Weight and radioactivity of the organs of progeny after 5-HT and/or Na2H32PO4 treatment of pregnant mice.

In 30-day-old offspring of mice treated with 5-HT and/or Na2H32PO4, there was a decrease in the fresh weight of the organs, being more noticeable in males than in females. In relation to a group injected with Na2H32PO4 and that to which both 5-HT and Na2H32PO4 were administered, a greater fresh weight of the organs in mice treated with 5-HT only was found. No differences in the radioactivity of 32P in the organs of 30-day-old offspring between the group of mice treated with Na2H32PO4 only and that injected with both 5-HT and Na2H32PO4, were observed.     

Embryonic survival in mice treated with AET, MEA or 5-HT on the first day of pregnancy

The embryotoxicity of AET, MEA, and 5-HT was investigated in Porton mice. Female mice on the first day of gestation were injected intraperitoneally with 2-amino-ethylisothiouronium bromide hydrobromide (AET), cysteamine hydrochloride (MEA) or serotonin-creatinine sulphate (5-HT) in a dose of 40 mg/kg body weight. Uterine contents were examined on the nineteenth day of pregnancy. As compared with controls, in mice treated with AET, MEA or 5-HT, a smaller number of live fetuses and a greater number of non-implanted embryos, resorptions, and dead fetuses were found. Not all females which were injected with these compounds had live fetuses. Among the compounds, MEA appeared to be more toxic than AET and 5-HT.     

Quantity of DNA in the organs of 19-day-old foetuses after AET, MEA, or 5-HT treatment of mice on the first day of gestation

On the first day of gestation, Porton mice were injected intraperitoneally with AET (2-aminoethylisothiouronium bromide hydrobromide), MEA (cysteamine hydrochloride,) or 5-HT (serotonin-creatinine sulphate), in a dose of 40 mg/kg of bodyweight. On the nineteenth day of pregnancy, the fresh weight of both heart and kidneys of foetuses, as well as DNA content in 25 mg of fresh tissue and in these whole organs were analysed. DNA was extracted from the foetal organs by means of Burton's method, which is based on the estimation of deoxiribose content in the colour reaction with diphenylamine. As compared to controls, in the remaining groups of mice lower fresh weight of both heart and kidneys of foetuses, greater DNA content in 25 mg of fresh tissue and smaller total amounts of DNA in the whole organs were found. Among the experimental groups of mice, statistically significant differences in the analysed values were observed between the group of animals treated with 5-HT and the remaining groups, with the exception of statistically non-significant difference in the DNA content of the whole kidneys between those injected with 5-HT and MEA.     

Placental changes due to administration of diethylstilbestrol (DES)

Pregnant mice were injected with 12.5 micrograms DES/kg body weight or 25 micrograms DES/kg body weight daily from gestation day 9 through day 12 or 16 and sacrificed on day 13 or 17. Placentas of DES treated animals were smaller than controls, the effect being dose dependent. Histologic changes in 13 gestation day placentas regional thinning of the labyrinth associated with an apparent inhibition of trophoblast maturation and development of fetal blood vessels. Knots of mononuclear cells form in the labyrinthine region of 13 day placentas exposed to the higher dose of DES. By 17 days gestation, coagulative necrosis is common in the decidua basalis, being most severe in those animals receiving 25 micrograms DES/kg. In many placentas the labyrinthine region is absent. The only remaining elements are trophoblast cells, giant cells and glycogen-containing cells. Fetal deaths associated with the lower dose of DES increased with time whereas 100% fetal mortality was associated with the higher dose.     

Quantity of glycogen in the liver of 19-day-old foetuses after AET, 5-HT, MEA, or GSH treatment of pregnant mice on the first day of gestation

Swiss mice were treated intraperitoneally with AET, 5-HT, MEA, or GSH, in a dose of 80 mg/kg of body weight, on the first day of gestation. On the 19th day of pregnancy, the fresh weight of liver of the foetuses, as well as glycogen content in 1 g of fresh tissue and in the whole organ were analysed. The determination of glycogen content in the foetal liver were made according to the anthrone method. As compared with controls, in the remaining groups of mice a lower fresh weight of foetal liver less glycogen per g of fresh tissue and a smaller total amount of glycogen in the whole organ were found. Among the compounds, AET appeared to be more toxic than 5-HT, MEA, and GSH.     

