Synaptic vesicle recycling in synapsin I knock-out mice.

The synapsins are a family of four neuron-specific phosphoproteins that have been implicated in the regulation of neurotransmitter release. Nevertheless, knock-out mice lacking synapsin Ia and Ib, family members that are major substrates for cAMP and Ca2+/ Calmodulin (CaM)-dependent protein kinases, show limited phenotypic changes when analyzed electrophysiologically (Rosahl, T.W., D. Spillane, M. Missler, J. Herz, D.K. Selig, J.R. Wolff, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1995. Nature (Lond.). 375: 488-493; Rosahl, T.W., M. Geppert, D. Spillane, D., J. Herz, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1993. Cell. 75:661-670; Li, L., L.S. Chin, O. Shupliakov, L. Brodin, T.S. Sihra, O. Hvalby, V. Jensen, D. Zheng, J.O. McNamara, P. Greengard, and P. Andersen. 1995. Proc. Natl. Acad. Sci. USA. 92:9235-9239; see also Pieribone, V.A., O. Shupliakov, L. Brodin, S. Hilfiker-Rothenfluh, A.J. Czernik, and P. Greengard. 1995. Nature (Lond.). 375:493-497). Here, using the optical tracer FM 1-43, we characterize the details of synaptic vesicle recycling at individual synaptic boutons in hippocampal cell cultures derived from mice lacking synapsin I or wild-type equivalents. These studies show that both the number of vesicles exocytosed during brief action potential trains and the total recycling vesicle pool are significantly reduced in the synapsin I-deficient mice, while the kinetics of endocytosis and synaptic vesicle repriming appear normal.

Aggresome formation and neurodegenerative diseases: therapeutic implications.

Accumulation of misfolded proteins in proteinaceous inclusions is a prominent pathological feature common to many age-related neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. In cultured cells, when the production of misfolded proteins exceeds the capacity of the chaperone refolding system and the ubiquitin-proteasome degradation pathway, misfolded proteins are actively transported to a cytoplasmic juxtanuclear structure called an aggresome. Aggresome formation is recognized as a cytoprotective response serving to sequester potentially toxic misfolded proteins and facilitate their clearance by autophagy. Recent evidence indicates that aggresome formation is mediated by dynein/dynactin-mediated microtubule-based transport of misfolded proteins to the centrosome and involves several regulators, including histone deacetylase 6, E3 ubiquitin-protein ligase parkin, deubiquitinating enzyme ataxin-3, and ubiquilin-1. Characterization of the molecular mechanisms underlying aggresome formation and its regulation has begun to provide promising therapeutic targets that may be relevant to neurodegenerative diseases. In this review, we provide an overview of the molecular machinery controlling aggresome formation and discuss potential useful compounds and intervention strategies for preventing or reducing the cytotoxicity of misfolded and aggregated proteins.

Comparative analyses of linac and Gamma Knife radiosurgery for trigeminal neuralgia treatments.

Dedicated linac-based radiosurgery has been reported for trigeminal neuralgia treatments. In this study, we investigated the dose fall-off characteristics and setup error tolerance of linac-based radiosurgery as compared with standard Gamma Knife radiosurgery. In order to minimize the errors from different treatment planning calculations, consistent imaging registration, dose calculation and dose volume analysis methods were developed and implemented for both Gamma Knife and linac-based treatments. Intra-arc setup errors were incorporated into the treatment planning process of linac-based deliveries. The effects of intra-arc setup errors with increasing number of arcs were studied and benchmarked against Gamma Knife deliveries with and without plugging patterns. Our studies found equivalent dose fall-off properties between Gamma Knife and linac-based radiosurgery given a sufficient number of arcs (>7) and small intra-arc errors (<0.5 mm) were satisfied for linac-based deliveries. Increasing the number of arcs significantly decreased the variations in the dose fall-off curve at the low isodose region (e.g. from 40% to 10%) and also improved dose uniformity at the high isodose region (e.g. from 70% to 90%). As the number of arcs increased, the effects of intra-arc setup errors on the dose fall-off curves decreased. Increasing the number of arcs also reduced the integral dose to the distal normal brain tissues. In conclusion, linac-based radiosurgery produces equivalent dose fall-off characteristics to Gamma Knife radiosurgery with a high number of arcs. However, one must note the increased treatment time for a large number of arcs and isocentre accuracies.

