doi:10.1093/brain/awh681 Brain (2006), 129, 352–365

Contralateral hemimicrencephaly andclinical–pathological correlations in childrenwith hemimegalencephaly

Noriko Salamon,2 Marissa Andres,1 Dennis J. Chute,3 Snow T. Nguyen,1 Julia W. Chang,1

My N. Huynh,1 P. Sarat Chandra,1 Veronique M. Andre,6 Carlos Cepeda,6 Michael S. Levine,6

Joao P. Leite,8 Luciano Neder,7 Harry V. Vinters3,4,5,6 and Gary W. Mathern1,5,6

Divisions of 1Neurosurgery, 2Neuroradiology, and 3Neuropathology and 4Department of Neurology, 5The Brain ResearchInstitute and 6The Mental Retardation Research Center, David Geffen School of Medicine, University of California,Los Angeles, CA, USA and 7Departments of Pathology and 8Neurology, Ribeirao Preto School of Medicine,University of Sao Paulo, Ribeirao Preto, SP, Brazil

Correspondence to: Gary W. Mathern, MD, Reed Neurological Research Center, 710 Westwood Plaza, Room 2123,Los Angeles, CA 90095-1769, USAE-mail: gmathern@ucla.edu

In paediatric epilepsy surgery patients with hemimegalencephaly (HME; n = 23), this study compared clinical,neuroimaging and pathologic features to discern potential mechanisms for suboptimal post-hemispherectomydevelopmental outcomes and structural pathogenesis. MRImeasured affected and non-affected cerebral hemi-sphere volumes forHME and non-HME cases, includingmonozygotic twins where one sibling hadHME. Stainingagainst neuronal nuclei (NeuN) determined grey and white matter cell densities and sizes in HME and autopsycases, including the non-affected side of a HME surgical/autopsy case. By MRI, the affected hemisphere waslarger and the non-affected side smaller in HME compared with non-HME children. The affected HME sideshowed enlarged abnormal deep grey and white matter structures and/or T2-weighted hypointensity in thesubcortical white matter in 75% of cases, suggestive of excessive pre-natal neurogenesis and heterotopias.Histopathological examination of the affected HME side revealed immature-appearing neurons in 70%, poly-microgyria (PMG) in 61% and balloon cells in 45% of cases. Comparedwith autopsy cases, inHME childrenNeuNcell densities on the affected side were increased in the molecular layer and upper cortex (+244 to +18%),decreased in lower cortical layers (�35%) and increased in the white matter (+139 to +149%). Deep greymatterMRI abnormalities and/or T2-weighted white matter hypointensity correlated with the presence of immature-appearing neurons and PMG on histopathology, decreased NeuN cell densities in lower cortical layers and apositive history of infantile spasms. Post-surgery seizure control was associated with decreased NeuN densitiesin the molecular layer. In young children with HME and epilepsy, these findings indicate that there are bilateralcerebral hemispheric abnormalities and contralateral hemimicrencephaly is a likely explanation for poorerpost-surgery seizure control and cognitive outcomes. In addition, our findings support the hypothesis that HMEpathogenesis probably involves somatic mutations that affect each developing cerebral hemisphere differentlywith more neurons than expected on the HME side.

Keywords: seizures; MRI volumetric; cortical dysplasia; malformations of cortical development; unilateral megalencephaly;cell cycle; neurogenesis; corticogenesis; tuberous sclerosis complex

Abbreviations: HME = hemimegalencephaly; NeuN = neuronal nuclei; PMG = polymicrogyria

Received May 23, 2005. Revised September 7, 2005. Accepted September 28, 2005. Advance Access publication November 16, 2005

IntroductionHemimegalencephaly (HME; also termed unilateral megalen-

cephaly) is a relatively rare but clinically impressive

malformation of cortical development characterized by

marked cerebral asymmetry (Friede, 1989). Originally

described by Sims (Sims, 1835), HME can occur in isolation

or in association with neurocutaneous syndromes or

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developmental disorders such as Klippel–Trenaunay syn-

drome, Ito’s hypomelanosis, neurofibromatosis and tuberous

sclerosis complex (Vigevano et al., 1996). Non-syndromic

HME occurs in multiple ethnic groups without a gender

preference and the clinical presentation typically consists of

early onset epilepsy, psychomotor retardation and contralat-

eral hemiparesis and hemianopia (Flores-Sarnat, 2002). The

aetiology of HME is unknown, although it is presumed to be

from abnormalities of neuroglial differentiation and cell

migration involving a single hemisphere (De Rosa et al.,

1992; Vinters et al., 1992; Arai et al., 1999; Hoffmann et al.,

2000).

An approach in determining HME pathogenesis is by

evaluating clinical, neuroimaging and histopathological find-

ings for both cerebral hemispheres based on an understanding

of normal developmental neurobiology (Rakic, 1988; Uher

and Golden, 2000; Volpe, 2000; Miyata et al., 2004; Andres

et al., 2005). In mammals, cerebral cortical development

begins with progenitor cell proliferation in the periventricular

germinal zones, and neural cell divisions and cell cycling are

tightly controlled (Marin-Padilla and Marin-Padilla, 1982;

Kostovic and Rakic, 1990; Marin-Padilla, 1999; Zecevic and

Rakic, 2001; Rakic and Zecevic, 2003). In mice, for example,

the number of progenitor cell divisions is limited to 10 or 11,

and each successive cell cycle becomes progressively longer

(G1-phase) as a larger proportion of progenitor cells differ-

entiate into neurons (Caviness et al., 1995, 2003; Sommer and

Rao, 2002). The earliest differentiated cells migrate to the

cortical surface forming the preplate, which is partitioned

by subsequent generations of neurons into a primordial plexi-

form layer (eventual molecular layer or Layer 1) and subplate.

The pyramidal neurons that form the hexalaminar post-natal

neocortex migrate in successive waves to form the cortical

grey matter from the inside out (i.e. earliest neurons in lower

grey matter). After pyramidal cell migration, cells in the

molecular layer and subplate degenerate (probably via apop-

tosis), and this coincides with secondary gyral folding in the

last trimester of human gestation (deAzevedo et al., 2003;

Rakic, 2003; Zecevic, 2004).

