Interactions of caffeine and restraint stress during pregnancy in mice

The maternal and developmental toxicity of combined exposure to restraint stress and caffeine was assessed in mice. On gestational Days 0-18, three groups of plug-positive females (n = 13-15) were given by gavage caffeine at 30, 60, and 120 mg/kg/day. Three additional groups received the same caffeine doses and were restrained for 2 hr/day. Control groups included restrained and unrestrained plug-positive mice not exposed to caffeine. All animals in the group concurrently exposed to 120 mg/kg/day of caffeine and restraint died during the experimental period. In the remaining groups, cesarean sections were performed on Day 18 of gestation, and the fetuses were weighed and examined for external, internal, and skeletal malformations and variations. Although maternal and embryo/fetal toxicity were observed at all caffeine doses, the adverse maternal and developmental effects were significantly enhanced in the groups concurrently exposed to caffeine and restraint. It was especially remarkable at 60 and 120 mg/kg/day. The results of this study suggest that maternal and developmental toxic effects might occur if high amounts of caffeine were consumed by women under a notable stress during pregnancy.     

Effects of restraint stress in gestation: implications for rodent developmental toxicology studies

Restraint has been used as a procedure to study the effects of stress on gestation outcome in rodents. The effects of restraint could potentially be used as a model for the impact of general stress produced by high doses of toxicants and other interventions. In mice, restraint in the peri-implantation period leads to implantation failure, and restraint at appropriate times in organogenesis produces cleft palate, supernumerary ribs, and resorption. In rats, there is some evidence for an association with restraint for implantation failure, but not for the morphological anomalies. Restraint in late gestation alters adult sexual behavior of male rat offspring, but consequences for their fertility are not known. Intrauterine growth retardation is not commonly associated with gestational restraint. In the few studies where they have been directly compared, different restraint procedures produced graded, qualitatively different, or no effects. Adrenocortical hormones have been implicated as mediating the effect of restraint on cleft palate, but not on supernumerary ribs, implantation failure, or sexual differentiation. Given the variety of restraint procedures and the varying species-dependent consequences, it is not possible to infer a generalizable pattern of developmental effects due to gestational stress from the restraint literature. As an alternative approach, contemporary methods in gene expression and developmental biology could profitably be applied to understanding different patterns of stress-mediated effects of toxicant exposures on intrauterine development.     

Maternal restraint stress diminishes the developmental potential of oocytes

Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs.     

The effect of restraint stress in early pregnancy in mice

Mice were exposed to 5 h of restraint stress on Days 1-3, 4-6, or 1-6 of pregnancy in the morning (08:30-13:30 h, a.m.) or afternoon (13:30-18:30 h, p.m.). Stress reduced the pregnancy rate from 90 to 52% (P less than 0.005) and average litter size on Day 18 from 8.2 to 5.2 young (P less than 0.005). Stress for 6 days was more effective than for 3 days (P less than 0.005) and an a.m. stress was more effective than a p.m. stress (P less than 0.005) in reducing the average litter size. Animals examined on Day 7 after 6 days of a.m. stress had decreased numbers of normal corpora lutea (CL), increased numbers of abnormal CL, decreased serum progesterone concentrations and tended to have fewer implantation sites. Abnormalities of embryo transport and implantation were also present. Changes in CL morphology and embryo transport and development were evident on Day 4 after only 3 days of restraint stress. These results show that many reproductive events of early pregnancy can be disrupted by restraint stress.     

Energetic response to repeated restraint stress in rapidly growing mice

There is a cost of stress that may result in the loss of normal biological function (e.g., growth). Repeated, and even single, applications of stressors have been shown to induce negative energy balance in rodents. However, here we addressed whether this energetic response changes during multiple stress exposure and whether there is complete recovery subsequent to the cessation of stress exposure. These questions were addressed in growing C57Bl/6 mice (31 day) by determining at different times the energetic and endocrine responses after the exposure to restraint (R) stress for 4 h applied once (R1), repeatedly over 3 days (R3), or repeatedly over 7 days (R7). Compared with control values, R elevated (P<0.05) plasma corticosterone and reduced plasma insulin-like growth factor I on all days of exposure to the stressor. Seven days, but not 1 or 3 days of R, decreased the net growth (126%, P<0.05) and deposition of fat (71%, P<0.05) and lean (60%, P<0.05) energy over the 7 days. Only R7 depressed the 7-day metabolizable energy intake (P<0.05), and R7, but not R1 or R3, increased the overall energy expenditure (10%, P<0.05). Our results demonstrate that repeated episodes of stress are energetically costly to the rapidly growing animal, but compensatory mechanisms mitigate this cost of repeated stress exposure and permit complete recovery of energy balance after the cessation of stress application.     

