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

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

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

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

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

MR Spectroscopic Analysis of Neonatal Rat Striatum: An Early Biomarker for Adult Hyperemotionality Following Perinatal Asphyxia

In vivo proton (1H) MR spectroscopy (MRS) can be used to identify neurological impairments in human and animal subjects. We hypothesized that neurochemical profiles acquired from one-week-old perinatally asphyxiated (PA) rats could be used to predict adult hyperemotionality. Gestational day 22 pregnant rat dams were administered spinal anesthesia and the uterus externalized into a heated (37.5 C) saline bath. Controlled asphyxia was produced by occluding the blood supply feeding one uterine horn (12 min). The other uterine horn remained undisturbed (non-occluded control). Fetuses were immediately delivered by cesarean section, then fostered to newly parturient dams. On Postnatal day 7, high resolution in vivo proton MR spectra were acquired from the striatum using a 7T small animal MRI scanner using single-voxel (3 x 3 x 3 cu mm) MRS imaging. The point-resolved spectroscopy (PRESS) sequence was applied, and positioning of MRS voxels based on T2-weighted images. Neurometabolites derived from spectral analysis were corrected for creatine + phosphocreatine (Cr + pCr). Significant changes in N-acetyl aspartate (NAA) and glutamate and glutamine (Glx) were observed in PA rats as compared to non-asphyxiated controls. At 8-weeks of age, subjects were singly housed (24 hrs), placed in an open field and exposed to a 52 min test comprised of a concatenation of novel stimuli. Videographic analysis revealed magnified emotional responses to novelty in PA rats as measured by position within the open field, responses to novel stimuli, and social interactions. In vivo MRS is a sensitive imaging tool for detecting neurochemical changes in early life that can be used to predict adult hyperemotionality

MR Spectroscopic Analysis of Neonatal Rat Striatum: An Early Biomarker for Adult Hyperemotionality Following Perinatal Asphyxia

In vivo proton (1H) MR spectroscopy (MRS) can be used to identify neurological impairments in human and animal subjects. We hypothesized that neurochemical profiles acquired from one-week-old perinatally asphyxiated (PA) rats could be used to predict adult hyperemotionality. Gestational day 22 pregnant rat dams were administered spinal anesthesia and the uterus externalized into a heated (37.5 C) saline bath. Controlled asphyxia was produced by occluding the blood supply feeding one uterine horn (12 min). The other uterine horn remained undisturbed (non-occluded control). Fetuses were immediately delivered by cesarean section, then fostered to newly parturient dams. On Postnatal day 7, high resolution in vivo proton MR spectra were acquired from the striatum using a 7T small animal MRI scanner using single-voxel (3 x 3 x 3 cu mm) MRS imaging. The point-resolved spectroscopy (PRESS) sequence was applied, and positioning of MRS voxels based on T2-weighted images. Neurometabolites derived from spectral analysis were corrected for creatine + phosphocreatine (Cr + pCr). Significant changes in N-acetyl aspartate (NAA) and glutamate and glutamine (Glx) were observed in PA rats as compared to non-asphyxiated controls. At 8-weeks of age, subjects were singly housed (24 hrs), placed in an open field and exposed to a 52 min test comprised of a concatenation of novel stimuli. Videographic analysis revealed magnified emotional responses to novelty in PA rats as measured by position within the open field, responses to novel stimuli, and social interactions. In vivo MRS is a sensitive imaging tool for detecting neurochemical changes in early life that can be used to predict adult hyperemotionality

Magnetic Resonance Spectroscopy Detects Neuroanatomically Specific Alterations in Brain Metabolites from Infancy to Adulthood in Birth Asphyxiated Rats

