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Keywords:

  • Focal cortical dysplasia;
  • Paired-pulse stimulation;
  • Intracortical inhibition;
  • Subdural electrode;
  • Epileptogenesis

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Summary: Purpose: Alternation of the intracortical inhibitory and excitatory mechanisms in focal cortical dysplasia (FCD) has not been well elucidated in vivo in humans. We investigated in vivo alternation of these mechanisms in epileptogenesis of FCD by means of paired-pulse direct cortical electrical stimulation.

Methods: A 31-year-old man with FCD at the left foot primary somatosensory (SI) and motor areas who underwent invasive monitoring with subdural electrodes was studied. By means of subdural electrodes, paired-pulse electrical stimulation was performed at the epileptic focus (foot SI) and control cortex (hand SI) with interstimulus interval (ISI) of 1–100 ms. Instead of using motor evoked potentials to investigate the degree of cortical excitability in response to motor cortex stimulation, we evaluated the size change of corticocortical evoked potentials (CCEPs), which are elicited at the adjacent cortex by direct cortical stimulation via fiber projection and thus reflect direct and indirect excitation of corticocortical projection neurons at the site of stimulation.

Results: During the interictal state, paired-pulse stimulation of the focus revealed abnormally enhanced intracortical inhibition at ISI of 1–10 ms (maximum, 22%) compared with control stimulation of the hand SI (ISI of 1–2 ms; maximum, 18%) (p < 0.01). While the patient was having the somatosensory aura that later evolved into the left-leg clonic seizure, single and paired stimulation at the focus showed increased cortical excitability (enlarged CCEP) and decreased intracortical inhibition, respectively.

Conclusions: During the aura, interictally enhanced intracortical inhibition at the focus was replaced by increased cortical excitability and decreased intracortical inhibition, suggesting increased net intrinsic epileptogenicity during seizure generation in this patient with FCD.

Focal cortical dysplasia (FCD) is a developmental abnormality of cerebral cortical cytoarchitecture histologically characterized by disorganized cortical lamination with an excess of large, aberrant neurons, and it has been increasingly recognized as a major cause for intractable partial epilepsy (1). Although intensive studies using animal models and human FCD tissues pointed to a high degree of intrinsic epileptogenicity due to imbalance between increased excitatory and decreased inhibitory mechanisms (2), little is known about in vivo epileptogenicity, especially transition from the interictal to ictal state.

Only a few techniques allow the investigation of excitatory and inhibitory mechanisms of the cortex in vivo in humans. The paired-pulse stimulation paradigm, either noninvasively by transcranial magnetic stimulation (TMS) or invasively by direct electrical cortical stimulation, can assess intracortical excitatory and inhibitory mechanisms within the primary motor cortex (MI) by analyzing the motor evoked potential (MEP) in the target muscle (3,4). This method, however, could not apply to other cortices where the final common pathway cannot be assessed by MEPs. We recently reported a new method, corticocortical evoked potentials (CCEPs), to track the corticocortical connections in vivo in humans (5,6). By means of subdural electrodes chronically implanted for the presurgical evaluation for epilepsy surgery, a single electrical stimulus is applied on the cortex to look at CCEPs that are produced at an adjacent or distant cortex via fiber projection by direct and indirect excitation of corticocortical projection neurons. We explored the notion that, by analogy to the motor system, the intracortical inhibitory and excitatory mechanisms could be evaluated at the site of cortical stimulation by applying subthreshold conditioning and suprathreshold test electrical pulses to analyze the size change of CCEPs. Although with much longer interstimulus intervals (ISIs), similar paired-pulse stimulation has been infrequently used to study the in vivo epileptogenicity in patients with mesial temporal lobe epilepsy by means of depth electrodes placed in the hippocampus (7). By applying paired-pulse stimulation directly to the focus, we, for the first time, documented in vivo alternation of intracortical inhibitory and excitatory mechanisms in epileptogenesis or ictogenesis of human FCD. Owing to the incidental seizure, we had an opportunity to demonstrate dynamic alternation of the cortical excitability during transition from the interictal to the ictal state. Details of the surgical procedures for focus resection with awake craniotomy will be reported elsewhere.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Case study

The patient was a 31-year-old right-handed man who had had intractable partial epilepsy since age 6 years. The seizures started with somatosensory aura of the left foot, followed by left foot/leg clonic or tonic seizures with occasional secondary generalization. The somatosensory aura could last for several minutes without its evolution to the ictal motor manifestation. Neurologic examination on admission was unremarkable except for mild impairment of the left foot fine movements. Brain magnetic resonance imaging (MRI) revealed blurring of the gray/white-matter junction at the foot portion of the right central sulcus with mildly high-intensity fluid-attenuated inversion recovery (FLAIR) abnormality (Fig. 1a). The patient finally underwent invasive monitoring with subdural electrodes for epilepsy surgery (Fig. 1b). The ictal-onset zone was identified at the left-foot primary somatosensory (SI, electrode A18, 19) and motor (MI, electrode B3) areas. The patient underwent resection of the focus, and the pathology was focal cortical dysplasia [type IIB of Palmini et al. (8)].

