Hydrocephalus shunt therapy: current titanium shunt valve implants obstructed by internal tissue proliferations identified as extracellular matrix membranes.

PURPOSE: Shunt valves, required for treatment of hydrocephalus, demand for high performance rates and lifelong excellent function. To overcome problems with traditional silicone materials, adjustable and gravity-adapted titanium valves were developed. Even modern shunt valve systems are still subject to occlusion. The aim of the present study was to investigate dysfunctional silicone and titanium valves for presence of cellular and proteinous materials inside the housings by means of histopathology. METHODS: A total of 19 explanted shunt valves from children between 2 and 182 months of age were investigated following dysfunction. After fixation in formalin and embedding in hard resin, slices were ground to a thickness of 5-30 µ. Besides standard histology, immunohistochemistry was performed using antibodies with markers for microglia, astrocytes, platelets, monocytes, and the proteins laminin, fibronectin, and collagen IV. RESULTS: Traces, layers, and plaques could be demonstrated in every investigated silicone or titanium valve with an implantation time of more than 6 days. Most of the tissue was found adjacent to silicone and titanium surfaces of the inner housing, the adjustment rotor, and ball-in-cone core. Markers for micro and astroglia stained positive in 40-60% of the specimen, mostly demonstrating a proteinous layer positive for laminin (80%), fibronectin (30%), and collagen IV (30%). CONCLUSIONS: Tissue reactions with formation of cellular and proteinous matrix components are common in obstructed silicone and titanium shunt valves. The tissue mimics astrocytic repair mechanisms genuine for basilar membrane matrix. The knowledge of these typical arachnoid patterns of colonization is a prerequisite for developing future shunt devices.

Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation.

Heavy riboflavin synthase of Bacillus subtilis was purified by a simplified procedure. The enzyme is a complex protein containing about 3 alpha-subunits (23.5 X 10(3) Mr) and 60 beta-subunits (16 X 10(3) Mr). The 10(6) Mr protein dissociates upon exposure to pH values above neutrality. Phosphate ions increase the stability at neutral pH. The dissociation induced by exposure of the enzyme to elevated pH is reversible in phosphate buffer at neutral pH. The stability of the enzyme at elevated pH values is greatly enhanced by the substrate analogue, 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. Electron micrographs of negatively stained enzyme specimens show spherical particles with a diameter of 15.6 nm. Various immunochemical methods show that the alpha-subunits are not accessible to antibodies in the native molecule. The native enzyme is not precipitated by anti-alpha-subunit serum, and riboflavin synthase activity is not inhibited by the serum. However, these tests become positive at pH values that lead to dissociation of the enzyme. Subsequent to dissociation of the native enzyme at elevated pH values, the beta-subunits form high molecular weight aggregates. These aggregates form a complex mixture of different molecular species, which sediment at velocities of about 48 S and 70 S. The average molecular weight was approximately 5.6 X 10(6). Homogeneous preparations have not been obtained. Electron micrographs show hollow, spherical vesicles with diameters of about 29 nm. The substrate analogue 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione can induce the reaggregation of isolated beta-subunits with formation of smaller molecules, which are structurally similar to native riboflavin synthase. A homogeneous preparation of reaggregated molecules was obtained by renaturation of beta-subunits from 6.4 M-urea in the presence of the ligand. The sedimentation velocity of this aggregate is about 7% smaller than that of the native enzyme. The molecular weight is 96 X 10(4). Electron micrographs show spherical particles with a diameter of about 17.4 nm. Inspection of the micrographs tentatively suggests the presence of a central cavity. It appears likely that these molecules, which are devoid of alpha-subunits, have the same number and spatial arrangement of beta-subunits as the native enzyme. All data are consistent with the hypothesis that the native enzyme consists of a central core of alpha-subunits surrounded by a capsid-like arrangement of beta-subunits. The number of beta-subunits and the shape of the protein suggest a capsid-like arrangement of beta-subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Heavy riboflavin synthase from Bacillus subtilis. Particle dimensions, crystal packing and molecular symmetry.

