Role of the nitric oxide-cGMP system in the regulation of ductus arteriosus patency in fetal rats

The purpose of this study was to examine the role of the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) system in the regulation of the ductus arteriosus (DA) patency in fetal rats. Pregnant rats were administered N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg, ip), an NO synthase (NOS) inhibitor; methylene blue (30, 50 and 100 mg/kg, ip), a soluble guanylate cyclase inhibitor; or indomethacin (3 mg/kg, po), a cyclooxygenase inhibitor, at various times before cesarean section. Dams were decapitated to obtain the fetuses by cesarean section, and fetuses were rapidly frozen in an acetone-dry ice mixture. Using rapid freezing and shaving methods, the calibers of the DA, pulmonary artery (PA) and descending aorta (Ao) were measured to evaluate the effects of treatment. L-NAME reduced the DA calibers to 86% of the initial values, but recovery to the control levels occurred 6 hr after the injection. Indomethacin decreased the DA calibers to 34% of the control values and sustained the DA constriction until 24 hr after the treatment. Methylene blue caused DA constriction to almost the same degree as indomethacin, but the levels normalized within 24 hr after the treatment. We conclude that L-NAME caused a slight constriction of the DA, whereas methylene blue and indomethacin caused marked constriction of the vessels, suggesting that the NO-cGMP system as well as prostaglandins contribute to the DA patency.     

百草枯引起胎鼠动脉管收缩与内皮素及其受体的关系研究

本文研究了百草枯对胎儿动脉管的毒性作用机理。取妊娠21d大鼠随机分成5组,在不同时间皮下注射25mg/kg百草枯,观察胎儿动脉管收缩情况;取妊娠21d大鼠随机分成4组,试验第1组母体皮下注射25mg/kg百草枯,第2组和第3组胎儿分别注射生理盐水和TAK-044后,母体皮下注射25mg/kg百草枯,观察胎儿动脉管收缩情况;用BQ-123代替TAK-044重复上述操作。结果表明:百草枯对妊娠末期胎儿动脉管具有毒性作用;由百草枯引起的胎儿动脉管收缩与ET有关,且以ETA受体作为媒介参与动脉管收缩。     

Role of nitric oxide in regulating the ductus arteriosus caliber in fetal rats

The role of nitric oxide (NO) on the ductus arteriosus (DA) patency was examined in fetal rats at various stages of gestation. N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg, i.p.), an NO synthase (NOS) inhibitor, or indomethacin (3 mg/kg, p.o.), a cyclooxygenase inhibitor, was administered at 3 hr before cesarean section to pregnant rats ranging from day 17 to day 21 of gestation. Dams were decapitated and the fetuses were obtained by cesarean section. The fetuses were rapidly frozen in an acetone-dry ice mixture. Using rapid-freezing and shaving methods, the calibers of the DA and pulmonary artery were measured. The constrictive effect of L-NAME on the fetal DA caliber was stronger than that of indomethacin in 19-day-old and immature fetuses. In near-term fetuses, the constrictive effects of L-NAME were reduced, while indomethacin caused marked DA constriction. We conclude that endogenous NO may play a major role in regulating the patency of the DA in earlier fetal stages, while dilator prostaglandins may play a greater role in regulating the ductal patency in the near-term fetus.     

Smooth muscle cell proliferation in the ductus arteriosus and the descending aorta, and effects of enalapril on SMC proliferation in perinatal rats

This study was carried out to determine the proliferation profile of the smooth muscle cells (SMC) in the media of the ductus arteriosus (DA) and the descending aorta (Ao), and to examine the effects of the angiotensin-converting enzyme inhibitor enalapril on the proliferation of these cells in perinatal rats. The proliferating cell nuclear antigen (PCNA) index of the DA peaked in 19-day-old fetuses at 75%, and the index significantly declined in 20-day-old fetuses. The PCNA index of the Ao showed a similar profile until pups reached 1 day of age; however, the index of the Ao then increased in 3-day-old pups. The PCNA indices of the DA and Ao decreased significantly after maternal oral treatment with enalapril (10 mg/kg for 7 days), with a more marked decline in the DA than in the Ao. The PCNA indices of these vessels in 20-day-old fetuses were not altered by maternal treatment with enalapril. These results indicate that the SMC proliferation rate in the DA was similar to that in the Ao until pups reached the age of 1 day, and that the inhibitory effect of enalapril on the SMC proliferation was age-dependent and more prominent in the DA than in the Ao.     

