Interrelations of laser doppler flowmetry and brain tissue oxygen pressure during ischemia and reperfusion induced by an experimental mass lesion.

The objective of this study was to assess interrelations between bilateral changes of cortical laser doppler flowmetry and intraparenchymal, subcortical partial tissue oxygen tension in the course of an experimental trauma. Ten animals served as a sham group, 8 Sprague-Dawley rats received an unilateral, focal parietal mass lesion. The bilateral course of cortical blood flow measured by laser doppler flowmetry (LDF) was correlated with subcortical, intraparenchymal partial tissue oxygen tension [p(ti)O2]. In the sham-operated group, laser doppler mean flow values drifted between 9.0% and 9.5% and showed no significant changes over time neither between the hemispheres nor within each hemisphere. Absolute mean p(ti)O2 in sham-operated animals was 32.4 mm Hg in the left and 30.5 mm Hg in the right hemisphere. In the trauma group, mean laser doppler flow values during maximum brain compression decreased ipsilateral to 20.3% and contralateral to 34.4% of the baseline values. P(ti)O2 decreased ipsilateral from 25.9 to 6.6 mm Hg (25.4%) and contralateral from 22.6 to 9.8 mm Hg (43.6%). After balloon deflation, cortical LDF was restored much faster compared to p(ti)O2, but did not reach baseline values [ipsilateral 61.6% (p < 0.05); contralateral 75.8% of baseline values]. The p(ti)O2 values reached 25.2 mm Hg (97%) ipsilateral and 23.7 mm Hg (105%) contralateral. A temporary phase of reactive hyperemia occurred sporadically shortly after decompression. Both parameters showed a significant but rather weak correlation (r = 0.56; p < 0.001). Based upon these findings, we conclude that intraparenchymal, subcortical p(ti)O2 measurements supplemented on-line cortical CBF monitoring and score out discontinuous alternative measurement techniques in detecting hemodynamically relevant events. The small spatial resolution of LDF and p(ti)O2 probes, however, which in the small animal model may be of negligible influence, does raise the question whether the values obtained represent the microcirculatory situation of the human brain.

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.

Fast blue, a fluorescent tracer in glioma cell culture, affects cell proliferation and motility.

The azo-dye, Fast Blue (FB), initially employed for retrograde neuronal tracing is increasingly used in cell invasion and migration studies to detect living cells in monolayer and glioma tumor cell spheroid models. As yet, it is assumed that a cell stained with a tracker dye retains the characteristics of the original cell. The following experiments compared the adhesion, migration and proliferation properties of the cell lines U373 and GaMG with and without FB staining. FB staining reduced cell adhesion (P < 0.01) and proliferative activity (P < 0.01) and also had a significant inhibitory effect on cell migration (P < 0.001). From the results presented it follows that FB staining markedly influences basic cell characteristics.

Cervical disk syndromes: value of metrizamide myelography and diskography.

This paper reports on the respective diagnostic values of myelography with water-soluble contrast media and diskography in a study of 100 patients examined between 1979 and 1981 and operated on because of cervical disk disease. The results of the study led to a change of the diagnostic procedures formerly applied in radicular syndromes (i.e., diskography, and then perhaps myelography) and in cervical myelopathy (myelography, rarely followed by diskography). Now cervical metrizamide myelography is always performed first. Diskography is only indicated in radicular syndromes to determine the segment causing clinical symptoms when there is a polysegmental space-occupying lesion on the myelogram in combination with a mono- or oligoradicular neurologic symptomatology; or in the case of a normal myelogram with complaints resistant to conservative treatment.

Clinical experience with 118 brain tissue oxygen partial pressure catheter probes.

