invasive Imaging Laboratory, Radiation Medicine Department, Loma Linda University, Loma Linda, California, U.S.A., and †Biological Imaging Center, Division of Biology, California Institute of Technology, Pasadena, California, U.S.A.

Summary: Neuroimaging has greatly assisted the diagnosis and treatment of epilepsy. Volumetric analysis, diffusion-weighted imaging, and other magnetic resonance imaging (MRI) modalities provide a clear picture of altered anatomical structures in both focal and nonfocal disease. More recently, advances in novel imaging methodologies have provided unique insights into this disease. Two examples include manganese-enhanced MRI (MEMRI) and diffusion tensor imaging (DTI). MEMRI involves injection of MnCl2 to evaluate neuronal activity where it is actively transported. Areas of neuronal hyperactivity are expected to have altered uptake and transport. Mapping of activation along

preferential uptake pathways can be confirmed by T1 -weighted imaging. DTI uses the intrinsic preferential mobility of water movement along axonal pathways to map anatomical regions. DTI has been used to investigate white matter disease and is now being applied to clinical and, to a lesser extent, animal investigations of seizure disorders. These two diverse MRI methods can be applied to animal models to provide important information about the functional status of anatomical regions that may be altered by epilepsy. Key Words: Diffusion tensor— Manganese—Epilepsy—Tract tracing—Rat—MRI.

The application of magnetic resonance imaging (MRI) to assess tissue anatomy and function has lead to its widespread use for clinical diagnosis of disease. Neuroanatomical investigations of disease, such as epilepsy, have benefited greatly from anatomical localization of altered brain structures. Although there are numerous reviews where MRI techniques are used for the diagnosis and treatment monitoring of epilepsy (Duncan, 2002,2003; Briellmann et al., 2005), this review will focus on two novel MRI techniques to assess functional anatomical changes after epilepsy and in particular their application to the study of small animal models.

within the brain that can be performed using a transported agent (MnCl2 ) and (2) diffusion tensor imaging (DTI). Manganese-enhanced MRI Overview Because Mn2+ is paramagnetic and an excellent MRI contrast agent, its tissue location can be visualized as areas of positive contrast enhancement in T1 -weighted MRI images (Geraldes et al., 1986; Fornasiero et al., 1987). Mn2+ has physicochemical similarities to Ca2+ and therefore mimics this important ion in some biological contexts. Recent work using both rodent and avian model systems demonstrate the usefulness of Mn2+ -enhanced MRI (MEMRI) in mapping functional pathways in the brain (Leergaard et al., 2003; Tindemans et al., 2003; Silva et al., 2004; Van der et al., 2004; Watanabe et al., 2004a, 2004b). Work and a review by Watanabe and coworkers (Watanabe et al., 2004a, 2004b) compare several routes on administration of Mn2+ (systemic as well as focal) and discuss many of the confounds concerning interpretation of MEMRI results in terms of neuronal tract tracing. Morita et al. (2002) used systemic application of Mn2+ to investigate activation of hypothalamic nuclei after application of hypertonic NaCl. They found that central NaClactivated areas were clearly identified by in vivo MEMRI and corresponded to those positive for Fos immunoreactivity. Similar positive activity correlations between MEMRI

FUNCTIONAL ANATOMY AND MRI Functional anatomy can be defined as the parcellation of anatomical structures within the brain into distinct parts that are electrically and metabolically active that can either participate in disease propagation or allow “normal” communication between brain structures. There are a number of MRI methods that can be used to assess functional anatomy following seizure activity, but we will review two relatively new and exciting methods: (1) tract-tracing Address correspondence and reprint requests to Andre Obenaus, PhD, Department of Radiation Medicine, Loma Linda University, Loma Linda, CA 92354, U.S.A. E-mail: [email protected] doi: 10.1111/j.1528-1167.2007.01237.x