Studies on closure of the ductus arteriosus in perinatal rats.

Measurements of the inner diameters (calibers) of the ductus arteriosus (DA) and pulmonary artery (PA) were made in late fetal rats and newborn rats, the latter being obtained by spontaneous or caesarean delivery. The fetal and newborn pups were frozen instantly with an acetone-dry ice mixture. The chests of these whole-body frozen pups were shaved with a surgical knife gradually from the back toward the ventral side to expose the DA and PA for measurements of their calibers. As a result, it was revealed that the DA was almost closed 180 min after birth, but that the closure and shrinkage of the DA were accelerated to some extent by caesarean delivery. On the other hand, there was no remarkable change in the PA throughout the postnatal period observed, regardless of the type of delivery, spontaneous or caesarean.     

Stereological and histological analysis of the developing rat heart

We studied with quantitative and microscopical methods the heart of rats divided into five age groups: embryos at the age of 11 days, fetuses at the age of 16 days and 20 days and also heart samples of 3-day-old pups and young adults (5 weeks of age) were used (n = 10 samples in each group). At the end of the study; heart samples were obtained from all animals. Stereological estimations were performed on heart volume, volume of heart lumen (ventricles and atria), volume of myocardium, numerical density of the myocyte nuclei and mean nuclear diameter of myocytes. Volumetric values and numerical data were estimated via Cavalieri method and physical dissector, respectively. In this study, histological examination was performed at light and electron microscopic levels. The numerical density of the myocyte nuclei increased from fetuses to young adults. Differences between embryos and fetuses, between fetuses and 3-day-old pups, and between 3-day-old pups and young adults were statistically significant. These results indicate that myogenesis continued in the rat myocardium during prenatal life and after birth.     

Early neurobehavioral disorders in the micrencephalic offspring induced by prenatal treatment with N-methyl-N-nitrosourea or methylazoxymethanol in the rat.

Micrencephalic neonatal pups were obtained from pregnant Crj:CD (SD) rats once treated with 5 mg/kg of N-methyl-N-nitrosourea (MNU) or 40 mg/kg of methylazoxymethanol (MAM) on day 12, 13, 14 or 15 of gestation (vaginal plug = day 0). They were reared by their own mothers and were subjected to various neurobehavioral tests during the suckling period, days 0 to 22 after birth. The brain weights in the MNU- and MAM-treated pups on postnatal day 22 were significantly less than those in the control pups. These micrencephalic pups were retarded in neurobehavioral ontogeny. By several tests, each of them showed an impaired performance such as paired limb movement, clumsy locomotion or hyperreflexive reaction. These behavioral disorders appeared different according to the day of treatment, without any substantial difference between the test compounds, MNU and MAM. The findings suggest that the different neurobehavioral characteristics in the micrencephalic pups may reflect their different brain disorders induced by the test compounds given on the different period of the treatment.     

Evaluation of biochemical evidence of congenital nutritional myopathy in two-week prepartum fetuses from selenium-deficient ewes

Muscle damage attributable to selenium (Se)/vitamin E deficiencies is known to develop at birth or later in lambs. The purpose of this study was to determine whether and when muscle damage develops in utero. Thirty pregnant ewes maintained on Se-deficient forages from birth were allotted to 3 equal groups. Half of each group was given a single IM injection of 0.056 mg of Se/kg of body weight, 1 month before parturition. At 3 weeks before parturition, cesarean section-derived fetuses from Se-deficient ewes did not have evidence of muscle damage. At 2 weeks before parturition, fetuses from Se-deficient ewes had biochemical evidence of congenital nutritional myopathy, as evidenced by low blood Se concentration (P less than 0.05) and by increased plasma creatinine kinase (P less than 0.001) and lactate dehydrogenase (P less than 0.01) activities, compared with fetuses from Se-treated ewes. Thus, for optimal protection of fetuses and newborn lambs in Se-deficient areas, Se should be administered to ewes at least 1 month before parturition.     

