Metab Brain Dis DOI 10.1007/s11011-015-9680-2

RESEARCH ARTICLE

Cytochrome c oxidase deficit is associated with the seizure onset zone in young patients with focal cortical dysplasia Type II Lili Miles 1 & Hansel M. Greiner 2 & Francesco T. Mangano 3 & Paul S. Horn 4 & James L. Leach 5 & Michael V. Miles 2

Received: 15 January 2015 / Accepted: 4 May 2015 # Springer Science+Business Media New York 2015

Abstract It has been postulated that mitochondrial dysfunction may be an important factor in epileptogenesis of intractable epilepsy. The current study tests the hypothesis that mitochondrial Complex IV (CIV) or cytochrome c oxidase dysfunction is associated with the seizure onset zone (SOZ) in patients with focal cortical dysplasia (FCD). Subjects were selected based on: age 0.20). All analyses were conducted using SAS® version 9.3 (SAS Institute, Inc., Cary, NC).

Results A total of 17 subjects met the inclusion criteria, including six with FCDI, nine with FCDIIa, and two with FCDIIb (ages ranging from 0.5y to 18y). SOZ and SSZ specimens were identifiable for all participants. Factors used to establish the SOZ vs. SSZ specimens are detailed in Table 1. Characteristics of the FCD study groups are described in Table 2. Minor clinical differences were noted between FCDI and FCDII groups (Table 2). Neuroimaging differences, related to MRIpositive lesional characteristics of FCD, were significantly more common in the FCDIIa/b group (Table 2). MRIpositive features in FCDII are frequent and well described (Leach et al. 2014; Lerner et al. 2009; Sisodiya et al. 2009). A representative example of electrographic results identifying SOZ and SSZ regions in an 8 year old female are shown in Fig. 1. Intraoperative and postoperative photographs illustrating the pre- and post-resection of the SOZ and the adjacent SSZ region for the same patient are provided in Fig. 2a-d. Histopathology features of the resected SSZ and SOZ regions for this patient are provided in Fig. 2e-f. Although the FCDI group failed the Shapiro-Wilk test for Normality (P-value0.20). Thus, paired data for FCDII were evaluated with the Student’s t test. Paired comparisons of SOZ vs. SSZ differences for SDH and CIII (normalized to both NCP and CS), and CS (normalized to NCP) were not statistically significant for each FCD study group (Wilcoxon Signed-Rank and Student’s t tests agreed in that all P-values were>0.25). Next, SOZ vs. SSZ differences were compared for CIVprot, CIVSDH, CIVCIII, and CIVCS for all participants (N=17), but failed to reach a statistically significant level. However, comparison of CIV in SOZ vs. SSZ tissues based on individual FCD subtype showed that CIVprot was significantly decreased in SOZ of the FCDII group using both Student’s t (P=0.003) and Wilcoxon Signed-Rank (P=0.007) tests (Fig. 3b). Similar trends in the FCDII group were seen for CIVSDH (Fig. 3d), CIVCIII (Fig. 3f), and CIVCS (Fig. 3h), as Student’s t test Pvalues wereFCDI

Table 2 Comparison of clinical factors associated with focal cortical dysplasia Type I (FCDI, N=6) and Type IIa/b (FCDII, N= 11) study groups. Mean±SD unless specified otherwise

Factors

FCDI

FCDII

P-value

Gender (F) Age at surgery (y) Duration of epilepsy (y) Age at epilepsy onset (y) Seizure frequency: Daily Weekly or sporadic Number AEDs at surgery: 1 >1 Lesional FCD (MRI) FDG-PET: Hypometabolism Scan not completed

1 (17 %) 8.3±6.1 3.4±2.2 4.9±4.2

5 (45 %) 10.6±4.3 6.8±3.8 3.8±4.2

0.33 0.32 0.06 0.37

3 (50 %) 3 (50 %)

6 (55 %) 5 (45 %)

1.0

1 (17 %) 5 (83 %) 1 (17 %)

3 (27 %) 8 (73 %) 8 (73 %)

1.0

2 (40 %) 1 (17 %)

6 (60 %) 1 (9 %)

0.61

4 (67 %) 2 (33 %)

5 (45 %) 6 (55 %)

