BRIEF COMMUNICATION
Thalamic structural connectivity in medial temporal lobe epilepsy *†Daniel S. Barron, ‡Nitin Tandon, *§Jack L. Lancaster, and *§¶#**Peter T. Fox Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
SUMMARY
Dr. Daniel Barron earned his doctorate in Human Imaging and currently is continuing his medical training.
The thalamus has been implicated in various stages of medial temporal lobe epilepsy (MTLE) seizure evolution. The relative density and functional significance (in epileptogenesis) of thalamic projections to MTL subregions, however, remains to be determined. This study used structural and diffusion magnetic resonance imaging (MRI) to evaluate thalamic connection density with distinct MTL subregions in terms of location and volume. Nineteen MTLE patients with unilateral hippocampal sclerosis (HS; 12 right; 10 female) were compared to 19 age-matched controls. Five regions of interest (ROIs) per hemisphere were created in native space: thalamus, amygdala, entorhinal cortex, hippocampus, and parahippocampus. Separate probabilistic tractography analyses were performed between the thalamus and each ipsilateral MTL subregion (four per hemisphere). Individual connectivity profiles and regional volumes were assessed. The medial pulvinar consistently showed the highest connection density with the hippocampus in healthy controls and in MTLE patients. Decreased thalamic connected volume was observed for thalamohippocampal pathways in patients with MTLE, and indicates pathway-specific deafferentation. Regional hippocampal and thalamic atrophy was also observed, indicating gray and white matter loss in the thalamohippocampal pathway. Consistent localization of dense medial pulvinar (PuM) connectivity with the hippocampus suggests chronic PuM stimulation could modulate the MTLE seizure network. Decreased thalamic connected volume is a promising biomarker for epileptogenesis that merits longitudinal validation. KEY WORDS: Hippocampal sclerosis, Magnetic resonance imaging, Diffusionweighted imaging, Thalamic connectivity.
Medial temporal lobe epilepsy (MTLE) is associated with network pathology in gray and white matter regions connected to the epileptogenic hippocampus.1 The thalamus is the most consistent site of extrahippocampal gray
matter loss in structural magnetic resonance imaging (MRI) studies of MTLE with hippocampal sclerosis (MTLE-HS).2 White matter pathologies detected with diffusion MRI include reduced axonal number,3 membrane circumference,4 and myelin thickness;4 the latter two confirmed with histopathology. Thalamic nuclei have been implicated in various stages of MTL seizure evolution.2 Reciprocal connections couple the medial temporal lobe (MTL) with multiple thalamic nuclei including the anterior nucleus, medial pulvinar, and medial dorsal nucleus.1,5,6 The relative density and functional significance (in epileptogenesis) of individual thalamo-MTL projections, however, remain to be determined. This study used structural and diffusion MRI to evaluate thalamic connection density with distinct MTL subregions in terms of location and volume. In particular, we aimed to do the following: (1) localize thalamic connection density
Accepted March 19, 2014. *Research Imaging Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, U.S.A.; †Yale University School of Medicine, New Haven, Connecticut, U.S.A.; ‡Department of Neurosurgery, University of Texas Health Science Center at Houston, Houston, Texas, U.S.A.; §Department of Radiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, U.S.A.; ¶South Texas Veterans Health Care System, San Antonio, Texas, U.S.A.; #State Key Lab for Brain and Cognitive Sciences, University of Hong Kong, Hong Kong, China; and **Department of Neurology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, U.S.A. Address correspondence to Daniel S. Barron, Yale School of Medicine, Student Affairs, 367 Cedar Street, New Haven, CT 06510, U.S.A. E-mail: [email protected] Wiley Periodicals, Inc. © 2014 International League Against Epilepsy
1
2 D. S. Barron et al. with distinct MTL subregions in healthy controls, (2) evaluate pathologic changes in thalamic connection density and connected volume patients with right and left MTLE-HS, and (3) relate connectivity changes to regional volume loss. We hypothesized that disease-related decreases in each would be observed.
