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Brain edema in gliomas is an epiphenomenon related to blood-brain-barrier (BBB) breakdown in which endothelial nitric oxide synthase (eNOS) plays a key role. When induced by vascular endothelial growth factor (VEGF), eNOS synthesizes nitric oxide that increases vascular permeability. We investigated the relationship between eNOS, VEGF and BBB dysfunction in experimental gliomas.Tumors were produced in Sprague-Dawley rats by transplacentary administration of Ethylnitrosourea (ENU). Immunoexpression of eNOS and VEGF(165) was studied to identify locations of vascular permeability. BBB permeability was evaluated using gadolinium and intravital dyes and BBB integrity by endothelial barrier antigen (EBA), glucose transporter-1 (GluT-1) and occludin immunostaining. Low grade gliomas displayed constitutive eNOS expression in endothelial cells and in VEGF-positive astrocytes surrounding vessels. Malignant gliomas overexpressed eNOS in aberrant vessels and displayed numerous adjacent reactive astrocytes positive for VEGF. Huge dilated vessels inside tumors and glomeruloid vessels on the periphery of the tumor showed strong immunopositivity for eNOS and a lack of occludin and EBA staining in several vascular sections. BBB dysfunction on these aberrant vessels caused increased permeability as shown by Gadolinium contrast enhancement and intravital dye extravasation.These findings support the central role of eNOS in intra- and peritumoral edema in ENU-induced gliomas.

Glioma growth depends on microvascular adaptation and angiogenesis. Our study focused on the structural changes that occur in the microvasculature to adapt to glioma growth. Vascular morphology, morphometry and permeability studies were performed in induced rat gliomas. Tumours were identified by magnetic resonance imaging and histopathology. Blood brain barrier integrity was examined by EBA and GluT-1 immunostaining and correlated with vascular permeability for gadolinium and intravital dyes. VEGF165 immunoexpression was also analyzed. Tumours were grouped in microtumours (6.69+/-0.99 mm3) displaying a homogeneous T2-w hyperintense signal corresponding to low-grade gliomas, and macrotumours (900.79+/-332.39 mm3) showing gadolinium contrast enhancement, intravital dye extravasation and histopathological features of high-grade gliomas. Results show that the microvascular network becomes aberrant as we move from micro to macrotumours. Vessel density decreases, whereas the relative area occupied by the vascular network increases. Microtumours display homogeneous angioarchitecture composed of simple and mildly dilated vessels similar to normal tissue. Macrotumours show different patterns, following a gradient from the neoangiogenic border to the hypoxic core. The tumour core contains scarce, huge, dilated vessels with some profiles co-expressing GluT-1 and VEGF165, the peripheral tissue shows light dilated vessels co-expressing EBA and GluT-1, and the border area displays glomeruloid vessels strongly positive for VEGF. Glucose uptake was maintained for some vascular endothelial sections in areas where BBB function was lost. In conclusion, during development of gliomas the microvasculature becomes aberrant, undergoing a sequence of adaptive changes which involve the distribution and permeability of vessels. This explains the disturbances of blood flow and the increased permeability.

Growth of solid tumors is highly dependent on angiogenesis. During tumor development, neoplastic cells switch to an angiogenic phenotype, playing a significant role in the expression of the vascular endothelial growth factor (VEGF). Seventy-two brain gliomas were induced in Sprague Dawley rats by prenatal exposure to ethylnitrosourea (ENU). Screening and location of tumors was carried out using magnetic resonance imaging (MRI). Conventional histology and immunocytochemistry for antibodies against glial fibrillary acidic protein (GFAP), S-100, NF, oligodendrocyte Ab-2, Ki-67, and VEGF165 were performed. The proliferation index (PI) was calculated from the Ki-67 labeling index, and the concentration of VEGF165 was quantified by enzyme-linked immunosorbent assay (ELISA). In vivo identification of macro- and microtumor appears to be useful to lead morphological and biochemical studies. Histopathology allows us to identify microtumors as classic oligodendrogliomas (CO; mean PI of 6.01 +/- 2.8%) and macrotumors as anaplastic oligodendrogliomas (AO; mean PI of 14.06 +/- 5%). Classic oligodendrogliomas show scarce VEGF165 expression whereas anaplastic ones display VEGF165 protein level 100-fold increased respect to CO. Astrocytes, neoplastic, and endothelial cells show differential immunostaining patterns from the border to the core of neoplasm. Positive structures for VEGF and their distribution vary according to PI increase. Anaplastic gliomas displaying VEGF-positive intratumor capillaries correspond to the highest PI values. To identify the "angiogenic switch," we propose the glioma stage characterized by VEGF immunopositive neoplastic cells inside the tumor and positive endothelial cells surrounding it.

