Developmental Research in Space: Predicting Adult Neurobehavioral Phenotypes via Metabolomic Imaging

As human habitation and eventual colonization of space becomes an inevitable reality, there is a necessity to understand how organisms develop over the life span in the space environment. Microgravity, altered CO2, radiation and psychological stress are some of the key factors that could affect mammalian reproduction and development in space, however there is a paucity of information on this topic. Here we combine early (neonatal) in vivo spectroscopic imaging with an adult emotionality assay following a common obstetric complication (prenatal asphyxia) likely to occur during gestation in space. The neural metabolome is sensitive to alteration by degenerative changes and developmental disorders, thus we hypothesized that that early neonatal neurometabolite profiles can predict adult response to novelty. Late gestation fetal rats were exposed to moderate asphyxia by occluding the blood supply feeding one of the rats pair uterine horns for 15min. Blood supply to the opposite horn was not occluded (within-litter cesarean control). Further comparisons were made with vaginal (natural) birth controls. In one-week old neonates, we measured neurometabolites in three brain areas (i.e., striatum, prefrontal cortex, and hippocampus). Adult perinatally-asphyxiated offspring exhibited greater anxiety-like behavioral phenotypes (as measured the composite neurobehavioral assay involving open field activity, responses to novel object, quantification of fecal droppings, and resident-intruder tests of social behavior). Further, early neurometabolite profiles predicted adult responses. Non-invasive MRS screening of mammalian offspring is likely to advance ground-based space analogue studies informing mammalian reproduction in space, and achieving high-priority multigenerational research that will enable studies of the first truly space-developed mammals

Outcomes of Intrauterine Asphyxia in the Full Term Rat

Intrauterine asphyxia, observed in 1-6 out of every 1,000 live full term births and nearly 60% of preterm births, is a component of many obstetric complications, including infection, preeclampsia, maternal diabetes, and intrauterine growth restriction. Numerous population-based studies show risk for developing schizophrenia is more than doubled following intrauterine asphyxia. The mechanistic link between intrauterine asphyxia and schizophrenia has yet to be elucidated. Intrauterine asphyxia causes an acute lack of oxygen in the blood, resulting in metabolic acidosis, hypercapnia, and hypoxia. Preclinical models have identified postnatal sequelae involving oxidative stress and inflammation, as well as alterations in the dopamine (DA) system that parallel abnormalities found in the schizophrenic brain. In particular, preclinical studies have identified an increased sensitivity to DA agonists, consistent with observed increases in DA activity in prodromal schizophrenic populations, suggesting a key DA system disturbance observed in the brain at the onset of schizophrenia can be induced by asphyxia at birth. How DA supersensitivity arises from birth asphyxia is unknown, but it is developmentally regulated, meaning that DA sensitivity may be preventable with early identification and intervention. However, there is no way to identify an individual that will develop DA supersensitivity prior to symptom onset. The aim of this experiment was to identify changes in the neurochemistry of forebrain dopamine targets usingin vivo magnetic resonance spectroscopy at ages prior to the emergence of DA supersensitivity. We observed developmental changes in the dorsal striatum (Str) and medial prefrontal cortex. In Str, the ratio of glutamine (Gln) to glutamate (Glu) measure on postnatal day (P) 7 was significantly increased in birth asphyxiated rats (APX) compared to non-asphyxiated (NON) littermates (p=.030), normalizing thereafter. On P35, APX show reduced Glu+Gln in the Str (p=.018) and increased Glu in the mPFC, relative to NON (p=.028). Str GABA was increased in APX at P7 (p=.023) and then decreased at P60 (p=.007) relative to NON. Further experiments are identifying in vitro dopamine metabolite and receptor levels in scanned brains

Perinatal Asphyxia Induces Hyperemotionality and Elevates Corticosterone (CORT) Release in Response to Novel Stimuli in Later Life