Fumonisins: current research trends in developmental toxicology

Fumonisin B₁ (FB₁) is a mycotoxin produced byFusarium verticillioides that is found in maize and maize-based foods. Reproductive studies in CD1 mice, rats and rabbits initially found no evidence that fumonisins are teratogenic. However, more recent findings suggest that they might increase the risk of neural tube defects (NTDs) in populations consuming large amounts of fumonisin-contaminated corn. When ≥15 mg/kg body weight fumonisin B₁ (FB₁) was given to pregnant LM/Bc mice by intraperitoneal (ip) injection, all litters were positive for NTDs. To determine if NTD induction is unique to the inbred LM/Bc mouse strain, NTD induction in LM/Bc and CD1 mice was compared: (a) in a study in whichF. verticillioides culture material providing ≤150 ppm FB₁ was fed to female mice before and during gestation, and (b) in a study in which FB₁ was given by ip injection to CD1 dams on gestation days 7 and 8, the critical time for NTD development. In the feeding study, one of five LM/Bc litters from dams fed the 150 ppm FB₁ diet was positive for NTDs whereas no NTDs were found in the CD1 litters. In the ip injection study, 40% of the litters at the highest dose tested, 45 mg/kg body weight, were positive for NTDs and one of nine low-dose (15 mg/kg body weight) litters was also positive. Thus, FB₁ induced NTDs in both LM/Bc and CD1 mice although the latter strain appears less sensitive. Comparative investigations using these strains will be useful for elucidating the mechanisms underlying fumonisin-induced NTDs in mice and determining the suitability of mouse models for studying the relationships between fumonisins and NTDs in humans.     

Mutagenic action of caffeine in an ascites tumour strain of mice: Cytogenetic investigation

S₂-sarcoma of white mice, treated in vivo with caffeine (0,25 g/kg i.p.) or caffeine (0,25 g/kg i.p.) and oxygen (0,5 ml i.p.) were evaluated for structural chromosome anomalies. Controls received aqua dest. (0,5 ml i.p.) or oxygen (0,5 ml i.p.). Caffeine treatment gave the same results as the control i.e. less than 1% cells with aberrations. Combination of caffeine and oxygen treatment yielded significant increase of chromatid breaks i.e. 4,4%, while reunion figures were as seldom as in the control group. Cells with gaps only were not scored.     

Cytogenetic investigations of mutagenic action of caffeine in premeiotic spermiogenesis in mice

14 male mice (C3H) were treated with maximal tolerated doses of 1-3-7-trimethylxanthine (caffeine). Animals were sacrificed and preparations of testicular tissue examined in intervals after treatment. The meiotic chromosomes at Metaphase I (Diakinesis) were studied by phase contrast microscopy for breaks, translocations and univalent chromosomes. No unusual chromosome changes could be found.     

对香豆酸对慢性束缚应激诱导小鼠抑郁样行为的作用

目的:探索对香豆酸(p-CA)对慢性束缚应激(CRS)诱导小鼠抑郁样行为的作用。方法:实验分两批进行,第一批小鼠随机分成对照组(Control),慢性束缚应激组(CRS)和慢性束缚应激+p-CA组(CRS+p-CA),每组8只,其中慢性束缚应激小鼠每天接受4 h的束缚应激,连续束缚21 d,而对照组小鼠留在笼中不被打扰。第22日小鼠腹腔注射溶媒(10%吐温80)或p-CA(100mg/kg),注射后1 h进行自发活动测试(LMA),注射后4 h进行强迫游泳测试(FST),注射后24 h进行悬尾测试。第二批小鼠随机分成慢性束缚应激组(CRS),慢性束缚应激+ANA-12(原肌球蛋白激酶B拮抗剂)组(CRS+ANA-12)和慢性束缚应激+p-CA组(CRS+p-CA)慢性束缚应激+p-CA+ANA-12组(CRS+p-CA+ANA-12),每组8只,4组小鼠每天接受4 h的束缚应激,连续束缚21d。第22日小鼠腹腔注射溶媒(10%吐温80)或p-CA(100 mg/kg),ANA-12(0.5 mg/kg)在p-CA注射前30 min给药。注射后1 h进行自发活动测试(LMA),注射后2 ...     

Effect of maternal restraint stress on fetal development of ICR mice.

The present study was conducted to elucidate the susceptibility of embryos and fetuses at different gestational stages to the maternal stress in mice. Groups of pregnant ICR mice were subjected to daily 12-h restraint stress, taped in the supine position on a plastic board, on gestational days (GD) 1-4, 5-8, 9-12 and 13-16, respectively. Caesarean sections were performed on gestational day 18, and the fetuses were weighed and examined for morphological defects. During the daily restraint for 4 days, the maternal body weights markedly decreased. Although the body weights recovered gradually after termination of the stress, the recovery was not full until the final stage of pregnancy. Interestingly, restraint stress caused growth retardation of the fetuses, leading to a significant decrease in their body weights, and increased early and late resorptions of embryos and fetuses according to the stress periods. Although the preceding (GD1-4) and concurrent (GD5-8) stresses did not affect embryonic implantation, restraint stress on GD9-12 caused cleft palate. Whereas vertebral abnormalities, mainly bipartite ossification, were observed only in animals stressed on GD5-8, abnormalities of sternebrae, exhibiting asymmetric or bipartite ossification, were enhanced by the stress at all of the gestational stages. On the other hand, the incidence of other malformations including renal malposition and costal abnormalities was not increased by stress at any of the 4 stages. Taken together, the results suggest that intensive restraint stress influences the maternal body weight resulting in growth retardation and increased mortality of embryos and fetuses, in addition to gestational stage-specific ventricular dilatation, cleft palate and sternal abnormalities.     

