Yasunori Shibutani

Repeated administration of low-dose lipopolysaccharide induces preterm delivery in mice: a model for human preterm parturition and for assessment of the therapeutic ability of drugs against preterm delivery.

OBJECTIVE Our purpose was to establish a new animal model for human preterm delivery for assessment of the protective effect of drugs against preterm delivery. STUDY DESIGN C3H/HeN, C3H/HeN, and BALB/c female mice impregnated by C3H/HeN, B6D2F1, and B6D2F1 male mice, respectively, were treated intraperitoneally with Escherichia coli lipopolysaccharide (0 to 100 microgram/kg, single dose or repeated doses at 1- to 6-hour intervals) on days 12 through 17 of pregnancy. On day 15 of pregnancy, the C3H/HeN females that had been impregnated by B6D2F1 males and administered lipopolysaccharide were treated intraperitoneally with indomethacin (1000 microgram/kg), ritodrine hydrochloride (1000 microgram/kg), urinary trypsin inhibitor (25 x 10(4) units/kg), or gabexate mesylate (100 mg/kg); preterm or term delivery was recorded for these mice. RESULTS C3H/HeN females impregnated by B6D2F1 males revealed the highest (100%) incidence of preterm delivery when the females were treated with 50 microgram/kg lipopolysaccharide twice at a 3-hour interval on day 15 or 17 of pregnancy. Indomethacin and urinary trypsin inhibitor used separately significantly decreased the incidence of preterm delivery, but only urinary trypsin inhibitor, and not any of the other drugs, significantly increased the incidence of term delivery in the mice. CONCLUSION A new animal model for investigation of preterm delivery was established, and its usefulness for assessment of the protective effect of drugs against preterm delivery was demonstrated.

Lipoteichoic acid induces preterm delivery in mice

The purpose of this study was to determine whether or not lipoteichoic acid (LTA) could induce preterm delivery in mice. On days 15 and 17 of pregnancy, female C3H/HeN mice impregnated by male B6D2F1 mice were given intraperitoneal injections of LTA (12.5-75 mg/kg, single dose or repeated doses at a 3-h interval). We examined the changes in cervix, placental trophoblasts, and plasma and amniotic fluid concentrations of interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) after dosing with LTA. In addition, the effect of LTA on the contraction of isolated uterine muscle from pregnant mice was also measured. The incidence of preterm delivery was highest (100%), when the pregnant animals were treated with 75 mg/kg LTA twice on day 15 of pregnancy or with 25 mg/kg LTA twice on day 17 of pregnancy. LTA-accelerated cervical ripening and placental abruption preceding the onset of preterm delivery, as well as increased plasma and amniotic fluid concentrations of IL-1alpha, IL-6, and TNF-alpha. Also, LTA increased contraction of uterine muscle strips. In conclusion, LTA induced preterm delivery in mice in the same manner as lipopolysaccharide (LPS), but the effective dose of LTA was larger than that of LPS.

Trophoblastic apoptosis in mice with preterm delivery and its suppression by urinary trypsin inhibitor

Objective To investigate histopathologic changes of the placenta in mice with preterm delivery induced by lipopoly-saccharide and the effect of urinary trypsin inhibitor. Methods Female C3H/HeN mice impregnated by male B6D2F1 mice were treated with lipopolysaccharide (50 μg/kg, intraperitoneally) or lipopolysaccharide plus urinary trypsin inhibitor (25 × 104 U/kg, intraperitoneally). At 3, 6, 9, and 18 hours after the second dose of lipopolysaccharide, and at delivery in the control and urinary trypsin inhibitor-treated groups, the concentrations of interleukin-1α and tumor necrosis factor-α were determined in serum and amniotic fluid. Subsequently, the placentas were examined. In the same manner, we examined mice treated with interleukin-1α (250 μg/kg, subcutaneously) on day 15 of pregnancy and intact mice on days 15 and 18 of pregnancy as well as at delivery. To assess the direct action of cytokines, we cultured placental slices with tumor necrosis factor-α, interleukin-1a, or tumor necrosis factor-α plus urinary trypsin inhibitor and examined them morphologically. Results Control mice were characterized by trophoblastic apoptosis and increased serum levels of tumor necrosis factor-α and interleukin-1α. In contrast, urinary trypsin inhibitor-treated mice showed suppression of apoptosis and lower cytokine levels. Interleukin-1α induced trophoblastic apoptosis and increased the serum level of tumor necrosis factor-α. The in vitro study showed that tumor necrosis factor-α directly induced trophoblastic apoptosis in placental slices. Conclusion We demonstrated that trophoblastic apoptosis occurs in the placentas of a mouse model with preterm delivery induced by lipopolysaccharide. We postulated that apoptosis may lead to placental abruption, and its development may be prevented by treatment with urinary trypsin inhibitor.