We tested the hypothesis that perinatal asphyxia (PA) augments adult hyperemotionality and increases corticosterone (CORT) release. Term fetuses were exposed to PA (12 min) by occluding (OCCL) the arteries feeding one uterine horn. Control subjects were derived from the opposite, non-occluded (N-OCCL) horn, and from Vaginal delivery groups. Newborn pups were fostered to newly parturient dams. Adult male and female rats were singly housed for 24hr, placed in an open field and videographed for the duration of a 52min novelty test comprised of a concatenation of novel environmental stimuli including exposure to the open field, sustained darkness, presentation of a novel object, startle stimuli, introduction of an unfamiliar same-gender intruder, and punctate acoustic stimuli. As compared to controls, OCCL adult rats showed a tendency to spend time near the wall rather than the center of the arena (thigmotaxis). Approaches to a novel object were slower in OCCL offspring relative to controls. Behavioral testing was associated with a significant increase in CORT, with greater magnitudes of response in OCCL offspring. Collectively, our findings provide clear evidence for magnified emotional responses and HPAA activity in response to novelty in adult offspring exposed to PA

Outcomes of Intrauterine Asphyxia in the Full Term Rat

Intrauterine asphyxia, observed in 1-6 out of every 1,000 live full term births and nearly 60% of preterm births, is a component of many obstetric complications, including infection, preeclampsia, maternal diabetes, and intrauterine growth restriction. Numerous population-based studies show risk for developing schizophrenia is more than doubled following intrauterine asphyxia. The mechanistic link between intrauterine asphyxia and schizophrenia has yet to be elucidated. Intrauterine asphyxia causes an acute lack of oxygen in the blood, resulting in metabolic acidosis, hypercapnia, and hypoxia. Preclinical models have identified postnatal sequelae involving oxidative stress and inflammation, as well as alterations in the dopamine (DA) system that parallel abnormalities found in the schizophrenic brain. In particular, preclinical studies have identified an increased sensitivity to DA agonists, consistent with observed increases in DA activity in prodromal schizophrenic populations, suggesting a key DA system disturbance observed in the brain at the onset of schizophrenia can be induced by asphyxia at birth. How DA supersensitivity arises from birth asphyxia is unknown, but it is developmentally regulated, meaning that DA sensitivity may be preventable with early identification and intervention. However, there is no way to identify an individual that will develop DA supersensitivity prior to symptom onset. The aim of this experiment was to identify changes in the neurochemistry of forebrain dopamine targets usingin vivo magnetic resonance spectroscopy at ages prior to the emergence of DA supersensitivity. We observed developmental changes in the dorsal striatum (Str) and medial prefrontal cortex. In Str, the ratio of glutamine (Gln) to glutamate (Glu) measure on postnatal day (P) 7 was significantly increased in birth asphyxiated rats (APX) compared to non-asphyxiated (NON) littermates (p=.030), normalizing thereafter. On P35, APX show reduced Glu+Gln in the Str (p=.018) and increased Glu in the mPFC, relative to NON (p=.028). Str GABA was increased in APX at P7 (p=.023) and then decreased at P60 (p=.007) relative to NON. Further experiments are identifying in vitro dopamine metabolite and receptor levels in scanned brains

A New Method for Inducing Perinatal Asphyxia in Rats Incorporating Within-Litter, Non-Ischemic Controls