Effect of prenatal gamma irradiation during the late fetal period on the postnatal development of the mouse

Pregnant Swiss albino mice were exposed to 0.3, 0.5, 1.0, or 1.5 Gy of gamma radiation on day 17 of gestation. Sham-exposed controls were examined for comparison. Exposed mice as well as controls were left to complete gestation and parturition. Pups were observed up to age 6 weeks; appearance of physiological markers (pinna detachment, eye opening, fur development, vaginal opening, and testes descent), postnatal mortality, body weight, body length, head length, head width, and tail length were recorded. A significant delay in fur development was observed at 0.3 Gy and in other physiological markers at doses above 0.3 Gy, while a significant increase in mortality and growth retardation occurred only at 1.0 and 1.5 Gy. Although congenital anomalies such as syndactyly and bent tail were observed at doses of 0.5-1.5 Gy, only syndactyly showed a statistically significant increase in frequency. A statistically significant lower body weight was observed during the first week of postnatal life, but body weights increased to normal levels by the second week in animals exposed to doses less than 1.0 Gy. At higher doses, low body weight persisted throughout the postnatal period. Head length and tail length showed a significant decrease from controls at 0.5-1.5 Gy, and the effect was evident from birth to age 6 weeks. But a similar effect on body length and head width was noticed only at 1.0 and 1.5 Gy. These studies indicate that even in the absence of any major morphological changes, normal development of physiological landmarks and postnatal growth can be impaired by fetal irradiation at 17 days p.c. (post coitus). Morphological changes appear to have a threshold between 0.3-0.5 Gy, while physiological marker effects may occur with a lower threshold.     

Maternal dietary tryptophan deficiency alters cardiorespiratory control in rat pups

Malnutrition during pregnancy adversely affects postnatal forebrain development; its effect upon brain stem development is less certain. To evaluate the role of tryptophan [critical for serotonin (5-HT) synthesis] on brain stem 5-HT and the development of cardiorespiratory function, we fed dams a diet ～45% deficient in tryptophan during gestation and early postnatal life and studied cardiorespiratory variables in the developing pups. Deficient pups were of normal weight at postnatal day (P)5 but weighed less than control pups at P15 and P25 (P < 0.001) and had lower body temperatures at P15 (P < 0.001) and P25 (P < 0.05; females only). Oxygen consumption (Vo(2)) was unaffected. At P15, deficient pups had an altered breathing pattern and slower heart rates. At P25, they had significantly lower ventilation (Ve) and Ve-to-Vo(2) ratios in both air and 7% CO(2). The ventilatory response to CO(2) (% increase in Ve/Vo(2)) was significantly increased at P5 (males) and reduced at P15 and P25 (males and females). Deficient pups had 41-56% less medullary 5-HT (P < 0.01) compared with control pups, without a difference in 5-HT neuronal number. These data indicate important interactions between nutrition, brain stem physiology, and age that are potentially relevant to understanding 5-HT deficiency in the sudden infant death syndrome.     

Susceptibility to postnatal growth retardation induced by 5-AZA-2'-deoxycytidine in utero: gender specificity and correlation with reduced insulin-like growth factor 1

The DNA demethylating agent 5-AZA-2'-deoxyxytidine (5-AZA-CdR) alters gene expression in mice exposed during developmental stages and causes malformations and growth suppression. The aim of this study was to determine if 5-AZA-CdR-induced growth retardation is associated with alterations in energy metabolism or in serum IGF-1 levels. Mice were exposed in utero to 5-AZA-CdR at gestation day 10. At postnatal day 21, exposed pups were weaned and body weights recorded. At 3 months of age, reproductive capacity was studied. At 5 months old, after body weight was recorded mice were killed and serum was collected to determine serum glucose, corticosterone, and IGF-1 levels. The body weights of both treated males and females were reduced at weaning compared with controls, but by 5 months of age, only the male body weight was affected. Reproductive capacity of males and females was reduced with males being more affected. Levels of corticosterone and glucose were not altered. Serum IGF-1 levels were lower in males exposed in utero to 5-AZA-CdR when compared to controls, but not in females, and correlated significantly with body weights. Our data suggest that the decreased levels of IGF-1 associated with the treatment could be the cause of the observed growth retardation in the in utero-exposed mice. A gender dimorphic effect, where males are more affected, is evident.     