Methods Subjects Nineteen MTLE patients with unilateral hippocampal sclerosis (12 right; 10 female) were compared to 19 agematched controls (9 female). Each MTLE patient was evaluated with ictal and interictal video–electroencephalography (EEG) and had a longstanding, multiple drug–refractory history of epilepsy prior to surgical candidacy. HS was determined by radiologic interpretation and subsequently by postsurgical histopathology. Further clinical information (e.g., age at seizure onset, surgical outcome, and so on) can be found in Table S1. All participants were enrolled using protocols approved by the University of Texas Health Science Center at Houston’s Institutional Review Board. Image acquisition Each subject underwent a single MRI session with a 3.0 T Phillips Intera (Philips Medical Systems, Bothell, WA, U.S.A.) Sequences included a 32-direction diffusionweighted (DW) sequence (high angular resolution, “overplus on” [a Phillips-specific setting wherein the image and gradient reference frame are not the same; this was
corrected for in our analysis], Repetition Time/Echo Time (TR/TE) = 8,500/67 msec; fractional anisotropy (FA) = 90 degrees; matrix size 128 9 128; field of view (FOV) = 224 mm; 2 mm thick axial slices, max. b-value = 1,000 s/mm2) and a T1-weighted sequence (TR/TE = 8.4/3.9 msec; FA = 8 degrees; matrix size = 256 9 256; FOV = 240 mm; slice thickness = 1.0 mm). Seed and target definition Five regions of interest (ROIs) were created per hemisphere: thalamus (seed), amygdala, entorhinal cortex, hippocampus, and parahippocampus (targets; see Fig. 1A). ROIs were created using the FREESURFER (5.1; http://surfer.nmr.mgh.harvard.edu) recon-all (for T1 images) and dt_recon (for DW images) functions. In this procedure, image volumes are resampled from native space to 1 mm isotropic space; segmentations are generated in 1 mm space then mapped back onto native image space. These tools have been validated by reference to manual thalamus7 and hippocampus8 labeling in healthy subjects and patients with epilepsy. Accuracy of ROI placement was visually verified in native patient space before subsequent analyses (also in native space). The volume of each ROI was calculated for subsequent comparison. Probabilistic tractography Connection probability between each thalamic seed voxel and each of the four medial temporal lobe targets was computed for each hemisphere for each participant, using the DW image volumes. For each thalamic voxel, 5,000 trials
A
B
Figure 1. Structural and diffusion MRI image volumes were used to create ROIs for subsequent probabilistic tractography. (A) depicts an individual subject diffusion weighted scan with five regions per hemisphere: thalamus (green), hippocampus (yellow), parahippocampus (red), amygdala (blue), and entorhinal cortex (orange). Probabilistic tractography was performed in native space from thalamic seeds to each ipsilateral MTL target. (B) depicts individual subject center of gravity of maximum thalamic connection density with the hippocampus, located consistently within the medial pulvinar (B, outlined in blue) across all subject groups (center of gravity plotted in MNI152 space; control = blue; L-MTLE = green; R-MTLE = red). Although center of gravity did not vary between groups, individual variations were observed. Epilepsia ILAE Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
3 Thalamus and MTLE were performed and the number of successful ipsilateral seed-to-target connections was recorded, resulting in eight seed-to-target images per patient (four per hemisphere, one per MTL target) that report the probabilistic connectivity of each thalamic voxel to a given MTL target. Connected volumes were computed for each seed-to-target image as the number of thalamic voxels with >20% probabilistic connectivity (or >1,000 successful connections out of 5,000 trials) to a given MTL target. The location of maximal connection density was calculated for each seedto-target image as the center of gravity (in Montreal Neurological Institute (MNI-305) space) of thalamic voxels with >80% probabilistic connectivity (>4,000 connections). All probabilistic tractography transformations and computations were performed using the FMRIB’s (The Oxford Centre for Functional Magnetic Resonance Imaging of the Brain) Diffusion Toolbox (http://fsl.fmrib.ox.ac.uk/fsl/ fslwiki/FDT).9 This pipeline includes eddy current correction and tractography modeling of 5,000 samples per voxel at a fiber threshold of 0.01. Individual patient results were normalized to MNI305 space for group comparisons using FREESURFER. Statistical methods Group differences in probable connectivity, connected volume, and regional subcortical volume were detected with Mann-Whitney10 tests, implemented in MATLAB (2012a; MathWorks, Natick, MA, U.S.A.). A nonparametric test was used to avoid assumptions about the distribution normality within or between groups. The false discovery rate (FDR) correction for multiple comparisons was used.11 Although the small sample size did not permit sufficient power to use clinical variables as covariates, we plotted individual subject’s clinical variables against their respective thalamic connected volume and regional volume for regions that showed significant decrease (p < 0.05, FDR corrected) on the group level (cf. Table S1 and Figs. S1 and S2).
Results Thalamic connection density The medial pulvinar (PuM) consistently showed the highest density of probabilistic connections with the ipsilateral hippocampus. This was observed for both hemispheres in all three groups (control, L-MTLE, and RMTLE). The average location of thalamohippocampal maximum connection density did not vary between groups; however, individual variations in this x, y, and z location were observed (group and individual results in Fig. 1B and Table S2). Thalamic connected volume In L-MTLE, thalamic connected volume reduction (vs. controls) was observed between the left thalamus and ipsi-
lateral hippocampus, and between the right thalamus and ipsilateral hippocampus and entorhinal cortex. In R-MTLE, thalamic connected volume reduction (vs. controls) was observed between the right thalamus and ipsilateral hippocampus and entorhinal cortex, and between the left thalamus and ipsilateral amygdala. Thalamic connected volume between the right thalamus and ipsilateral hippocampus in L-MTLE patients was the only reduction to survive FDR correction for multiple comparisons. See Figure 2 and Table 1 for details. ROI volumetry In L-MTLE, regional volume decrease (vs. controls) was observed in the left hippocampus, left amygdala, and bilateral thalamus. In R-MTLE, volume decrease (vs. controls) was observed in the right hippocampus and bilateral thalamus. All of these regions survived FDR correction for multiple comparisons. See Figure 2 and Table 1 for details. Clinical variables Exploratory plots of individual subjects’ clinical variables against their respective thalamic connected volume and regional volume can be found in Figures S1 and S2. The exploratory plots indicate that there are reasonably strong associations between age at surgery and volumetric measures; these associations require further confirmation in larger sample sizes.
