Calbindin D28K expression in relation to granule cell dispersion, mossy fibre sprouting and memory impairment in hippocampal sclerosis: a surgical and post mortem series.

Reorganisation of the dentate gyrus, including granule cell dispersion (GCD) and mossy fibre sprouting, typically accompany hippocampal sclerosis (HS) in temporal lobe epilepsy. Calbindin (CB) expression in granule cells increases during infancy, influences granule cell excitability, vulnerability to excitotoxicity in addition to important physiological functions in memory. Our aim was to study CB patterns in relation to dentate gyrus re-organisation, epilepsy history and memory function. Forty-five surgical cases and 11 post mortems were examined from patients with drug-resistant epilepsy in addition to three post mortem controls. In the surgical cases, CB expression, and the degree of GCD and mossy fibre sprouting were measured. In post mortem cases, CB expression was assessed in relation to the pattern of HS along the rostral-caudal axis of the hippocampus, and compared to PM controls. Three patterns were identified. In Group 1 (40%), the most dispersed granule cells were CB-positive and basal cells negative imitating developmental patterns. In Group 2 (47%), normal CB expression was retained and in Group 3 (13%), CB was predominantly lost in granule cells. These patterns correlated with GCD, the presence of mossy fibre sprouting and the pattern of HS. Group 1 was associated with early onset of seizures but not independently predictive of outcome. In post mortem cases, altered CB expression lateralised to the side of HS and persisted despite seizure remission in some patients. No significant correlation between CB expression and memory function was identified. CB expression patterns in HS may indicate developmental dysmaturation and are associated with the extent of GCD and mossy fibre sprouting in HS. The functional significance of CB loss, in terms of epileptogenesis and effects on memory, remain uncertain.

Bilateral reorganization of the dentate gyrus in hippocampal sclerosis: a postmortem study.

BACKGROUND: Hippocampal sclerosis (HS) is the most common surgical pathology associated with mesial temporal lobe epilepsy (MTLE). HS is typically characterized by mossy fiber sprouting (MFS) and reorganization of neuropeptide Y (NPY) fiber networks in the dentate gyrus. One potential cause of postoperative seizure recurrence following temporal lobe surgery may be the presence of seizure-associated bilateral hippocampal damage. We aimed to investigate patterns of hippocampal abnormalities in a postmortem series as identified by NPY and dynorphin immunohistochemistry. METHODS: Analysis of dentate gyrus fiber reorganization, using dynorphin (to demonstrate MFS) and NPY immunohistochemistry, was carried out in a postmortem epilepsy series of 25 cases (age range 21-96 years). In 9 patients, previously refractory seizures had become well controlled for up to 34 years prior to death. RESULTS: Bilateral MFS or abnormal NPY patterns were seen in 15 patients including those with bilateral symmetric, asymmetric, and unilateral HS by conventional histologic criteria. MFS and NPY reorganization was present in all classical HS cases, more variably in atypical HS, present in both MTLE and non-MTLE syndromes and with seizure histories of up to 92 years, despite seizure remission in some patients. CONCLUSION: Synaptic reorganization in the dentate gyrus may be a bilateral, persistent process in epilepsy. It is unlikely to be sufficient to generate seizures and more likely to represent a seizure-induced phenomenon.

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

AIMS: Glial fibrillary acidic protein (GFAP)-delta 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-delta is speculated to have roles in regulation of astrocyte size and motility and a subpopulation of GFAP-delta-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 sclerosis (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-delta 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 astrocytic component of gangliogliomas showed immunoreactivity, colocalizing with conventional GFAP. No neuronal expression for GFAP-delta was seen in any of the pathologies. Quantitative analysis in 10 FCDIIB and five TSC cases revealed greater numbers of GFAP-delta-positive balloon cells than conventional GFAP. There was no GFAP-delta expression within nodular heterotopia. CONCLUSIONS: GFAP-delta expression 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-delta within heterotopia supports their composition from cells destined for deeper cortical layers.

Quantitative grey matter histological measures do not correlate with grey matter probability values from in vivo MRI in the temporal lobe.

