Hiroshi Tokimura

Ryk is essential for Wnt-5a-dependent invasiveness in human glioma

Glioblastoma is characterized by marked invasiveness, but little is known about the mechanism of invasion in glioblastoma cells. Wnts are secreted ligands that regulate cell proliferation, differentiation, motility and fate at various developmental stages. In adults, misregulation of the Wnt pathway is associated with several diseases. Recently, we reported that Wnt-5a was overexpressed and correlated with cell motility and infiltrative activity through the regulation of matrix metalloproteinase (MMP)-2 in glioma-derived cells. Although several receptors for Wnt-5a were identified, the receptors of Wnt-5a that mediate cellular responses of glioma were not clearly identified. Knockdown of receptor-like tyrosine kinase (Ryk) but not that of Ror2 suppressed the activity of MMP-2 and Wnt-5a-dependent invasive activity in glioma cells. These results suggest that Ryk is important for the Wnt-5a-dependent induction of MMP-2 and invasive activity in glioma-derived cells and that Ryk might have a novel patho-physiological function in adult cancer invasion. Furthermore, not only the expression of Wnt-5a but also that of Frizzled (Fz)-2 and Ryk was correlated with the WHO histological grade in 38 human glioma tissues. Taking these findings together, Fz-2 and Ryk could be therapeutic or pharmacological target molecules for the control of Wnt-5a-dependent invasion of human glioma in the near future.

Low distribution of synaptic vesicle protein 2A and synaptotagimin-1 in the cerebral cortex and hippocampus of spontaneously epileptic rats exhibiting both tonic convulsion and absence seizure

The spontaneously epileptic rat (SER) is a double mutant (zi/zi, tm/tm) which begins to exhibit tonic convulsions and absence seizures after 6 weeks of age, and repetitive tonic seizures over time induce sclerosis-like changes in SER hippocampus with high brain-derived neurotrophic factor (BDNF) expression. Levetiracetam, which binds to synaptic vesicle protein 2A (SV2A), inhibited both tonic convulsions and absence seizures in SERs. We studied SER brains histologically and immunohistochemically after verification by electroencephalography (EEG), as SERs exhibit seizure-related alterations in the cerebral cortex and hippocampus. SERs did not show interictal abnormal spikes and slow waves typical of focal epilepsy or symptomatic generalized epilepsy. The difference in neuronal density of the cerebral cortex was insignificant between SER and Wistar rats, and apoptotic neurons did not appear in SERs. BDNF distributions portrayed higher values in the entorhinal and piriform cortices which would relate with hippocampal sclerosis-like changes. Similar synaptophysin expression in the cerebral cortex and hippocampus was found in both animals. Low and diffuse SV2A distribution portrayed in the cerebral cortex and hippocampus of SERs was significantly less than that of all cerebral lobes and inner molecular layer (IML) of the dentate gyrus (DG) of Wistar rats. The extent of low SV2A expression/distribution in SERs was particularly remarkable in the frontal (51% of control) and entorhinal cortices (47%). Lower synaptotagmin-1 expression (vs Wistar rats) was located in the frontal (31%), piriform (13%) and entorhinal (39%) cortices, and IML of the DG (38%) in SER. Focal low distribution of synaptotagmin-1 accompanying low SV2A expression may contribute to epileptogenesis and seizure propagation in SER.

Identification of CD133+ Cells in Pituitary Adenomas

Stem-like cells in tumors are capable of self-renewal and pluri-differentiation; they are thought to play important roles in tumor initiation and maintenance. Stem-like cells in malignant glioma express CD133. We examined samples from human pituitary adenoma, a generally benign neoplasm, for CD133 expression using routine immunohistochemical and biochemical methods. Our study of 70 pituitary adenomas (clinically nonfunctioning adenomas and growth hormone-, prolactin-, adrenocorticotropic hormone-, and thyroid-stimulating hormone-producing adenomas) showed that 18 (25.7%) expressed CD133. This rate was higher in clinically nonfunctioning (33.3%) than functioning adenomas (12.0%) (p = 0.085). Real-time PCR assay revealed the expression of CD133 mRNA in samples immunohistochemically positive for CD133. Neither the patient age and gender, nor the tumor size or postoperative recurrence rate correlated with CD133 positivity. CD133+ cells ubiquitously coexpressed CD34, nestin, and VEGFR2 (KDL1). S-100 and GFAP were not coexpressed with CD133. Chromogranin A, Pit-1, SF-1, and NeuroD1 were immune-negative, indicating that CD133+ cells did not have the potential to differentiate into functional endocrine cells. Our data suggest that the expression of CD133 in pituitary adenomas is related to immature endothelial progenitor cells that may play a role in the neovascularization of pituitary adenomas. Further studies are needed to elucidate the significance of CD133+ cells with respect to neovascularization and their sustainable growth in pituitary adenomas.