K252a inhibits proliferation of glioma cells by blocking platelet-derived growth factor signal transduction.

Growth factors are known to regulate glioma proliferation. The glioma cell lines U87 and T98G were examined for evidence of an autocrine stimulatory loop involving the neurotrophin family of growth factors. Although neurotrophin-3 and TrkC RNA were detected by reverse transcription-PCR, there was no evidence of significant interaction between neurotrophin-3 and its cognate receptor TrkC. The microbial alkaloid K252a has been described to inhibit both Trk tyrosine kinase activity and neuroblastoma cell proliferation. K252a inhibited proliferation in U87 (IC50 = 1170 nM) and T98G (IC50 = 529 nM) but induced apoptosis in U87 cells only. At concentrations of 500 nM to 1 microM, K252a blocked only platelet-derived growth factor (PDGF)-mediated receptor autophosphorylation. These results suggest that an autocrine loop involving PDGF is functional and important for maintaining tumor growth. There is no evidence to support the existence of a neurotrophin-mediated autocrine loop. K252a, through inhibition of PDGF signal transduction, may be a novel therapeutic agent in the treatment of human gliomas.

Transcriptional regulation of gene expression of sec6, a component of mammalian exocyst complex at the synapse.

Sec6, an essential component of the mammalian brain exocyst complex, is believed to function in synapse formation and synaptic plasticity. During neuronal development, the expression of the Sec6 gene correlates temporally with neurite outgrowth and synaptogenesis. To understand the mechanisms that regulate the Sec6 gene expression, we have cloned and characterized the 5'-terminal region of the murine Sec6 gene. We have shown that the 5'-untranslated region of the murine Sec6 gene is encoded by two exons that are separated by a 1560-bp intron. Primer extension analysis demonstrates that Sec6 gene transcription is initiated from a unique site. The Sec6 promoter is embedded in a CpG island and lacks canonical TATA or CAAT boxes. Sequence analysis of the 5'-flanking region and the first intron reveals the presence of a number of binding sites for transcription factors AP-1, AP-2, AP-4, ATF, C/EBPbeta, GATA-1, Oct 1, SP1, STAT, and NRSF. Transfection experiments using Sec6-luciferase fusion genes demonstrate that the 5'-flanking sequence functions as a strong promoter in neuronal but not in nonneuronal cells. Deletion analysis reveals the presence of a core promoter between nucleotide position -139 and +53, and two enhancer and four silencer elements within the 5'-flanking region and the first intron sequence. These results indicate that neuronal expression of the Sec6 gene involves a relatively specific core promoter and interplay between multiple positive and negative regulatory elements.

Neuron-specific and developmental regulation of the synapsin II gene expression in transgenic mice.

Synapsin II, a major phosphoprotein of synaptic vesicles, is believed to function in neurotransmitter release as well as in synapse formation. The expression of the synapsin II gene is neuron-specific, and correlates temporally with synaptogenesis. To understand the mechanisms by which the expression of the synapsin II gene is regulated in vivo, we generated transgenic mice carrying a 5.1-kb 5'-flanking sequence of the murine synapsin II gene fused to the firefly luciferase reporter gene. The synapsin II-luciferase transgene is specifically expressed in neural tissues, such as brain and spinal cord, but not in non-neural tissues. Throughout the brain, the expression of the transgene is widely distributed, and restricted only to neuronal cells. Moreover, the expression of the transgene is developmentally regulated, with a temporal profile similar to that of endogenous synapsin II expression. These results indicate that the 5.1-kb flanking sequence of the murine synapsin II gene contains cis-regulatory elements that are required for directing neuron-specific and synaptogenesis-regulated expression in vivo.

