Megan L. Cock

White Matter Injury after Repeated Endotoxin Exposure in the Preterm Ovine Fetus

Intrauterine infection has been linked to neurologic injury in preterm infants. However, a reproducible model of white matter injury in the preterm fetus in a long gestation species that can be monitored in utero is currently unavailable. Thus, our objective was to determine the effects of bacterial endotoxin (lipopolysaccharide, LPS) on physiologic and inflammatory responses and brain structure in the preterm ovine fetus. At 0.7 of gestation, six catheterized fetuses received three to five intravenous injections of LPS (1 μg/kg) over 5 d; seven fetuses served as controls. Fetal responses were monitored and brain tissue examined 10–11 d after the initial LPS injection. After LPS on d 1 and 2, fetuses became transiently hypoxemic and hypotensive and blood IL-6 levels were increased, but these responses were smaller or absent after subsequent LPS exposures. Neural injury was observed in all LPS-exposed fetuses, most prominently in the cerebral white matter. Injury ranged from diffuse subcortical damage to periventricular leukomalacia, and in the brainstem the cross-sectional area of the corticospinal tract was reduced by 30%. Thus, repeated exposure of the preterm ovine fetus to LPS causes neuropathology resembling that of cerebral palsy and provides a robust model for exploring the etiology, prevention, and treatment of white matter damage.

Effects of chronic placental insufficiency on brain development in fetal sheep

Clinical evidence has linked intrauterine compromise such as fetal hypoxemia to poor neurologic outcome in the newborn. In this study we examined the effects of inducing chronic fetal hypoxemia by impairment of placental function on brain development in fetal sheep. Placental insufficiency was induced from 120 to 140 d of gestation (term = 145-148 d) by injection of microspheres into the umbilical circulation in five fetal sheep. Fetal partial pressure of oxygen, Pao2, was reduced from 24.1 ± 0.5 mm Hg before embolization to 14.8 ± 0.4 mm Hg after embolization (p< 0.05). In another three fetuses a similar level of hypoxemia(Pao2, 13.8 ± 0.4 mm Hg) occurred spontaneously. At 140 d of gestation the fetal brains were perfused with fixatives and compared with five control fetuses for the assessment of structural and immunohistochemical alterations. Hypoxemic fetuses demonstrated severe gliosis in the cerebral cortex and reduced myelination of subcortical white matter as visualized by glial fibrillary acidic protein and myelin basic protein staining, respectively (p < 0.05). White matter lesions were observed in two fetuses. The diameter of cerebral capillaries was increased in hypoxemic fetuses (p < 0.05), but there was no change in the number of nitric oxide synthase immunoreactive cells. Growth of neuronal processes was affected in the cerebellum, where there was also a reduction in the number of Purkinje neurons (p < 0.05). These results show that a prolonged period of placental insufficiency, resulting in moderate fetal hypoxemia during the last third of gestation, can affect neurodevelopmental processes that occur late in gestation such as myelination and growth of the cerebellum. This prenatal damage could affect neural connectivity and have functional consequences after birth.

Intrauterine growth restriction delays cardiomyocyte maturation and alters coronary artery function in the fetal sheep

