Thirteen right-handed subjects with r-MTLE-HS (n = 7) or l-MTLE-HS (n = 6) and 13 healthy subjects underwent recording of 3 MGS and 30 MGS and brain MRI for VBM quantitative analysis.

Mesial temporal lobe epilepsy with hippocampal sclerosis patients were recruited among the outpatients consecutively referred to the Epilepsy Centre of the Neurological Institute C. Mondino of Pavia.

We excluded patients unable to participate due to difficulties in understanding the experimental procedures or in maintaining attention for a long time, those older than 60 years, those with a seizure frequency more than 2 per week, and those who modified antiepileptic treatment in the previous month or experienced an epileptic seizure in the previous 36 h. Exclusion criteria were chosen in order to avoid gray mater reduction associated with age (21) and biases in eye movement test performance possibly due to post-ictal dysfunction.

Patients’ demographical and clinical data are shown in Table 1.

Epilepsy diagnosis was supported by clinical, electroencephalography (EEG), and MRI criteria. More in detail, conventional brain MRI showed atrophy and T2 signal increase that were limited to the hippocampal formation in each patient, and did not show parahippocampal atrophy in any of them. EEG traces, ictal symptoms, and signs suggested a mesial temporal lobe seizure onset in each patient (6).

All patients were on treatment with antiepileptic drugs with serum levels in therapeutic range.

Thirteen right-handed healthy subjects (seven women and six men; mean age: 38.4 years, SD: 10.5, range: 27–60 years) age-matched with the patient group (mean age: 43.8 years, SD 11.0, range: 29–60 years) were included in the control group.

The research protocol received approval from the local Ethics Committee and all the procedures were conducted in accordance with the Declaration of Helsinki. All the subjects gave written informed consent before participating in the study.

The eye movements were calibrated and recorded monocularly from the right eye with the scleral search coil technique (22) (SKALAR system S3020: spatial resolution better than 0.1°; sampling rate 250 Hz, bandwidth 0–70 Hz).

The eye movement recording sessions, data acquisition, and analysis were the same as reported in our previous study (20).

The subjects were seated in a dark room with their head in the upright position on a chinrest. For every subject, we recorded the reflexive saccades (RS), the 3 MGS, and the 30 MGS in three separate sessions. In each session, every subject performed 18 trials in both directions (leftward and rightward saccades) for a total of 108 trials each.

In the RS paradigm, a horizontally presented lateral target with an unpredictable direction and amplitude (10°, 15°, or 20°) was lit for 2 s, while the subject was staring at a central point. The subjects were instructed to look at this light immediately after its appearance and until it disappeared. The next trial began at the central fixation point.

In the MGS paradigm, the subjects tried to memorize the location of a horizontally presented lateral target lit for 200 ms, while they were staring at the central point. The target had unpredictable direction and amplitude (10°, 15°, or 20°). After the memorization delay of 3 or 30 s, the central fixation point was switched off, which was the signal for the subject to perform a saccade toward the memorized location. The previously flashed target was shown again after 2 s and the subject had to make a corrective saccade if necessary. The next trial began at the central fixation point.

Memory-guided saccades trials with prosaccades, namely erroneous RS directed at the flashed target, were excluded from analysis. We used a custom-made program developed with LabView software (National Instruments, Austin, TX, USA) to analyze the saccades offline by identifying the beginning and the end of each saccade based on threshold velocity criteria; the difference in the eye position at these two points corresponded to the pulse amplitude. The operator positioned one additional mark that identified the final position, namely the position the eye reached after all the corrective saccades and before the reappearance of the target; the difference between the starting and final positions corresponded to the final amplitude.

We computed the following equations:

the saccade accuracies (SA) as pulseSA = pulse amplitude/target amplitude,finalSA = final amplitude/target amplitude

A logarithmic transformation was needed in order to approximate a normal distribution of the values, and we used the absolute value of the ǀ 1 − SA ǀ differences to express a scatter of the MGS endpoints despite the presence of both hypometric and hypermetric saccades.

