Expression patterns of glial fibrillary acidic protein (GFAP)-delta in epilepsy-associated lesional...

Expression patterns of glial fibrillary acidicprotein (GFAP)-delta in epilepsy-associatedlesional pathologies

L. Martinian*, K. Boer†, J. Middeldorp‡, E. M. Hol‡, S. M. Sisodiya*, W. Squier§, E. Aronica†¶ and

M. Thom*

*Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College, London, §Department of

Neuropathology, John Radcliffe Hospital, Oxford, UK, ‡Netherlands Institute for Neuroscience, Royal Netherlands

Academy of Arts and Sciences, Amsterdam, †Department of Neuropathology, Academic Medical Center, Amsterdam, and

¶Stichting Epilepsie Instellingen Nederland, Heemstede, the Netherlands

L. Martinian, K. Boer, J. Middeldorp, E. M. Hol, S. M. Sisodiya, W. Squier, E. Aronica and M. Thom (2009)

Neuropathology and Applied Neurobiology 35, 394–405_ 394..405

Expression patterns of glial fibrillary acidic protein (GFAP)-delta in epilepsy-associated lesional

pathologies

Aims: Glial fibrillary acidic protein (GFAP)-d is a novel

isoform that differs in its C-terminal sequence from other

GFAP isoforms. Previous studies suggest restriction of

expression to the subpial layer, subventricular zone and

the subgranular zone astrocytes, with an absence in

pathological conditions causing reactive gliosis. GFAP-d is

speculated to have roles in regulation of astrocyte size and

motility and a subpopulation of GFAP-d-positive glia may

be multipotent stem cells. The aim of this study was to

investigate its expression in common causes of lesion-

related refractory epilepsy. Methods: Hippocampal sclero-

sis (HS), focal cortical dysplasia (FCD) type IIB, cortical

tuberous sclerosis (TSC) lesions, gangliogliomas, grey

matter heterotopias and hemimegalencephaly from a

wide age range of patients using both surgical and post

mortem tissue specimens were studied. Results: GFAP-d

expression was observed in CA4 and CA1 astrocytes in HS

with less frequent labelling in the granule cell layer, even

where granule cell dispersion was present. No significant

labelling was noted in the subiculum in HS cases or in any

subfields in non-HS epilepsy cases. Balloon cells in FCDIIB

and hemimegalencephaly, giant cells in TSC and the astro-

cytic component of gangliogliomas showed immunoreac-

tivity, colocalizing with conventional GFAP. No neuronal

expression for GFAP-d was seen in any of the pathologies.

Quantitative analysis in 10 FCDIIB and five TSC cases

revealed greater numbers of GFAP-d-positive balloon cells

than conventional GFAP. There was no GFAP-d expression

within nodular heterotopia. Conclusions: GFAP-d expres-

sion patterns in HS overall appears to mirror regional

reactive gliosis. It is a useful marker for the demonstration

of balloon cells in FCD and TSC, which may be relevant to

their abnormal size and localization. The lack of GFAP-d

within heterotopia supports their composition from cells

destined for deeper cortical layers.

Keywords: balloon cells, focal cortical dysplasia, GFAP-delta, hippocampal sclerosis

Introduction

Glial fibrillary acidic protein (GFAP) is an intermediate

filament of which several isoforms have been described,

including a, b, g, d [1], D135, D164, Dexon 6 [2] and more

Correspondence: Maria Thom, Division of Neuropathology, Depart-ment of Clinical and Experimental Epilepsy, Institute of Neurology,Queen Square, London WC1N 3BG, UK. Tel: +44 020 7837 3611;Fax: +44 020 7916 9546; E-mail: [email protected]

394 © 2009 Blackwell Publishing Ltd

Neuropathology and Applied Neurobiology (2009), 35, 394–405 doi: 10.1111/j.1365-2990.2008.00996.x

mailto:[email protected]

recently k [3]. The predominant splice form, GFAP-a, is

expressed in the majority of astrocytes, is highly specific

for astroglial lineage, upregulated in response to injuries

and ageing and is widely used as a diagnostic indicator

of reactive gliosis. GFAP-b is predominantly found in

peripheral nervous system [4], whereas GFAP-g has been

identified in and outside the central nervous system, in

mouse bone marrow and spleen [5,6].