Perinatal asphyxia results in impairment in blood-gas exchange and occurs in approximately 1-6 per 1,000 live full-time births, leading to increased rates of infant mortality and long-term neurological deficits. Animal models of perinatal asphyxia leading to hypoxic ischemia continue to be developed. However, relatively few studies actually describe true intrauterine hypoxic ischemia. Here we report on a new method for controlled perinatal asphyxia in the rat that induces a moderate (12 minute) period of hypoxic ischemia and provides within-litter, non-asphyxiated controls. On the day of parturition (gestational day 22), pregnant Sprague-Dawley dams were situated with lower bodies immersed in buffered isotonic saline bath (37.5° C) and the uterus externalized. Surgical silk was used to ligate one uterine artery for 12 min, a duration shown to produce significant effects on brain and behavior. Because of the rat’s duplex uterus with independent arterial flow to each horn, ligation of blood flow and, thus, perinatal asphyxiation of fetuses in one horn was achieved without deleterious effects on the second horn. Blood-gas was analyzed immediately following caesarian delivery. Our results show a significant reduction in blood pH and disruption of CO2/O2 blood-gas levels for perinatal asphyxiated animals, as compared to intrauterine controls. Functional assessments, such as neonatal movement and rate of respiration, were also analyzed. Neuronal damaged was assessed via measurement of whole brain lactate and identification of necrotic foci. Collectively our results demonstrate the expected effects of an intrauterine event that leads to hypoxic ischemia, validating a new model of perinatal asphyxia that provides within-litter, intrauterine controls

A New Method for Inducing Perinatal Asphyxia in Rats Incorporating Within-Litter, Non-Ischemic Controls

Perinatal asphyxia results in impairment in blood-gas exchange and occurs in approximately 1-6 per 1,000 live full-time births, leading to increased rates of infant mortality and long-term neurological deficits. Animal models of perinatal asphyxia leading to hypoxic ischemia continue to be developed. However, relatively few studies actually describe true intrauterine hypoxic ischemia. Here we report on a new method for controlled perinatal asphyxia in the rat that induces a moderate (12 minute) period of hypoxic ischemia and provides within-litter, non-asphyxiated controls. On the day of parturition (gestational day 22), pregnant Sprague-Dawley dams were situated with lower bodies immersed in buffered isotonic saline bath (37.5° C) and the uterus externalized. Surgical silk was used to ligate one uterine artery for 12 min, a duration shown to produce significant effects on brain and behavior. Because of the rat’s duplex uterus with independent arterial flow to each horn, ligation of blood flow and, thus, perinatal asphyxiation of fetuses in one horn was achieved without deleterious effects on the second horn. Blood-gas was analyzed immediately following caesarian delivery. Our results show a significant reduction in blood pH and disruption of CO2/O2 blood-gas levels for perinatal asphyxiated animals, as compared to intrauterine controls. Functional assessments, such as neonatal movement and rate of respiration, were also analyzed. Neuronal damaged was assessed via measurement of whole brain lactate and identification of necrotic foci. Collectively our results demonstrate the expected effects of an intrauterine event that leads to hypoxic ischemia, validating a new model of perinatal asphyxia that provides within-litter, intrauterine controls

Perinatal Asphyxia Induces Hyperemotionality and Elevates Corticosterone (CORT) Release in Response to Novel Stimuli in Later Life

We tested the hypothesis that perinatal asphyxia (PA) augments adult hyperemotionality and increases corticosterone (CORT) release. Term fetuses were exposed to PA (12 min) by occluding (OCCL) the arteries feeding one uterine horn. Control subjects were derived from the opposite, non-occluded (N-OCCL) horn, and from Vaginal delivery groups. Newborn pups were fostered to newly parturient dams. Adult male and female rats were singly housed for 24hr, placed in an open field and videographed for the duration of a 52min novelty test comprised of a concatenation of novel environmental stimuli including exposure to the open field, sustained darkness, presentation of a novel object, startle stimuli, introduction of an unfamiliar same-gender intruder, and punctate acoustic stimuli. As compared to controls, OCCL adult rats showed a tendency to spend time near the wall rather than the center of the arena (thigmotaxis). Approaches to a novel object were slower in OCCL offspring relative to controls. Behavioral testing was associated with a significant increase in CORT, with greater magnitudes of response in OCCL offspring. Collectively, our findings provide clear evidence for magnified emotional responses and HPAA activity in response to novelty in adult offspring exposed to PA

Neurometabolic Alterations in the Acute Postnatal Phase of Perinatal Asphyxia in the Rat