Heavy riboflavin synthase from Bacillus subtilis is an enzyme complex consisting of approximately three alpha-subunits (Mr 23.5 X 10(3)) and 60 beta-subunits (Mr 16 X 10(3)). The enzyme has been crystallized from phosphate buffer in a hexagonal crystal modification that belongs to space group P6(3)22. The asymmetric unit of the crystal cell contains ten beta-subunits. The structure of this unusual 10(6) Mr protein has been studied by small-angle X-ray scattering, electron microscopy of three-dimensional crystals, and crystallographic methods. The scattering curves can be interpreted in terms of a hollow sphere model with a ratio of inner and outer radius of 0.3:1. A diameter of 168 A was estimated from the scattering curves, in close agreement with electron microscopic studies. An aggregate with the stoichiometry beta 60, which was obtained by ligand-driven reaggregation of isolated beta-subunits, showed similar shape and dimensions, but a larger value for the ratio Ri/Ra. Electron micrographs of freeze-etched enzyme crystals showed approximately spherical molecules, which were arranged in hexagonal layers. The lattice constants found from the micrographs are in good agreement with the values derived from X-ray diffraction data. Rotation function calculations in Patterson space showed a set of peaks for 2-fold, 3-fold and 5-fold local rotation axes, accurately consistent with icosahedral symmetry and with the particle orientation A shown in the Appendix. The crystal packing can be described as follows: enzyme particles with icosahedral symmetry (point group 532) are located at points 32 of the hexagonal cell, corresponding to positions (0, 0, 0) and (0, 0, 1/2) on the 6-fold screw axes. From the data reported, it may be concluded that the enzyme structure can be described as an icosahedral capsid of 60 beta-subunits with the triangulation number T = 1. The alpha-subunits are located in the central core space of the capsid, but their spatial orientation is incompletely understood.

Brain metastases in neurosurgery: indications, surgical procedures and outcome.

Between July 1987 and June 1995, 1897 patients underwent operative therapeutic procedures for resection of tumours of the central nervous system in our department. 252 patients (13.3%) suffered from a metastatic disease in the brain or spinal cord. They comprised 113 females and 139 males. The age distribution ranged between 14 and 86 years with a median age of 58 years. The histology of resected tumours was distributed among lung-(54), breast-(33), melanoma-(27), kidney-(23) and prostate carcinoma (19). Other metastizing tumour sites were present in 96 cases. The age distribution of breast carcinoma ranged between 32 and 78 years with the median located at 54 years. 5 patients underwent a surgical resection of a local recurrence with a median latency of 9 months. Median survival time was estimated as 13 months with a one year survival of 63%. The aim of the study was to reflect on incidences, therapeutic procedures and outcome of brain metastatis.

Expression of CD 73 (ecto-5'-nucleotidase) in 165 glioblastomas by immunohistochemistry and electronmicroscopic histochemistry.