The GFAP-d or -delta, discovered by Condorelli [7], is

also referred to as GFAP-e [1,3]. It differs from other iso-

forms in its C-terminal tail sequence, the last two exons

being replaced with an alterative terminal exon, resulting

in a unique 41-amino-acid sequence [3]. Recent studies of

the expression patterns of GFAP-d in adult human tissues

have demonstrated specific localization in subpial astro-

cytes of the cerebral cortex, the subgranular zone of

hippocampus and subependymal layer of the cerebral

ventricles [8], the latter regions harbouring neural stem

cells in the normal adult human brain. The specific func-

tions of GFAP-d are not well characterized, but its expres-

sion influences filament stability and probably cell motility

[8–11]. Recently, GFAP-d expression has been demon-

strated in balloon cells (BC) in cortical dysplasia from

patients with epilepsy [12]. The aim of the present study

was to extend these studies and more widely investigate

the expression patterns of GFAP-d in common causes of

lesion-related refractory epilepsy, many of which are

either maldevelopmental in aetiology or elicit a reactive

gliosis. By comparison with canonical GFAP expression,

the potential of GFAP-d as a discriminative marker in the

diagnosis of these pathologies can be assessed.

Material and method

Case selection

Cases were selected from the neuropathology archives at

the Institute of Neurology and the National Hospital for

Neurology and Neurosurgery, London (UK), the John

Radcliffe Hospital, Oxford (UK), Academic Medical Center

(University of Amsterdam) in Amsterdam and University

Medical Center in Utrecht (Netherlands). The study was

approved by the Joint Research Ethics Committee of the

Institute of Neurology and the National Hospital for Neu-

rology and in Amsterdam where informed consent was

obtained for the use of brain tissue and for access to

medical records for research purposes. For all surgical

cases, patients underwent therapeutic surgical resection

for refractory epilepsy and the pathological tissue was

surplus to diagnostic requirement. The post mortem tissues

were from patients with long histories of focal epilepsy and

era-appropriate consent for retention of post mortem tissue

was granted by the next of kin.

In all, 74 epilepsy lesions were studied and included

surgical and post mortem tissues from a wide age range

as detailed in Table 1. Pathologies included focal cortical

dysplasia (FCD) type IIB (13 cases), hippocampal scle-

rosis (HS) [seven with granule cell dispersion (GCD),

seven without GCD, eight with bilateral or asymmetrical

damage at post mortem and six paediatric cases with HS

associated with cortical infarct), hemimegalencepahly

(HMEG) (five cases), cortical tubers from patients with

tuberous sclerosis (TSC) (six cases), ganglioglioma (GG)

(six cases) and laminar and nodular grey matter heteroto-

pia (five cases). In addition, temporal lobes from 10 epi-

lepsy surgical patients with no HS were studied as well as

control cases from 15 patients without history of epilepsy

and no significant neuropathology at surgery or autopsy,

from a wide patient age range.

Immunohistochemistry

Seven-micron-thick paraffin wax-embedded sections were

dewaxed and rehydrated through graded alcohols and

taken to water. Endogenous peroxidase was blocked with

3% hydrogen peroxide in deionized water for 10 min.

Sections were microwaved in Vector Antigen Retrieval

Solution (Vector, Burlington, CA, USA) for 12 min and

cooled for 20 min. For the study of FCD, HS and heteroto-

pias cases, sections were incubated with polyclonal

primary antibody against GFAP-d (1:5000, Chemicon

International, Temecula, CA USA) for 40 h at 4°C. For

TS, HMEG and GG cases, we used the identical rabbit

polyclonal GFAP-d antibody (first raised at the Netherlands

Institute for Neuroscience [8]; immunocytochemistry was

carried out as previously described [13]).

In addition, staining in each case was carried out with

a conventional GFAP antibody (which also recognizes

GFAP-d in addition to more abundant GFAP-a and

henceforth referred to as GFAP). Sections were digested

with Proteinase K enzyme and incubated with primary

rabbit polyclonal anti-GFAP (1:1500, DAKO, Glostrup,

Denmark) for 1 h. Labelling was detected with DAKO

ChemMate Envision (DAKO) and staining was visualized

with DAKO DAB + Chromogen. Tissue from a glioblas-

toma multiforme was used as a positive control. Negative

GFAP-delta expression in epilepsy lesional pathologies 395

© 2009 Blackwell Publishing Ltd, Neuropathology and Applied Neurobiology, 35, 394–405

controls were treated identically except that the primary

antibody was replaced with normal rabbit immunoglobu-

lin fraction from DAKO. Between all steps, sections were

washed with PBS and 0.05% Tween 20. Antibodies were

diluted in DAKO ChemMate Diluent.