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A. OBENAUS AND R. E. JACOBS

and BOLD fMRI have been determined (Duong et al., 2000). Recordings of the dynamic response of MEMRI to somatosensory stimulation have been used to produce region specific maps of neuronal activity (Aoki et al., 2002). Small focal injections of manganese ions deep within the mouse central nervous system combined with in vivo high-resolution MRI can delineate neuronal tracts originating from the site of injection (Pautler et al., 2003). The current model of the mechanism underlying Mn2+ tract tracing is that the Mn2+ ions enter neurons through voltage and ligand-gated Ca2+ channels. Upon entry into the activated neuron, Mn2+ is transported along microtubules, most likely in vesicles, and some of it is released at the synaptic terminal. In vivo and in vitro biochemical data support this model (Narita et al., 1990; Lucaciu et al., 1997; Pautler et al., 1998; Takeda et al., 1998; Pautler, 1999). Furthermore, Mn2+ washes out of tissues after several days as evidenced by a return of MRI signal back to basal levels (Pautler et al., 1998). This opens up the possibility of using the same animal repeatedly in developmental and time series studies (Fig. 1). MEMRI has been used to trace olfactory and visual pathways in the rodent (Pautler et al., 1998), the frontal cortical connections in the rhesus macaque (Pautler, 1999; Pautler et al., 1999), and to characterize regional differences in a subset of connections in the olfactory bulbs of Dickie’s Small Eyes (Sey) mice and the primary olfactory cortex in the mutant Reeler mouse (Pautler, 1999; Pautler et al., 1999). Validation of MEMRI with coinjection of traditional tracers has been performed in the macaque (Saleem et al., 2002). Studies using a rat model for cerebral ischemia revealed focal enhancement in a region much smaller than that revealed by apparent diffusion coefficient images (Aoki et al., 2003, 2004). Turnbull and coworkers have employed MEMRI to map regions of accumulated sound-evoked activity in awake, normally behaving mice (Yu et al., 2005).

Toxicity Manganese toxicity in humans is associated with parkinsonism and dystonia (Pal et al., 1999) and has also been well documented in animal models systems (Aschner and Aschner, 1991; Sloot and Gramsbergen, 1994; Silva et al., 2004). This is a major drawback for human studies, but careful dosage and short-term protocols make MEMRI a viable and revealing methodology for animal studies. As a paramagnetic T1 contrast agent, significant local concentration of the ion is needed to induce measurable MRI signal enhancement. Reasonable estimates place the level of agent needed to be on the order of 10 to 100s of micromolar (Ahrens et al., 1998). A final issue may be the need in some studies to utilize mannitol to open the blood–brain barrier to obtain adequate signal (Duong et al., 2000). Epilepsia, Vol. 48, Suppl. 4, 2007

FIG. 1. Mn2+ was stereotaxically injected into the posterior hippocampus (5nl of 400mM MnCl2) of a normal C57B/L6J mouse. High resolution MR images were recorded before and at 24 hours post injection. Locations with positive differences (Postminus Preinjection) indicate the presence of Mn2+. It is transported from the injection site through the fimbria to the septal nucleus. Gray background showing anatomy is the preinjection image.

Requirements and example Van der Linden and Koretsky et al. (Koretsky and Silva, 2004; Van der et al., 2004) emphasize that MEMRI is a new technology for which methodological as well as interpretation issues are still under development. Placement, timing, amount, and route of Mn2+ introduction are important methodological factors. Changes in Mn2+ induced hyperintensity can be influenced by many factors (e.g., differences in neuronal electrical activity, differences in axon density, differences in rates of transport, differences in rates of uptake, etc.). Much current work employs a two-pronged approach: (a) investigate details of how Mn2+ interacts with the neuron (uptake mechanisms, transport mechanisms, efflux mechanisms); and (b) use the MEMRI technology and what is currently known about mechanisms to investigate in vivo workings of the brain in model systems. The actual MR image acquisition is relatively straightforward, typically involving a high-field (4.7 T or greater) small animal scanner, T1 -weighted imaging protocols to highlight areas with significant concentrations of Mn2+ , image acquisition before and at several time points after introduction of Mn2+ , and postprocessing of the image data to quantify where, when, and how much image intensity is modulated by the experimental paradigm. For example, Nairismagi and coworkers have