The larger forebrain and gyral folds of the human cerebral

cortex are thought to be from an increase in the number

and/or cell cycles of progenitor cells (Rakic, 1995; Haydar

et al., 1999; Kuan et al., 2000; Chan et al., 2002). The number

of pre-neurogenesis precursors is influenced by mitotic rates,

programmed cell death and the proportion of cells that

terminally differentiate into neurons at the end of each cell

cycle (Rakic, 1988; Hayakawa et al., 1991; Uher and

Golden, 2000; Kuzniecky and Barkovich, 2001; Chenn and

Walsh, 2002; Caviness et al., 2003). Consequently, small

alterations in the number of progenitor cells, especially at

later cell cycles, have a dramatic impact on final cerebral

size, gyral shapes and cell densities in different layers of the

cortex (Chenn and Walsh, 2003; Putz et al., 2005; Tarui et al.,

2005).

In a previous study of less severe paediatric epilepsy surgery

patients with cortical dysplasia, we found increased upper

cortical neuronal packing densities without megalencephaly

supporting the hypothesis that non-HME dysplasia is prob-

ably due to increased neurogenesis in later progenitor cell

cycles and partial failure of post-neurogenesis programmed

cell death in the molecular layer and subplate (Andres et al.,

2005). We further hypothesized that intractable epilepsy was

the consequence of incomplete cortical maturation with pre-

servation of ‘pro-epileptic’ immature neurons and synaptic

circuitry (Mathern et al., 2000; Cepeda et al., 2003, 2005a, b;

Andre et al., 2004). The purpose of the current study was to

apply similar clinical–pathological comparisons to HME

cases, a more severe form of cortical dysplasia, to discern if

the probable developmental aetiology involved similar or dif-

ferent abnormalities of cerebral development than non-HME

dysplasia cases.

MethodsClinical cohort and pre-surgery evaluationPatients with HME (n = 23) were identified through the University of

California, Los Angeles (UCLA) Paediatric Epilepsy Surgery Program

database. The cases had surgery between 1990 and 2004, and the

clinical protocols have been previously published (Mathern et al.,

1999; Jonas et al., 2004, 2005). For this study, HME patients were

defined as those surgical cases where most (at least three lobes) or all

of one cerebral hemisphere was larger compared with the opposite

hemisphere on MRI. Informed consent was obtained to use clinical

data for research studies. The standardized pre-surgery evaluation

included detailed history and neurological examinations, and inter-

ictal and ictal scalp EEG recordings. Neuroimaging studies included

the aforementioned MRI and cerebral 2-[18F]fluoro-2-deoxy-D-

glucose PET.

MRI and cerebral volume measurementsQuantitative assessments were performed on MRI scans obtained

since 2000 using the same inpatient machine and sequence protocols,

and were available for 11 HME cases and were compared with 6

non-HME cases. At UCLA, there were very few scans performed in

children under 2 years of age without neurological injury or disease

on the same MRI machine. Hence, three of the non-HME cases were

children without epilepsy, including the monozygotic twin of a HME

case (see Fig. 3), and the other three were undergoing epilepsy

pre-surgery evaluations for non-HME pathologies (stroke and

mild cortical dysplasia) with ages similar to the HME group

(Andres et al., 2005). The MRI scans were performed on a General

Electric 1.5 tesla Signa scanner (Milwaukee, WI, USA). Sequences

included high-resolution coronal T1-weighted spoiled gradient-

recalled echo pulse sequences [SPGR; repetition time (TR) 13 ms,

echo time (TE) 2.8 ms, inversion time 300 ms, flip angle 25�, field of

view (FOV) 24 cm, 1.5 mm coronal thickness slices, 78–124 slices per

patient, matrix 256 · 256, number of excitations (NEX) = 1], and

coronal T2-weighted images (TR 2000 ms, TE 120 ms, FOV 24 cm,

5 mm thickness slices, matrix 192 · 192, NEX = 2). Volumetric MRI

analysis was performed with custom-designed commercial software

(Silhouette; CEDARA, Ontario, Canada; www.cedera.com), and cor-

onal T1 SPGR images were used for the analyses. The images were

transferred into the Silhouette program and each coronal section was

segmented into CSF and brain parenchyma. The volumes from each

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MRI coronal section were summated and the cerebellum and brain-

stem regions removed to obtain cerebral hemispheric volumes. For

data analysis, each hemisphere was defined as the one affected by the

HME (i.e. side of surgery) or the non-affected non-operated side

(Andres et al., 2005). For the non-HME cases, the same assignments

were applied for the three pre-surgery patients except for the

perinatal stroke case where the affected side was excluded, and

the affected side was randomly selected for the other three non-

HME/non-epilepsy cases.

In addition to the volumetric assessments, the MRI scans of 16

HME cases were qualitatively assessed for neuroimaging abnormal-

ities (11 cases for volumetric plus 5 scans on older MRI machines).

The qualitative assessments included (i) the lobes involved with

megencephaly; (ii) ventricular enlargement; (iii) a straightened

frontal horn of the lateral ventricle; (iv) deep grey matter abnormal-

ities of the caudate nucleus, thalamus, basal ganglia and olfactory

tract; (v) enlargement of the cortical grey and white matter at the

anterior corpus callosum; and (vi) T2-weighted signal changes

(hypo- and hyper-intensity) in the hemispheric white matter.

HistopathologyBrain tissue from surgery for all 23 HME cases was routinely fixed in

buffered formalin and multiple serial sections reviewed as previously

published (De Rosa et al., 1992; Vinters et al., 1992; O’Kusky et al.,

1996). Two neuropathologists (D.J.C. and H.V.V.) reviewed each

case and assessed for histopathological signs of cortical dyslamina-

tion, polymicrogyria (PMG), dysmorphic cytomegalic neurons and

balloon cells, immature-appearing neurons, excessive white matter

neurons, glial/neuronal heterotopias and calcifications. PMG was

defined as an abnormal arrangement of cell layers with excessive

folding of upper layers, and fusion of gyral surfaces (Friede, 1989)

(see Fig. 4B and C). Immature-appearing neurons were defined as

cells with neuroblast-like features consisting of round to oval nuclear

configurations with a thin rim of cytoplasm (Prayson et al., 2002)

(see Fig. 4F). One previously reported HME patient died intra-

operatively, and homotopic tissue blocks from both hemispheres

were available for histopathological review and neuronal nuclei

(NeuN) immunocytochemistry (ICC) (Jahan et al., 1997).

Tissue selection for NeuN stainingFrom 14 HME cases operated since 1998, 1.5–2 cm blocks of neo-

cortex and adjoining white matter involving the crown of a gyrus in

an area of severe dysplasia were microsurgically removed at surgery

and the blocks immersion fixed for ICC. The remaining brain tissue

from the surgical resection was processed for histopathology as

described above. Neocortical blocks from 11 autopsy cases of similar

ages without known neurological disease were also collected for

comparison. Death in the autopsy group was from acute cardiac,

septic or traumatic causes, and brain tissue was collected between

3 and 11 h after death (mean 6 SD; 6.6 6 2.3 h).