Discussion The medial pulvinar consistently showed the highest density of connections with the ipsilateral hippocampus in healthy controls and in MTLE patients. Decreased thalamic connected volume was observed for thalamohippocampal pathways in MTLE patients and indicates pathway-specific deafferentation. Regional hippocampal and thalamic atrophy indicate gray and white matter loss in the thalamohippocampal pathway. Thalamic connectivity Thalamic nuclei associated with the MTLE seizure network include the anterior nucleus (AN), the medial dorsal nucleus (MDN), and the medial pulvinar (PuM).1,2 Bilateral stimulation of the AN modestly reduced seizure activity in a randomized trial of 110 patients, 66% of whom had temporal lobe epilepsy.12 The MDN is a central contributor to seizure onset and spread.13 The PuM is strongly correlated with ictal activity in patients with temporal lobe epilepsy.14 Atrophy in all three nuclei has been correlated with MTLE disease duration and severity, further supporting their involvement in the epileptogenic network.15 Our report that the PuM consistently showed the highest density of connections with the ipsilateral hippocampus strongly suggests that the PuM is a component of the MTLE epileptogenic network. Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
4 D. S. Barron et al.
Figure 2. Reduced regional volume (above) and thalamic connected volume (below) in MTLE patients compared to controls. L, left; R, right; Amyg, amygdala; Ento, entorhinal cortex; Hipp, hippocampus; Para, parahippocampus; Thal, thalamus; *p < 0.05 with false discovery rate correction for multiple comparisons (Mann-Whitney test of specified MTLE group vs. controls, see also Table S1). Volume is reported as mm3. Epilepsia ILAE
Table 1. Reduced regional volume (above) and reduced thalamic connected volume (below) in MTLE patients compared to controls. Regional volume is based on FREESURFER segmentation. Connected volume is measured as voxels with >20% probable connectivity between ipsilateral thalamic seed to specified MTL target. L-MTLE vs. control
Amyg Entorhinal Hipp Parahipp Thal
Left hemi
Right hemi
2.37 (0.008)* 1.503 (0.066) 3.41 (0)* 2.138 (0.016)* 2.427 (0.007)*
2.052 (0.02)* 1.936 (0.026)* 0.953 (0.17) 0.722 (0.235) 2.861 (0.002)*
R-MTLE vs. control
Amyg Entorhinal Hipp Parahipp Thal
L-MTLE vs. control
Amyg Entorhinal Hipp Parahipp
Left hemi
Right hemi
0.635 (0.262) 1.416 (0.078) 2.081 (0.018) 1.069 (0.142)
0.924 (0.177) 1.676 (0.046) 2.717 (0.003)* 0.462 (0.321)
Left hemi
Right hemi
0.344 (0.365) 0.912 (0.18) 0.953 (0.17) 0.385 (0.35) 2.453 (0.007)*
1.52 (0.064) 1.48 (0.069) 3.305 (0)* 1.135 (0.128) 2.413 (0.007)*
R-MTLE vs. control
Amyg Entorhinal Hipp Parahipp
Left hemi
Right hemi
1.764 (0.038) 1.642 (0.05) 1.338 (0.09) 0.385 (0.35)
0.02 (0.491) 2.109 (0.017) 1.845 (0.032) 1.541 (0.061)
Data shown is Mann-Whitney t-score with uncorrected p-values reported in parentheses; significant comparisons (overall p < 0.05) after FDR correction for multiple corrections are marked with asterisks.
Decreased thalamic connected volume Pathway-specific changes in MLTE have been suggested from histopathologic,4 volumetric,2 and network analyses.3 Decreased thalamic connected volume with the hippocampus in MTLE patients with confirmed hipEpilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
pocampal and thalamic atrophy corroborates and extends these observations. Although other MTL subregions showed reduced thalamic connected volume and ROI volume, consistent decreases in both measurements were not detected. This suggests that epileptogenesis
5 Thalamus and MTLE primarily affects thalamohippocampal connections; however, it is uncertain whether this pathway-specific deafferentation is a cause or effect of epileptogenesis. Notwithstanding, thalamohippocampal connected volume may prove to be a useful biomarker of the epileptogenic process. Therapeutic implications Consistent localization of dense PuM connectivity with the hippocampus suggests chronic PuM stimulation could modulate the MTLE seizure network. This corroborates previous indications for PuM targeting in MTLE network modulation.6,14 Although there was considerable overlap in thalamohippocampal maximum connection density in patients and controls (cf. Results, Fig. 1), variable individual maximum connection density within the PuM suggests modulatory targets based on connection density could be tailored to a patient’s unique connectivity fingerprint.9,16 In addition, decreased thalamic connected volume with the ipsilateral hippocampus is a promising biomarker of epileptogenesis, and merits subsequent longitudinal validation.