Toxicity of ultrasound in mice: Neonatal studies

Summary Pregnant mice were exposed to ultrasound (continuous wave, 2 MHz) on Day 8 of gestation to determine effects on the progeny. The most significant finding was a decrease in mean uterine weight of the female progeny. The thresholds for this effect were 140 s at 0.5 W/cm² and 60 s at 1 W/cm², which were below the thresholds previously reported for other effects in mice. We suggest that this indicates a delay or impairment of maturation of the mice exposed in utero.Exposure of the dams to spatial average intensity of 1 W/cm² for 40 and 60 s had no effect on body weight of the progeny, compared with sham-treated controls. In this experiment the body weights of progeny from sham-treated controls were significantly lower than those from untreated controls on Days 10, 17, and 25 of age. After exposure in utero to 0.5 W/cm² for 180 s, statistically significant decreases in mean body weights of the neonates were observed, but only on Day 25 of age, in both sexes compared with sham-treated controls. At necropsy at Day 25 of age, neonatal organ weights relative to body weights were not significantly affected for the thymus in either sex or for the seminal vesicles and testes in comparison with sham-treated controls.     

Thyroid hormone plasmatic levels in rats treated with serotonin in acute and chronic way

Many experiments show that serotonin (5-HT) controls thyroidal function at hypothalamic level, inhibiting the TRH secretion. The majority of experiments are done in an acute way, consisting of a single serotonin dose injected intraperitoneally (ip) or intracerebroventricularly (ic) with the effect registered after a short time (usually 1 h) as in normal environmental conditions similar to the TSH stimulation test, that consists of transfer of the experimental animals from 30°C to 4°C for 30 min, thus inducing stimulation of the hypothalamus-hypophysis-thyroid axis. The aim of the present research was to study the correlation between 5-HT and the thyroidal function, measuring plasmatic thyroid hormone levels in rats ip treated in chronic (injected daily for 10 days with different doses of 5-HT), and in acute way (after 1 h from a single 2.0 mg/kg bw 5-HT dose) in normal environmental conditions to evidence the serotonin site action activity outside the blood-brain barrier. The results of the chronic experiment show an inhibitory effect of 5-HT, on T3 and T4 plasmatic level, only when it is injected at medium doses (0.2 and 0.4 mg/kg bw for T3, and 0.2 for T4), while the results of the acute experiment do not evidence any modification. These results show that in normal environmental conditions the outside 5-HT site action is active only when the 5-HT is injected chronically at defined doses, probably for a down-regulation phenomenon.     

Different Diabetogenic Response to Moderate Doses of Streptozotocin in Pregnant Rats,and Its Long-Term Consequences in the Offspring

Diabetes during pregnancy results in congenital malformations and long-term postnatal diseases. Experimental models are still needed to investigate the mechanism responsible for these alterations. Thus, by the administration of different doses of streptozotocin (STZ) (0, 25, 30, or 35 mg/kg body weight, intravenous) at the onset of pregnancy in rats, the present study sought an appropriate animal model for this pathology. At day 6 of pregnancy, plasma glucose was progressively higher with an increasing STZ dose, and in rats receiving the 35-mg dose, 2 subgroups were detected: some animals had plasma glucose levels above controls but below 200 mg/dL (mildly diabetic, MD), whereas others had levels above 400 mg/dL (severely diabetic, SD). At day 20 of pregnancy, the MD rats had normal glycemia, but after an oral glucose load (2 g/kg body weight), plasma glucose increased more and insulin increased less than in controls. The SD rats maintained their hyperglycemia and had a greatly impaired oral glucose tolerance. At day 20, fetuses of SD dams were fewer, weighed less, and had enhanced plasma glucose and triglycerides and decreased insulin, whereas those from MD dams did not differ from controls. At birth, newborns from MD dams had higher body weight, plasma insulin, and liver triglycerides as well as total body lipid concentrations than controls, and on day 21, remained macrosomic and showed higher plasma glucose and liver triglyceride concentrations. At 70 days of age, offspring of MD dams had impaired oral glucose tolerance but normal plasma insulin change in the case of females, whereas plasma insulin increased less in males. These alterations were manifest more in those offspring from dams that had > 50% macrosomic newborns than in those from dams that had < 50% macrosomic newborns. In conclusion, whereas our MD rats mimic the changes taking place in gestational diabetic women and show the long-term risk of macrosomia, the SD rats are more similar to uncontrolled diabetics. Thus these two rat models, obtained with moderate amounts of STZ, could be used to study the pathophysiological consequences of these different diabetic conditions.     