NeuN ICC processingNeuN was chosen over traditional Nissl stains because of the speci-

ficity of this antibody in identifying differentiated neurons, and the

fixation protocols were regimented so that autopsy and surgical

tissue were processed in a similar fashion. Surgical and autopsy

ICC tissue blocks were immediately immersion fixed in freshly pre-

pared phosphate-buffered 4% paraformaldehyde for 24–48 h, and

then cryoprotected overnight in 20% buffered sucrose and stored at

�80�C. Cryostat-cut sections (30 mm) were collected and placed in

individual 3 ml wells containing 0.05 mol/l Tris–HCl-buffered saline

(TBS; pH 7.4). The free-floating sections were processed the same

day as follows with 10 min TBS rinses (three changes) between each

step. Five minutes in 3% hydrogen peroxide/10% methanol in TBS;

60 min in a blocking solution of 2% normal horse serum in TBS;

overnight in primary antisera against NeuN (mouse anti-neuronal

nuclei; Chemicon International; Temecula, CA, USA; Catalog #

MAB377; 1 : 2000 dilution) diluted in 2% normal blocking

serum; 60 min in diluted biotinylated anti-mouse antibody (ABC

kit, Vector Laboratories, Burlingame, CA, USA); and 30 min in a

solution of excess avidin and biotinylated horseradish peroxidase

(ABC Kit, Vector Laboratories). The sections were developed for

7–8 min in 0.5 mg/ml 3,30-diaminobenzidine tetrahydrochloride

and 0.01% hydrogen peroxide. After sufficient colourization, the

reaction was halted by washing in several rinses of cold PBS, the

sections were mounted on subbed slides, air dried, treated for 35 s in

0.1% osmium tetroxide in 0.1 mol/l phosphate buffer (pH 7.4),

dehydrated and coverslipped (Mathern et al., 1995, 1997). For the

single autopsy HME case with matched bilateral formalin fixed

paraffin blocks of the frontal, parietal and temporal neocortex,

10 mm sections were placed on slides and processed for NeuN

ICC as noted above.

NeuN defined cell densitiesNine regions per tissue section were selected in a standardized

manner for NeuN cell counts as previously published (Andres

et al., 2005). Because of the neocortical dyslamination associated

with HME, we selected cell density sample sites based on pre-

determined distances from the pial surface or bottom of the cortical

ribbon instead of identified neocortical cell layers (see Fig. 5A, D and

G). An ocular grid was positioned over the tissue section with the pial

surface at the top. For Layer 1, a 10 · 10 box at ·40 magnification

(31 mm · 31 mm) was positioned with the superior line parallel to the

pial surface, and all NeuN labelled cells within the box were counted

except those touching the upper and right borders of the grid. The

neocortical grey matter sample sites were labelled Levels 1 (superior)

to 6 (inferior), and their location determined by measuring the

distance from the bottom of Layer 1 to the junction of the neocortex

and white matter and dividing by 6. A 5 · 5 box at ·40 magnification

(15.2 mm · 15.2 mm) was positioned at each location, and NeuN-

positive cells within the box counted. In the grey matter, the distance

between each 5 · 5 box varied by 18.6–21.7 mm from case to case. For

the NeuN cells in the superficial white matter, a 3 · 10 box at ·10

magnification (37.2 mm · 124 mm) was positioned just below the

neocortical-white matter junction, and cells were counted. At a

distance of 24.8 mm below the bottom of that box another 5 · 10

box (62 mm · 124 mm) was positioned, and cell counts performed in

the deep white matter. Cell counts were calculated as the number of

NeuN cells/10 000 mm2.

It must be emphasized that NeuN-labelled cell densities are estim-

ates of the number of neurons per unit area (i.e. packing density) and

not an absolute calculation of the total number of neurons per

hemisphere. Experimental techniques used to determine absolute

neuronal quantities within a hemisphere or brain region of autopsy

cases were not practical in this surgical study because the entire

cerebral hemisphere or area of pathology was not available for sam-

pling. Likewise, it is nearly impossible to correct for tissue volume

changes that occur from fixation shrinkage, although it can be

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assumed that it should be the same for HME and autopsy cases with

our protocol. However, neuron densities, as used in this study,

are reliable relative estimates of packing densities, and statistical

differences between groups of patients that are similarly processed

and counted can be accurately determined (Mathern et al., 1995,

1997).

Cortical thicknessTo assess the average thickness of the neocortex, an image computer

was used as previously described (Mathern et al., 1997). The same

NeuN stained sections used for neuronal counts were imaged using

a video charge-coupled device camera (SPOT RT CCD; v3.2;

Diagnostic Instruments, Inc.; Sterling Heights, MI, USA) attached

to a Zeiss microscope interfaced with a PC. Once captured, the image

was analysed using image system software (Image-Pro Plus, v4.1;

Media Cybernetics, Silver Spring, MD, USA). The operator imaged

the tissue section at low magnification and outlined for the computer

straight portions of the cortical ribbon in a shape as close as possible

to a rectangle or trapezoid. Once outlined, the computer measured

the perimeter (P) and area (A). The average cortical thickness (CT)

was calculated using the quadratic equation: CT = [P � (P2 � 16 ·A)1/2]/4. One investigator performed these measures blinded to the

pathology classification, and as previously indicated for neuronal

densities neocortical thickness measurements should be considered

as relative estimates.

NeuN cell sizeThe same imaging system was used to assess NeuN labelled cell size.

Images at ·50 were captured sampling regions from Layer 1, the

upper (Levels 2 and 3) and lower (Levels 5 and 6) grey matter and

superficial and deep white matter regions. The operator outlined for

the computer all individual NeuN labelled cells within the captured

image and the computer calculated the average area per cell for that

section and region (mm2). Typically, 20 or more NeuN labelled cells

were measured for each sample site.

Data analysisMRI volumes, histopathological findings and NeuN measurements

were entered into a database and analysed using a statistical program

(StatView 5; SAS Institute, Inc., Cary, NC, USA). Differences bet-

ween autopsy or non-HME cases and HME patients involving con-

tinuous dependent variables were statistically compared using an

analysis of covariance (ANCOVA) that included the log of age at

surgery or autopsy as co-independent variables. Comparisons using

nominal variables were performed using Chi-square tests.

Results were considered different at a minimal level of significance

of P < 0.05.