image

Figure 1. a: T1-weighted brain magnetic resonance imaging (MRI). Note blurring of the gray/white matter junction at the foot portion of the right central sulcus (arrowhead). Fluid-attenuated inversion recovery image showed mildly high intensity abnormality in the same region. The pathology was focal cortical dysplasia. b: Arrangement of subdural electrodes and results of the invasive presurgical evaluation. The central sulcus was identified by short-latency somatosensory evoked potentials and coregistration with 3D MRI (19) and further confirmed by intraoperative inspection. CS, central sulcus; na, no data of cortical function available because of high impedance of the electrode.

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Single- and paired-pulse stimulation

Informed consent was obtained according to the Clinical Research Protocol No. 443 approved by the Ethics Committee of our institute. All studies were done on a single day with the patient lying awake on the bed after completion of the standard presurgical evaluations. The antiepileptic medication (carbamazepine, phenobarbital, phenytoin, clobazam, and zonisamide) was not changed during the study. Single-pulse stimulation was first performed to determine the threshold intensity to elicit CCEPs (THCCEP) in the cortices adjacent to the stimulation site. The electrical stimulus used for this purpose consisted of a constant-current square-wave pulse of 0.3-ms duration, which was given bipolarly to a pair of adjacent electrodes at 1 Hz in alternating polarity (MEB-2200/MS-201B; Nihon Kohden, Tokyo, Japan). Stimulation of the focus was performed at the foot SI (electrode pair A18-19), and that of the control cortex at the hand SI (A3-4), a functionally homologous area away from the focus. Electrocorticograms (ECoGs) were recorded with a bandpass filter of 0.5–1,500 Hz in reference to a scalp electrode placed on the skin at the left mastoid process (Biotop; NEC-Sanei, Tokyo, Japan). CCEPs were obtained by averaging ECoGs recorded from the surrounding areas, time-locked to the stimulus. Current intensity was gradually increased by 0.5 mA, and THCCEP was determined when two trials of 20 averaged responses first showed reproducible CCEPs. Details of this method have been described elsewhere (5,6).

After determining THCCEP, paired-pulse stimulation was performed at the focus and control cortex. The intensity of the conditioning stimulus was set at (THCCEP× 50%) mA, and that of the test stimulus at (THCCEP+ 0.5∼1) mA. Apart from the intensity difference, the two stimuli were the same in polarity and pulse duration (0.3 ms). Each set of paired stimuli was given at 1 Hz in alternating polarity for safety considerations and to reduce stimulus artifacts. Averaging was made time-locked to the test stimulus. Two sets of paired stimulation were performed with the following ISIs: 1, 2, 4, 8, 10, 20, 40, 80, and 100 ms. These sets were randomly intermixed with single-pulse or unconditioned trials, which were given in six sets to confirm the reproducibility of waveforms against possible polarization of platinum electrodes. Each set of stimulations was separated by a 1- to 3-min interval. CCEPs were obtained by averaging two trials of 16–20 responses within a set after confirming the reproducibility, and then were averaged across the two sets for each ISI. Paired-pulse stimulation was first performed at the control cortex and then at the focus. To evaluate the effect of paired stimulation on the cortical excitability, the change of CCEP amplitude, measured from peak to trough in each individual set, was investigated at the electrode showing the maximal CCEP in comparison with that of single-pulse stimulation. The Mann–Whitney U test was used to compare statistically the cortical excitability between the focus and control stimulations.

As an incidental seizure occurred during paired-pulse stimulation of the focus with the test stimulus at (THCCEP+ 1) mA, the interictal study was performed 4 h later at a weaker but suprathreshold intensity (THCCEP+ 0.5 mA).

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Single-pulse stimulation at the epileptic focus (foot SI) elicited CCEPs from the surrounding areas with its maximum at the foot MI (electrode B3), most likely via projection from the SI to MI (Fig. 2a). Similarly, the maximal CCEP was recorded at the hand MI (electrode A7) in response to control stimulation at the hand SI (Fig. 2b). Paired stimulation at the focus showed the decrease of CCEP amplitude (intracortical inhibition) at ISI of 1–10 ms with the maximum inhibition (22%) at ISI of 4 ms (Figs. 3a and 4). At these ISIs (1–10 ms), stimulation at the control cortex, where no interictal spikes were observed, showed less intracortical inhibition (maximum of 18% at ISI of 2 ms) compared with focus stimulation (p < 0.01) (Fig. 4). Among six sets of single-pulse stimulations, no consistent decline suggestive of electrode polarization was observed in CCEP amplitude (Fig. 2a and b).

