Stressed Pups Research Articles

Prenatal restraint stress affects early neurobehavioral response and oxidative stress in mice pups.

Prenatal stress (PS), in both humans and animals, presents a potential risk to the mother and her fetus throughout gestation. PS is always associated with physiological changes that alter embryonic development and predispose the individual to lifelong health problems, including susceptibility to mental illness. This study aims to identify the harmful effects of prenatal restraint stress (PRS), commonly employed to induce stress painlessly and without any lasting debilitation during gestation. This stress is applied to pregnant Swiss albino mice from E7.5 to delivery for three hours daily. Our results show that PS affects dams' weight gain during the gestational period; moreover, the PS dams prefer passive nursing, exhibit a lower percentage of licking and grooming, and impair other maternal behaviors, including nesting and pup retrieval. Concerning the offspring, this stress induces neurobehavioral impairments, including a significant increase in the time of recovery of the young stressed pups in the surface righting reflex, the latency to avoid the cliff in the cliff avoidance test, longer latencies to accomplish the task in negative geotaxis, and a lower score in swimming development. These alterations were accompanied by increased Malondialdehyde activity (MDA) at PND17 and 21 and downregulation of AchE activity in the whole brain of pups on postnatal days 7 and 9. These findings demonstrated that PS causes deleterious neurodevelopmental impairments that can alter various behaviors later in life.

Prenatal administration of heparin-binding epidermal growth factor-like growth factor in an experimental model of necrotizing enterocolitis decreased both incidence and severity of the disease.

Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of death in premature infants and causes long-term disabilities. Previously, enteral heparin-binding epidermal growth factor-like growth factor (HB-EGF) administered after birth demonstrated decreased incidence and severity of NEC in a neonatal animal model of NEC. We investigated the potential prophylactic strategy of preventing NEC using prenatally administered HB-EGF. An HB-EGF (800 µg/kg/dose) dose was injected into pregnant rats via tail vein or intraperitoneal route 2 hours prior to delivery. After cesarean section (C-section) at 21 days' gestation, the rat pups were subjected to the NEC protocol by inducing stressors: hypoxia, hypothermia, hypertonic feeds, and orogastric gavage of lipopolysaccharide (2 mg/kg). Postnatally, pups were monitored for 96 hours and assessed for the development of clinical and postmortem histological NEC. The experimental NEC incidence in untreated, stressed rat pups was 66%. Compared with untreated pups, the maternal administration of HB-EGF correlated with a significant NEC incidence and severity decrease in rat pups. The strongest decrease was seen when HB-EGF was administered via the intraperitoneal route 2 hours prior to C-section (66% vs 31%, *p<0.05). Prenatal HB-EGF administration significantly increased pups' survival after NEC protocol exposure, with the greatest benefit observed in the group that received HB-EGF intraperitoneally 2 hours before delivery. Prenatal administration of HB-EGF decreases the incidence and severity of NEC, preserves gut barrier function and increases survival. This may represent a novel prophylactic clinical strategy for NEC offered to mothers at risk of delivering a premature infant.

