Combined intra-operative monitoring of hearing by means of auditory brainstem responses (ABR) and transtympanic electrocochleography (ECochG) during surgery of intra- and extrameatal acoustic neurinomas.

BACKGROUND: Although being established as a standard procedure in intra-operative monitoring in acoustic neurinoma surgery, auditory brainstem responses (ABR) represent a far-field technique bearing some technical limitations. This prospective study was designed to evaluate electrocochleography (ECochG) as a supplementary tool for hearing preservation. METHOD: 84 patients with unilateral intra-/extrameatal acoustic neurinomas (extrameatal diameter: 5-55 mm) preserving serviceable hearing, were operated on using a combined (neuro-/otosurgical) suboccipital approach. ECochG was recorded simultaneously to ABR following transtympanic insertion of a steel needle electrode into the promontory under otoscopic view. FINDINGS: Serviceable hearing (Class 1-3 according to Gardner/Robertson) was preserved in 43 out of 84 patients (51.2%), of whom 40 showed both ECochG and ABR being preserved. All 24 patients with loss of both modalities became deaf. Hearing preservation was observed in 4 out of 12 patients with preserved ECochG but loss of ABR (waves III-V). The reverse was observed in 2 cases with postoperative deafness. While both ECochG and ABR amplitudes were significantly correlated with pre- and postoperative hearing, latencies of ECochG summating (SP) and action potential (AP) proved to be more reliable indicators for preserved hearing than ABR (peak I/III/V) latencies. The predictive value of baseline ABR amplitudes for postoperative hearing, however, was superior to ECochG parameters. Only in large neurinomas (extrameatal diameter: >2 cm) tumour size was found to be a significant predictor for the preservation of hearing. Apart from three cases with postoperative otoliquorrhea and one further case presenting with local bleeding within the external acoustic meatus, no side effects were observed. CONCLUSIONS: In combination with ABR monitoring, ECochG proved to be a useful supplementary tool for hearing preservation in acoustic neurinoma surgery. It is particularly helpful during electrocautery and drilling, since no averaging is required. Special applications are: (1) small tumours with good serviceable hearing; (2) and/or a large intrameatal portion; (3) cases with lost or endangered contralateral hearing (e.g. bilateral acoustic neurinomas), when the preservation of poor or even non-functional hearing is desirable.

Intra-operative electromyographic monitoring of the lower cranial motor nerves (LCN IX-XII) in skull base surgery.

The functional preservation of lower (motor) cranial nerves (LCN) is endangered during skull base surgery. Intra-operative EMG monitoring of the LCN IX-XII was investigated in 78 patients undergoing 80 operations on various skull base tumors with regard to technical feasibility and clinical efficacy. Ongoing 'spontaneous muscle activity' (SMA) and 'compound muscle action potentials' (CMAP) following supramaximal bipolar stimulation were intra-operatively recorded applying needle electrodes into the soft palate (CN IX: n=76), the vocal cord (CN X: n=72), the trapezius muscle (CN XI: n=18), and the tongue (CN XII: n=71). From 24/22/8 cases with LCN IX/X/XII deficits (despite monitoring) only 5/6/4 remained unchanged (3-6 months postoperative). An irreversible plegia of the LCN IX/X/XII occurred in three (1/1/1) patients. In 7/6/1 patients postoperative (3-6 months) LCN IX/X/XII function was better than preoperatively. In all patients accessory nerve function remained unchanged. 'Pathological' SMA of the LCN IX/X/XII occurred in 12/16/8 cases, but in only 6/5/3 cases corresponded to postoperative LCN deficits. Corresponding 'pathological' SMA patterns were found in 18/17/5 out of 24/22/8 cases with postoperative LCN IX/X/XII dysfunction. Reproducible CMAP of LCN IX/X/XI/XII could be recorded in 59/56/11/32 patients. Approximate 'normal' values were calculated and compared to (very few) data so far given in the literature. Electromyographic monitoring proved to be a safe tool for the intra-operative identification and localization of the LCN contributing to their anatomical and functional preservation. The predictive value of standard neurophysiological parameters for functional outcome, however, is limited.

Vascular endothelial growth factor-driven glioma growth and vascularization in an orthotopic rat model monitored by magnetic resonance imaging.