Confocal immunofluorescence

For the study of FCD and HS pathologies, 7-mm sections

were dewaxed and rehydrated and washed in water.

Endogenous peroxidase was quenched with 3% hydrogen

peroxide and deionized water. Sections were microwaved

in antigen retrieval buffer (Vector). Protein blocking was

done with normal horse serum (Vector) followed by incu-

bation of primary antibodies overnight at 4°C. The com-

binations were rabbit polyclonal anti-GFAP-d (1:5000

dilution) with monoclonal anti-GFAP (1:30 dilution,

DAKO), CD34 (1:30 dilution, DAKO), anti-NeuN (1:1500

dilution, Chemicon), monoclonal anti-human nestin

(1:500, R&D systems) and polyclonal doublecortin

(1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Sections were washed and incubated with secondary anti-

rabbit ImmPRESS (Vector) for polyclonal GFAP-d followed

by Cy3 tyramide signal amplification (PerkinElmer Life

and Analytical Sciences, Boston, MA, USA). Sections then

were washed and quenched in 1% hydrogen peroxide in

PBS for 20 min in order to prevent any deposited tyramide

combining with the second tyramide signal that followed.

The sections were incubated with anti-mouse ImmPRESS

for monoclonals GFAP and NeuN; for doublecortin, the

detection system was Avidin Biotin Kit from Santa Cruz

followed by fluorescein tyramide (Perkin Elmer, Boston,

MA, USA). Sections were mounted on Vectashield with

Dapi (Vector) and visualized with a Zeiss (Gena, Germany)

LSM 510 Meta confocal laser microscope. For TS cases

sections, after incubation with the primary antibodies,

were incubated for 2 h at room temperature with Alexa

Fluor® 568-conjugated anti-rabbit or Alexa Fluor® 488

anti-mouse IgG (1:1000, Molecular Probes, Eugene, OR,

USA). Sections were then analysed by means of a laser

scanning confocal microscope (Bio-Rad, Hercules, CA,

USA; MRC1024) equipped with an argon-ion laser.

Table 1. Details of cases studied according to pathology group

Pathology type (PM or S)

Number of

cases in group

Mean age at surgery or

death (range) (years) Localization Mean duration of epilepsy (range)* (years)

FCD IIB (S) 12 33 (14–52) Temporal (6)

Frontal (6)

20.5 (9–41)

FCD IIB (PM) 1 59 Frontal

HS with GCD (S) 7 24 (10–40) Temporal 19.6 (9–31)*

HS without GCD (S) 7 33.2 (18–55) Temporal 21 (7–36)*

Bilateral HS (PM) 8 69.75 (38–90) Bilateral

Temporal

62.8 (31–74)

Paediatric HS with neonatal infarct (S) 6 14 (9–20) Temporal All childhood onset of seizures*

No HS (S) 10 27.2 (14–36) Temporal 18 (14–25)*

HMEG (S) 6 18 (2–24) months CH 4.5 (2–7) months

Cortical tubers (TSC) (S) 6 20.3 (6–35) Temporal (2)

Frontal (4)

15.1 (4–34)

Ganglioglioma (S) 6 30 (16–60) Temporal 15.8 (9–25)

Grey matter heterotopias (PM) 5 48.25 (25–69) Bilateral (3)

Temporal (1)

Localized (1)

37.25 (23–69)

Controls adult (S) 4 37 (27–57) Temporal (4) NA

Controls adult (PM) 6 51.6 (26–79) Temporal (6) NA

Controls paediatric (PM) 5 4.2 (1–15) months Temporal (5) NA

Clinical details of the groups of pathologies studied. *In some cases, accurate data on duration of seizures in all cases in group were not available.

CH, cerebral hemisphere; FCD, focal cortical dysplasia; GCD, granule cell dispersion; HMEG, hemimegalencephaly; HS, hippocampal sclerosis;

NA, not applicable; PM, Post mortem tissues; S, surgical tissues; TSC, tuberous sclerosis.

396 L. Martinian et al.


Quantitative analysis in FCD cases

In 10 cases of FCDIIB and five TS cases, comparative

quantitative analysis was carried out for the numbers of

immunoreactive (IR) BC in the white matter on adjacent

sections stained with GFAP-d and GFAP. Using Histometrix

software (Kinetic Imaging, Liverpool, UK) and image

acquisition and analysis software (Image Pro Plus vs 5.02;

Media Cybernetics, Silver Spring, MD, USA), an identical

region of interest in the white matter, beneath the region

of dysplasia or tuber, was outlined at low magnification

(¥2.5 objective). One slide per case was selected and the

mean area for quantitative analysis was 5.95 mm2. All BC

within this region were counted systematically at high

magnification (¥40 objective) as positive IR (including

strong or intermediate intensity of labelling) or negative.