Limitations and Future Directions Diffusion MRI has been shown to detect reduced axonal number,3 membrane circumference,4 deafferentation,3 and myelin thickness;4 the latter by reference to human histopathology. Notwithstanding, both diffusion MRI and probabilistic tractography present methodologic limitations concerning image artifact distortions and spatial resolution, tract directionality, and the ability to model multidirectional tract fibers; these have been detailed9,16 and investigated4 elsewhere. A major limitation of the present study is the limited sample size, which we hope to expand in future work and thereby further investigate the relationship of clinical variables to volumetric and thalamic connected volume reductions. The present findings would be most effectively extended by applying higher-direction (>60 directions, as opposed to 32-direction applied here) diffusion MRI to a larger sampling of a comparable population. Targeting patient-specific seizure-onset zones with longitudinal probabilistic tractography could further elucidate the effects of MTLE progression on the thalamohippocampal pathway and determine whether probabilistic tractography could inform modulatory therapies in MTLE-HS. Future work could also investigate the relationship of clinical covariates suggested by the exploratory plots in Supporting Materials. In addition, because the present findings do not include lateralized effects, subsequent analyses in other methods (e.g., resting-state fMRI) could evaluate whether the thalamohippocampal pathways show lateralized effects in patients with MTLE. As suggested earlier, longitudinal validation is required to investigate the utility of
thalamohippocampal connected volume as a biomarker for epileptogenesis.
Conclusion The medial pulvinar consistently showed the highest density of connections with the hippocampus in healthy controls and in MTLE-HS patients. Thalamohippocampal pathways show decreased thalamic connected volume in MTLE patients with regional thalamic and hippocampal atrophy, indicating disease-related neuronal and axonal loss in this pathway. Further investigation of MTLE connectivity with probabilistic tractography is promising.
Acknowledgments The authors thank Jose E. Cavazos (UTHSC-SA, edits), Kristin S. Budde (UTHSC-SA, edits), Vatche G. Baboyan (UTHSC-H, data transfer), and the patients who participated in this study. The authors also thank the anonymous reviewers for their time and helpful directives. This study was funded by 1F31 NS083160-01 (D.S.B.) and RO1 MH074457 (P.T.F.).
Disclosure None of the authors has any conflicts of interest to disclose. The authors confirm that they have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References 1. Spencer SS. Neural networks in human epilepsy: evidence of and implications for treatment. Epilepsia 2002;43:219–227. 2. Barron DS, Fox PM, Laird AR, et al. Thalamic medial dorsal nucleus atrophy in medial temporal lobe epilepsy: a VBM meta-analysis. Neuroimage Clin 2013;2:25–32. 3. Bonilha L, Nesland T, Martz GU, et al. Medial temporal lobe epilepsy is associated with neuronal fibre loss and paradoxical increase in structural connectivity of limbic structures. J Neurol Neurosurg Psychiatry 2012;83:903–909. 4. Concha L, Livy DJ, Beaulieu C, et al. In vivo diffusion tensor imaging and histopathology of the fimbria-fornix in temporal lobe epilepsy. J Neurosci 2010;30:996–1002. 5. Guye M. The role of corticothalamic coupling in human temporal lobe epilepsy. Brain 2006;129:1917–1928. 6. Rosenberg DS, Mauguiere F, Catenoix H, et al. Reciprocal thalamocortical connectivity of the medial pulvinar: a depth stimulation and evoked potential study in human brain. Cereb Cortex 2009;19:1462–1473. 7. Keller SS, Gerdes JS, Mohammadi S, et al. Volume estimation of the thalamus using freesurfer and stereology: consistency between methods. Neuroinformatics 2012;10:341–350. 8. Pardoe HR, Pell GS, Abbott DF, et al. Hippocampal volume assessment in temporal lobe epilepsy: how good is automated segmentation? Epilepsia 2009;50:2586–2592. 9. Johansen-Berg H, Gutman DA, Behrens TEJ, et al. Anatomical connectivity of the subgenual cingulate region targeted with deep brain stimulation for treatment-resistant depression. Cereb Cortex 2008;18:1374–1383. 10. Glantz SA. Primer of biostatistics. 7th Ed. McGraw Hill Medical: New York City, NY, USA, 2012. 11. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B 1995;57(1):289–300. Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
6 D. S. Barron et al. 12. Fisher R, Salanova V, Witt T, et al. Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy. Epilepsia 2010;51:899–908. 13. Bertram EH, Zhang D, Williamson JM. Multiple roles of midline dorsal thalamic nuclei in induction and spread of limbic seizures. Epilepsia 2008;49:256–268. 14. Rosenberg DS, Mauguiere F, Demarquay G, et al. Involvement of medial pulvinar thalamic nucleus in human temporal lobe seizures. Epilepsia 2006;47:98–107. 15. Bernhardt BC, Bernasconi N, Kim H, et al. Mapping thalamocortical network pathology in temporal lobe epilepsy. Neurology 2012;78:129– 136. 16. Pouratian N, Zheng Z, Bari AA, et al. Multi-institutional evaluation of deep brain stimulation targeting using probabilistic connectivity-based thalamic segmentation. J Neurosurg 2011;115:995–1004.
Supporting Information Additional Supporting Information may be found in the online version of this article:
Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
Figure S1. Exploratory graphs of regions with significant volume reduction with clinical information in L-MTLE (green), R-MTLE (red) and healthy control (blue) subjects. Figure S2. Exploratory graphs of regions with significant thalamic connected volume reduction with clinical information in L-MTLE (green), R-MTLE (red) and healthy control (blue) subjects. Table S1. Individual patient clinical characteristics. Table S2. Individual patient variability of maximum thalamo-hippocampal connection density.