356 Microbial origins in the developmental programming of offspring health

Abstract Adverse parental lifetime experiences, such as stress and malnutrition, are implicated in the risk for offspring metabolic, cardiovascular, and neurodevelopmental disorders. In recent years, the maternal microbiome has emerged as an important factor that affects maternal health and infant development during pregnancy and beyond. Using mouse models of maternal stress experience during pregnancy, we examined the hypothesis that maternal stress experience alters the composition of the maternal microbiota and this altered community is transmitted to offspring to mediate effects of prenatal stress. As maternal stress exposure exhibits disruptive effects on both in utero environment and maternal microbiota, it has been difficult to assess the mechanistic involvement of the maternal microbiome to the prenatal stress phenotype independent of stress effects on the fetal environment. To circumvent this, we developed a maternal microbiota transplantation method in which embryonic day 18.5 mouse pups were delivered by caesarean section, thereby preventing natural colonization, and then transplanted with various maternal microbiota communities via orogastric gavage. Transplantation of maternal microbiota from stressed dams into naïve pups delivered by caesarean section recapitulated phenotypes that resembled those seen in prenatally stressed males, including reduced body weight and increased neuroendocrine response to acute stressors. However, transplantation of control maternal vaginal microbiota into prenatally stressed pups delivered by caesarean section did not rescue the prenatal stress phenotype. We showed that the inability to rescue the prenatal stress phenotype is related to transcriptional reprogramming to pathways involved in the regulation of innate immunity in the fetal gut and brain prior to birth and colonization by maternal microbiota. These results are interesting in light of recent studies demonstrating a critical role of the maternal microbiome on homeostasis of immune cell populations in the offspring brain. As an important requirement for normal growth and development, metabolites produced by the maternal gut microbiota cross the placental barrier and gain access to fetal circulation. Thus, we examined the hypothesis that maternal stress experience during pregnancy significantly alters the availability of maternal gut microbiota-derived metabolites, thereby shifting the availability of these metabolites required for the developing brain. Metabolomic profiling of the maternal compartment and fetal brain showed stress-induced reduction in a class of microbiota-derived metabolites involved in immune homeostasis and chromatin remodeling. Indeed, functional profiling of immune populations in the fetal brain revealed an association between reduced metabolite availability and increased infiltration of proinflammatory monocytes in the fetal brain. Further, as the gut microbiome is fairly accessible within clinical settings, we are now also asking how our mouse model translates to humans in a cohort of pregnant women exposed to childhood adversity. Collectively, our results suggest that intergenerational transmission of stress exposure occur via the maternal microbiota through two novel mechanisms. First, stress alterations during pregnancy impact the available pool of maternal gut microbiota-derived metabolites necessary for normal prenatal growth and development. Second, transmission of stress-altered vaginal microbiota may alter critical immune and metabolic processes underlying neurodevelopment.

Prenatal stress increased γ2 GABAA receptor subunit gene expression in hippocampus and potentiated pentylenetetrazol-induced seizure in rats.

Objective(s):Stress during pregnancy is able to bring extensive effects on neurobehavioral development in offspring. The GABAergic system plays a pivotal role in neuronal excitability, which can be affected by prenatal stress (PS). This study aimed to evaluate impact of the PS on γ2 subunit of gamma-aminobutyric acid A (GABAA) receptor gene expression in the hippocampus and seizure induced by pentylenetetrazol (PTZ) in developing rats.Materials and Methods:In this experimental study, female Wistar rats were exposed to restraint stress during gestation and their offspring were studied on postnatal days 14 and 21 (P14 and P21, respectively) for epileptic behaviors and γ2 GABAA receptor subunit gene expression. Quantitative real-time PCR was used for evaluating the γ2 GABAA receptor subunit gene expression in rat pups. Meanwhile, PTZ was injected into the pups, and seizure behaviors were recorded for 60 min. Results:The results showed that γ2 subunit mRNA expression significantly increased in the hippocampus of the stressed pups. The expression level of γ2 subunit was higher on P21 compared to that on P14 in both groups. Number of seizures with tonic–clonic features increased in pups of stressed group compared to the control group. Prenatal stress significantly caused an increase in the total score of seizure on P21.Conclusion:The effect of PS on seizure susceptibility is age-specific; the increased γ2 subunit level in the hippocampus might be, at least in part, the underlying mechanism for PS-induced augmentation of seizures in immature rats.

Early life stress and high fat diet: From comfort food to nutritional stress

The perinatal period is a critical period during which the quality of the environment has long lasting effects on adult phenotype. Early adversity in childhood has been associated with higher risks to develop neuropsychiatric disorders later in life. Nowadays, modern societies are characterized by a high consumption of fat. Although these changes also affect nutritional environment during the perinatal period, their impacts on brain functioning and behavior remain still unknown. Our study is aiming to determine the programming effects of maternal high-fat diet (HFD, western diet) and early stress (maternal separation) on offspring behaviour in particular emotional behavior, cognition and food intake. Our results reveal that maternal separation enhances motivation for palatable food and combined with maternal HFD exacerbates vulnerability to diet induced obesity. In contrast, maternal HFD protects the offspring against the emotional and cognitive impairments induced by maternal separation. This protective effect of maternal HFD could be related to the increase of maternal care in stressed dams exposed to HFD and to the attenuation of neurodevelopmental alterations in the prefrontal cortex of stressed pups. In conclusion, our work demonstrates that stress and nutrition during early life interact and influence later adult phenotype.