The density of BC (or giant cells) per mm2 and percentage

of labelled BC (or giant cells) compared with the total

number was compared with both antibodies.

Results

In the control tissues, GFAP-d IR-glial cells were present in

the subpial region of layer I. Small cells, with short bi- or

multipolar processes, sometimes extending horizontally

along the limiting margin of the brain, were identified

discontinuously along the subpial region with occasional

cells deeper in layer I (Figure 1a). Colocalization of GFAP-d

was seen with GFAP in a proportion of small glial cells

in cortical layer I, including the subpial region. GFAP-d

reactivity was confined to the cell bodies whereas GFAP

showed formation of extensive fibre networks and was

present in greater numbers of cells (Figure 1a). GFAP-d

IR-glia was not present in the deeper cortex, but small

numbers of GFAP-d IR astrocytic-appearing cells were

present in the white matter.

Focal cortical dysplasia

Immunostaining patterns with GFAP-d were consistent

in all the cases studied. The majority of the BC showed dif-

fuse cytoplasmic positivity with variable intensity of label-

ling, many of the cells showing multipolar processes

(Figure 1b,c). IR-BC were present both in the cortex and

white matter. Labelling of small astrocytic cells was also

noted, some with multiple nuclei, present in the region of

dysplasia as well as in cortical layer I in the subpial region.

Dysplastic neurones were not labelled with GFAP-d

(Figure 1d), which was confirmed by double labelling with

NeuN (Figure 1h). In contrast to staining with conven-

tional GFAP, where BC were often obscured by dense mesh-

works of IR-glial fibres, GFAP-d distinctly demonstrated

BC (Figure 1e–g). Quantitative analysis revealed that

lower numbers per mm2 of IR-BC were consistently

present in the white matter with conventional GFAP com-

pared with GFAP-d. GFAP-d also showed a higher mean

percentage positivity of BC of 82% compared with 64%

with GFAP (Table 2). There was no correlation between

the density of GFAP-d IR-BC and patient age, sex, age of

onset or duration of seizures.

Hippocampal sclerosis (surgical cases)

A similar regional staining pattern was present in all

cases of classical HS. Prominent numbers of distinct,

single IR-glial cells were present in the CA1 subfield in

regions of neuronal loss in the stratum pyramidale and

adjacent white matter (stratum radiatum) (Figure 2a).

The junction between CA1 and subiculum showed an

abrupt transition, with the subiculum being devoid of

GFAP-d-positive cells (Figure 2b). The IR-cells were multi-

polar in morphology with short cytoplasmic processes and

frequent binucleate and multinucleate cells were identified

(Figure 2c). The dense fibrillary meshworks of fibres

typically present in chronic HS with conventional GFAP

were not visualized with GFAP-d staining. Frequent IR-

cells with similar morphology were present in the hilus

(Figure 2c), in some cases particularly in the subgranular

zone (Figure 2d). The characteristic radial glial fibre

pattern in the dentate gyrus, as observed with conven-

tional GFAP, was not prominent with GFAP-d (Figure 2h).

Furthermore, HS cases with GCD did not show qua-

litatively different patterns to those without GCD

(Figure 2d,e). Cases with no HS showed small numbers of

IR-cells in the hilus, some in the subgranular zone, but

little positivity in CA1 sector. The temporal lobe adjacent

to HS showed varying numbers of GFAP-d-positive in the

white matter (Figure 2f). In all cases, IR-cells were present

along the ventricular border of the temporal horn.

Hippocampal sclerosis (post mortem cases)

In cases with unilateral HS, the morphology and distribu-

tion of IR-cells was similar to that observed in the surgical

cases of classical HS. Small IR-glial cells were noted in the

hilus, subgranular zone and CA1 region, but they were



less conspicuous in the dentate gyrus. However, in many

of the post mortem cases, the overall staining reaction in

the hippocampus was weaker compared with surgical

cases of HS, although subventricular zone astrocytes often

retained strong labelling intensity. In the contralateral

preserved hippocampus, occasional IR-hilar cells and

immunopositive fibres were observed.

