Exposure Of Pups Research Articles

Chronic in utero plasma hyperosmolality alters hypothalamic arginine vasopressin synthesis and pituitary arginine vasopressin content in newborn lambs

Objective: Arginine vasopressin is synthesized in the hypothalamus and secreted by the posterior pituitary gland in response to plasma hypertonicity. Previous studies suggest that in utero and neonatal exposure of rat pups to prolonged alterations of plasma osmolality may permanently alter (imprint) arginine vasopressin synthesis and secretion, thus adult responses to osmotic challenges. Little is known, however, of the potential for imprinting of neuroendocrinologic systems in precocial species. In view of the frequent occurrence of altered maternal and fetal plasma tonicity (eg, maternal dehydration, hyperemesis), we sought to determine the effect of prolonged maternal hypertonicity on arginine vasopressin gene expression and pituitary gland content in neonatal sheep. Study Design: Pregnant ewes at 119 ± 3 days of gestation were water restricted to achieve and maintain plasma hypertonicity (10-20 mOsm/kg above baseline level) until normal term delivery. Newborns were provided maternal nursing ad libitum. Within 24 hours after birth, study neonatal lambs (n = 6) and age-matched control neonatal lambs (n = 5) were killed, and the pituitary gland and hypothalamus were removed and frozen immediately. Pituitary arginine vasopressin content was determined by radioimmunoassay, and hypothalamic arginine vasopressin gene expression was quantified with Northern blot. Differences in pituitary arginine vasopressin content and hypothalamic arginine vasopressin gene expression (arginine vasopressin/ β-actin ratio) between study and control newborn lambs were analyzed by unpaired t test. Results: In response to maternal water restriction, maternal plasma osmolality increased from 307 ± 0.9 mOsm/kg to 325 ± 1.3 mOsm/kg, and plasma sodium increased from 147 ± 1.3 mEq/L to 156 ± 1.2 mEq/L. The maternal plasma hyperosmolality and hypernatremia were maintained until normal term delivery. At the time of death, study (in utero dehydrated) lambs had higher plasma sodium (150 ± 0.4 mEq/L vs 146.5 ± 1.5 mEq/L; P <.05) and chloride (112.8 ± 1.0 mEq/L vs 108.5 ± 1.5 mEq/L; P <.05) levels, and lower potassium (4.5 ± 0.2 mEq/L vs 5.5 ± 0.3 mEq/L; P <.05) concentrations than control newborn lambs. Both newborn groups had similar plasma osmolality (320.0 ± 1.3 mOsm/kg vs 318.0 ± 3.4 mOsm/kg). Total pituitary arginine vasopressin content was significantly greater in the study than in the control newborn lambs (8.3 ± 2.8 μg vs 1.6 ± 1.3 μg; P <.05). Conversely, hypothalamic arginine vasopressin messenger RNA levels were lower in the study newborn lambs than in the control newborn lambs (arginine vasopressin/β-actin ratio: 0.29 ± 0.01 vs 0.68 ± 0.15; P <.05). Conclusion: Despite the presence of plasma hypernatremia, prolonged elevation of fetal plasma tonicity increases newborn pituitary arginine vasopressin content yet decreases hypothalamic arginine vasopressin gene expression. The present study suggests that prolonged prenatal exposure to plasma hypertonicity may imprint the hypothalamic-pituitary arginine vasopressin regulatory system. (Am J Obstet Gynecol 2002;187:191-6.)

Increased insulin sensitivity and decreased body weight in female rats after postnatal corticosterone exposure.

