Takeo Uzuka

Therapeutic efficacy of targeting chemotherapy using local hyperthermia and thermosensitive liposome: evaluation of drug distribution in a rat glioma model

A method was developed of targeting chemotherapy using thermosensitive liposomes to treat malignant gliomas. Using the brain heating system, when the tumour core is heated to >43°C, the tumour infiltrating zone is exposed to mild hyperthermia (40–43°C). Thermosensitive liposomes were designed to release their contents at 40°C to target both the tumour core and tumour infiltrating zone. The present study investigated the anti-tumour effect on rat glioma models in tumour drug uptake and tumour growth delay studies. Elevated accumulation of ADR in the rat C6 glioma after treatment was obtained in the area heated to >40°C. However, there was no significant difference between the areas heated to 40–42°C and >43°C. Furthermore, it was found that ADR concentrations in the mildly hyperthermic areas were significantly higher following treatment with liposomal ADR than with free ADR. The animals treated with the new combination therapy had significantly longer overall survival time in comparison to those receiving other treatments. Thus, thermosensitive liposomes release their contents in response to mild hyperthermia and this combination therapy has a greater therapeutic efficacy for malignant brain tumours. This method is a promising approach for the treatment of malignant glioma patients.

Induction of autophagy in temozolomide treated malignant gliomas

Autophagy is a dynamic process of protein degradation. Induction of autophagy by temozolomide (TMZ) has been noted in glioma cell lines. Twenty‐eight specimens, obtained from 14 patients before and after TMZ treatment, were analyzed to investigate whether induction of autophagy could be detected in surgical specimens by immunohistochemical analysis. Macroautophagy was monitored by immunohistochemical analysis employing anti‐light chain 3 isoform B (LC3B) and anti‐lysosome‐associated membrane protein 1 (LAMP1) antibodies; chaperone‐mediated autophagy was monitored by anti‐LAMP2A antibody immunostaining. Furthermore, detection of LC3B protein by Western blotting was performed on six specimens obtained from the preserved frozen tissues of three patients. All specimens showed dot‐like staining for each immunostain in the cytoplasm of glioma cells, indicating induction of autophagy. LC3B, LAMP1 and LAMP2A immunostains were semiquantitatively scored from 1 to 3 points. Combination of the three scores after TMZ treatment (6.4 ± 1.2) showed a significant increase (P = 0.020) compared to pre‐treatment scores (5.2 ± 1.5). Western blotting for LC3B showed increased LC3B‐I and LC3B‐II expression after TMZ treatment. The present study proved that autophagy monitoring by immunohistochemical staining of surgical specimens was feasible. These results suggest that autophagy is induced by TMZ.

Intraventricular pleomorphic xanthoastrocytoma with anaplastic features

Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytic tumor that usually occurs in the superficial cerebral hemispheres of children and young adults and has a relatively favorable prognosis. We report an unusual case of supratentorial, intraventricular tumor in a 52‐year‐old man. The tumor was composed of pleomorphic cells, including giant cells, most of which were multinucleated, and small cells. In addition, frequent xanthic changes in the cytoplasm of the tumor cells, and widespread reticulin deposits and lymphocytic infiltrates in the stroma were characteristic features. Large areas of necrosis were also evident. However, mitotic figures were rare (1–2 mitoses per 10 high‐power fields). Many tumor cells were positive for GFAP, and a number were positive for neurofilament protein and synaptophysin, indicating their neuronal differentiation. In addition, occasional tumor cells were positive for CD34. p53 protein was entirely negative in the tumor cells. In diagnosing this tumor histopathologically, differentiation between PXA and giant cell glioblastoma (GCG), a rare variant of glioblastoma, was problematic. However, considering the overall histopathological picture, a final diagnosis of PXA with anaplastic features was made. The present case indicates that PXA can occur as an intraventricular tumor, and suggests that in some instances, it would be very difficult to differentiate PXA and GCG histopathologically.

