Brain tumour diagnostics using a DNA methylation-based classifier as a diagnostic support tool.

AIMS: Methylation profiling (MP) is increasingly incorporated in the diagnostic process of central nervous system (CNS) tumours at our centres in The Netherlands and Scandinavia. We aimed to identify the benefits and challenges of MP as a support tool for CNS tumour diagnostics. METHODS: About 502 CNS tumour samples were analysed using (850 k) MP. Profiles were matched with the DKFZ/Heidelberg CNS Tumour Classifier. For each case, the final pathological diagnosis was compared to the diagnosis before MP. RESULTS: In 54.4% (273/502) of all analysed cases, the suggested methylation class (calibrated score ≥0.9) corresponded with the initial pathological diagnosis. The diagnosis of 24.5% of these cases (67/273) was more refined after incorporation of the MP result. In 9.8% of cases (49/502), the MP result led to a new diagnosis, resulting in an altered WHO grade in 71.4% of these cases (35/49). In 1% of cases (5/502), the suggested class based on MP was initially disregarded/interpreted as misleading, but in retrospect, the MP result predicted the right diagnosis for three of these cases. In six cases, the suggested class was interpreted as 'discrepant but noncontributory'. The remaining 33.7% of cases (169/502) had a calibrated score <0.9, including 7.8% (39/502) for which no class indication was given at all (calibrated score <0.3). CONCLUSIONS: MP is a powerful tool to confirm and fine-tune the pathological diagnosis of CNS tumours, and to avoid misdiagnoses. However, it is crucial to interpret the results in the context of clinical, radiological, histopathological and other molecular information.

Genetic variations in VEGF and VEGFR2 and glioblastoma outcome.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are central components in the development and progression of glioblastoma. To investigate if genetic variation in VEGF and VEGFR2 is associated with glioblastoma prognosis, we examined blood samples from 154 glioblastoma cases collected in Sweden and Denmark between 2000 and 2004. Seventeen tagging single nucleotide polymorphisms (SNPs) in VEGF and 27 in VEGFR2 were genotyped and analysed, covering 90% of the genetic variability within the genes. In VEGF, we found no SNPs associated with survival. In VEGFR2, we found two SNPs significantly associated to survival, namely rs2071559 and rs12502008. However, these results are likely to be false positives due to multiple testing and could not be confirmed in a separate dataset. Overall, this study provides little evidence that VEGF and VEGFR2 polymorphisms are important for glioblastoma survival.

The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat.

We have used immunocytochemical methods to localize the homeodomain LIM protein Isl-1 in the adult rat. Isl-1 immunoreactivity is expressed in polypeptide hormone-producing cells of the endocrine system, in neurons of the peripheral nervous system, and in a subset of brain nuclei. Isl-1 is also expressed in a subset of motoneurons in the spinal cord and brain stem, but not in regions of the central nervous system involved in sensory function or in neocortical areas. The pattern of expression of Isl-1 suggests that this gene may be involved in the specification and maintenance of differentiated phenotypical properties of these cells.

Spontaneous regression of two putative supratentorial haemangioblastomas in one patient.

Supratentorial haemangioblastomas are exceedingly rare lesions. We report a patient with spontaneous regression of two suspected supratentorial haemangioblastomas after removal of one lesion. The patient was a 61-year-old man who had a generalised seizure. Investigation with MRI revealed three supratentorial lesions situated in the trigone, occipital and frontal locations. The lesion in the occipital area was surgically removed and the histopathology was consistent with a haemangioblastoma. MRI investigations performed 6 months and one year after the operation confirmed that the two remaining lesions had totally disappeared.

Extracellular superoxide dismutase deficiency and atherosclerosis in mice.

