一氧化氮合酶抑制剂L-NAME对大鼠脑缺血耐受诱导的影响

采用大鼠四血管闭塞全脑缺血耐受模型和脑组织切片形态学方法,观察应用一氧化氮合酶(NOS)抑制剂L—NAME对大鼠海马CAl区脑缺血耐受(BIT)诱导的影响,在整体水平探讨一氧化氮(NO)在BIT诱导中的作用。54只Wistar大鼠凝闭双侧推动脉后分为6组：(1)假手术组(n＝6);分离双侧颈总动脉,但不阻断脑血流;(2)损伤性缺血组(n＝6)：全脑缺血10min;(3)预缺血 损伤性缺血组(n＝6)：脑缺血预处理(CIP)3min,再灌注72h后行全脑缺血10min;(4)L—NAME组;分别于CIP前1h和后1、12及36h腹腔注射L—NAME(5mg／kg),每个时间点6只动物,其余步骤同预缺血 损伤性缺血组;(5)L—NAME L—精氨酸组(n＝6)：于CIP前1h腹腔注射L—NAME(5mg／kg)和L—精氨酸(300mg／kg),其它步骤同L—NAME组;(6)L—NAME 损伤性缺血组(n＝6)：于腹腔注射L—NAME(5mg／kg)72h后行全脑缺血10min。实验结果表明,(1)单纯10min全脑缺血可使海马CAl区组织学分级增加(表明损伤加重),神经元密度降低(P＜0．01);(2)预缺血 损伤性缺血组的海马CAl区组织学分级、神经元密度与假手术组相比,无显著性差别(P＞0．05);(3)L—NAME组中,应用L—NAME后海马CAl区组织学分级增加,神经元密度降低,与预缺血 损伤性缺血组相比有显著性差异(P＜0．05),表明L—NAME可阻断CIP对神经元的保护作用;(4)L—NAME L—精氨酸组与L—NAME组相比,海马CAl区组织损伤明显减轻(P＜0．05),但与预缺血 损伤性缺血组相比仍有显著性差别(P＜0．05),提示L-精氨酸可部分逆转L—NAME的作用;(5)L—NAME 损伤性缺血组的组织学表现与损伤性缺血组相同(P＞0．05)。这些结果表明,在整体情况下N0参与BIT的诱导。与CIP前1h及后1、12h给予L—NAME组相比,CIP后36h给予L—NAME对CIP保护作用的阻断效应明显减弱,提示N0在CIP后较早阶段即开始参与BIT的诱导。     

Nω-硝基-L-精氨酸甲酯抑制吗啡镇痛耐受大鼠中脑和脊髓一氧化氮合酶/N-甲基-D-天冬氨酸受体表达上调

采用热甩尾测痛法观察全身应用非特异性一氧化氮合酶（nitric oxide synthase,NOS）抑制剂——N^ω-硝基-L-精氨酸甲酯（L-NAME）对吗啡镇痛耐受形成的影响,并通过观察脊髓和中脑神经元型NOS（nNOS）和N-甲基-D-天冬氨酸（NMDA）受体亚单位表达的变化来阐释NO／NMDA受体在吗啡镇痛耐受形成中的作用。将36只健康成年Sprague-Dawley大鼠平均分为6组（每组6只）：1组为对照组,皮下注射生理盐水1ml;2、3、4、5和6组为处理组,分别皮下注射L-NAME10mg／kg、L-NAME20mg／kg、吗啡10mg／kg、L-NAME10mg／kg＋吗啡10mg／kg、L-NAME20mg／kg＋吗啡10mg／kg,每天2次。在注射前测量大鼠的热甩尾潜伏期（tail-flick latency,TFL）基础值,随后每天第一次给药50min后测量其TFL。第8天最后一次给药80min后（除2组和5组之外）断头取脊髓和中脑,采用RT-PCR技术测量nNOS以及NMDA受体1A（NR1A）和2A（NR2A）亚单位的表达。结果显示,2组大鼠第1天至第7天的TFL与基础值相比无显著差异;3组第7天时的TFL仍显著高于基础值;4组的TFL在第1天时最高,第2至第6天期间逐渐降低,第6天时与基础值相比无显著差异：5组的TFL在实验过程中呈下降趋势,虽然第7天时较第1天有所降低,但是仍然显著高于基础值;6组的TFL变化趋势与5组相同。PT—PCR分析结果显示,与1组相比,3组脊髓和中脑的nNOS mRNA表达显著降低,但NR1A mRNA和NR2A mRNA表达无显著改变;4组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著高于1组。与4组相比,6组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著降低。结果提示,吗啡镇痛耐受大鼠脊髓和中脑的nNOS和NMDA受体表达增加,联合应用L—NAME可抑制长期应用吗啡所致的nNOS表达增加和NMDA受体上调,延缓吗啡镇痛耐受的形成。本研究结果提示,脊髓和中脑的NO／NMDA受体与吗啡镇痛耐受形成密切相关。     