0.62

Extent of surgery: Single lobe Multilobar or hemispherotomy

0.0498

Metab Brain Dis Fig. 1 An example of the method for electrographic determination of the seizure onset zone (SOZ) and secondary spread zone (SSZ) regions in an 8 year old female with FCD Type IIa in the SOZ and FCD Type I in the SSZ. Top: Time frequency analysis at initial electrographic onset, 150–300 Hz bandwidth. Bottom: Electrocorticography, high-pass filter 1 Hz with no low-pass filter. Select electrodes shown in a referential montage. An initial change in power is noted in RSF at 150–200 Hz (channel selected blue), corresponding spectral power change shown with red arrow pointing to RSF 24. As seizure continues, there is spread of rhythmic activity to electrodes RSF 7–8 and 15–16 (green arrows)

reports (Hauptman and Mathern 2012; Iyer et al. 2010; Krsek et al. 2009; Leach et al. 2014; Lerner et al. 2009; Miles et al. 2013; Sisodiya et al. 2009). FCDI is generally considered to be associated with structural features such as cortical dyslamination and normal neuronal morphology (Aronica et al. 2012; Blümcke et al. 2011). FCDII involves more cytopathology which is characterized by large, dysmorphic neurons with balloon cells (FCDIIb) or without (FCDIIa) balloon cells (Blümcke et al. 2011). The current results agree, in that most of the subjects with lesional abnormalities were in the FCDII group (Table 2). MRI-positive features in FCDII are also frequent and well-described (Leach et al. 2014; Lerner et al. 2009; Sisodiya et al. 2009). Patients with hypometabolism on FCD-PET were also more common in the FCDII group, however the difference did not reach statistical significance (Table 2).

Importance of Complex IV The importance of ETC CIV dysfunction in disorders of energy metabolism in the CNS is widely recognized (Arnold 2012; Böhm et al. 2006). Significantly decreased CIVactivity, an important diagnostic criterion for mitochondrial disease, has been reported to have the strongest association with mitochondrial oxidative phosphorylation capacity (Larsen et al. 2012). Dysfunctional mitochondria in neurons, which are the major source of reactive oxygen species and oxidative stress, may be a factor in epileptogenesis (Aguiar et al. 2012; Bourens et al. 2012). CIV has been characterized as the ratelimiting enzyme of the mitochondrial respiratory chain, and considered a primary target for investigation of mitochondrial dysfunction in many diseases (Arnold 2012). The function of CIV in the ETC is the central site of regulation of oxidative phosphorylation, proton pumping

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Fig. 2 Intraoperative and postoperative photographs showing the relationship between the seizure onset zone (associated with FCD Type IIa) and the secondary spread zone (associated with FCD Type I) regions in an 8 year old female. In addition, histological differences between the SSZ and SOZ regions are also shown for these resections. a Intraoperative grid photograph after right craniotomy showing seizure onset zone (SOZ) in red and secondary spread zone (SSZ) in green. The picture is oriented anatomically with the patient’s nose to the right. RSF=right superior frontal. AIH= anterior interhemispheric (electrodes represented in approximate locations). Hand motor was found overlapping SOZ at RSF contacts 21–22 and 30. Therefore the cortex underneath these

electrodes was not resected. SOZ resection region shown by dashed line, and SSZ shown in solid line. b Preoperative MRI (T2-weighted axial) demonstrating locations of the SOZ (dashed line) and SSZ (solid line). Area of ill-defined gray matter–white matter junction compatible with Type II cortical dysplasia is noted in the SOZ (arrow). Intraoperative image c and postoperative MRI d after resection demonstrating the positions of the SOZ (dashed line) and SSZ (solid line). e A representative hematoxylin-eosin (H&E) slide of the SSZ region with FCS Type I dyslamination for this 8 year old patient. f A representative H&E slide of the SOZ region with FCD Type IIa showing a dysmorphic neuron (arrow) for this 8 year old patient

efficiency, ATP production, and reactive oxygen species production, which in turn affects cell signaling and survival (Arnold 2012). In the brain CIV is a sensitive and reliable metabolic biomarker for neurons, in part, because glial contribution is minimal (Wong-Riley 1989). Biochemical, histochemical, immunohistochemical, and molecular measures of CIV correlate well with neuronal functional activity (Wong-Riley 2012).

