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To obtain commensurate numerical data of neuronal network morphology in vitro, network analysis needs to follow consistent guidelines. Important factors in successful analysis are sample uniformity, suitability of the analysis method for extracting relevant data and the use of established metrics. However, for the analysis of 3D neuronal cultures, there is little coherence in the analysis methods and metrics used in different studies. Here, we present a framework for the analysis of neuronal networks in 3D. First, we selected a hydrogel that supported the growth of human pluripotent stem cell-derived cortical neurons. Second, we tested and compared two software programs for tracing multi-neuron images in three dimensions and optimized a workflow for neuronal analysis using software that was considered highly suitable for this purpose. Third, as a proof of concept, we exposed 3D neuronal networks to oxygen-glucose deprivation- and ionomycin-induced damage and showed morphological differences between the damaged networks and control samples utilizing the proposed analysis workflow. With the optimized workflow, we present a protocol for preparing, challenging, imaging and analysing 3D human neuronal cultures., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Extracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge., Objective: To probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells., Methodology: First, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM)., Results: A peak in cup-shaped particles was observed in SEC fractions 7-10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield., Conclusions: We demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objective: To evaluate changes in the learning attitudes of primary care physicians., Design: Qualitative study through one focus group interview with the programme's participants. Analysis of the focus group content using the Steps for Coding and Theorization method., Setting: Japan., Participants: Eight primary care physicians who completed a 2-year continuing professional development (CPD) programme using a problem-based learning (PBL) approach, focused on acquiring the skills needed to practise as primary care physicians in the community., Results: Participants described positive changes in their attitudes and behaviours as a result of the training programme. These changes were grouped into three main themes: 'changes in learning methods regarding medical practice', 'encounters with diverse perspectives and values, and confidence gained from those encounters', and 'showing one's attitude towards learning and its influence on others'. The experienced practitioners participating in this study reported that the programme helped them apply their skills more broadly; for example, searching the literature for psychosocial aspects of practice and engaging more comfortably with diverse perspectives. They reported the positive impact of their learning on their coworkers., Conclusion: A 2-year CPD programme using PBL can influence primary care physicians' attitudes and learning-related behaviours. Further research is needed to determine which specific aspects of the programme are the most effective and whether the changes in attitudes and behaviours described affect patient care., Competing Interests: Competing interests: MM received lecture fees and lecture travel fees from the Centre for Family Medicine Development of the Japanese Health and Welfare Co-operative Federation. MM is an adviser for the Centre for Family Medicine Development Practice-Based Research Network. The other authors report no conflicts of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The cardiac autonomic nervous system (cANS) regulates cardiac function by innervating cardiac tissue with axons, and cardiomyocytes (CMs) and neurons undergo comaturation during the heart innervation in embryogenesis. As cANS is essential for cardiac function, its dysfunctions might be fatal; therefore, cardiac innervation models for studying embryogenesis, cardiac diseases, and drug screening are needed. However, previously reported neuron-cardiomyocyte (CM) coculture chips lack studies of functional neuron-CM interactions with completely human-based cell models. Here, we present a novel completely human cell-based and electrophysiologically functional cardiac innervation on a chip in which a compartmentalized microfluidic device, a 3D3C chip, was used to coculture human induced pluripotent stem cell (hiPSC)-derived neurons and CMs. The 3D3C chip enabled the coculture of both cell types with their respective culture media in their own compartments while allowing the neuronal axons to traverse between the compartments via microtunnels connecting the compartments. Furthermore, the 3D3C chip allowed the use of diverse analysis methods, including immunocytochemistry, RT-qPCR and video microscopy. This system resembled the in vivo axon-mediated neuron-CM interaction. In this study, the evaluation of the CM beating response during chemical stimulation of neurons showed that hiPSC-neurons and hiPSC-CMs formed electrophysiologically functional axon-mediated interactions.

