Kimiyoshi Hirakawa

Invasion of experimental rat brain tumor: early morphological changes following microinjection of C6 glioma cells

SummaryWe present morphological data of the early stage of tumor invasion in the central nervous system. C6 rat glioma cells were injected into the caudate-putamen of rat brain using glass micropipettes to minimize traumatic reactions. Four days after the inoculation, we examined the tumor-brain interface using light and electron microscopy. Ultrastructurally the tumor processes were attached to the perivascular basement membrane instead of the astroglial end-feet. At the tumor periphery, the vessel walls were in contact with both tumor processes and astroglial end-feet. Astrocytes withdrew their processes from the vascular walls and changed into a reactive phenotype, while the neuronal cells remained virtually intact, even when surrounded by tumor cells. Immunohistochemical study using C6 cells labeled with bromodeoxyuridine showed migration of the cells toward the perviascular space that was distant from the site of injection. These observations represent the earliest morphologically detectable changes of the tumor-brain interface, and suggest that the C6 cells possess the characteristics of high affinity to the endothelial basement membrane and invade along the preexisting blood vessels with brain parenchymal infiltration.

Postoperative hemodynamic and metabolic changes in patients with subarachnoid hemorrhage.

Positron emission tomography was performed using an oxygen-15 gas inhalation technique to measure regional cerebral blood flow, metabolic rate for oxygen, oxygen extraction fraction, and cerebral blood volume in 13 patients with subarachnoid hemorrhage during the period of delayed vasospasm after surgery as well as in 10 volunteers as controls. Compared with the controls, the patients showed decreased hemoglobin concentration and decreased total arterial oxygen content due to postoperative hemodilution. Global reductions in the metabolic rate for oxygen and in the tissue oxygen supply were noted even in the apparently normal cortex of the patients in spite of adequate blood flow and adequate oxygen extraction fraction. In addition, regional reductions in blood flow and in perfusion reserve were seen in the cortical territory corresponding to cerebral vasospasm. Our results indicate that two processes are involved in the pathophysiology of cerebral vasospasm: 1) generalized impairment of oxygen metabolism with a reduced tissue oxygen supply, even in the apparently normal cortex, and 2) additional impairment of regional perfusion in the territory of vasospasm.

In vivo measurement of energy metabolism and the concomitant monitoring of electroencephalogram in experimental cerebral ischemia

The energy metabolites in rat brain in vivo were measured by using topical magnetic resonance (TMR) during the whole course of ischemia, in combination with the concomitant monitoring of electroencephalogram (EEG). Immediate loss of high energy phosphorus compounds, phosphocreatine (PCr) and ATP, resulted in the flattening of EEG after the induction of ischemia. PCr and ATP returned to almost normal level 30 min after recirculation of the ischemic brain, but EEG showed no recovery and the abnormality lasted for 12 h. The measurement of in vivo 31P-NMR is essential for the decision of the convalescence of cellular function in the brain.

Saccular Aneurysms of the Distal Anterior Cerebral Artery

We report a series of 42 consecutive patients with aneurysms of the distal anterior cerebral artery (ACA). Of these, 36 patients had one aneurysm, 5 had two aneurysms, and one had three aneurysms. Thirty patients had a ruptured distal ACA aneurysm; among these patients, the size of the aneurysm was less than 5 mm in diameter in 20, 6 to 10 mm in 7, and larger than 11 mm in 3. Eighteen patients (42.9%) had multiple aneurysms, and distal ACA aneurysms were responsible for a subarachnoid hemorrhage in 10. Thirty-four patients underwent direct surgery, and 30 of these had excellent outcomes 3 months after surgery. The treatment of patients with distal ACA aneurysms is often technically difficult, because of their broad neck configuration and the coexistence of other aneurysms. Nevertheless, the present study emphasizes that distal ACA aneurysms tend to bleed, irrespective of their size, and that excellent outcomes are obtainable by direct surgery.

Surgically induced angiogenesis to compensate for hemodynamic cerebral ischemia.