Craniofacial and axial skeletal defects induced by the fungicide triadimefon in the mouse

BACKGROUND: Triadimefon is an antifungal derived from triazole. In in vitro whole-rodent embryo cultures, triazole-derivatives showed specific teratogenic effects at the branchial apparatus. The aim of the present work was to test in vivo triadimefon (FON), in order to verify a relationship between triazole exposure, embryonic abnormalities, and/or fetal malformations. METHODS: Pregnant CD-1 mice were treated with 0-300 mg/kg FON by gavage on day 8 post coitum (p.c.) at 10:00 AM, and sacrificed on day 8 p.c. at 1:00 PM, on day 9 p.c. at 10:00 AM, on day 10 p.c. at 10:00 AM, and at term of gestation (day 18 p.c.). At midgestation, the embryos were processed for specific immunostainings to visualize the hindbrain segmentation (day 8 p.c.) and the neural crest cell migration (days 8 and 9 p.c.). Fetuses explanted at term were all processed for skeletal examination after double-staining of osseous and cartilaginous tissues. RESULTS: At midgestation, the immunostaining of rhombomeres 3 and 5 showed a light scattering of the immunostained areas; the neural crest cell migration was unaffected, but their localization at the branchial arch level was abnormal. At term, several severe malformations were observed at the craniofacial and at the axial skeletal level. Ectopic cartilage was observed at the upper jaw. CONCLUSIONS: Triadimefon is teratogenic. The observed craniofacial malformations could be explained by an alteration of the rhombomeric organization and neural crest migration to the branchial arches; the axial abnormalities could be explained by the abnormal segmental identity specification.     

Sensitive Windows of Skeletal Development in Rabbits Determined by Hydroxyurea Exposure at Different Times throughout Gestation

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo–fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day,GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio‐facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.     

Nitric oxide and superoxide radical are involved in both initiation and development of adjuvant arthritis in rats

Nitric oxide synthase(NOS) inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME, 10–300 mg/kg) and L-N^G-monomethyl-arginine (L-NMMA, 30–300 mg/kg) suppressed the swellings of adjuvant-injected paw of rats (25–54%) at day 2 and 8 when dosed intraperitoneally and orally for 4 days from day -1 to day 2 after adjuvant. L-NAME (30–300 mg/kg) also suppressed the edema of the non adjuvant-injected paws (15–42%) at day 28. Local injection of this inhibitor (2 and 10 mg/kg) was without effect. L-arginine (1 g/kg, i.p.), impaired the suppression by L-NAME. Bovine blood Cu, Zn-superoxide dismutase (SOD, 3 mg/kg, i.p.: 28% suppression) and L-NAME (30 mg/kg i.p.: 36% suppression) showed additive effect (52%) in adjuvant-injected paws at day 8 when co-injected. As the effect of 30 mg/kg L-NAME corresponded nearly to that of 10 mg/kg Voltaren^R, this NOS inhibitor would be worth considering as an anti-inflammatory agent. Sodium nitroprusside (NO-donor) and methylene blue (guanylate cyclase inhibitor) had no effect. L-NAME was also suppressive when dosed after adjuvant inoculation and NO is involved in the development and maintenance of swelling.     