Early-life stress, microbiota, and brain development: probiotics reverse the effects of maternal separation on neural circuits underpinning fear expression and extinction in infant rats

Early-life stress has pervasive, typically detrimental, effects on physical and mental health across the lifespan. In rats, maternal-separation stress results in premature expression of an adult-like profile of fear regulation that predisposes stressed rats to persistent fear, one of the hallmarks of clinical anxiety. Probiotic treatment attenuates the effects of maternal separation on fear regulation. However, the neural pathways underlying these behavioral changes are unknown. Here, we examined the neural correlates of stress-induced alterations in fear behavior and their reversal by probiotic treatment. Male Sprague-Dawley rats were exposed to either standard rearing conditions or maternal-separation stress (postnatal days [P] 2–14). Some maternally-separated (MS) animals were also exposed to probiotics (Lactobacillus rhamnosus and L. helveticus) via the maternal drinking water during the period of stress. Using immunohistochemistry, we demonstrated that stressed rat pups prematurely exhibit adult-like engagement of the medial prefrontal cortex during fear regulation, an effect that can be prevented using a probiotic treatment. The present results add to the cross-species evidence that early adversity hastens maturation in emotion-related brain circuits. Importantly, our results also demonstrate that the precocious neural maturation in stressed infants is prevented by a non-invasive probiotic treatment.

The maternal vaginal microbiome partially mediates the effects of prenatal stress on offspring gut and hypothalamus.

Early prenatal stress disrupts maternal-to-offspring microbiota transmission and has lasting effects on metabolism, physiology, cognition, and behavior in male mice. Here we show that transplantation of maternal vaginal microbiota from stressed dams into naive pups delivered by cesarean section had effects that partly resembled those seen in prenatally stressed males. However, transplantation of control maternal vaginal microbiota into prenatally stressed pups delivered by cesarean section did not rescue the prenatal-stress phenotype. Prenatal stress was associated with alterations in the fetal intestinal transcriptome and niche, as well as with changes in the adult gut that were altered by additional stress exposure in adulthood. Further, maternal vaginal transfer also partially mediated the effects of prenatal stress on hypothalamic gene expression, as observed after chronic stress in adulthood. These findings suggest that the maternal vaginal microbiota contribute to the lasting effects of prenatal stress on gut and hypothalamus in male mice.

Sex-Specific Consequences of Neonatal Stress on Cardio-Respiratory Inhibition Following Laryngeal Stimulation in Rat Pups.

The presence of liquid near the larynx of immature mammals triggers prolonged apneas with significant O2 desaturations and bradycardias. When excessive, this reflex (the laryngeal chemoreflex; LCR) can be fatal. Our understanding of the origins of abnormal LCR are limited; however, perinatal stress and male sex are risk factors for cardio-respiratory failure in infants. Because exposure to stress during early life has deleterious and sex-specific consequences on brain development it is plausible that respiratory reflexes are vulnerable to neuroendocrine dysfunction. To address this issue, we tested the hypothesis that neonatal maternal separation (NMS) is sufficient to exacerbate LCR-induced cardio-respiratory inhibition in anesthetized rat pups. Stressed pups were separated from their mother 3 h/d from postnatal days 3 to 12. At P14–P15, pups were instrumented to monitor breathing, O2 saturation (Spo2), and heart rate. The LCR was activated by water injections near the larynx (10 µl). LCR-induced apneas were longer in stressed pups than controls; O2 desaturations and bradycardias were more profound, especially in males. NMS increased the frequency and amplitude of spontaneous EPSCs (sEPSCs) in the dorsal motor nucleus of the vagus (DMNV) of males but not females. The positive relationship between corticosterone and testosterone observed in stressed pups (males only) suggests that disruption of neuroendocrine function by stress is key to sex-based differences in abnormal LCR. Because testosterone application onto medullary slices augments EPSC amplitude only in males, we propose that testosterone-mediated enhancement of synaptic connectivity within the DMNV contributes to the male bias in cardio-respiratory inhibition following LCR activation in stressed pups.

