Cilostazol, an inhibitor of cyclic nucleotide phosphodiesterase 3 (PDE3), has been used clinically for the treatment of chronic arterial occlusive diseases and secondary prevention of cerebral infarction. The beneficial effect of cilostazol is attributed to both anti-platelet aggregation and vasodilation. Effects of cilostazol on resistance-sized cerebral arteries, however, have not been well elucidated. In this study, we investigated the mode of action of cilostazol on pressurized cerebral arterioles. Cerebral arterioles were cannulated and monitored with an inverted microscope. The vessels developed myogenic tone by constricting approximately 50% from the maximum passive diameter. Cilostazol (10⁻⁹–10⁻⁴ M) produced concentration-dependent vasodilation. The agent also produced a similar relaxation in the arterioles precontracted by 5-hydroxytryptamine or U46619, a stable analog of thromboxane A₂. Cilostazol (10⁻⁶ M) augmented the adenosine-induced vasodilation of the arterioles. Adenosine (10⁻⁷ M) also caused a potentiation of the cilostazol-mediated vasodilation. Nω-nitro-L-arginine methyl ester (L-NAME) (10⁻⁴ M), a nitric oxide synthase inhibitor, or aspirin (10⁻⁵ M), a cyclooxygenase inhibitor, did not significantly alter the cilostazol-induced arteriolar dilation. Furthermore, vasodilation by cilostazol was observed with endothelial-denudated arterioles. These results suggest that cilostazol produces the vasodilation of cerebral arterioles independent on the functional endothelium. [Jpn J Physiol 55 Suppl:S91 (2005)]

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We examined the behavior of factor XI ( F. XI) in Watanabe-hereditable hyperlipidemic (WHHL ), to study the participation of F. XI in atherosclerosis. F. XI was isolated with use of anti-human F. XI monoclonal antibody-Sepharose. Purified F. XI was single band in sodium dodecylsulfate polyacrylamide gel electrophoresis (M. W.: 80, 000), and the specific coagulant activity was 75unit/mg protein. ¹²⁵I- F. XI, iodinated with Bolton-Hunter reagent, was administered intravenously to WHHL and Japanese white (normal). Then blood was taken from ear vein and the trichloroacetic acid precipitate of plasma was counted. Turnover of ¹²⁵I- F. XI was significantly faster in WHHL (T1/2=2.84±0.44 days) than in normal (T1/2=4.44±0.42 days). By en-face autoradiography, specific accumulation of ¹²⁵I- F. XI was observed in thoracic aorta of WHHL . In contrast, no radioactivity was observed in normal. These results suggest that activation of F. XI proceeds on the atherosclerotic lesion of WHHL .

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Utility of Japanese White mutants with hypotrichosis (htr strain) was investigated in dermal toxicity studies. In the primary irritation study, the primary irritation indices (P.I.I.) by application of hexachlorophene and 3, 3’, 4’, 5-tetrachlorosalicylanilide (TCSA) were higher in the htr rabbits than in the control, haired rabbits. The P.I.I. by application of Tween 80 and sodium lauryl sulfate (SLS) were similar between the htr rabbits and the control rabbits. In the 16-day cumulative irritation study, irritation scores induced by SLS and sodium hydroxide were lower in the htr rabbits than in the control rabbits throughout the observation periods. However, approximately 30% of the dorsal skin of the control rabbits underwent the anagen phase of the hair cycle during the cumulative periods, resulting in difficulty to apply the chemicals and to estimate the skin reactions. In the phototoxicity study, irritation scores of the htr rabbits treated with 8-methoxypsoralen and TCSA were similar to those of the control rabbits. These results revealed advantages of the htr rabbits in the administration of test chemicals and the evaluation of skin reactions, suggesting the usefulness of htr rabbits for dermal toxicity studies.