Planning of hyperthermic treatment for malignant glioma using computer simulation

Interstitial hyperthermia was applied using a radiofrequency generator in the treatment of four malignant glioma patients who had especially deep seated brain tumours or were at high risk. Prior to heating tumours, treatment planning based on an accurate prediction of temperature distribution is essential. The present paper introduces a novel treatment planning method and discusses its clinical efficacy. The two-dimensional finite element method was used for simulation of temperature distribution, which was calculated using the bioheat transfer equation. This technique was applied to plan treatment. Temperature was measured at two points during heating and these values were compared with those estimated by the simulation. In addition, the area of the contrast enhanced (CE) rim on the pre-heating computed tomography (CT) image was compared with the low density area of the CE rim on the post-heating CT image, which was obtained within 2 months after heating. The optimal position and number of radiofrequency (RF) electrodes to include the outside of the CE rim in the simulated area above 42 degrees C contour could be easily determined using this planning system in all cases. The temperature estimated by the simulation was in good agreement with the actual values obtained (within 0.4 degrees C). The post-heating CT image revealed that the hyperthermic procedure described herein achieved more than an 80% low density area within the CE rim in all cases (mean 86.0%). These results demonstrate that this novel treatment planning method may prove to be a clinically valuable tool in the treatment of malignant glioma with RF electrodes.Interstitial hyperthermia was applied using a radiofrequency generator in the treatment of four malignant glioma patients who had especially deep seated brain tumours or were at high risk. Prior to heating tumours, treatment planning based on an accurate prediction of temperature distribution is essential. The present paper introduces a novel treatment planning method and discusses its clinical efficacy. The two-dimensional finite element method was used for simulation of temperature distribution, which was calculated using the bioheat transfer equation. This technique was applied to plan treatment. Temperature was measured at two points during heating and these values were compared with those estimated by the simulation. In addition, the area of the contrast enhanced (CE) rim on the pre-heating computed tomography (CT) image was compared with the low density area of the CE rim on the post-heating CT image, which was obtained within 2 months after heating. The optimal position and number of radiofrequency (RF) electrodes to include the outside of the CE rim in the simulated area above 42°C contour could be easily determined using this planning system in all cases. The temperature estimated by the simulation was in good agreement with the actual values obtained (within 0.4°C). The post-heating CT image revealed that the hyperthermic procedure described herein achieved more than an 80% low density area within the CE rim in all cases (mean 86.0%). These results demonstrate that this novel treatment planning method may prove to be a clinically valuable tool in the treatment of malignant glioma with RF electrodes.

Near-infrared spectroscopic study and the Wada test for presurgical evaluation of expressive and receptive language functions in glioma patients: With a case report of dissociated language functions

Near-infrared spectroscopy (NIRS) has proven to be useful for the evaluation of language lateralization in healthy subjects, infants, and epileptic patients. This study for the first time investigated the expressive and receptive language functions separately, using NIRS in presurgical glioma patients. We also describe a special case with dissociated pattern of language functions. Ten glioma patients were examined. Using NIRS, the hemodynamic changes during a verb generation task or story listening task were measured in the cerebral hemisphere on either side covering the language areas. Following the NIRS study, the Wada test was performed in all the patients. The NIRS study revealed increases of oxyhemoglobin and decreases of deoxyhemoglobin in the language areas elicited by both tasks. In 9 patients, who were all right-handed, the expressive and receptive language functions were lateralized to the left hemisphere. The results of the NIRS study were completely consistent with those of the Wada test. In the remaining 1 patient with a right sided insular glioma, who was right-handed, the NIRS study revealed stronger activation of the right inferior frontal region during the verb generation task, and stronger activation of the left superior temporal region during the story listening task. This dissociated language function was validated by the Wada test and the postoperative neurological course. These results demonstrate that a NIRS study using our technique is extremely valuable for preoperative assessment of the language functions and exemplifies how a preoperative NIRS study can allow detection of unforeseen language lateralization.