Evidence of nitric oxide and angiotensin II regulation of circulation and cutaneous drinking in Bufo marinus

The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (l-NAME) increased vascular resistance (VR) 10% above baseline of 3.08+/-0.08 (n=11) mmHg/mL/min at 10 mg/kg and 20% above 3.05+/-0.08 (n=9) at 50 mg/kg in anesthetized toads (Bufo marinus). Blood pressure was unaffected by either dose of L-NAME. Blood flow decreased at the higher dose of L-NAME. L-arginine (300 mg/kg) reversed the effects of L-NAME on VR and blood flow in toads treated with 10 mg/kg but not with 50 mg/kg. Injection of 50 mg/kg L-NAME into empty-bladder toads produced a 10% decrease in water uptake, J(v), resulting in a J(v) of 1,267+/-11 cm(3)/cm(2)/s x 10(-7) (n=9) compared to 1,385+/-12 (n=8) for controls. Injection of 10 microg/kg angiotensin II (ANG II) increased J(v) 15% across the pelvic patch (J(v), cm(3)/cm(2)/s x 10(-7)), resulting in a J(v) of 1,723+/-12 cm(3)/cm(2)/s x 10(-7) (n=8) compared to 1,471+/-12 (n=8) for controls. It is hypothesized that during cutaneous drinking blood flow into the capillary bed of the pelvic patch is regulated by nitric oxide and ANG II.     

Reversible effects of triamcinolone and lack of effects with aspirin or L-656,224 on external genitalia of male Sprague-Dawley rats exposed in utero.

Inhibitors of the arachidonic acid cascade were given to pregnant rats during the critical period for morphogenesis of the external genitalia. Groups treated subcutaneously (s.c.) with 0.1 or 0.25 mg/kg/day of triamcinolone acetonide (TA) on gestational days (GD) 14-19 had male fetuses on GD 20 with moderate decreases in absolute anogenital distance (AGD), but gross and histological examinations revealed no alterations to the genital tubercle (i.e., no hypospadias). The s.c. coadministration of arachidonic acid at 100 mg/kg/day had minimal to no effect on AGD in the TA-exposed groups. No effect on AGD was observed in male fetuses from groups administered aspirin orally at 150 mg/kg/day, and only a 6% decrease was observed in the 300-mg/kg/day group. Neither TA nor aspirin adversely affected AGD of female fetuses. In another study, TA was administered on GD 11-19 at dose levels of 0.05 and 0.1 mg/kg/day, and dams were allowed to deliver. High-dose male offspring examined on postcoitum day (PCD) 23, had moderate decreases in AGD. In both studies with TA, there were also significant decreases in offspring weights. The contribution of the decreased weight to the decrease in absolute AGD was examined by a variety of methods (ratio of AGD to cube root of weight or biparietal distance, comparison to weight-matched controls, and covariance analysis). We conclude that TA caused a specific decrease in AGD on GD 20 that was largely reversed by PCD 23. When examined as adults (8 weeks old), the external genitalia of TA-exposed offspring were normal. Thus, the TA-induced decreases in AGD on GD 20 did not predict irreversible malformation. TA also caused other effects, which included a somewhat flattened genital tubercle and apparently thinned and glossy skin between the tubercle and the anus in both sexes on GD 20 and PCD 23, but not as adults. In addition, there were high pup mortality and high incidences of micrognathia and omphalocele (in the 0.25-mg/kg/day group only). Aspirin at 75 or 150 mg/kg/day and a specific lipoxygenase inhibitor (L-656,224) at 1,000 or 2,000 mg/kg/day were also administered from GD 14 to 19, and no offspring effects were observed. Thus, of the three agents that potentially inhibit the arachidonic acid cascade, only triamcinolone produced moderate effects on rat external genitalia that were largely reversible.(ABSTRACT TRUNCATED AT 400 WORDS)     

Critical period for a teratogenic VLA-4 antagonist: Developmental effects and comparison of embryo drug concentrations of teratogenic and non-teratogenic VLA-4 antagonists