Experimental transplacental transmission of canine herpesvirus in pregnant bitches during the second trimester of gestation

The effects of canine herpesvirus (CHV) on fetuses were studied after IV inoculation of pregnant bitches in the 2nd trimester of gestation. Cesarean sections were performed on 2 bitches that were inoculated with CHV on the estimated 30th day of gestation. Bitch M-1 had 2 mummified fetuses and bitch M-2 had 4 mummified and 2 dead fetuses and 3 live-born pups. Infection by CHV was confirmed histopathologically by the presence of focal areas of necrosis associated with intranuclear inclusion bodies in heart muscle sections of 1 dead fetus; CHV was not recovered from other organs. Abortion occurred between the 2nd and 3rd week after inoculation of another pregnant bitch inoculated with CHV on the estimated 30th day of gestation. Two bitches inoculated with CHV on the estimated 40th day of gestation gave birth prematurely to 10 pups. The detection of characteristic herpesviral lesions in various organs and the reisolation of CHV from the liver, spleen, kidneys, and lungs of premature pups indicated CHV infection. Transplacental infection of fetal pups by CHV resulted in their death and subsequent mummification. It appears that abortion and premature birth also may occur in pregnant bitches infected during the 2nd trimester of gestation.     

Gestational exposure to nonylphenol causes precocious mammary gland development in female rat offspring

This study examined whether or not exposure to 4-nonylphenol (NP) during late gestation affects reproductive and mammary development in the offspring of female rats. Time pregnant Long Evans rats were gavaged with NP (10 or 100 mg/kg), atrazine (ATR, 100 mg/kg), or corn oil on gestation days 15-19. The uterus weights of the NP (100 mg/kg/d)-exposed pups were higher than those of the controls but the weights of the other organs were unchanged. Delayed mammary gland (MG) development was detected in the ATR pups on PND 4 and persisted through to PND 66. The high dose NP pups had advanced lobular development of their MG on PND 22, while the glands from the low dose NP pups were no different morphologically from the controls. Immunohistochemical comparisons of the mammary sections from PND 41 demonstrated low levels of estrogen receptor (ER) staining in the control gland stroma and epithelium but higher levels in the tissue of the pups exposed to NP and ATR. ATR also elevated ER in the stroma surrounding the epithelial layer of the terminal end buds. The level of progesterone receptor (PR) staining was markedly lower in the epithelium of the 100 mg/kg NP glands vs. the control glands. However, PR was present at high levels in the epithelium of the 10 mg/kg NP glands and was even more prominent in the ATR-exposed ductal epithelium and fat cell nuclei. The level of prolactin staining was only elevated in glands containing lobule areas (NP-exposed) compared with the control levels. These results suggest that NP and ATR have opposite effects on the development of MG after gestational exposure. Exposure to them during the critical period of epithelial outgrowth altered the receptor levels of mammary progesterone and prolactin and might contribute to the differences in the mammary morphology at PND 41.     

Persistence of porcine parvovirus in swine infected in utero and followed through maturity