Background: Three-dimensional (3D) in vitro models have been developed into more in vivo resembling structures. In particular, there is a need for human-based models for neuronal tissue engineering (TE). To produce such a model with organized microenvironment for cells in central nervous system (CNS), a 3D layered scaffold composed of hydrogel and cell guiding fibers has been proposed., New Method: Here, we describe a novel method for producing a layered 3D scaffold consisting of electrospun poly (L,D-lactide) fibers embedded into collagen 1 hydrogel to achieve better resemblance of cells' natural microenvironment for human pluripotent stem cell (hPSC)-derived neurons. The scaffold was constructed via a single layer-by-layer process using an electrospinning technique with a unique collector design., Results: The method enabled the production of layered 3D cell-containing scaffold in a single process. HPSC-derived neurons were found in all layers of the scaffold and exhibited a typical neuronal phenotype. The guiding fiber layers supported the directed cell growth and extension of the neurites inside the scaffold without additional functionalization., Comparison With Existing Methods: Previous methods have required several process steps to construct 3D layer-by-layer scaffolds., Conclusions: We introduced a method to produce layered 3D scaffolds to mimic the cell guiding cues in CNS by alternating the soft hydrogel matrix and fibrous guidance cues. The produced scaffold successfully enabled the long-term culture of hPSC-derived neuronal cells. This layered 3D scaffold is a useful model for in vitro and in vivo neuronal TE applications., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Takotsubo cardiomyopathy is characterized by transient regional systolic dysfunction of the left ventricle, mimicking myocardial infarction. Although systolic left ventricular (LV) function normalizes in most cases, the outcome is not always favourable. Recently, a rare electrocardiogram (ECG) finding, lambda wave ST elevation or 'triangular QRS-ST-T waveform', was suggested as a possible marker of poor outcome in Takotsubo patients., Case Summary: After a brief episode of chest pain and shortness of breath, a 67-year-old woman developed cardiogenic shock. Her resting ECG showed widespread ST elevations, which soon evolved into a pattern of triangular QRS-ST-T waveforms in the inferior leads and V3-V6. Emergent coronary angiography was normal. The ejection fraction was 20% with apical ballooning and an LV thrombus. At 1-month follow-up, the patient was asymptomatic and the ECG showed only T-wave inversions., Discussion: The triangular QRS-ST-T waveform ECG pattern has recently been introduced as a high-risk marker in the Takotsubo syndrome., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objectives: This study aimed to identify training needs among primary care physicians in Japan who had no formal primary care training., Methods:  We conducted a focus group interview with seven Japanese primary care physicians who had not previously undergone specialist training in primary care and had been recruited to a family medicine training program that used a problem-based learning approach. At the start of the program, the physicians attended the interview. The discussion was recorded, and the transcribed interview was analyzed using the Steps for Coding and Theorization method., Results:  Three main themes emerged. First, there is a lack of standard re-education programs for physicians who move away from their specializations into primary care. Second, there is insufficient training on primary care in undergraduate and postgraduate medical education in Japan. Third, continuing professional development programs should cover the communication skills, attitudes, and behaviors necessary for primary care practice., Conclusions:  This study clarified the needs to be addressed in our training program for primary care physicians involved in retraining in primary care. It is important to consider how to best include the communication skills, attitudes, and behaviors necessary for primary care among the topics covered in the program. As the program undergoes further iteration, it will be important to check whether it meets the needs of primary care practitioners. It will be necessary to investigate the needs of re-education programs for more physicians in many areas, and to emphasize the importance of primary care re-education in these abilities in undergraduate and postgraduate medical education.

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell-, biomaterial-, and environment-related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan-poly(vinyl alcohol) (HA1-PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1-PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1-PVA-col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As the complex structure of nervous tissue cannot be mimicked in two-dimensional (2D) cultures, the development of three-dimensional (3D) neuronal cell culture platforms is a topical issue in the field of neuroscience and neural tissue engineering. Computer-assisted laser-based fabrication techniques such as direct laser writing by two-photon polymerization (2PP-DLW) offer a versatile tool to fabricate 3D cell culture platforms with highly ordered geometries in the size scale of natural 3D cell environments. In this study, we present the design and 2PP-DLW fabrication process of a novel 3D neuronal cell culture platform based on tubular microtowers. The platform facilitates efficient long-term 3D culturing of human neuronal cells and supports neurite orientation and 3D network formation. Microtower designs both with or without intraluminal guidance cues and/or openings in the tower wall are designed and successfully fabricated from Ormocomp. Three of the microtower designs are chosen for the final culture platform: a design with openings in the wall and intralumial guidance cues (webs and pillars), a design with openings but without intraluminal structures, and a plain cylinder design. The proposed culture platform offers a promising concept for future 3D cultures in the field of neuroscience.

Stem cell transplantations for spinal cord injury (SCI) have been studied extensively for the past decade in order to replace the damaged tissue with human pluripotent stem cell (hPSC)-derived neural cells. Transplanted cells may, however, benefit from supporting and guiding structures or scaffolds in order to remain viable and integrate into the host tissue. Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. All cell types are shown to efficiently spread over the nanofiber platform and orient according to the fiber alignment. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment. In this work, clinically relevant materials and substrates for nanofibers, fiber coatings, and hydrogel scaffolds are used and combined with cells suitable for developing functional cell grafts for SCI repair., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neural tissue engineering and three-dimensional in vitro tissue modeling require the development of biomaterials that take into account the specified requirements of human neural cells and tissue. In this study, an alternative method of producing biomimetic hydrogels based on gellan gum (GG) was developed by replacing traditional crosslinking methods with the bioamines spermidine and spermine. These bioamines were proven to function as crosslinkers for GG hydrogel at +37 °C, allowing for the encapsulation of human neurons. We studied the mechanical and rheological properties of the formed hydrogels, which showed biomimicking properties comparable to naïve rabbit brain tissue under physiologically relevant stress and strain. Human pluripotent stem cell-derived neuronal cells demonstrated good cytocompatibility in the GG-based hydrogels. Moreover, functionalization of GG hydrogels with laminin resulted in cell type-specific behavior: neuronal cell maturation and neurite migration.