ResultsClinical characteristicsTwenty-three HME cases were identified for this

clinical–pathological study, and, with the exception of one

case of tuberous sclerosis complex, none of the other patients

had neurocutaneous or other developmental syndromes asso-

ciated with HME. In other words, in this HME cohort all

but one patient were non-syndromic clinical presentations.

All 23 HME cases underwent hemispherectomy, which

included 6 anatomical, 5 functional and 12 modified

functional procedures (Cook et al., 2004). The HME patients

constituted 17% of hemispherectomy cases (n = 138), 9% of

extratemporal resections (hemispherectomy + multilobar +

non-temporal lobar; n = 255) and 7% of all paediatric epilepsy

surgery procedures at our institution (above + temporal lobe;

n = 316). There were 12 females and 12 right-sided cases (52%

respectively). Mean age (years 6 SD) at seizure onset was

0.1 6 0.15 (range 0–0.5), age at surgery was 1.5 6 1.2 (range

0.2–4.1) and seizure duration before surgery was 1.4 6

1.2 (range 0.2–4). A history of infantile spasms was noted

in 15 cases (65%), and post-surgery seizure control at last

follow-up (mean 4.3 years; range 1–10) was noted in 15 out of

22 (68%) cases. Perioperative complications were noted in

four (17%) HME cases, and included one intraoperative death

(Jahan et al., 1997), a second case that survived cardiac arrest

in the operating room from excessive blood loss, a permanent

third nerve palsy and a post-surgery cranial infection success-

fully treated with antibiotics (Di Rocco and Iannelli, 2000).

Four (17%) HME children underwent re-operations for

recurrent seizures and possible incomplete disconnections

with successful seizure control in one case after repeat surgery.

Cerebral hemisphere volumes and neuronal packing dens-

ities are known to dramatically change during the early

post-natal period as the brain rapidly grows (Andres et al.,

2005). To statistically control for post-natal brain growth we

performed an analysis of covariance (ANCOVA), and the

results (F/P-values) are shown for MRI-assessed cerebral

hemisphere volumes and NeuN-defined cell densities and

sizes (Table 1). The ANCOVA incorporated the pathology

groups (MRI, HME versus non-HME; NeuN, HME versus

autopsy) and log of age at surgery as the independent

variables. Statistically significant interactions would indicate

different changes with age between pathology groups. No

significant interactions were identified meaning that we

found either age-related change for all categories and/or

differences between HME and non-HME (or autopsy)

comparison groups. The remainder of the Results section

will sequentially discuss clinical–pathological findings for

HME cases.

MRI and cerebral hemisphere volumesPre-surgical studies for quantitative analysis were available on

11 (48%) HME cases using the same in-patient MRI machine

and scanning sequences. These 11 MRI scans were compared

with 6 scans from infants without epilepsy (n = 3) or children

undergoing pre-surgery epilepsy evaluation with different

pathologies (n = 3; one stroke and two with subtle

non-HME cortical dysplasia). The mean age (years 6 SD)

at MRI for all 17 cases was 0.70 6 0.5 (range 0.1–1.87), and

there were no statistically significant differences for age

between the HME and non-HME groups (P = 0.58). An

additional five MRI scans (total of 16) were available for

qualitative assessments.

Qualitatively, all 16 HME cases showed enlargement of the

affected compared with non-affected cerebral hemisphere

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with apparent thickening of the grey matter, and poor grey/

white matter differentiation (Fig. 1). HME involved all four

lobes of the brain in nine cases (56%). In the remaining seven

cases there was no preference for which lobes of the brain were

enlarged with the frontal, temporal and occipital lobes

involved in five patients, and the parietal lobe in six cases

(21 out of 28 lobes; 75%). Enlargement of the ipsilateral lateral

ventricle (Fig. 1D and I) and a straightened appearance of the

ipsilateral frontal horn of the lateral ventricle was noted in

11 out of 16 HME cases (69%; Fig. 1C arrow). In six HME

cases (37%), deeper grey matter structures, such as the caud-

ate nucleus, thalamus, basal ganglia and olfactory tract, were

larger than expected, often displacing neighbouring white

matter structures or the ventricular system (Fig. 1D arrow,

E and F arrowhead). Likewise, in eight HME cases (50%) there

was enlarged abnormal grey and white matter structures at

the anterior corpus callosum medial to the ventricle that

displaced the lateral frontal horn (Fig. 1A and C arrow, G

and I). Thus, a total of 12 out of 16 (75%) HME patients

showed enlargement of deep grey and white matter structural

abnormalities on MRI. Seven HME cases (44%) demonstrated

a thinner corpus callosum than would be expected for age

(data not shown). Two HME cases showed normal white

matter signal symmetric to the contralateral side, three

cases had hypointense T2-weighted signal in the affected

white matter (Fig. 1D), four cases demonstrated hyperintense

T2-weighted foci and seven cases showed both hypo- and

hyper-intense T2-weighted white matter abnormalities in

the central or subcortical regions on the affected

side (Fig. 1). Ten cases showed a band-like appearance of

T2-weighted hypointense signal in the periventricular white

matter suggestive for heterotopia (Fig. 1B arrow and D). No

statistically significant correlations were found comparing the

presence and absence of the above qualitative MRI abnormal-

ities with each other (Chi-square; P > 0.09).

For the quantitative MRI analysis, results of the ANCOVA

found three MRI-assessed cerebral volume measures that

logarithmically increased with age and three factors that

were different between HME and non-HME patient groups

(Fig. 2 and Table 1). Total cerebral volumes along with affec-

ted and non-affected hemisphere volumes logarithmically

increased with age (Fig. 2B; P = 0.0001). The averaged

volumes were increased in the affected cerebral hemispheres

(+18%) and decreased in the non-affected hemispheres

(�13%) of HME cases compared with non-HME patients

(Fig. 2A, left; P < 0.039). Consequently, when the volumes

of the affected were subtracted from the non-affected cerebral

hemispheres (side-to-side comparison) there were greater

differences in HME (33%) compared with non-HME cases

(7%; Fig. 2A, right; P = 0.015). Seven of 11 HME cases had

affected minus non-affected cerebral hemisphere volume dif-

ferences that exceeded +2 SDs of the mean for the non-HME

group (>63 cc; range 75–293). Cerebral hemispheric asym-

metry with contralateral hemimicrencephaly is further illus-

trated in a presentation of monozygotic twins where the child

with HME had a larger affected and smaller non-affected

cerebral hemisphere compared with the normal sibling

(Fig. 3). In this HME cohort, affected, non-affected and affec-

ted minus non-affected MRI-assessed cerebral hemisphere

volumes did not correlate with gender (t-tests; P > 0.19),

Table 1 ANCOVA statistical results (F/P-values) comparing autopsy (NeuN) or non-HME (MRI) cases withHME patients