In post mortem cases with bilateral HS, GFAP-d expres-

sion was seen in both sides in the hilar region and CA1.

Where the severity of HS appeared asymmetrical, GFAP-d

IR-cells were more frequent on the side, showing the more

severe cell loss.

Paediatric temporal lobectomies

In two patients, classical patterns of HS were present and

GFAP-d IR patterns were similar to adult cases. In three

cases with end folium HS, GFAP-d IR-cells were present in

the CA4 region only and in a single case without HS, no

GFAP-d-positive cells were observed in the hippocampus,

even in the subgranular region. In all cases, GFAP-d-

positive cells were present in the subventricular zone,

around the hippocampal fissure (Figure 2g) and also

extensively in the region of cortical scarring at the site of

the old infarcts.

Figure 1. Focal cortical dysplasia (FCD) type IIB. (a) Glial fibrillary acidic protein (GFAP)-d-positive astrocytes in the marginal subpial zone

of the cortex (arrow) in a control case with expression relatively limited to the cell body. Inset shows double labelling of GFAP-d (cy3: red)

with conventional GFAP (FitC: green) in the same region which demonstrates colocalization in a few glial cells, but more extensive processes

and fibre formation are visualized with GFAP (FitC: green). (b) GFAP-d showed intense labelling of balloon cells in FCD type IIB cases, many

showing striking multipolar cytoplasmic processes. (c) A variability of intensity of immunolabelling with GFAP-d was noted, with some

balloon cells appearing weak or negatively labelled (arrow). (d) Dysplastic and hypertrophic neurones appeared negative with GFAP-d;

however, prominent numbers of small, bi- and multinucleate cells were present throughout the region of the dysplasia (inset). (e) White

matter beneath region of dysplasia clearly delineates the balloon cell populations whereas labelling with conventional GFAP (FitC: green)

(f) in the same region demonstrates that the balloon cells are partially obscured by the dense glial fibres. (g) Combination of GFAP-d with

conventional GFAP (FitC: green) confirms colocalization of labelling in balloon cells with delta isoform being largely restricted to the cell

body and absent in the fibres of the gliosis. (h) GFAP-d (cy3: red) with NeuN (cy3: red) in FCD IIB showed no colocalization of staining (scale

bars in a,c,d,e,f = 100 microns, b = 10 microns, g = 20 microns, h = 30 microns).



TSC lesions, HMEG and GG

In cortical tubers, moderate to strong intensity of labelling

of a proportion of giant cells (Figure 3a,c) was seen in all

cortical layers for GFAP-d. Many of these cells exhibited

multipolar processes and in addition, multinucleate cells

and smaller reactive astrocytic cells were seen. In HMEG,

similar labelling of balloon-like cells was noted, although

a proportion was negative or weakly labelled (Figure 3d).

Larger GFAP-d IR astrocytes were noted in GG, but

were infrequent. In all these pathologies, no labelling of

dysplastic or abnormal neuronal cell types was seen

(Figure 3b,e,f). Quantification of giant cells in TSC lesions

also showed a higher proportion and number per mm2

with the GFAP-d compared with conventional GFAP.

Grey matter heterotopia

The GFAP-d showed labelling of small astrocytes with short

processes in the subpial region of the overlying cortex and

in the periventricular tissue beneath the ependymal lining.

There were no IR-cells within the heterotopia, even where

rudimentary laminar architecture was present. In only one

case, small numbers of IR-glia were noted around the

margin of some of the nodules (Figure 3g). Comparison

with conventional GFAP staining patterns in this case

showed marked gliosis surrounding nodules with glial

fibres extending into the nodules (Figure 3h), and focal

variable astrocytic gliosis was also confirmed within the

heterotopia in all other cases.

Co-expression of GFAP-d with

developmental markers

The GFAP-d IR-BC in FCD cases showed evidence of

co-expression with developmentally regulated protein

nestin (Figure 4a–c); co-expression with CD34 and dou-

blecortin in BC was not identified. Giant cells in TS and

HMEG also show co-expression of GFAP-dand nestin. In HS

cases, co-expression of GFAP-dwith nestin was noted in the

small and binucleate astrocytes in the region of sclerosis

(Figure 4d–f), but not with doublecortin or CD34 in these

cell types (Figure 4j–l). In control tissues, a lack of colocal-

ization between GFAP-d and doublecortin-expressing cells

in the subpial region was noted (Figure 4g–i).