MRI OF FUNCTIONAL ANATOMY IN EPILEPSY MODELS employed MEMRI to examine mossy fiber tracking in a rat kainic acid (KA) model of epileptogenesis (Nairismagi et al., 2006). Two weeks after KA treatment, Mn2+ was injected into layers II and III of the caudal entorhinal subfield of the entorhinal cortex, and MRI was performed 3 days later using a T1 -weighted 3D MRI protocol on a 4.7T animal scanner. The same animals were subsequently killed, the brains fixed and processed with Nissl staining (to identify anatomy and tissue damage) and Timm histochemistry (to assess mossy fiber sprouting). Mn2+ induced hyperintensity was observed in the dentate gyrus and the CA3 subregion of the hippocampus in both normal and KA treated animals, but was more extensive in KA-treated cohort. Furthermore, differential Mn enhancement correlated with the extent of mossy fiber sprouting. Lack of complete understanding of the details of Mn2+ uptake, trafficking, and accumulation by neurons hampers detailed interpretation of these types of experiment; nevertheless, these studies do demonstrate that MEMRI can be used to examine brain alterations associated with epileptogenesis. Diffusion tensor imaging Overview DTI is an advanced variant of diffusion-weighted imaging (DWI) that can assess tissue microstructure in vivo. DWI, as well as DTI, is based on the Brownian motion of water molecules within the tissues of interest (Le Bihan, 2003). Within the brain, the microstructure of the white matter fibers preferentially limits the diffusion of water molecules in a parallel fashion but tends to restrict perpendicular motion (Fig. 2). This privileged water motion is termed anisotropy. Conversely, water motion is considered isotropic when unrestricted by physical boundaries. Anisotropic water movement can be described by a tensor determined with a minimum of six independent diffusion measurements (Neil et al., 2002; Barboriak, 2003). Once the MR DTI datasets have been collected, the diffusion tensor elements are derived and matrix diagonalization is typically performed to transform the coordinate system describing the water molecule diffusion to a local frame of reference coinciding with anatomical structures. Parameters derived are rotationally invariant enabling cross laboratory comparison of results. The trace (diffusivity or apparent diffusion coefficient, ADC) is a summary parameter that independently reports generalized changes in brain water diffusivity (Fig. 2). Fractional anisotropy (FA) constitutes a measure of the anisotropy present within the diffusion tensor, while relative anisotropy (RA) is a mathematically derived ratio of anisotropic and isotropic components within the diffusion tensor (Fig. 2) (Basser and Jones, 2002). These measures taken together can provide insight into dynamic changes within the brain (both white and gray matter).

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Tractography utilizes the direction of the eigenvector corresponding to the largest eigenvalue to depict the underlying white matter tracts. Although tractography is useful for presurgical evaluation of tumor locations and other diseases (Basser and Pierpaoli, 1996; Arfanakis et al., 2002a; Li and Noseworthy, 2002; Sinha et al., 2002; Field et al., 2004; Sundgren et al., 2004), there are only a few reports for the study of epilepsy.

Is DTI useful in epilepsy? While currently not routinely used in the epilepsy clinic, there are several studies that attempt to correlate changes in DTI with other more conventional diagnostic modalities. DTI comparison with electroencephalograms (EEGs) has been performed. DTI diffusivity (trace) has been shown to correlate primarily with the location of the EEG focus, whereas FA changes also found distant from the focal origin (Rugg-Gunn et al., 2001; Thivard et al., 2006). Interestingly, extratemporal lobe epilepsy had a stronger correlation (DTI vs. EEG) than temporal lobe epilepsy (TLE) (Thivard et al., 2006). In TLE patients, the hippocampal formation exhibited increased diffusivity and decreased FA when ipsi- vs. contralateral comparisons were made (Assaf et al., 2003). However, the FA results were not significantly different when compared with those in the hippocampal formations of control patients. Other investigators have found significant bilateral changes in the FA of TLE patients (Concha et al., 2005; Salmenpera et al., 2006). A recent study highlights the heterogeneity of the DTI findings in TLE (Thivard et al., 2005), in which there was increased diffusivity in the epileptic hippocampus, decreased diffusivity in the contralateral hippocampus and decreased FA in ipsi- and contralateral temporal lobe structures. These abnormalities did not correlate with duration, onset, or frequency of seizures. Similar findings have also been reported by others (Arfanakis et al., 2002b; Diehl et al., 2005). Taken together, it is clear that DTI provides some insight into diagnosis of epilepsy, but it does not yet provide an independent method for diagnosis of this disease. In the past, anatomical MRI has been helpful in defining regions of putative neuropathology. Histopathological correlation with DTI is typically only available after surgical resection. When surgical resection does occur, histological and DTI findings are often supportive in showing microstructural anatomical changes that are consistent with neuronal death, gliosis, and myelin loss (Dumas et al., 2005; Gross et al., 2006), but additional research is required to confirm these findings. One of the key features of MRI is the ability to temporally and spatially track changes within the brain. Currently, there are no reports of using DTI for temporal evaluation of epilepsy progression. Several research groups have noted DTI changes after surgical resection, but these are potentially complicated by tissue Epilepsia, Vol. 48, Suppl. 4, 2007