MRI/cortical measure Pathology category* Log of age Interaction Figures

MRI assessmentTotal cerebral volumes 1.33/0.273 64.3/<0.0001 2.70/0.126 1–3Affected hemisphere 5.75/0.035 30.6/0.0001 2.84/0.117 1–3Non-affected hemisphere 5.65/0.039 50.7/<0.0001 1.13/0.309 1–3Aff-non-aff hemisphere 7.97/0.015 1.62/0.227 1.0/0.336 1–3Cortical thickness 1.47/0.241 0.034/0.857 0.382/0.549 4–6

Neuronal densitiesLayer 1 NeuN 5.61/0.040 0.156/0.695 0.001/0.979 4–6Level 1 NeuN 1.06/0.317 2.62/0.124 0.489/0.494 4–6Level 2 NeuN 0.009/0.924 2.08/0.168 0.382/0.545 4–6Level 3 NeuN 0.492/0.493 2.34/0.145 1.39/0.254 4–6Level 4 NeuN 7.72/0.013 3.67/0.072 0.066/0.801 4–6Level 5 NeuN 1.03/0.325 3.10/0.096 1.40/0.253 4–6Level 6 NeuN 5.39/0.033 1.24/0.281 2.37/0.142 4–6Superficial WM NeuN 1.23/0.281 3.94/0.063 0.576/0.458Deep WM NeuN 1.68/0.211 3.62/0.073 0.235/0.634

Neuronal sizeLayer 1 NeuN cell size 2.38/0.141 0.559/0.465 0.124/0.729Upper GM NeuN cell size 0.658/0.429 1.71/0.208 0.820/0.378Lower GM NeuN cell size 0.877/0.361 0.109/0.745 1.68/0.211Super. WM NeuN cell size 1.89/0.185 0.593/0.451 0.131/0.722Deep WM NeuN cell size 0.529/0.477 0.205/0.656 0.027/0.872

Significant values indicated in bold type. *For MRI assessments: HME (n = 11) versus non-HME (n = 6). For all otherscategories the comparison is HME (n = 14) versus autopsy controls (n = 11).

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side resected (P > 0.36), history of infantile spasms (P > 0.33)

and seizure control post-surgery (P > 0.58). However, T2-

weighted hypointensity in the white matter by MRI correlated

with a positive history of infantile spasms (Chi-square; P =

0.032).

HistopathologyHME was often diagnosed in the freshly cut surgical specimen

by the obvious blurring of the cortex–white matter junction,

and variably sized gyri (Fig. 4A). Tissue sections from all

23 HME surgical specimens were reviewed, and every case

showed severe cortical dysplasia with cortical dyslamination,

excessive cells in the molecular layer, the presence of dys-

morphic neurons and excessive heterotopic neurons in the

white matter (Mischel et al., 1995). More variables were other

histopathological features of severe cortical dysplasia on the

affected HME side (Fig. 4 and Table 2). Balloon cells were

detected in 45% and cytomegalic neurons in 96% of HME

Fig. 1 Representative MRI of HME cases with intractable epilepsy. Right/left orientation of all panels is shown in A. (A and B) An 8-monthold presented in status epilepticus from right HME. Note the diffuse increase in cortical grey matter thickness and hemispheric size on theright relative to the non-affected left hemisphere. Arrow in B indicates periventricular signal change consistent with white matterheterotopia. (C) A 0.33 year old with seizures since birth. In addition to the increased size of the right hemisphere compared with the leftnotice the thickened cortex and white matter near the corpus callosum (arrow), enlarged and upward straightened tilt of the rightfrontal horn of the lateral ventricle. (D–F) A 5-month old with seizures since birth and left HME. The deep central grey matter is increasedin size including the caudate nucleus (arrow) with corresponding increased T2 signal changes, an open sylvian fissure (arrowhead) andwhite matter changes in the temporal lobe. The left olfactory tract is also enlarged (arrowhead). This patient was seizure free at lastfollow-up 1-year post-hemispherectomy. (G and H) A 9-month old with seizures since birth and right HME. The enlarged frontal lobe withthickened grey matter is associated with loss of the underlying white matter and enlargement of the lateral ventricle. Again, note theupward tilt of the right frontal horn. (I) A 1-year old with a diagnosis of tuberous sclerosis complex and seizures since birth. There is leftHME with increased grey matter thickness and white matter changes.

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cases (Fig. 4D, E and Table 2). By comparison, PMG was

observed in 61% and immature-appearing neurons in 70%

of cases (Figs. 4B, C, F and Table 2). In this HME cohort,

PMG, balloon cells, immature-appearing neurons and

glial/neuronal heterotopias did not correlate with MRI-

assessed total, affected or non-affected cerebral hemisphere

volumes (t-test; P > 0.11), gender (Chi-square; P > 0.25), side

resected (P > 0.37), age at surgery (P > 0.20), age at seizure

onset (P > 0.51), seizure duration (P > 0.22) or post-surgery

seizure control (P > 0.15). However, PMG on histopathology

was associated with enlarged deep grey matter MRI abnor-

malities (Fig. 1D–F; Chi-square; P = 0.035). In addition, the

presence of immature-appearing neurons in the surgical spe-

cimen correlated with a positive history of infantile spasms

(P = 0.019), and T2-weighted white matter hypointensity on

MRI scans (P = 0.047; Fig. 4F). By comparison, balloon cells in

the surgical specimen did not correlate with neuroimaging

abnormalities or clinical variables (P > 0.14).

NeuN densities, cortical thickness andcell sizeTissue blocks prospectively collected at surgery were available

from 14 (61%) HME cases, and were compared with 11 aut-

opsy cases (controls). The mean age (years 6 SD) of all 25

cases was 2.3 6 2.4, and there were no statistically significant

differences between the HME and autopsy groups (t-test;

P = 0.23). Visually, NeuN packing densities in the upper

cortical layers were variable in HME cases, some patients

showing more and others fewer cells per unit area than

aged comparable autopsy cases (Fig. 5A–C). Middle cortical

layers were not generally different in most HME compared

Fig. 2 Bar graphs (A; mean 6 SEM) showing differences in hemispheric MRI-assessed cerebral volumes and scatter plot B indicating volumechanges with age. The P-values from the ANCOVA (Table 2) are indicated for each plot. (A) Controlling for age (ANCOVA), total cerebralvolumes for both hemispheres were not statistically different between HME and non-HME cases (P = 0.271). However, for HME cases,the affected cerebral hemisphere was larger (P = 0.035), and the non-affected side was smaller (P = 0.039) compared with non-HMEcases. The differences in affected minus non-affected cerebral hemisphere volumes were greater in HME cases compared with non-HMEcases (P = 0.015). (B) For both HME and non-HME cases, total cerebral volumes logarithmically increased from birth to age 2 years(P < 0.0001). Similar data showed a logarithmic increase for affected and non-affected cerebral hemispheres (Table 1; data not shown).