BACKGROUND: Integrins such as VLA-4 (Very late antigen 4, integrin alpha4beta1) play key roles in cell-cell interactions that are critical for development. Homozygous null knockouts of the VLA-4 alpha4-subunit or VCAM-1 (VLA-4 cell surface ligand) in mice result in failure of the allantois and chorion to fuse leading to interrupted placentation and cardiac development and embryo lethality. Embryo-fetal studies of three VLA-4 antagonists, IVL745, IVL984, and HMR1031 [Crofts et al., Birth Defects Res B 71:55-68 (this issue), 2004] with exposure on gestation days (GD) 6-17 (rat), 6-18 (rabbit) or 6-15 (mouse) showed that only IVL984 treatment resulted in embryo lethality and cardiac defects. Objectives of the current study were to determine the critical period for inducing IVL984-related embryo-fetal effects, and to test the hypothesis that these effects were due to higher embryo drug concentrations. METHODS: IVL984 was administered at 40 mg/kg/day to pregnant rats on GD 4 and 5, GD 6 and 7, GD 8 and 9, GD 10 and 11, or GD 12 and 13. Animals were euthanized on GD 21 and uteri and fetuses were examined. A treatment period of GD 10-12 was selected for subsequent toxicokinetic (TK) studies in which IVL984, HMR1031, or IVL745 was administered to pregnant rats and rabbits. On GD 12, maternal plasma, extra-embryonic tissue (placenta and amniotic fluid), and embryonic tissue were collected and analyzed for drug concentrations. RESULTS: In the IVL984 critical period study in pregnant rats, treatment on GD 10 and 11 resulted in increased post-implantation loss, skeletal variations, and spiral septal defects similar to those observed in standard embryo-fetal development studies with treatment throughout organogenesis. There were no embryo-fetal effects after treatment on GD 4 and 5, GD 6 and 7, or GD 8 and 9. There was a single aorta malformation after treatment on GD 12 and 13. In the TK studies, IVL745, HMR1031, and IVL984 were all detectable in embryonic tissue and there was no evidence for accumulation. Rat and rabbit embryo exposures (AUC or dose-adjusted AUC) on GD 12 could not explain the observed teratology (IVL984相似文献   

Effects of (R)-deoxycoformycin (pentostatin) on intrauterine nucleoside catabolism and embryo viability in the pregnant mouse.

The viability of early mouse embryos is acutely sensitive to (R)-deoxycoformycin (pentostatin), a tight-binding inhibitor of adenosine deaminase (ADA). Previous studies have shown that a single 5-mg/kg dose on day 7 (plug = day 0) of gestation fully inhibits uteroplacental ADA activity within 0.5 h; causes massive cell death in the neural plate and primary mesenchyme by 6 h, major craniofacial anomalies by day 10, and resorption by day 12 (Knudsen et al., '89; Airhart et al., '91). The present study has examined further the developmental toxicity and early effects of this inhibitor on ADA metabolism. (R)-Deoxycoformycin was administered to pregnant CD-1 (ICR) mice as a single intraperitoneal dose of 0.5-10 mg/kg total body weight on days 6-11 of gestation. The major adverse effect, early resorption, was dose dependent and specific to day 7-8 exposure. Treatment with 5 mg/kg on day 7 resulted in 85% resorptions, 15% malformations, and a 24% reduction in mean fetal weight, whereas the same dose of (S)-deoxycoformycin had no effect. Levels of adenosine and 2'-deoxyadenosine, which are the endogenous substrates of ADA, were monitored in the embryo/decidual unit (E/D) by reversed-phase high-performance liquid chromatography (RP-HPLC). In response to the inhibitor, both nucleosides increased transiently in the antimesometrial compartment (antimesometrial decidua + embryo). Peak levels (Cmax) of adenosine and 2'-deoxyadenosine were dose dependent over the range tested (0.05-10 mg/kg). Exposure to 5 mg/kg on day 7 raised adenosine levels within 0.5 h to 42-fold over the basal level of 0.06 nmol/mg protein. There was an even stronger effect on 2'-deoxyadenosine levels, which were elevated 674-fold over the detection limit of 0.0005 nmol/mg protein. Direct exposure to the inhibitor in serum-free E/D culture produced similar results: 50 microM (R)-deoxycoformycin within 1 h raised adenosine levels 26-fold and 2'-deoxyadenosine levels 410-fold. In vivo studies also showed a general correlation between embryolethality and the length of adenine nucleoside pool expansion, apparent for exposure on day 7, 8, or 9 but not on day 6, suggesting that the embryo becomes sensitive to adenosine or 2'-deoxyadenosine once the neural plate has formed.     