Hippocampal NR3C1 DNA methylation can mediate part of preconception paternal stress effects in rat offspring

Many studies have been shown that maternal stress during pregnancy and in early life period influences offspring in the behavioral and molecular aspects in human and animal models. Recent research has indicated that the environmental condition of males before conception has effects on next generations. We evaluated whether preconception paternal stress (PPS) could influence on hippocampal glucocorticoid receptor gene (GR), (NR3C1) expression, corticosterone response and behavioral outcomes of their offspring. For this purpose, adult male rats were subjected to daily 10min session of forced swimming for 21 consecutive days. Then, two parental breeding groups were formed: stressed father (SF) and non-stressed father (NSF) or control group. 30-day-old pups were tested for anxiety-like behavior by using the elevated plus maze (EPM). Serum corticosterone level was also measured by ELISA. Hippocampal NR3C1 DNA methylation, gene and protein expression were respectively assayed by methylation sensitive restriction enzymes (Real Time PCR), Reverse Transcriptase PCR (RT-PCR) and Western-blotting in all groups. More anxiety-related behavior, serum corticosterone concentration, DNA methylation levels of NR3C1 and lower expression of this gene were significantly observed in paternally stressed pups compared to control pups. As well, molecular changes were more pronounced in male pups compared to female pups. Our results revealed that paternal stress prior to conception has a negative effect on molecular, hormonal and behavioral outcomes in their offspring.

Prenatal Exposure to Continuous Constant Light Alters Endochondral Ossification of the Tibiae of Rat Pups

Clinical and experimental studies suggest that prenatal exposure to stress can impact the growth and development of offspring. The effect of prenatal exposure to constant light, applied as a chronic stressor, on endochondral ossification of the tibiae of 3-day-old and 15-day-old pups was histomorphometrically evaluated. Pregnant rats were divided into 2 groups: mothers chronically exposed to a 12:12-hour light/light cycle (LL) and control mothers maintained on a 12:12-hour light/dark cycle on days 10-20 of pregnancy. On postnatal days 3 and 15, the pups were weighed and euthanized. The tibiae were resected and histologically processed to obtain sections for hematoxylin and eosin staining (HE) and tartrate-resistant acid phosphatase (TRAP) histochemistry, in order to perform histomorphometric determinations. The data were statistically analyzed. A significant decrease in hypertrophic cartilage thickness was observed in the tibiae of the 3-day-old (LL: 0.134 ± 0.02 vs. controls: 0.209 ± 0.023 mm; p < 0.01) and 15-day-old (LL: 23.32 ± 3.98 vs. controls: 22.96 ± 1.93 mm; p < 0.05) prenatally stressed pups. The subchondral bone volume was significantly lower in the tibiae of the 3-day-old LL pups (38.83 ± 6.14%) than in the controls (62.83 ± 10.67%; p < 0.01). The decrease in subchondral bone volume and hypertrophic cartilage thickness shows that the normal growth process of the tibia is impaired in prenatally stressed pups.

Prenatal stress is a vulnerability factor for altered morphology and biological activity of microglia cells.

Several lines of evidence suggest that the dysregulation of the immune system is an important factor in the development of depression. Microglia are the resident macrophages of the central nervous system and a key player in innate immunity of the brain. We hypothesized that prenatal stress (an animal model of depression) as a priming factor could affect microglial cells and might lead to depressive-like disturbances in adult male rat offspring. We investigated the behavioral changes (sucrose preference test, Porsolt test), the expression of C1q and CD40 mRNA and the level of microglia (Iba1 positive) in 3-month-old control and prenatally stressed male offspring rats. In addition, we characterized the morphological and biochemical parameters of potentially harmful (NO, iNOS, IL-1β, IL-18, IL-6, TNF-α, CCL2, CXCL12, CCR2, CXCR4) and beneficial (insulin-like growth factor-1 (IGF-1), brain derived neurotrophic factor (BDNF)) phenotypes in cultures of microglia obtained from the cortices of 1–2 days old control and prenatally stressed pups. The adult prenatally stressed rats showed behavioral (anhedonic- and depression-like) disturbances, enhanced expression of microglial activation markers and an increased number of Iba1-immunopositive cells in the hippocampus and frontal cortex. The morphology of glia was altered in cultures from prenatally stressed rats, as demonstrated by immunofluorescence microscopy. Moreover, in these cultures, we observed enhanced expression of CD40 and MHC II and release of pro-inflammatory cytokines, including IL-1β, IL-18, TNF-α and IL-6. Prenatal stress significantly up-regulated levels of the chemokines CCL2, CXCL12 and altered expression of their receptors, CCR2 and CXCR4 while IGF-1 production was suppressed in cultures of microglia from prenatally stressed rats. Our results suggest that prenatal stress may lead to excessive microglia activation and contribute to the behavioral changes observed in depression in adulthood.