Magnetic resonance spectroscopy (MRS) provides a safe, non-invasive means of directly assessing biochemistry in localized brain regions of pediatric and adult populations. This technology holds much promise for early identification of developmental neural dysfunction, enabling early intervention. Herein, we report longitudinal, neuroanatomically specific metabolic profiles from neonatal life to adulthood in birth asphyxiated rats. Epidemiological evidence identifies obstetric complications, such as birth asphyxia, as risk factors in the emergence of psychiatric and cognitive dysfunction in later life. A single, moderate (12-15min) bout of experimentally controlled asphyxia in a rat fetus at term can exert persistent changes in brain morphology and behavior. On postnatal days (P) 7, 35 and 60, we acquired MRS metabolite profiles from male rats exposed to 12 min asphyxia (APX) at term and non-asphyxiated (NON) littermates. We hypothesized that APX rats would show signs of ongoing metabolic dysfunction at P7 (i.e. increased lactate) and in adulthood, decreased N-Acetylaspartate and N-acetylaspartylglutamate (NAA+NAAG) would indicate striatal damage and neuron loss. P7 profiles of a 27mm3 area (voxel) of subcortical tissue including striatum and surrounding tissue revealed a decrease in NAA+NAAG/total creatine (tCr) (t=2.061; p=0.0584) for APX compared to NON littermates (N=8). This difference did not persist at P35 or P60. However P60 APX rats showed reduced striatal taurine/tCr (t=2.34; p=0.035). In a second study underway (N=4), we increased asphyxia duration to 15 min and are conducting a longitudinal comparison of metabolites exclusively in dorsal striatum of APX and NON rats. Our preliminary findings show that on P7, but not P35 or P60, APX rats have significantly increased striatal lactate/tCr (t=2.51; p=0.046) and guanine/tCr (t=3.66; p=0.011), indicating delayed metabolic deficit following birth asphyxia. APX rats show increased striatal NAA+NAAG/tCr (t=2.33; p=0.058) on P35 and significantly reduced NAAG/tCr (t=-2.55; p=0.044) on P60, suggesting persistent striatal dysfunction in adulthood. These studies show different metabolite patterns based on precise voxel placement. In our second study, a smaller voxel (10-15mm3) solely incorporating dorsal striatum uncovered a broader pattern of changes, supporting our hypothesis of ongoing striatal metabolic disruption in neonates, with long term striatal damage in adulthood. This work highlights the need for specificity of voxel placement and identifies unique, tissue-specific pattern of metabolite changes throughout early life and into adulthood following birth asphyxia

Magnetic Resonance Spectroscopy Detects Neuroanatomically Specific Alterations in Brain Metabolites from Infancy to Adulthood in Birth Asphyxiated Rats

Magnetic resonance spectroscopy (MRS) provides a safe, non-invasive means of directly assessing biochemistry in localized brain regions of pediatric and adult populations. This technology holds much promise for early identification of developmental neural dysfunction, enabling early intervention. Herein, we report longitudinal, neuroanatomically specific metabolic profiles from neonatal life to adulthood in birth asphyxiated rats. Epidemiological evidence identifies obstetric complications, such as birth asphyxia, as risk factors in the emergence of psychiatric and cognitive dysfunction in later life. A single, moderate (12-15min) bout of experimentally controlled asphyxia in a rat fetus at term can exert persistent changes in brain morphology and behavior. On postnatal days (P) 7, 35 and 60, we acquired MRS metabolite profiles from male rats exposed to 12 min asphyxia (APX) at term and non-asphyxiated (NON) littermates. We hypothesized that APX rats would show signs of ongoing metabolic dysfunction at P7 (i.e. increased lactate) and in adulthood, decreased N-Acetylaspartate and N-acetylaspartylglutamate (NAA+NAAG) would indicate striatal damage and neuron loss. P7 profiles of a 27mm3 area (voxel) of subcortical tissue including striatum and surrounding tissue revealed a decrease in NAA+NAAG/total creatine (tCr) (t=2.061; p=0.0584) for APX compared to NON littermates (N=8). This difference did not persist at P35 or P60. However P60 APX rats showed reduced striatal taurine/tCr (t=2.34; p=0.035). In a second study underway (N=4), we increased asphyxia duration to 15 min and are conducting a longitudinal comparison of metabolites exclusively in dorsal striatum of APX and NON rats. Our preliminary findings show that on P7, but not P35 or P60, APX rats have significantly increased striatal lactate/tCr (t=2.51; p=0.046) and guanine/tCr (t=3.66; p=0.011), indicating delayed metabolic deficit following birth asphyxia. APX rats show increased striatal NAA+NAAG/tCr (t=2.33; p=0.058) on P35 and significantly reduced NAAG/tCr (t=-2.55; p=0.044) on P60, suggesting persistent striatal dysfunction in adulthood. These studies show different metabolite patterns based on precise voxel placement. In our second study, a smaller voxel (10-15mm3) solely incorporating dorsal striatum uncovered a broader pattern of changes, supporting our hypothesis of ongoing striatal metabolic disruption in neonates, with long term striatal damage in adulthood. This work highlights the need for specificity of voxel placement and identifies unique, tissue-specific pattern of metabolite changes throughout early life and into adulthood following birth asphyxia