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FIG. 2. Diffusion tensor imaging concepts and sample results. A: A schematic illustration of white matter (WM) tracts (blue = axons, yellow = myelin sheath) that can be represented as diffusion ellipsoids (blue ellipsoid). The diffusivity in the three principal directions can be described by λ1, λ2 and λ3. B: The axial diffusivity, radial diffusivity, ADC and relative anisotropy (RA) are derived mathematically as shown. C: In vivo DTI scalar index maps of a representative mouse brain after focal ischemia. CA: Focal ischemia did not alter relative anisotropy (RA) values. CB: An apparent decrease of ADC () is seen in the injured cortex (red arrow) and external capsule in vivo. Note the interesting rim of lower ADC values surrounding the central area of injured cortex. CC: Focal ischemia reduces axial diffusivity, λ|| and, CD: similar reductions were also observed in the radial diffusivity, λ⊥ .

voids, hemorrhage, and other issues (Concha et al., 2006; Gross et al., 2006). There have been no reports of using DTI to evaluate the effectiveness of pharmacological intervention in epilepsy. Epilepsia, Vol. 48, Suppl. 4, 2007

However, these data should be forthcoming as DTI enters the clinical mainstream. It is worth noting that many patients are typically on some form of anti-epileptic or seizure medication at the time of DTI measurements which

MRI OF FUNCTIONAL ANATOMY IN EPILEPSY MODELS can be a potentially confounding factor. Thus, while DWI and DTI are important tools for the assessment and progression of disease, DTI has not emerged as a key imaging modality for epilepsy, nor has it yet been able to provide routine clinical diagnosis.

DTI issues The advantage of DTI is its ability to probe the microenvironment to evaluate local changes in water movement. The potentially superior sensitivity of DTI may aid in the visualization of subtle changes within the brain as a consequence of epilepsy that may not be observed using other imaging methodologies (i.e., DWI, T2). Another important clinical advantage is the lack of an injected reporter molecule or contrast agent; DTI utilizes the intrinsic water signal within the brain to evaluate changes after disease. However, significant disadvantages also exist with clinical and research applications of DTI. DTI is demanding technically, as it requires longer imaging times, is very sensitive to movement and postacquisition analysis of datasets are computationally intensive. There are also no standardized method(s) for analysis, which makes comparisons across patients difficult although there is some progress in this area (Hagmann et al., 2006). Finally, interpretation of the DTI results (trace, FA, and RA) are often difficult without correlative histopathology, and thus, the physiological importance of altered DTI parameters after epilepsy remains to be fully explored.

DTI in animal models of epilepsy At the present time there are very few DTI studies using animal models of epilepsy. This is striking given that there are numerous studies using DWI for analysis and visualization of altered anatomical structures, particularly within the hippocampus in a wide variety of epilepsy models (Wall et al., 2000; Bhagat et al., 2001; Eidt et al., 2004). In large part, this lack of study has been due to the long acquisition times required on animal research scanners, postprocessing software, and quantification of the DTI analysis. However, both in vivo and ex vivo studies in other animal models of neurodegenerative disease have shown the utility of DTI for tracking white matter changes within the brain. Axial and radial diffusivity measurements after demyelination injury in mice have been reported to be excellent surrogate markers (Song et al., 2005; Sun et al., 2006). More recently, high resolution ex vivo tractography of the hippocampus has reported alterations in mean diffusivities between dendritic and cell layers within the dentate gyrus (Shepherd et al., 2006). Thus, while there are technical hurdles to overcome, DTI for epilepsy research in animal models is likely to be useful in visualization of altered neuronal pathways within the brain.

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SUMMARY AND CONCLUSIONS MEMRI has real potential for aiding in the understanding of the etiology of epilepsy. It can provide an assessment of relative neuronal activity via preferential uptake in specific locations in the brain after systemic application or by analysis of specific neuronal circuits after stereotaxic injection. For the foreseeable future, this technology will be limited to animal model systems. Nevertheless, the ability to repeatedly image the same specimen with MEMRI before, during, and after the onset of the epileptic condition will provide valuable information to the goal of understanding the mechanisms of this devastating disease. While DWI has been used extensively for studies of animal models of epilepsy, little work in using DTI for investigation of altered neuronal pathways has been performed. In contrast, DWI and DTI are starting to become important tools for the diagnosis and evaluation of epileptic patients, but DTI methods are not robust enough to solely rely on this imaging modality for focal or non-focal seizures/epilepsies localization. Currently, DTI is useful as an adjunct imaging method that can elucidate the underlying microstructural changes within the suspected brain region. Clearly, more clinical and basic science work is required to determine the significance of DTI and its role in delineating the progression of disease. Acknowledgments: The authors acknowledge Dr. ShengKwei Song for a critical review of the manuscript and for providing Fig. 2. The Non-Invasive Imaging Laboratory is supported in part by a NASA Cooperative Agreement NCC9-149 to Loma Linda University. The Biological Imaging Center is supported by the Beckman Institute at Caltech.

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