C-type natriuretic peptide modulates permeability of the blood-brain barrier

C-type natriuretic peptide (CNP) is abundant in brain and is reported to exert autocrine function in vascular cells, but its effect on blood–brain barrier (BBB) permeability has not been clarified yet. Here, we examined this effect. Transendothelial electrical resistance (TEER) of in vitro BBB model, composed of bovine brain microvascular endothelial cells and astrocytes, was significantly dose dependently decreased by CNP (1, 10, and 100 nmol/L). C-type natriuretic peptide treatment reduced both the messenger RNA (mRNA) and protein expressions of tight junction (TJ) protein zonula occludens-1 (ZO-1). The effects on TEER, mRNA, and protein expressions of ZO-1 were mimicked by cyclic GMP (cGMP) analog 8-bromo-cGMP (1 μmol/L) and reversed by protein kinase G (PKG) inhibitor Rp-8-CPT-cGMPS (100 μmol/L), thus implying the role of PKG and cGMP signaling in BBB function. Transcription factor JunD knockdown by small interfering RNA resulted in no change of permeability by CNP. In vivo study of mouse brain by fluorimetric analysis with intravenous administration of sodium fluorescein (40 mg/kg) also showed a significant increase in BBB permeability by CNP (10 nmol/kg, intravenously). These findings suggest that CNP modulates the BBB permeability by altering ZO-1 expression.

Cranial surgery without head shaving

OBJECTIVE Based on a series of 632 patients who underwent craniotomy without head shaving, we report the efficacy and safety of our simplified procedure and document the usefulness of the electrosurgical scalpel. METHODS After brushing a chlorhexidine-alcohol solution onto the craniotomy site, the hair was parted from the incision line and fixed with adhesive paper drapes. In recent cases, electrosurgical scalpels were used for scalp- and subcutaneous dissection. At the end of surgery, the wound was closed in the usual manner, taking care that no hair was in the wound and the hair and wound were rinsed with clean water in the operating room. We did not apply disinfectant for postoperative wound care, rather, the hair was shampooed on the 2nd, 4th, and 6th postoperative day. RESULTS Among 632 patients who underwent cranial surgery without head shaving, only 7 (1.1%) developed postoperative wound infections. None of the 34 patients who underwent craniotomy using the electrosurgical scalpel developed wound infections. CONCLUSIONS Our simplified cranial surgery without head shaving does not increase the risk of wound infection. Because the use of the electrosurgical scalpel for skin and soft tissue dissection minimizes bleeding, the probability of wound infection appears to be reduced.

Development and pharmacological verification of a new mouse model of central post-stroke pain.

Central post-stroke pain (CPSP) including thalamic pain is one of the most troublesome sequelae that can occur after a cerebrovascular accident. Although the prevalence of CPSP among stroke patients is relatively low, the persistent, often treatment-refractory, painful sensations can be a major problem and decrease the affected patients quality of life. To better understand of the pathophysiological basis of CPSP, we developed and characterized a new mouse model of thalamic CPSP. This model is based on a hemorrhagic stroke lesion with collagenase in the ventral posterolateral nucleus of the thalamus. Histopathological analysis indicated that the thalamic hemorrhage produced a relatively confined lesion that destroys the tissue within the initial bleed, and also showed the presence of activated microglia adjacent to the core of hemorrhagic lesions. Behavioral analysis demonstrated that the animals displayed diclofenac-, morphine- or pregabalin-resistant mechanical allodynia and thermal hyperalgesia of the hind paw contralateral to the lesion for over 112 days. However, we found that minocycline, a microglial inhibitor, significantly ameliorated mechanical allodynia and thermal hyperalgesia. These results suggest that this model might be proved as a useful animal model for studying the neuropathology of thalamic syndrome, and developing improved therapeutics for CPSP.

Identification of the Cerebral Motor Cortex by Focal Magnetic Stimulation: Clinical Application to Neurosurgical Patients

A figure-8 coil was designed that was capable of generating currents over a relatively limited area. In this study, we used the figure-8 coil to localize human cerebral motor cortex by transcranial magnetic stimulation and assessed its value as a diagnostic tool for neurosurgical disease. The subjects were 10 normal volunteers and 12 neurosurgical patients. The points obtaining a response were plotted and the response area was delineated for each stimulus output. Rectangular coordinates were subsequently determined for the C3-Cz-C4 line with the stimulation sites set 1.0 cm apart. Then amplitude and latency maps were displayed by a computer wave map program. In normal volunteers, the posterior margin of the site where a threshold +5% stimulus obtained a response from the THE or ADM muscles almost coincided with the central sulcus. The optimal areas were relatively narrow on the amplitude and latency maps. In the patient with a left frontal convexity meningioma, the left threshold +5% area for the THE and ADM muscles showed marked anterolateral shift. No threshold map could be obtained on the affected side in patients with metastatic tumor and astrocytoma. Low perfusion areas caused by moyamoya disease and arteriovenous malformations produced narrowing of the optimal zone on the threshold map of the affected side. Hyperexcitability of the motor cortex was recognized in the arteriovenous malformation patients. Transcranial focal magnetic stimulation is a useful noninvasive diagnostic method for identification of the human cerebral motor cortex. It provides important information about changes in the localization and excitability of the motor cortex in neurosurgical patients and should be helpful for treatment planning.