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with autopsy cases (Fig. 5D–F), and in the lower cortex there

were fewer neurons per unit area in most HME cases

(Fig. 5G–I).

ANCOVA found no statistically significant NeuN density

or size measurements that changed within the limited age

range of this cohort, but three variables were found that

were statistically different between the HME and autopsy

cases (Table 1, Figs 5 and 6). On the affected side, NeuN

cell counts showed increased densities in Layer 1 (molecular

layer; +244%; P = 0.04), and decreased densities for lower

cortical Levels 4 (�35%; P = 0.013) and 6 (�35%; P = 0.033;

Fig. 6A). Numerically, NeuN densities were increased in

upper cortical Levels 2 (+18%) and 3 (+56%) and decreased

in cortical Levels 1 (�9%) and 5 (�11%), but the differences

did not reach statistical significance (P > 0.32). Similarly,

NeuN densities in the superficial (+139%) and deep white

matter (+149%) were numerically increased in HME

compared with autopsy cases (Tables 1 and 3). Cortical thick-

ness was increased in HME compared with autopsy cases

(+27%), but the differences were not statistically significant

(Fig. 6A; P = 0.24). While again not statistically significant,

NeuN cell size measurements were increased for HME com-

pared with autopsy cases in Layer 1 (+42%), lower grey matter

(+11%), superficial white matter (+23%) and deep white

matter (+20%), but not upper grey matter (�1%; Table 3).

In one surgical/autopsy HME case, three paired formalin-

fixed tissue blocks from the affected HME and non-affected

apparently ‘normal’ side were available for NeuN density and

cell size measurements (Fig. 6B) (Jahan et al., 1997). Analysis

found that there were numerically decreased neuronal

densities in the upper cortex of the HME compared

with the apparently ‘normal’ side (Level 1, �31%; Level 2,

�16% and Level 3, �29%), and an increase in cortical

thickness on the HME side (+38%). In addition, NeuN-

assessed cell sizes were numerically increased on the HME

compared with ‘normal’ side for Layer 1 (+26%), upper grey

matter (+79%) and lower grey matter (+87%; data not

shown).

Several NeuN measurements correlated with MRI, histo-

pathological features or clinical variables (Table 3). In this

HME cohort, a history of infantile spasms was associated with

a statistically significant decrease in NeuN cell densities for

cortical Level 4 (�55%), Level 5 (�58%) and Level 6 (�51%),

but not Layer 1 (P = 0.95), Levels 1–3 (P > 0.076), or super-

ficial or deep white matter regions (P > 0.70). Likewise, deep

MRI grey matter abnormalities (Fig. 1D–F) correlated with

decreased NeuN cell densities in cortical Level 4 (�75%), and

Level 6 (�79%). Similarly, PMG at histopathology was asso-

ciated with lower NeuN densities in Level 6 (�56%). Finally,

patients with post-hemispherectomy seizure control were

associated with decreased Layer 1 molecular layer NeuN dens-

ities on the HME side (�68%). No statistically significant

associations were found comparing cortical thickness and

NeuN cell sizes with histopathological, MRI and clinical

variables.

DiscussionThis study of children with HME undergoing hemispherec-

tomy for medically refractory epilepsy identified several

findings unappreciated in previous clinical, neuroimaging

and pathological appraisals. By volumetric MRI, the affected

cerebral hemisphere was larger (ipsilateral HME) and the

non-affected side smaller (contralateral hemimicrencephaly)

than non-HME cases (Figs 1–3 and Table 1). The HME

hemisphere demonstrated deep grey and white matter MRI

abnormalities and/or T2-weighted hypointensity in the sub-

cortical white matter in 75% of cases, suggestive for excessive

pre-natal neurogenesis and heterotopias (Fig. 1). Balloon

cells, considered in some classification systems to be essential

histopathological characteristics of HME (Barkovich et al.,

2001; Flores-Sarnat et al., 2003; Palmini et al., 2004) were

Fig. 3 Axial T2-weighted MRI and volumetric findings frommonozygotic twins with one sibling presenting with HME andseizures. Twin A developed seizures within days of birth, and bothinfants underwent MRI within 2-weeks of each other at age 4months. (A and B) The twin with seizures showed enlargement ofthe left compared with the right cerebral hemisphere (A) whilethe twin without seizures had a normal scan without hemisphereasymmetry. (C and D) Calculated volumes for each cerebralhemisphere and twin are shown (note the difference in right/leftorientation compared with panels A and B). Total cerebralvolume for the twin with HME is 539 and 568 cm3 for the normaltwin. However, there were differences in hemisphere volumeswith the ‘normal’ side of the HME case being smaller than eitherhemisphere of the normal twin.

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identified in 45% of surgical specimens. By comparison,

immature-appearing neurons were identified in 70% and

PMG in 61% of paediatric HME cases (Table 2 and Fig. 4).

Compared with autopsy cases, in HME children NeuN dens-

ities on the affected side were significantly increased in the

molecular layer (+244%), decreased in the lower cortex

(�35%) and numerically increased in upper cortical layers

(+56 to +18%) and superficial and deep white matter (+139 to

+149%; Figs 5 and 6; Table 1). The decrease in lower cortical

NeuN densities (average �20%) was similar to the increase in

cortical thickness (+27%) on the affected HME side. Deep

grey matter MRI abnormalities and/or T2-weighted white

matter hypointensity positively correlated with the presence

of immature-appearing neurons and PMG on histopathology,

Fig. 4 Histopathological features of HME. (A) Freshly cut surface from a HME surgical specimen showing thickened grey matter withindistinct grey–white matter border (upper), and subcortical heterotopia (lower). (B and C) NeuN stained region of PMG. The box in B isshown at higher magnification (C). The density of NeuN stained cells was visibly increased in the superficial cortical layers compared withthe deeper layers. (D) H and E stained section through the cortex shows cortical dyslamination with numerous dysmorphic cells includingenlarged cytomegalic neurons and balloon cells. (E) Magnified views of the grey matter showing dysmorphic cytomegalic neurons andballoon cells. Balloon cells were most often found in either upper cortical layers or white matter, and cytomegalic neurons were mostfrequently observed in the lower grey and upper white matter regions. (F) A cluster of immature-appearing cells in the middle of thegrey matter. These neuroblast-like cells are characterized by their round to oval nuclear configurations with a thin rim of cytoplasm inwhich there are typically no dysmorphic features. Immature-appearing cells were observed in the white and grey matter, and mostfrequently in the perisylvian tissue blocks. Calibration bars as indicated. Panels E and F of equal magnification.