Lipoprotein peroxidation in the arterial wall has been implicated in atherogenesis. The superoxide radical is formed in arteries and can induce such oxidation. Extracellular superoxide dismutase (EC-SOD) occurs in high concentration in the vascular wall interstitium, and in this study, we examined the importance of the enzyme in atherogenesis. On an apolipoprotein E-null background, the limited aortic lesions induced by a 1-month atherogenic diet were larger in EC-SOD wild-type mice than in EC-SOD-null mice, whereas there were no differences between the EC-SOD genotypes in the larger lesions seen after 3 months on the diet or after 8 months on normal chow. Despite smaller or equal lesions in the EC-SOD-null mice, their cholesterol levels were somewhat higher. Also, on a wild-type background, there were no effects produced by the absence or presence of EC-SOD on atherogenic diet-induced aortic root lesions. The urinary excretion of the lipid peroxidation biomarker 8-isoprostaglandin F(2alpha) was related to the rates of atherogenesis in the mice but was not influenced by the EC-SOD genotype. Likewise, the EC-SOD status had no effect on the staining for oxidized low density lipoprotein epitopes in aortic root sections. Our findings suggest that EC-SOD has little influence on atherogenesis in mice.

Cortical neurogenesis in adult rats after transient middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: This study explored the possible occurrence of newly generated nerve cells in the ischemic cortex of adult rats after middle cerebral artery occlusion and reperfusion. METHODS: Nine- to 10-week-old male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion by the monofilament method. Rats received repeated intraperitoneal injections of the cell proliferation-specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Brain sections were processed for immunohistochemistry with an avidin-biotin complex-alkaline phosphatase and/or -peroxidase method. Brain sections processed with double-immunofluorescent staining were further scanned by confocal microscopy. RESULTS: Interspersed among the predominantly newly formed glial cells, some cells were double labeled by BrdU and 1 of the neuron-specific markers, Map-2, beta-tubulin III, and Neu N, at 30 and 60 days after stroke onset. These cells were randomly distributed throughout cortical layers II through VI, occurring with highest density in the ischemic boundary zone. Three-dimensional confocal analyses of BrdU and the neuron-specific marker Neu N confirmed their colocalization within the same cortical cells. CONCLUSIONS: This study suggests that new neurons can be generated in the cerebral cortex of adult rats after transient focal cerebral ischemia. Cortical neurogenesis may be a potential pathway for brain repair after stroke.

Cortical neurogenesis in adult rats after reversible photothrombotic stroke.

Neurogenesis occurs throughout life in the dentate gyrus of hippocampus and subventricular zone, but this phenomenon has rarely been observed in other brain regions of adult mammals. The aim of the current study was to investigate the cell proliferation process in the ischemically challenged region-at-risk after focal cerebral ischemia in the adult rat brain. A reversible photothrombotic ring stroke model was used, which features sustained hypoperfusion followed by late spontaneous reperfusion and a remarkable morphologic tissue recovery in the anatomically well defined somatosensory cortical region-at-risk. Twelve-week-old male Wistar rats received repeated intraperitoneal injections of the cell proliferation specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Immunocytochemistry of coronal brain sections revealed that the majority of BrdU-positive cells were of glial, macrophage, and endothelial origin, whereas 3% to 6% of the BrdU-positive cells were double-labeled by BrdU and the neuronspecific marker Map-2 at 7 and 100 days after stroke onset in the region-at-risk. They were distributed randomly in cortical layers II-VI. Three-dimensional confocal analyses of BrdU and the neuronal-specific marker Neu N by double immunofluorescence confirmed their colocalization within the same cells at 72 hours and 30 days after stroke induction. This study suggests that, as a potential pathway for brain repair, new neurons can be generated in the cerebral cortex of adult rats after sublethal focal cerebral ischemia.

Quantitative synaptology of functionally different types of cat medial gastrocnemius alpha-motoneurons.

The aim of this ultrastructural investigation was to study quantitatively the synaptology of the cell bodies and dendrites of cat medial gastrocnemius (MG) alpha-motoneurons of functionally different types. In electrophysiologically classified and intracellularly HRP-labelled MG alpha-motoneurons of the FF (fast twitch, fatigable), FR (fast twitch, fatigue resistant) and S (slow twitch, very fatigue resistant) types, the synaptic covering of the soma as well as that of dendritic segments located within 100 microns and at 300, 700, and 1,000 microns distance, respectively from the soma, was analyzed. The synaptic boutons were classified into the L-(apposition length > 4 microns) and S-types (< 4 microns) with spherical synaptic vesicles, and the F-type with flat or pleomorphic synaptic vesicles. The length of apposition towards the motoneuron membrane was measured for each bouton profile. Approximately 1,000 boutons contacted the soma and a similar number of boutons contacted the proximal dendrites within 50 microns from the soma. The number of dendritic boutons was larger at the 300 microns distance than at the 100 and 700 microns distances. The three types of motoneurons showed similar values for percentage synaptic covering and synaptic packing density in the proximal dendrites, while in the most distal dendritic regions the S motoneurons had more than 50% higher values for percentage covering, packing density and total number of boutons. The S motoneurons also exhibited a larger preponderance of F-type boutons on the soma. The ratio between the F- and S-types of boutons decreased somatofugally along the dendrites in the type FF and FR motoneurons, while in the S motoneurons it remained fairly constant.