The Inhibitory Effects of Nω-Nitro-L-Arginine Methyl Ester on Nitric Oxide Synthase Activity Vary Among Brain Regions in Vivo but Not in Vitro

We studied the effects of intracerebroventricular and intraperitoneal injection and the in vitro effects of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the nitric oxide synthase activities of the cerebellum, brainstem, hypothalamus, hippocampus, and the remainder of the brain after dissections. Male rats were chronically implanted with lateral icv guide cannula. L-NAME was injected in doses of 0.2, 1, and 5 mg intracerebroventricularly, and 50 mg/kg intraperitoneally. L-NAME induced dose-dependent suppression of NOS activities in each brain region. The threshold dose was 0.2 mg; 1 mg L-NAME completely abolished brain nitric oxide synthase activity 90 min after the injection. Brain NOS activities returned to baseline level 48 h after the injection of 5 mg L-NAME. There were significant differences between the sensitivity of various regions to L-NAME after in vivo but not in vitro administration of the enzyme inhibitor. These findings indicate that intracerebroventricular injection of L-NAME is a useful tool for inhibiting brain nitric oxide synthase activities in vivo. The differences between the sensitivity of different brain regions to L-NAME as well as the relative fast recovery of nitric oxide synthase activities must be taken into account when L-NAME is administered intracerebroventricularly to rats.     

Assessment of female and male fertility in Sprague-Dawley rats administered vorinostat, a histone deacetylase inhibitor

BACKGROUND: Histone deacetylase (HDAC) inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation, and apoptosis of tumor cells. These compounds are now marketed or are in clinical development. One such HDAC inhibitor, vorinostat (suberoylanilide hydroxamic acid [SAHA], Zolinza), was assessed for its potential effects on fertility in Sprague–Dawley rats. METHODS: Female rats were administered oral dose levels of 0 (vehicle only), 15, 50, or 150 mg/kg/day of vorinostat for 14 days before cohabitation, during cohabitation, and through Gestation Day (GD) 7. In a separate study, male rats were administered oral dose levels of 0 (vehicle only), 20, 50, or 150 mg/kg/day for 10 weeks before cohabitation, during cohabitation, and until the day before scheduled sacrifice (approximately 14 weeks total). In both studies, % peri‐implantation loss and % postimplantation loss were evaluated on GD 15–17. Testicular weight and histomorphology, cauda epididymal sperm count, and sperm motility were evaluated in the male rat study at termination. RESULTS: There were treatment‐related decreases in body weight gain at 150 mg/kg/day in both studies. There were no effects on mating or fertility indices in either study. In the female study there were increased numbers of corpora lutea in all drug‐treated groups (only 1 or 2 affected dams in low and mid‐dose groups), and a marked increase in percent postimplantation loss only in the high‐dose group. No treatment‐related effects were observed on litter or sperm parameters of the male study. CONCLUSIONS: Vorinostat had no effects on mating or fertility in rats up to 150 mg/kg/day. There were no indications of reproductive toxicity in drug‐treated male rats. Increases in corpora lutea or resorptions were observed in treated female rats. Birth Defects Res (Part B) 80:1–8, 2007. © 2007 Wiley‐Liss, Inc.     

