Prophylactic intra-arterial administration of fasudil hydrochloride for vasospasm following subarachnoid haemorrhage.

PURPOSE: We evaluated patients treated with prophylactic intra-arterial administration of fasudil hydrochloride (IAF) after subarachnoid haemorrhage (SAH). MATERIALS AND METHODS: Between August 1998 and December 2012, 92 patients with aneurysmal SAH were treated with IAF for angiographic vasospasm without ischemic symptoms after their follow-up angiography. Patients comprised 50 women and 42 men, aged 24-83 (mean 56.6) years. IAF consisted of 15 mg of fasudil hydrochloride dissolved in 20 ml physiological saline and injected through a catheter during approximately 15 min, after diagnostic angiography. The clinical outcome was evaluated using the Glasgow Outcome Scale (GOS) at discharge and ischemic lesions resulting from vasospasm were assessed on computed tomography (CT) scan at discharge. RESULTS: Forty-eight patients underwent surgical clipping and 44 patients underwent endovascular coiling. Angiographic improvement was observed in all patients (100 %). At discharge, 76 (83.0 %) of 92 patients showed good recovery on GOS. Nine patients developed progression of delayed ischemic neurological deficits (DIND) and three of these patients had ischemic lesions on CT scans. No patient had any significant changes in vital signs or any other adverse effects resulting from IAF. CONCLUSION: IAF therapy was safe and effective for patients with vasospasm following SAH. Prophylactic IAF therapy may prevent symptomatic vasospasm.

Quantitative studies on the polarization optical properties of living cells. I. Microphotometric birefringence detection system.

A method of polarization optical analysis is described in which phase retardation attributable to birefringence of a minute area in a microscopic object is determined. The optical system consists of a polarizing microscope with "rectified" strain-free lenses, a photoelectric detector to determine the intensity of the light passing through a minute window located at the image plane of the specimen, and a stage that moves the specimen at appropriate velocities for scanning. The error resulting from any flare of light emerging from outside of the area to be measured is minimized by limiting the illuminated area. The specimen can be observed during the measurement of light intensity by illuminating the whole microscope field at a wavelength different from that of the light used for the measurement. The retardation of the specimen is determined by comparing the specimen and background intensities as functions of the azimuth of a Brace-Köherl compensator. Alternatively, retardation is obtained directly from the light intensity at a fixed compensator angle, using the theory of polarization optics. The basal noise level for the present apparatus is approximately 0.03 nm when measuring birefringence of a 4-micron2 area in 0.1 s, using a X 40, NA 0.65 objective. The noise decreases in inverse proportion to the square root of the area times the duration of measurement.

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage.

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

Studies on the microtubules in heliozoa. 3. A pressure analysis of the role of these structures in the formation and maintenance of the axopodia of Actinosphaerium nucleofilum (Barrett).

Electron microscope preparations were made of specimens of Actinosphaerium nucleofilum fixed in glutaraldehyde before, during, and after exposure to high pressures (4,000 to 8,000 psi). A study of this material showed that, although other organelles were relatively stable, the microtubular elements of the axopodia and cytosome became unstable under pressure. Their rapid disintegration under pressure was correlated with beading and retraction of the axopodia. Moreover, after the release of pressure, microtubules reappeared as soon as, or sooner than the reextension of the axopodia. The rate of disintegration increased as the pressure was raised. At 4,000 psi, few if any tubules remained after 10 min, whereas at 6,000 and 8,000 psi the disintegration was much more rapid. Some adaptational reorganization of the microtubules and axopodia occurred while relatively low pressures were maintained. This was accompanied by an actual elongation of the axopodia in specimens maintained for 20 min at 4,000 psi, but was confined to knoblike axopodial remnants in animals kept at 6,000 psi. No regeneration of tubules or axopodia occurred at 8,000 psi. The presence of fibers and a finely fibrillar material in pressurized animals suggests that these may be derivatives of microtubular disintegration. This evidence, though purely morphological, is consistent with the hypothesis that microtubules play an important role not only in maintaining the formstability of the axopodia, but also in the active process by which the axopodia reextend themselves after retraction.

Quantitative studies on the polarization optical properties of living cells II. The role of microtubules in birefringence of the spindle of the sea urchin egg.