Changes in size and dendritic arborization patterns of adult cat spinal alpha-motoneurons following permanent axotomy.

This study was performed to analyse quantitatively the changes in dimensions and dendritic branching patterns of adult cat spinal alpha-motoneurons following permanent axotomy, i.e., in a situation in which the transected motoraxons are prevented from reinnervating their peripheral target muscle. After transection and ligation of the medial gastrocnemius nerve of adult cats, homonymous alpha-motoneurons were intracellularly labelled with horseradish peroxidase and subjected to quantitative light microscopic analyses. The cell bodies and proximal dendrites were studied at 3, 6, and 12 weeks after the axotomy. An initial increase in cell body size at 3 weeks was followed by a gradual return towards normal values. The mean diameter of the stem dendrites was decreased at all time periods studied, and the combined diameter of the stem dendrites was reduced at 12 weeks after the axotomy. Entire dendritic trees were reconstructed at 12 weeks postoperatively, and the regression equations describing the correlations between dendritic stem diameter, on one hand, and the size of the entire dendrite, on the other, were used to calculate the total dendritic length, volume, and membrane area of whole axotomized motoneurons. The dendritic branching patterns were also analysed. In comparison with normal medial gastrocnemius alpha-motoneurons, the dendritic membrane area and volume of the axotomized cells had decreased by 36% and 29%, respectively, at 12 weeks after the axotomy. This reduction in dendritic size was due to a loss of preterminal and terminal dendritic segments. Abnormal dendritic elongations were observed in 2 of 16 completely reconstructed dendrites.

Restorative effects of reinnervation on the size and dendritic arborization patterns of axotomized cat spinal alpha-motoneurons.

In a preceding paper [Brännström, et al. (1992) J. Comp. Neurol. 318:439-451] a marked reduction in dendritic size was observed in cat spinal motoneurons following permanent axotomy. The aim of the present study was to analyse the possible restorative effects of peripheral reinnervation on the size and dendritic branching patterns of cat spinal motoneurons which had been deprived of neuromuscular contact for an extended period of time. In adult cats the medial gastrocnemius (MG) nerve was transected and ligated. After 6 weeks the nerve was allowed to reinnervate its muscle through a nerve graft. With approximately 6 weeks needed for muscle reinnervation [Foehring, et al. (1986) J. Neurophysiol. 55:947-965], the MG motoneurons were devoid of neuromuscular contact for altogether about 12 weeks. Two years later reinnervated MG alpha-motoneurons were intracellularly labelled with horseradish peroxidase to allow quantitative analyses of the cell bodies and dendritic trees. Comparisons were made with previous data from normal and permanently axotomized MG motoneurons. The reinnervated motoneurons exhibited positive correlations between dendritic stem diameter, on one hand, and combined length, volume, membrane area, and number of end branches of the whole dendrite, on the other. By using the regression equations for these correlations, the total dendritic size of whole reinnervated motoneurons could be estimated. Such calculations showed that in comparison with the reduction in dendritic size found at 12 weeks after permanent axotomy (Brännström et al., see above), peripheral reinnervation caused the dendritic volume and membrane area to return to normal values. However, the values for combined dendritic length and number of dendritic end branches were still reduced by more than 25% as compared to the normal situation. The results indicate that following reinnervation of the target muscle, the axotomized motoneurons did not recover their original number of dendritic branches. The normalization of dendritic membrane area and volume was instead accomplished by two other mechanisms, namely an increase in dendritic diameters and an increased number of dendrites per neuron.