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In the present study we attempted to establish specific-pathogen-free (SPF) breeding colonies with two groups of limited-flora (LF) rabbits, both ex-germfree rabbits, and their offspring. Two groups of LF rabbits associated with cecal flora of conventional (CV) rabbits produced in a previous study [Exp. Animals, submitted], were transferred to individual barrier rooms and some of the LF rabbits were accomodated in isolators to maintain the basic flora for SPF rabbits. The composition of the cecal flora of LF rabbits was stable for a long period; bacteroides remained predominant and clostridia dominant. From the SPF rabbits, different types of bacteria, e.g., enterobacteriaceae and streptococci, which could not be isolated in the isolator were detected at a low population level at an early stage in the establishment of the SPF colonies, but the basic composition of the cecal flora was mainly bacteroidaceae and clostridia and did not change over a long period, and the floral composition became similar to that of CV rabbits. The fertility and weaning rates of the SPF rabbits were satisfactory for a SPF colony. In addition, these SPF colonies were free of more than one year -specific pathogens.

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Twenty-three of 42 European rabbits (Oryctolagus cuniculus), belonging to the same

colony, died in March 2020 (55% mortality) in Chiba prefecture, Japan. The disease course was extremely acute without indicators of death or hemorrhage. Necropsy revealed liver swelling, discoloration, cloudiness and fragility, and pulmonary edema. Histologically, severe hepatocellular necrosis (mainly peripheral) and intra-glomerular capillary hyalin thrombi were observed. On molecular-biological examination, reverse transcription polymerase chain reaction analysis of RNA from tissues detected a hemorrhagic disease virus, confirmed as a RHDV-2 VP60 fragment, which shared 99.42% nucleotide identity with the homologous fragment of RHDV-2 German isolate by nucleotide sequence analysis. This report shows the outbreak of hemorrhagic disease caused by RHDV-2, an emerging infectious disease, in Japan.

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遺伝性高脂血症(WHHL)ウサギにおいて, 粥状硬化の血圧およびその安定性に対する影響を調べた. 6-30ヵ月齢の正常な日本白色種ウサギ25匹および12-35ヵ月齢のWHHLウサギ12匹について, 麻酔下で血圧測定用カテーテルを左鎖骨下動脈より大動脈弓に慢性的に留置し, 数日後, ゲージ内で自由行動下にて, 平均血圧を約6時間連続的にコンピュータで記録した. 平均血圧の6時間の平均値は, WHHLウサギでは85.8～131.4mmHgにあり, 正常ウサギより有意に高い値を示したが, いずれのウサギにおいても加齢による上昇は観察されなかった. WHHLウサギの平均血圧の6時間の標準偏差は5.6～12.6mmHgで, 加齢により有意な増加を示したが, 正常ウサギでは有意な変化はみられなかった. WHHLウサギの血清総コレステロールおよびトリグリセリド濃度は, それぞれ475および328mg/dlで, それぞれ正常ウサギの9, 7倍であった. 上記生理学的所見は大動脈病理所見をよく反映していた. 以上の結果より, WHHLウサギにおける血圧安定性の低下は, 加齢に伴う粥状硬化の進展による動脈コンプライアンスの減少ならびに圧受容器の応答性の低下に起因するものと解釈した.

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Objective: Severe cerebral contusion will cause a functional defect in partial central nerve and will lower patients' living quality. The above problem is still a difficult problem unsolved in clinical treatment. This study is going to clarify the methodology of brain tissue transplantation, including the way of operation and the law of microcirculatory formation. Method: Twenty healthy male, Japanese white rabbits with long ears (about three months after birth) of clean degree, weighting between 1.2kg and 1.3kg, were anaesthetized by 3% pentobarbital sodium in vein before receiving an intracerebral transplantation operation. A window was opened on their parietal bone symmetrically, and the cortical brain tissues on the symmetrical areas (on the left and right side) of the rabbits' parietal cortex areas were exchanged and transplanted. Gentamycin sulfate was injected each day to resist infection. Ten and twenty days later, an observation was made as to the survival of the transplanted area and host brain tissue. A microcirculation color camera system was used to analyze the pictures of angiogenesis. With regard to the survival of transplanted brain tissues, their changes in micromorphology were observed. Besides, pathological sections were also prepared to determine their surviving conditions on cell level. Results: (1) Surgical operation has contributed to a satisfactory morphological anastomosis between transplanted brain tissues and host brain tissues. (2) Micro-blood vessel loops were observed to have formed on the section (host side) of rabbits' parietal cortex areas, which had been excised partially when transplantation was not filled. Marker was implanted into the transplanted brain tissues to confirm the possibility of the regeneration of a microcirculation among brain tissues, which had been exsomatized completely. (3) Analysis of the pathological sections of the transplanted brain tissues showed traces of surviving nervous cells. Conclusions: Under given conditions, nervous cells' survival can be maintained by transplanted brain tissues and can be nourished by angiogenesis and characteristic microcirculation connections with host brain tissues.