Overexpression of PACAP in the pancreas failed to rescue early postnatal mortality in PACAP-null mice

PACAP exerts multiple activities as a hormone and neurotransmitter, and has been proposed to play vital roles in a variety of neuronal functions. PACAP is also involved in insulin secretion from pancreatic beta-cells. Recently, we and other groups demonstrated that PACAP-deficient mice (PACAP(-/-)) are viable, but suffer from increased postnatal mortality. To ascertain whether this high mortality is rescued by overexpression of PACAP in peripheral tissue (such as pancreas), we performed a genetic cross between PACAP(-/-) and our recently developed transgenic mice overexpressing PACAP in pancreatic beta-cells; and then examined the survival rate of their F2 progeny. PACAP(-/-) mice were segregated into two groups based on mortality as well as body weight gain: PACAP(-/-) that survived >20 days of age with normal weight gain and PACAP(-/-) that died before 20 days with a marked weight loss. Kaplan-Meier survival analysis demonstrated that PACAP(-/-) mice and those carrying the PACAP transgene have similarly lower survival probability compared with their heterozygous littermates that served as positive controls. Further study using additional tissue-specific transgenic or knockout mouse models will be required to determine the causative defects underlying the high mortality of PACAP(-/-) mice.     

Altered precursor availability and metabolism of monoamines in the brain caused by the injection of physiologic saline to suckling rats: On the reliability of saline “controls” in neurochemical studies

The effect of subcutaneous injections of saline (0.9% NaCl, 10–40 μl/g b. wt) to 5- and 20-day old rats on the concentrations of tyrosine (Tyr) and tryptophan (Trp) in the serum and the brain and on the levels of biogenic amines and their metabolites in the developing brain at 6 h p.i. is described. At day 5 the concentration of Tyr in the blood was decreased (dose-dependent), but the brain concentrations of Tyr and of its amine-metabolites, dopamine (DA), norepinephrine (NE), homovanillic acid (HVA) and dihydroxyphenylacetate (DOPAC) were unaffected. In contrast, in the 20-day old rat, serum Tyr was unaffected by the saline injections, but the Tyr concentration in the brain decreased markedly at the highest saline dose. The concentrations of NE (only at maximum dose) and of DA (independent on the amount of saline injected) were elevated in the brains of saline injected 20-day old rats. The concentrations of Trp and indoles were more affected at day 5 than at day 20: slightly decreased concentration of Trp in the serum but markedly increased concentrations of brain Trp (only at maximum dose), elevated serotonin (5-HT, independent on the amount of saline injected) and 5-hydroxyindoleacetic acid (5-HIAA, at maximum dose) in the brain. If the maximum dose of 40 μl/g body weight was injected to suckling rats repeatedly during the whole suckling period (in 12 h intervals), some effects caused by one single injection of 40 μl/g disappeared (Tyr—depletion in blood or brain, increase in brain NE, DA and Trp), but other additional effects appeared (decreased DA and increased DOPAC, decreased 5-HT and 5-HIAA). The results show that saline injections do cause characteristic, age-dependent alterations of precursor availability as well as of the rate of synthesis and degradation of catecholamine and 5-HT. Repeated treatments have different effects than one single treatment on the precursor availability and the metabolism of monoamines. These alterations must be taken into account if the effects of certain “specific” treatments are compared and discussed in relation to saline “controls”.     

