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Background: Hypoxia-Ischemia (HI) is a condition in which the brain is deprived of adequate blood and oxygen supplies, resulting in a variety of neurodevelopmental morbidities in newborns. Inter-Alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins that have anti-inflammatory properties in systemic inflammatory disorders. The neuroprotective effects of treatment with IAIPs on apoptosis resulting from HI-related injury have not been examined yet. Objective: To investigate the effects of systemic treatment with IAIPs on apoptosis in HI-exposed neonatal rats. Design/Methods: Postnatal day 7 rats were randomly assigned to one of three groups: Placebo-Sham (PL-Sham, n=8), Placebo-HI (PL-HI, n=8) and IAIP-HI (n=8) groups. The PL-Sham group was not exposed to HI and received placebo intraperitoneally (0.9% NaCl); the PL-HI group was exposed to HI and received placebo (0.9% NaCl); the IAIP-HI group was exposed to HI and received IAIPs treatment (30 mg/kg). The Rice-Vannucci model was utilized to induce HI-related brain injury: unilateral carotid artery ligation followed by hypoxia (90 min in 8% O 2 ). Rat sex was recorded. Placebo or IAIP treatment was given at 0, 24 and 48 hours after HI, and brains were perfused and fixed at 72 hours. Fixed brain tissues were immunohistochemically stained with TUNEL apoptotic cellular markers. Apoptotic cells were counted without knowledge of the group assignment using the StereoInvestigator 10.0, Fractionator probe (MBF Bioscience). Results: There were more TUNEL positive cells in the PL-HI group than in the PL-Sham group overall (female+male) as well as in males in the cortex, total hemisphere and hippocampus (all P P =0.11, P =0.30 and P =0.12, respectively). There appeared to be less apoptosis in the male IAIP-treated HI rats compared with the male placebo-treated HI rats ( P =0.29 for hemisphere and cortex, P =0.51 for hippocampus). Conclusion: Apoptosis is significantly increased after HI in the total hemisphere, cortex, and hippocampus of male neonatal rats, but not in females. Further investigation is needed to determine the neuroprotective effects of IAIPs on apoptosis in HI-exposed neonatal rats, although preliminary data suggests a protective trend for male rats.

Background: Inter-alpha inhibitor proteins (IAIPs) are immunomodulatory proteins that act as anti-inflammatory agents in hypoxic-ischemic (HI) injury. We have recently shown that administering IAIP after HI improves histopathological brain injury, and behavioral outcomes in neonatal rats. Microglia are glial cells that act as macrophages in the brain and are important constituents in the neuroinflammatory response. Objective: To determine the effect of IAIP treatment on microglial expression in specific brain regions of neonatal rats after HI brain related injury. Methods: The Vannucci model was used to induce neonatal HI brain injury. Postnatal day 7 rats were assigned to a non-ischemic sham-control group (Sham, n=11), a right carotid ligation and hypoxia exposed (8% oxygen for 90 min) placebo-treated group (IschPL, n=12), or a right carotid ligation and hypoxia IAIP-treated group (Isch-IAIP, n=12). The sex of the rats was recorded. IAIP (30 mg/kg) or PL was given intraperitoneally at 0, 24 and 48 h after HI. 72 h after HI, brains were collected, sectioned, and prepared for slides. We performed immunohistochemistry by staining with Iba-1 (selective for microglia) and DAPI. Stereological analyses were then performed with the StereoInvestigator 10.0 Fractionator probe without knowledge of group assignment to quantify microglia present in the whole hemisphere, cortex, corpus callosum, and hippocampus. Results: The number of Iba-1 positive microglial cells per area of tissue was lower in Sham than in the Isch-PL animals in the cortex, hippocampus, and hemisphere of female and in the hippocampus and corpus callosum of male rats (P

Deleterious effects result from both glucocorticoid insufficiency and excess glucocorticoid tissue exposure in the developing brain. Accumulating evidence suggests a net benefit of postnatal glucocorticoid therapy when administered shortly after the first week of life to premature infants with early and persistent pulmonary dysfunction, particularly in those with evidence of relative adrenal insufficiency. The decision to treat with steroids should ensure maximum respiratory benefit at the lowest possible neurologic risk, while avoiding serious systemic complications. Ongoing clinical trials must validate this approach.