Discussion

We have described consistent expression patterns of

GFAP-d, an isoform of GFAP, in a large series of

Table 2. Quantification of BC in FCD IIB and GC in TSC with GFAP and GFAP-d immunohistochemistry

Case

(remove numbers)

Percentage of GFAP-d

BC/GC of total number (n)

Percentage of GFAP

BC/GC of total number (n)

Density of GFAP-d

positive BC/GC (mm2)

Density of GFAP-positive

BC/GC (mm2)

FCD

1 95 (110) 87 (41) 10.53 1.8

2 75 (62) 77 (54) 2.53 2.27

3 97 (47) 86 (22) 2.09 0.86

4 93 (322) 86 (45) 14.7 1.88

5 98 (397) 87 (245) 19.45 11.23

6 69 (74) 45 (45) 4.56 0.95

7 74 (69) 52 (62) 8.23 2.32

8 67 (83) 45 (34) 7.56 1.67

9 75 (121) 41 (38) 9.32 2.89

10 76 (115) 38 (48) 10.65 2.54

TSC

1 74 (97) 49 (66) 9.87 2.76

2 71 (78) 38 (75) 8.10 2.56

3 78 (103) 41 (32) 11.67 1.78

4 63 (65) 35 (34) 6.94 2.67

5 67 (74) 26 (24) 7.89 1.82

The number in parentheses in columns 2 and 3 is the total number of cells counted in region of interest. BC, balloon cells in focal cortical

dysplasia (FCD) (cells with both intense and intermediate staining were included and expressed as a percentage of all balloon cells in the white

matter); GC, giant cells in tuberous sclerosis (TSC) lesion (similar analysis was carried out on white matter); GFAP-d, glial fibrillary acidic

protein-delta compared with conventional GFAP (DAKO), which recognizes both a and d isoforms.



pathological lesions associated with focal epilepsy from a

wide age range of patients. GFAP intermediate filaments

are part of the cytoskeleton of glial cells and all share a

common structure of a highly conserved a-helical rod-

like domain with nonhelical head and tail domains. The

delta isoform of GFAP (which has also been termed

GFAP-e [3]) with its unique tail domain has been little

studied in human disease processes to date. It is consid-

ered to have roles in intermediate filament assembly and

stability [3], cell motility and migration [8] and may

interact with other cytoplasmic and nuclear proteins,

including histones [7].

Hippocampal sclerosis is the commonest pathology

associated with temporal lobe epilepsy [14]. In HS, a

subfield-specific dense fibrillary gliosis, as highlighted with

conventional GFAP labelling, accompanies the neuronal

loss in CA1 and CA4 in the classical pattern. In our series,

GFAP-d expression consistently showed a similar distribu-

tion mirroring this pattern of gliosis. The morphology of

the GFAP-d IR astrocytes differed from that shown by con-

ventional GFAP, with predominantly a perinuclear stain-

ing pattern and frequently in binucleate cells and showed

colocalization with nestin. This later novel finding could

suggest a role of GFAP-d in glial cell proliferation and a

Figure 2. Hippocampal sclerosis. (a) Surgical case of hippocampal sclerosis with glial fibrillary acidic protein (GFAP)-d expression

highlighting the regions of gliosis and neuronal loss, in particular CA1 and the hilus with an abrupt transition of staining appreciated

between CA1 and the subiculum (S) and shown at higher magnification in (b). (c) Hilus showing small, distinct glial cell populations with

short cellular processes labelling with GFAP-d; inset showing binucleate GFAP-d-positive cells, which were frequently observed. (d) GFAP-d

cells were more frequent in the hilus than in the granule cell layer (GCL) in case without granule cell dispersion (GCD). (e) In cases with HS

and GCD, radial glial fibres were also not highlighted and (e) illustrates the case with maximal staining in the GCL, which was always less

than the hilus. (f) The white matter of the temporal lobe adjacent to hippocampal sclerosis showed GFAP-d-positive cells. (g) In paediatric

hippocampal sclerosis cases, similar regional staining patterns with GFAP-d were observed and, as in controls, prominent glial labelling

around the ventricle and along the hippocampal fissure (arrowed) was noted. (h) Double labelling with GFAP (FITC: green) and GFAP-d (cy3:

red) in the GCL in hippocampal sclerosis confirmed that the numerous radial glial fibres did not label with delta that colocalized only to a

small number of glial cells in this region (scale bars in a,g = 1000 microns, b = 100 microns, c–f = 10 microns, h = 20 microns).