image

Figure 2. Corticocortical evoked potentials (CCEPs) elicited by single-pulse stimulation at the focus and control cortex. a: CCEPs in response to the focus (foot SI) stimulation. The electrical pulse was given at 3.0 mA. The vertical bar corresponds to the time of the single-pulse stimulus. Subaverage waveforms are plotted for the first (black) and second (gray) half of the whole six sets to show the reproducibility of the responses. CCEPs were recorded in the adjacent cortices with the maximum response at the foot MI (electrode B3). STIM, a pair of electrodes stimulated in a bipolar fashion. Other conventions are the same as for Fig. 1. b: CCEPs in response to the control (hand SI) stimulation. The electrical pulse was given at 4.5 mA. Conventions are the same as for Fig. 2a. CCEPs were recorded in the adjacent cortices with the maximum response at the hand MI (electrode A7). Note the similarity of waveforms (maximum response at MI) between the focus and control stimulation.

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image

Figure 3. Interictal and ictal corticocortical evoked potentials (CCEPs) elicited by the stimulation of the epileptic focus. a: Comparison of interictally recorded CCEPs between single-pulse (unconditioned) and paired-pulse (conditioned at ISI 4-ms) stimulation. An unconditioned or test stimulus was given at 3.0 mA. Gray and black waveforms denote CCEPs elicited by unconditioned and conditioned stimuli, respectively. Note decrease of CCEP size (intracortical inhibition) with paired-pulse stimulation, being maximum at the foot MI (electrode B3, left bottom). The vertical bar corresponds to the time of the unconditioned or conditioned test stimulus. b: Comparison of CCEPs between interictal and ictal states, elicited by single-pulse (unconditioned) stimulation at 3.5 mA. Gray and black waveforms denote CCEPs recorded during interictal and ictal states, respectively. Enlarged CCEPs (140% at electrode B3, left bottom) were recorded when the patient was having the left foot somatosensory aura, which evolved into the left foot clonic seizure in a minute. ISI, interstimulus interval. Other conventions are the same as for Fig. 2.

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image

Figure 4. Cortical excitability studied by paired-pulse stimulation at the epileptic focus (foot SI) and control (hand SI) cortex. At each interstimulus interval (ISI), the mean size of the conditioned responses is shown as a percentage of that of the unconditioned CCEP. Interictally, focus stimulation showed intracortical inhibition throughout the 1- to 10-ms range of ISIs (maximum of 22% at ISI 4 ms), whereas intracortical inhibition was seen only at ISI of 1–2 ms in the control stimulation (maximum of 18% at ISI 2 ms) (p < 0.01 at ISIs of 1–10 ms; Mann–Whitney U test). When the patient was having the left foot somatosensory aura, intracortical inhibition was not observed, although tested for limited ISIs (4, 8, 100 ms). Solid circle, open circle, and solid triangle denote results of interictal focus stimulation, ictal focus stimulation, and interictal control stimulation, respectively.

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In another series of paired-pulse stimulations at the focus, subjective paresthesia of the left foot occurred spontaneously between stimulus sessions. Because (a) no ictal discharges were observed in the ECoG at and around the focus and (b) this sensation of a variable degree frequently occurred (one to five per day) without evolution to ictal motor phenomena since functional mapping with 50-Hz electrical stimulation, paired-pulse stimulation was continued in the presence of this sensation under the notion that this protocol did not at least provoke the somatosensory aura. The patient, however, had a left-foot/leg clonic seizure 8 min after the sensation started, and it was concluded at that time that the frequent somatosensory sensation was actually the somatosensory aura. This series of stimulations was cancelled with limited investigation. Single-pulse stimulation in the presence of this left-foot somatosensory aura showed an enlarged CCEP at electrode B3, to the magnitude of 140% relative to that elicited with the identical intensity in the absence of the sensation (Fig. 3b). Because of this incidental motor seizure, paired stimulation was partly done, and no intracortical inhibition was observed with stimulation at ISI of 4 and 8 ms, at which interval inhibition was noted interictally (Fig. 4).

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

TMS at the MI has been used to explore cortical excitability in vivo in patients with epilepsy. The attempts, however, had been limited because the cortical excitability could be evaluated only through the motor system by analyzing the EMG of the target muscle. Findings of a cortical silent period and intracortical inhibition/facilitation have been inconsistent among studies, mainly because of different locations of the focus and perhaps of the use of various antiepileptic drugs (AEDs) (9,10). Even in patients with partial epilepsy arising from the MI, judged from the MRI or semiology, the intracortical inhibitory mechanism was enhanced in one study (11) and disrupted in another (12). This is probably due to inaccurate focus localization or stimulation. The present study is unique in that focal cortical stimulation was applied directly to the focus (foot SI) and control cortex (hand SI).