OBJECTIVE: We assessed the technical and diagnostic reliability of partial pressure of oxygen (PO2) of brain tissue (P(ti)O2) monitoring. The monitoring system and the catheter probes were tested in vitro, and clinical experiences obtained with 118 brain P(ti)O2 catheter probes, used in 101 patients, are reported. METHODS: The polarographic (LICOX; Medical Systems Corp., Greenvale, NY) P(ti)O2 catheter probe lies 22 to 27 mm below the dura level; its PO2-sensitive surface is 7.1 mm2. For 10 patients, the adaptation time (with initially unreliable signals after insertion) was determined. For 27 patients, the probe was removed in a stepwise fashion (three increments of 5 mm) and the heterogeneity of brain P(ti)O2 levels was investigated. After removal of the catheter probes, their PO2 and zero display error values were determined and compared with probe performance data obtained in vitro with unused PO2 catheter probes. RESULTS: Small iatrogenic hematomas were observed for two patients (1.7%). No infection occurred after 6.7 +/- 3.9 days (mean +/- standard deviation) of monitoring. The technical complication (dislocation or defect) rate was 13.6%. The mean adaptation time was 79.0 +/- 51.7 min. A flow chart is presented, which helps to rule out artifacts. The mean P(ti)O2 measured at 22 to 27 mm below the dura was 23.8 +/- 8.1 mm Hg, at 17 to 22 mm was 25.7 +/- 8.3 mm Hg, at 12 to 17 mm was 33.0 +/- 13.3 mm Hg (P < 0.01, compared with the initial value), and at 7 to 12 mm was 33.3 +/- 13.3 mm Hg (P < 0.01). Recent catheter probe versions exhibited a PO2 display error of -1.2 +/- 5.1% (mean +/- standard deviation, n = 38) and a mean zero display error of 1.1 +/- 0.9 mm Hg (n = 34). The greatest PO2 display errors were measured during the first 4 days of continuous monitoring. In the in vitro test (of 12 unused catheter probes), the maximal probe display error was 1.07 +/- 2.14%, tested at temperatures between 22 degrees C and 37 degrees C and tested at oxygen pressures of 0, 44, and 150 mm Hg. In vitro, the zero display error was -0.21 +/- 0.25 mm Hg. CONCLUSION: Brain P(ti)O2 monitoring, reflecting an area 17 to 27 mm below the dura, is a safe and reliable technique for monitoring cerebral oxygenation. Excluding the first 1 hour after insertion, data are reliable, with almost 100% good data quality.

Arachnoid cysts associated with subdural hematomas and hygromas: analysis of 16 cases, long-term follow-up, and review of the literature.

OBJECTIVE: Subdural hematomas and hygromas are infrequently encountered complications of arachnoid cysts of the middle cranial fossa and are particularly rare with cysts of other regions. Reports in the literature focus on casuistic observations. Therapeutic recommendations often include fenestration or extirpation of the cyst wall, in addition to evacuation of the space-occupying lesion. This study evaluates the results of and rationale for a more conservative approach, usually without cyst removal. METHODS: Sixteen cases of complicated arachnoid cysts, from a total of 658 patients with subdural hematomas or hygromas, were analyzed retrospectively together with 75 other cases reported in the literature. Additionally, 94 magnetic resonance imaging scans from 89 patients with untreated arachnoid cysts, from a total of 11,487 examinations, were reviewed for signs of hemorrhagic complications. RESULTS: Arachnoid cysts of the middle cranial fossa were found in 2.43% of patients with chronic subdural hematomas or hygromas. This indicated a fivefold greater prevalence of arachnoid cysts, compared with our magnetic resonance imaging-examined patient group. Only two patients with untreated cysts showed signs of hemorrhage in magnetic resonance imaging scans. An excellent or good therapeutic result was achieved with evacuation of the subdural fluid by drainage or craniotomy in 13 cases and with conservative treatment in two cases. Only one patient underwent additional fenestration of the cyst wall. No additional symptoms from the arachnoid cysts occurred in a follow-up period of up to 14 years after therapy. CONCLUSIONS: We do not generally consider it necessary to perform cyst diversion or fenestration at the time of drainage of a hematoma or hygroma in previously asymptomatic arachnoid cysts.

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.

Inverse correlation of cell proliferation and expression of progesterone receptors in tumor spheroids and monolayer cultures of human meningiomas.