BACKGROUND: CD 73 (5'-nucleotidase) is an ectoenzyme, which is expressed on normal and neoplastic glial plasma membranes. The enzyme binds to intracellular filamentous actin and the extracellular matrix proteins laminin and fibronectin. CD 73 is a signalling pathway metabolite in the immune response of lymphocytes. The ectoenzyme catalyzes the conversion of purine and pyrimidine ribo- and deoxyribo-nucleoside monophosphates (AMP, GMP, IMP) and leads to elevation of the corresponding nucleosides (adenosine) in the extracellular space and might therefore modulate neuronal signalling and vascular perfusion. CD 73 has also been called a cellular motility factor. There is an increasing amount of evidence for the modulatory role of PKC-mediated CD 73 activity in ischemia, regeneration and repair, glioma cell proliferation and a possible invasion promoting feature of the ectoenzyme. The aim of the present study was to investigate the expression patterns of CD 73 together with the labelling of PKC and EGFR. The latter is known as a marker for primary glioblastomas. PATIENTS AND METHODS: We investigated the expression of CD 73 in 165 glioblastoma specimens together with the expression patterns of PKC and EGFR by immunocytochemistry on cryosections with a 4-step grading evaluation by two independent observers. CD 73 was further investigated morphologically by electron-microscopic histochemistry in cell cultures of glioblastoma specimens. RESULTS: With these methods it was possible to demonstrate a dense labelling pattern of glioblastoma specimens with anti-CD 73. 95.7% of the glioblastomas were identified with staining products, 63% with labelling grades 2 and 3. The dense staining of the endoplasmatic reticulum, vesicles, caveolar structures and glial membranes was demonstrated by electron-microscopic histochemistry. Some free enzymatic activity was located bound to the ECM components. We observed a significant coexpressions of CD 73 with PKC (p = 0.001) and CD 73 with EGFR (p = 0.022), which is a prospective marker for a high rate of early recurrency. CONCLUSIONS: The CD 73 activity was densely distributed on the membranes of glioblastoma cells in vivo and in cell cultures. The electron-microscopic histochemical studies could demonstrate enzymatic activity at the cell membranes and in vesicular structures and caveolae. Free staining deposits located on ECM components may result in a migration- and infiltration-promoting activity. The CD 73 expression could be correlated with the expression grades of PKC and EGFR. The latter has been identified as a prognostic factor which is expressed mainly on primary glioblastomas. PKC is a known tumour metabolite in several proliferation promoting pathways of EGF receptor signalling.

Expression of nitric oxide synthase isozymes (NOS I-III) by immunohistochemistry and DNA in situ hybridization. Correlation with macrophage presence, vascular endothelial growth factor (VEGF) and oedema volumetric data in 220 glioblastomas.

BACKGROUND: Nitric oxide (NO) is synthesized from arginine by three different isozymes of nitric oxide synthase (NOS I-III). NO has been identified as a powerful metabolite of vascular smooth muscle cell function, cerebral blood circulation and oedema induction. NOS induction by different cytokines has been shown previously in glioblastoma cell cultures and NOS III expression due to astrocytoma grading has been shown in several tumors recently. The aim of the present study was to study the coexpression of NOS I-III, macrophage and capillary presence with VEGF, EGF and their receptors and to investigate a possible mechanism in peritumoral oedema generation. MATERIALS AND METHODS: We have investigated the expression (4-grade values, blinded assay by two observers) of NOS I-III together with those of VEGF, VEGF- R (Flt-1), EGF-R1, von-Willebrand-factor (VWF) and a pan-macrophage marker (Ki-M1P) immunohistochemically in tumor specimens from 220 patients and performed tumor volume morphometry by image analysis in a subgroup of 32 cases to test for any correlation with the peritumoral oedema volumes. Inducible NOS II was further investigated by in situ labelling with a DNA oligonucleotide probe cocktail. RESULTS: All of the specimens revealed some NOS expression, NOS II was expressed in macrophages, microglia and endothelial cells, NOS III and I was localized in glioblastoma cells, NOS III in endothelial cells as well. The highest degrees of expression were observed in 46% (NOS I), 22% (NOS II) and 75% (NOS III) of all specimens. Inducible NOS II in any expression grade was observed in 47.5% of the specimens. Significant correlations were observed for the expression of the macrophage marker Ki-M1P with NOS II (p = 0.024), endothelial NOS III with NOS I (p = 0.0003), VEGF-R1 with NOS II (p = 0.0008) and NOS III (p = 0.011) The oedema volumes could not be correlated significantly with NOS or VEGF-R1 expression values but with those of endothelial staining (p = 0.02). We observed a trend towards higher Ki-M1P expression values together with higher oedema volume extensions. In situ hybridization demonstrated reaction products in endothelial and perivascular regions and sometimes scattered throughout the specimens revealing the labelling of macrophages. CONCLUSIONS: The main source of NO is NOS I and NOS III. The latter is located in endothelial cells and glioblastoma cells. The expression of NOS II in glioblastomas is restricted to infiltrating macrophages. NOS II and III expressions were observed significantly together with that of VEGF-R1. Neither NOS I-III nor VEGF-R expression could be correlated with the extension of the peritumoral oedema.