The transitional physiology during the perinatal period facilitates adaptation from fetal to neonatal life and allows the gastrointestinal tract to assume essential postnatal functions. Intrinsic hemodynamic control in the developing intestines matures as a function of postnatal age. Immature vascular regulation in the newborn gastrointestinal tract is offset by an enhanced ability to extract oxygen from blood, and to some extent by increases in intestinal perfusion. Vasodilatation in the neonatal intestine is near maximal. Therefore, additional stressors particularly during the postprandial period increase the risk of mismatches in the oxygen supply to demand ratio, potentially predisposing intestinal tissue to hypoxia and ischemic injury.

Objectives: The blood-brain barrier is a selective diffusion barrier between brain parenchyma and the intravascular compartment. Tight junctions are integral components of the blood-brain barrier. Pro-inflammatory cytokines are important in the pathogenesis of brain injury and could modify the protein constituents of tight junctions. We hypothesized that interleukin-6 (IL-6) downregulates key protein constituents of endothelial tight junctions (e.g. occludin and claudin-5). Methods: We examined the effects of IL-6 on tight junction protein expression using an in vitro blood-brain barrier model. We isolated microvessels from yearling and adult ovine cerebral cortex and placed them into culture with IL-6 concentrations of 0 (control, phosphate-buffered saline), 1, 10, and 100 ng/ml for 24 h. Cerebral microvessels were harvested, Western immunoblot performed for occludin and claudin-5, densitometry performed, and results expressed as a ratio to control values. Results: Western immunoblot analysis showed that treatment with 100 ng/ml of IL-6, but not the lower concentrations, reduced (p < 0.05) occludin expression in microvessels from yearling and adult sheep and claudin-5 in microvessels from adult sheep. However, treatment with 10 ng/ml of IL-6 increased claudin-5 in microvessels from yearling sheep. The percent of lactate dehydrogenase released from the microvessels into the surrounding media was not increased by IL-6 treatment, suggesting that the reductions in tight junction proteins did not result from cell death. Treatment of adult cerebral cortical microvessels with IL-6 preincubated with anti-IL-6 monoclonal antibodies partially attenuated the reduction in claudin-5. Conclusion: We conclude that IL-6 modulates tight junction protein expression in cerebral cortical microvessels from yearling and adult sheep.

The blood-brain barrier maintains central nervous system homeostasis and limits the entry of blood-borne substances that could alter neuronal function and survival. The barrier exists predominantly at the endothelium of cerebral vascular microvessels. The cerebral vascular endothelium becomes highly specialized during the formation of the neurovascular unit early in embryonic development. The blood-brain barrier is present and functional early in fetal life. The tightness of the barrier gradually increases throughout gestation and in the newborn period. Alterations in the basolateral environment of the cerebral microvasculature can modify the blood-brain barrier properties by modulating the expression of the endothelial tight junctions and other biochemical properties of the cerebral vascular endothelium. Maturation of the blood-brain barrier late in gestation correlates with increases in endogenous corticosteroids and with exposure to exogenous corticosteroids. Several adverse fetal and neonatal conditions can alter the structure and function of the blood-brain barrier. Impairment of blood-brain barrier function in the perinatal period could increase the entry of bilirubin and other neurotoxic substances from the systemic circulation into the brain, thereby exacerbating and/or causing damage to the developing brain.