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We studied the effect of hypoxia on lipid accumulation in cultured fibroblasts from normal rabbits (normal fibroblasts) and in low density lipoprotein (LDL) receptor-negative fibroblasts from WHHL (Watanabe heritable hyperlipidemic) rabbits (WHHL fibroblasts). These cells were incubated in medium with normolipemic serum (NRS) or hyperlipemic serum (HRS). The cells were incubated in a humidified atmosphere of either 20% O₂, 75% N₂, and 5% CO₂ (control cells) or 2% O₂, 93% N₂, and 5% CO₂ (hypoxic cells).
After 48h incubation of normal fibroblasts in medium with 20% NRS, free fatty acid (FFA) levels were increased slightly and the triglyceride (TG) level markedly in hypoxic cells. In the medium with 20% HRS, in addition to the increased FFA and TG levels, the free cholesterol level was increased slightly and the esterified cholesterol level markedly in hypoxic cells. Moreover, in WHHL fibroblasts, which lack LDL receptors, cellular lipid accumulation was also observed after 48h incubation in the medium with 20% HRS, and hypoxic incubation enhanced the cellular cholesterol and TG accumulation, as in normal fibroblasts.
These results suggest that under hyperlipemic conditions, non LDL receptor-mediated uptake of lipoproteins plays a major role in cellular lipid deposition and that tissue hypoxia promotes lipid accumulation in peripheral cells by the LDL receptor-independent mechanisms.

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The bile of homozygous WHHL- (Watanabe Heritable Hyperlipidemic ) was analyzed and compared with Japanese White rabbits. There is no significant difference in bile acid and cholesterol concentration of both hepatic and gallbladder bile. And among the bile acids composition of hepatic bile, deoxycholic acid occupied the dominant portion, about 85 per cent. In the rest small portion, a small quantity of cholic acid, allodeoxycholic acid and lithocholic acid were existed. The composition of bile acids was almost the same in the two groups except for the decrease of cholic acid in WHHL-. In perfusion experiment of isolated liver, no difference was observed in the serial bile flow, biliary cholesterol and bile acid concentrations in the two groups. Lipid concentrations of perfusate and cholesterol contents of liver, which were measured after perfusion, showed no difference in the two groups. Synthesis of cholesterol was measured in WHHL- by the sterol balance technique. Newly cholesterol synthesis decreased by one thirds in WHHL-, but not significantly. These results revealed that hepatic handling of bile acids in WHHL- was almost the same as in normal , while it is well known that homozygous human subjects with familial hypercholesterolemia show decrease of biliary cholic acid. WHHL-, which possess a nearly complete deficiency of LDL-receptor, tend to reduce cholesterol synthesis with little influence on catabolism of cholesterol to bile acid.

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We have recently isolated macroglobulin Xa inhibitor from human plasma. Human plasma was heated for 10 minutes at 56°C. Heated plasma was chromatographed on a DEAE cellulose column with step wise elution technique using 0.04M Tris-HCl buffer. Activated factor X inhibitor was eluted with 0.03M NaCl in same buffer. This inhibitor was fractionated on a Sephadex G-200 column. Activated factor X inhibitor was observed in the first peak (void volume). On Ouchterlony's method, this inhibitor (M-XaI) made precipitin lines with antihuman serum serum, anti-human β-lipoprotein serum and anti-M-XaI serum. Anti-Xa activity of M-XaI was not neutralized with anti-human α₂ macroglobulin serum, anti-human AT III serum and anti-β-lipoprotein serum. However, anti-M-XaI serum and anti-human serum serum neutralized completely anti-Xa activity of M-XaI.
This is the hitherto undescribed macroglobulin inhibitor of activated factor X.