Thalamic structural connectivity in medial temporal lobe epilepsy *†Daniel S. Barron, ‡Nitin Tandon, *§Jack L. Lancaster, and *§¶#**Peter T. Fox Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
SUMMARY
Dr. Daniel Barron earned his doctorate in Human Imaging and currently is continuing his medical training.
The thalamus has been implicated in various stages of medial temporal lobe epilepsy (MTLE) seizure evolution. The relative density and functional significance (in epileptogenesis) of thalamic projections to MTL subregions, however, remains to be determined. This study used structural and diffusion magnetic resonance imaging (MRI) to evaluate thalamic connection density with distinct MTL subregions in terms of location and volume. Nineteen MTLE patients with unilateral hippocampal sclerosis (HS; 12 right; 10 female) were compared to 19 age-matched controls. Five regions of interest (ROIs) per hemisphere were created in native space: thalamus, amygdala, entorhinal cortex, hippocampus, and parahippocampus. Separate probabilistic tractography analyses were performed between the thalamus and each ipsilateral MTL subregion (four per hemisphere). Individual connectivity profiles and regional volumes were assessed. The medial pulvinar consistently showed the highest connection density with the hippocampus in healthy controls and in MTLE patients. Decreased thalamic connected volume was observed for thalamohippocampal pathways in patients with MTLE, and indicates pathway-specific deafferentation. Regional hippocampal and thalamic atrophy was also observed, indicating gray and white matter loss in the thalamohippocampal pathway. Consistent localization of dense medial pulvinar (PuM) connectivity with the hippocampus suggests chronic PuM stimulation could modulate the MTLE seizure network. Decreased thalamic connected volume is a promising biomarker for epileptogenesis that merits longitudinal validation. KEY WORDS: Hippocampal sclerosis, Magnetic resonance imaging, Diffusionweighted imaging, Thalamic connectivity.
Medial temporal lobe epilepsy (MTLE) is associated with network pathology in gray and white matter regions connected to the epileptogenic hippocampus.1 The thalamus is the most consistent site of extrahippocampal gray
matter loss in structural magnetic resonance imaging (MRI) studies of MTLE with hippocampal sclerosis (MTLE-HS).2 White matter pathologies detected with diffusion MRI include reduced axonal number,3 membrane circumference,4 and myelin thickness;4 the latter two confirmed with histopathology. Thalamic nuclei have been implicated in various stages of MTL seizure evolution.2 Reciprocal connections couple the medial temporal lobe (MTL) with multiple thalamic nuclei including the anterior nucleus, medial pulvinar, and medial dorsal nucleus.1,5,6 The relative density and functional significance (in epileptogenesis) of individual thalamo-MTL projections, however, remain to be determined. This study used structural and diffusion MRI to evaluate thalamic connection density with distinct MTL subregions in terms of location and volume. In particular, we aimed to do the following: (1) localize thalamic connection density
Accepted March 19, 2014. *Research Imaging Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, U.S.A.; †Yale University School of Medicine, New Haven, Connecticut, U.S.A.; ‡Department of Neurosurgery, University of Texas Health Science Center at Houston, Houston, Texas, U.S.A.; §Department of Radiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, U.S.A.; ¶South Texas Veterans Health Care System, San Antonio, Texas, U.S.A.; #State Key Lab for Brain and Cognitive Sciences, University of Hong Kong, Hong Kong, China; and **Department of Neurology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, U.S.A. Address correspondence to Daniel S. Barron, Yale School of Medicine, Student Affairs, 367 Cedar Street, New Haven, CT 06510, U.S.A. E-mail: [email protected] Wiley Periodicals, Inc. © 2014 International League Against Epilepsy
1
2 D. S. Barron et al. with distinct MTL subregions in healthy controls, (2) evaluate pathologic changes in thalamic connection density and connected volume patients with right and left MTLE-HS, and (3) relate connectivity changes to regional volume loss. We hypothesized that disease-related decreases in each would be observed.