Glucocorticoids are important for normal brain development. Elevation or removal of these hormones can permanently modify the structure and function of the fetal brain. The purpose of this study was to examine the effects of postnatal corticosterone exposure of female pups on metabolic, endocrine and anthropometric variables in adulthood. Female pups were given subcutaneous injections of corticosterone (5 mg/kg, CORT) or vehicle 3 and 5 days after birth. From 6 weeks of age, the CORT rats weighed significantly less than did controls, with diminished fat depots, decreased serum levels of leptin and reduced food intake. Adult CORT rats showed increased insulin sensitivity, measured by hyperinsulinemic, euglycemic clamp (5 mU/kg/min), as compared with controls. CORT rats had lower basal corticosterone levels and lower corticosterone levels 15 and 90 min after exposure to stress. The results indicate that postnatal exposure to corticosterone leads to increased insulin sensitivity, low body weight with diminished fat depots, leptin and food intake. This suggests that postnatal exposure to corticosterone induces specific programming, with consequences in adult life.

Postnatal endotoxin exposure results in increased insulin sensitivity and altered activity of neuroendocrine axes in adult female rats.

Severe postnatal infection leads to a systemic inflammatory response with release of cytokines and glucocorticoids, representing a stressful event for the newborn child. The purpose of this study was to mimic this situation and to study the effects of early postnatal endotoxin exposure of female rat pups on metabolic, endocrine and anthropometric variables in adulthood. Female pups were given subcutaneous injections of lipopolysaccharides (LPS; Salmonella enteriditis, 0.05 mg/kg) or vehicle 3 and 5 days after birth. Six hours after injection, LPS-treated rats had higher corticosterone levels than controls. As adults, LPS-exposed female rats showed increased insulin sensitivity (P<0.05), measured with the hyperinsulinemic euglycemic clamp (5 mU/kg per min). They exhibited a higher locomotor activity (P<0.05) and increased skeletal muscle mass in comparison with controls (P<0.05). Basal ACTH and corticosterone levels in LPS-treated rats were elevated (P<0.05), as were corticosterone levels after exposure to a novel environment stress (P<0.05). The adrenals were morphologically changed and enlarged (P<0.05) in LPS-exposed rats at 11 weeks of age, and a higher density of hypothalamic but not hippocampal glucocorticoid receptor protein was found in the LPS-treated rats (P<0.05). Furthermore, circulating progesterone levels were lower (P<0.05) and testosterone tended to be higher. The results indicate that postnatal exposure to LPS leads to increased insulin sensitivity in the adult female rat. In addition, LPS-treated rats showed changes in the regulation of hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes. This study suggests that postnatal exposure to an endotoxin such as LPS can induce specific programming of neuroendocrine regulation, with long-term consequences in adult life.

TCDD-mediated oxidative stress in male rat pups following perinatal exposure.

2,3,7,8-tetrachlorododibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known. Exposure to TCDD has been shown to cause oxidative stress in a variety of animal models. In this study, pregnant Long Evans rats were dosed with 1 microg TCDD/kg on gestational day (GD) 15 so as to investigate oxidative stress in the liver of male pups following gestational exposure to TCDD. Lipid peroxidation (TBARS), production of reactive oxygen species (ROS), and total glutathione (GSH) were assayed to identify changes in oxidative stress parameters in the pup liver at GD 21 and postnatal days (PND) 4, 25, 32, 49, and 63. Mean ROS levels in pups were elevated at all time points tested with a significant elevation at PND 4 and PND 25. However, pup hepatic lipid peroxidation was unchanged throughout the time course. In addition, hepatic total GSH levels were not significantly changed although the means for the TCDD-treated groups were less than those of the controls at all time points except PND 49. The results indicate that although the levels of ROS are increased following gestational/lactational exposure, this increase does not translate to direct oxidative damage or significant changes to endogenous antioxidant defense mechanisms. Further investigation into the effect of gestational/lactational exposure in pups should include additional endpoints for further characterization of the time course of the response, the effect upon extrahepatic tissues, and investigation of differences between male and female offspring.