Birefringence of the mitotic apparatus (MA) and its change during mitosis in sea urchin eggs were quantitatively determined using the birefringence detection apparatus reported in the preceding paper (Hiramoto el al., 1981, J. Cell Biol. 89:115-120). The birefringence and the form of the MA are represented by five parameters: peak retardation (delta p), through retardation (delta t), interpolar distance (D1), the distance (D2) between chromosome groups moving toward poles, and the distance (D3) between two retardation peaks. Distributions of birefringence retardation and the coefficient of birefringence in the spindle were quantitatively determined in MAs isolated during metaphase and anaphase. The distribution of microtubules (MTs) contained in the spindle is attributable to the form birefringence caused by regularly arranged MTs. The distribution coincided fairly well with the distribution of MTs in isolated MAs determined by electron microscopy. Under the same assumption, the distribution of MTS in the spindle in living cells during mitosis was determined. The results show that the distribution of MTs and the total amount of polymerized tubulin (MTs) in the spindle change during mitosis, suggesting the assembly and disassembly of MTs as well as the dislocation of MTs during mitosis.

Effect of glucocorticoid replacement on tumor growth after adrenalectomy in mice.

We studied the effects of glucocorticoid replacement on tumor growth after adrenalectomy of Meth A sarcoma in mice. Tumor growth was inhibited in the adrenalectomized mice when a minimum dose of corticosterone, 0.3 mg/day, was given for replacement, and higher doses led to an even greater inhibition. Corticosterone had no effect on tumor growth in the irradiated mice. Sinecomitant immunity in the case of growth of the retransplanted excised tumor was compromised in the adrenalectomized mice. In vivo neutralization and immunosuppressive activities were absent in the spleen cells of the adrenalectomized mice. It would thus appear that adrenalectomy suppresses tumor growth by mechanisms other than glucocorticoid ablation. For optimum tumor control, glucocorticoid replacement after adrenalectomy should be in excess of the minimum daily requirements.

Centrifuge microscope as a tool in the study of cell motility.

The centrifuge microscope (CM) is composed of a centrifuge and a microscope optical system designed to observe minute objects, especially living cells, during the application of centrifugal acceleration. Structures and characteristics of various types of CM designed and constructed up to the present and studies done with the CM on cell biology, especially cell motility, are reviewed. These studies include observations of the behavior of cells and cell components in a centrifugal field, determination of the mechanical properties of the cell surface and cytoplasm, microsurgical operations on cells with centrifugal force, and determination of the magnitude and the site of generation of motive force for cell motility.

A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus.

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.

Coxsackie viral infection of human myocardium.

The myocardiu m of a young patient who had clinically established cardiomyopathy and suspected Coxsackie virus B4 infection was studied. Coxsackie B4 viral antigen was found in the ventricular and atrial myocardium by specific immunofluorescent antibody staining. Histologic examination revealed varying degrees of myocardial damage. Interstitial fibrosis and edema, swelling and deterioration of hypertrophic muscle fibers, connective tissue proliferation, stasis of small coronary blood vessels, atrophy of myocardial fibers with pyknosis of nuclei, and lytic deterioration were observed. Electron microscopic examination showed portions of the Z bands to be either widened or displaced into the sarcomere. Adjacent cell membranes in the region of the intercalated disc in the myocardium of both the ventricle and the atrium were separated, forming large gaps. Morphologic changes were most pronounced in the atrium adjacent to the mitral valve, in which the mitochondria were grossly swollen, and large vesicles were present in the sarcoplasm. The pathologic changes found in the myocardium of all chambers of the heart apparently were due to Coxsackie B4 viral infection.

MECHANICAL PROPERTIES OF STARFISH OOCYTES.

The stiffness of the starfish oocyte was determined from the degree of deformation when it was compressed by a definite force between a pair of parallel plates. The deformation of the oocyte increases during continued application of a constant force, indicating visco-elasticity of the cell. A cyclic change in stiffness of the oocyte accompanying meiotic divisions was found: the stiffness of the oocyte decreases during early stage of meiotic division, increases before the onset of the first polar body formation, then decreases, increases again before the onset of the second polar body formation, and decreases thereafter. Deuteration causes increase in stiffness of the oocyte.

MECHANICAL PROPERTIES OF SEA URCHIN EGGS III. VISCO-ELASTICITY OF THE CELL SURFACE.