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decreased lower cortical NeuN cell densities and a history of

infantile spasms. In HME cases, seizure control post-surgery

was associated with decreased NeuN densities in the molecu-

lar layer on the affected side (Table 3). These findings indicate

the existence of bi-hemispheric abnormalities in HME cases

that may explain the suboptimal developmental outcomes

observed in these children post-hemispherectomy. Further-

more, based on an understanding of normal developmental

neurobiology, our neuroimaging and neuropathological find-

ings support the hypothesis that HME pathogenesis probably

involves somatic mosaic mutations that affect cortical

development differently for each side.

Previous clinical studies have noted worse post-surgery

seizure control along with poorer cognitive and language

outcomes in HME cases compared with other children under-

going epilepsy neurosurgery (Vigevano et al., 1996; Battaglia

et al., 1999; Carreno et al., 2001; Curtiss et al., 2001; Maehara

et al., 2002; Jonas et al., 2004). The reasons for the suboptimal

surgical outcomes in HME cases are unknown, but our data

support the concept that one reason may be because the

contralateral apparently ‘normal’ cerebral hemisphere

shows hemimicrencephaly. Consistent with this notion, in

our cohort the HME cases with the greatest pre-surgery

side-to-side cerebral asymmetries by MRI (>100 cc) were

post-surgery developmentally doing the worst, especially

for language, despite being seizure free. In our previous

quantitative MRI study of less severe paediatric cortical

dysplasia patients we reported minimal increases (+3%) in

cerebral hemisphere volumes comparing the affected with the

non-affected side (Andres et al., 2005). This contrasts with the

33% side-to-side asymmetry in HME cases in our current

report. Hence, our findings would agree with prior proposals

using qualitative MRI assessments that poorer developmental

outcomes in HME children are associated with the severity

of cerebral asymmetry (Barkovich and Chuang, 1990;

Sener, 1995).

Our neuroimaging and histopathology findings also sug-

gest that previous hypotheses involving HME pathogenesis

should be revised to consider developmental mechanisms that

affect both hemispheres differently (Barkovich and Chuang,

1990; Barkovich et al., 2001; Flores-Sarnat et al., 2003; Palmini

et al., 2004). Previous classification systems, based on

qualitative neuroimaging or histopathological findings on a

smaller number of patients have speculated that HME patho-

genesis is from a very early cortical developmental abnor-

mality on one side that alters neuronal migration and/or

neuroglial differentiation to explain the presence of dys-

morphic balloon cells on histopathology. If these hypotheses

are correct then in HME one would expect (i) a normal sized

contralateral cerebral hemisphere because the aetiological

process affects one side; (ii) decreased trans-cortical neuronal

densities and thickness because of reduced neurogenesis from

abnormal neuroblast formation and/or incomplete neuronal

migration; and (iii) balloon cells to be present in all HME

cases.

While decreased lower cortical NeuN packing densities in

our study could be envisioned as a sign of abnormal early

cortical neuron migration consistent with prior proposals,

when all the neuroimaging and neuropathological data are

collectively reviewed an alternative ontogenetic hypotheses

should be considered. The decrease in lower cortical NeuN

packing densities (average Level 4–6; �20%) was associated

with an increase in MRI cerebral volumes (+18%) and cortical

thickness (+27%). By comparison, on the affected side NeuN

cell densities were numerically increased in the upper cortex

(average Level 1–3; +22%). Thus, compensating for changes

in cortical thickness and cerebral hemisphere volume, total

neuronal numbers in the lower cortex are probably similar to

autopsy cases and increased in the upper cortex of the HME

hemisphere. Furthermore, in the majority of HME cases we

found enlarged deep grey matter MRI abnormalities in regions

normally associated with pre-natal neurogenesis suggesting

excessive neuronal production (Haydar et al., 1999; Rakic

and Zecevic, 2000; Chan et al., 2002), and T2-weighted hypo-

intensity indicative of excess heterotopic neurons in the sub-

cortical white matter. Taken together, these neuroimaging

and histopathological findings support the view that there

appears to be more neurons than expected throughout the

ipsilateral hemisphere of HME patients. Assuming that neur-

ons in the upper cortex are the latest born, the pattern

of histopathological abnormalities is most consistent

with the concept that neurons were overproduced late in

corticoneurogenesis, and there is partial failure of post-

neurogenesis programmed cell death in the remnants of

the molecular layer and subplate. These conclusions are sim-

ilar to our previous findings in less severe non-HME forms of

cortical dysplasia, and support the concept that paediatric

cortical dysplasia associated with intractable epilepsy, includ-

ing HME, may have comparable ontogenetic mechanisms

(Andres et al., 2005). In contrast, balloon cells, thought to

be products of abnormal neuroglial differentiation, were iden-

tified in less than half of our HME cases (Table 2), and their

presence in the surgical specimen did not correlate with

clinical, MRI or neuropathological variables. Such findings

support the notion that abnormal mechanisms that produce

balloon cells are not essential for HME pathogenesis (Mischel

et al., 1995; Cepeda et al., 2005a).

Table 2 Frequency of histopathological features on theaffected side of HME cases with intractable epilepsy(n = 23)

Pathologic feature None (%) Mild (%) Severe (%)

PMG 39 39 22Cortical dyslamination 0 43 57Balloon cells 65 22 13Cytomegalic neurons 4 39 57Immature-appearing cells 30 70 0Excessive white matter neurons 0 48 52Calcifications 17 74 9Glial/neuronal heterotopias 39 61 0

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Of equal relevance was our observation that immature-

appearing neurons were frequent histopathological findings

in HME patients, and correlated with T2-weighted MRI white

matter hypointensity and a history of infantile spasms. Such

clinical–pathological correlations suggest that mechanisms

that over produce immature neurons in the later stages of

cortical development maybe more representative of the onto-

genetic process leading to HME and seizures (Caviness et al.,

2003; Tarui et al., 2005). In other words, molecular mechan-

isms that affect pre-neurogenesis progenitor cell cycling may

be cellular pathways to investigate as possible ‘causes’ of

non-syndromic paediatric HME and cortical dysplasia asso-

ciated with epilepsy (Cotter et al., 1999a, b). Such conclusions

support our previous hypothesis that epileptogenesis in cor-

tical dysplasia and HME is probably due to incomplete cor-

tical maturation with excessive immature-appearing neurons

and under developed synaptic circuitry (Cepeda et al., 2005a,b).