Background: This trial was designed to evaluate the safety and clinical responses to a combination of temozolomide (TMZ) chemotherapy and immunotherapy with fusions of DCs and glioma cells in patients with glioblastoma (GBM)., Method: GBM patients were assigned to two groups: a group of recurrent GBMs after failing TMZ-chemotherapy against the initially diagnosed glioma (Group-R) or a group of newly diagnosed GBMs (Group-N). Autologous cultured glioma cells obtained from surgical specimens were fused with autologous DCs using polyethylene glycol. The fusion cells (FC) were inoculated intradermally in the cervical region. Toxicity, progression-free survival (PFS), and overall survival (OS) of this trial were evaluated. Expressions of WT-1, gp-100, and MAGE-A3, recognized as chemoresistance-associated peptides (CAP), were confirmed by immunohistochemistry of paraffin-embedded tumor samples. Patient's PBMCs of pre- and post-vaccination were evaluated by tetramer and ELISPOT assays., Results: FC-immunotherapy was well tolerated in all patients. Medians of PFS and OS of Group-R (n = 10) were 10.3 and 18.0 months, and those of Group-N (n = 22) were 18.3 and 30.5 months, respectively. Up-regulation and/or cytoplasmic accumulation of CAPs was observed in the recurrent tumors of Group-R patients compared with their initially excised tumors. Specific immune responses against CAPs were observed in the tetramer and ELISPOT assays., Conclusions: The combination of TMZ-treatment leading to up-regulation and/or cytoplasmic accumulation of CAPs, with FC-immunotherapy as a means of producing specific immunity against CAPs, may safely induce anti-tumor effects in patients with GBM.

Recent advances in genomic technology and genome-wide analysis have identified key molecular alterations that are relevant to the diagnosis and prognosis of brain tumors. Molecular information such as mutations in isocitrate dehydrogenase (IDH) genes or 1p/19q co-deletion status will be more actively incorporated into the histological classification of diffuse gliomas. BRAF V600E mutations are found frequently in circumscribed low-grade gliomas such as pleomorphic xanthoastrocytoma (PXA) and extra-cerebellar pilocytic astrocytoma, or epithelioid glioblastomas (E-GBM), a rare variant of GBM. This mutation is relatively rare in other types of diffuse gliomas, especially in adult onset cases. Here, we present an adult onset case of IDH wild-type/BRAF V600E-mutated diffuse glioma, evolving from grade III to grade IV. The tumor displayed atypical exophytic growth and had unusual histological features not fully compatible with, but indicative of PXA and E-GBM. We discuss differential diagnosis of the tumor, and review previously described diffuse gliomas with the BRAF V600E mutation.

Background: Total removal of craniopharyngioma is the most acceptable therapeutic modality; however, there are cases in which radical excision is not possible. To reduce the cystic component volume, an Ommaya reservoir catheter can be placed endoscopically. However, there are certain complications and risks with this type of maneuver, such as misplacement of the catheter, which may result in leakage of cyst contents or installed fluids such as bleomycin. Thus, accurate placement of intracystic catheter is extremely important., Patients and Methods: The authors placed Ommaya reservoir catheters running over the outer surface of a transparent endoscopic sheath in three cases., Results: This neuroendoscopic procedure permits easier manipulation of the catheter and precise placement of the catheter tip. This technique was useful for placement of Ommaya reservoir catheters., Conclusions: This new technique of catheter placement with neuroendoscopy is more accurate, safer, and less invasive., (Georg Thieme Verlag KG Stuttgart · New York.)

The future of tissue engineering applications for neuronal cells will require a supportive 3D matrix. This particular matrix should be soft, elastic and supportive for cell growth. In this study, we characterized the suitability of a 3D synthetic hydrogel matrix, PuraMatrix™, as a growth platform for human embryonic stem cell (hESC)-derived neural cells. The viability of the cells grown on top of, inside and under the hydrogel was monitored. The maturation and electrical activity of the neuronal networks inside the hydrogel were further characterized. We showed that cells stayed viable on the top of the PuraMatrix™ surface and growth of the neural cells and neural processes was good. Further, hESC-derived neurons, astrocytes and oligodendrocytes all grew, matured and migrated when cultured inside the hydrogel. Importantly, neuronal cells were able to form electrically active connections that were verified using microelectrode array. Thus, PuraMatrix is a good supportive growth matrix for human neural cells and may serve as a matrix for neuronal scaffolds in neural tissue engineering., (Copyright © 2012 John Wiley & Sons, Ltd.)