OBJECTIVE: The goal of the present study was to develop an orthotopic in vivo model for the investigation of vascular endothelial growth factor (VEGF)-dependent glioma growth and vascularization. METHODS: C6 glioma cells were infected with viruses encoding sense or antisense VEGF. Expression of the transgene was controlled by Northern blot analysis, Western blot analysis, and immunohistochemistry. Spheroids generated from both clones as well as from wild-type and mock-transfected cells were implanted in the brains of Sprague-Dawley rats. Growth and vascularization were assessed using magnetic resonance imaging after 7 and 11 days. Histology was studied using hematoxylin and eosin staining, immunohistochemistry with anti-von Willebrand staining, anti-VEGF, anti-CD8, and assessment of vessel density. RESULTS: Cell proliferation, migration, and invasion in vitro were very similar in all cell clones. Sense gliomas demonstrated by far the fastest growth in vivo, with intense contrast enhancement meeting criteria for highly malignant tumors. Histological examination revealed masses of von Willebrand- and VEGF-positive tumor vessels with a high vessel density. Antisense gliomas depicted the radiological features of low-grade gliomas, with slow growth and poor vascularization, although they were highly infiltrative. Wild-type and mock-transfected gliomas demonstrated similar growth and vascularization patterns intermediate between sense and antisense gliomas. Any influence of the allogeneic response of the hosts on different tumor sizes could be excluded. CONCLUSION: Our model elucidates glioma growth and vascularization as strongly VEGF dependent, which is consistent with human gliomas. Thus, this model is suitable for testing antiangiogenic strategies to interfere with the VEGF/VEGF receptor system, as well as for exploring VEGF-independent mechanisms using the antisense-transfected clone.

Quantitative parameters of intraoperative electromyography predict facial nerve outcomes for vestibular schwannoma surgery.

OBJECTIVE: Facial nerve monitoring is an established method that is routinely used during cerebellopontine angle tumor surgery. The aim of this study was to determine quantitative electromyographic (EMG) parameters that were predictive of facial nerve outcomes. METHODS: In 137 patients with intra-/extrameatal vestibular schwannomas, the most proximal (the exit from the brainstem) and distal (the fundus of the internal auditory canal) parts of the facial nerve were stimulated after total tumor removal. A quantitative analysis of absolute values and ratios (proximal/distal) of evoked EMG parameters (amplitude, latency, and duration) was performed, and parameters were correlated with postoperative (1 and 6 wk and 6 mo) facial nerve function (FNF). RESULTS: Absolute values of EMG amplitudes were statistically correlated with FNF (P < 0.05). Amplitude ratios (proximal/distal) demonstrated an even greater predictive power. The risk of exhibiting facial palsy 6 months after surgery increased from 1.6% (amplitude ratio of >0.8) to 75% (ratio of <0.1). For EMG latencies, only the ratios revealed a significant correlation with FNF. The latency ratio-dependent risk of facial palsy after 6 months increased from 2.9% (ratio of <1.05) to 33% (ratio of >1.35). The durations of the muscle responses were not significantly correlated with clinical outcomes. CONCLUSION: The predictive power of the amplitudes and latencies of electrically evoked muscle responses could be improved by calculating proximal/distal ratios. The proximal/distal amplitude ratio proved to be the most powerful parameter for intraoperative assessment of postoperative FNF.

Models for assessment of angiogenesis in gliomas.

In the last two decades, much attention has been focussed on mechanisms of glioma vascularization including the investigation of growth factors and receptors involved. Recently, these efforts resulted in various approaches for antiangiogenic treatment of experimental brain tumors. These basic science and preclinical trials need an assortment of models, which should allow investigating a variety of questions. Several objectives concerning basic endothelial cell (EC) characteristics can adequately be studied in vitro using EC monolayer assays. Three-dimensional spheroid techniques respect the more complex cell-cell and cell-environment interplay within a three-dimensional culture. To optimize the imitation of the crucial interaction of human gliomas with host endothelial cells, immunological cells and extracellular matrix, animal models are mandatory. An essential rule is to utilize an orthotopic model, since tumor-host interaction is organ specific. To avoid alloimmunogenic responses, it is desirable to use weakly or not immunogenic glioma grafts, what is best accomplished in a syngeneic model. However, since rat gliomas poorly resemble human glioma growth patterns, human glioma xenografting into immunocompromized animals should be considered. In vivo monitoring techniques like videoscopy via a cranial window or magnetic resonance imaging (MRI) allow for functional studies and improve the validity of the model employed. Finally, it is essentially to recognize the limitations of each model considered and to select that model, which seems to be most appropriate for the objectives to be investigated.

Heterogeneous regional expression patterns of matrix metalloproteinases in human malignant gliomas.