When a sea urchin egg was compressed between two parallel plates, the force required to keep the distance between the plates constant gradually decreased with time. The contours of the compressed egg were different from the contours expected from the assumption that the surface forces are uniform over the entire surface. The surface forces of the egg without deformation computed from the area of the cell surface in contact with the substratum, the density of the egg and its size were 0.02-0.04 dynes/cm in Hemicentrotus pulcherrimus. Larger values were obtained in eggs during compression. Surface forces, which were computed from measurements of the form of the egg and the applied force when the egg was deformed by a rod and a plate supporting the egg, increased as the deformation increased. From these results, it was concluded that the cell surface is visco-elastic in sea urchin eggs.

FORCE EXERTED BY THE CLEAVAGE FURROW OF SEA URCHIN EGGS.

A drop of ferrofluid injected into the center of a dividing sea urchin egg is deformed into the shape of an hourglass when the cleavage furrow advances. The force applied to the drop is determined from the deformation of the drop and the interfacial tension between the ferrofluid and the protoplasm. The interfacial tension is determined from the deformation of a spherical drop in the protoplasm when a magnetic field is applied, and the force applied to the drop, which is estimated from the deformation by magnetic field of a similar drop in 2 per cent aqueous solution of Triton X-100 and the interfacial tension between the ferrofluid and this solution. The force applied to the drop in the dividing egg increases during an early stage of cleavage and decreases during a later stage. The force attained a maximum of 9 × 10-3 dyne in an egg of Temnopleurus toreumaticus which pinched the drop into two when it divided. Smaller maximum forces, 3.9 × 10-3 dyne in the eggs of Temno-pleurus toreumaticus and 2.0 × 10-3 dyne in the eggs of Clypeaster japonicus (mean values), were obtained when the furrowing was arrested by the drop. The magnitude of the maximum tension developed in the contractile element located in the furrow cortex is discussed.

MECHANICAL PROPERTIES OF THE CELL SURFACE IN STARFISH EGGS.

The mechanical properties of the cell surface of the starfish egg at various stages of maturation have been investigated using the cell elastimeter. When constant negative pressure was applied to a part of the cell with a micropipette closely in contact with it, it bulged out, and the bulge rapidly increased at first and then gradually reached a steady value within one min. The relation between the deformation of the cell surface (i.e. degree of bulging) and applied negative pressure was almost linear in both oocytes at the germinal vesicle stage and mature eggs. The surface force and the elasticity value: i.e., the product of the elastic modulus of the surface membrane (layer) and its thickness, were determined from the relation between the deformation and the negative pressure. The elasticity value was about 5 times the surface force in both oocytes at the germinal vesicle stage and mature eggs. When maturation of the oocyte was induced by 1-methyladenine, the stiffness of the cell surface decreased shortly before the breakdown of the germinal vesicle. The stiffness transiently increased at the time of formation of the first and second polar-bodies.

FERTILIZATION PROCESS IN THE HEART-URCHIN, CLYPEASTER JAPONICUS OBSERVED WITH A DIFFERENTIAL INTERFERENCE MICROSCOPE.

The observations of the fertilization process in the heart-urchin, Clypeaster japonicus with a differential interference microscope indicate that the sperm pronucleus is carried to the center of the egg by the growth of the sperm aster as stated by Chambers (5), and that the egg pronucleus is carried to the center of the aster by a filamentous structure formed between them. The curved path of egg pronucleus in the fertilized egg is interpreted as the combination of the movement of the center of the aster and the movement of the egg pronucleus toward the center of the aster. The movement and the rotation of the sperm head result from pushing by the tail being engulfed in the egg.

REFRACTIVE INDEX OF THE PROTOPLASM IN SEA URCHIN EGGS.

The distribution of the refractive index (RI) of the protoplasm in sea urchin eggs was determined from the optical path differences at various regions of the cell measured by interference microscopy assuming that the cell structure is symmetrical about the line passing through the center of the cell and that of the nucleus in unfertilized eggs and about the spindle axis in fertilized eggs during mitosis and cleavage. The RI of the cytoplasm in the unfertilized egg was uniform except for the cortical region, which had the RI higher than that of the underlying endoplasm. The RI of the cortex was generally higher than that of the underlying endoplasm, which did not appreciably change during mitosis and cleavage. The RI of the nucleus was lower than that of the cytoplasm. The RI of the mitotic apparatus was lower than that of the surrounding cytoplasm. The fertilization membrane had a thickness of about 0.6 µm in hydrated state and about 25 nm in dried state (mean values). The RI of the perivitelline space was about 0.00015 higher than that of seawater, equivalent to 0.08 g/100 ml of contents.