Objective: We report our initial experience of real-time intraoperative C-arm computed tomography (C-arm CT: DynaCT)-guided navigation surgery for pituitary tumors., Methods: Recent advancement in flat panel technology makes it possible to obtain CT-like images by using rotation of the C-arm of a digital subtraction angiography (DSA) system. A specially designed new suite, which has C-arm CT-imaging-capable DSA in combination with a navigation system (VectorVision Sky, BrainLAB AG, Munich, Germany), allows neurosurgeons to perform endoscopic transsphenoidal procedures under real-time navigation support. Thirty-one pituitary tumor patients were examined. During or after tumor removal, contrast-enhanced DynaCT was conducted to rule out residual tumor in 12 cases. When enhanced tumor was confirmed, additional removal was continued without moving the patients., Results: DynaCT and subsequent image transfer to navigation system was performed in all cases without difficulties, requiring only an additional 15 minutes of surgical time. Sellar fenestration in relation to tumors and absence of hidden hematomas was confirmed in all cases. The contrast-enhanced DynaCT was found to be contributing to a better handling of the residual tumor. In 9 of these 12 cases (75%), residual tumor was detected on DynaCT; consequently, further removal was considered. In 2 cases (16%) there was no enhanced lesion, indicating complete removal., Conclusions: The proposed technique of intraoperative visualization in the hybrid operating room can be easy to perform and may be a useful adjunct to conventional transsphenoidal surgery for an improved resection rate and less cavernous sinus and internal carotid artery injury., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The aim of this study was to clarify the relationship between the histological features of GH-producing adenomas surgically resected at the Toranomon Hospital and the clinical features of the patients. Histological examinations, including immunohistochemistry for anterior pituitary hormones and cytokeratin (CK), were performed on 242 consecutively excised GH-producing pituitary adenomas. Immunohistochemistry showed 45% of the adenomas to be monohormonal and 55% to be plurihormonal, producing GH-PRL (77%), GH-TSH (13%), and GH-PRL-TSH (10%). One-fourth of the monohormonal GH adenomas had a dot-like pattern of CK immunoreactivity in the majority of the tumor cells (>80%); they were significantly more common in female or younger patients and usually tended to be larger and more invasive than monohormonal GH adenomas with perinuclear CK. Interestingly, CK-immunonegative adenomas were found in only 5% of the patients; they also showed a tendency to be larger, suggesting that they are a distinct type of GH adenoma with clinically aggressive features. Serum hormone levels correlated well with tumor size only in GH-producing adenomas with a perinuclear pattern of CK immunoreactivity. Each histological subtype of adenoma, classified according to the pattern of CK immunoreactivity, was associated with distinct clinical characteristics. This information is useful for understanding the pathophysiology of acromegaly-causing GH-producing adenomas.

Cerebral aneurysms are the predominant cause of spontaneous subarachnoid hemorrhage (SAH). However, if an aneurismal cause has been excluded, there remains but a short list of meningiomas or metastatic lesions as possible causes. This article details a case of neoplasm that presented exclusively with SAH. A 31-year-old male presented with a SAH with normal cerebral angiography. The initial magnetic resonance image (MRI) revealed a lesion in the left uncus thought to be recovering hemorrhage. Subsequent MRI, however revealed the mass to be expanding. A neuroendoscopical biopsy of the lesion established a diagnosis of glioblastoma. An affirmation is made that patients experiencing "angiographically-negative" SAH should undergo MRI, occasionally on a serial basis, to exclude other etiologies for hemorrhage, including neoplasma.

We report a Japanese patient with a complaint of unilateral watery nasal discharge. Analysis of the nasal discharge showed it to contain high levels of sugar and transferrin, which indicated cerebrospinal fluid (CSF) rhinorrhea. A diagnosis of sphenoid sinus meningoencephalocele was easily made on the basis of the CT, MRI and nasal discharge findings. We performed surgery by an image-guided endoscopic endonasal approach (IGEEA). An image guidance system (IGS) was used to confirm the position of the bone defect and the prolapsed brain lobe. We resected the brain lobe, and used fat tissue and fascia to create an extracranial-intracranial blockade. As of 18 months after the operation, there is no evidence of infection or CSF leakage. The IGEEA enabled us to successfully repair the middle skull base using a multi-layer sealing technique, while the IGS allowed us to confirm the anatomical structures and successfully avoid causing collateral damage to the surrounding tissues. This case exemplifies the beneficial effect that of the development of surgical support equipment on the operative approach that is now indicated for sphenoid sinus meningoencephaloceles: the endonasal approach has largely replaced other approaches, such as lateral rhinotomy., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Fully equipped high-end digital subtraction angiography (DSA) within the operating room (OR) environment has emerged as a new trend in the fields of neurosurgery and vascular surgery., Objective: To describe initial clinical experience with a robotic DSA system in the hybrid OR., Methods: A newly designed robotic DSA system (Artis zeego; Siemens AG, Forchheim, Germany) was installed in the hybrid OR. The system consists of a multiaxis robotic C arm and surgical OR table. In addition to conventional neuroendovascular procedures, the system was used as an intraoperative imaging tool for various neurosurgical procedures such as aneurysm clipping and spine instrumentation., Results: Five hundred one neurosurgical procedures were successfully conducted in the hybrid OR with the robotic DSA. During surgical procedures such as aneurysm clipping and arteriovenous fistula treatment, intraoperative 2-/3-dimensional angiography and C-arm-based computed tomographic images (DynaCT) were easily performed without moving the OR table. Newly developed virtual navigation software (syngo iGuide; Siemens AG) can be used in frameless navigation and in access to deep-seated intracranial lesions or needle placement., Conclusion: This newly developed robotic DSA system provides safe and precise treatment in the fields of endovascular treatment and neurosurgery.