Background and Purpose The ischemic brain may stimulate angiogenesis to compensate for impaired circulation. We examined the conditions promoting such angiogenesis to provide the basis for surgical treatment. Methods The degree of cerebral hemodynamic stress was studied in patients with moyamoya disease using the stable xenon-enhanced computed tomographic acetazolamide tolerance test and positron emission tomography. Patients were subjected to surgery in which scalp arteries were placed on the cerebral cortex without vessel-to-vessel anastomosis. Formation of the newly vascularized collateral network connecting the implanted artery to cortical arteries was assessed angio-graphically 12 to 17 months after surgery. Results Preoperative average resting cerebral blood flow for cortex that developed revascularization of cortical arteries was not significantly different from that for cortex that did not. However, cortex that developed revascularization had an average preoperative increase of blood flow by acetazolamide tiveincreasetreatment of −3.29±4.6 mL/min per 100 cm3 (n=20), which was significantly less (P=.0034) than that of cortex that did not show revascularization (20.7±4.3 mL/min per 100 cm3; n=9). Good revascularization developed when the cortex showed increase of blood flow by acetazolamide treatment of less than 0 (steal phenomenon). Preoperative positron emission tomography data indicated that revascularization developed when the cortex was under “misery perfusion.” Postoperative hemo-dynamics were ameliorated by revascularization. Conclusions Angiogenesis to connect the implanted scalp arteries to the cerebral cortical arteries was selectively initiated when ischemia of hemodynamic origin existed.

The role of superoxide anions in the pathogenesis of cerebral vasospasm.

Background and Purpose To determine the role of superoxide anions in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage, we studied the preventive effect of human recombinant copper‐zinc superoxide dismutase (h‐r SOD) in a rabbit subarachnoid hemorrhage (SAH) model. Methods Forty‐five rabbits receiving intracisternal injection of 3 mL autologous nonheparinized blood or 3 mL saline were divided into four groups as follow: (1) saline injected and no treatment (control group, n=6); (2) blood injected and no treatment (SAH group, n=20); (3) blood injected and treated by multiple intracisternal injections of 30 000 U of h‐r SOD in 0.5 mL saline (SOD group, n=9); and (4) blood injected and treated by multiple intracisternal injections of 0.5 mL saline (saline group, n=10). Serial angiograms were performed after the blood injection, and the diameter of the basilar artery was measured. Three animals from the control group and five animals from the SAH and SOD groups each were killed 2 days after SAH, and their basilar arteries were processed for transmission electron microscopic observations. Results In the SAH and saline groups, the diameter of the basilar arteries was significantly reduced (28±14% and 27±9%, respectively) at 2 days after the blood injection, then recovered to pre‐SAH levels until 11 days. In the SOD group, the diameter of the basilar artery was only minimally changed during the follow‐up period. Transmission electron microscopy revealed endothelial injury in all basilar arteries in the SAH group, whereas endothelial injury was minimal in the SOD group. Conclusions We determined that h‐r SOD prevents the occurrence of vasospasm, possibly as a result of preventing endothelial injury initiated by superoxide anions. (Stroke. 1994;25:864‐868.)

Immunohistochemical detection of progesterone receptors and the correlation with Ki-67 labeling indices in paraffin-embedded sections of meningiomas

Female sex steroids may play a role in the proliferation of meningiomas. We investigated the progesterone receptor (PgR) immunoreactivities and the Ki-67 labeling indices in the formalin-fixed, paraffin-embedded sections of meningiomas from 39 patients. After autoclave pretreatment of the sections (which were immersed in a citrate buffer), the sections were incubated with the monoclonal antibody for the PgR and the MIB-1 monoclonal antibody for the Ki-67 antigen. In the meningiomas studied, the immunoreactivity for the PgR was moderately to strongly positive in 51%, weakly positive in 21%, and negative in 28%. The nuclear staining for the PgR was clear, and no tumors were positive for the estrogen receptor. The Ki-67 labeling indices of the PgR-positive meningiomas (mean +/- standard deviation, 2.35 +/- 2.12%) were significantly lower than those of the PgR-negative meningiomas (6.53 +/- 4.83%) (P < 0.05). Two meningiomas that had recurred more than once showed high Ki-67 labeling indices and negative immunostaining for the PgR. These findings indicate that the PgR status may be closely related to the growth potentials of the meningiomas. Our results confirm that the immunodetection of the PgR and the Ki-67 antigen on the paraffin sections of meningiomas provides a practical tool for estimating the biological behavior of the meningiomas.

