Oligodendrocyte lineage and myelination are compromised in the gray matter of focal cortical dysplasia type IIa.

OBJECTIVES: Focal cortical dysplasias (FCDs) are local malformations of the human neocortex and a leading cause of medically intractable epilepsy. FCDs are characterized by local architectural disturbances of the neocortex and often by a blurred gray-white matter boundary indicating abnormal white matter myelination. We have recently shown that myelination is also compromised in the gray matter of dysplastic areas, since transcripts encoding factors for oligodendrocyte differentiation and myelination are downregulated and myelin fibers appear fractured and disorganized. METHODS: Here, we characterized the gray matter-associated myelination pathology in detail by in situ hybridization, immunohistochemistry, and electron microscopy with markers for myelin, mature oligodendrocytes, and oligodendrocyte precursor cells in tissue sections of FCD IIa and control cortices. In addition, we isolated oligodendrocyte precursor cells from resected dysplastic tissue and performed proliferation assays. RESULTS: We show that the proportion of myelinated gray matter is similar in the dysplastic cortex to that in controls and myelinated fibers extend up to layer III. On the ultrastructural level, however, we found that the myelin sheaths of layer V axons are thinner in dysplastic specimens than in controls. In addition, the density of oligodendrocyte precursor cells and of mature oligodendrocytes was reduced. Finally, we show for the first time that oligodendrocyte precursor cells isolated from resected dysplastic cortex have a reduced proliferation capacity in comparison to controls. SIGNIFICANCE: These results indicate that proliferation and differentiation of oligodendrocyte precursor cells and the formation of myelin sheaths are compromised in FCD and might contribute to the epileptogenicity of this cortical malformation.

Whole Transcriptome Screening Reveals Myelination Deficits in Dysplastic Human Temporal Neocortex.

Focal cortical dysplasias (FCDs) are local malformations of the human neocortex with strong epileptogenic potential. To investigate the underlying pathomechanisms, we performed a whole human transcriptome screening to compare the gene expression pattern of dysplastic versus nondysplastic temporal neocortex. Tissue obtained from FCD IIIa cases (mean age 20.5 years) who had undergone surgical treatment, due to intractable epilepsy, was compared with nondysplastic specimens (mean age 19.9 years) by means of Affymetrix arrays covering 28 869 genes. We found 211 differentially expressed genes (DEX) among which mainly genes important for oligodendrocyte differentiation and myelination were downregulated in FCD IIIa. These findings were confirmed as functionally important by Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis. The reduced expression of myelin-associated transcripts was confirmed for FCD Ia, IIa, and IIIa by real-time RT-qPCR. In addition, we found that the density of myelin basic protein mRNA-expressing oligodendrocytes and of 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive myelin fibers was significantly reduced in dysplastic cortex. Moreover, high-resolution confocal imaging and 3D reconstruction revealed that the myelin fiber network was severely disorganized in dysplastic neocortex, indicating a disturbance of myelin sheath formation and maintenance in FCD.

Characterization of focal cortical dysplasia with balloon cells by layer-specific markers: Evidence for differential vulnerability of interneurons.

OBJECTIVE: Focal cortical dysplasia (FCD) is a major cause of pharmacoresistant focal epilepsy. Little is known about the pathomechanisms underlying the characteristic cytoarchitectural abnormalities associated with FCD. In the present study, a broad panel of markers identifying layer-specific neuron subpopulations was applied to characterize dyslamination and structural alterations in FCD with balloon cells (FCD 2b). METHODS: Pan-neuronal neuronal nuclei (NeuN) and layer-specific protein expression (Reelin, Calbindin, Calretinin, SMI32 (nonphosphorylated neurofilament H), Parvalbumin, transducin-like enhancer protein 4 (TLE4), and Vimentin) was studied by immunohistochemistry on paraffin sections of FCD2b cases (n = 22) and was compared to two control groups with (n = 7) or without epilepsy (n = 4 postmortem cases). Total and layer-specific neuron densities were systematically quantified by cell counting considering age at surgery and brain region. RESULTS: We show that in FCD2b total neuron densities across all six cortical layers were not significantly different from controls. In addition, we present evidence that a basic laminar arrangement of layer-specific neuron subtypes was preserved despite the severe disturbance of cortical structure. SMI32-positive pyramidal neurons showed no significant difference in total numbers, but a reduction in layers III and V. The densities of supragranular Calbindin- and Calretinin-positive interneurons in layers II and III were not different from controls, whereas Parvalbumin-expressing interneurons, primarily located in layer IV, were significantly reduced in numbers when compared to control cases without epilepsy. In layer VI, the density of TLE4-positive projection neurons was significantly increased. Altogether, these data show that changes in cellular composition mainly affect deep cortical layers in FCD2b. SIGNIFICANCE: The application of a broad panel of markers defining layer-specific neuronal subpopulations revealed that in FCD2b neuronal diversity and a basic laminar arrangement are maintained despite the severe disturbance of cytoarchitecture. Moreover, it showed that Parvalbumin-positive, inhibitory interneurons are highly vulnerable in contrast to other interneuron subtypes, possibly related to the epileptic condition.