OBJECTIVE: The progesterone receptor (PgR) can be detected in 60 to 70% of meningiomas using immunohistochemistry] in situ. Whereas in monolayer tissue cultures the PgR is only rarely expressed, we were able recently to demonstrate the preservation of the PgR in fragment spheroid cultures of meningiomas. The aim of the present study was to evaluate the stability of PgR expression in meningioma spheroids in vitro and the correlation of PgR expression and cell proliferation in spheroids and whether meningioma cells reaggregated to spheroids from monolayer cultures to reexpress the PgR again. METHODS: Tumor fragment spheroids (Weeks 1-6) and cell monolayers (Passages 1 and 3) of 15 PgR-positive meningiomas were investigated by immunohistochemistry for the expression of PgRs and their proliferative activity, as demonstrated by positivity for the proliferation-related antigen Ki-67. To study PgR reexpression in reaggregated spheroids, Northern blots were performed. In addition, a reverse transcriptase-polymerase chain reaction technique was established and evaluated in combination with immunohistochemistry. Growth of meningioma spheroids was quantified in the presence of progesterone and the specific antagonist onapristone. RESULTS: The PgR remained stable in spheroids for 6 weeks in 9 of 13 cases that were able to be evaluated. All tumor fragment spheroids exhibited a proliferation index of 5 to 40% Ki-67-positive cells. Monolayer cell cultures, on the other hand, failed to express PgRs but revealed higher proliferation indices (40-90%) to a significant extent. The detection of PgR messenger ribonucleic acid in reaggregated spheroids by means of reverse transcriptase-polymerase chain reaction correlated to the nuclear expression of PgR in immunohistochemistry. Neither progesterone nor its antagonist onapristone altered spheroid growth in vitro. CONCLUSION: The expression of the PgR in meningiomas is preserved in spheroid cultures with low proliferation indices for at least 6 weeks, whereas monolayer cell cultures with a high proliferative activity lack PgR expression. The inverse pattern of Ki-67-positive cells in the outer regions of the spheroids and PgR-expressing tumor cells in the spheroid centers leads us to the conclusion that proliferating meningioma tumor cells do not express PgRs. This might also explain why tumor cell growth in vitro was neither affected by progesterone nor by onapristone. Monolayer cell cultures can be reaggregated to spheroids, the consequence being a reexpression of PgRs and, therefore, a down-regulation of proliferation.

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.

Is there a benefit of preoperative meningioma embolization?

OBJECTIVE: To evaluate the effect of preoperative embolization of meningiomas on surgery and outcomes. METHODS: In a prospective study, 60 consecutive patients with intracranial meningiomas who were treated in two neurosurgical centers were included. In Center A, embolization was performed for none of the patients (n = 30). In Center B, 30 consecutive patients with embolized meningiomas were treated. Preoperatively, tumor size and location, neurological status, and Barthel scale score were recorded. In Center B, the extent of tumor devascularization was evaluated using angiography and postembolization magnetic resonance imaging. Intraoperatively, blood loss, the numbers of blood units transfused, and the observations of the neurosurgeon concerning hemostasis, tumor consistency, and intratumoral necrosis were recorded. Postoperatively, the neurological status and duration of hospitalization were recorded. Six months after surgery, the outcomes were assessed using the Barthel scale and neurological examinations. RESULTS: The mean tumor sizes were 22.9 cc in Center A and 29.6 cc in Center B (P > 0.1). The mean blood losses did not differ significantly (646 ml in Center A versus 636 ml in Center B; P > 0.5). However, for a subgroup of patients with subtotal devascularization (>90% of the tumor) on postembolization magnetic resonance imaging scans in Center B, blood loss was less, compared with the entire group in Center A (P < 0.05). The observations of the neurosurgeon regarding hemostasis, tumor consistency, and intratumoral necrosis did not differ significantly. There were no surgery-related deaths in either center. The rates of surgical morbidity, with permanent neurological worsening, were 20% (n = 6) in Center A and 16% (n = 5) in Center B. There was one permanent neurological deficit (3%) caused by embolization. CONCLUSION: In this preliminary study, only complete embolization had an effect on blood loss. The value of preoperative embolization for all meningiomas must be reconsidered, especially in view of the high costs and risks of embolization.

Retinoids inhibit human glioma cell proliferation and migration in primary cell cultures but not in established cell lines.