对香豆酸上调BDNF及改善慢性束缚应激诱导小鼠记忆障碍的作用

目的:探索对香豆酸(p-CA)对慢性束缚应激(CRS)小鼠记忆障碍的作用及其可能机制。方法:实验分两批进行,第一批小鼠随机分成对照组(Control)、慢性束缚应激+溶媒组(CRS)和慢性束缚应激+p-CA组(CRS+p-CA),每组8只,其中CRS小鼠每天接受4 h的束缚应激,连续束缚10 d,而对照组小鼠留在笼中不被打扰。第11日小鼠腹腔注射溶媒(10%吐温80)或p-CA(100 mg/kg),注射后2 h进行Y迷宫测试,注射后24 h进行新颖物体识别(NOR)采样,采样后2 h进行新颖物体识别测试。实验结束后断头取脑剥离海马并检测BDNF蛋白表达。第二批小鼠随机分成慢性束缚应激组(CRS),慢性束缚应激+ANA-12(原肌球蛋白激酶B拮抗剂)组(CRS+ANA-12),慢性束缚应激+p-CA组(CRS+p-CA)和慢性束缚应激+p-CA+ANA-12组(CRS+p-CA+ANA-12),每组8只,四组小鼠每天接受4 h的束缚应激,连续束缚10 d。第11日小鼠腹腔注射溶媒(10%吐温80)或p-CA(100mg/kg),ANA-12在p-CA注射前30 min给药。注射后2 h...     

Effect of restraint stress on plasma concentrations of cortisol, progesterone and pregnancy associated-glycoprotein-1 in pregnant heifers during late embryonic development

The aim of the present study was to evaluate the effect of restraint stress, which is commonly practised in the field, on plasma concentrations of cortisol, progesterone (P4) and bovine pregnancy-associated glycoprotein-1 (boPAG-1) in pregnant heifers between Days 30 to 40 of gestation. Twelve Holstein-Friesian heifers between Days 30 (Day 0 of experiment) and 40 (Day 10 of experiment) of pregnancy in a Hungarian dairy farm were used in the present study. The heifers were exposed to an acute stressor consisting of immobilisation (restraint stress) in a crush for 2 h (Group 1, n = 6) on Day 2 (Hour 48) and for 2 × 2 h (Group 2, n = 6) on Days 2 and 3 (Hour 72) of the experiment.Transrectal ultrasonography (7.5 MHz linear-array rectal transducer) was performed daily from Day 0 to Day 10 of the experiment to detect embryonic heartbeat or the fate of the conceptus. Blood samples were withdrawn before each ultrasonographic examination. Additional blood samples were withdrawn by 1 and 2 h (at Hours 49 and 50 in Groups 1 and 2 and Hours 73 and 74 in Group 2) of the onset of applying the stressor. Plasma cortisol, P4 and boPAG-1 concentrations were measured by radioimmunoassay. Acute restraint stress significantly (P < 0.001) increased the plasma cortisol level in pregnant heifers at 1 h of the exposure to the stressor at Days 2 (48 h) and 3 (72 h) of the experiment. On the other hand, the restraint stress did not affect the concentration of P4 and boPAG-1 concentrations in both groups. In conclusion, restraint stress for 2 h during early pregnancy in heifers increased blood cortisol, but it did not affect the concentrations of P4 and boPAG-1 between Days 30 to 40 of gestation.     

Effects of oolong tea on metabolism of plasma fat in mice under restraint stress

We investigated the effects of oolong tea on the basic metabolism of plasma lipids in mice under restraint stress. When a lipid emulsion (Intralipid 20%; a lipid emulsion containing 20% soybean oil) was injected intravenously into mice, the restraint stress prolonged the half-life (T 1/2) of elimination for plasma triglyceride (TG) from 28.7 to 55.5 min. The elimination rate per minute was 48.2% in stressed mice with the rate in starved control mice as 100%. Therefore, TG metabolism was disrupted by the stress, and the use of TG as an energy source decreased. We found that the metabolism of lipids significantly response to the restrained stress in the present study. Plasma TG was 515.9 +/- 29.9mg/dl 35min after Intralipid administration in control stressed mice, 478.7 +/- 26.7 mg/dl in the stressed group given caffeine 100 mg/kg of body weight, and 418.3 +/- 18.4 mg/dl in the stressed group given 1,000 mg/kg oolong tea, an improvement by 7.2% and 18.9%, respectively, with the value for the untreated control group. The intake of oolong tea alleviated the stress-induced decrease in the rate of blood lipid metabolism; this effect may have arisen from some non-specific stress-relieving property of the tea or from acceleration of lipid metabolism by properties of polyphenols, etc. in tea. Oolong tea had anti-stress effects on plasma TG metabolism, and the effects did not depend on caffeine.