Apart from generating T-helper (Th) effector responses, dendritic cells (DCs) are capable of initiating tolerance against the inciting antigens. Therefore, successful DC-based immunotherapy against malignant tumors requires an additional strategy to activate antigen-processing DCs. We studied the antitumor immune responses conferred by fusions of DCs and glioma cells in vitro. Fusion cells (FCs) were stimulated with polyriboinosinic polyribocytidylic acid [Poly(I:C)] and/or small interference RNA (siRNA) of IL-10 (IL-10-siRNA). Increased IFN-β expression induced by Poly(I:C) transfection was accompanied by enhanced production of IL-10 and IL-12p70 in the FCs. We also found that the ability of Poly(I:C)-transfected FCs to produce IL-12p70, but not IFN-β, was preserved when endogenous IL-10 was suppressed by IL-10-siRNA. To analyze the antigen-presenting function further, DCs, glioma cells, and peripheral lymphocytes were established from patients newly diagnosed with glioma. In this experiment, peripheral lymphocytes were stimulated with autologous FCs and restimulated with autologous glioma cells. CD4T cells isolated from the stimulated lymphocytes were subjected to the ELISPOT and WST-1 assays, which revealed that the IL-10-siRNA/Poly(I:C)-cotransfected FCs elicit an efficient tumor-specific Th1 response. These findings support the relevance of using Poly(I:C) and IL-10-siRNA in clinical immunotherapy protocols with an FC-based vaccine for patients with malignant glioma as a means of promoting Th1-induced tumor antigen presentation.

Background: The purpose of our study was to evaluate the application of thermoreversible gelation polymer (TGP) as a local drug delivery system for malignant glioma., Materials and Methods: Polymeric microspheres or liposomes loaded with doxorubicin (sphere-dox or lipo-dox) were combined with TGP to provide continuous drug delivery of doxorubicin (dox) for kinetic release studies and cell viability assays on glioma cell lines in vitro. For in vivo studies, TGP loaded with dox alone (TGP-dox) was combined with sphere-dox or lipo-dox. Their antitumor effects on subcutaneous human glioma xenografts were evaluated in nude mice., Results: In vitro, TGP combined with sphere-dox or lipo-dox released dox for up to 30 days. In vivo, TGP-dox combined with sphere-dox or lipo-dox inhibited subcutaneous glioma tumor growth until day 32 and day 38, respectively., Conclusion: TGP in combination with microspheres or liposomes successfully prolonged the release of dox and its antitumor effects.

The authors describe a case of rhabdoid meningioma (RM) in a 17-year-old boy that was determined by measuring the tumor volume during preoperative follow-up. The volume of the tumor located in the left occipital lobe, was measured every 1-5 months, using an image analysis software. The tumor volume doubling time (Td) ranged from 1.0 to 4.9 years in the first 11 months, but became 0.3 years in the last two months. The tumor grew rapidly in the last two months at which time surgery was performed. Pathological examination of the surgical specimen showed that the tumor contained rhabdoid cells (RCs). RCs were heterogeneously distributed in the tumor admixed with spindle-shaped cells. The areas where RCs were predominant had malignant histological features, with necrosis and high proliferation indices, whereas the areas with few RCs lacked the malignant features. The tumor grew slowly in the initial phase, possibly because components with low proliferation rates occupied most of the tumor. The tumor began to grow rapidly when the malignant component containing abundant RCs became predominant. To the authors' knowledge, this is the first report monitoring the volumetric change of RM periodically. Our investigation indicated that volumetric analysis is useful to decide surgical intervention of the meningiomas with potential malignancy.

Objective: The fluorescence-guided resection using 5-aminolevulinic acid (5-ALA) is a well established method for the treatment of brain tumor, especially malignant glioma. However, there is no report on photodynamic diagnosis (PDD) for spinal tumor. In the present study, we evaluated the usefulness of PDD for spinal ependymoma using 5-ALA., Methods: Three patients with spinal ependymoma received oral doses of 5-ALA (20 mg/kg body weight) 2 hours before anesthesia induction. Intraoperatively, fluorescence was observed with a 420 nm sharp cut filter after excitation with a violet semiconductor laser (405 nm) and was verified by analysis of fluorescent spectra. Residual fluorescent samples taken from the tumor cavity were examined histologically, Results: Fluorescence peaked at 636nm in the removed tumors in all cases. Fluorescent tissue tended to exist at the cranial and caudal portion in the tumor cavity or around the anterior median fissure. The residual fluorescent tissue was not detected after removal of the tumor in case 1. The residual fluorescent tissue was composed of tumor cells and ependymal lining in case 2 or the infiltrated inflammatory cells and vascular endothelial cells in case 3. Postoperative magnetic resonance (MR) imaging showed no residual tumor in any of the cases., Conclusion: The results of this study indicate the usefulness of 5-ALA-induced tumor fluorescence in guiding resection of spinal ependymoma. 5-ALA-induced porphyrin fluorescence may label spinal ependymomas easily and clearly enough to enhance the completeness of tumor removal.