HYPEROXIA INCREASES PROTEIN MASS OF 5-LIPOXYGENASE AND ITS ACTIVATING PROTEIN, FLAP, AND LEUKOTRIENE B 4 OUTPUT IN NEWBORN RAT LUNGS

In this study, the authors examined in newborn rat lung tissues the release of leukotriene B 4 (LTB 4) from tissue explants in vitro, the protein expression of the LT-synthesizing enzyme, 5-lipoxygenase (5-LO), and its activating protein (FLAP), and the effects of in vivo hyperoxic exposure (>95% O 2) on these parameters. Basal LTB 4 output increased from 0.98 ng/mgDNA/30 min at day 1 to 3.3 ng/mgDNA/30 min at day 28 (P <. 05). Exposure of rat pups to >95% O 2 from days 1 to 7 and 60% O 2 from days 7 to 28 stimulated a 1.6-fold (P <. 05) increase in LTB 4 output, compared to normoxic pups (to 1.6 ng/mgDNA/30 min) by day 1 and on day 7. The calcium ionophore, A23187, caused an increase in LTB 4 output from both exposure groups, but LTB 4 output was consistently greater (P <. 05) from hyperoxia-exposed pups. Western immunoblotting of lung tissue showed that 5-LO and FLAP protein mass increased (P <. 05) from days 4 to 14. Hyperoxia exposure increased the mass of both proteins (P <. 05). Immunohistochemistry localized 5-LO and FLAP mainly to alveolar macrophages on day 14, but some staining was evident in parenchymal tissue. These data show that hyperoxia increases LTB 4 output, as well as protein levels of 5-LO and FLAP, in newborn rat lungs during early postnatal life. Elevated LTB 4 may contribute to the etiology of newborn lung disease.

Effects of prenatal exposure to deltamethrin on forced swimming behavior, motor activity, and striatal dopamine levels in male and female rats

The effects of prenatal exposure of rat pups to 0.08 mg/kg deltamethrin (DTM) on physical, reflex and behavioral developmental parameters, on forced swimming and open-field behaviors, and on striatal monoamine levels at 60 days of age were observed. Maternal and offspring body weight, physical and reflex development were unaffected by the exposure to the pesticide. At 21 days of age, open-field locomotion frequency and immobility duration of male and female offspring were not different between control and exposed animals. However, male rearing frequency was increased in experimental animals. A decreased immobility latency to float and in general activity after the swimming test in male offspring was observed at adult age; no interference was detected in the float duration during the swimming test. In addition, these animals presented higher striatal 3,4-dihydroxyphenylacetic acid (DOPAC) levels without modification in dopamine (DA) levels and an increased DOPAC/DA ratio. These data indicate a higher activity of the dopaminergic system in these animals. Noradrenaline (NA) levels were increased, while MHPG levels were not detectable in the system studied. Serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels, as well as the homovanillic acid (HVA)/DA ratio, were not modified by the exposure to the pesticide. No changes were observed in swimming and open-field behaviors nor were there any changes in striatal monoamines or their metabolites in the female experimental group. In relation to the pesticide formula, the present data showing that prenatal exposure to DTM alters latency to float and the activity of striatal dopaminergic system might reflect a persistent effect of the pesticide on animal motor activity, mainly in males. On the other hand, the decrease in general activity observed in experimental male rats suggests higher levels of emotionality induced by previous exposure to the swimming behavior test in relation to control animals. Data gathered in the present study may be important for the assessment of the safety of pyrethroid insecticides.

Timing of hyperoxic exposure during alveolarization influences damage mediated by leukotrienes.

Hyperoxic exposure of rat pups during alveolarization (postnatal days 4-14) severely retards alveolar development. Some aspects of this inhibition are mediated by leukotrienes (LTs) and may be time sensitive. We determined 1) the effects of exposure to hyperoxia (O(2)) during discrete periods before and during alveolarization on developing alveoli and 2) whether a relationship exists between O(2) and LTs in these periods. Pups were exposed to >95% O(2) from days 1 to 4, 4 to 9, 9 to 14, or 4 to 14 in the absence and presence of the LT synthesis inhibitor MK-0591. Both the level of in vitro lung tissue LT output on days 4, 9, and 14 and the degree of alveolarization on day 14 were determined. Pups exposed to O(2) from days 4 to 9 had a more profound inhibition of alveolarization on day 14 compared with those exposed to O(2) from days 1 to 4 or 9 to 14. Peptido-LT levels were significantly higher in pups exposed to O(2) on days 9 and 14 compared with pups in air and returned to normal once normoxia was restored. LT inhibition from days 4 to 14, 4 to 9, or 9 to 14 in pups exposed to O(2) from days 4 to 14 prevented the O(2)-induced inhibition of alveolarization. These data suggest that developing alveoli are sensitive to LTs shortly before and after day 9, significantly retarding certain parameters of alveolarization on day 14. We conclude that some of the effects of O(2) are not uniform throughout different stages of alveolarization and that this is likely related to the timing of LT exposure.