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The effect of the heat treatment, which was applied under various conditions, on δ-aminolevulinic acid dehydrase (ALAD) activities was investigated by means of determination of ALAD activities in the peripheral blood of normal rabbits (N-) and intravenously lead-injected rabbits (Pb-). Furthermore, lead was added in vitro to N- or Pb-rabbits' blood, then ALAD activities were assayed with and without heat treatment. The following findings were obtained:
(1) ALAD activity of the N- blood decreased monotonously with increase of temperature and time of the heat treatment. (2) Decrease of ALAD activity of the Pb- blood is shown in Table 1. (3) In both N- and Pb-rabbits' blood, the ALAD activities after heat treatment at 60°C for 5min converge to a definite value, which is about 1/3 of ALAD activity of the N- blood, the value being nearly constant and independent of the doses of lead administration. It was revealed that an increase of the activity by the heat treatment in the Pb- blood takes place only in cases in which the initial activity is lower than this value. (4) When lead was added to the N- or Pb- blood in vitro, (a) ALAD activity was not affected by 2.9×10^-10M Pb concentration in incubation mixture in both cases. (b) By 2.9×10^-8M Pb ALAD activity fell to about 50% in the N- blood and to 40∼20% in the Pb- blood, the latter depending upon the doses of lead previously administered in vivo. (c) Decrease of ALAD activity by heat treatment was enhanced by adding lead in vitro, and this effect was more remarkable in the blood which was affected by previous administration of lead. (5) Preincubation of the N- or Pb- blood with lead only enhanced the decrease of ALAD activity, without showing any increase in the activity.

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In the present report, the author gives the result of his attempt to solve the questions whether the labrinthine fluid contains agglutinin or not, and further whether the transfusion of the agglutiniu of the blood serum into the labyrinthine fluid occurs in an immunised animal. Using as the test animals normal or rabbits immunised with the typhoid bacillus or the choleravivrio, he obtained the under-mentioned results:
1) Though the typhoid bacillus or the choleravivrio is often agglutinated in the serum of the normal , no agglutination was found in the labyrinthine fluid of the normal .
2) No agglutination occured with the typhoid bacillus or the choleravivrio either in the cerebrospinal fluid or in the aqueous humour of the eye of the normal .
3) The proportion of agglutinin transfered into the labyrinthine fluid of a immunised with the typhoid bacillus, was as 1: 100-1: 200 compared with that in the serum.
4) The quantity of agglutinin in the cerebrospinal fluid and in the aqueous humour of the eye of a immunised with the typhoid bacillus, was almost the same as that in the labyrinthine fluid.
5) The proportion of agglutinin transfered into the labyrinthine fluid of a immunised with the choleravivrio, was as 1: 200-1: 400 compared with that in the serum.
6) The quantity of agglutinin in the cerebrospinal fluid and in the aqueous humour of the eye of a immunised with the choleravivrio, was almost the same as that in the labyrinthine fluid.
7) When the value of agglutinin in the serum is low, it does not pass into the labyrinthine fluid, the cerebrospinal fluid or the aqueous humour of the eye of the .
8) It may be assumed that the transfusion of the agglutinin of the blood serum into the labyrinthine fluid, has great significance in connection with the physiology of the labyrinth.

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A novel lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) was recently identified in bovine aortic endothelial cells. It is strongly suggested to have a potential role in the initiation and development of atherosclerosis. In this study, we have isolated cDNA clones encoding the homologue of LOX-1 by screening a placenta cDNA library. In amino acid sequence and domain structure organization, the LOX-1 is highly conserved with the human counterpart. Transfection of LOX-1 cDNA to HEK-293 cells conferred the activity to bind and internalize oxidized low density lipoprotein. LOX-1 was identified as a 45-kDa protein by Western blot analysis with a specific monoclonal antibody. Notably, analyses by reverse trascription-polymerase chain reaction and Western blot revealed that LOX-1 was accumulated in 8-week-old Watanabe heritable hyperlipidemic aortas compared with normal aortas. Immunostaining confirmed that the augmented expression of LOX-1 was primarily localized within the intima at the earliest stages of atherogenesis. The most prominent staining was in the endothelial cells of lesions. Furthermore, the distinctive staining of LOX-1 was identified in the endothelium of nonlesion areas of Watanabe heritable hyperlipidemic aortas. Taken together, these findings support the possibility that LOX-1 might be involved in the initiation of atherosclerosis.