We examined the effects of development, exogenous, and endogenous glucocorticoids on Na(+),K(+)-ATPase activity and subunit protein expression in ovine cerebral cortices and renal cortices. Ewes at 60%, 80%, and 90% gestation, newborns, and adults received 4 dexamethasone or placebo injections. Cerebral cortex Na(+),K(+)-ATPase activity was higher (P < .05) in placebo-treated newborns than fetuses of placebo-treated ewes and adults, α(1)-expression was higher at 90% gestation than the other ages; α(2)-expression was higher in newborns than fetuses; α(3)-expression was higher in newborns than 60% gestation; β(1)-expression was higher in newborns than the other ages, and β(2)-expression higher at 60% than 80% and 90% gestation, and in adults. Renal cortex Na(+),K(+)-ATPase activity was higher in placebo-treated adults and newborns than fetuses. Cerebral cortex Na(+),K(+)-ATPase activity was higher in dexamethasone- than placebo-treated adults, and α(1)-expression higher in fetuses of dexamethasone- than placebo-treated ewes at 60% and 80% gestation. Renal cortex Na(+),K(+)-ATPase activity and α(1)-expression were higher in fetuses of dexamethasone- than placebo-treated ewes at each gestational age, and β(1)-expression was higher in fetuses of dexamethasone- than placebo-treated ewes at 90% gestation and in dexamethasone- than placebo-treated adults. Cerebral cortex Na(+),K(+)-ATPase activity, α(1)-expression, β(1)-expression, and renal cortex α(1)-expression correlated directly with increases in fetal cortisol. In conclusion, Na(+),K(+)-ATPase activity and subunit expression exhibit specific developmental patterns in brain and kidney; exogenous glucocorticoids regulate activity and subunit expression in brain and kidney at some ages; endogenous increases in fetal cortisol regulate cerebral Na(+),K(+)-ATPase, but exogenous glucocorticoids have a greater effect on renal than cerebral Na(+),K(+)-ATPase.

Although the placenta is the primary organ responsible for fetal clearance and electrolyte homeostasis, fetal kidneys contribute to amniotic fluid production and fetal hemodynamics. Maternal factors can significantly influence fetal urinary output and blood pressure. Maturation of neonatal glomerular filtration rate (GFR) depends on the development of renal blood flow (RBF). After birth, a marked increase in systemic blood pressure and decrease in renal vascular resistance results in elevated RBF and consequent increases in GFR. Vasoactive factors, including renin, angiotensin II, glucocorticoids, nonsteroidal anti-inflammatory drugs, nitric oxide, prostaglandins, bradykinin, and endothelin, each play vital roles in the regulation and development of neonatal GFR. Prematurity and intrauterine growth restriction (IUGR) may affect renal endowment and place infants at risk for hypertension and accelerated loss of renal function later in life.

Objectives: The cytokines interleukin (IL)-1β and IL-6 are modulators of the neuroimmune axis and have been implicated in neuronal cell death cascades after ischemia or infection. Previous work has shown that some cross-species conservation exists between human and rodent blood-brain barrier (BBB) transport systems. To further assess cross-species conservation of cytokine transport across the BBB, the current studies investigated permeability and inhibition of ovine IL-1β and IL-6 in the mouse. Methods: IL-1β or IL-6 was radioactively labeled with 131I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 µg/mouse of an unlabeled ovine or murine cytokine (IL-1β or IL-6) to assess self- and/or cross-inhibition of transport. Permeability was assessed using multiple-regression analysis. Results: There was a significant linear relationship for both ovine 131I-IL-1β and 131I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 µg/mouse unlabeled ovine IL-1β or IL-6 significantly reduced the transport of ovine 131I-IL-1β or 131I-IL-6, respectively, across the BBB. Transport of both ovine 131I-IL-1β and 131I-IL-6 was significantly inhibited by 1 µg/mouse of murine IL-1β or IL-6, respectively. In contrast, 1 µg/mouse of unlabeled ovine IL-1β or IL-6 did not inhibit the transport of murine 131I-IL-1β or 131I-IL-6. Conclusions: Ovine IL-1β and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

Gap junctions are specialized membrane structures that mediate intercellular communication and facilitate passage of ions and small molecules between adjacent cells. Connexins comprise a multigene family of transmembrane proteins that form gap junctions. Connexin-32 and connexin-43 are among the most abundant connexins in brain and are highly expressed during development. Connexin-32 is expressed primarily in oligodendrocytes and connexin-43 in astrocytes in adult brain. However, both connexins are expressed in neurons during development. We examined the effects of ontogeny on connexin-32 and connexin-43 protein abundance in cerebral cortices of sheep during development. Western immunoblot was used to measure connexin-32 and connexin-43 expression in cerebral cortices of fetuses at 60%, 80%, and 90% of gestation, in newborn lambs and adult sheep. Values were expressed as ratios to a single adult control cerebral cortical sample. Connexin-32 abundance was higher (P

Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day-18 mice and postnatal (P) day-10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIPs proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS.