Synthesis of 6-acetamido-5-amino- and -5-guanidino-3, 4-dehydro-N-(2-ethylbutyryl)- 3-piperidinecarboxylic acids related to zanamivir and oseltamivir, inhibitors of influenza virus neuraminidases.

[reaction: see text] 6-Acetamido-5-amino- and -5-guanidino-3, 4-dehydro-N-(2-ethylbutyryl)-3-piperidinecarboxylic acids (8 and 9) have been synthesized starting from natural siastatin B, a bacterial neuraminidase inhibitor isolated from Streptomyces culture in a stereospecific fashion. These compounds are related to zanamivir and oseltamivir, inhibitors of influenza virus neuraminidases.

Mechanical properties of the cortex before and during cleavage.

The changes in stiffness of the cell before and during cleavage reported previously for various kinds of echinoderm eggs are classified into three types. There is no general rule that cleavage starts when the stiffness attains a maximum or a minimum. Because the stiffness of the cell surface is much greater than that of the mitotic apparatus, the change in stiffness of the cell represents mainly the change in stiffness of the cell surface. The tension at the cell surface changes in parallel over the entire surface before the onset of cleavage. After the onset of cleavage, the tension at the furrow surface becomes greater in the direction parallel to the furrow than perpendicular to it. A remarkable change in mechanical properties occurs at the furrow surface simultaneously with the onset of cleavage. Judging from the temporal and spatial coincidence between this change and the formation of the contractile ring reported previously in sea urchin eggs at the onset of cleavage, it is concluded that this change is due to the formation of the contractile ring that generates the motive force for cleavage.

Lymphadenectomy for cure in patients with early gastric cancer and lymph node metastasis.

The anatomic distribution, size, and histologic mode of involvement of 98 metastatic lymph nodes in 49 of 370 patients were examined to determine to what extent lymphadenectomy should be performed in addition to gastrectomy in patients with early gastric cancer. Nodal involvement in the marginal sinus (30 nodes) and partial medullary sinus (37 nodes) were commonly seen, and the lymph nodes of those types were enlarged compared with 1,086 patients with no metastatic lymph nodes (control group). Lymph nodes of the wide medullary sinus (11 nodes), small nodule (3 nodes), and massive involvement types (17 nodes) did not enlarge compared with those of the other types and those of the control group. Most of the metastatic sites (76.6 percent) were in the perigastric lymph nodes along the lesser and greater curvatures, about a fifth were in the extraperigastric nodes along the left gastric, common hepatic, celiac, and splenic arteries, and the least were in the extraperigastric nodes (3.1 percent) along the hepatoduodenal ligament. Since the rate of macroscopic diagnosis during operation was so poor, regardless of the histologic modes of nodal involvement, and also in cases of metastatic lymph nodes less than 15 mm in widest diameter, for curative operation of patients with early gastric cancer, perigastric and extraperigastric lymph nodes along the main arteries near the stomach should be completely dissected, in addition to resection of the stomach.

Rheological properties of echinoderm eggs during cell division.

Mechanical properties of the cell before and during cleavage in sea urchin eggs (a typical equal symmetrical cytoplasmic division) and polar-body formation (a typical unequal cytoplasmic division) are reviewed with special reference to the mechanism of cell division. Both in sea urchin eggs and starfish oocytes, the tension at the cell surface gradually increases before the onset of cytoplasmic division. The tension gradually decreases during division in starfish oocytes and in some sea urchin eggs. Some other sea urchin eggs display two peaks of the tension, one just before the onset of division and the other during division. The cleavage furrow is formed by the active contraction of a layer formed in the equatorial cortex of the cell in sea urchin eggs and of a ring-shaped layer formed in the cortex around the animal pole of the cell in starfish oocyte.

[Anatomical quantification of the tibial part of the plantar aponeurosis].

The metrical analysis of the anatomical characteristics is important because of its objectiveness. As it is concerned with the organs belonging to the locomotor system, the metrical method of the bones has already been systematized by Martin (1928), whereas the same kind of method for use on other organs remains undeveloped. The author aims to establish the metrical method of the plantar aponeurosis. The method for measuring the tibial part of the aponeurosis developed in this paper is sufficiently applicable for obtaining its principal anatomical characteristics. The results show that the tibial portion of the plantar aponeurosis becomes statistically significantly wider and thinner in its anterior part, and that the thickness of the tibial portion of the aponeurosis in the anterior part is larger on the right side than on the left side.