Two primitive neuroectodermal tumor cell lines require an activated insulin-like growth factor I receptor for growth in vitro.

OBJECTIVE: To determine the expression of the insulin-like growth factors (IGFs) and the IGF-I receptor in primitive neuroectodermal tumor cell lines and to assess the importance of these proteins in the growth of cell lines in vitro. METHODS: Ribonucleic acid blotting and reverse transcriptase-polymerase chain reaction were used for detection of IGF and IGF-I expression. Ribonucleic acid blotting was used for detection of up-regulation of c-fos in the presence of exogenous growth factor. Immunoprecipitation was used to demonstrate autophosphorylation of the receptor in the presence of exogenous growth factor. Ligand binding analysis was used to determine the binding affinity of the receptor and the number of receptors per cell. Growth of curves in the presence of monoclonal antibody that blocks binding of ligand to receptor was measured to determine the requirement for an activated receptor during growth. RESULTS: Expression of IGF-II was identified in one cell line. No expression of IGF-I was seen in any cell line. Expression of IGF-I receptor was detected in all three cell lines. Immunoprecipitation experiments demonstrated autophosphorylation of the receptor after addition of IGF-I to growing cells. Ligand binding analysis revealed 9.2 x 10(4) and 4 x 10(4) receptors per cell in the Daoy and PFSK cell lines, respectively. Addition of either IGF alone or in combination to serum-starved cells was not able to restore growth of the cell lines. A blocking monoclonal antireceptor antibody decreased growth of Daoy and PFSK cells in a dose-dependent fashion. Complete arrest of growth occurred at 1 microgram/ml antibody in both cell lines. CONCLUSION: The IGF-I receptor is expressed by primitive neuroectodermal tumor cell lines in vitro. An activated receptor is important for cell proliferation in vitro. Additional work will establish the importance of these findings for tumors in vivo.

Neurotrophins and Trk receptors in primitive neuroectodermal tumor cell lines.

OBJECTIVE: Primitive neuroectodermal tumors (PNETs) are thought to be derived from early central nervous system precursors. Therefore, we hypothesized that the neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) and their receptors (TrkA, TrkB, and TrkC), which are involved in the proliferation, differentiation, and survival of neuronal cells, might be important in regulating tumor growth. METHODS: Using ribonucleic acid (RNA) blotting and reverse transcription-polymerase chain reaction analysis, we investigated the expression of these ligands and their receptors in six PNET cell lines (Daoy, PFSK, D283 Med, UW288-1, CHP707m, and D341 Med). Neurotrophin protein levels were measured using enzyme-linked immunosorbent assay procedures. Receptor function was demonstrated by autophosphorylation. Induction of c-Fos expression and effects on cell proliferation were assessed after the addition of exogenous neurotrophin. RESULTS: Three cell lines expressed messenger RNA for all neurotrophins, whereas the other three expressed two of the three neurotrophins. Neurotrophin protein levels were low. All cell lines expressed trkA messenger RNA. Five expressed the amino terminus of trkB, but three of these did not express the carboxyl terminus. All cell lines contained trkC messenger RNA, but the receptor was truncated in two cell lines. No cell line contained message for a receptor containing an insertion in the tyrosine kinase domain. The addition of neurotrophin to PNET cells resulted in phosphorylation of a protein that was immunoprecipitated with an anti-pan-Trk antibody. c-Fos expression and cell growth were increased by preincubation with neurotrophins, but only in the cell lines expressing the relevant full-length receptors. CONCLUSION: The expression of neurotrophins and neurotrophin receptors by PNET cell lines is variable. The presence of activated Trk receptors in these cell lines may be required for rapid growth, via an autocrine loop mechanism. This will require further investigation.

The use of lobectomy in the management of severe closed-head trauma.