Basic study of brain injury mechanism caused by cavitation

The purpose of this study is to discuss the mechanism of brain injury experimentally, with respect to the pressure changes on the surface of a brain agar phantom by cavitation. First, an experimental system to perform an impact experiment is presented. We present some images taken by a high-speed camera of the behavior of a simple physical head model with and without the brain agar phantom during impact. From the photographs of the high-speed camera, we can confirm that cavitation bubbles occur at the contrecoup side, irrespective of the usage of the brain agar phantom. Second, two experimental systems to perform impact and strike experiments are presented. The pressure changes on the surface of the brain agar phantom at contrecoup side were measured by two kinds of experiments and impact velocities. Frequency analysis of the measured pressure changes was conducted by FFT software. From these results, we found that the collapse of cavitation bubbles at the contrecoup side can strongly affect the characteristics of pressure changes on the surface of the brain agar phantom.

Temperature distributions of developed needle type applicator

This paper discusses radio frequency (RF) interstitial hyperthermia for a brain tumor with needle type applicators. In this method, it is necessary to make appropriate thermal distribution to the region of the brain tumor. However, it is difficult to predict the thermal distribution. We estimated temperature distribution inside an agar phantom by the finite element method (FEM), and heated agar phantom by the developed system. First, we compared computer simulation results and experimental results. Next, we discussed the heating properties of changing the inserted direction of a single type needle applicator. Finally, the temperature distribution of agar phantom heated by a multi type needle applicator was presented

Mild hyperthermia plus adenoviral p53 over-expression additively inhibits the viability of human malignant glioma cells

Adenoviral replacement of the p53 gene has already been proved effective for the treatment of various tumours, including malignant gliomas. However, it is difficult to treat malignant glioma with p53 gene therapy alone because of problems with resistance or a less-than-satisfactory response to the treatment. This study investigated whether heat shock at 43°C (mild hyperthermia) augments the cytotoxic effect of p53 gene transfer on malignant glioma cells expressing wild-type p53 (D54) or mutant p53 (U373-MG and U251-MG). The combination of mild hyperthermia and adenoviral p53 over-expression had an additive inhibitory effect on cellular proliferation in all three cell lines studied. Further, both cell cycle analysis and a DNA fragmentation assay showed that apoptosis was induced by p53 over-expression alone but not by heat shock at 43°C alone. However, p53 over-expression followed by mild hyperthermia additively increased the proportion of cells in which apoptosis was induced, regardless of the endogenous p53 status of the tumour cells. Interestingly, a caspase-independent mechanism was observed to be involved in the p53-induced apoptosis in U251-MG and D54 cells. Taken together, the findings showed that combining adenoviral p53 transfer with mild hyperthermia inhibits the proliferation of malignant glioma cells in an additive manner, irrespective of their endogenous p53 status, suggesting a novel treatment strategy for this malignancy.

Finite element analysis and experimental study on mechanism of brain injury using brain model.

The aim of this study is to discuss the occurrence mechanism of the brain injury analytically and experimentally. In this paper, first, an experimental system to do an impact experiment was presented. The pressure changes inside a brain agar phantom were measured. Second, a three-dimensional FEM model of the impact experiment was constructed. From the results of the fundamental analysis, the transmitted pressure inside the brain agar phantom could be presented. The comparison of the computer simulation and experimental results showed that the negative pressure values, same as the positive pressure occurred in the coup side region of the agar, also appeared in the contrecoup side region of the agar

Identification and validation of a gene expression signature that predicts outcome in malignant glioma patients

Better understanding of the underlying biology of malignant gliomas is critical for the development of early detection strategies and new therapeutics. This study aimed to define genes associated with survival. We investigated whether genes selected using random survival forests model could be used to define subgroups of gliomas objectively. RNAs from 50 non-treated gliomas were analyzed using the GeneChip Human Genome U133 Plus 2.0 Expression array. We identified 82 genes whose expression was strongly and consistently related to patient survival. For practical purposes, a 15-gene set was also selected. Both the complete 82 gene signature and the 15 gene set subgroup indicated their significant predictivity in the 3 out of 4 independent external dataset. Our method was effective for objectively classifying gliomas, and provided a more accurate predictor of prognosis. We assessed the relationship between gene expressions and survival time by using the random survival forests model and this performance was a better classifier compared to significance analysis of microarrays.