Age-dependent induction of aberrant crypt foci in rat colon by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine and azoxymethane.

Pups and adult rats received seven oral exposures (three time weekly) of the food mutagen PhIP (50 mg/kg), or two subcutaneous exposures (once weekly) of the experimental carcinogen azoxymethane (3.75 mg/kg). Aberrant crypt foci (ACF) were scored 8 weeks after the first exposure. In addition, lactating dams with suckling pups were orally exposed to 50 mg/kg of PhIP, three times weekly for three weeks. Direct PhIP exposure of pups induced 2.2 times more ACF than similar exposure of adult rats (2.0+/-0.0 versus 0.9+/-0.8, P<0.05). The growth of ACF, expressed as crypt multiplicity AC/ACF, was 3.5 times larger in neonatally exposed rats than in rats exposed in adulthood (8.0+/-7.3 versus 2.3+/-1.6, P<0.05). PhIP exposure via breast milk induced ACF in 3 of 25 animals. However, the difference versus controls, which had no ACF, did not reach statistical significance. Contrary to PhIP, azoxymethane induced more ACF in adult rats than in pups (2.8+/-1.9 versus 4.8+/-1.7, P<0.05). Similarly to PhIP however, azoxymethane induced 3.2 times larger ACF (AC/ACF) in pups than in adult rats (11.9+/-8.4 versus 3.7+/-1.9, P<0.001). Whereas no PhIP-induced ACF (0/15) were observed in the lymphoid follicles, approximately 60% of the azoxymethane-induced ACF (32/56) were located in these structures. This difference was statistically significant (P<0.001). The density of azoxymethane-induced ACF was 80 times larger in the lymphoid follicles than in the surrounding mucosa (P<0.01). Based on the assumption that the formation of ACF with high multiplicity is predicative for the tumour development we conclude that neonatal rats are more susceptible to PhIP and azoxymethane than adult rats.

Testicular abnormalities in male rats after lactational exposure to nonylphenols.

Lactational exposure of male rat pups to nonylphenols (NPs) decreased the size of their testes and male accessory glands. At 31 d of age, NP-treatment of male rats resulted in less cellular differentiation of the seminiferous tubules (STs) and increased intertubular space compared to controls. At maturity, NP-treated males showed varying degrees of abnormalities in the affected testes. In the moderately affected ones, about 20-30% of their STs had poorly differentiated germinal elements. Cell lineage was less organized. In extreme cases, all STs of the affected testis failed to differentiate into germinal elements. These abnormalities in germinal element differentiation might be the primary cause for a number of the NP-treated males having a lower epididymal sperm count and a lower percentage of motile sperm compared to age-matched control males. Zymogram analysis of testis homogenates by sodium dodecyl sulfate gelatin gels revealed two major forms (64-66 kDa and 50-52 kDa) of gelatinases. Only the 50-52-kDa form was greatly reduced or absent in the affected testis. Lactational exposure of male pups to NPs thus leads to various testicular abnormalities including lack of differentiation of STs, lowering of sperm count, and reduction in the percentage of motile sperm and modulation of a specific form of testicular proteinases.

Effects of hyperoxia on nitric oxide synthase expression, nitric oxide activity, and lung injury in rat pups.