Perinatal hypoxic-ischemic encephalopathies represent a range of intrauterine and birth complications that, in cases most severe, can result in mental retardation, cerebral palsy, or death. More recent clinical studies have indicated that perinatal hypoxic-ischemia may play a causal role in etiology of cognitive deficits in the absence of motor disorders. To further evaluate this role, we have utilized an intrauterine model of a one-time perinatal asphyxic event in the rat that provides normoxic, within-litter controls and preserves many clinically-relevant features of this dynamic, late-gestational time period. Our current findings show significant disturbances immediately following birth in pH, pCO2, and pO2, as measured in mixed arterial and venous blood samples from asphyxiated, non-asphyxiated, and vaginal-born rat pups an indication of metabolic acidosis. We have additionally found a significant increase in the concentration of brain lactate levels (a marker hypoxic-ischemic encephalopathy) immediately following birth in asphyxiated (56.5 ± 2.9 μg/dl) as compared to non-asphyxiated (24.4 ± 1.1 μg/dl) and vaginal-born rats (29.6 ± 3.2 μg/dl). At one hour postnatal, average whole brain lactate concentrations were measurably lower, but remained significantly elevated in the asphyxiated group (23.9 ± 3.6 μg/dl) versus non-asphyxiated (11.7 ± 3.1 μg/dl) and vaginal-born (7.5 ± 0.74 μg/dl). Linear regression analysis revealed low blood pH to be an accurate indicator of elevated brain lactate concentration at one hour postnatal (R2 = 0.495; p=0.003). One important consideration in the interpretation of these findings is the role of lactate metabolism in the neonatal mammal as a secondary energy source. This consideration necessitates a more detailed investigation of neural high-energy phosphate levels during the acute postnatal phase of increased whole-brain lactate concentration. Current work with this model is aimed at just such an investigation utilizing 31P NMR spectroscopy, a technique with increasing clinical relevance in the assessment of neonatal hypoxic-ischemic encephalopathy

Neurometabolic Alterations in the Acute Postnatal Phase of Perinatal Asphyxia in the Rat

Perinatal hypoxic-ischemic encephalopathies represent a range of intrauterine and birth complications that, in cases most severe, can result in mental retardation, cerebral palsy, or death. More recent clinical studies have indicated that perinatal hypoxic-ischemia may play a causal role in etiology of cognitive deficits in the absence of motor disorders. To further evaluate this role, we have utilized an intrauterine model of a one-time perinatal asphyxic event in the rat that provides normoxic, within-litter controls and preserves many clinically-relevant features of this dynamic, late-gestational time period. Our current findings show significant disturbances immediately following birth in pH, pCO2, and pO2, as measured in mixed arterial and venous blood samples from asphyxiated, non-asphyxiated, and vaginal-born rat pups an indication of metabolic acidosis. We have additionally found a significant increase in the concentration of brain lactate levels (a marker hypoxic-ischemic encephalopathy) immediately following birth in asphyxiated (56.5 ± 2.9 μg/dl) as compared to non-asphyxiated (24.4 ± 1.1 μg/dl) and vaginal-born rats (29.6 ± 3.2 μg/dl). At one hour postnatal, average whole brain lactate concentrations were measurably lower, but remained significantly elevated in the asphyxiated group (23.9 ± 3.6 μg/dl) versus non-asphyxiated (11.7 ± 3.1 μg/dl) and vaginal-born (7.5 ± 0.74 μg/dl). Linear regression analysis revealed low blood pH to be an accurate indicator of elevated brain lactate concentration at one hour postnatal (R2 = 0.495; p=0.003). One important consideration in the interpretation of these findings is the role of lactate metabolism in the neonatal mammal as a secondary energy source. This consideration necessitates a more detailed investigation of neural high-energy phosphate levels during the acute postnatal phase of increased whole-brain lactate concentration. Current work with this model is aimed at just such an investigation utilizing 31P NMR spectroscopy, a technique with increasing clinical relevance in the assessment of neonatal hypoxic-ischemic encephalopathy