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It is noted that there are two types of WHHL rabbits; the one with high incidence of coronary atherosclerosis (type 1), and the other with low incidence of coronary atherosclerosis (type 2). In order to analyze the difference between type 1 and type 2 WHHL rabbits, in this study, the atherogenesity of some classes of lipoproteins were estimated by incubation with mouse peritoneal macrophages.
Among the several classes of lipoproteins, VLDL from type 1 WHHL could stimulate cholesteryl [14C]-oleate synthesis 10.5 fold than did VLDL from type 2 WHHL . VLDL from normal showed no significant stimulatory effect. LDL did not stimulate esterification of cholesterol in macrophages, unless it became modified form.
Mass ratios (cholesterol/protein) of these lipoproteins were calculated for their characterization. The calculated ratios of β-VLDL, VLDL from type 1 WHHL , from type 2 WHHL and from normal were 13.6, 5.69, 2.05 and 0.82, respectively.
These data suggest that the high incidence of coronary atherosclerosis in type 1 WHHL is partially due to the cholesteryl ester rich VLDL, and that the atherosclerosis observed in WHHL is caused not only by high concentration of plasma LDL, but also by cholesteryl ester rich VLDL.

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Species-specificity and reacting site of antiglobulin factors, non-neutralizable by homologous γ-globulin, were studied and the following results were obtained:
1) Many of human sera showing only weak antiglobulin activity of anti-antibody nature against human red cells sensitized with human whole Rh-antibody were found to be capable of agglutinating the red cells sensitized with pepsin fragment —F(ab′)2— of the Rh-antibody.
2) In serum of a exhibiting antiglobulin activity of anti-antibody nature, there was found, in addition to a factor reacting only with antibodies, a factor capable of cross-reacting with both (homologous) and human (heterologous) antibodies.
3) anti-antibodies were found to agglutinate sheep red cells sensitized with non-agglutinating pepsin fragment of anti-sheep erythrocyte antibody. This finding seems to indicate that reacting site against anti-antibody must be located in the Fab portion of IgG.
4) Cross-reacting antiglobulin factor of anti-antibody nature was also found in a antiserum against HB antigen, prepared by immunization with specific immune complex.

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The facial artery and its ramifications in the were studied by the acryl plastic injection method. The facial artery arose independently from the external carotid artery or the maxillary inferomedial to the origin tendon of the digastricus muscle, or in common with the lingual artery. It curved inferomedially lateral to this muscle, but did not curve when its origin was located on the maxillary, where it gave rise to the styloglossal and the digastric branches, then the parotid branch medial to the ventral portion of the gland and the submandibular branch between this gland and the pterygoideus medialis muscle. Both glandular branches in some cases supplied the submandibular lymph nodes and the skin. The artery then passed forwards on the medial surface of the pterygoideus medialis and gave rise to the muscular branches for it and the maxillomandibular branch. The facial artery appeared on the face passing through the vascular notch of the mandible. The submental artery, giving off the infradigastric, the posterodigastric and the mylohyoid branches, curved forwards on the medial surface of the mandible and terminated at the insertion of the digastricus after giving rise to the supradigastric branches. Sometimes it supplied the lymph nodes and the skin covering an area beneath the symphysis and the submandibular region. The primary masseteric branch, giving off twigs to the maxillomandibularis muscle, passed through the vascular notch together with the facial artery and supplied the masseter muscle from its anteroinferior end. The facial passed along the anterior margin of this muscle and gave rise to the inferior labial depressor, the zygomaticoauricular branches and the inferior labial artery. It ascended between the zygomaticus and the buccinator, and finally divided into the superior labial artery and the oral angular branch in the middle of the buccinator, after giving off the secondary and the tertiary masseteric branches and the branch to the masseteric gland. The inferior labial artery passed anterosuperiorly between the buccinator and the zygomaticoauricularis, giving off the branches to the pars molaris of the buccinator and to the superficial mandibular gland and the inferior labial depressor branch up to the angle of the mouth, where it gave rise to the branch to the pars buccalis of the buccinator, the diastema and the gingival branches. It passed medially within the orbicularis oris and formed the inferior labial arterial arch by anastomosing with the opposite fellow. The secondary masseteric supplied the pars molaris and the the masseter from the medial side and the tertiary masseteric from the anterior margin. The branch to the masseteric gland supplied the pars molaris of the buccinator and divided into the anterior and posterior, which supplied the buccal mucosa and gingivae posterior to P₁.

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