Although hyperoxic exposure is an important contributor to the development of bronchopulmonary dysplasia and nitric oxide (NO) has been implicated in the pulmonary response to oxygen, the role of NO in mediating chronic neonatal lung injury is unclear. Therefore, rat pups were exposed to normoxia or hyperoxia (>95% O2) from d 21 to 29. After the rats were killed, their lungs were removed for analysis of nitric oxide synthase (NOS) expression, NO activity as measured by 3',5'-cyclic guanosine monophosphate (cGMP) assay, and lung pathology. Hyperoxia caused 5-fold and 2-fold increases in inducible (i) NOS and endothelial (e) NOS levels, respectively. NO activity was assessed by measuring cGMP levels after normoxic or hyperoxic exposure in the presence and absence of NOS blockade with either aminoguanidine (AG) or Nomega-nitro-L-arginine (L-NNA). cGMP levels were elevated in hyperoxic versus normoxic rats (287+/-15 versus 106+/-9 pmol/mg protein, respectively, p < 0.001), and this increase in cGMP was attenuated after NOS blockade with either AG or L-NNA. Hyperoxic exposure significantly increased lung/body weight ratios and induced histologic changes of interstitial and alveolar edema; however, these hyperoxia-induced histologic changes were not altered by NOS blockade with AG or L-NNA. We conclude that hyperoxic exposure of rat pups up-regulated both iNOS and eNOS and increased NO activity as measured by cGMP levels derived from both iNOS and eNOS. Blockade of NOS reduced cGMP levels in the hyperoxic rat pups; however, it did not seem to reverse the pathologic consequences of hyperoxic exposure.

Regulation of hypothalamic arginine vasopressin messenger ribonucleic acid and pituitary arginine vasopressin content in fetal sheep: Effects of acute tonicity alterations and fetal maturation

Objective: Fetal arginine vasopressin contributes to fetal and amniotic fluid homeostasis by increasing water resorption in the kidney and, at higher plasma levels, circulatory homeostasis by vasopressor effects. In utero and neonatal exposure of rat pups to prolonged alterations in plasma osmolality may permanently alter (imprint) pituitary arginine vasopressin content and adult responses to osmotic challenges. Our objective was to investigate fetal developmental changes and the impact of maternal dehydration and maternal hyponatremia on fetal pituitary arginine vasopressin content and hypothalamic arginine vasopressin messenger ribonucleic acid expression. Study Design: Ten pregnant ewes with singleton fetuses (135 ± 1 day) were chronically prepared with maternal vascular catheters. Ewes were assigned to receive water deprivation (n = 4) [desamino, d -Arg8]-arginine vasopressin–induced plasma hyponatremia (n = 3), or 4 days of observation (n = 3). Three additional pregnant ewes with preterm (110 ± 1 day) singleton fetuses were also included for a study of maturational effects. Daily maternal blood samples were analyzed for determination of plasma arginine vasopressin, electrolytes, and osmolality. After the study protocol, fetuses were operatively delivered, umbilical blood samples obtained, and fetuses put to death for pituitary and hypothalamic tissues. Pituitary arginine vasopressin content was determined by radioimmunoassay, and hypothalamus arginine vasopressin messenger ribonucleic acid expression was detected by Northern blotting. Results: Dehydration significantly (P < .05) increased, and hyponatremia significantly decreased maternal plasma sodium concentration compared with controls. Fetal plasma sodium concentration significantly changed in parallel with maternal values (dehydration: 139 ± 1 to 150 ± 1 mEq/L; hyponatremia: 138 ± 1 to 128 ± 5 mEq/L). Fetal hypothalamic arginine vasopressin messenger ribonucleic acid expression and pituitary content did not change in relation to these relatively acute alterations in plasma tonicity. However, among all animals, arginine vasopressin messenger ribonucleic acid expression was significantly negatively correlated with pituitary arginine vasopressin content (r2 = 0.563; P = .02). Arginine vasopressin messenger ribonucleic acid expression was significantly lower in both preterm and near-term fetuses (P < .05) than that in the maternal ewe, although pituitary arginine vasopressin content (in micrograms per milligram of protein) was significantly greater in preterm fetuses (P < .01, vs maternal; P < .05, vs near term). Conclusions: The significant inverse relation between arginine vasopressin content and arginine vasopressin messenger ribonucleic acid suggests a dynamic arginine vasopressin synthesis–content feedback relationship is functional in the near-term fetus. Although relatively acute periods of maternal hypertonicity or hypotonicity do not alter fetal pituitary arginine vasopressin content or hypothalamic arginine vasopressin messenger ribonucleic acid expression, longer-term plasma tonicity alterations may potentially have an impact on the fetal arginine vasopressin hypothalamic-pituitary axis. (Am J Obstet Gynecol 1998;179:899-905.)