The effect of co-administration of restraint stress and morphine in prenatal period and re-exposure to stress at the end of infancy on corticosterone blood levels and pentylentetrazol-induced epileptic behaviors in rat

Stressful events during gestation have important effects on the later physical and mental health of the offspring. The present study aimed to identify effects of co-administration of stress and morphine in prenatal period and re-exposure to stress at the end of infancy on corticosterone blood levels and pentylentetrazol-induced epileptic behaviors in rat. Materials and Methods: Pregnant rats were divided to six groups of control, stress, saline, morphine, stress-saline and stressmorphine. In the stressed group, rats were stressed and held immobile for three consecutive days started on day 15 of pregnancy. The rats in saline and morphine groups received saline and morphine in the same days. In the morphine/saline-stress groups, rats were exposed to stress and received morphine/saline simultaneously. On postnatal day 22, half of the pups re-exposed to stress, and pentylentetrazol-induced epileptic behaviors of each rat were assessed. Blood samples were collected to determine corticosterone level. Results: Latency of first epileptic behavior decreased significantly in stress-morphine group compared to other groups. Re-exposure to stress significantly led to a decrease in number of clonic seizure. There was no significant difference between the experimental groups in duration of clonic attacks and mortality rate. The level of corticosterone showed a significant increase in stressed pups and decreased in morphine group. Conclusion: these results indicated that co-administration of morphine and restraint stress during late pregnancy had profound impact on neurochemical development and might alter vulnerability to PTZ-induced epileptic behaviors. Prenatal stress is more powerful than postnatal stress on influencing neural development and seizure susceptibility in rats

Changes induced by prenatal stress in behavior and brain morphology: can they be prevented or reversed?

This chapter presents a critical analysis of the behavioral alterations reported in the offspring of women exposed to stress and/or depression during pregnancy and the neurochemical and structural changes underlying them. Among the alterations attributed to prenatal stress in humans and experimental rats of both sexes is impaired regulation of the hypothalamic-pituitary-adrenal (HPA) axis, anxiety and exaggerated fear of novelty, and decreased social interaction. Learning and attention deficits are more prevalent in boys and male rats. Fear of novelty and anxiety are associated with enlargement of the amygdala and its corticotropin-releasing factor content, and decreased socialization, with lower oxytocin activity in the amygdala. Learning deficits are associated with a decrease in neurogenesis, dendritic complexity, and spine number in the dorsal hippocampus. Fostering prenatally stressed (PS) pups onto control mothers prevents the dysregulation of the HPA axis and heightened anxiety, indicating a role for postnatal factors in their etiology. By contrast, learning impairment and decreased socialization are not affected by this fostering procedure and are therefore prenatally mediated.In spite of their widespread use in depressed pregnant women, selective serotonin reuptake inhibitor (SSRI) antidepressants do not normalize the behavior of their children. When administered during gestation to stressed rats, SSRIs do not reduce anxiety or learning deficits in their offspring. Moreover, when given to unstressed mothers, SSRIs induce anxiety in the offspring. The detrimental effect of SSRIs may result from inhibition of the serotonin transporter exposing the brain to excess amounts of 5-hydroxytryptamine (5-HT) at a critical time during fetal development.

Behavioural pattern of stressed rat pups in an elevated plus maze following treatment of tablet mentat

Behavioural pattern of stressed rat pups in an elevated plus maze following treatment of tablet mentat

Prenatal effects of buspirone and stress on behavioral reactions in rat pups of different sexes during period of ontogeny with low level of brain serotonin

Previously we revealed for the first time pain response exacerbation caused by inflammation in rats born to dams exposed to stress during pregnancy (prenatal stress). The present study is devoted to tinvestigation of prenatal stress effects on psychoemotional and tonic pain reactions in rat pups during the individual development period that is characterized with a dramatic reduction of the brain serotonin level. Effects of maternal buspirone before stress during pregnancy on functional indices of psychoemotional and tonic pain systems in the offspring were also investigated. Prenatal stress increased the number of pain patterns (flexing + shaking) during different phases of the time-course of formalin-induced pain in females and males to a greater extent in males. Prenatlly stressed rat pups of both sexes failed to show reliable changes in the index of psychoemotional behavior in the forced swim test. With the aim to decrease pain response exacerbation found in prenatally stressed offspring, pregnant dams were exposed to chronic injections of serotoninergic anxiolytic and antidepressant buspirone which is an agonist of 5-HT1A receptors; prenatal effect of buspirone on psychoemotional behavior in prenatally stressed rat pups was also evaluated. Maternal buspirone normalized pain behavior and decreased considerably the time of immobility, the index of depressive behavior in the forced swim test. The present results indicate analgesic and antidepressive effects of maternal buspirone in prenatally stressed 10-day old rat pups and demonstrate sexual dimorphism in effects of prenatal stress on the time-course of formalin-induced pain. Differences in effects of prenatal influences on pain respone during the interphase in males and females indicate earlier maturation of the descending serotonergic inhibitory system of afferent pain signals modulation in males than in females and demonstrate that 5-HT1A receptors are involved in this process.