Tight junctions of the blood-brain barrier are composed of transmembrane and associated cytoplasmic proteins. The transmembrane claudin proteins form the primary seal between endothelial cells and junctional adhesion molecules (JAMs) regulate tight junction formation. We have previously shown that claudin-1, claudin-5, zonula occludens (ZO)-1, and ZO-2 exhibit differential developmental regulation from 60% of gestation up to maturity in adult sheep. The purpose of the current study was to examine developmental changes in claudin-3, -12, and JAM-A protein expression in cerebral cortices of fetuses at 60%, 80%, and 90% gestation, and in newborn and adult sheep. We also examined correlations between changes in endogenous cortisol levels and tight junction protein expression in cerebral cortices of the fetuses. Claudin-3, -12 and JAM-A expressions were determined by Western immunoblot. Claudin-3 and -12 were lower (P

Pro-inflammatory cytokines contribute to hypoxic–ischemic brain injury. Blood–brain barrier (BBB) dysfunction represents an important component of hypoxic–ischemic brain injury in the fetus. Hypoxic–ischemic injury could accentuate systemic cytokine transfer across the fetal BBB. There has been considerable conjecture suggesting that systemic cytokines could cross the BBB during the perinatal period. Nonetheless, evidence to support this contention is sparse. We hypothesized that ischemia–reperfusion increases the transfer of systemic interleukin-1β (IL-1β) across the BBB in the fetus. Ovine fetuses at 127 days of gestation were studied 4 hours after 30 minutes of bilateral carotid artery occlusion and compared with a nonischemic group. Recombinant ovine IL-1β protein was expressed from an IL-1β pGEX-2 T vector in E. coli BL-21 cells and purified. The BBB function was quantified in 12 brain regions using a blood-to-brain transfer constant with intravenous 125I-radiolabeled IL-1β (125I-IL-1β). Interleukin-1β crossed the intact BBB in nonischemic fetuses. Blood-to-brain transport of 125I-IL-1β was higher ( P < 0.05) across brain regions in fetuses exposed to ischemia–reperfusion than nonischemic fetuses. We conclude that systemic IL-1β crosses the intact fetal BBB, and that ischemia–reperfusion increases transfer of this cytokine across the fetal BBB. Therefore, altered BBB function after hypoxia–ischemia facilitates entry of systemic cytokines into the brain of the fetus.

We examined the effects of hyperosmolality on blood-brain barrier (BBB) permeability during development to test the vulnerability of the immature barrier to stress. The BBB response to hyperosmolality was quantified using the blood-to-brain transfer constant ( Ki) with α-aminoisobutyric acid in fetuses at 60% and 90% gestation, premature, newborn, and older lambs. Kiplotted against osmolality increased as a function of increases in osmolality in all groups and brain regions. The relationship was described ( P < 0.05) by a segmented regression model. At lower osmolalities, changes in Kiwere minimal, but after a break point (threshold) was reached, the increase ( P < 0.05) was linear. We examined the responses of Kito hyperosmolality within each brain region by comparing the thresholds and slopes of the second regression segment. Lower thresholds and higher slopes imply greater vulnerability to hyperosmolality in the younger groups. Thresholds increased ( P < 0.05) with development in the thalamus, superior colliculus, pons, and spinal cord, and slopes of the second regression segment decreased ( P < 0.05) in the cerebellum, hippocampus, inferior colliculus, medulla, and spinal cord. BBB resistance to hyperosmolality increased ( P < 0.05) with development in most brain regions. The pattern of the Kiplotted against osmolality was ( P < 0.05) heterogenous among brain regions in fetuses and premature and newborn lambs, but not in older lambs. We conclude that 1) BBB permeability increased as a function of changes in osmolality, 2) the barrier becomes more resistant to hyperosmolality during development, and 3) the permeability response to hyperosmolality is heterogenous among brain regions in fetuses and premature and newborn lambs.