Many approaches to local tumor treatment have been reported and their efficacy demonstrated in patients with malignant glioma. We studied thermoreversible gelation polymer (TGP) as a novel drug delivery system (DDS) for treating this type of tumor. TGP exhibits sol-gel transition i.e., is water-soluble in the sol phase below the chosen sol-gel transiting temperature and water-insoluble in the gel phase above this temperature. We conjugated doxorubicin with TGP to prepare doxorubicin-TGP (DXR-TGP), then studied the kinetics of doxorubicin release from TGP and the antitumor activity of DXR-TGP in vitro and in vivo. The diffusive speed of doxorubicin from TGP was 9.4x10(-7) cm(2)/s and doxorubicin was reliably released from TGP. DXR-TGP showed antitumor activity against the human glioma cell lines T98G and U87MG and in a subcutaneous tumor model in nude mice. Pathologically, detection of the proliferation marker Ki-67 was considerably lower in the DXR-TGP group than in the control group (30-40% vs. 60-70%, respectively). This is to the best of our knowledge the first report of TGP as a novel drug delivery system, and further we provide evidence that TGP exhibits potential for use as a novel DDS for malignant glioma.

Histoplasma capsulatum infection is, for the most part, asymptomatic or of little clinical consequence. Disseminated infection due to H. capsulatum is rather uncommon. Clinically apparent infection of central nervous system (CNS) is rare, and involves in 10 to 50% of patients with disseminated histoplasmosis. Although CNS histoplasmosis is frequently fetal or only discovered as an autopsy, some patients can be effectively treated with anti-fungal agents. We describe a 44-year-old civil man who is engineering contractor with headache without evidence of systemic infection. Magnetic resonance imaging showed enhancing masses in the third and forth ventricles, right interpeduncular cistern, and right cerebello-pontine angle. After biopsy of what was presumed to be a malignant lymphoma, the patient died of rapidly progressive multiple cerebral infarctions. The autopsy revealed the CNS histoplasmoma disseminating systemically. And we finally diagnosed him as histoplasmoma by gene analysis. It was extremely difficult to make a diagnosis based on his physical and radiological findings because it should be included in the differential diagnosis of a well or ring enhanced lesion. It is very important to ask patients about their birthplace, past illness, occupation, and where they had traveled. In the present case, the patient working for the construction has visited many countries including the African Continent and Central America. Clinicians should maintain a high index of suspicion in patients who are from any area endemic for histoplasmosis. The clinical, radiological and pathological features of this infection were reviewed in this report.

Introduction: Total removal of the tumor is the most acceptable therapeutic modality in the management of craniopharyngioma; however, there are innumerable factors that can upset treatment plans. Unresectable lesions are often treated with gamma knife surgery (GKS). Reduction of the cystic volume is necessary, to decrease the area to be treated with GKS. An Ommaya reservoir system is usually placed during open surgery or by stereotactic access., Materials and Methods: The authors use a neuroendoscope for safer and less invasive placement of the Ommaya reservoir into deep-seated cystic lesions. The cystic component is aspirated, and the Ommaya reservoir tube is precisely guided and placed into the cyst cavity under neuroendoscopic control with a newly developed two-burr-hole technique. This neuroendoscopic procedure could make it easier to reduce cystic volume prior to GKS as the final procedure. This technique may also be used for instillation of chemotherapeutic agents and for repeat aspirations, making the achievement of cystic control more likely. This type of neuroendoscopic management is a safe and effective procedure and could be considered as an alternative management technique for some stubborn cystic craniopharyngiomas.

The therapeutic modalities for pineal region tumors in Western countries differ from those in far-eastern countries, that is, Japan and Korea, mainly because of the different patient populations. The majority of pineal region tumors in Japan and Korea are radio sensitive and/or chemosensitive, and adjuvant therapy rather than extensive surgery plays the main part in the treatment of these tumors. The authors have applied minimally-invasive preferential management in pineal region tumors in last 8 years. For the therapeutic regimen, if the tumor markers alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) were not detected in serum and there was significant ventricular dilation visualized on neuroimages, neuroendoscopic surgery was first applied for tumor debulking with tissue diagnosis and gross morphological analysis of the tumor and the intraventricular structures, followed by third-ventriculostomy. In the results, our minimally-invasive preferential regimen clarified the precise indication for neuroendoscopic procedures, and the majority of our patients with dilated ventricles and no evidence of tumor markers were treated satisfactorily with effective neuroendoscopic procedures as the initial procedure. Then avoided unnecessary craniotomy and radiotherapy and promised excellent therapeutic outcomes. Neuroendoscopic procedures have a great advantage in the management of chemo- or radiosensitive tumors, such as germinoma, pineoblastoma, or primitive neuroectodermal tumor. The neuroendoscopic anatomy including the lateral and third ventricles with a pineal region tumor with or without tumor dissemination was described in detail, together with the neuroendoscopic surgical technique.