The potential of porcine parvovirus (PPV) to persistently infect swine exposed in utero was studied. Forty eight 80- to 95-day-old fetuses from 5 PPV seropositive sows were inoculated intramusculary with a virulent strain of PPV or with cell culture medium (controls). Blood samples were collected at birth prior to nursing and at monthly intervals thereafter and tested for antibodies to PPV. Virus-inoculated and control pigs were euthanized at either 1 week before birth (-1), at birth (0) and at weeks 2, 4, 6, 8, 10, 22, and 28 after birth. Presence of viral DNA and antigen was evaluated using slot blot DNA hybridization and indirect FA techniques, respectively. All inoculated fetuses (n = 26) and 7 control fetuses (n = 22) seroconverted in utero, and these pigs maintained antibody titers greater than log10 2 for the period of testing (0-38 weeks after birth). After passive antibody titers had reached subdetectable levels in control animals, animals remained seronegative through an additional 14 weeks of testing in spite of close contact with infected pigs. Virus antigen was not detected in any tissues examined from pigs euthanized at term. In contrast, PPV DNA was detected consistently from pigs at birth from various tissues, and from the lung of one pig at 6 weeks of age and from the lymph nodes of one pig euthanized at 28 weeks of age. The results indicate that pigs infected with PPV in utero may be persistently infected, however the likelihood of shedding to contact animals is minimal.     

Pathogenicity of minute virus of canines (MVC) for the canine fetus.

Minute Virus of Canines (MVC) was shown to cause transplacental infections with embryo resorptions, especially in dams given parenteral inoculations prior to gestational day 30. Responses to oral-nasal inocula were inconsistent. Direct exposures of embryos or fetuses to MVC via the amniotic sac, however, resulted in their deaths after an incubation period of approximately 2 weeks. Virus was prominent in fetal lung and small intestinal villi, but it could not be demonstrated in fetal or uterine tissues greater than 2 weeks after the estimated time of embryonic/fetal death. With 1 exception, dams inoculated during the last trimester of pregnancy gave birth to normal pups; some pups had developed active immune responses. The exceptional dam whelped dead or dying pups with anasarca or myocarditis. Antibody levels to MVC were generally higher in dams with infected fetuses than in bitches with normal pups at term. Failures to isolate virus, or demonstrate viral antigens in tissues by immunostaining, other than near the time of embryo/fetal death, indicate the need for more sensitive viral detection methods.     

氯羟吡啶的特殊毒性——致畸作用

观察了国产氯羟吡啶对 Ｗ ｉｓｔａｒ 大鼠的致畸作用。氯羟吡啶以 ４、２０、１００、２００ ｍ ｇ／ｋ ｇ ４ 个剂量进行试验。结果,氯羟吡啶 １００、２００ ｍ ｇ／ｋ ｇ 组鼠妊娠率极显著低于阴性对照组 （ Ｐ ＜ ００１）, １００ ｍ ｇ／ｋｇ 的氯羟吡啶还对胎鼠平均窝重有显著不利影响,２００ ｍ ｇ／ｋｇ 组胎鼠平均身长及平均体重均显著小于阴性对照组 （ Ｐ ＜００５）。另外,２００ ｍ ｇ／ｋｇ 的氯羟吡啶可导致骨化迟缓增多和绝大部分胎鼠（１５／１６）多肋（１４ 对肋骨）,还引起母鼠体重下降,且胎仔平均畸形出现率显著高于阴性对照组。由此认为,氯羟吡啶有一定的致畸作用和胚胎毒作用。     

Transmissible congenital demyelinating encephalopathy of lambs

Twenty-two fetal lambs were inoculated in utero with tissue suspension prepared from lambs born with weakness, incoordination and clonic tremor. Clinically, affected newborn lambs had clonic tremor, were generally weak and had abnormally pigmented hairy fleece. The inoculation resulted in a disseminated encephalomyelitis with secondary teratologic changes in a significant number of fetuses. The mononuclear inflammatory changes were most obvious 14 days after inoculation, after which there was rapid resolution. Changes seen at birth were chronic astrocytosis with neuron loss in the spinal cord and cerebellar dysplasia. Single radioimmunodiffusion studies showed consistently low IgG in infected fetuses and high IgG in lambs at birth.     