The aim of the study was to assess the differential intra- and intertumoral heterogeneity and patterns of matrix metalloproteinase expression in human glioblastomas in vivo. 12 glioblastoma samples were analyzed for MMP expression by semi-quantitative RT-PCR. A total of 56 samples (8 adjoining regions of 6 glioblastoma tumors) were immunohistochemically examined for the expression and regional distribution of gelatinase-A (MMP-2), gelatinase-B (MMP-9), matrilysin (MMP-7) and stromelysin-1 (MMP-3). Gelatinase-A mRNA was detected in all samples, gelatinase-B was found in numerous samples. Correspondingly, strong expression levels of both gelatinase protein was seen in immunohistochemistry. Gelatinase-A was expressed by both tumor cells and endothelium while gelatinase-B was found to be restricted to endothelial cells. Stromelysin-1 protein was not detected in any of the samples. Matrilysin was found around tumor cells of three samples from one patient only. The strong immunoreactivity seen for gelatinase-A around tumor cells and blood vessels suggests a role in both tissue degradation and tumor neoangiogenesis which is in accordance with previously published in vitro data. The marked localization of gelatinase-B to the endothelium and its presence in non-infiltrative benign lesions, however, makes a direct proteolytic role of gelatinase-B on ECM components during glioma invasion appear unlikely. Its close association with vascular structures, however, might indicate a link to neoangiogenesis. The significance of matrilysin which was only seen in tumor cells in three samples remains unclear. Stromelysin-1, though strongly expressed in cell lines, does not appear to play a role in glioblastoma tumors in vivo.

Different vascular patterns of medulloblastoma and supratentorial primitive neuroectodermal tumors.

Astrocytoma vasculature patterns differ according to histological grade of malignancy with glioblastoma multiforme (WHO grade IV) showing most extensive endothelial proliferation. Here, we determined whether the vascular patterns of medulloblastoma and supratentorial primitive neuroectodermal tumors (PNETs), which can be hardly distinguished histopathologically, differ. We evaluated the spatial organization of vessels in medulloblastomas and PNETs using antibodies to von Willebrand factor (vWF) and CD34. Medulloblastoma capillaries showed slight endothelial cell hyperplasia. Microvessels sprouted from the capillaries and formed glomeruloid clusters. There were areas with chains of unopposed endothelial cells (3-10 cells). Supratentorial PNETs had highly branched capillaries with extensive endothelial cell hyperplasia. Glomeruloid arrays of microvessels extended from the capillaries. Small fragments of endothelial tubes were scattered throughout the tumor. Therefore, medulloblastomas and supratentorial PNETs showed different spatial organization of tumor vessels which can be used for differentiation of each tumor entity. These vascular patterns may reflect different tumor derived angiogenic stimuli.

Cell-extracellular matrix interaction in glioma invasion.

Astrocytic tumours of the central nervous system express cell adhesion receptors of the integrin superfamily, CD44 and adhesion receptors of the immunoglobulin superfamily. Glioma cells utilize these receptors to adhere to and migrate along components of the extracellular matrix (ECM), which is uniquely distributed and regulated within the brain and the spinal cord. For penetration into healthy brain tissue a number of proteases are expressed, which degrade proteins of the extracellular matrix. Thus, glioma cell invasion into the adjacent brain tissue is dependent on the interaction of glioma cells with the extracellular matrix and the subsequent destruction of matrix barriers. There is a critical balance between expression of various adhesion receptors and proteases. The tight regulation of critical levels of proteases and receptors expressed by glioma cells or other cells is necessary for the "physiological" behaviour of glioma cells. Shifts in the balance of protein expression determine glioma cell behaviour in their micro-environment and can initiate or influence the complex process of glioma cell invasion. The complex receptor-ECM interaction in glioma cell invasion is discussed focussing upon the role of integrin receptors and matrix-metalloproteinases. Influencing these molecules or their regulation may lead to novel therapeutic approaches in the treatment of malignant glioma.

Vascularization of human glioma spheroids implanted into rat cortex is conferred by two distinct mechanisms.

Aim of this study was to develop and characterize an applicable in vivo model to investigate angiogenesis of human gliomas. An established glioblastoma spheroid model was used to investigate the neovascularization of a standardized avascular solid tumor mass. Spheroids of two human glioma cell lines were labeled with an in vivo fluorescent dye. Single spheroids were implanted into the cortex of athymic rats. After 1, 3, 7, 14, and 21 days, brain sections containing the spheroid were immunostained for endothelial cells or vascular endothelial growth factor (VEGF). The dye-stained glioma spheroid and the endothelial cells were visualized by confocal microscopy. Two distinct mechanisms of tumor vascularization could be observed. (1) "Classical" angiogenesis with new vessels sprouting from existing host vessels into the spheroid was seen. (2) Individual endothelial cells were found to migrate towards and into the center of the spheroid where they coalesced to form new vessels. This process occurred as early as 24 hr after spheroid implantation. Spheroid vascularization was accompanied by an increase of VEGF expression, which peaked 7 days after implantation and returned to normal patterns by 14-21 days. Besides the "classical" angiogenesis by angiogenic blood vessels, the recruitment of individual endothelial cells seems to be an additional mechanism in early glioma vascularization. Our model proves to be a reliable, reproducible system to study in vivo angiogenesis of human gliomas.