Research studies suggest that tumor-related angiogenesis contributes to the phenotype of malignant gliomas. We assessed the effect of local delivery of the angiogenesis inhibitor endostatin on human glioma cell line (U-87MG) xenografts. Baby hamster kidney (BHK) cells were stably transfected with a human endostatin (hES) expression vector and were encapsulated in alginate-poly L-lysine (PLL) microcapsules for long-term delivery of hES. The release of biologically active endostatin was confirmed using assays of bovine capillary endothelial (BCE) proliferation and of tube formation. Human endostatin released from the microcapsules brought about a 67. 2% inhibition of BCE proliferation. Furthermore, secreted hES was able to inhibit tube formation in KDR/PAE cells (porcine aortic endothelial cells stably transfected with KDR, a tyrosine kinase) treated with conditioned U-87MG medium. A single local injection of encapsulated endostatin-secreting cells in a nude mouse model resulted in a 72.3% reduction in subcutaneous U87 xenografts' weight 21 days post treatment. This inhibition was achieved by only 150.8 ng/ml human endostatin secreted from 2 x 10(5) encapsulated cells. Encapsulated endostatin-secreting cells are effective for the treatment of human glioblastoma xenografts. Continuous local delivery of endostatin may offer an effective therapeutic approach to the treatment of a variety of tumor types.

Objective: We used complementary deoxyribonucleic acid expression microarrays to assess the effects of radiotherapy on gene expression in glioblastoma multiforme. We hypothesized that postradiation recurrent tumors may demonstrate alterations in gene expression from the primary tumor specimen., Methods: Patients were diagnosed with glioblastoma multiforme at resection of the initial tumor, and they received 60 Gy of fractionated radiotherapy before recurrence. Ribonucleic acid samples from both the primary and the postradiation recurrent tumor in each patient were screened and compared using complementary deoxyribonucleic acid expression arrays and Northern blot analysis., Results: Messenger ribonucleic acid levels of growth factors participating in paracrine loops, such as vascular endothelial growth factor and platelet-derived growth factor receptor beta, were decreased in postradiation recurrent tumors as compared with primary tumors in three of four patients. However, messenger ribonucleic acid levels of growth factors involved in autocrine loops, such as epidermal growth factor receptor, platelet-derived growth factor alpha, platelet-derived growth factor A, and basic fibroblast growth factor, were decreased in two of four, two of four, three of four, and three of four patients' recurrent tumors, respectively. Microvessel counts demonstrated that blood vessel growth was decreased significantly in postradiation recurrent tumor specimens., Conclusion: After radiotherapy of glioblastoma multiforme, levels of paracrine-acting growth factors are diminished in correspondence with the reduction in vascular density. In contrast, growth factors that participate in autocrine loops demonstrate elevated levels of gene expression. These results suggest that maintenance of autocrine loops may be important in tumor regrowth after radiotherapy.

The up-regulation of cyclooxygenase 2 (COX-2) expression is a frequent occurrence in a variety of different tumors. In this study, COX-2 protein expression was investigated in 50 glioma and 3 normal brain specimens by immunohistochemistry. Expression of COX-2 protein was observed in all normal brain and glioma specimens by immunohistochemistry, regardless of histological grade. The immunoreactive score was significantly higher in high-grade glioma than low-grade glioma and normal brain specimens. For a subset of these tumors (nine gliomas and three normal brain), Western blot analysis was also performed. COX-2 protein was detected in all specimens by Western blot analysis. The effect of the specific COX-2 inhibitor NS-398 on monolayer cell cultures and three-dimensional glioma spheroids was investigated using U-87MG and U-251MG human glioblastoma cell lines. The proliferation rate was assessed in monolayer cultures. In addition, a growth assay, a migration assay, an apoptosis assay, and a tumor invasion assay were performed in a three-dimensional spheroid culture system. NS-398 was able to reduce the proliferation of monolayer cell cultures, as well as the growth of spheroids and tumor cell migration, in a dose-dependent manner. There was also a moderate increase in the number of apoptotic cells in the treated spheroids. NS-398 did not have an inhibitory effect on tumor invasion in the coculture spheroid system. Our study provides evidence that COX-2 is up-regulated in the majority of high-grade gliomas and that a potential role of COX-2 inhibitors as an adjuvant therapy for brain tumors may exist.

Interleukin-18 (IL-18) exhibits antitumor activity in various laboratory models. In the current study, brain tumors in naive mice regressed after an intratumoral injection of a single dose of recombinant IL-18 (rIL-18). Intraperitoneal rIL-18 substantially delayed the growth of subcutaneously inoculated gliomas but not gliomas located in the brain. Efficacy was reduced when studies were performed in mice depleted of natural killer cells. Although intracerebral administration of rIL-18 increased the serum interferon-gamma concentration, the antitumor effect of IL-18 was not mediated by interferon-gamma. These data suggest the therapeutic potential for control of tumor growth by intratumoral administration of rIL-18 in patients with glioma.