Voxel-based morphometry (VBM) is commonly used to study systematic differences in brain morphology from patients with various disorders, usually by comparisons with control subjects. It has often been suggested, however, that VBM is also sensitive to variations in composition in grey matter. The nature of the grey matter changes identified with VBM is still poorly understood. The aim of the current study was to determine whether grey matter histopathological measurements of neuronal tissue or gliosis influenced grey matter probability values that are used for VBM analyses. Grey matter probability values (obtained using both SPM5 and FSL-FAST) were correlated with neuronal density, and field fraction of NeuN and GFAP immunopositivity in a grey matter region of interest in the middle temporal gyrus, in 19 patients undergoing temporal lobe resection for refractory epilepsy. There were no significant correlations between any quantitative neuropathological measure and grey matter probability values in normal-appearing grey matter using either segmentation program. The lack of correlation between grey matter probability values and the cortical neuropathological measures in normal-appearing grey matter suggests that intrinsic cortical changes of the type we have measured do not influence grey matter probability maps used for VBM analyses.

Balloon cells associated with granule cell dispersion in the dentate gyrus in hippocampal sclerosis.

Granule cell dispersion (GCD) is a common finding in hippocampal sclerosis in patients with intractable focal epilepsy. It is considered to be an acquired, post-developmental rather than a pre-existing abnormality, involving dispersion of either mature or newborn neurones, but the precise factors regulating it and its relationship to seizures are unknown. We present two cases of GCD with associated CD34-immunopositive balloon cells, a cell phenotype associated with focal cortical dysplasia type IIB, considered to be a developmental cortical lesion promoting epilepsy. This observation opens up the debate regarding the origin of balloon cells and CD34 expression and their temporal relationship to seizures.

Correlation of quantitative MRI and neuropathology in epilepsy surgical resection specimens--T2 correlates with neuronal tissue in gray matter.

Newer MRI methods can detect cerebral abnormalities not identified on routine imaging in patients with focal epilepsy. Correlation of MRI with histopathology is necessary to understand the basis of MRI abnormalities and subsequently predict histopathology from in vivo MRI. The aim of this study was to determine if particular quantitative MR parameters were associated with particular histological features. Nine patients with temporal lobe epilepsy were imaged at 1.5 T using standard presurgical volumetric and quantifiable sequences: magnetization transfer and FFT2. The resected temporal lobe was registered with the volumetric MRI data according to our previously described method to permit correlation of the modalities. Stereologically measured neuronal densities and field fraction of GFAP, MAP2, synaptophysin and NeuN immunohistochemistry were obtained. Analyses were performed in the middle temporal gyrus and compared with quantitative MRI data from the equivalent regions. There was a significant Spearman Rho negative correlation between NeuN field fraction and the T2 value in gray matter (correlation coefficient -0.72, p=0.028). There were no significant correlations between any neuropathological and MR measures in white matter. These preliminary findings suggest that T2 in gray matter is sensitive to the proportion of neuronal tissue. Novel quantitative MRI measures acquired with higher field strength magnets, and so with superior signal to noise ratios, may generate data that correlate with histopathological measures. This will enable better identification and delineation of the structural causes of refractory focal epilepsy, and will be of particular benefit in patients in whom current optimal MRI does not identify a relevant abnormality.

Methodological aspects of 3D and automated 2D analyses of white matter neuronal density in temporal lobe epilepsy.

White matter neuronal density has been correlated with clinical outcome after temporal lobectomy for refractory epilepsy. Both morphometric 2D (two-dimensional) and stereological 3D (three-dimensional) analyses of neuronal density have been performed. 3D analyses are thought to be more accurate than 2D counts, but more time-consuming. We compared 3D and automated 2D measurements in the same specimens. Adjacent 20-microm (for 3D analyses) and 5-microm (for 2D analyses) sections from 10 temporal lobectomies were stained for NeuN immunohistochemistry. Analysis of 100% of a region of interest (ROI) in deep white matter was performed using an image analysis system (Histometrix, Kinetic Imaging, UK). 3D analyses were undertaken using x 63 magnification (6 h/case). Automated 2D analyses were undertaken using automatic neuronal identification at x 10 magnification with three to four repeats (1.5 h/case). The range of neuronal densities for 3D measurements was 2120-4910 neurones/mm(3), and for automated 2D measurements 17.4-47.1 neurones/mm2. There was a linear correlation between the two methods with an r2 of 0.58. [corrected] Count-recount variability was 1.4-9.9% for the 3D and 5.1-36.6% for the automated 2D measurements. We found a wide range of white matter neuronal densities using either analysis. The low agreement between methods, and the high count-recount variability for the automated 2D analyses, indicate that despite being more time-consuming, rigorous 3D stereological analyses have to be performed to obtain reliable results. These findings have implications for studies requiring neuronal counts in normal and disease states.