P1E134 - On the characteristic features of behavioural teratogenicity after prenatal aluminum exposure in rat pups

P1E134 - On the characteristic features of behavioural teratogenicity after prenatal aluminum exposure in rat pups

Thyroid hormones as biomarkers in grey seals

Changes in grey seal ( Halichoerus grypus) serum thyroid hormones with age, sex and during lactation were investigated. Concentrations were related to polychlorinated biphenyl (PCB) uptake and exposure in pups and females. There was no effect of sex or stage of lactation on either free or total thyroxine (T 4) and triiodothyronine (T 3), but significant age-related variability was seen. Pups had significantly higher concentrations of total T 4 than adults ( p <0.0001) and serum levels declined with age. In general, there was no significant relationship between PCB exposure to pups through female milk or between PCB blubber concentrations and thyroid hormone levels, when the effect of age was controlled for. However, the ratio T 3:T 4 was significantly correlated with the concentration of one congener, CB169 ( p = 0.048), when stage of lactation was used as a covariate. In conclusion, serum thyroid hormone levels may be less useful biomarkers of contaminant exposure in seals than other biochemical measures. Although concentrations vary predictably with age, other factors, such as condition and nutritional status, may compromise their usefulness in assessing contaminant-induced hormone disruption in pinnipeds.

Stress inhibits the proliferation of granule cell precursors in the developing dentate gyrus

The granule cell population of the dentate gyrus is produced predominantly during the postnatal period in rats. Previous studies have shown that experimental increases in the levels of adrenal steroids suppress the proliferation of granule cell precursors during the first postnatal week, the time of maximal neurogenesis in the dentate gyrus. These findings raise the possibility that stressful experiences that elevate adrenal steroid levels may inhibit the production of granule neurons, and thus alter the development of the dentate gyrus. To test this possibility, we exposed naive rat pups to the odors of a known predator, adult male rats, and examined both plasma corticosterone levels and the number of [3]H-thymidine labeled cells in the dentate gyrus. A single exposure of rat pups to adult male rat odor elevated corticosterone levels immediately and diminished the number of [3]H-thymidine labeled cells in the granule cell layer by 24 h later. These results suggest that stressful experiences suppress the production of granule neurons in the developing dentate gyrus.