ECM-mediated glioma cell invasion.

Cell adhesion receptors of the integrin superfamily, CD44, and adhesion receptors of the immunoglobulin superfamily are expressed by high-grade astrocytic tumors of the central nervous system. These receptors are critical for the invasion of these tumors in the nervous system. Glioma cells utilize these receptors to adhere to and migrate along the components of the extracellular matrix, which is uniquely distributed and regulated within the brain and the spinal cord. For this reason, glioma cell invasion into the adjacent brain tissue is dependent on the interaction of glioma cells with the extracellular matrix. The receptor-ECM component interaction is discussed, focusing on the role of cell adhesion molecules of the integrin family and CD44 in glioma cell adhesion and invasion.

Guanidinobenzoatase and UPA in high-grade human astrocytomas and after xenografting cell suspensions into the rat cerebral cortex: proteases for metastasis and disease progression.

BACKGROUND: Invasion and metastasis is aided by the secretion of guanidinobenzoatase, that cleaves the link peptide to fibronectin, and urokinase plasminogen activator (uPA), which initiates a molecular cascade to activate plasmin and collagenases. This process permits malignant cell migration through the extracellular matrix. MATERIALS: Original human astrocytomas were examined for guanidinobenzoatase and uPA. Suspensions of high-grade human astrocytomas were xenografted into pockets in host cerebral cortex for 1-7 days. RESULTS: A class of guanidinobenzoatase positive cells was observed in the original human astrocytomas and in tumor masses formed in the implantation pocket and around blood vessels. Secondary foci containing guanidinobenzoatase positive cells formed around blood vessels and individual positive astrocytoma cells migrated on the glia limitans along parallel and intersecting nerve fiber fascicles and the corpus callosum. uPA and GFAP were colocalized with guanidinobenzoatase. CONCLUSION: The high-grade astrocytomas reestablish themselves and maintain their characteristics as a tissue although grafted as individual cells.

Transfection and dye premarking of human and rat glioma cell lines affects adhesion, migration and proliferation.

It is assumed that a cell that is transfected for any gene addition or replacement or was premarked with a cell tracker dye retains the characteristics of the original cell. The following experiments compare the original C6 rat glioma cell line with C6 cells transfected with the retroviral plasmid LacZ, and the human glioma cell lines GaMG, U373, U251, and D54 with cells stained with tracker dyes (Dil and DiO). We tested adhesion, migration and proliferation. C6 cell transfection did not affect adhesion but decreased (p < 0.05) migration. Dil staining resulted in a significant decrease (p < 0.01) in adhesion in all cell lines but U251. After DiO staining human cell lines U373 and D54 displayed a decrease in adhesion (p < 0.01) whereas U251 and GaMG cells had enhanced adhesion (p < 0.01). Dye marking of C6, GaMG and U373 cells did not alter migratory capacity. In contrast, Dil and DiO reduced migration of U251 and D54 cells (p < 0.05). There was a decrease (p < 0.01) in proliferation of the human cell lines after Dil staining. Transfection or membrane dyes can alter basic cell characteristics. The assumption that a transfected or dye marked cell is the same as the original cell but with an additional gene or the presence of a dye in the membrane is untenable.

ECM dependent and integrin mediated tumor cell migration of human glioma and melanoma cell lines under serum-free conditions.

Collagen IV, laminin and fibronectin are constituents of the cerebral extracellular matrix (ECM), which is critical in glioma cell invasion. The aim of the present study was to evaluate the integrin dependent cell-matrix interactions of two tumors with different invasive properties under matrixfree conditions. Two human glioma (GaMG, U373) and melanoma (MV3, BLM) cell lines were grown in serum free medium. Immunofluorescence microscopy of collagen IV, laminin, and fibronectin was performed. The adhesion of monolayer cells and their migration out of multicellular spheroids was quantified for these ECM components. Integrin chains known to act as laminin receptors were blocked by specific antibodies in additional migration assays. All cell lines expressed all the ECM components under serum free conditions. Tumor cell adhesion and migration in both glioma and melanoma cell lines was increased by all the ECM components, laminin being the strongest promotor of migration. However, migration was dose dependent in gliomas, whereas melanomas revealed a dose optimum of 10 micrograms/ml laminin. Antibodies against alpha 3 integrins significantly reduced migration on laminin in all cell lines, anti-beta 1 in all cell lines except U373. Anti-alpha 2 in BLM showed a strong effect, anti-alpha 6 was a stronger inhibitor in glioma than in melanoma cells. Integrins are functionally involved in tumor cell locomotion on laminin. The blocking of laminin related integrin chains markedly reduces cell motility in a varying manner between the cell lines. Moreover, different cell lines utilize different integrins as the laminin receptor.