吡喹酮非水溶液注射剂的研制--日本血吸虫病治疗试验

利用本课题组研制的吡喹酮非水溶液注射剂，分别进行了小鼠和牛的日本血吸虫病治疗试验。结果表明，感染日本血吸虫的小鼠按每千克体质量20mg和30mg的剂量肌肉注射吡喹酮的减雌率均达100％，人工感染日本血吸虫的牛按每千克体质量10mg和12mg的剂量肌肉注射吡喹酮的减雌率均达100％，每千克体质量8mg的剂量的减雌率为96．41％。自然感染血吸虫病牛按每千克体质量10mg的剂量肌肉注射后30d，粪便转阴率达90．50％。这一结果说明，研制的吡喹酮注射剂具有良好的治疗效果。     

Decreased sperm number and motile activity on the F1 offspring maternally exposed to butyl p-hydroxybenzoic acid (butyl paraben)

Butyl p-hydroxybenzoic acid (butyl paraben, BP) is widely used as a preservative in food and cosmetic products. Routledge et al showed that BP is weakly estrogenic in both in vitro and in vivo (rat uterotrophic) analyses. We investigated whether maternal exposures to BP during gestation and lactation periods affected the development of the reproductive organs of the F1 offspring. Pregnant Sprague-Dawley rats were injected subcutaneously with 100 or 200 mg/kg of BP from gestation day (GD) 6 to postnatal day (PND) 20. In the group exposed to 200 mg/kg of BP, the proportion of pups born alive and the proportion of pups surviving to weaning were decreased. The body weights of female offspring were significantly decreased at PND 49. The weights of testes, seminal vesicles and prostate glands were significantly decreased in rats exposed to 100 mg/kg of BP on PND 49. In contrast, the weights of female reproductive organs were not affected by BP. The sperm count and the sperm motile activity in the epididymis were significantly decreased at doses of 100 and 200 mg/kg of BP. In accordance with the sperm count in the epididymis, the number of round spermatids and elongated spermatids in the seminiferous tubule (stage VII) were significantly decreased by BP. Testicular expression of estrogen receptor (ER)-alpha and ER-beta mRNA was significantly increased in 200 mg/kg of BP treated group at PND 90. Taken together, these results indicated that maternal exposure of BP might have adverse effects on the F1 male offspring.     

Kinetics of detection of blastogenic responses of neonatal calves inoculated in utero with tetanus toxoid, killed Mycobacterium bovis, and killed Brucella abortus.

Nine pregnant cows were laparotomized and their fetuses were immunized with tetanus toxoid, killed Brucella abortus, and killed Mycobacterium bovis. Blastogenesis assays and total leukocyte and differential counts were done when the calves were 1, 2, 3, 7, 14, 21, 28, and 60 days of age. Initial blastogenesis responses to antigens, phytohemagglutinin, and concanavalin A were not positive as frequently as were the responses obtained when the calves were 2 to 3 weeks of age. The probability of obtaining a positive response to an antigen was positively correlated with the magnitude of the response, as determined by delayed-type hypersensitivity skin reactions. Leukocyte and differential WBC counts in immunized calves were similar to those of unimmunized calves. The mean leukocyte count for the immunized calves remained near 16,000 cells/mm3; blood obtained in the first few days after birth contained a greater number of neutrophils than lymphocytes, whereas lymphocyte-to-neutrophil ratio gradually approached those of adult cattle, in which lymphocytes predominate.     

N -ethyl- N -nitrosourea induces retinal photoreceptor damage in adult rats

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.     

碘醚柳胺对大鼠胚胎毒性和致畸性

以23．0,51.8,116.4mg/kg碘醚柳胺在Wistar大鼠妊娠后7－15d口服，各剂量组吸收胎和死胎率增加，活胎率减少，但对胎鼠外观，骨骼和内脏无致畸性，高剂量组对胎鼠生长发育和孕体重增长有明显的负影响，表明碘西边柳胺对大鼠有胚胎毒性，无致畸作用，提出，在临床上怀孕动物应慎用。     

Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1

OBJECTIVE: To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1-induced responses. ANIMALS: 36 horses without gastrointestinal tract disease. PROCEDURE: To determine cumulative concentration-response relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10(-9) to 10(-6)M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10(-9), 10(-7), and 10(-5)M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared. Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. RESULTS: ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10(-5)M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system.