For each subject, paradigm, and saccade direction, we computed the mean value of latency, SA, E, and ED.

The patients were compared with the controls as a group by using repeated measure analyses of variance on all the parameters listed before. The RS and the 3- and 30-s MGS were analyzed separately. The analyses considered one intra-individual factor (saccade direction: right or left), one inter-individual factor (group: controls or patients), and their interactions.

The significance value was set at p = 0.05.

We used chi-square test to evaluate the occurrence of patients whose mean value exceeded the normal range, which was calculated on the control group as the mean ± 2.5 SDs.

All the subjects’ MRI were performed with the same machine (Philips Intera Gyroscan 1.5 T), using coronal T1 Gradient Echo sequences, with identical acquisition parameters [echo time (TE) 4.6 ms, repetition time (TR) = 25 ms, thickness = 1.6 mm, slice gap = 0.8 mm, matrix = 256 × 256, voxel = 0.9 mm × 0.9 mm × 0.8 mm, means = 1].

The VBM study was performed through a between-groups comparison by dividing the subjects in three groups on the basis of presence and side of the hippocampal atrophy: controls (Ctrls), r-MTLE-HS (seven subjects), and l-MTLE-HS (six subjects). The areas of reduced gray-matter volume were identified first, then a study on the hippocampal region was performed: the mean volume of the hippocampal gray matter on each side and the difference in volume between the two sides were correlated with the clinical data (age at epilepsy onset, epilepsy duration, seizure frequency, antiepileptic drugs) and the results of MGS recording.

Neuroimage processing was done by statistical parametric map (SPM) (discrete cosine transform cutoff 8 mm) and MATLAB 7.4 programs. Acquired images were normalized on a whole-brain standard template MNI 152, with masking of the hippocampal region (7). Normalized images were segmented through the standard gray and white matter (GM/WM) templates from SPM. Filter value was set at 6-mm smoothing kernel, as suggested for studying structures with dimensions comparable with the hippocampus (7).

Normalized, segmented, and smoothed images were weighted regarding confounding variables (sex, age, and total brain volume). After the described preprocessing and normalization operations, anatomical images underwent a voxel-wise statistical analysis aimed at identifying differences between the three groups.

Statistical parametric maps of the whole brain were created for several comparisons: Ctrls vs. r-MTLE-HS, Ctrls vs. l-MTLE-HS, and l-MTLE-HS vs. r-MTLE-HS. All contrasted images were created using a p < 0.005.

We correlated the hippocampal and parahippocampal gray-matter volumes with the clinical and MGS parameters, and compared hippocampal and parahippocampal volumes of MTLE-HS patients with and without abnormal MGS.

Accuracy parameters’ mean and SE values are shown in Table 2.

The effects of saccade direction and experimental group on all saccadic parameters are shown in Table 3.

The peak velocity values were significantly influenced by saccade direction being larger for rightward than for leftward saccades for all kinds of saccades, with no significant effect of group or group*direction interaction.

The latency values of RS, 3 MGS, and 30 MGS were not significantly influenced by direction, group, or group*direction interaction.

The pulseE values of both RS and 3 MGS were not significantly influenced by saccade direction, group, or group*direction interaction, whereas the pulseE of 30 MGS was significantly influenced by saccade direction being larger for rightward than for leftward saccades, with no significant effect of group or group*direction interaction.

The finalE values showed a different behavior depending on the different kinds of saccades.

For RS it proved to be smaller to the right than to the left, showing a significant direction effect with no significant effect of group and direction*group interaction. The finalE values of 3 MGS were not significantly influenced by saccade direction, group, or group*direction interaction. The finalE values of 30 MGS showed the same behavior detectable for pulseE of the same kind of saccades, since they were significantly influenced by saccade direction being larger to the right than to the left, with no significant effect of group or group*direction interaction.