Methods Subjects Nineteen MTLE patients with unilateral hippocampal sclerosis (12 right; 10 female) were compared to 19 agematched controls (9 female). Each MTLE patient was evaluated with ictal and interictal video–electroencephalography (EEG) and had a longstanding, multiple drug–refractory history of epilepsy prior to surgical candidacy. HS was determined by radiologic interpretation and subsequently by postsurgical histopathology. Further clinical information (e.g., age at seizure onset, surgical outcome, and so on) can be found in Table S1. All participants were enrolled using protocols approved by the University of Texas Health Science Center at Houston’s Institutional Review Board. Image acquisition Each subject underwent a single MRI session with a 3.0 T Phillips Intera (Philips Medical Systems, Bothell, WA, U.S.A.) Sequences included a 32-direction diffusionweighted (DW) sequence (high angular resolution, “overplus on” [a Phillips-specific setting wherein the image and gradient reference frame are not the same; this was
corrected for in our analysis], Repetition Time/Echo Time (TR/TE) = 8,500/67 msec; fractional anisotropy (FA) = 90 degrees; matrix size 128 9 128; field of view (FOV) = 224 mm; 2 mm thick axial slices, max. b-value = 1,000 s/mm2) and a T1-weighted sequence (TR/TE = 8.4/3.9 msec; FA = 8 degrees; matrix size = 256 9 256; FOV = 240 mm; slice thickness = 1.0 mm). Seed and target definition Five regions of interest (ROIs) were created per hemisphere: thalamus (seed), amygdala, entorhinal cortex, hippocampus, and parahippocampus (targets; see Fig. 1A). ROIs were created using the FREESURFER (5.1; http://surfer.nmr.mgh.harvard.edu) recon-all (for T1 images) and dt_recon (for DW images) functions. In this procedure, image volumes are resampled from native space to 1 mm isotropic space; segmentations are generated in 1 mm space then mapped back onto native image space. These tools have been validated by reference to manual thalamus7 and hippocampus8 labeling in healthy subjects and patients with epilepsy. Accuracy of ROI placement was visually verified in native patient space before subsequent analyses (also in native space). The volume of each ROI was calculated for subsequent comparison. Probabilistic tractography Connection probability between each thalamic seed voxel and each of the four medial temporal lobe targets was computed for each hemisphere for each participant, using the DW image volumes. For each thalamic voxel, 5,000 trials
A
B
Figure 1. Structural and diffusion MRI image volumes were used to create ROIs for subsequent probabilistic tractography. (A) depicts an individual subject diffusion weighted scan with five regions per hemisphere: thalamus (green), hippocampus (yellow), parahippocampus (red), amygdala (blue), and entorhinal cortex (orange). Probabilistic tractography was performed in native space from thalamic seeds to each ipsilateral MTL target. (B) depicts individual subject center of gravity of maximum thalamic connection density with the hippocampus, located consistently within the medial pulvinar (B, outlined in blue) across all subject groups (center of gravity plotted in MNI152 space; control = blue; L-MTLE = green; R-MTLE = red). Although center of gravity did not vary between groups, individual variations were observed. Epilepsia ILAE Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
3 Thalamus and MTLE were performed and the number of successful ipsilateral seed-to-target connections was recorded, resulting in eight seed-to-target images per patient (four per hemisphere, one per MTL target) that report the probabilistic connectivity of each thalamic voxel to a given MTL target. Connected volumes were computed for each seed-to-target image as the number of thalamic voxels with >20% probabilistic connectivity (or >1,000 successful connections out of 5,000 trials) to a given MTL target. The location of maximal connection density was calculated for each seedto-target image as the center of gravity (in Montreal Neurological Institute (MNI-305) space) of thalamic voxels with >80% probabilistic connectivity (>4,000 connections). All probabilistic tractography transformations and computations were performed using the FMRIB’s (The Oxford Centre for Functional Magnetic Resonance Imaging of the Brain) Diffusion Toolbox (http://fsl.fmrib.ox.ac.uk/fsl/ fslwiki/FDT).9 This pipeline includes eddy current correction and tractography modeling of 5,000 samples per voxel at a fiber threshold of 0.01. Individual patient results were normalized to MNI305 space for group comparisons using FREESURFER. Statistical methods Group differences in probable connectivity, connected volume, and regional subcortical volume were detected with Mann-Whitney10 tests, implemented in MATLAB (2012a; MathWorks, Natick, MA, U.S.A.). A nonparametric test was used to avoid assumptions about the distribution normality within or between groups. The false discovery rate (FDR) correction for multiple comparisons was used.11 Although the small sample size did not permit sufficient power to use clinical variables as covariates, we plotted individual subject’s clinical variables against their respective thalamic connected volume and regional volume for regions that showed significant decrease (p < 0.05, FDR corrected) on the group level (cf. Table S1 and Figs. S1 and S2).
Results Thalamic connection density The medial pulvinar (PuM) consistently showed the highest density of probabilistic connections with the ipsilateral hippocampus. This was observed for both hemispheres in all three groups (control, L-MTLE, and RMTLE). The average location of thalamohippocampal maximum connection density did not vary between groups; however, individual variations in this x, y, and z location were observed (group and individual results in Fig. 1B and Table S2). Thalamic connected volume In L-MTLE, thalamic connected volume reduction (vs. controls) was observed between the left thalamus and ipsi-
lateral hippocampus, and between the right thalamus and ipsilateral hippocampus and entorhinal cortex. In R-MTLE, thalamic connected volume reduction (vs. controls) was observed between the right thalamus and ipsilateral hippocampus and entorhinal cortex, and between the left thalamus and ipsilateral amygdala. Thalamic connected volume between the right thalamus and ipsilateral hippocampus in L-MTLE patients was the only reduction to survive FDR correction for multiple comparisons. See Figure 2 and Table 1 for details. ROI volumetry In L-MTLE, regional volume decrease (vs. controls) was observed in the left hippocampus, left amygdala, and bilateral thalamus. In R-MTLE, volume decrease (vs. controls) was observed in the right hippocampus and bilateral thalamus. All of these regions survived FDR correction for multiple comparisons. See Figure 2 and Table 1 for details. Clinical variables Exploratory plots of individual subjects’ clinical variables against their respective thalamic connected volume and regional volume can be found in Figures S1 and S2. The exploratory plots indicate that there are reasonably strong associations between age at surgery and volumetric measures; these associations require further confirmation in larger sample sizes.