There is now extensive evidence suggesting that intrauterine perturbations are linked with an increased risk of developing cardiovascular disease. Human epidemiological studies, supported by animal models, have demonstrated an association between low birth weight, a marker of intrauterine growth restriction (IUGR), and adult cardiovascular disease. However, little is known of the early influence of IUGR on the fetal heart and vessels. The aim of this study was to determine the effects of late gestational IUGR on coronary artery function and cardiomyocyte maturation in the fetus. IUGR was induced by placental embolization in fetal sheep from 110 to 130 days of pregnancy (D110–130); term ∼D147; control fetuses received saline. At necropsy (D130), wire and pressure myography was used to test endothelial and smooth muscle function, and passive mechanical wall properties, respectively, in small branches of left descending coronary arteries. Myocardium was dissociated for histological analysis of cardiomyocytes. At D130, IUGR fetuses (2.7 ± 0.1 kg) were 28% lighter than controls (3.7 ± 0.3 kg; P= 0.02). Coronary arteries from IUGR fetuses had enhanced responsiveness to the vasoconstrictors, angiotensin II and the thromboxane analogue U46619, than controls (P < 0.01). Endothelium‐dependent and ‐independent relaxations were not different between groups. Coronary arteries of IUGR fetuses were more compliant (P= 0.02) than those of controls. The incidence of cardiomyocyte binucleation was lower in the left ventricles of IUGR fetuses (P= 0.02), suggestive of retarded cardiomyocyte maturation. We conclude that late gestational IUGR alters the reactivity and mechanical wall properties of coronary arteries and cardiomyocyte maturation in fetal sheep, which could have lifelong implications for cardiovascular function.

Fetal growth restriction has long-term effects on postnatal lung structure in sheep.

We have previously shown that fetal growth restriction (FGR) during late gestation in sheep affects lung development in the near-term fetus and at 8 wk after birth. In the present study, our aim was to determine the effects of FGR on the structure of the lungs at 2 y after birth; our hypothesis was that changes observed at 8 wk after birth would persist until maturity. FGR was induced in sheep by umbilicoplacental embolization, which was maintained from 120 d until delivery at term (approximately 147 d); birth weights of FGR lambs were 41% lower than in controls. At 2 y after birth, body and lung weights were not different, but there were 28% fewer alveoli and alveoli were significantly larger than in controls; hence there was a 10% reduction in the internal surface area relative to lung volume in FGR sheep compared with controls. The lungs of FGR sheep, compared with controls, had thicker interalveolar septa as a result of increased extracellular matrix deposition; the alveolar blood–air barrier was also thicker, largely because of an 82% increase in basement membrane thickness. These changes are qualitatively similar to those observed at 8 wk. Our data show that structural alterations in the lungs induced by FGR that were apparent at 8 wk were still evident at 2 y after birth, indicating that FGR may result in permanent changes in the structure of the lungs of the offspring and may affect respiratory health and lung aging later in life.

Placental insufficiency and fetal growth restriction lead to postnatal hypotension and altered postnatal growth in sheep.

Low birth weight has been associated with elevated arterial pressure in later life but mechanisms are unknown. Our aim was to determine the effects of low birth weight resulting from intrauterine growth restriction (IUGR) on fetal and postnatal arterial pressures and the potential roles of circulating cortisol and renin. We induced IUGR by umbilico-placental embolization (UPE) in fetal sheep from 120 d of gestation until birth (approximately 147 d); postnatal lambs (8 IUGR, 8 controls) were studied for 8 wk. Fetal and postnatal arterial pressures were measured and blood samples taken for measurement of gas tensions, cortisol concentrations and renin activity. In IUGR fetuses, mean arterial pressure (MAP) initially increased with UPE, but near term was not different to values in controls. IUGR lambs weighed 33% less than controls at birth and remained lighter than controls during the 8 postnatal weeks; their growth pattern was different to that of controls. IUGR lambs had lower MAP than controls, and this relative hypotension (−4 mm Hg) persisted throughout the 8 postnatal weeks. Covariate analysis showed that the relative hypotension of IUGR lambs could have resulted from their smaller size. Plasma cortisol concentrations were not different between IUGR and control animals before or after birth. Plasma renin activity was not different in postnatal IUGR lambs compared with controls. Thus, postnatal cortisol and renin levels were not consistent with the development of hypotension or hypertension. We conclude that late gestational IUGR in sheep leads to relative hypotension in the early postnatal period, probably a result of reduced body size.