Diagnostic Accuracy of Epilepsy-dedicated MRI with Post-processing.

PURPOSE: To evaluate the diagnostic accuracy of epilepsy-dedicated 3 Tesla MRI including post-processing by correlating MRI, histopathology, and postsurgical seizure outcomes. METHODS: 3 Tesla-MRI including a magnetization-prepared two rapid acquisition gradient echo (MP2RAGE) sequence for post-processing using the morphometric analysis program MAP was acquired in 116 consecutive patients with drug-resistant focal epilepsy undergoing resection surgery. The MRI, histopathology reports and postsurgical seizure outcomes were recorded from the patient's charts. RESULTS: The MRI and histopathology were concordant in 101 and discordant in 15 patients, 3 no hippocampal sclerosis/gliosis only lesions were missed on MRI and 1 of 28 focal cortical dysplasia (FCD) type II associated with a glial scar was considered a glial scar only on MRI. In another five patients, MRI was suggestive of FCD, the histopathology was uneventful but patients were seizure-free following surgery. The MRI and histopathology were concordant in 20 of 21 glioneuronal tumors, 6 cavernomas, and 7 glial scars. Histopathology was negative in 10 patients with temporal lobe epilepsy, 4 of them had anteroinferior meningoencephaloceles. Engel class IA outcome was reached in 71% of patients. CONCLUSION: The proposed MRI protocol is highly accurate. No hippocampal sclerosis/gliosis only lesions are typically MRI negative. Small MRI positive FCD can be histopathologically missed, most likely due to sampling errors resulting from insufficient harvesting of tissue.

Surgical Treatment of Extratemporal Epilepsy: Results and Prognostic Factors.

BACKGROUND: Surgery is a widely accepted option for the treatment of pharmacoresistant epilepsies of extratemporal origin. OBJECTIVE: To analyze clinical and epileptological results and to provide prognostic factors influencing seizure outcome. METHODS: This retrospective single-center study comprises a consecutive series of 383 patients, most of whom had an identifiable lesion on MRI, who underwent resective surgery for extratemporal epilepsy. Data including diagnostic modalities, surgical treatment, histopathology, prognostic factors, and epileptological outcome were analyzed. RESULTS: Resective procedures were located as follows: frontal (n = 183), parietal (n = 44), occipital (n = 24), and insular (n = 24). In 108 cases resection included more than 1 lobe. Histopatholological evaluation revealed focal cortical dysplasias (n = 178), tumors (n = 110), cavernomas (n = 27), gliosis (n = 42), and nonspecific findings (n = 36). A distinct epileptogenic lesion was detected in 338 (88.7%) patients. After a mean follow-up of 54 mo, 227 (62.5%) patients remained free from disabling seizures (Engel class I), and 178 (49%) were completely seizure free (Engel class Ia). There was no perioperative mortality. Permanent morbidity was encountered in 46 cases (11.8%). The following predictors were significantly associated with excellent seizure outcome (Engel I): lesion visible on magnetic resonance imaging (MRI; P = .02), noneloquent location (P = .01), complete resection of the lesion (P = .001), absence of epileptic activity postoperatively (P = .001), circumscribed histological findings (P = .001), lower age at surgery (P = .008), and shorter duration of epilepsy (P = .02). CONCLUSION: Surgical treatment of extratemporal epilepsy provides satisfying epileptological results with an acceptable morbidity. Best results can be achieved in younger patients with circumscribed MRI lesions, which can be resected completely.

The stereotactic suboccipitaltranscerebellar approach to lesions of the brainstem and the cerebellum.