Dienogest, a synthetic steroid, suppresses both embryonic and tumor-cell-induced angiogenesis

Orally administered dienogest (17alpha-cyanomethyl-17beta-hydroxy-estra-4,9-diene-3-one) is efficacious against human hormone-dependent cancer xenografts in severely immunodeficient mice and in rats with experimental endometriosis, but its mechanisms of action remain unclear. We assessed the effect of dienogest on angiogenesis, because these two diseases that are sensitive to dienogest are known to be angiogenesis-dependent. Topical dienogest treatment dose-dependently inhibited embryonic angiogenesis, the ID(50) value being 6.4 nmol/egg. Oral administration of dienogest (1 mg kg(-1) day(-1)) for 5 consecutive days significantly suppressed angiogenesis induced by S-180 mouse tumor cells in the mouse dorsal air sac assay. In vitro experiments showed that dienogest at concentrations up to 10 microM had little or no effect on the proliferation of plasminogen activator activity or formation of tube-like structures by microvascular endothelial cells. These results suggest that dienogest is a new, orally active antagonist of angiogenesis, and that its anti-angiogenic action may be involved in its therapeutic effects on cancer xenografts and endometriosis that we observed previously.

Acute Toxicity of an Anti-Fas Antibody in Mice

By histopathologic examination of various organs in 3 normal strains, C3H/HeN, ICR, and DBA/1J, of mice treated intravenously once with anti-Fas antibody (Jo2), we failed to determine any target organ, except the liver, responsible for the acute lethality induced by the Fas/anti-Fas antibody interaction. However, we could show the presence of Fas-mediated apoptosis in other organs aside from the liver and normal mouse strain differences in susceptibility to anti-Fas antibody. Among these strains, C3H/HeN was the most susceptible to the antibody, followed by ICR and DBA/1J. We observed Fas-mediated apoptosis in the liver, spleen, thymus, lymph nodes, Peyers patch, intestine, skin, coagulation glands, ovary, uterus, and vagina in all 3 strains and additionally in the epididymides and seminal vesicles in the DBA/1J strain. We also demonstrated that Fas-mediated apoptosis of small lymphocytes in the mantle zone of splenic lymphatic follicles preceded that of the hepatocytes or thymic cells. Since cellular damage was most severe in the liver among all the apoptotic organs in the 3 mouse strains, liver injury induced by anti-Fas antibody is speculated to play a significant role in the death.

Age-Related Changes in Coagulation, Fibrinolysis, and Platelet Aggregation in Male WBN/Kob Rats

We investigated the age-related changes in blood coagulation, fibrinolysis, and platelet aggregation in male WBN/Kob rats, animals that exhibit spontaneously diabetes mellitus at more than 6 months of age. The rats aged 6 months or more showed significant hyperglycemia, hypoinsulinemia, and hyperlipidemia. As changes in coagulation parameters, the data indicated significant increases in factors II, V, VII, VIII, IX, X, and XII activities; a significant decrease in antithrombin III activity in rats more than 6 months of age; significant increases in fibrinogen level and factor XI activity; and significant decreases in prothrombin time and activated partial thromboplastin time in those more than 9 months of age. As changes in fibrinolytic parameters, the animals showed significant decreases in plasminogen and tissue-type plasminogen activator, and significant increases in alpha2-plasmin inhibitor and plasminogen activator inhibitor at more than 6 months of age. In addition, there were significant correlations between the plasma levels of coagulation/fibrinolytic markers and the 4-hour fasting glucose or lipids. Furthermore, they displayed significant increases in ADP- or collagen-induced platelet aggregation and in cholesterol/phospholipid molar ratio in platelets at more than 9 months of age. The increase in cholesterol/phospholipid ratio may be responsible for hyperaggregation of platelets in diabetic animals. These findings suggest that WBN/Kob rats are suitable for research on blood coagulation abnormalities in diabetes. However, further studies are needed to clarify the details of the mechanisms involved.