Expression of focal adhesion kinase (p125 FAK) and proline-rich tyrosine kinase 2 (PYK2/CAKb) in cerebral metastases, correlation with VEGF-R-, ecNOS III-labelling and morphometric data.

BACKGROUND: Cerebral metastasis occurs in about 20% of all neurosurgical patients. Cerebral metastases have a typical spherical morphology with a common central necrosis and perifocal oedema. It has been proposed that oedema extension, tumour volumes and infiltrative behaviour are partially mediated by vascular endothelial growth factor (VEGF) and nitric oxide (NO). In several systemic tumour entities NO is suggested as a factor which influences the metastatic potential. VEGF has recently been reported to influence the matrix related migratory activity by interaction with focal adhesion kinase (p125FAK) and proline-rich tyrosine kinase beta (PYK2/CAK beta). Nitric oxide, which is produced in metastases by three different NOS isozymes is capable of antagonizing the binding of FAK to matrix integrins. NO, VEGF and FAK/PYK 2 are therefore considered to be important mediators of the cerebral metastatic incidence, growth, infiltration and oedema extension. The aim of our present study was to investigate the expression of p125FAK and the coexpression with PYK2/CAK beta, VEGF-receptor FLT-1, NOS isozymes NOS I-III, capillary density and the histology in 130 specimens of resected cerebral metastatic tumours. A further analysis was performed to morphometrically evaluate tumour and oedema volumes and to correlate the immunohistochemical data in a subgroup of 40 patients. MATERIALS AND METHODS: Cryosections (N = 130) of metastatic resections were investigated immunohistologically using a 4-step scoring evaluation for the expression of NOS I-III, VEGF-receptor FLT-1, and capillary vessel presence by endothelial Von-Willebrand-Factor (VWF) staining. Tumour and oedema extension was measured in preoperative MRI (N = 40) scans by an image-processing device (Kontron) and the ratios of oedema volumes to total tumour volumes were calculated. The data were analysed statistically (Spearman rank order correlation and Kruskal-Wallis ANOVA) and correlated with the clinical data. RESULTS: FAK immunoexpression was observed in 50% of the specimens (31.2% gradings 2 and 3). We observed a significant coexpression (p = 0.0001) with PYK 2 labelling which occurred frequently in 74% of the specimens (42% gradings 2 and 3). The VEGF receptor FLT-1 could be detected in 70% of them, 24% at higher expression values 2 and 3. The expression of NO synthase was frequently observed. NOS I was detected in 83.6% of the specimens, values 2 and 3 in 40.5%. NOS III, the endothelial isoform, was observed in 39.4% of the specimens (gradings 2 and 3) and inducible NOS II in 29.4% (grading 2 and 3) of them. Coexpressions were statistically significant for FAK and NOS III (Spearman p = 0.008) and FAK and VEGF-R (p = 0.03). The morphometric evaluation resulted in tumour volumes between 2.0 and 83 cm3 (mean 22.5 +/- 19.1 SD) with oedema ratios between 0 and 100% (mean 62.2 +/- 22.5 SD). FAK expression correlated significantly (p = 0.06) with tumour volumes and histology. CONCLUSION: The frequent histotypic occurrence of FAK and PYK2 in metastases could be an important factor in the modulation of metastatic capacity and infiltrative behaviour and might influence the disease course. Judging from its frequent expression PYK2 may generate the more relevant signals. A further aspect is the possible interaction with endothelial NOS III and VEGF receptor, which could be important for the infiltrative behaviour in a latent hypoxic scenery and environment.

Oedema extension in cerebral metastasis and correlation with the expression of nitric oxide synthase isozymes (NOS I-III).