A retrospective review is presented of 20 patients with traumatic brain injury who were treated during the course of their illness by lobectomies either after a herniation or other significant deterioration or to reduce elevated intracranial pressure. All the patients suffered from blunt head trauma. Patient ages ranged from 19 to 59 years (average, 34 yr). The initial Glasgow Coma Scale score ranged from 3 to 15 (average, 8.2). There were 14 frontal lobectomies, 2 temporal, 3 frontal and temporal, and 1 occipital. Surgery was performed between 0 and 8 days after injury (average, 2.8). Outcome was favorable (good or moderately disabled) in 11 patients and unfavorable (severely disabled, persistently vegetative, or dead) in 9. No patients survived in a persistently vegetative state. A higher initial Glasgow Coma Scale score was positively correlated with a more favorable outcome (P < 0.03). Younger patients also showed a significant positive relationship to outcome (P < 0.0005). Better pupillary reactivity showed a significant trend toward a more favorable outcome (P < 0.04). The type of lesions identified on computed tomographic scans had no association with outcome. A lobectomy can be a useful adjuvant in the management of severe brain injury, especially in younger patients with relatively higher initial Glasgow Coma Scale scores who subsequently deteriorate or develop elevated intracranial pressure.

Hemangiopericytoma of the temporal bone presenting as a retroauricular mass.

An unusual case of a hemangiopericytoma arising from the temporal bone is presented. The patient was noted to have a postauricular mass and was neurologically asymptomatic. A preoperative magnetic resonance image and an angiogram revealed the tumor to be highly vascular. Preoperative embolization facilitated the surgical removal of the tumor by rendering it avascular. Current therapy consists of radical resection of the tumor with postoperative radiation therapy. Patients must be monitored carefully for local recurrence and systemic metastasis.

Diffuse arteriovenous malformations: a clinical, radiological, and pathological description.

In a review of our series of patients with arteriovenous malformations (AVMs), a group with atypical angiographic and histopathological characteristics was discovered. Unlike the typical AVM, these lesions contained normal cerebral tissue between the abnormal vessels. We call these lesions diffuse AVMs, and think that this AVM represents one end of the AVM spectrum from a tight nidus to a diffuse lesion. The mean age of these patients was 18.1 years. Eight patients presented with an intracerebral hemorrhage, two with seizures, one with headache without hemorrhage, and one with ischemic symptoms compatible with vascular steal. Cerebral angiography revealed three AVMs to be 2 to 4 cm in diameter, four were 4 to 6 cm in diameter, and five were > 6 cm in diameter. Characteristic angiographic features included multiple small arterial feeders, small ectatic vessels in the malformation itself, multiple small draining veins, and a diffuse, puddling appearance of the contrast dye. Despite 16 operations in 11 patients, complete resection of the AVM was accomplished in only 8. The four patients with residual disease have received radiation therapy. Histopathology of the surgical specimens found AVM vessels interspersed among normal appearing neurons and white matter. Leptomeningeal angiodysplasia was noted when the cerebral cortex was involved. Gliosis was noted in some cases. Diffuse AVMs represent a difficult surgical challenge and recognition of the lesion aids in surgical planning.

Cytokine expression in radiation-induced delayed cerebral injury.

Radiation-induced delayed brain injury is a well-documented complication of both standard external beam radiation (teletherapy) and interstitial brachytherapy; however, the cause of this damage has not been determined. Cytokines and growth factors are important regulatory proteins controlling the growth and differentiation of normal and malignant glial cells, which have been implicated in the tissue response to radiation injury. Six snap-frozen brain biopsies showing radiation injury were obtained from four patients harboring malignant gliomas who underwent either postoperative external beam and/or stereotactic interstitial brachytherapy at standard dosages. The specimens showed variable amounts of gliosis, tissue necrosis, calcification, inflammation, and vascular proliferation and hyalinization. Frozen tissue sections were examined for the presence of infiltrating lymphocytes, macrophages, cytokines, and other immunoregulatory molecules by the use of a panel of specific monoclonal and polyclonal antibodies. All specimens showed diffuse T cell infiltration with both CD4+ and CD8+ cells. Infiltrating activated macrophages (CD11c+, HLA-DR+) were prominent in five of six cases. Tumor necrosis factor-alpha and interleukin-6 immunoreactivity was prominent in four of six cases and was predominately localized to macrophages. Transforming growth factor-beta astrocytic and macrophage immunoreactivity was present at moderate levels in all cases. This study suggests that in radiation necrosis, interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6 are expressed, predominately by infiltrating macrophages.