Blockade of corticotropin‐releasing hormone receptor 1 attenuates early‐life stress‐induced synaptic abnormalities in the neonatal hippocampus

Adult individuals with early stressful experience exhibit impaired hippocampal neuronal morphology, synaptic plasticity and cognitive performance. While our knowledge on the persistent effects of early-life stress on hippocampal structure and function and the underlying mechanisms has advanced over the recent years, the molecular basis of the immediate postnatal stress effects on hippocampal development remains to be investigated. Here, we reported that repeated blockade of corticotropin-releasing hormone receptor 1 (CRHR1) ameliorated postnatal stress-induced hippocampal synaptic abnormalities in neonatal mice. Following the stress exposure, pups with fragmented maternal care showed retarded dendritic outgrowth and spine formation in CA3 pyramidal neurons and reduced hippocampal levels of synapse-related proteins. During the stress exposure, repeated blockade of glucocorticoid receptors (GRs) by daily administration of RU486 (100 µg g(-1) ) failed to attenuate postnatal stress-evoked synaptic impairments. Conversely, daily administration of the CRHR1 antagonist antalarmin hydrochloride (20 µg g(-1) ) in stressed pups normalized hippocampal protein levels of synaptophysin, postsynaptic density-95, nectin-1, and nectin-3, but not the N-methyl-d-aspartate receptor subunits NR1 and NR2A. Additionally, GR or CRHR1 antagonism attenuated postnatal stress-induced endocrine alterations but not body growth retardation. Our data indicate that the CRH-CRHR1 system modulates the deleterious effects of early-life stress on dendritic development, spinogenesis, and synapse formation, and that early interventions of this system may prevent stress-induced hippocampal maldevelopment.

Early Prenatal Stress Increases Body Weight and Reduces Nociception in Adult Male Rats

Aims: Behavioral responses of 3-month-old male pups from female Wistar rats exposed to daily 1 hour or 3 hour restraint stress during the first 7 days of pregnancy were studied by tail flick and formalin test. Methodology: Eighteen mature virgin female albino rats (140g -160g) were randomly allocated in a blinded fashion to 3 groups (n=6 each) and mated. Group 1 rats were the control and did not undergo restraint stress. Groups 2 and 3 rats were restrained for 1 hour and 3 hours respectively during the first 7 days of pregnancy. Six male rats only served purpose of copulation. At 3 month of age, 34 pups consisting of 10, 11 and 13 pups delivered by rats in groups 1, 2 and 3 respectively were randomly selected and studied for nociception. Results:Body weights were higher in both 1 hour and 3 hour prenatally stressed pups compared to that of the control. The latency period during the tail immersion test in the pups prenatally stressed for 3 hour daily but not those stressed for 1 hour daily was significantly higher (p<0.05) compared to that of control. While there were no significant differences in the formalin score in pups prenatally stressed for 1 hour and 3 hour compared to the score of the control during the ea rly phase, the formalin score of the pups OriginalResearch Article