ENDOCRINE MECHANISMS UNDERLYING THE GROWTH EFFECTS OF DEVELOPMENTAL LEAD EXPOSURE IN THE RAT

A dose-response study was conducted to examine the growth suppression associated with developmental lead exposure in a rat model and to determine the endocrine mechanisms underlying these effects. Ad libitum intake of lead acetate (0.05% to 0.45% w/v) was initiated in time-impregnated female Sprague-Dawley rats (n = 10-15/ group) at gestational day 5. At birth, pups were culled to four male and four females per litter. Lead exposure of dams continued until weaning, following which lead exposure of pups was continued until sacrifice at age 21, 35, 55, and 85 days. Birth weight and prepubertal and pubertal growth rates were significantly suppressed. Growth rates were suppressed to a much greater degree in male as compared to female pups. Decreased growth rates were accompanied by a significant decrease in plasma insulin-like growth factor 1 (IGF1) concentrations and (1) a significant increase in pituitary growth hormone (GH) content during puberty in pups of both sexes, (2) a delay in the developmental profiles of the GH-dependent male-specific liver enzymes cytochrome P-450 CYP2C11 and N -hydroxy-2-acetylaminofluorene sulfotransferase, and (3) continued suppression of these enzymes in lead-exposed adult male pups. In addition, significant decreases in plasma sex steroids, testosterone (male) and 17 -estradiol (female), were observed during puberty. Postpuberty, at age 85 d, both IGF1 and sex steroid levels were indistinguishable from control pups despite continued lead exposure. Growth rates were also similar in control and lead-exposed pups between age 57 and 85 d. Data suggest that the mechanism underlying lead-induced sex-independent suppression of growth observed in these studies involves disruption of GH secretion during puberty. It is possible that the mechanisms underlying the greater suppression of somatic growth observed at puberty in lead-exposed male offspring may be due to the additional hypoandrogenization produced by the action of lead on the hypothalamic-pituitary-testicular axis.

Oxygen has an Indirect Effect on Alveolar Development that is Mediated by Leukotrienes (LTs). † 1938

We have previously shown that exposure of rat pups to >95% O2 during alveolarization (postnatal d4-14) causes an increase in LT production along with severe inhibition of alveolarization measured on d14. Preventing the formation of LTs using the FLAP inhibitor, MK-0591, during O2 exposure (d4-14) allows alveolar development to occur normally. We wanted to investigate whether or not there was a critical period during alveolarization for the O2 effect on the developing lung and if so, whether this could be connected with a critical period for LT effects.

DEVELOPMENT OF AN INHALATION SYSTEM FOR THE SIMULTANEOUS EXPOSURE OF RAT DAMS AND PUPS DURING DEVELOPMENTAL NEUROTOXICITY STUDIES

Due to mass flux constraints, the exposure of pups along with dams to particles cannot be easily done in traditional whole-body inhalation systems. Typical inhalation developmental neurotoxicity studies rely on maternal separation during exposure, resulting in pup stress that may alter behavioral development, thereby confounding study results. We developed an individual-rat, whole-body inhalation exposure cylinder that allows for the simultaneous exposure of rat dams and their pups to vapors, aerosols, and particles. We evaluated pup growth and development following gestational and lactational 6-h/ day exposures to HEPA-filtered air using the novel exposure cylinder, in which dams and pups were maintained together, versus rat pups for which only the dams were exposed or control animals that remained in the domiciliary cage with the dams. Rat pups that were separated from their dams during neonatal exposure to HEPA-filtered air demonstrated significant delays in several developmental landmarks such as surface righting, negative geotaxis, and eye opening, as well as decreased terminal body and brain weights. Increased spontaneous motor activity was observed on postnatal day (PND) 13, 17, and 60 in pups that were separated from their dams. Malnutrition is the likely cause for the observed hyperactivity and delayed development. We then compared maternal blood levels of tertiary amyl methyl ether (TAME) and its metabolite tertiary amyl alcohol (TAA) in the 2 exposure systems following exposure to 500 ppm TAME for 6 h. Dams exposed in the novel exposure cylinder had higher blood levels of TAME compared with those exposed in stainless steel caging. We also compared levels of particulate manganese phosphate within the novel exposure cylinder to determine if levels achieved within the exposure cylinder were similar to levels generated within a 1-m3 chamber. The novel exposure cylinder is an ideal system for exposure of dams and pups to particles, due to the high horizontal flow rates, which result in an adequate mass flux flowing past the breathing zone of the animals with retained normal growth and development.

Neonatal treatment with tamoxifen causes immediate alterations of the sexually dimorphic nucleus of the preoptic area and medial preoptic area in male rats.