Vascular colocalization of P-glycoprotein, multidrug-resistance associated protein 1, breast cancer resistance protein and major vault protein in human epileptogenic pathologies.

Multidrug transporters, such as P-glycoprotein (P-gp), multidrug-resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP), are associated with multidrug resistance in cancers; other molecules, such as major vault protein (MVP), have a similar association with drug-resistant cancer. These proteins are postulated to generate drug resistance in epilepsy. They have been shown individually to be up-regulated in epileptogenic brain tissue. In any consideration of the function, inhibition or evasion of the activity of such proteins, the colocalization of such proteins needs to be understood. We systematically determined the presence of such colocalization, focusing on microvascular endothelium from epileptogenic human brain tissue. Double labelling immunofluorescence and confocal laser scanning microscopy were used to determine colocalization of P-gp, MRP1, BCRP and MVP in one case of hippocampal sclerosis and two cases of focal cortical dysplasia type IIb. Endothelial colocalization was examined with double labelling using antibodies to CD34 and Factor VIII. The presence of P-gp, BCRP and MVP in microvascular endothelium was confirmed. P-gp, BCRP and MVP colocalized in microvascular endothelium, though not all proteins appeared to be identically distributed within this tissue. MRP1 did not colocalize to endothelium. These findings were not unexpected but required formal confirmation. The demonstrated colocalization of P-gp, BCRP and MVP in microvascular endothelium in epileptogenic human brain tissue has important implications for functional experiments (including single knock-out mice studies), work with specific and broad-spectrum inhibitors of transport function, and any eventual trials of treatment of refractory epilepsy involving modulation of the function of these proteins.

Mcm2 labelling of balloon cells in focal cortical dysplasia.

Balloon cells (BC) are the prominent and defining cellular component of type IIB Focal Cortical Dysplasia (FCD), a common cause of focal epilepsy in patients undergoing surgical treatment. BC are considered immature cells of uncommitted cellular differentiation having immunophenotypical characteristics of both neurones and glia. They are often located in the lower cortical layers and white matter underlying the dysplastic cortex, suggesting migratory arrest during development. We investigated the proliferative potential of BC in 15 cases of FCD from patients with a wide range of ages using immunohistochemistry for Mcm2 (mini chromosome maintenance protein) and Ki67. In the majority of cases, BC showed Mcm2 nuclear positivity. In addition, cells with intermediate neuronal-glial characteristics were labelled whilst the dysmorphic or hypertrophic pyramidal neuronal components of FCD were not. Ki67 labelled only occasional BC. These findings support the view that BC cells represent a pool of less differentiated glial cells with proliferative capacity which may have potential for delayed neuronal differentiation. Furthermore, as Mcm2 specifically identifies BC populations, this marker may be of diagnostic value in the subtyping of FCD lesions in patients with epilepsy.

Cortical neuronal densities and lamination in focal cortical dysplasia.

Focal cortical dysplasia (FCD) is considered to represent a malformation due to abnormal cortical development (MCD) and is an important cause of focal epilepsy. The histopathological features include abnormal laminar architecture, the presence of hypertrophic and dysmorphic neurones in FCD type IIA and additional balloon cells in FCD type IIB. The events causing these sporadic lesions are unknown, but abnormal progenitor cell proliferation occurring late in corticogenesis has been proposed. FCD-like lesions have, however, also been described following a cerebral injury early in life. We carried out a stereological assessment on 15 cases of FCD on NeuN- and Nissl-stained sections from patients with a wide age range, and identified a significant reduction in the neuronal density in all cases in the region of dysplasia compared to the adjacent unaffected cortex (mean neuronal densities 19.2x10(3)/mm3 in the region of dysplasia; 42.8x10(3)/mm3 in the adjacent cortex). Relative differences in neuronal density and size in FCD cases between the superficial (layer I and II) and deep cortical laminae (layer V and VI) were similar to that observed in other pathologies including mild MCD, temporal neocortex adjacent to hippocampal sclerosis as well as in a non-epilepsy surgical control group. The lower overall neuronal densities observed in FCD may reflect neuropil expansion, a local failure of neuronal migration, proliferation or secondary neuronal loss. The preservation of relative differences in neuronal densities between cortical layers and laminar patterns of neurofilament staining in FCD would support the view that the temporal sequence of lamination is not affected.