Although the generator mechanism of CCEPs is not exactly known, it is plausible to speculate by analogy to the motor cortex stimulation (3,4) that cortical surface stimulation generates direct and indirect orthodromic discharges in corticocortical projection neurons, thus activating the corticocortical short or long association fibers to convey the impulses to the cortical area where they project (see ref. 5 for detailed discussion). In the present study, single-pulse stimulation of the foot (focus) and hand (control) SI elicited CCEPs in the adjacent cortex with the maximum response at the foot (electrode B3) and hand (A7) MI, respectively, most likely via short association fibers. Although the foot MI (B3) is also a part of the epileptic focus, the similar waveforms observed at electrodes A7 and B3 are indicative of the normal anatomic and functional connectivity within the focus (the foot SI–MI circuit). Indeed, it has been reported that normal cortical function (13) and corticocortical connection (5) can coexist in FCD. Our aim in the present study was to investigate cortical excitability at the focus. Similar to motor cortex stimulation (3,4), we hypothesized that intracortical inhibition of the projection neurons occurs via interneurons at the site of paired-pulse stimulation. Because the degree of inhibition is evaluated by the size change of CCEP, which reflects corticocortical excitability, it may well be termed “corticocortical inhibition” in this sense.

Paired-pulse stimulation revealed increased intracortical inhibition at the focus in the interictal state, compared with the control cortex. At ISI of 4–10 ms, no inhibition or even facilitation was observed in control stimulation, whereas inhibition was seen in focus stimulation. It implies the existence of abnormally enhanced inhibition at the focus. Moreover, we incidentally had an opportunity to study intracortical mechanisms during transition from the interictal to the ictal state. While the patient was having the somatosensory aura, cortical excitability was increased, as judged from the enlarged CCEP in response to single-pulse stimulation. This was accompanied by a decrease of the interictally enhanced intracortical inhibition, albeit studied at limited ISIs. In summary, it seems that net intrinsic epileptogenicity at the focus increased in association with ictogenesis with increased excitatory and decreased inhibitory mechanisms. We, however, cannot rule out the possibility that increased cortical excitability during the aura is an epiphenomenon rather than the mechanism of ictogenesis. Even if this were the case, the observation would be nevertheless valuable because it was shown that the electrophysiologic changes with our method preceded the ictal epileptiform activity during the aura.

Although immunocytochemical studies of human FCD tissue suggest an increase in excitatory amino acid neurotransmission and an overall decrease in the number of γ-aminobutyric acid (GABA)ergic interneurons and receptors (14,15), clusters of increased GABAergic terminals were reported to impinge on glutamatergic neurons (14). Because intracortical inhibition is known to reflect GABAA-mediated circuits (10,16), the “overexpression” of the terminals could be an anatomic substrate accounting for functionally enhanced inhibition or GABAA function, seen interictally in the present study, as a compensatory mechanism to maintain interictal homeostasis (17). Recently an in vitro electrophysiologic study of the human FCD tissue showed that the GABA system also plays a paradoxical role in ictogenesis through GABAA receptor–mediated synchronization when neuronal excitability is enhanced by treatment with 4-aminopyridine (18). Although it is mostly a speculation, once the milieu was switched into the “ictal state” with increased cortical excitability, the interictally observed enhanced GABAA function might have resulted in the precipitation of seizures through GABAA receptor–mediated synchronization.

A few limitations exist in the present study. First, the interictal focus stimulation was performed 4 h after the seizure. Considering frequent auras (one to five per day), we think the 4-h interval was reasonable for the limited schedule of the invasive monitoring. Second, the control study was performed at the ipsilateral hand SI. It would be more desirable to investigate the contralateral foot SI as a control, but was not feasible because of the limited coverage with subdural electrodes. Because the intracortical inhibition could differ between the affected and intact hemispheres in patients with partial epilepsy involving the MI (11), the use of the ipsilateral hand SI might be a confounding factor for the disagreement with TMS studies.

Because the study was made in a single patient in the limited circumstances, accumulation of further cases will help delineate whether the current technique and observation is valid for various kinds of FCD or for partial epilepsy in general. In particular, detailed comparison between the in vivo excitability at the focus electrodes and immunocytochemical findings in the corresponding surgical specimen will be helpful for understanding the epileptogenicity in FCD.

Acknowledgments

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Acknowledgment:  This work was partly supported by the Research Grant from the Japan Epilepsy Research Foundation (RM), Grants-in-Aid for Young Scientists (B) 17790578 (RM) and Scientific Research Grant on Priority Areas 17022023 (HF) from the Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT), and the Research Grants for the Treatment of Intractable Epilepsy (16-1) from the Japan Ministry of Health, Labour, and Welfare (AI).

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES
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