Although tumor-specific T lymphocytes recognize tumor-associated antigens (TAA) present on their cell surface via major histocompatibility complex (MHC) molecules, T cells require other activating signals. These are provided by costimulatory molecules, including B7-1 (CD80), B7-2 (CD86) and intercellular adhesive molecule 1 (ICAM-1; CD54). Transfecting mouse tumor cell lines with the B7 gene can lead to primary tumor rejection and the establishment of protective immunity. However, some studies have shown that the B7 effect upon T-cell-dependent tumor immunity is limited. Therefore, we examined the antitumor effects of recombinant interleukin 12 (IL-12) and genetically engineered glioma cells expressing B7-1 or both B7-1 and ICAM-1. Vaccination of mice with B7-1-expressing tumor cells substantially inhibited the growth of subcutaneously inoculated gliomas but not those located in the brain. Vaccination with B7-1-expressing tumor cells and systemic recombinant IL-12 (rIL-12) was more effective than either B7-1-expressing tumor cells or rIL-12 alone. Our murine brain tumor model also showed that vaccination with tumor cells expressing both B7-1 and ICAM-1 combined with rIL-12 prolonged survival. We have demonstrated the therapeutic potential of vaccination with rIL-12 and tumor cells expressing both B7-1 and ICAM-1 in the control of glioma growth., (Copyright 1999 Wiley-Liss, Inc.)

Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor or cytotoxic lymphocyte maturation factor, has potential for use as an immunomodulator in cancer therapy because it significantly retards the growth of some murine tumors. In this study, we analyzed the antitumor effects of lymphocytes stimulated in vitro with both recombinant IL-2 (rIL-2) and rIL-12. When IL-12 was added to mouse splenocytes (SPCs) or human peripheral blood monocytes (PBMCs) incubated with IL-2 for > 4 days, IL-2-induced cytotoxicity against glioma cells was augmented. In contrast, IL-12 inhibited IL-2-induced lymphokine-activated killer (LAK) cell activity when added concurrently to cultures. The concentration of IL-10 induced by IL-12 increased in the supernatant of human PBMCs costimulated with IL-2 and IL-12. Endogenous IL-10 augmented the cytotoxicity of SPCs stimulated with IL-2 or IL-12 or both. However, tumor-bearing mice treated with PBMCs stimulated with both IL-2 and IL-12 did not survive longer than those treated with PBMCs stimulated with IL-2 alone (LAK cells).

Interleukin 12 (IL-12) exhibits anti-tumor activity in a variety of laboratory models. Although IL-12 itself activates strong anti-tumor activity, the combination of vaccine therapy with IL-2-transduced tumor cells and systemic rIL-12 has been shown to cure tumor-bearing mice more effectively than either rIL-12 or IL-2-transduced tumor vaccines alone. In the present study, regression of brain tumors established in naive mice was obtained by combined administration of an intratumoral injection of a single dose of IL-2-producing glioma cells (SR/IL-2 cells) and recombinant IL-12. Intraperitoneal rIL-12 administration substantially delayed the growth of s.c. inoculated gliomas, but not of gliomas located in the brain. Although vaccination with SR/IL-2 cells alone was not effective against s.c. inoculated gliomas, the combination therapy of vaccination with irradiated SR/IL-2 cells and systemic rIL-12 was more effective than rIL-12 alone. In our brain-tumor model, intratumoral administration of irradiated SR/IL-2 cells and of rIL-12 remarkably prolonged survival as compared with untreated mice. Efficacy was reduced when studies were performed in mice depleted of CD8+ cells or NK cells. Mice cured of their intracerebral tumors by combined administration of SR/IL-2 cells and rIL-12 demonstrated protective immunity upon rechallenge. In summary, the therapeutic potential for control of tumor growth by intratumoral administration of IL-2-producing glioma cells and rIL-12 may be useful in the development of treatment for patients with glioma.

We subcutaneously inoculated parental and glioma cells genetically engineered to express interleukin-2 (SR/IL-2) into syngeneic mice. The tumor growth of the transfectants was slower than that of the parental cells. We then stereotactically inoculated transfectants into the brains of mice. The survival of the mice injected with parental cells was shorter than that of the mice inoculated with transfectants. SR/IL-2 cells were inoculated subcutaneously into the flank of mice, after which rmIL-12 was administered intraperitoneally (i.p.). The resultant transient tumor growth was followed by regression. rmIL-12 or saline were then injected i.p. into mice that had been inoculated in the brain with SR/IL-2 cells. There was no significant difference in survival time between the treated and control groups.

Herein we describe experiments showing that the early growth response gene 1 (EGR-1) promoter is sufficient to confer selective expression of the luciferase gene (Luc) in glioma cell lines exposed to ionizing radiation. Activity of the EGR-1 promoter was investigated in human glioblastoma cells using the plasmid vector, pEGR-Luc. The EGR-1 promoter gene directed radiosensitive expression of luciferase. This promoter showed high levels of activity (10-fold) in irradiated glioma cell lines as compared to basal levels of activity in nonirradiated cell lines. Maximum activation was detectable at 1-3 hr after stimulation with 20 Gy. The results also demonstrate that cells modified to contain the herpes simplex virus-thymidine kinase (HSV-tk) gene under control of the EGR-1 promoter become sensitive to treatment with the antiviral agent ganciclovir (GCV), whereas nonirradiated cells and nontransfected cells were unaffected by this agent. This results suggest that therapeutic genes can be expressed selectively in irradiated glioma cells. The results also indicate that the EGR-1 promoter can be used to induce exogenous genes selectively in radiation fields used for the treatment of malignant brain tumors.