BACKGROUND: The development of a peritumoral oedema is a common radiological sign in preoperative CT- and MRI scans of patients with cerebral metastasis. Large tumours can be accompanied by a marginally extended oedema and vice versa. Several cytokines (VEGF) have been identified as mediators of vascular induction and permeability. Transmitters such as nitric oxide (NO) have been identified as specific mediators of vascular dilation and tumour blood flow in primary brain tumours in which different NOS isozymes (NOS I and III) are induced as a result of the latent hypoxic metabolic scenery. Other authors have considered NO as an endothelial stabilising metabolite. Inducible NOS II is expressed by microglia and macrophages invading during tumour growth. At present, no data exist on NO synthesising enzymes in cerebral metastasis. MATERIALS AND PATIENTS: Cryosections (N = 96) of metastatic resections were investigated immunohistologically using a 4-step grading evaluation for the expression of NOS I-III, VEGF-receptor FLT-1, a pan-macrophage marker Ki-M1P, and capillary vessel presence by endothelial Von-Willebrand-Factor staining. The tumour and oedema extension was measured in preoperative MRI scans by an image processing device (Kontron) and calculated for the ratios of oedema volumes to total tumour volumes. The data were analysed statistically (Pearson Chi2 and Kruskal-Wallis analysis of variances) and correlated with the clinical data. Inducible NOS II was further investigated by in situ hybridization with a (4x30 mer) DNA oligoprobe cocktail. RESULTS: Between 1987 and 1996 289 patients in our department suffered from a metastatic disease in the brain or spinal cord. In 96 cases resected tumour material was processed for the immunohistological investigation. The age distribution ranged from 14 to 85 years with a median age of 58 years. The mean duration of symptoms before diagnosis was estimated as 53 days. The expression of NO synthase was frequently observed. NOS I was detected in 83.6%, gradings 2 and 3 in 40.5% of them. NOS III, the endothelial isoform, was observed in 39.4% (gradings 2 and 3), inducible NOS II in 29.4% (grading 2 and 3) of the specimens. The VEGF receptor FLT-1 could be detected in 70% of them, 24% in higher expression 2 and 3. The pan macrophage marker Ki-M1P was observed in 72% of all cases. Fifty seven percent of the specimens exhibited strong labelling with antibodies against VWF. Coexpressions were statistically significant for the VEGF receptor and NOS I-III (p < 0.01), Ki-M1P and NOS I and II (p < 0.05). A negative correlation was detected for the oedema index (oedema volume/total volume) and the labelling data for NOS III (r = -0.44, p = 0.13) and VEGF-R (r = -0.42, p = 0.022). No correlation existed for Ki-M1P, VWF and NOS I. CONCLUSIONS: The objective of the study was to investigate oedema morphometry, expression of NOS I-III and VEGF-R, presence of capillary vessels and macrophages in cerebral metastasis. A further aim was to investigate a putative oedema induction by NO producing isozymes. Nitric oxide synthase expression was statistically significantly correlated with the expression of the VEGF receptor and the presence of macrophages and microglia. There was a negative correlation between oedema extension and the presence of NOS III and VEGF-R. The results seem to indicate a specific oedema modulating role of NO in cerebral metastasis.

Elevated cerebral perfusion pressure and low colloid osmotic pressure as a risk factor for subdural space-occupying hygromas?

BACKGROUND: Space-occupying subdural hygromas are a late complication of severe traumatic brain injury (TBI) and may delay the patient's recovery. To evaluate the risk factors involved, we performed a semiretrospective, -prospective analysis of three groups of patients, which differed with regard to the techniques used in the management of their cerebral perfusion pressure (CPP) and colloid osmotic pressure (COP) to determine the occurrence of space-occupying subdural hygromas. PATIENTS AND METHODS: Between 1989 and 1997 we examined 696 patients after a severe TBI: Group 1. 1989-1994 mean CPP: 67 (elevated for therapeutic reasons by catecholamines, if necessary), mean COP: 19. Group 2. January 1995-October 1996, mean CPP: 77, mean COP: 20. Group 3. November 1996-December 1997, mean CPP: 79, mean COP: 23 (elevated for therapeutic reasons by infusions of colloids). The groups were comparable for other criteria. RESULTS: Compared to Group 1, Group 2, with a high CPP but lower COP, showed a significantly higher (p < 0.01; chi2-test with correction of Yates) percentage of posttraumatic subdural hygromas with space-occupying aspects, clinical signs of bradycardia, hypertension and impaired consciousness requiring surgery (Group 1: 1.75%; Group 2: 10.46%; Group 3: 0%). In Group 3 we saw no patient with a space-occupying hygroma. CONCLUSION: We conclude that iatrogenic elevated CPP, which has been reported to be helpful in preventing secondary ischemic damage after a severe TBI, may be harmful to a patient if the COP is not maintained within physiological ranges.