Finally, whether genetic and/or environmental in origin the

aetiology of HME seems to begin after zygote separation as

illustrated in our study of monozygotic twins (Fig. 3).

It is important to consider the potential limitations of our

human study when interpreting the results. For example, our

Fig. 5 NeuN-stained sections illustrating differences in cell densities for layer 1 and upper cortex (levels 1 and 2; A–C), lower cortex (levels3–5; D–F) and white matter (G–I) from an autopsy case (A, D and G), and two HME cases (B, C, E, F, H and I) of similar ages. Boxes inleft column indicate approximate locations for NeuN cell counts. Compared with the autopsy case, the first HME patient (middle column)shows increased NeuN densities in the upper cortex and white matter with similar densities in the lower cortex. The increased uppercortex densities were associated with infolding of the neuronal layer near the pial surface (panel B arrow). In contrast, the second HME case(right column) shows lower NeuN densities throughout the cortex with minimal distinction between the grey and white matter. Allmicrographs are at identical magnification.

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findings are based on neuroimaging and histopathology

obtained from mostly non-syndromic HME patients with

epilepsy who were surgical candidates. Our results and inter-

pretations may or may not be applicable to syndromic HME

cases, non-surgical HME patients with bi-hemispheric sei-

zures and the few HME cases without intractable epilepsy.

Likewise, our MRI studies did not include a very large sample

of non-seizure normal controls because such a group was

nearly impossible to recruit. However, based on our prior

MRI study of children with milder forms of cortical dysplasia

and non-dysplasia pathologies, non-affected cerebral hemi-

sphere volumes were only minimally different, and thus

non-HME cases are a reasonable comparison group for the

HME cohort (Andres et al., 2005). Furthermore, our cohort

consisted of very young HME children (less than age 4 years).

We do not know if there could be progressive pathological

abnormalities with longer seizure histories (Mathern et al.,

2002). In addition, we measured NeuN cell densities from a

limited number of tissue blocks per patient and did not sys-

tematically sample the entire brain (Cook et al., 2004). Despite

the expected increased variability due to the limited number

of sample sites, we still found statistically significant differ-

ences between HME and autopsy patient groups supporting

the view that our methods were sufficiently sensitive for this

study. Moreover, we counted NeuN positive cells which

should accurately identify differentiated neurons and avoid

potential confusion with glia or other non-neuronal cell types.

However, we cannot discern whether the cells were excitatory

or inhibitory. Finally, our hypothesis concerning HME patho-

genesis is based on the presumption that the developmental

neurobiology of cortical development and migration are func-

tional in these morphologically abnormal brains.

Fig. 6 Bar graphs showing mean (6SEM) NeuN cell densities and cortical thickness for autopsy (control) and HME cases usingparaformaldehyde fixed 30 mm cryostat sections (upper), and affected and non-affected side of a single HME case that died during surgeryusing 10 mm formalin fixed blocks (lower). The P-values are indicated. (A) For the paraformaldehyde fixed 30 mm cryostat sections,statistically significant differences were found for Layer 1, Level 4 and Level 6 (P < 0.04). (B) For the single case that was formalin fixed,NeuN densities from three homotopic neocortical regions per side were averaged. Numerically, there were increased densities for Level 1and cortical height in the affected hemisphere compared with the non-affected ‘normal’ side. Because of the different fixation techniquesand slice thickness, NeuN density measurement cannot be directly compared between the upper and lower graphs.

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ConclusionsExamining paediatric epilepsy surgery patients with HME,

this study found changes in cerebral hemisphere volumes

by MRI and NeuN cell densities that correlated with clinical

and histopathological variables that in turn provide clues to

HME pathogenesis and an explanation for poorer cognitive

and seizure control outcomes post-hemispherectomy. Based

on our results, we propose that HME pathogenesis is the

consequence of over proliferation of progenitor cells in

later cell cycles. We also propose that HME pathogenesis

involves mechanisms that interfere with late corticoneurogen-

esis with partial failure of post-neurogenesis apoptosis in the

molecular layer and subplate (Andres et al., 2005). Finally, we

propose that poorer post-surgery seizure control and cognit-

ive outcomes are due to contralateral hemimicrencephaly in

most HME patients. It should be noted that these proposed

concepts do not exclude other possible explanations, such as a

defect in neuronal migration in later cortical development,

and the mechanisms of inducing contralateral hemimicren-

cephaly with ipsilateral HME will require additional studies.

However, our data provide a conceptual framework for

proposing hypothesis-driven studies directed toward

molecular mechanisms that affect progenitor cell cycling,

migration and pre- and post-neurogenesis to explain HME

and seizures.

AcknowledgementsThis study was supported by the National Institutes of

Health grants R01 NS38992 and P05 NS02808 to G.W.M.

and FAPESP (CInAPCe-Project-05/56447-7) to J.P.L.

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Table 3 Clinical, MRI and histopathological correlationswith NeuN cell density and size measurements in HMEpatients

Cells/10 000 mm2 6 SD HME Autopsy P-value

HME versus autopsy controlsSuperficial white matter 117 6 97 49 6 28 0.28Deep white matter 92 6 68 37 6 26 0.21

Cell size (mm2)Layer I 108 6 61 76 6 10 0.14Upper grey matter 152 6 46 153 6 76 0.43Lower grey matter 176 6 76 158 6 61 0.36Superficial white matter 199 6 81 162 6 49 0.18Deep white matter 194 6 111 162 6 49 0.48

Cells/10 000 mm2 6 SD Infantilespasms

No infantilespasms

History of infantile spasmsLevel 4 748 6 384 1654 6 368 0.0091Level 5 719 6 423 1717 6 986 0.0221Level 6 613 6 389 1249 6 353 0.049

Cells/10 000 mm2 6 SD Positive MRI Negative MRI

Enlarged deep grey/white matter by MRILevel 4 270 6 217 1088 6 461 0.021Level 6 187 6 59 895 6 355 0.0038

Cells/10 000 mm2 6 SD PMG No PMG

PMGLevel 6 389 6 300 887 6 421 0.037

Cells/10 000 mm2 6 SD Seizure free Not seizure free

Post-surgery seizure controlLayer 1 (molecular layer) 368 6 180 1136 6 574 0.0026

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