Much clinical and biologic data have been processed in the search for useful objective parameters to predict brain tumor behavior. Seventy cases of astrocytic glioma collected by a single clinical team were studied using a full complement of clinical procedures: follow up (7 years), histologic analysis, DNA content estimation, and cell kinetics by flow cytometry. Proliferating cell nuclear antigen (PCNA) was determined by immunocytochemical-coupling flow cytometry (PFC) and also by counting under light microscopy (PIHC). A statistical evaluation was carried out to establish the usefulness of several parameters for glioma prognosis. The cases were histologically classified as 14 low-grade astrocytomas, 20 anaplastic astrocytomas, and 36 glioblastomas multiforme. The survival curve showed significant differences between histologic groups. Diploid populations were more frequent in low-grade astrocytomas, and aneuploid tumors often had increased S-phase and proliferative fractions. The PCNA-labeled index (PCNA-LI) increased with malignancy and correlated with histologic grading (P = 0.01). The PCNA-LI and age segregated low- from high-grade astrocytomas (including anaplastic astrocytoma and glioblastoma multiforme), but none of the variables considered differentiated anaplastic astrocytoma from glioblastoma multiforme. The Cox regression test displayed significant values for age, histologic diagnosis, and PCNA determinations when considered in tandem. Discriminant analysis obtained a function integrating age and specifically PIHC-LI to help in the prognosis of doubtful cases. The results emphasize the importance of parameters integrating different variables in an attempt to provide an accurate prognosis, the most significant being age, histopathologic diagnosis, and the proliferative fraction determined by PCNA.

Introduction: The homeostasis of tissues depends on a strict control of cell growth, differentiation and death. Several proteins, which are involved on the regulation of the cell cycle, can suffer diverse alterations and produce an uncontrolled cell proliferation and the genesis of a neoplastic process. The assessment of cell proliferation is an useful method applied to Neuro-oncology in order to know the behavior of gliomas., Development: This work is focussed on the analysis of different methods, all of them employed to study the cell proliferation: immunostaining of proliferating cell nuclear antigen (PCNA) and Ki-67, DNA content and ploidy by flow cytometry, in vitro incorporation of bromodeoxyuridine (BrdU) and the identification of apoptotic cells. The study of the DNA by flow cytometry establishes a relationship between ploidy and the prognostic of gliomas. The assessment of PCNA provides us with objective data about the proliferative activity of gliomas. Both Ki-67 expression and BrdU incorporation are also useful methods in the study of gliomas., Conclusions: In short, the most malignant gliomas are characterized by a high frequency of aneuploidies and high PCNA, Ki-67 and BrdU labelling indexes. All of these described methods can be used as prognostic markers complementary to the classic criteria employed nowadays.

The growth of solid tumors is highly dependent on vascular proliferation. Vascular endothelial growth factor (VEGF), the main mediator of angiogenesis, and platelet-derived growth factor receptor-beta (PDGFR-beta), receptor for the potent mitogen PDGF, are two indicators of the angiogenic potential of human gliomas. We studied a series of 57 surgical biopsies of astrocytic neoplasms by immunohistochemistry to elucidate the relationship between tumor proliferation, quantified as Ki67-LI, and the expression of these two proteins. Ki67-LI increases throughout histological malignancy, although staining in endothelial cells has rarely been recorded. Elevated amounts of VEGF-positive tumor cells (VEGF-LI) were found in anaplastic astrocytomas and glioblastomas, mainly around areas of necrosis, cysts, or edema. Endothelium of blood vessels was consistently stained. PDGFR-beta positivity was found in glomeruloid formations and in tumor cells, excluding pilocytic astrocytomas. Multinucleated giant cells and perivascular tumor cells were positive in glioblastomas. In addition, peritumoral microglia-like cells were also stained in some cases. Statistical correlation was only found between PDGFR-beta and Ki67 LIs. In conclusion, VEGF as permeability factor is involved in the development of secondary neoplastic changes, whereas PDGFR-beta is directly correlated to proliferation indexes. Strong expression of VEGF and PDGFR-beta found in endothelium and tumor cells would seem to support a combined role in tumoral neoangiogenesis.

The III ventricle is an uncommon location for choroid plexus papilloma at any age. We describe three new cases of choroid plexus papillomas of the III ventricle (CPPs). All children were boys under 4 months of age and all presented with increased intracranial pressure, hydrocephalus and macrocephaly. The three were examined by preoperative computed tomography (CT) and ultrasonography. Two of them were investigated with magnetic resonance imaging (MRI). The first case was treated with a right corticofrontal transventricular approach and subtotal resection, so that he required a second operation through a transcallosal approach. In the other two cases a transcallosal approach was used. Two children needed permanent ventriculo-peritoneal shunts. The average follow-up of 4.3 years has revealed no neurological deficits in any case. The timing of and the need for shunting are major considerations. Clinical and imaging follow-up (CT and/or ultrasonography) are very helpful in controlling postoperative hydrocephalus and subdural effusion, avoiding unnecessary shunting in many cases. The operative approaches, transcortical and transcallosal, are discussed.