Dose-response for retinoic acid-induced forelimb malformations and cleft palate: a comparison of computerized image analysis and visual inspection

BACKGROUND: The objectives of this study were to (1) compare two techniques (computerized image analysis and visual morphological evaluation) for the assessment of fetal forelimb malformations and (2) increase the robustness of the dose-response curve for forelimb and cleft palate malformations resulting from all-trans retinoic acid (RA) exposure in GD 11 mice. METHODS: Pregnant CD-1 mice were administered a single oral dose of all-trans RA (0, 2.5, 10, 30, 60, or 100 mg/kg) on GD 11. GD 18 fetuses were examined for malformations using visual morphological scoring and computerized image analysis. RESULTS: Dose-dependent changes occurred in the size and shape of the humerus, radius, and ulna based on both assessment methodologies. The most sensitive indicators for the lowest effect level (10 mg/kg) on forelimbs were roundness, a shape measurement determined by image analysis, and visual morphological scoring. For all other bone measurements (proximal and distal width, area, length, and perimeter), the lowest effect level was 30 mg/kg. The maximum effect for limb defects and total malformed fetuses was seen at 60 mg/kg and higher. Incidence of cleft palate increased over the entire range of administered doses reaching a maximum of 74% (100 mg/kg). CONCLUSIONS: Overall, results indicate that computerized image analysis was no more sensitive in detecting changes in the humerus, radius, and ulna than gross visual examination. Dose-response modeling of developmental endpoints yielded comparable benchmark dose levels for long bones and cleft palate that ranged from 0.24 to 7.6 mg/kg all-trans RA. Birth Defects Res B 71:289-295, 2004. Copyright 2004 Wiley-Liss, Inc.     

MAPK信号途径在一氧化氮抑制大鼠心肌肥大中的作用

实验观察了一氧化氮（NO）前体L－精氨酸对肾性高血压大鼠心肌组织eNOS蛋白表达及亚硝酸盐／硝酸盐含量、MKP－1蛋白表达及MAPK活性的影响,以及与心肌肥厚的关系,采用两肾一夹Goldblatt肾性高血压模型,随机分为5组：L－精氨酸高、中、低剂量组,分别于术后第5周给予L－精氨酸50、150及450mg/kg;L－NAME组,腹腔注射L－NAME 10mg/kg,同时给予L－精氨酸150mg/kg;高血压对照组,正常饮水,以及另设的一假手术对照组。用药8周后,用插管法测量大鼠动脉血压、左心室重与体重比值,用胶内原位磷酸化法测MFAPK活性、免疫印迹法检测心肌组织eNOS及MKP－1蛋白表达、酶还原法测定心肌组织亚硝酸盐／硝酸盐－硝酸盐含量。结果表明：（1）L－精氨酸可明显抑制肾动脉狭窄术后的血压升高、左心室重与体重比增加,增加心肌组织eNOS、MKP-1蛋白表达及亚硝酸盐－硝酸盐含量,降低心肌组织MAPK活性,其中以150mg/kg组作用最为明显;（2）NOS抑制剂L－NAME可明显抑制－精氨酸的以上作用,肾性高血压大鼠心肌组织eNOS蛋白表达下降。NO生成减少及MKP－1蛋白表达下降以及MAPK活性增强可能与高血压及心肌厚形成有关,L－精氨酸通过促进心肌组织eNOS蛋白表达、增加NO产生和MKP－1表达、减弱MAPK活性而发挥抗高血压及心肌肥厚的作用。     

Development in the cynomolgus macaque following administration of ustekinumab,a human anti‐il‐12/23p40 monoclonal antibody,during pregnancy and lactation