A few studies have evaluated CIV in epilepsy models. In one report decreased Complex IV activity was noted in rat cortex of a kainic acid-induced status epilepticus model 3 days after injury (Milatovic et al. 2001). Other investigators used CIV histochemistry, estimated by optical density, to demonstrate neuronal loss and a non-homogenous decrease in CIV activity in piriform cortex of adult rats after pilocarpine-

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Fig. 3 Complex IV (CIV) activity in seizure onset zone (SOZ) and secondary spread zone (SSZ) resections in FCDI and FCDII groups. Comparison of CIV activity results from adjacent surgical resections from 17 patients who had resections identified as the SOZ and an adjacent specimen identified as the SSZ. a CIVprot activity from six subjects with focal cortical dysplasia (FCD) Type I, and b CIVprot activity from 11 subjects with FCD Type II. c CIVSDH activity from six

subjects with FCD Type I, and d CIVSDH activity from 11 subjects with FCD Type II. e CIVCIII activity from six subjects with FCD Type I, and f CIVCIII activity from 11 subjects with FCD Type II. g CIVCS activity from six subjects with FCD Type I, and (H) CIVCS activity from 11 subjects with FCD Type II. (box: median and inter-quartile ranges; vertical lines: range of minimum and maximum observations)

induced status epilepticus (Otáhal et al. 2005). Others have shown that interictal FDG-PET hypometabolism in adult patients with temporal lobe epilepsy was correlated with decreased respiratory chain glucose-oxidation capacity, suggesting the possibility of an intrinsic metabolic abnormality (Vielhaber et al. 2003). Although ETC biochemistry was not determined by the investigators, their histological examination suggested CIV deficiency in pyramidal neurons in the CA3 region of hippocampal resections (Vielhaber et al. 2003). More recently investigators evaluated changes in mitochondrial function in rat hippocampus after pilocarpinetreated status epilepticus (Gao et al. 2014). CIV and SDH activities were determined in isolated mitochondria by spectrophotometry (Gao et al. 2014). A significant decrease in CIV activity was measured 7 days and 45 days after status epilepticus, but SDH activity was nearly constant throughout the study (Gao et al. 2014). The results of these previous studies support the importance of CIV as a biomarker in epilepsy models and the current findings.

Neurons are known to be particularly susceptible to oxidative stress because of high oxygen requirement and limited antioxidant defenses in the CNS (Aguiar et al. 2012). Some have postulated an epileptogenic cycle may ensue, which leads to energy production failure, decreased efficiency of ATP-dependent processes, such as neuronal Ca++ homeostasis, impaired uptake of excitotoxic neurotransmitters, and further promulgation of seizures (Kang et al. 2013; Khurana et al. 2013; Martinc et al. 2012). The current results provide data supporting the possibility that CIV activity is selectively reduced in the mitochondrial respiratory chain of SOZ neurons. A deficit of CIV in SOZ neurons would correspond with a deficit in energy production, and support the theory linking energy production failure to promulgation of seizures. Limitations Because of the retrospective design the current study cannot conclude whether CIV deficit in FCDII subjects is a cause or effect. Also the study only included epilepsy

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surgery patients, and did not permit comparison with a control group. Because there was only one investigator for each segment of the study, it was not possible to evaluate inter-rater reproducibility. Biochemical results obtained from analysis of tissue homogenates may be less sensitive for the detection of mitochondrial dysfunction than evaluation of individual cells. Brain tissues from normal controls were not available. Because of the delay in sample processing, autopsy specimens were unacceptable for ETC complex functional testing. Other investigators have acknowledged the limited availability of appropriate control brain tissue for neuropathology studies in epilepsy patients (Sarnat et al. 2011). The current authors took an alternative approach, i.e., using subjects as their own controls. This approach showed decreased CIV activity in SOZ regions resected from FCDII subjects, but not from FCDI individuals (Fig. 3). Given that some SSZ specimens demonstrated abnormalities consistent with cortical dysplasia and an epileptogenic zone, a comparison between SOZ and SSZ tissues biases the study towards a false negative result. In addition, because resections from adjacent SSZ regions are potentially affected at some level by chronic seizures, a truly normal control group may have shown more significant ETC differences. Future research considerations These results support the use of adjacent cortical resections to distinguish differences between SOZ and SSZ groups. The results also suggest the possibility that a CIV deficit in SOZ specimens of the FCDII group may be an intrinsic ETC abnormality, and not simply a result of decreased mitochondrial content. These results support the study hypothesis. Recent studies have demonstrated the marked susceptibility of the brain to mitochondrial respiratory chain dysfunction (Pickrell et al. 2011; Srinivasan and Avadhani 2012). Because of the importance of neuronal dependence on oxidative metabolism and the relationship of CIV as the rate-limiting enzyme in the mitochondrial respiratory chain, a CIV deficit associated with the SOZ of patients with FCDII may be an important consideration for future epilepsy research.

Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For this type of study formal consent is not required. Conflict of interest The authors declare that they have no conflict of interest.

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