Clinical presentation and treatment of aneurysms associated with basilar artery fenestration

Aneurysms associated with a fenestrated basilar artery are rare, and treatment strategies have yet to be established. A direct surgical approach to the basilar artery is challenging because the surrounding anatomy is complex. We retrospectively compared the clinical features and treatment outcomes of eight patients (seven female, one male) with aneurysms associated with a fenestrated basilar artery after clipping or coil embolisation and reviewed the literature. Of the eight aneurysms, four were ruptured; seven aneurysms were located at the proximal part of the basilar artery and one aneurysm was located at the middle of the basilar artery. Six aneurysms were surgically treated. Four aneurysms were embolised with Guglielmi detachable coils, two aneurysms were clipped via the transcondylar or temporopolar approach, and two aneurysms were not treated. All six surgically treated patients had good outcomes. We found that both coil embolisation and direct clipping to treat aneurysms associated with a fenestrated basilar artery have advantages and disadvantages. To obtain favourable outcomes, the selected treatment modality must consider the patients age and clinical condition, the aneurysm size and shape, the direction of the dome, the relationship with perforators, and the neurosurgeons expertise.

Efficacy and safety of the pterional keyhole approach for the treatment of anterior circulation aneurysms.

Keyhole surgery is partly replacing the standard pterional approach in patients undergoing surgery to treat aneurysms of the anterior circulation. We describe the pterional keyhole approach for the clipping of anterior circulation aneurysms and discuss the efficacy and safety of our keyhole craniotomy procedure. We treated 103 patients with 111 intracranial aneurysms by surgical clipping via the pterional keyhole approach and retrospectively compared the characteristics and clinical outcomes of the keyhole procedure and the standard pterional approach. We also compared the surgical results of the keyhole approach when the operator was an experienced neurosurgeon or a less experienced neurosurgeon guided by an experienced colleague. All keyhole operations were carried out successfully without enlargement of the craniotomy or a change to a different approach. The outcomes of the keyhole and the standard pterional approach in patients with subarachnoid hemorrhage were not significantly different. Favorable outcomes were obtained in patients with unruptured aneurysms treated by either experienced or less experienced surgeons. The pterional keyhole approach offers the same surgical possibilities as conventional pterional approaches for the treatment of anterior circulation aneurysms. It is safe and simple and yields favorable outcomes even if the operators are less experienced neurosurgeons. Careful patient selection and sufficient opening of the sylvian fissure are the key points for good outcomes and the prevention of intraoperative complications.

Pituitary metastases: current practice in Japan

OBJECT With advancement of cancer treatment and development of neuroimaging techniques, contemporary clinical pictures of pituitary metastases (PMs) must have changed from past reports. The goal of this paper was to elucidate the clinical features of PMs and current clinical practice related to those lesions. In this retrospective study, questionnaires were sent to 87 physicians who had treated PMs in Japan. RESULTS Between 1995 and 2010, 201 patients with PMs were treated by the participating physicians. The diagnosis of PM was histologically verified in 69 patients (34.3%). In the other 132 patients (65.7%), the PM was diagnosed by their physicians based on neuroimaging findings and clinical courses. The most frequent primary tumor was lung (36.8%), followed by breast (22.9%) and kidney (7.0%) cancer. The average interval between diagnosis of primary cancer and detection of PM was 2.8 ± 3.9 (SD) years. Major symptoms at diagnosis were visual disturbance in 30.3%, diabetes insipidus in 27.4%, fatigue in 25.4%, headache in 20.4%, and double vision in 17.4%. Major neuroimaging features were mass lesion in the pituitary stalk (63.3%), constriction of tumor at the diaphragmatic hiatus (44.7%), hypothalamic mass lesion (17.4%), and hyperintensity in the optic tract (11.4%). Surgical treatment was performed in 26.9% of patients, and 74.6% had radiation therapy; 80.0% of patients who underwent radiotherapy had stereotactic radiotherapy. The median survival time was 12.9 months in total. Contributing factors for good prognosis calculated by Cox proportional hazard analysis were younger age, late metastasis to the pituitary gland, smaller PM size, and radiation therapy. The Kaplan-Meier survival was significantly better in patients with breast cancer and renal cell cancer than in those with lung cancer. CONCLUSIONS At the time of this writing, approximately 60% (120/201) of PMs had been treated by stereotactic radiation therapy in Japan. The median survival time was much longer than that reported in past series. To confirm the changes of clinical features and medical practice, a prospective and population-based survey is mandatory.