Altered distribution and toxicity of digitoxigenin in fasted mice

Intravenous administration of digitoxigenin (DTXGN) evokes seizure episodes in mice which may be dependent on brain biogenic amines such as serotonin (5-HT). Fasting is known to have effects on both drug toxicity and brain 5-HT synthesis. The purpose of this study was to assess the effects of overnight fasting on DTXGN toxicity. The i.v. LD-50 of DTXGN was increased by 61% in fasted mice. Adjustment of DTXGN dose for the decrease in body weight of fasted mice did not alter the fasting induced protection. A loading dose of 1-tryptophan (25 mg/kg, i.p.) did not alter mortality rates in either fed or fasted mice. Cortical levels of 3H-DTXGN were decreased significantly by 25% in fasted mice. Liver and blood levels were elevated significantly. These data suggest that decreased DTXGN toxicity is associated with a decrease in its distribution to the cerebral cortex and emphasize the importance of acute dietary status in the expression of drug toxicity.     

Abnormal development of blood pressure and growth in rats exposed to perinatal injection stress

Subcutaneous injections of alkaline saline were made perinatally in Sprague-Dawley rats according to two schedules. In a pre-/postnatal group, dams were treated from 19th gestational day to 9th day postpartum and pups from day 0–9. In a postnatal group, pups alone were injected from day 0–6. At 19–23, 50–56 and 82–86 days of age, injected rats and uninjected controls were anesthetized and arterial blood pressure measured. Rats from the pre-/postnatal group had higher blood pressures (58%) and body weights at 19–23 days and lower blood pressure (35%) and body weight at 82–86 days of age. Blood pressure and body weight were comparable to control at all ages in the postnatal injection group. It is concluded that as a result of the maternal stress produced by the injections there was a generalized disturbance of growth processes resulting in hypotension and decreased body weight in adulthood.     

Hormone synthesis and storage in the thyroid of human preterm and term newborns: effect of thyroxine treatment.

Iodine and thyroglobulin concentrations, as well as iodine, T3, T4 and sialic acid contents of thyroglobulin, were measured in thyroid glands collected postmortem from 42 human premature or term newborns and infants. Three groups were considered: very preterm newborns (24-32 postmenstrual weeks, < 5 days postnatal life), preterm and term newborns (34-41 postmenstrual weeks, < 5 days postnatal life) and infants (born at term, postnatal age 1-8 months). Five very preterm and seven preterm newborns received a daily dose of 10 microg/kg L-T4 for at least 3 days. Thyroid weight and sialic acid content of thyroglobulin progressed with maturation. Intrathyroidal concentrations of iodine and thyroglobulin did not increase significantly before the 42nd week of postmenstrual age. The level of thyroglobulin iodination increased during the postnatal life, except in the very preterm neonates. T4 and T3 content of thyroglobulin was directly proportional to its degree of iodination and positively related to its sialic acid content. L-T4 treatment of preterm newborns increased thyroglobulin iodination and T4-T3 content, without increasing thyroglobulin concentration in the thyroid. It was concluded that the storage of thyroglobulin and iodine in the thyroid develops around term birth. This, associated with the resulting rapid theoretical turnover of the intrathyroidal pool of T4 in Tg, could be an important factor of increased risk of neonatal hypothyroxinemia in the premature infants. The L-T4 treatment of preterm newborns does not accelerate the maturational process of the thyroid gland.     

Early Postnatal In Vivo Gliogenesis From Nestin-Lineage Progenitors Requires Cdk5