Immunohistochemical and electronmicroscopic effects of a new 2.1 microns Ho:YAG laser on the rat brain.

Using an experimental animal model, the thermal single-pulse lesion derived from a mid-infrared 1.0 Joule 300 microns fibre-conducted Holmium: Yttrium-Aluminum-Garnet (Ho:YAG) laser was examined, with special emphasis on the orientation and depth of the tissue reaction. Performing biparietal craniotomy in Sprague-Dawley rats weighing 250-300 g, both hemispheres were targeted by different radiant exposures from 20 to 140 J/cm2 derived from a 600-800 microsecond single pulse. After survival periods of one to 30 days, the animals were sacrificed and both hemispheres were processed for light- and electronmicroscopic investigations. To resolve the depth and orientation of the tissue reaction regarding the localization of reactive astrocytes, we looked for the expression of glial proteins like glial fibrillary acidic protein (GFAP), Vimentin and S 100 with a three-step biotin-avidin immunoperoxidase method. Neuronal and secondary axonal damage was investigated by labelling Neurofilament and Synaptophysin. The tissue reaction beneath the ablated material, consisting of a vacuolation and coagulation zone resulting from heat diffusion, was further elucidated by localization of the heat shock protein (HSP 72 kilo Dalton). Revealing the extension of reactive astrocytes and the degree of the electronmicroscopically depicted glial oedema, the depth of the tissue damage was estimated to reach about 700 microns beneath laser excision. Since McKenzie predicted the depth of tissue damage beneath CO2 and YAG laser excisions in a theoretical mathematical model, the authors were able to develop a sensitive model for testing new laser systems and as a promising instrument for neurosurgery.

First experiences with a 2.0-microm near infrared laser system for neuroendoscopy.

Nd:YAG, argon and diode lasers have been used in neurosurgical procedures including neuroendoscopy. However, many neurosurgeons are reluctant to use these lasers because of their inappropriate wavelength and uncontrollable tissue interaction, which has the potential to cause serious complications. Recently, a 2.0-microm near infrared laser with adequate wavelength and minimal tissue penetration became available. This laser was developed for endoscopic neurosurgical procedures. It is the aim of the study to report the initial experiences with this laser in neuroendoscopic procedures. We have performed 43 laser-assisted neuroendoscopic procedures [multicompartmental congenital, posthaemorrhagic or postinfectious hydrocephalus (n = 17), tumour biopsies (n = 6), rescue of fixed and allocated ventricular catheters (n = 2), endoscopic third ventriculostomy (ETV, n = 17) and aqueductoplasty (n = 1)] in 41 patients aged between 3 months and 80 years. The laser beam was delivered through a 365-microm bare silica fibre introduced through the working channel of a rigid endoscope. It was used for the opening of cysts, perforating the third ventricular floor, and for coagulation prior to and after biopsy. The therapeutic goals [creating unhindered cerebrospinal fluid (CSF) flow between cysts, ventricles and cisterns, sufficient tissue samples for histopathological diagnosis and catheter rescue] were achieved in 40 patients by the first and in 2 patients by a second neuroendoscopic operation. In one child, a CSF shunt was later required despite patency of the created stoma proven by magnetic resonance imaging (MRI). In another patient ETV was abandoned due to a tiny third ventricle. There was neither mortality nor transient or permanent morbidity. The authors conclude that the use of the 2.0-microm near infrared laser enables safe and effective procedures in neuroendoscopy.

Infratentorial meningioma in an 8-year-old child as first sign of neurofibromatosis type 2.