OBJECTIVE: Retinoids are known to exhibit a broad spectrum of biological activities, and they participate in the onset of differentiation and the inhibition of growth in a wide variety of cancer cells. Some of these vitamin A derivatives are already in clinical use. However, data on retinoid actions in glial tumors are rather sparse. Therefore, we studied the effects of the natural retinoic acid (RA) forms all-trans-RA, 9-cis-RA, and 13-cis-RA on glioma cell lines and primary cultures from patients with glioblastomas multiforme. METHODS: Six human glioma cell lines, one rat glioma cell line, and 20 primary cultures established from biopsies from patients with glioblastomas multiforme were investigated. Tumor cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell-counting assays. Random migration out of tumor spheroids was quantified using a video-morphometry system. Invasion was investigated using a confrontational coculture test system. Retinoid receptor (RA receptor [RAR]alpha, -beta, and -gamma and retinoid X receptor [RXR]alpha, -beta, and -gamma) expression status was determined using reverse transcription-polymerase chain reaction studies. RESULTS: Treatment of five human glioma cell lines with the different retinoids at concentrations up to 10(-5) mol/L produced no reduction of proliferation, using various incubation times. For one human glioma cell line (U343MG-A) and one rat glioma cell line (C6), which were previously reported to be sensitive to retinoids, we could confirm strong inhibitory effects on proliferation and clear changes in morphological features after retinoid treatment. Application of the different retinoids to low-passage primary cultures of human glioblastomas resulted in marked inhibition of proliferation (30-95%) for all tested samples. Using three-dimensional spheroid cultures, we detected retinoid-induced decreases in cell migration (24-65%). Invasion was not affected by these vitamin A derivatives. In an analysis of the expression patterns for retinoid receptors (RARs and RXRs), all primary culture samples yielded positive results for RAR gamma and RXR alpha and negative results for RAR alpha, RAR beta, and RXR gamma, whereas the results of RXR beta expression were heterogeneous among different patients. The cell lines, irrespective of their RA sensitivities, did not exhibit any major differences in receptor expression. CONCLUSION: Retinoids strongly inhibit proliferation and migration in primary cultures of human glioblastomas multiforme. Our data support a clinical trial of retinoids for the treatment of human malignant gliomas. We observed that most established cell lines were not sensitive to RA. This difference between long-term cell lines and primary cultures cannot be explained by different retinoid receptor expression patterns.

Surgical management of tethered spinal cord in adults: report of 54 cases.

OBJECT: The clinical features specific to tethered cord syndrome (TCS) in adults as well as factors determining outcome and prognosis have rarely been addressed systematically. The authors studied 56 patients, 54 of whom were treated surgically over the last 16 years. METHODS: In 17 patients who had been asymptomatic during childhood, TCS was diagnosed 8 years after onset of symptoms. Tethered cord syndrome was diagnosed 4 years after worsening in 39 patients with neurological signs or symptoms since childhood. The patients were followed for an average of 8 years. Features specific to adult-age presentation included nondermatomal pain aggravated by movement in 34 patients and conditions such as pregnancy and childbirth (in five of 11 pregnant patients). The most frequent tethering lesions were lipoma in 32, tight terminal filum in 28, and split cord malformation and secondary adhesions in 12 patients, respectively. Improvement or stabilization of symptoms at 6 months after surgery was noted in 46 (85%) of 54 patients. Improvement in pain status was most frequent (86%) followed by improvements in spasticity (71%), bladder dysfunction (44%), and sensorimotor deficits (35%). Factors associated with adverse outcome included preoperative duration of neurological deficits more than 5 years and incomplete untethering. On average, 8 (80%) of 10 patients with incomplete untethering developed recurrent symptoms 5 years after surgery compared with only seven (16%) of 44 patients in whom complete untethering was achieved. Seven patients underwent reoperation and in five of them stabilization of symptoms was attained. At a mean follow up of 8 years, 46 (85%) of the 54 surgically treated patients were in stable neurological condition, including those in whom reoperation was performed. CONCLUSIONS: Surgery for TCS is as beneficial in adults as it is in children. Its success depends on early diagnosis and complete untethering of the spinal cord.

Differential effects of vincristine and phenytoin on the proliferation, migration, and invasion of human glioma cell lines.

The aim of this study was to investigate the antimigratory and antiinvasive potential of vincristine sulfate (VCR) on human glioma cells and to analyze whether phenytoin (5,5-diphenylhydantoin; DPH) might act synergistically with VCR. Vincristine affects the cytoplasmic microtubules; DPH has been reported to enhance VCR cytotoxicity in murine cells. In two human glioma cell lines, GaMG and D-37MG, we found VCR to reduce monolayer growth and colony formation in a dose-dependent fashion at concentrations of 10 ng/ml and above. Phenytoin increased the cytotoxic and cytostatic effects of VCR in monolayer cells but not in spheroids. Multicellular spheroids were used to investigate directional migration. A coculture system of GaMG and D-37MG spheroids with fetal rat brain aggregates was used to analyze and quantify tumor cell invasion. A dose-dependent inhibition of migration and invasion by VCR was observed in both cell lines without further enhancement by DPH. Immunofluorescence microscopy with antibodies against alpha-tubulin revealed dose-dependent morphological alterations in the microtubules when the cells were exposed to VCR but not after incubation with DPH. Based on the combination of standardized in vitro model systems currently in use and the present data, the authors strongly suggest that VCR inhibits migration and invasion of human glioma cells. This is not altered by DPH, which inhibits cell proliferation in combination with VCR.