Discussion The medial pulvinar consistently showed the highest density of connections with the ipsilateral hippocampus in healthy controls and in MTLE patients. Decreased thalamic connected volume was observed for thalamohippocampal pathways in MTLE patients and indicates pathway-specific deafferentation. Regional hippocampal and thalamic atrophy indicate gray and white matter loss in the thalamohippocampal pathway. Thalamic connectivity Thalamic nuclei associated with the MTLE seizure network include the anterior nucleus (AN), the medial dorsal nucleus (MDN), and the medial pulvinar (PuM).1,2 Bilateral stimulation of the AN modestly reduced seizure activity in a randomized trial of 110 patients, 66% of whom had temporal lobe epilepsy.12 The MDN is a central contributor to seizure onset and spread.13 The PuM is strongly correlated with ictal activity in patients with temporal lobe epilepsy.14 Atrophy in all three nuclei has been correlated with MTLE disease duration and severity, further supporting their involvement in the epileptogenic network.15 Our report that the PuM consistently showed the highest density of connections with the ipsilateral hippocampus strongly suggests that the PuM is a component of the MTLE epileptogenic network. Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
4 D. S. Barron et al.
Figure 2. Reduced regional volume (above) and thalamic connected volume (below) in MTLE patients compared to controls. L, left; R, right; Amyg, amygdala; Ento, entorhinal cortex; Hipp, hippocampus; Para, parahippocampus; Thal, thalamus; *p < 0.05 with false discovery rate correction for multiple comparisons (Mann-Whitney test of specified MTLE group vs. controls, see also Table S1). Volume is reported as mm3. Epilepsia ILAE
Table 1. Reduced regional volume (above) and reduced thalamic connected volume (below) in MTLE patients compared to controls. Regional volume is based on FREESURFER segmentation. Connected volume is measured as voxels with >20% probable connectivity between ipsilateral thalamic seed to specified MTL target. L-MTLE vs. control
Amyg Entorhinal Hipp Parahipp Thal
Left hemi
Right hemi
2.37 (0.008)* 1.503 (0.066) 3.41 (0)* 2.138 (0.016)* 2.427 (0.007)*
2.052 (0.02)* 1.936 (0.026)* 0.953 (0.17) 0.722 (0.235) 2.861 (0.002)*
R-MTLE vs. control
Amyg Entorhinal Hipp Parahipp Thal
L-MTLE vs. control
Amyg Entorhinal Hipp Parahipp
Left hemi
Right hemi
0.635 (0.262) 1.416 (0.078) 2.081 (0.018) 1.069 (0.142)
0.924 (0.177) 1.676 (0.046) 2.717 (0.003)* 0.462 (0.321)
Left hemi
Right hemi
0.344 (0.365) 0.912 (0.18) 0.953 (0.17) 0.385 (0.35) 2.453 (0.007)*
1.52 (0.064) 1.48 (0.069) 3.305 (0)* 1.135 (0.128) 2.413 (0.007)*
R-MTLE vs. control
Amyg Entorhinal Hipp Parahipp
Left hemi
Right hemi
1.764 (0.038) 1.642 (0.05) 1.338 (0.09) 0.385 (0.35)
0.02 (0.491) 2.109 (0.017) 1.845 (0.032) 1.541 (0.061)
Data shown is Mann-Whitney t-score with uncorrected p-values reported in parentheses; significant comparisons (overall p < 0.05) after FDR correction for multiple corrections are marked with asterisks.
Decreased thalamic connected volume Pathway-specific changes in MLTE have been suggested from histopathologic,4 volumetric,2 and network analyses.3 Decreased thalamic connected volume with the hippocampus in MTLE patients with confirmed hipEpilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
pocampal and thalamic atrophy corroborates and extends these observations. Although other MTL subregions showed reduced thalamic connected volume and ROI volume, consistent decreases in both measurements were not detected. This suggests that epileptogenesis
5 Thalamus and MTLE primarily affects thalamohippocampal connections; however, it is uncertain whether this pathway-specific deafferentation is a cause or effect of epileptogenesis. Notwithstanding, thalamohippocampal connected volume may prove to be a useful biomarker of the epileptogenic process. Therapeutic implications Consistent localization of dense PuM connectivity with the hippocampus suggests chronic PuM stimulation could modulate the MTLE seizure network. This corroborates previous indications for PuM targeting in MTLE network modulation.6,14 Although there was considerable overlap in thalamohippocampal maximum connection density in patients and controls (cf. Results, Fig. 1), variable individual maximum connection density within the PuM suggests modulatory targets based on connection density could be tailored to a patient’s unique connectivity fingerprint.9,16 In addition, decreased thalamic connected volume with the ipsilateral hippocampus is a promising biomarker of epileptogenesis, and merits subsequent longitudinal validation.