presence in more immature astrocytes, which requires

further study. In addition, the lack of staining of fibrillary

meshworks of fibres with GFAP-d allowed clearer delinea-

tion of the astrocyte cell bodies, which could potentially

facilitate quantitative analysis. Colocalization with con-

ventional GFAP confirmed that GFAP-d expression was

present in a subset of glial cells in the region of gliosis. As

the conventional GFAP antibody we use recognizes both

GFAP-a and -d, it was not possible to determine if expres-

sion of GFAP-d isoform in HS occurs in a distinct glial

subpopulation. Previous cell transfection studies have

shown that GFAP-a has a better intrinsic filament-

forming capacity than GFAP-d, which is less able to form

multimers [3,8,15]. This could explain the lack of demon-

stration of GFAP-d in fibrillary processes in HS, including

in the radial glial fibres in the dentate gyrus.

It is known that GFAP-d is expressed at 100-fold lower

levels than conventional GFAP isoforms in normal brain

[3]. In astrocytic gliosis associated with multiple sclerosis

or Alzheimer’s disease, GFAP-d was barely detectable

using immunohistochemistry, using the same antibody

as used in our study [8]. The ratio of GFAP-a : -d tran-

scripts is highly correlated in the subventricular astro-

cytes where GFAP-d transcripts are disproportionally

more abundant [8]. Whether a strict ratio is maintained

in other anatomical regions and in conditions of patho-

logical gliosis, or if selective splicing of GFAP-d isoforms

occurs in hippocampal astrocytes in epilepsy remains to

be studied. Furthermore, expression patterns of GFAP-d

may alter in the developing brain and with cell matura-

tion [3]. In paediatric cases of HS, as well as in some

adults, it is likely that the gliotic process commences

Figure 3. Tuberous slerosis and other pathologies. (a) Giant cells (top) and multinucleate cells (bottom) were labelled with glial fibrillary

acidic protein (GFAP)-d in tuberous sclerosis cortical lesions, but (b) dysmorphic neurones were negative. (c) GFAP and GFAP-d colocalized in

the giant cells. (d) Abnormal balloon-like glia cells in hemimegalencepahly cases also showed variable intensity of labelling with GFAP-d, but

abnormal neurones were negative (e) and similarly in ganglioglioma (f). (g) Heterotopia showed mild marginal staining at the edge of

nodules in one case with GFAP-d (arrowed), although no positive labelling was seen within the nodule whereas GFAP immunolabelling on

adjacent section (h) confirmed gliosis at the margins of the nodules (arrow) as well as within the nodules in all cases (inset) (scale bars in

a = 27 microns, b,c,e = 40 microns, d,f = 80 microns and g,h = 100 microns).



Figure 4. Balloon cell in focal cortical dysplasia labelled with glial fibrillary acidic protein (GFAP)-d (a) and nestin (b), showing

colocalization within the cytoplasm (c). Similarly in HS cases, small and binucleate GFAP-d-positive astrocytic cells colocalized with nestin

(e; merged in f ). In control tissues, GFAP-d cells in the supial region (g) did not show colocalization with the small number of

doublecortin-positive cells identified (h; merged in i). In the hilus in HS cases, similarly GFAP-d-positive astrocytes (j) did not label with

doublecortin that labelled occasional hilar neurones (k; merged in l). Bars in a–c = 23 microns, in d–f = 40 microns and in g–l = 20 microns.



early in life, possibly following an initial injury. Therefore,

the time of onset of gliosis could be a factor explaining

the discrepancy between our finding of GFAP-d in gliosis

associated with HS, but not in Alzheimer’s disease

and multiple sclerosis, acquired during adulthood [8].

Examination of post mortem cases with long histories of

epilepsy also allowed us to assess GFAP-d expression

patterns over time in comparison with surgical cases

with shorter histories. We identified strongly IR-glial cells

in CA1 in patients with HS with over 70 years of dura-

tion of epilepsy, although in other cases subventricular

astrocytes were labelled more intensely than in the scle-

rotic hippocampus.