OBJECTIVE: The stereotactic suboccipital-transcerebellar approach is widely regarded as technically demanding requiring substantial modifications of the standard stereotactic methods thus often making a transfrontal approach preferable. In this comprehensive series we aim to present our experience with the stereotactic suboccipital-transcerebellar approach to lesions of the brainstem or cerebellum using two standard stereotactic systems. PATIENTS AND METHODS: In the period of 2000-2015 overall 80 patients (mean age 43.95 ±â€¯23.76 years) with lesions of the brainstem or cerebellum underwent stereotactic surgery for diagnostic or therapeutic purposes via a suboccipital approach. In 59 patients stereotactic surgery was performed using the Riechert-Mundinger Stereotactic Frame, the Leksell Stereotactic Frame was used in 21 patients. For both frames standard systems were used without modification. Retrospective analysis of intraoperative stereotactic technique, achievement of the predefined surgical objectives and perioperative complications was carried out. RESULTS: In this series, the stereotactic suboccipital-transcerebellar approach proved to be feasible with two standard stereotactic systems. Using either frame the predefined surgical objective was achieved in 90.0%. A verified neuropathological diagnosis was obtained in 89.6%. Minor transient perioperative complications occurred in 8.75%. There was no surgery-related permanent morbidity or mortality. CONCLUSION: In this comprehensive series the stereotactic suboccipital-transcerebellar approach using a standard stereotactic system proved to be a favorable stereotactic approach with a high diagnostic success rate and no surgery-related permanent morbidity.

Disorganization of neocortical lamination in focal cortical dysplasia is brain-region dependent: evidence from layer-specific marker expression.

BACKGROUND: Focal cortical dysplasias (FCD) are local disturbances of neocortical architecture and a common cause of pharmaco-resistant focal epilepsy. Little is known about the pathomechanisms leading to architectural abnormalities associated with FCD. RESULTS: In the present study we compared 52 FCD cases originating from the frontal or temporal lobe with or without Ammon's horn sclerosis (AHS) with regard to structural and molecular differences. We applied layer-specific (ER81, RORß, SMI32, TLE4) and interneuron (calbindin, parvalbumin) markers by means of immunohistochemistry, in situ hybridization (ISH), and real time RT-PCR and correlated our findings with clinical parameters. We found that: (1) Structural abnormalities were most prominent in layers III-VI including changed morphology of individual neurons or dispersion, blurring and thinning of layers. These alterations were most pronounced in isolated frontal FCD, whereas the most homogeneous group was FCD IIIa. (2) Numbers of calbindin- and parvalbumin-positive interneurons varied considerably within the different FCD groups, but were not generally reduced. A significant decrease was only found for calbindin-positive interneurons in frontal FCD, and for parvalbumin-positive interneurons in FCD IIIa. (3) Interestingly, FCD IIIa presented with significant changes in the numbers of calbindin- or TLE4-positive neurons when compared to isolated FCD or controls. (4) Correlations between clinical and cellular parameters strongly depended on FCD localisation and age of the patients. CONCLUSIONS: In summary, our data suggest that late cortical development is disturbed in FCD, yet most likely by different causes depending on brain region, FCD type and FCD severity.

Evaluation of the automated hematology analyzer Sysmex XT-2000iV ™ compared to the ADVIA ® 2120 for its use in dogs, cats, and horses. Part II: Accuracy of leukocyte differential and reticulocyte count, impact of anticoagulant and sample aging.

The automated laser-based hematology analyzer Sysmex XT-2000iV™ provides a 5-part differential count and specific cytograms that are of great interest for large veterinary laboratories. The aim of the study was to validate the Sysmex XT-2000iV compared to the laser-based hematology analyzer ADVIA® 2120 and manual differential in dogs, cats, and horses as well as the impact of anticoagulant (heparin, ethylenediamine tetra-acetic acid [EDTA], and citrate) and storage at 22°C and 4°C. Consecutive fresh K(3)-EDTA blood samples from 216 cats, 314 dogs, and 174 horses were included. The impact of anticoagulant and sample storage was assessed in specimens obtained from an additional 9 cats, 10 dogs, and 10 horses. Agreement between both analyzers was excellent to good except for monocytes and canine reticulocytes. Spearman rank correlation coefficients (r (s)) between Sysmex XT-2000iV and manual differential were good to fair and ranged from 0.91 (cat lymphocytes) to 0.44 (cat monocytes). Hematocrit value (Hct), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC; all: P < 0.001), and mean corpuscular volume (MCV; P < 0.01) were higher in canine citrated blood compared to heparin and EDTA. In cats, lymphocytes and monocytes were lower in heparinized blood compared to EDTA (P < 0.05), whereas in horses no significant effect was seen. Regarding storage time and temperature, white and red blood cell counts, hemoglobin, and MCH were stable. Hct, MCV, and MCHC were influenced by erythrocyte swelling. Differential count remained stable for 24 hr (22°C) and nearly 72 hr (4°C) except for monocytes. The overall performance of the Sysmex XT-2000iV was excellent and compared favorably with that of the ADVIA 2120. A special strength was the excellent detection of feline eosinophils.