BACKGROUND: Ustekinumab is a human monoclonal antibody that binds to the p40 subunit of interleukin (IL) 12 and IL‐23 and inhibits their pharmacological activity. To evaluate potential effects of ustekinumab treatment during pregnancy, developmental studies were conducted in cynomolgus macaques. METHODS: Ustekinumab was tested in two embryo/fetal development (EFD) studies and in a combined EFD/pre and postnatal development (PPND) study. In the EFD studies, pregnant macaques (12/group) were dosed with saline or ustekinumab (9 mg/kg IV, 22.5 mg/kg SC, or 45 mg/kg IV or SC during the period of major organogenesis, gestation day [GD] 20–50). Fetuses were harvested on GD100–102 and examined for any effects on development. In the EFD/PPND study, pregnant macaques were injected with saline or ustekinumab (22.5 or 45 mg/kg SC) from GD20 through lactation day 33. Infants were examined from birth through 6 months of age for morphological and functional development. Potential effects on the immune system were evaluated by immunophenotyping of peripheral blood lymphocytes and immunohistopathology of lymphoid tissues in fetuses and infants and by T‐dependent antibody response (TDAR) to KLH and TTX and by DTH response in infants. Ustekinumab concentrations were measured in serum from dams, fetus, and infants and in breast milk. RESULTS: Ustekinumab treatment produced no maternal toxicity and no toxicity in the fetuses or infants, including no effects on the TDAR or DTH responses. Ustekinumab was present in serum from GD100 fetuses and was present in infant serum through day 120 post‐birth. Low levels of ustekinumab were present in breast milk. CONCLUSIONS: Exposure of macaque fetuses and infants to ustekinumab had no adverse effects on pre‐ and postnatal development. Birth Defects Res (Part B) 89:351–363, 2010. © 2010 Wiley‐Liss, Inc.     

Real-time monitoring of nitric oxide and blood flow during ischemia-reperfusion in the rat testis

In the present study, we attempted to clarify the role of nitric oxide (NO) and its release during the ischemia-reperfusion rat testis. Eight-week-old male Sprague-Dawley rats were divided into seven groups: age-matched control rats, ischemia (30 minutes)-reperfusion (30 minutes) rats without N^G-nitro-L-arginine methyl ester (L-NAME) and L-arginine (L-Arg) treatment, ischemia (30 minutes)-reperfusion (30 minutes) rats treated with L-NAME (10, 30, and 100 mg/kg), ischemia-reperfusion rats treated with L-Arg (10 and 30 mg/kg). Sixty minutes prior to induction of ischemia, L-NAME or L-Arg was administrated intraperitoneally. Real-time monitoring of blood flow and NO release were measured simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. NO₂-NO₃ and malonaldehyde (MDA) concentrations were measured in the experimental testes. Furthermore, we investigated possible morphological changes in the testis. Clamping of the testicular artery decreased blood flow to 5–20% of the basal level measured before clamping. Immediately following clipping of the artery, NO release rapidly increased. After removing the clip, NO release gradually returned to the basal level. This phenomenon was enhanced by treatment with L-Arg and inhibited by treatment with L-NAME. NO₂-NO₃ concentrations were increased by treatment with L-Arg and decreased by treatment with L-NAME, while MDA concentrations were increased by treatment with L-NAME and were decreased by treatment with L-Arg. In histological studies, the ischemia-reperfusion caused infiltration of leukocytes and a rupture of microvessels in the testis. Our data suggest that NO has cytoprotective effects on ischemia-reperfusion injury in the rat testis.     

Transplacental exposure to methylene blue initiates teratogenesis in the mouse: preliminary evidence for a mechanistic implication of cyclic GMP pathway disruption

BACKGROUND: The vital dye methylene blue (MB) has been shown to be teratogenic when injected into the amnion in the second trimester. On the other hand, the teratogenic potential of transplacental exposure to MB has not been determined. METHODS: MB was administered subcutaneously to ICR (CD-1) mice at 0, 35, 50, 60, or 70 mg/kg on gestation day 8 (plug day = day 0). Teratological assessments were carried out at term gestation, on gestation day 18. Since MB inhibits soluble guanylate cyclase enzyme activity, zaprinast (ZPN), a selective cGMP-phosphodiesterase type V inhibitor, was administered to prevent developmental disorders initiated by MB at 50 mg/kg. RESULTS: There was a dose-dependent increment of embryolethality. MB treatment also produced axial skeleton and neural tube defects. Coadministration of ZPN (20 mg/kg per three times) abolished completely MB-induced neural tube defects and reduced by one-half the incidence of fetuses exhibiting axial skeletal defects. ZPN did not provide protection against the embryocidal effects of MB. CONCLUSIONS: This study showed that transplacental exposure to MB is teratogenic in the mouse. Coadministration of ZPN prevented partly MB-induced teratogenesis, which supports the hypothesis that imbalance of cGMP pathway accounts, in part, for the teratogenicity of MB.     