MRI of neonatal necrotizing enterocolitis in a rodent model

Neonatal necrotizing enterocolitis (NEC) is a poorly understood life-threatening illness afflicting premature infants. Research is hampered by the absence of a suitable method to monitor disease progression noninvasively. The primary goal of this research was to test in vivo MRI methods for the noninvasive early detection and staging of inflammation in the ileum of an infant rat model of NEC. Neonatal rats were delivered by cesarean section at embryonic stage of day 20 after the beginning of pregnancy and stressed with formula feeding, hypoxia and bacterial colonization to induce NEC. Naturally born and dam-fed neonatal rats were used as healthy controls. In vivo MRI studies were performed using a Bruker 9.4-T scanner to obtain high-resolution anatomical MR images using both gradient echo and spin echo sequences, pixel-by-pixel T2 maps using a multi-slice–multi-echo sequence, and maps of the apparent diffusion coefficient (ADC) of water using a spin echo sequence, to assess the degree of ileal damage. Pups were sacrificed at the end of the MRI experiment on day 2 or 4 for histology. T2 measured by MRI was increased significantly in the ileal regions of pups with NEC by histology (106.3 ± 6.1 ms) compared with experimentally stressed pups without NEC (85.2 ± 6.8 ms) and nonstressed, control rat pups (64.9 ± 2.3 ms). ADC values measured by diffusion-weighted MRI were also increased in the ileal regions of pups with NEC by histology [(1.98 ± 0.15) × 10–3 mm2/s] compared with experimentally stressed pups without NEC [(1.43 ± 0.16) × 10–3 mm2/s] and nonstressed control pups [(1.10 ± 0.06) × 10–3 mm2/s]. Both T2 and ADC values between these groups were found to be significantly different (p < 0.03). The correlation of MRI results with histologic images of the excised ileal tissue samples strongly suggests that MRI can noninvasively identify NEC and assess intestinal injury prior to clinical symptoms in a physiologic rat pup model of NEC. © 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.

Effect of Restraint Stress during Gestation on Pentylenetetrazol-Induced Epileptic Behaviors in Rat Offspring.

Epilepsy is a neurodevelopmental disorder which is strongly influenced by genetic and environmental factors. Gestational stress has been shown to be an important factor for affecting seizure susceptibility. The present study was conducted to address whether gestational stress may affect pentylentetrazol (PTZ)-induced epileptic behavior in rat offspring in a sex- and age- dependent manner. Pregnant rats were divided into control and stressed groups (n=6 in each). In the stressed group, pregnant rats were under restraint stress and held immobile in the Plexiglas tube twice per day one hour per session for three consecutive days started on day 17 of pregnancy. To induce seizure, on postnatal days 15 (P15) and 25 (P25), PTZ (40-50 mg/kg, IP) was injected to rat offspring (n=12, one male and one female from any litter for each group/day). Then, epileptic behaviors of each rat were recorded. Epileptic behaviors of stressed pups showed significant changes in comparison to control ones. The time to onset of the first epileptic behavior was shortened while mean duration and frequency of tonic-clonic attacks increased in stressed pups on both P15 and P25. Female offspring were different from male offspring in terms of epileptic behavior. Moreover, focal attacks were more obvious and significantly longer in the offspring of stressed group at the age of 25 days than those of 15 day old. Prenatal restraint stress potentiated PTZ-induced epileptic behavior, age and sex dependently, probably due to alteration of neural and endocrine pathways during developmental process. Male and younger rats were more sensitive to stress than female and older ones.

Prenatal Stress Produces Social Behavior Deficits and Alters the Number of Oxytocin and Vasopressin Neurons in Adult Rats

The present study investigated the long-lasting effects of prenatal repeated restraint stress on social behavior and anxiety, as well as its repercussions on oxytocin (OT) and vasopressin (VP)-positive neurons of the paraventricular (PVN) and supraoptic (SON) nuclei from stressed pups in adulthood. Female Wistar rats were exposed to restraint stress in the last 7 days of pregnancy. At birth, pups were cross-fostered and assigned to the following groups: prenatally non-stressed offspring raised by prenatally non-stressed mothers (NS:NS), prenatally non-stressed offspring raised by prenatally stressed mothers (S:NS), prenatally stressed offspring raised by prenatally non-stressed mothers (NS:S), prenatally stressed offspring raised by prenatally stressed mothers (S:S). As adults, male prenatally stressed offspring raised both by stressed mothers (S:S group) and non-stressed ones (NS:S group) showed impaired social memory and interaction. In addition, when both adverse conditions coexisted (S:S group), increased anxiety-like behavior and aggressiveness was observed in association with a decrease in the number of OT-positive magnocellular neurons, VP-positive magnocellular and parvocellular neurons of the PVN. The NS:S group exhibited a reduction in the amount of VP-positive magnocellular neurons compared to the S:NS. Thus, the social behavior deficits observed in the S:S and NS:S groups may be only partially associated with these alterations to the peptidergic systems. No changes were shown in the OT and VP cellular composition of the SON nucleus. Nevertheless, it is clear that a special attention should be given to the gestational period, since stressful events during this time may be related to the emergence of behavioral impairments in adulthood.