Evaluation of the impedance analyzer PocH-100iV Diff for analysis of canine and feline blood.

BACKGROUND: An automated impedance-based in-house hematology analyzer, the PocH-100iV Diff, which provides a 3-part leukocyte differential count that includes eosinophils, recently has been introduced. OBJECTIVES: The aims of this study were to validate results from the PocH-100iV Diff for dogs and cats and evaluate the impact of the anticoagulant used and sample storage conditions. METHODS: Blood samples collected in K(3) EDTA from 153 cats and 150 dogs were included in the comparison study. The reference analyzer was the ADVIA 2120 hematology analyzer, and manual differential leukocyte counts and PCV were the manual reference methods. RESULTS: Coefficients of variation were < 3% except for platelet counts and feline differential and eosinophil counts. Correlation between analyzers was good to excellent except for hemoglobin (HGB) concentration in dogs and RBC indices for both species. Biases were close to 0 except for MCHC and platelet counts. Correlation with manual counts was good for lymphocytes and OTHR cells (combined neutrophil and monocyte counts) and fair and poor for feline and canine eosinophil counts, respectively. Estimated sensitivity and specificity for detection of eosinophilia were, respectively, 50% and 98% for cats and 34% and 77% for dogs. A significant anticoagulant effect was seen for MCV in cats and for HCT, MCH, MCHC, and platelet, OTHR, and eosinophil counts in dogs. RBC and WBC counts, HGB concentration, and MCH were stable for 72 h. HCT, MCV, MCHC, and platelet counts were affected by sample storage (dogs > cats; 22°C > 4°C). CONCLUSIONS: The PocH-100iV Diff is a suitable in-house instrument. A strength is its specific, but moderately sensitive, detection of feline eosinophils.

Evaluation of the automated hematology analyzer Sysmex XT-2000iV™ compared to the ADVIA® 2120 for its use in dogs, cats, and horses: Part I--precision, linearity, and accuracy of complete blood cell count.

The automated laser-based hematology analyzer Sysmex XT-2000iV™ providing a complete blood cell count (CBC) and 5-part differential has been introduced in large veterinary laboratories. The aim of the current study was to determine precision, linearity, and accuracy of the Sysmex analyzer. Reference method for the accuracy study was the laser-based hematology analyzer ADVIA® 2120. For evaluation of accuracy, consecutive fresh blood samples from healthy and diseased cats (n = 216), dogs (n = 314), and horses (n = 174) were included. A low intra-assay coefficient of variation (CV) of approximately 1% was seen for the CBC except platelet count (PLT). An intra-assay CV ranging between 2% and 5.5% was evident for the differential count except for feline and equine monocytes (7.7%) and horse eosinophils (15.7%). Linearity was excellent for white blood cell count (WBC), hematocrit value, red blood cell count (RBC), and PLT. For all evaluated species, agreement was excellent for WBC and RBC, with Spearman rank correlation coefficients (r(s)) ranging from >0.99 to 0.98. Hematocrit value correlated excellently in cats and dogs, whereas for horses, a good correlation was evident. A good correlation between both analyzers was seen in feline and equine PLT (r(s) = 0.89 and 0.92, respectively), whereas correlation was excellent for dogs (r(s) = 0.93). Biases were close to 0 except for mean corpuscular hemoglobin concentration (4.11 to -7.25 mmol/l) and canine PLT (57 × 10(9)/l). Overall, the performance of the Sysmex analyzer was excellent and compared favorably with the ADVIA analyzer.