Meningiomas are rare intracranial tumors in pediatric patients. In contrast to meningiomas in adults, childhood ones have a poorer prognosis because of their high growth potential and tendency to recur. Meningiomas are often associated with neurofibromatosis type 2 (NF2) which is an autosomal-dominant disorder. In contrast to adults who primarily present with symptoms due to vestibular tumors, the initial symptoms in children with NF2 are subtle skin tumors, posterior capsular cataracts, or neurological signs secondary to cranial nerve(s) schwannoma excluding vestibular nerve, and/or brainstem or spinal cord compression. Here we report on the clinical, radiological, and histological findings in an 8-year-old boy who was diagnosed with an isolated infratentorial meningioma and a novel splice site mutation in the NF2 gene. The same mutation was detected in the boy's mother who suffered from hearing loss and tinnitus due to a bilateral vestibular schwannoma. Our patient demonstrates the need for molecular testing for NF2 gene mutations even in isolated childhood meningiomas although they do not fulfill the clinical criteria of NF2.

Expression of glutathione S-transferase T1 (GSTT1) in human brain tumours.

Glutathione S-transferases (GSTs) play a central role in a number of metabolic processes. Glutathione S-transferase T1 (GSTT1) is a polymorphic cytosolic enzyme and a member of the theta class of GSTs. Typical substrates for GSTT1 are industrial compounds, such as dichloromethane and ethylene oxide. It has been shown that also chemotherapeutic drugs such as BCNU [i.e. 1,3-bis(2-chloroethyl)-1-nitrosourea] are efficiently inactivated by GSTT1. BCNU is a drug which is increasingly used locally in the chemotherapy of glioblastoma multiforme WHO grade IV. Therefore, if GSTT1 were expressed in neoplastic cells of brain tumours it could be a factor for chemoresistance. In order to clarify a possible role of GSTT1 in chemoresistance, as a first step, we localized this enzyme in malignant gliomas such as glioblastoma multiforme WHO grade IV and oligodendroglioma WHO grade II. Because of its polymorphism we first genotyped the samples for GSTT1 by PCR. Using in situ hybridization, we then demonstrated that GSTT1 transcripts are expressed in neoplastic cells of both tumour types. Immunohistochemistry revealed then that whereas neoplastic cells in glioblastoma multiforme WHO grade IV contain GSTT1, it was not localized in oligodendroglioma cells. Given the polymorphism of GSTT1 and its potential activity towards BCNU, the localization of GSTT1 in glioblastoma cells can be considered as a possible factor of non-homogeneous chemotherapy response among patients with different GSTT1 genotypes.

Piezoelectric bone surgery: a revolutionary technique for minimally invasive surgery in cranial base and spinal surgery? Technical note.

OBJECTIVE: Piezoelectric surgery represents an innovative, ultrasonic surgery technique for performing a safe and effective osteotomy or osteoplasty that contrasts with the traditional hard and soft tissue management methods with rotating instruments. METHODS: Because of its physical and mechanical properties, the definitive clinical advantage of piezoelectric bone surgery with regard to precision cutting lies in the sparing of vital neurovascular bundles or general soft tissue and better visualization of the surgical field, thus suggesting its great safety. Piezoelectric bone surgery has been previously described only in oral and maxillofacial operative procedures in adults. RESULTS: Five children between the age of 6 and 84 months were operated on for craniosynostosis, tethered cord, and an extraconal intraorbital tumor. The usefulness of piezoelectric bone surgery during neurosurgical procedures is presented for these cases. This technique is especially recommended when there are anatomic difficulties because of poor intraoperative visibility or the presence of delicate anatomic structures. CONCLUSION: The present preliminary report (comprising illustrative case reports) demonstrates and introduces for the first time the utility of piezoelectric bone surgery in cranial base and spinal surgery in children. Until now, there has been no documented neurosurgical experience of this technique even in adults.

Ligand-binding studies on heavy riboflavin synthase of Bacillus subtilis.