In adult rats, when plasma osmolality increases, water flows across the blood-brain barrier down its concentration gradient from brain to plasma, and brain volume deceases. The brain responds to this stress by gaining osmotically active solutes, which limit water loss. This phenomenon is termed brain volume (water) regulation. We tested the hypothesis that brain volume regulation is more effective in young lambs and adult sheep than in fetuses, premature lambs, and newborn lambs. Brain water responses to acute hyperosmolality were measured in the cerebral cortex, cerebellum, and medulla of fetuses at 60 and 90% of gestation, premature ventilated lambs at 90% of gestation, newborn lambs, young lambs at 20–30 days of age, and adult sheep. After exposure of the sheep to increases in systemic osmolality with mannitol plus NaCl, brain water content and electrolytes were quantified. The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress. There were significant differences from an ideal osmometer in the cerebral cortex of fetuses at 90% of gestation, cerebral cortex, and cerebellum of newborn lambs, and cerebral cortex, cerebellum, and medulla of young lambs and adult sheep; however, there were no differences in the brain regions of fetuses at 60% of gestation and premature lambs, cerebellum and medulla of fetuses at 90% of gestation, and medulla of newborn lambs. We conclude that 1) brain water loss is maximal and brain volume regulation impaired in most brain regions of fetuses at 60 and 90% of gestation and premature lambs; 2) brain volume regulation develops first in the cerebral cortex of the fetuses at 90% of gestation and in the cerebral cortex and cerebellum of newborn lambs, and then it develops in the medulla of the lambs at 20–30 days of age; 3) brain water loss is limited and volume regulation present in the brain regions of young lambs and adult sheep; and 4) the ability of the brain to exhibit volume regulation develops in a region- and age-related fashion.

We examined the effects of maternal corticosteroid administration on water content in regional tissue in ovine fetuses at 60%, 80%, and 90% of gestation. After catheters were placed in the fetuses, the ewes were given four 6-mg doses of dexamethasone or placebo injections 12 hours apart over 48 hours. Water conent of fetal tissue was determined 18 hours after the last injection was given to the ewes. Tissue water ws determined by wet-to-dry weight ratio in brain (cerebral cortex, caudate nucleus, cerebellum, midbrain, and medulla) and non-neural tissues (kidney, liver, muscle, and skin) at each gestational age. Water content (P < .05) in brain regions was lower in fetuses from dexamethasone-treated than placebo-treated ewes at 60% but not 80% or 90% of gestation and in non-neural tissues at each gestational age. Maternal treatmet with a contricosteroid regimen similar to that used in the clinical setting was associated with small decreases in brain water content early but not later in gestation. This corticosteroid treatment regimen was also associated with decreased regional non-neural tissue water content at 60%, 80%, and 90% of gestation.

Antenatal glucocorticoids are routinely given to women at risk for preterm delivery. The fetus is protected from excessive glucocorticoids by the placental enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2), which catalyzes the conversion of cortisol to its biologically inactive metabolite, cortisone. We examined the effects of antenatal dexamethasone on the expression of placental 11beta-HSD-2 in fetal sheep. Ewes were randomized to receive repeated or single courses of dexamethasone or placebo beginning at 76-78 or 104-106 d of gestation, respectively. In the single course group, the ewes received dexamethasone (6 mg, n = 7) or placebo (n = 6) as four intramuscular injections over 48 h up to 18 h before placental harvest. In the repeated course group, the ewes received the same treatment (dexamethasone, n = 10, or placebo, n = 9) once a week for 5 consecutive weeks starting at 76-78 d of gestation. Placental harvest occurred at 106-108 d of gestation in the four groups. By semi-quantitative RT-PCR, we found that placental 11beta-HSD-2 expression was lower in the fetuses of ewes exposed to a single course of dexamethasone than placebo (p0.05). Placental 11beta-HSD-2 expression did not differ significantly between fetuses of ewes treated with repeated courses of dexamethasone compared with placebo, or a single course of dexamethasone. Fetuses of dexamethasone treated ewes weighed less than those of placebo treated ewes (ANOVA, main effects for dexamethasone versus placebo treatment: F = 14.5, p = 0.007). Fetuses of ewes exposed to repeated courses of dexamethasone weighed less than those of ewes exposed to placebo or a single course of dexamethasone (p0.05). We conclude that maternal antenatal dexamethasone treatment reduces placental 11beta-HSD-2 expression and fetal weight at mid-gestation in the ovine pregnancy.