Regional GFAP-d expression in subventricular, sub-

granular and subpial zone astrocytes in normal brains [8]

persists in disease states as shown in our present surgical

and post mortem epilepsy series. This could support an

important functional or regulatory role for specific glial

populations in these regions. For example, it has been

proposed that a subpopulation of GFAP-d-positive cells

represent multipotent stem cell [8]. In human adult

brain, a subventricular astrocyte ribbon has been identi-

fied that acts as a source of multipotent progenitor cells

[16]. In epilepsy, one possibility regarding the cause of

GCD, a common finding in HS [14], is excess proliferation

of subgranular zone progenitor cells as a result of sei-

zures [17,18]. We examined cases of HS with and

without GCD, but found no difference in GFAP-d expres-

sion patterns in the subgranular zone. An alternative

theory for GCD favours the formation of the radial glial

scaffold in the dentate gyrus as a key factor in inducing

the cell migration [19]. It has been shown that when

GFAP-d expression reaches a critical level, collapse of the

intermediate filament cytoskeleton occurs [8,15]. The

lack of GFAP-d that we noted in radial fibres in HS could

suggest that this relative reduction in these glial cells,

compared with other GFAP isoforms, is a factor confer-

ring stability of the glial network and thus promoting

GCD [20].

Our finding of GFAP-d expression in a large proportion

of BC in FCD IIB confirms the findings of a recent study

[12]. We have also confirmed similar expression in the

giant cells and balloon-like cells associated with TSC and

HMEG. As GFAP-d is largely absent in the fibrillary gliosis

associated with many FCD cases, it appears a superior

marker to conventional GFAP antibodies that also recog-

nize the more abundant GFAP-a-positive fibres, obscur-

ing many BC. This was supported by our quantitative

analysis of BC using the different GFAP antibodies and

may also reflect greater affinity or binding of the specific

GFAP-d antibody for this isoform, although there are no

data yet available to support this. This clearer identifica-

tion and delineation of BC with GFAP-d could be of diag-

nostic value in the classification of FCD subtypes [21]. It

has been suggested that GFAP-d expression in BC may

reflect their progenitor cell phenotype from ventricular

zone astrocytes [12]. As it has been proposed that

GFAP-d has roles in moulding the shape and size of the

cell through its effects on the cytoskeleton and in influ-

encing cell motility [8], any abnormal expression levels of

GFAP-d, or alteration in ratio of GFAP-a : -d could be a

factor determining the abnormal localization (white

matter rather than cortical plate) and morphology of BC

and giant cells. The expression of GFAP isoforms has

been shown to be tightly regulated [22], and as well as

developmental regulation, cell stress could influence the

relative ratio of GFAP transcripts. These possibilities, in

the context of any deregulation of GFAP-d expression in

BC in patients with refractory epilepsy require further

investigation. In addition, neuronal expression of some

GFAP isoforms has been shown in patients with Alzhe-

imer’s disease [2] and binding of GFAP-d with transmem-

brane proteins presenilin I and II has been shown [1]. In

the present series, which included FCD cases with dys-

morphic neurones bearing neurofibrillary tangles (as

previously reported [23]), no neuronal expression for

GFAP-d was observed as was confirmed on double label-

ling studies.

In patients with grey matter heterotopia, we demon-

strate an absence of GFAP-d glial cells within the hetero-

topia in all cases, in contrast to conventional GFAP that

showed variable astrocytic gliosis. This would support the

lack of contribution of subpial-layer or marginal-zone cell

types to the heterotopia and the proposal that heterotopia

arise from cells destined for deeper cortical layers (II–VI).

We have previously reported the lack of reelin-positive

Cajal–Retzius-like cells, also normally present in cortical

layer I, within heterotopia [24],which reinforces this

suggestion.

In conclusion, GFAP-d is expressed in a variety of

pathologies associated with epilepsy. Increased expression

is shown in regions of astrocytic gliosis in HS and in BC in

FCD IIB and allied lesions. Whether there is selective tran-

scription for GFAP-d isoform in epilepsy will be important

to establish in future studies due to the important role

this protein has in influencing the cell cytoskeleton and



cell motility, which may be relevant to many of the cyto-

architectural and developmental aberrations observed in

patient with focal epilepsy.

Acknowledgements

This work has been supported by a grant from the MRC

(grant number G79059) and the UCLH/UCL Biomed

Centre (LM, SS, MT). Also the National Epilepsy Fund,

NEF 05-11 (EA,KB); EU-FP7-project 202167, Stichting

Michelle M06.011, M07.016 (EA); Hersenstichting Ned-

erland 13F05.08, 15F07.40 (EH, JM); and ISAO 04511

(EH, JM). We would like to thank Dr. W.G.M. Spliet (neu-

ropathologist; Department of Pathology, UMCU) for the

collaboration in the collection of the cases.

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Received 11 June 2008

Accepted after revision 3 October 2008

Published online Article Accepted on 29 October 2008



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