Tamoxifen is an antiestrogen widely used for the treatment of breast cancer. Current evolutions in preventive strategies to include healthy premenopausal women warrant the study of its developmental toxicity. Perinatal treatment of male rodents with tamoxifen caused reproductive tract lesions and sexual behavior deficits similar to those induced by diethylstilbestrol (DES). Those abnormalities could originate, at least in part, from lesions of the hypothalamic-pituitary axis. The initial alterations caused by tamoxifen in the hypothalamic medial preoptic area (MPOA) and sexually dimorphic nucleus of the preoptic area (SDN-POA) were studied in 6-day-old male rat pups treated with 100 micrograms tamoxifen (group 1), 1 microgram DES (group 2) or vehicle (group 3) from PN1 to 5. In situ hybridization was performed to analyze the expression of the GAP-43 gene, a marker of neuronal differentiation, and morphometry was used to study the neuronal density in the SDN-POA and MPOA and the volume and number of neurons in the SDN-POA. Tamoxifen reduced severely the volume and neuron numbers in the SDN-POA (46.1% and 47.8% of controls, respectively). The neuronal density of the MPOA was not modified. GAP-43 gene expression was decreased as demonstrated by a greater percentage of unlabeled neurons (grade 0) mirrored by a lesser percentage of intensely labeled ones (grade 2) in the SDN-POA and MPOA. In contrast to the effects of the antiestrogen, DES did not affect the above endpoints. These data indicated that developmental exposure of male rat pups to tamoxifen-induced immediate neuronal loss in one and altered differentiation in two hypothalamic areas crucial to reproduction. How those initial alterations contribute to the pathogenesis of the reproductive disorders observed in the adult male needs further investigation.

Cochlear integrity in rats with experimentally induced audiogenic seizure susceptibility

While chronic susceptibility of rodents to audiogenic seizures (AGSs) is often accompanied by cochlear lesions, it has not been demonstrated whether cochlear hair cell losses are essential to pathogenesis in this epileptic disorder. An alternative possibility is that the neonatal timing of hearing losses is what unites various models of chronic AGS susceptibility. In the latter case, either transient or permanent hearing losses might induce susceptibility as long as they concur with a critical period of development. To address this issue, it was examined whether lesions were universally present in cochleas of adult rats after having been made susceptible to sound-triggered seizures by different types and severities of neonatal auditory trauma. Neonatal treatments included: (1) an 8 min exposure of rat pups to intense noise (125 dB SPL) on postnatal day (PND) 14; (2) injections of low doses of kanamycin (KM: 100 mg/kg) on PNDs 9–12; or (3) injections of high doses of KM (500 mg/kg) on PNDs 9–12. As adults, rats in all experimental groups, but not in sham-treated groups, exhibited sound-triggerable seizure responses. Nonetheless, this outcome did not depend on integrity of cochleas. Hair cells were rarely missing in the cochleas of noise-exposed, low-dosage KM-treated, or sham-treated rats. By contrast, all inner and outer hair cells were missing from the basal 75% of cochleas of adult rats which had been treated with high-dose KM on PNDs 9–12. Results indicate that cochlear lesions are not requisite for the induction or expression of AGS susceptibility. At the same time, however, significant hair cell losses do not necessarily preclude susceptibility. It appears that the neonatal timing rather than the permanence of hearing losses may be what engenders chronic AGS susceptibility.

The Effects of Neonatal Hypoxia on Kindled Seizure Development and Electroconvulsive Shock Profiles

Our previous research indicated that the exposure of rat pups to an hypoxic environment during a discrete developmental period (postnatal days 10-15) produces short-term seizures and confers an enduring increase in susceptibility to pentylenetetrazol- and flurothyl-induced seizures. In this study, we evaluated the effects of hypoxic insult in this neonatal period of susceptibility to electrical kindling and corneal electroconvulsive shock. Ten-day-old rat pups were exposed to a 3% O2 environment, as previously described, and were either kindled or exposed to corneal electroshock at adulthood (70 days old). Neither kindled seizure development from the septal nucleus or amygdala nor electroconvulsive shock profiles were significantly altered by hypoxic pretreatment. Results indicate that hypoxia produces increases in seizure susceptibility that are observable in only some experimental seizure models but not in others. This outcome serves to target some anatomic systems more than others in the mechanisms involved in hypoxia-induced neural reorganization.