Effect of human leukocyte interferon on malignant brain tumors.

The antitumor effect of human leukocyte interferon was investigated on ten patients with malignant brain tumor. In eight cases of primary tumor, IFN alone was administered when their recurrent sign was evident. A dose of 3 × 106 IU or 1 × 106 IU of IFN was injected intramuscularly two or three times a week in high‐dose group, while a dose of 5 × 104 IU once a week in low‐dose group. No remarkable side effects including bone marrow depression were noted. Natural killer activity was enhanced and immunologic skin reaction manifested. Partial remission of more than 50% decrease of tumor volume calculated on CT scan was seen in two cases in the low‐dose group for about 3–6 months. Complete remission could not be obtained by IFN alone. Our pilot study has shown that IFN alone will not be effective against progressive malignant brain tumors by general administration. Further investigation should be carried out to improve the use of IFN therapy in malignant brain tumor.

Significance of proton relaxation time measurement in brain edema, cerebral infarction and brain tumors

We examined the proton relaxation times in vitro in various neurological diseases using experimental and clinical materials, and consequently obtained significant results for making a fundamental analysis of magnetic resonance imaging (MRI) as followings. 1) In the brain edema and cerebral infarction, T1 prolonged and T2 separated into two components, one fast and one slow. Prolongation of T1 referred to the volume of increased water in tissue. The slow component of T2 reflects both the volume and the content of increased edema fluid in tissue. 2) In the edematous brain tissue with the damaged Blood-Brain-Barrier (BBB), the slow component of T2 became shorter after the injection of Mn-EDTA. Paramagnetic ion could be used as an indicator to demonstrate the destruction of BBB in the brain. 3) After the i.v. injection of glycerol, the slow component of T2 became shorter in the edematous brain with the concomitant decrease of water content. The effects of therapeutic drug could be evaluated by the measurement of proton relaxation times. 4) Almost all tumor tissue showed a longer T1 and T2 values than the normal rat brain, and many of them showed two components in T2. It was difficult to determine the histology of tumor tissue by the relaxation time alone because of an overlap of T1 and T2 values occurred among various types of brain tumors. 5) In vivo T1 values of various brain tumor were calculated from the data of MRIs by zero-crossing method, and they were compared with the in vitro T1 values which were measured immediately after the surgical operation. Though the absolute value did not coincide with each other due to differences in magnetic field strength, the tendency of the changes was the same among all kinds of tumors. It is concluded that the fundamental analysis of proton relaxation times is essentially important not only for the study of pathophysiology in many diseases but also for the interpretation of clinical MRI.

Antitumor effect induced by granulocyte/macrophage-colony-stimulating factor gene-modified tumor vaccination : comparison of adenovirus- and retrovirus-mediated genetic transduction

Irradiated tumor cells genetically modified to secrete granulocyte/macrophage-colony-stimulating factor (GM-CSF tumor vaccine) are potent stimulators of systemic antitumor immunity. For the preparation of a GM-CSF gene-modified tumor vaccine, it is important to achieve efficient genetic transduction of tumor cells, leading to an appropriate expression of the induced gene. In this report, with a view to developing a protocol for an effective cancer vaccination therapy, we examined the vaccination efficacies of tumor cells secreting GM-CSF by either adenovirus-or retrovirus-mediated genetic transduction. By using an adenoviral vector, Adex1CAmGMCSF, a highly efficient gene were achieved. Unexpectedly, animal vaccination studies showe that the GM-CSF tumor vaccine transduced with the Adex1CAmGMCSF recombinant adenovirus (adenoviral GM-CSF tumor vaccine) was less efficacious than that transduced with the MFGmGMCSF recombinant retrovirus (retroviral GM-CSF tumor vaccine). The GM-CSF serum concentration attained by the adenoviral GM-CSF tumor vaccine was much higher than that obtained by the retroviral GM-CSF tumor vaccine. Our findings indicate that an optimal level of GM-CSF production is important for the tumor vaccine to elicit an adequate response in the host antitumor immunity.