Progesterone receptors are detectable in tumor fragment spheroids of meningiomas in vitro.

Progesterone receptors (PgR) are hardly to be found in monolayer tissue culture of meningiomas although 60-70% of native tissue specimens are PgR positive. Thus we examined whether RgR might be detectable in fragment spheroids of meningiomas in vitro. 25 meningiomas of 17 women and 8 men were investigated as native tissue, monolayer cell culture (primary passage and passage 3) and as fragment spheroid culture after 1 and 3 weeks. 18/25 native tissue samples revealed the PgR. There was no prevalence of sex or histological subtypes discernible. PgR was preserved in cell clumps of the primary passage but was completely lost when cells grew as monolayers (primary passage and passage 3). 21 meningiomas formed spheroids in vitro. In 8 out of 15 native PgR positive tumors the fragment spheroids showed PgR on cryosections after 1 and 3 weeks of culture. We conclude that PgR are preserved in a considerable amount of tumor fragment spheroids of meningiomas in contrast to monolayer culture. Thus spheroids seem to be a suitable tool to investigate progesterone/anti-progesterone effects in meningiomas in vitro.

Invasive behaviour of human gliomas is mediated by interindividually different integrin patterns.

BACKGROUND: Glioma invasion is still a major obstacle for successful therapy. In the past we could demonstrate that glioma invasion is mediated by different adhesion molecules of the integrin family. Here we investigated whether a common pattern of integrin profiles might be involved, potentially providing a therapeutical avenue. MATERIAL AND METHODS: Multicellular spheroids were generated out of three human cell lines (GaMG, U373, U251) and from tumor biopsies of 9 human glioblastomas. After confrontation with rat brain aggregates, functional blocking antibodies against different integrin subunits (alpha 2, alpha 3, alpha v, alpha 1, alpha v beta 3, alpha v beta 5) or four different disintegrines (kistrin, echistatin, eristostatin, flavoridin) were added. Integrin patterns of the human cell lines/specimens were determined by FACScan or immunohistochemistry. RESULTS: In cell lines, antibodies against alpha 2, alpha 3, alpha v and alpha v beta 5 effectively reduced invasion into rat brain aggregates. Little effect could be observed with the anti-beta 1- or with anti-alpha v beta 3- antibodies. In primary tumor specimens, however, a different invasion pattern in regard to its integrin dependency emerged with antibodies against the alpha 3-chain or the alpha v beta 3 integrin being the most effective. Invasion of primary tumor tissue into the brain aggregates was by far more aggressive compared to that of the cell lines. Accordingly, it was less influenced by integrin antibodies. The disintegrines affected migration of glioma cells on purified ECM substrates in a heterogeneous matter, but had no impact on tumor invasion into brain aggregates. CONCLUSION: Although invasion of human gliomas is mediated by integrins, due to the heterogeneity of its dependency on different integrins this approach seems not to be appropriate to sufficiently alter glioma invasion in a therapeutical neuro-oncological setting.

Comparative amplification analysis of human glioma tissue and glioma derived fragment spheroids using reverse chromosome painting (RCP).

Tumor fragment spheroids (TFS) represent an organotypic in vitro model with preserved cytoarchitecture and matrix components of the native tumor in situ. In order to determine whether DNA amplifications within gliomas remain stable in spheroid culture, tumor fragment spheroids were established from 15 human gliomas including 14 glioblastoma and one anaplastic astrocytoma. Native tumor tissue, monolayers as well as TFS were evaluated for DNA amplification using reverse chromosome painting (RCP). A modified protocol for DNA isolation from TFS was established. Amplifications in the original tumor tissue were found on chromosomes 12q13-15, tel, 4q12-13 and 11p12-13, an amplification on 11p12-13 is reported for the first time. By RCP we could demonstrate that amplified domains on 12q13-15 and 4q12-13 in three tumors were maintained in TFS whereas the amplification on 11p12-13 could not be confirmed in TFS. In monolayer cultures, all amplifications which were detected in primary culture were lost until passage 5. The results of this first comparative study of DNA amplification in glioma by analyzing native tumor tissue and tumor fragment spheroids enables us to conclude that TFS seems to be a promising in vitro model for the study of DNA amplification under cell culture conditions.

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.

Inhibition of Ras farnesylation by lovastatin leads to downregulation of proliferation and migration in primary cultured human glioblastoma cells.