Limitations and Future Directions Diffusion MRI has been shown to detect reduced axonal number,3 membrane circumference,4 deafferentation,3 and myelin thickness;4 the latter by reference to human histopathology. Notwithstanding, both diffusion MRI and probabilistic tractography present methodologic limitations concerning image artifact distortions and spatial resolution, tract directionality, and the ability to model multidirectional tract fibers; these have been detailed9,16 and investigated4 elsewhere. A major limitation of the present study is the limited sample size, which we hope to expand in future work and thereby further investigate the relationship of clinical variables to volumetric and thalamic connected volume reductions. The present findings would be most effectively extended by applying higher-direction (>60 directions, as opposed to 32-direction applied here) diffusion MRI to a larger sampling of a comparable population. Targeting patient-specific seizure-onset zones with longitudinal probabilistic tractography could further elucidate the effects of MTLE progression on the thalamohippocampal pathway and determine whether probabilistic tractography could inform modulatory therapies in MTLE-HS. Future work could also investigate the relationship of clinical covariates suggested by the exploratory plots in Supporting Materials. In addition, because the present findings do not include lateralized effects, subsequent analyses in other methods (e.g., resting-state fMRI) could evaluate whether the thalamohippocampal pathways show lateralized effects in patients with MTLE. As suggested earlier, longitudinal validation is required to investigate the utility of
thalamohippocampal connected volume as a biomarker for epileptogenesis.
Conclusion The medial pulvinar consistently showed the highest density of connections with the hippocampus in healthy controls and in MTLE-HS patients. Thalamohippocampal pathways show decreased thalamic connected volume in MTLE patients with regional thalamic and hippocampal atrophy, indicating disease-related neuronal and axonal loss in this pathway. Further investigation of MTLE connectivity with probabilistic tractography is promising.
Acknowledgments The authors thank Jose E. Cavazos (UTHSC-SA, edits), Kristin S. Budde (UTHSC-SA, edits), Vatche G. Baboyan (UTHSC-H, data transfer), and the patients who participated in this study. The authors also thank the anonymous reviewers for their time and helpful directives. This study was funded by 1F31 NS083160-01 (D.S.B.) and RO1 MH074457 (P.T.F.).
Disclosure None of the authors has any conflicts of interest to disclose. The authors confirm that they have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References 1. Spencer SS. Neural networks in human epilepsy: evidence of and implications for treatment. Epilepsia 2002;43:219–227. 2. Barron DS, Fox PM, Laird AR, et al. Thalamic medial dorsal nucleus atrophy in medial temporal lobe epilepsy: a VBM meta-analysis. Neuroimage Clin 2013;2:25–32. 3. Bonilha L, Nesland T, Martz GU, et al. Medial temporal lobe epilepsy is associated with neuronal fibre loss and paradoxical increase in structural connectivity of limbic structures. J Neurol Neurosurg Psychiatry 2012;83:903–909. 4. Concha L, Livy DJ, Beaulieu C, et al. In vivo diffusion tensor imaging and histopathology of the fimbria-fornix in temporal lobe epilepsy. J Neurosci 2010;30:996–1002. 5. Guye M. The role of corticothalamic coupling in human temporal lobe epilepsy. Brain 2006;129:1917–1928. 6. Rosenberg DS, Mauguiere F, Catenoix H, et al. Reciprocal thalamocortical connectivity of the medial pulvinar: a depth stimulation and evoked potential study in human brain. Cereb Cortex 2009;19:1462–1473. 7. Keller SS, Gerdes JS, Mohammadi S, et al. Volume estimation of the thalamus using freesurfer and stereology: consistency between methods. Neuroinformatics 2012;10:341–350. 8. Pardoe HR, Pell GS, Abbott DF, et al. Hippocampal volume assessment in temporal lobe epilepsy: how good is automated segmentation? Epilepsia 2009;50:2586–2592. 9. Johansen-Berg H, Gutman DA, Behrens TEJ, et al. Anatomical connectivity of the subgenual cingulate region targeted with deep brain stimulation for treatment-resistant depression. Cereb Cortex 2008;18:1374–1383. 10. Glantz SA. Primer of biostatistics. 7th Ed. McGraw Hill Medical: New York City, NY, USA, 2012. 11. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B 1995;57(1):289–300. Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
6 D. S. Barron et al. 12. Fisher R, Salanova V, Witt T, et al. Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy. Epilepsia 2010;51:899–908. 13. Bertram EH, Zhang D, Williamson JM. Multiple roles of midline dorsal thalamic nuclei in induction and spread of limbic seizures. Epilepsia 2008;49:256–268. 14. Rosenberg DS, Mauguiere F, Demarquay G, et al. Involvement of medial pulvinar thalamic nucleus in human temporal lobe seizures. Epilepsia 2006;47:98–107. 15. Bernhardt BC, Bernasconi N, Kim H, et al. Mapping thalamocortical network pathology in temporal lobe epilepsy. Neurology 2012;78:129– 136. 16. Pouratian N, Zheng Z, Bari AA, et al. Multi-institutional evaluation of deep brain stimulation targeting using probabilistic connectivity-based thalamic segmentation. J Neurosurg 2011;115:995–1004.
Supporting Information Additional Supporting Information may be found in the online version of this article:
Epilepsia, **(*):1–6, 2014 doi: 10.1111/epi.12637
Figure S1. Exploratory graphs of regions with significant volume reduction with clinical information in L-MTLE (green), R-MTLE (red) and healthy control (blue) subjects. Figure S2. Exploratory graphs of regions with significant thalamic connected volume reduction with clinical information in L-MTLE (green), R-MTLE (red) and healthy control (blue) subjects. Table S1. Individual patient clinical characteristics. Table S2. Individual patient variability of maximum thalamo-hippocampal connection density.