Role of urinary trypsin inhibitor in the maintenance of pregnancy in mice

Objective To investigate the mechanisms whereby urinary trypsin inhibitor prevents lipopolysaccharide-induced preterm delivery in mice. Methods On day 15 of pregnancy, C3H/HeNCrg female mice impregnated by Crg:B6D2F1 male mice were treated intraperitoneally with lipopolysaccharide (50 μg/kg, twice at a 3-hour interval) to induce preterm delivery. Urinary trypsin inhibitor (2.5 × 104, 7.5 × 104, or 25 × 104 units/kg, ten times at 1-hour intervals) or saline solution was administered intraperitoneally to the animals. Results The incidence of preterm delivery was significantly decreased on a dose-related basis by urinary trypsin inhibitor treatment. Urinary trypsin inhibitor prevented the morphologic and functional changes in fetal membranes and cervical ripening preceding the onset of preterm delivery. Urinary trypsin inhibitor also suppressed the increase in plasma and amniotic fluid concentrations of interleukin-1α, interleukin-6, and tumor necrosis factor-α after the lipopoly-saccharide dosing in this animal model for preterm delivery. Conclusion Urinary trypsin inhibitor prevents the pathogenicity of preterm delivery through the suppression of cytokine production.

Spontaneous adrenal and hepatic myelolipomas in the common marmoset

Myelolipomas occurring simultaneously in the adrenal and liver were found in a 2.7-yr-old, bred female common marmoset (Callithrix jacchus). The animal bore single adrenal and multiple hepatic myelolipomas. The adrenal myelolipoma consisted of mature adipose cells and focal collections of normal hematopoietic elements and was unencapsulated. In the liver, the myelolipomas, which were partially encapsulated, included a large amount of hematopoietic tissue and adipose cells that resembled normal bone marrow cells in various ways. Additionally, one of the multiple nodules contained several bony spicules with osteoblasts. Furthermore, there were invasive figures of hematopoietic cells, such as myeloblasts, erythroblasts, and megakaryocytes, in the hepatic sinusoids around the lesions. Thus, this case has some unusual morphological characteristics and is the first report, to our knowledge, on multiorganic myelolipomas in common marmosets.

Histopathological and biochemical studies on pancreatic fibrosis in WBN/Kob rats

Abstract We investigated the time-course of changes in pancreatic fibrosis accompanied with pancreatitis in WBN/Kob rats. The areas of fibrosis and fatty replacement were analysed morphometrically, and biochemical measurements of pancreatic and plasma prolyl hydroxylase and of pancreatic collagenase were assessed. Male rats showed acute pancreatitis at 2–3 months of age, lesions that later underwent a transition to widespread fibrosis. The fibrosis then decreased, and the fibrotic tissue was replaced with adipose tissue. Morphometrically, the fibrotic area reached its maximal size when the rats were 4 months old, diminishing thereafter. The fibrosis occurred mainly in the intralobular space, and was principally attributable to type-III collagen. Type-I collagen scarcely appeared throughout the experimental period. α-Smooth muscle actin appeared in and around myofibroblasts that developed in an early stage and diminished later in accordance with the progressive manner of fibrosis. The plasma prolyl hydroxylase level was higher in males than in females from 4 through 10 months of age. Pancreatic collagenase activity in the males also increased during the same period. These findings suggest that pancreatic fibrosis in male WBN/Kob rats is affected by the balance between prolyl hydroxylase and collagenase.

Ability of intrauterine bacterial lipopolysaccharide to cause in situ uterine contractions in pregnant rabbits.

Background. To investigate the ability of bacterial lipopolysaccharide delivered by the intrauterine route to cause uterine contractions in rabbits, and to assess the suppressive effect of urinar trypsin inhibitor on them.