Fetal Brain Injury Following Prolonged Hypoxemia and Placental Insufficiency: A Review

It is well-established that severe, acute episodes of hypoxemia can damage the brain before birth, but the effects of more sustained hypoxemia are less well understood. We have used fetal fetal sheep in a series of studies aimed at determining the effects of prolonged hypoxemia, induced by placental insufficiency of differing severity and duration, on fetal brain structure. Restriction of placental, and hence fetal, growth by carunclectomy caused impaired development of neural processes and connections in the hippocampus, cerebellum, and visual cortex; neuronal migration and neuronal numbers did not appear to be affected. Twenty days of placental insufficiency during late gestation induced by umbilicoplacental embolisation also caused abnormalities in brain structure; the cerebellum, which develops late in gestation, was particularly affected. In the cortex, there was evidence of white matter lesions, an increase in the size of capillaries and a proliferation of astroglia. We also examined the effects of shorter periods of hypoxemia (6-12 hr) near mid-gestation on brain structure; fetuses were allowed to recover for 7 or 35 days after the hypoxemic challenge. The major changes were mild focal damage in the cortical white matter, a reduction in the number of Purkinje cells, a delay in the growth of neural processes in the cerebellum and proliferation of blood vessels. The hippocampus was also affected, in particular the areal density of pyramidal cells was reduced. The use of several classes of pharmacological agents with the potential to protect neurons from hypoxemic injury is discussed in relation to the developing brain.

Prolonged reductions in placental blood flow and cerebral oxygen delivery in preterm fetal sheep exposed to endotoxin: possible factors in white matter injury after acute infection.

Objective: Endotoxin causes hypoxemia and white matter injury in the preterm ovine fetus. Because cerebral hypoxia could contribute to brain injury, our objective was to determine the effects of endotoxin on regional cerebral oxygen (O2) delivery. To investigate causes of fetal hypoxemia, we also measured placental blood flow. Methods: We administered endotoxin (lipopolysaccharide, LPS) at 1 μg/kg (intravenously) to 11 catheterized fetal sheep at approximately 0.7 of term; controls (n = 7) received saline. We measured fetal cerebral blood flow (CBF) and placental blood flow using micropheres, arterial blood gases, arterial pressure, and heart rate. Results: Seven fetuses survived LPS administration (LPS-S) and four died. LPS-S fetuses were hypoxemic at 4-8 hours after LPS. Fetal hemoglobin concentration and hematocrit increased by about 14% at 4 hours after LPS exposure, and mean arterial pressure decreased significantly from 4-8 hours. After LPS, CBF did not change significantly, but total cerebral O2 delivery decreased by 35.7% at 4 hours and by 28.3% at 8 hours. O2 delivery to cerebral white matter decreased below pre-LPS values at 4 hours (-35.9%) and 8 hours (-28.6%) after LPS. Relative to pre-LPS values, placental blood flow decreased by 53.3% at 4 hours and 43.0% at 8 hours after LPS. Conclusions: Immature fetal sheep exposed to LPS had profound reductions in placental blood flow and cerebral O2 delivery, which could contribute to fetal brain injury. Reduced O2 delivery to white matter was similar to that in other brain regions. Mechanisms that enable fetal CBF to increase in hypoxemic conditions were apparently ineffective in the presence of LPS.

Chronic endotoxin exposure causes brain injury in the ovine fetus in the absence of hypoxemia.