Reduced-dose radiosurgery for vestibular schwannomas.

OBJECTIVE: To evaluate tumor control and complications associated with low-dose radiosurgery for vestibular schwannomas. METHODS: Between December 1993 and January 2000, 47 patients with vestibular schwannomas were treated at our center with gamma knife radiosurgery. The marginal tumor doses ranged from 7.5 to 14.0 Gy (median, 12.0 Gy) for patients treated after microsurgery and from 10.0 to 15.0 Gy (median, 12.0 Gy) for patients in whom radiosurgery was the primary treatment. The median maximum tumor diameter was 18 mm (range, 3-50 mm). Evaluation included audiometry, neurological examination, and serial imaging tests. A survey was conducted at the time of analysis. RESULTS: Follow-up data were available for 45 patients and ranged from 1 to 7 years (median, 3.6 yr). In 43 patients (96%), tumor control (no radiographic progression or surgical resection) was observed. All 33 previously untreated patients had tumor control. Transient facial weakness, experienced in two patients (4%), had resolved completely within 6 months. No patient developed trigeminal neuropathy. Hearing was diminished from baseline in 12% of patients with useful hearing (Gardner-Robertson Class III). However, all patients with pretreatment hearing Gardner-Robertson Class I or II maintained testable hearing (Class I to III) at the most recent examination. CONCLUSION: Low-dose radiosurgery in this series provided comparable local control and decreased incidences of complications in relation to other reports. Additional follow-up will allow more definitive conclusions to be reached regarding the ultimate rates of tumor control and hearing preservation. Nevertheless, the current dose used for vestibular schwannomas at the University of Maryland Medical Center is 12.0 Gy to the tumor periphery.

The standardized assessment of argyrophilic nucleolar organizer regions in meningeal tumors.

Tissue markers of cellular proliferation have been recently utilized as prognostic indicators in tumors of the central nervous system. Nucleolar organizer regions represent transcriptionally active sites of ribosomal deoxyribonucleic acid (DNA) and can be identified by a simple argyrophilic technique. The authors describe a standardized approach to the assessment of these argyrophilic nucleolar organizer regions in meningeal tumors. Twenty-five meningiomas were classified histologically into benign, atypical, or malignant groups. In addition, two hemangiopericytomas and one leptomeningeal melanoma were examined. Appropriate sections were silver stained and argyrophilic nucleolar organizer regions were counted in 200 nuclei. The mean argyrophilic nucleolar organizer region count was statistically different (p less than 0.001) between benign tumors (245 +/- 156, 1.23/cell), atypical tumors (497 +/- 135, 2.49/cell), and malignant tumors (921 +/- 59, 4.61/cell). The count for recurrent meningiomas (544 +/- 76) was also statistically different (p less than 0.02) from non-recurrent tumors (329 +/- 183). The standardized assessment of argyrophilic nucleolar organizer regions can be easily performed by any surgical pathology laboratory without specialized equipment and, in meningeal tumors, may be useful as an independent indicator of biological behavior.

Radiation necrosis following gamma knife surgery: a case-controlled comparison of treatment parameters and long-term clinical follow up.