BACKGROUND: Malignant astrocytomas are the most common primary intracranial human tumors. All therapeutic approaches are limited due to their high proliferative capacity and their ability to diffusely invade the brain. Amplification of tyrosine kinase receptors and their signaling pathways have been implicated as contributing to the molecular pathogenesis of astrocytomas, providing possible new targets for therapeutic intervention. In particular, astrocytomas, although lacking oncogenic Ras mutations, have elevated levels of activated Ras. Lovastatin, an inhibitor of the beta-hydroxy-beta-methylglutary CoA reductase (HMG-CoA-reductase), is currently used to treat patients with hypercholesterolemia. In addition, it inhibits isoprenylation of several members of the Ras superfamily of proteins and therefore has multiple cellular effects including the reduction of proliferation. MATERIALS AND METHODS: In this study, we investigated the impact of lovastatin on two human glioma cell lines and on 15 primary cell cultures established from biopsies of patients with glioblastoma multiforme (GBM,) Proliferation of glioma cell lines and primary tumor cells was determined by cell counting and by using the MTT assay. The cell morphology was analyzed by staining of actin filaments with phalloidin. Apoptosis was measured using the TUNEL assay. To investigate the influence of this drug on glioma cell motility, tumor cell migration was investigated using three dimensional spheroid disintegration assays. In addition, tumor cell invasion was analyzed with a confrontational assay between tumor spheroids and rat brain aggregates. RESULTS: Inhibition of farnesyl biosynthesis using lovastatin led to a block in Ras mediated signaling, indicated by lower MAPK activity. Consequently, tumor cell proliferation was reduced up to 80%. Lovastatin appeared to decrease glioma viability by inducing apoptosis, as indicated by morphological changes and increase of TUNEL positive cells. Lovastatin acts through isoprenoid depletion, because supplementation of the media with 50-100 microM mevalonate restored all tau eta epsilon effects. Invasion of tumor cells into brain tissue was not effected while migration was reduced beta upsilon about 30-40% in cells treated with high concentrations (> or = 100 microM) of lovastatin. This was surprising because drug treatment at lower concentrations led to a disruption of the actin cytoskeleton, as indicated by Phalloidin staining. CONCLUSION: Our data strongly suggest that inhibition of elevated Ras activity by lovastatin effectively targets the MAPK and probably other signaling pathways thus offering a pharmacological based approach for a potential treatment of human astrocytomas.

Brain tissue pO2-monitoring: catheterstability and complications.

The authors report on the stability and complications of 73 LICOX brain ti-pO2-microcatheters in 70 patients. Mean monitoring time was 7.5 +/- 4.0 days. Patients prone to cerebral hypoxia (after severe head injury (GCS < 9) or a subarachnoid hemorrhage) had a ti-pO2-microcatheter inserted next to the ICP-probe in the typical frontal position. After the first 15 insertions, instead of the 3-way-screw (needing a 6 mm burrhole), a 1-way-screw (needing a 2.7 mm burrhole) was used for fixation in the bone; by doing so, the procedure can be performed in the ICU and takes only 15 min. Whenever possible a calibration at room air (to determine the sensitivity-drift) and in oxygen free solution (to determine the zero-drift) was performed after removal of the catheters. Ideally the expected pO2 at room air was around 154 mmHg (temperature dependent) and at zero calibration 0 mmHg. Mean sensitivity-drift for 54 catheters was -8.5 +/- 15.4%. Dividing the catheters into groups, depending on the duration of monitoring (1-4, 5-8 and 9-16 days), revealed that the greatest part of the (negative) sensitivity-drift occurred during day 1-4 after insertion. After 1 week of monitoring sometimes a positive drift occurred (being far less than the negative drift during the first 4 days). Compared to the old catheters (-10.3 +/- 17.3%) (on the first half of the patients) the new ones showed a lower sensitivity-drift (-6.8 +/- 13.4%). The zero-drift of 56 catheters was low with mean drift after 7.5 +/- 4.0 days of 1.5 +/- 1.5 mmHg. Here also the highest drift occurred on day 1-4 after insertion. No infection was seen and 2 times (2.7%) a small hematoma, not needing evacuation occurred. As the ti-pO2-catheter (having a smaller diameter) and the ICP-catheter were inserted at the same time, one cannot distinguish which catheter caused the hematoma. A possible explanation for the occurrence of the two hematomas is the insertion of the catheters too close to the midline. The authors conclude that LICOX ti-pO2-monitoring is a safe and reliable method. Further decrease of the complication rate and increase of the catheter-stability may be expected.