Heavy riboflavin synthase is a complex enzyme consisting of three alpha subunits and approximately 60 beta subunits. Ligand-binding studies were performed with a variety of substrate and product analogues by analytical ultracentrifugation and by equilibrium dialysis. Nonlinear binding curves indicate the involvement of non-equivalent binding sites which could be assigned to the alpha and beta subunits by comparison with light riboflavin synthase (subunit composition alpha 3) and with aggregates of isolated beta subunits. The beta subunit binding site shows a high degree of stereospecificity. Tightly binding ligands must have a ribityl side chain and a pyrimidine or pteridine moiety with polar substituents.

A modified silver method for demonstrating developing nervous tissue in culture.

Dissociated explants of 8-day-old embryonic chick cerebrum were cultured for up to 18 days. By the beginning of the 2nd week and thereafter, primary cultures of neurons exhibited characteristic differentiated morphology with an interconnecting neurofibrillary network that became increasingly ramified. Neurons in bipolar, tripolar or multipolar form could be demonstrated positively using a short modified silver impregnation method with potassium ferrocyanide.

Glycerol gangliotomy of the second dorsal cervical root in rats: an experimental study to evaluate a minimal invasive approach for the treatment of the chronic cervicogenic headache.

Glycerol is a known agent in the therapy of chronic tic douloureux. It has been used for about 20 years in percutaneous, retrogasserian minimal-invasive rhizotomy, although the pharmacological mechanism of the pain relief involved remains unclear. To investigate glycerol treatment as a possible replacement for invasive approaches in the therapy of chronic cervicogenic headaches, we performed an experimental study on the pathomorphologic action of anhydrous glycerol injection into the second upper cervical dorsal root ganglion (DRG) of rats. Glycerol injections into the second cervical ganglion were investigated light- and electron-microscopically in a series of 40 rats for survival times of up to 30 days. We detected an unspecific overall effect on sensory neurons and satellite cells, as well as on myelinated and unmyelinated axons and Schwann cells. This could be detected after 5 days and sometimes led to degeneration of most of the neurons. Contralateral saline injections as a control showed no morphological effects. The loss of afferent fiber connections to the posterior horn of the myelon could be detected by immunohistochemical labeling of reactive astrocytes. Our results show a glycerol-induced deterioration of the cytoarchitecture of the neurons and their glial satellite cells. The effects on the ganglion cells appear to have been mediated by membrane disturbances and loss of glial integrity. These observations are contrary to previously reported results indicating the specific effect of glycerol on thin myelinated sensory axons.

Expression of tyrosine kinases FAK and Pyk2 in 331 human astrocytomas.

The progression of malignancy from astrocytomas to glioblastomas remains clinically as well as histopathologically unpredictable. The focal adhesion kinase (FAK) and the proline-rich tyrosine kinase (Pyk2) show a high expression in glioma cell lines and have an influence on increased cell proliferation and migration of glioma cells in vitro and in vivo. The aim of this study was to correlate the coexpression of FAK and Pyk2 to the WHO grade of malignancy in human astrocytomas. Immunohistochemical staining scores of FAK and Pyk2 were analyzed in 331 astrocytomas and correlated to each other and to the WHO grade. Significant coexpression of FAK and Pyk2 in astrocytomas was demonstrated. Pyk2 expression occurred much more frequently and with higher expression scores within the different WHO grades. Beyond this, a significant correlation between the WHO grade of malignancy of astrocytomas and the expression of FAK, as well as of Pyk2, was detected. This connection and the roles of these two tyrosine kinases in the progression of tumors should be confirmed by further studies.

Crystallization and preliminary X-ray diffraction study of heavy riboflavin synthase from Bacillus subtilis.

Crystals of heavy riboflavin synthase from Bacillus subtilis have been obtained from 1.3 M sodium/potassium phosphate, pH 8.7, containing 0.35 mM 5-nitroso-6-(1'-D-ribitylamino)-2,4(1H,3H)-pyrimidinedione. X-ray photographs indicate a hexagonal unit cell with dimensions of a = b = 156.4 A; c = 298.5 A; alpha = beta = 90 degrees; gamma = 120 degrees. The space group was established as P6(3)22 with 12 general positions.