OBJECT: Radiation necrosis is the only significant complication of gamma knife surgery (GKS). The authors studied treatment plan parameters in patients who had radiation necrosis to determine if risk factors for necrosis could be identified. METHODS: Between September 1994 and December 1998, 286 patients were treated with GKS by the senior author. Of the 243 patients who were suitable for analysis, 17 developed radiation necrosis and were prospectively followed. Concurrently, 17 patients without necrosis were randomly selected as case controls on the basis of histological findings in their lesions. Integral dose-volume histograms (DVHs) were calculated and dose-volume treatment parameters were determined. A comparison was made with both the established Kjellberg and Flickinger isonecrosis risk lines. Clinical outcome was assessed according to time to resolution of symptoms and return to normal radiographic appearance. CONCLUSIONS: Treatment plan variables associated with the risk of necrosis were increased tumor volume (TV) integral dose, increased TV, and increased 10-Gy volume. Other risk factors included repeated radiosurgery to the same lesion and glioma histological findings. The Kjellberg 1% risk line predicted a 5% risk of radiation necrosis and the Flickinger 3% risk line predicted a 3% risk. The median time to development of necrosis was 4 months, and symptomatic and radiographic recovery times were 7.5 and 10.5 months, respectively. The median survival time in patients with necrosis was 30 months. The authors recommend prospective TV determination and DVH calculation for all radiosurgical treatments and the avoidance of repeated radiosurgical treatments to the same lesion when possible.

Multiple thoracic disc herniations. Case report.

A patient with herniated thoracic discs in tandem is reported. The previous literature is reviewed. Difficulties with the preoperative diagnosis and the surgical approach to these lesions are discussed.

The effect of user-defined variables on dosimetry consistency in Gamma Knife planning.

We report a dosimetric variation caused by a user-defined variable for the Leksell Gamma Knife planning system. Treatment plans of 31 randomly selected patients were studied retrospectively to determine the dosimetric effects in the dose prescription and computation as a result of dose matrix positioning in the Leksell Gamma Plan (LGP, Version 4.12). Phantom studies with ion chamber measurements were carried out to validate the accuracy of the computation results. An average overdose of 2% was found due to the variations in the user-defined dose matrix position for the studied cases. In the extreme, the overdose value was as high as 5% with an over-treatment time exceeding 2 min. The phantom measurements were found to agree with the LGP calculation within 0.5%. An adaptive method was developed and demonstrated in this study to eliminate such dosimetry variations.

Acute complications following gamma knife radiosurgery are rare.

BACKGROUND: Gamma knife radiosurgery (GKR) is a safe and effective alternative to surgery for intracranial lesions. Most studies evaluating toxicity after GKR have concentrated on the delayed radiation effects. METHODS: We retrospectively reviewed 835 consecutive GKR cases for early (within 7 days) neurological complications or death. RESULTS: We identified a total of 18 patients (2.2%) who had a neurological event or death. Five (0.6%) patients developed new focal deficits, 12 (1.4%) patients experienced a seizure and there were three (0.4%) deaths. Two deaths were related to development of seizures and neurological deterioration. One death was caused by a respiratory arrest related to the patient's primary cancer. Of the five patients with neurological deficits, none had a persistent deficit. In two cases the neurological deficits were due to an increase in edema. Whether this occurred as a result of the gamma knife treatment or was the natural progression of the tumor is unclear. CONCLUSIONS: Complications after GKR are uncommon and the risk of a permanent deficit arising from an acute neurological event is exceedingly low.

Growth factors in central nervous system development and tumorigenesis.

Growth factors play an important role in the development of the normal central nervous system as well as in the genesis of central nervous system tumors. Some of the more important growth factors and growth factor receptors, as they pertain to neurooncology, are reviewed in this article.

Cell and molecular biology for neurosurgeons (or everything you wanted to know about molecular biology but were afraid to download).

We are fortunate to be physicians at a time when the molecular pathogenesis of disease is being unraveled. Beginning with the discovery of the structure of DNA to the Human Genome Project, molecular biology over the past 45 years has revolutionized medicine. Neurosurgery has a strong scientific tradition, but to remain active participants in this new era of medicine, we must understand the science of pathology at the molecular level, applying our unique perspective to its advancement. We will review most of the current techniques used today by cell and molecular biologists. Interwoven with these descriptions will be a brief discussion of pertinent molecular and cell biology concepts, a full review being beyond the scope of this article.