Objective: Intrauterine infection has been linked to brain injury in human infants, although the mechanisms are not fully understood. We recently showed that repeated acute exposure of preterm fetal sheep to bacterial endotoxin (lipopolysaccharide [LPS]) results in fetal hypoxemia, hypotension, increased systematic proinflammatory cytokines, and brain damage, including white matter injury. However, it is not clear whether this injury is caused by reduced cerebral oxygen delivery or inflammatory pathways independent of hypoxia. The aim of the present study was to determine the effects on the fetal brain and placenta of a chronic intrauterine inflammatory state, induced by LPS infusion into the fetal circulation, a model that did not cause hypoxia. Methods: At 0.65 of term, eight catheterized fetal sheep received intravenous infusions of LPS (5 to 15 μg) over 5 days; control fetuses received saline. Fetal physiologic responses were monitored throughout the infusion. Fetal brain and placental tissues were examined histologically 6 days after the conclusion of the infusion. Results: LPS infusions did not result in physiologically significant alterations to fetal blood gases or mean arterial pressure; however, plasma proinflammatory cytokine levels were elevated. Following LPS exposure there was no difference in fetal body or brain weights (P >.05); placental weight was reduced (P <.05), consistent with reduced placentome cross-sectional area (P <.05). In the cerebral hemispheres subcortical white matter injury was present in six LPS-exposed fetuses and included axonal damage, microgliosis, oligodendrycyte injury, and increased β amyloid precursor protein (β-APP) expression. Conclusions: Chronic, systemic exposure of the fetus to LPS resulted in fetal brain damage in the absence of hypoxemia or hypotension, although the resulting injury was less severe than following repeated acute exposure.

Compromised Respiratory Function in Postnatal Lambs after Placental Insufficiency and Intrauterine Growth Restriction

Epidemiologic studies have shown persistent effects of low birth weight on respiratory function and lung health, but underlying mechanisms are not understood. Our aim was to determine the effects of intrauterine growth restriction (IUGR), a major cause of low birth weight, on postnatal respiratory function. IUGR was induced by umbilico-placental embolization during late gestation in chronically catheterized sheep. Umbilico-placental embolization was performed between 120 d of gestation and term (∼146 d) during which fetuses were hypoxemic and hypoglycemic relative to controls. Umbilico-placental embolization led to a 48% reduction in birth weight compared with controls, and throughout the postnatal study period IUGR lambs (n = 8) remained lighter than controls (n = 8). Respiratory function was repeatedly studied in lambs for 8 wk after birth; during this period, IUGR lambs were mildly hypoxemic and tended to be hypercapnic compared with controls. In IUGR lambs, relative to controls, O2 consumption (mL/min/kg) and minute ventilation (mL/kg) were increased and pulmonary diffusing capacity (adjusted for functional residual capacity) was decreased. Functional residual capacity, measured by helium dilution, and total lung capacity (measured at 30 cm H2O) were smaller in IUGR lambs than in controls. When adjusted for functional residual capacity, static lung compliance was reduced and chest wall compliance was increased in IUGR lambs. At 8 wk, pulmonary DNA and protein concentrations were decreased in IUGR lambs relative to controls. We conclude that restriction of fetal growth by placental insufficiency induces alterations in the lungs and chest wall that result in persistent impairments in respiratory function during early postnatal life.

Nephron Endowment and Filtration Surface Area in the Kidney after Growth Restriction of Fetal Sheep

Low birth weight is associated with adult-onset diseases including hypertension and renal disease; altered renal development after intrauterine growth restriction (IUGR) may underlie such prenatal programming. Our aim was to investigate nephron endowment and renal filtration surface area in fetal sheep in which IUGR resulted from late gestational umbilico-placental embolization (UPE) or natural twinning. UPE was performed between 120 and 140 d of gestation (term ∼147 d). At autopsy (140 d), body weights of UPE and twin fetuses were, respectively, 34% and 28% lower than controls. Kidneys were sampled using a smooth fractionator approach and glomerular number was estimated using a physical disector/fractionator technique. Glomerular capillary length and filtration surface area were estimated using unbiased stereological techniques. Although relative kidney weights (grams per kilogram body weight) were not different between groups, nephron endowment was 40% lower in twin fetuses compared with controls (34.3 ± 10.6 × 104 and 55.9 ± 19.8 × 104, respectively; p < 0.05); UPE did not alter nephron number (50.7 ± 13.2 × 104). There was no difference in the glomerular capillary length or surface area between the UPE and control fetuses. IUGR due to twinning leads to reduced nephron endowment whereas late gestational IUGR does not, suggesting that reduced nephron endowment is dependent on the timing of the growth restriction. Our findings demonstrate that reduced birth weight per se does not necessarily imply reduced nephron endowment.