Effect of combination of nitric oxide synthase and cyclooxygenase inhibitors on carrageenan-induced pleurisy in rats

In the present study, we examined the effects of L-nitroarginine methylester (L-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, indomethacin (IND), a non-selective COX inhibitor and a combination of these agents (L-NAME+IND) on carrageenan-induced pleurisy in rats. Exudate volume, albumin leakage, leukocyte influx, exudate and plasma nitrite/nitrate (NO(x)) levels and exudate PGE(2) levels increased markedly 6 h after an intrapleural injection of 2% carrageenan. First, the effects of L-NAME and IND alone were investigated. L-NAME non-significantly reduced exudate volume by 26% at 10 mg/kg (i.p.), and significantly by 45% at 30 mg/kg. IND dose-dependently decreased the exudate volume at 0.3-10 mg/kg (p.o.) and the effect reached the maximal level at 1 mg/kg (33%). Second, the effects of L-NAME (10 mg/kg, i.p.), IND (1 mg/kg, p.o.) and L-NAME+IND were examined. L-NAME and IND alone at the dose employed significantly reduced the exudate volume and albumin levels by 21-26%. L-NAME but not IND tended to reduce the increased exudate and plasma NO(x) by 18% and 19%, respectively. IND but not L-NAME significantly reduced leukocyte numbers and PGE(2) levels in the exudates by 25% and 77%, respectively. L-NAME+IND significantly reduced exudate volume, albumin leakage, leukocyte number, PGE(2) and NO(x) by 43%, 41%, 31%, 80% and 37%, respectively. The inhibitory effects of L-NAME+IND on exudate volume, albumin leakage and NO(x) levels were greater than those of L-NAME and IND alone. In conclusion, a non-selective NOS inhibitor and COX inhibitor showed anti-inflammatory effects at the early phase of carrageenan-induced pleurisy, and a combination of both inhibitors had a greater effect than each alone probably via the potentiation of NOS inhibition. The simultaneous inhibition of NOS and COX could be a useful approach in therapy for acute inflammation.     

Modulatory effects of static magnetic fields on blood pressure in rabbits

Acute effects of locally applied static magnetic fields (SMF) on pharmacologically altered blood pressure (BP) in a central artery of the ear lobe of a conscious rabbit were evaluated. Hypotensive and vasodilator actions were induced by a Ca(2+) channel blocker, nicardipine (NIC). Hypertensive and vasoconstrictive actions were induced by a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME). The hemodynamic changes in the artery exposed to SMF were measured continuously and analyzed by penetrating microphotoelectric plethysmography (MPPG). Concurrently, BP changes in a central artery contralateral to that of the exposed ear lobe were monitored. SMF intensity was 1 mT and the duration of exposure was 30 min. A total of 180 experimental trials were carried out in 34 healthy adult male rabbits weighing 2.6-3.8 kg. Six experimental procedures were chosen at random: (1) sham exposure without pharmacological treatment; (2) SMF exposure alone; (3) decreased BP induced by a single intravenous (iv) bolus injection of NIC (100 microM/kg) without SMF exposure; (4) decreased BP induced by injection of NIC with SMF exposure; (5) increased BP induced by a constant iv infusion of L-NAME (10 mM/kg/h) without SMF exposure; (6) increased BP induced by infusion of L-NAME with SMF exposure. The results demonstrated that SMF significantly reduced the vasodilatation with enhanced vasomotion and antagonized the reduction of BP via NIC-blocked Ca(2+) channels in vascular smooth muscle cells. In addition, SMF significantly attenuated the vasoconstriction and suppressed the elevation of BP via NOS inhibition in vascular endothelial cells and/or central nervous system neurons. These results suggest that these modulatory effects of SMF on BP might, in part, involve a feedback control system for alteration in NOS activity in conjunction with modulation of Ca(2+) dynamics.