The early postnatal period is a unique time of brain development, as diminishing amounts of neurogenesis coexist with waves of gliogenesis. Understanding the molecular regulation of early postnatal gliogenesis may provide clues to normal and pathological embryonic brain ontogeny, particularly in regards to the development of astrocytes and oligodendrocytes. Cyclin dependent kinase 5 (Cdk5) contributes to neuronal migration and cell cycle control during embryogenesis, and to the differentiation of neurons and oligodendrocytes during adulthood. However, Cdk5’s function in the postnatal period and within discrete progenitor lineages is unknown. Therefore, we selectively removed Cdk5 from nestin-expressing cells and their progeny by giving transgenic mice (nestin-CreERT2/R26R-YFP/CDK5^flox/flox [iCdk5] and nestin-CreERT2/R26R-YFP/CDK5^wt/wt [WT]) tamoxifen during postnatal (P) days P2-P 4 or P7-P 9, and quantified and phenotyped recombined (YFP+) cells at P14 and P21. When Cdk5 gene deletion was induced in nestin-expressing cells and their progeny during the wave of cortical and hippocampal gliogenesis (P2-P4), significantly fewer YFP+ cells were evident in the cortex, corpus callosum, and hippocampus. Phenotypic analysis revealed the cortical decrease was due to fewer YFP+ astrocytes and oligodendrocytes, with a slightly earlier influence seen in oligodendrocytes vs. astrocytes. This effect on cortical gliogenesis was accompanied by a decrease in YFP+ proliferative cells, but not increased cell death. The role of Cdk5 in gliogenesis appeared specific to the early postnatal period, as induction of recombination at a later postnatal period (P7-P9) resulted in no change YFP+ cell number in the cortex or hippocampus. Thus, glial cells that originate from nestin-expressing cells and their progeny require Cdk5 for proper development during the early postnatal period.     

The effects of chronic manganese feeding on the activity of monoamine oxidase in various organs of the developing rat

1. Chronic manganese feeding of rat with doses as high as 10 mg/ml in drinking water had no effect on body weight increases during postnatal development. The organ weight increases in brain, liver, heart and kidney also remained unaffected, but spleen weight was consistently lower than in the age-matched controls after Mn-feeding, being more marked at the higher doses. An increase in the Mn concentration to 20 mg/ml led to drastic body weight losses not unlike that seen in malnutrition. 2. There were differential developmental changes in monoamine oxidase (MAO) with respect to tissue type and substrate used. Manganese feeding did not affect the developmental patterns of MAO in brain, heart and kidney. However, hepatic MAO activities towards 5-HT and BzNH2 were found to increase after 10--15 days of postnatal life. 3. In contrast, the activity in the spleen towards 5-HT was lower in the high Mn-treated group in the first few days post-partum.     

Splenocyte subsets in normal and protein malnourished mice after long-term exposure to cocaine or morphine

An experimental model which resembles human drug addiction was developed to study the effect of chronic drug (cocaine or morphine) administration on the immune system. As malnutrition has been associated with drug use, a low protein diet has been evaluated for its contribution to the impairment of the immune system during cocaine/morphine addiction. Female C57BL/6 mice that received a 20% or 4% casein diet were studied. Both drugs were administered intraperitoneally daily for 11 weeks and drugs were administered in increasing daily doses, beginning after 3 weeks of diet consumption. Doses of cocaine began with 5 mg/kg body weight and reached the maximum dose of 40 mg/kg/day at the fourth week. Doses of morphine gradually increased from 10 mg/kg to 75 mg/kg body weight with the maximum dose reached after 5 weeks of treatment. Cocaine administration reduced body weight, particularly in the low protein diet group, and spleen weight in protein malnourished mice. Cocaine as well as saline injected mice showed a decrease in the percentage of CD4+ CD8+ and Mac-1+ cells and an increase in B cells in the spleens of well nourished mice. Morphine-treated mice showed similar results to those observed in cocaine or saline treated mice. These results suggest that cocaine, morphine or saline injection can alter the percentage of cells that express a defined phenotype independently of the nutritional status of the subject. Moreover, the effect appears dependent on a stress mediated process.     

Chromosome aberrations in spermatocytes and oocytes of mice irradiated prenatally

5 pregnant mice were exposed to a single dose of 150 R whole body γ-irradiation on the 12th day of gestation. The ocytes and spermatocytes, collected from the F₁ progeny at ages 10–12 weeks, were examined for chromosome aberrations in metaphase I and compared with those of the progeny of non-irradiated controls. No differences were found in the type and frequency of aberrations between irradiated and controls nor between the sexes. It appears, therefore, that either primordial germ cells of both males and females are fairly resistant to radiation or an efficient selection or repair mechanism has eliminated the aberrant cells.