Cajal-Retzius cells, inhibitory interneuronal populations and neuropeptide Y expression in focal cortical dysplasia and microdysgenesis.

Focal cortical dysplasia (FCD) and microdysgenesis (MD) are likely to represent abnormalities of radial neuronal migration during cortical development. We investigated the distribution of reelin-positive Cajal-Retzius cells, known to be important in the later stages of radial neuronal migration and cortical organization, in 12 surgical cases of both MD and FCD. Quantitation revealed significantly higher numbers of these cells in MD cases compared to controls. As the majority of cortical interneurones arise via tangential rather than radial migration, we studied the distribution and morphology of inhibitory interneuronal subsets immunolabelled for calbindin, parvalbumin and calretinin within these malformations. Frequent findings were a reduction of inhibitory interneurones in the region of FCD and abnormally localised hypertrophic or multipolar calbindin-positive interneurones in both FCD and MD. Neuropeptide Y immunostaining showed a striking increase in the density of the superficial plexus of fibres in both MD and FCD cases in addition to labelling of dysplastic neurones, which may represent an adaptive anti-convulsant mechanism to dampen down seizure propagation.

Progesterone receptors are expressed with higher frequency by optic nerve sheath meningiomas.

Optic nerve sheath meningiomas are infrequent neuropathological specimens as conservative management of these benign tumors is often adopted. It is established that progesterone receptor expression in meningiomas may be of functional significance in the growth of these neoplasms and is related to the tumor grade and likelihood of recurrence. In addition, progesterone receptor expression can be indicative of a potential response of surgically less accessible meningiomas to hormonal treatments. We analyzed 30 surgically resected optic nerve sheath meningiomas and showed high levels of progesterone receptor expression compared to benign meningiomas from other intracranial sites suggesting these tumors may be amenable to hormonal therapy.

Regional distribution of amyloid-Bri deposition and its association with neurofibrillary degeneration in familial British dementia.

Familial British dementia (FBD), pathologically characterized by cerebral amyloid angiopathy (CAA), amyloid plaques, and neurofibrillary degeneration, is associated with a stop codon mutation in the BRI gene resulting in the production of an amyloidogenic fragment, amyloid-Bri (ABri). The aim of this study was to assess the distribution of ABri fibrillar and nonfibrillar lesions and their relationship to neurofibrillary pathology, astroglial and microglial response using immunohistochemistry, confocal microscopy, and immunoelectron microscopy in five cases of FBD. Abnormal tau was studied with immunoblotting. We present evidence that ABri is deposited throughout the central nervous system in blood vessels and parenchyma where both amyloid (fibrillar) and pre-amyloid (nonfibrillar) lesions are formed. Ultrastructurally amyloid lesions appear as bundles of fibrils recognized by an antibody raised against ABri, whereas Thioflavin S-negative diffuse deposits consist of amorphous electron-dense material with sparse, dispersed fibrils. In contrast to nonfibrillar lesions, fibrillar ABri is associated with a marked astrocytic and microglial response. Neurofibrillary tangles and neuropil threads occurring mainly in limbic structures, are found in areas affected by all types of ABri lesions whereas abnormal neurites are present around amyloid lesions. Immunoblotting for tau revealed a triplet electrophoretic migration pattern. Our observations confirm a close link between ABri deposition and neurodegeneration in FBD.

Expression of three oligosaccharide conjugates by neonatal rat dorsal root ganglion neurons: comparison with CGRP and GAP43 immunoreactivity.

Adult dorsal root ganglion neurons express oligosaccharides conjugated to lipids that may be involved in cell-cell recognition, and consequently in the laminar organisation of their central terminations. This paper describes an immunohistochemical study of the developmental expression of 2 lactoseries (LA4 and LD2) and 1 globoseries (SSEA4) oligosaccharide conjugates in rats from embryonic d 19 to postnatal d 60. The expression of calcitonin gene related peptide and the growth associated protein GAP43 was also examined for comparative purposes. We found that these oligosaccharide conjugates begin to be expressed after birth, suggesting that they may be involved in maturation of the central or peripheral terminations, rather than axonal guidance.