The ED values of both RS and 3 MGS were not significantly influenced by saccade direction, group, or group*direction interaction, whereas the ED of 30 MGS was significantly influenced by group. This effect was mainly attributed to r-MTLE-HS patients who were unable to reduce the pulseE, and hence to minimize ED, as effectively as controls for leftward saccades (Sheffè post hoc test: p = 0.035), but a similar trend was detectable in r-MTLE-HS patients for rightward saccades also.

Thereby, the results of group analyses can be summarized as follows: –

RS pulseE and ED values were independent from direction, group, and their interaction, while finalE values were smaller for rightward than for leftward saccades both in controls and in patients.

We also considered the patients individually; that is, we checked if accuracy values of their saccades fell within the normal limits defined as the mean ± 2.5 SDs computed in the control group (Figure 1).

The pulseE was invariably normal both in the control and in the patient groups for all kind of saccades.

The finalE was abnormal in a few subjects: two subjects (1 r-MTLE-HS and 1 control) for r-RS, 1 r-MTLE-HS patient for r-3MSG, no subjects for 30 MGS: the chi-square test showed that the distribution of abnormalities did not differ between patients and controls.

The ED was abnormal only in one l-MTLE-HS patient for r-3MGS: again, the chi-square test showed that the distribution of abnormalities did not differ in the patients as compared with controls.

The main finding derives from 30 MGS that showed an abnormal ED in four r-MTLE-HS patients (all of them for leftward saccades and in one of them for rightward saccades also) and in none of the l-MTLE patients or of the controls: the higher occurrence of abnormalities in the r-MTLE-HS group proved to be statistically significant (Fisher’ exact test p = 0.003).

Table 4 displays the results of gray-matter changes.

Right MTLE-HS patients showed right hippocampal atrophy (Figure 2A) as compared with healthy controls, and when the statistical significance threshold was lowered to p < 0.03, contralateral hippocampal atrophy was also detected (Figure 2B). r-MTLE-HS patients showed relative right hippocampal atrophy as compared with l-MTLE-HS patients (Figure 2C).

Regions with significantly reduced gray-matter volume involved several extrahippocampal brain regions in addition to the ipsilateral hippocampus.

Compared with healthy controls, r-MTLE-HS patients had regions with significantly reduced gray-matter volume also in the right-inferior temporal gyrus, and in the ipsi- and contralateral cerebellum. Compared with l-MTLE-HS patients, they showed reduced gray-matter volume of the left central sulcus and the left precentral gyrus.

Left MTLE-HS patients showed relative left hippocampal atrophy as compared with healthy controls and not compared with r-MTLE-HS patients. When the statistical significance threshold was lowered to p < 0.03, no contralateral hippocampal atrophy was detected.

Compared with healthy controls, l-MTLE-HS patients had regions with significantly reduced gray-matter volume also in the left-inferior temporal gyrus and corpus callosus and in the ipsi- and contralateral insula and cerebellum. Compared with r-MTLE-HS patients, they showed reduced gray mater volume of the left-inferior temporal gyrus, insula, and putamen.

Correlation analysis in MTLE-HS subjects showed no significant effects. In particular, no significant correlation was found between ipsilateral H and PH gray-matter volume and 30 MGS ED, number of anti-epileptic drugs, seizure frequency, age, age at epilepsy onset, and disease duration.

The PHC volumes were invariably larger in the right than in the left hemisphere both in controls and in patients (Table 5).

The right and the left PHC volumes were not different in patients with and in those without abnormal 30 MGS ED values (Table 6). By contrast, the patients with abnormal 30 MGS ED values showed a right–left PHC volume difference smaller than those with normal 30 MGS ED, namely abnormal 30 MGS ED values are associated with smaller right PHC volumes as expected on the basis of their left PHC volume. All the four patients with abnormal 30 MGS ED belonged to the r-MTLE-HS group, and three of them showed the smallest volume difference, whereas the other one showed the largest difference and the shortest disease duration.

Finally, concerning the hippocampus, none of the volume parameters, including the right–left difference, proved to be different depending on the abnormality of 30 MGS ED.