Hypoxic-ischemic (HI) brain injury is frequently associated with premature and/or full term birth related complications. HI injury often results in learning and processing deficits that reflect widespread damage to an extensive range of cortical and sub-cortical brain structures. Further, inflammation has been implicated in the long-term progression and severity of HI injury. Recently, Inter-alpha Inhibitor Proteins (IAIPs) have been shown to attenuate inflammation in models of systemic infection. Importantly, preclinical studies of neonatal HI injury and neuroprotection often focus on single time windows of assessment or single behavioral domains. This approach limits translational validity, given evidence for a diverse spectrum of neurobehavioral deficits that may change across developmental windows following neonatal brain injury. Therefore, the aims of this research were to assess the effects of human IAIPs on early neocortical cell death (72 hours post insult), adult regional brain volume measurements (cerebral cortex, hippocampus, striatum, corpus callosum) and long-term behavioral outcomes in juvenile (P38-50) and adult (P80+) periods across two independent learning domains (spatial and non-spatial learning), after postnatal day 7 HI injury in rats. Here, for the first time, we show that IAIPs reduce acute neocortical neuronal cell death and improve brain weight outcome 72 hours following HI injury in the neonatal rat. Further, these longitudinal studies are the first to show age, task and treatment dependent improvements in behavioral outcome for both spatial and non-spatial learning following systemic administration of IAIPs in neonatal HI injured rats. Finally, results also show sparing of brain regions critical for spatial and non-spatial learning in adult animals treated with IAIPs at the time of injury onset. These data support the proposal that Inter-alpha Inhibitor Proteins may serve as novel therapeutics for brain injury associated with premature birth and/or neonatal brain injury and highlight the importance of assessing multiple ages, brain regions and behavioral domains when investigating experimental treatment efficacy.

Inter-alpha inhibitor proteins (IAIPs) found in relatively high concentrations in human plasma are important in inflammation. IAIPs attenuate brain damage in young and adult subjects, decrease during sepsis and necrotizing enterocolitis in premature infants, and attenuate sepsis-related inflammation in newborn rats. Although a few studies have reported adult organ-specific IAIP expression, information is not available on age-dependent IAIP expression. Given evidence suggesting IAIPs attenuate brain damage in young and adult subjects, and inflammation in newborns, we examined IAIP expression in plasma, cerebral cortex (CC), choroid plexus (CP), cerebral spinal fluid (CSF), and somatic organs in fetal, newborn, and adult sheep to determine the endogenous expression patterns of these proteins during development. IAIPs (enzyme-linked immunosorbent assay) were higher in newborn and adult than fetal plasma ( P

Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV-enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.

article i nfo We have previously shown that increases in blood-brain barrier permeability represent an important compo- nent of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody at- tenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24 h of reperfusion. Groups were sham operated placebo-control- (n = 5), ischemia-placebo- (n = 6), ischemia-anti-IL-1β antibody- (n = 7), and sham-control antibody- (n = 2) treated animals. Systemic infusions of placebo (0.154 M NaCl) or anti- interleukin-1β monoclonal antibody (5.1 ± 0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4 h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin- 1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal ce- rebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (P b 0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (P b 0.001), brain anti- interleukin-1β monoclonal antibody levels were higher (P b 0.03), and interleukin-1β protein concentrations (P b 0.03) and protein expressions (P b 0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion- related increases in Ki across the brain regions (P b 0.04), and Ki showed an inverse linear correlation (r = �0.65, P b 0.02) with anti-interleukin-1β monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1β mono- clonalantibody infusionsafter ischemiaresult inbrainanti-interleukin-1βantibodyuptake, andattenuate ische- mia-reperfusion-related interleukin-1β protein up-regulation and increases in blood-brain barrierpermeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1β, contributes to impaired blood- brain barrier function after ischemia in the fetus.

Gap junctions facilitate intercellular communication and are important in brain development. Connexins (Cx) comprise a transmembrane protein family that forms gap junctions. Cx-32 is expressed in oligodendrocytes and neurons, Cx-36 in neurons, and Cx-43 in astrocytes. Although single antenatal steroid courses are recommended for fetal lung maturation, multiple courses can be given to women at recurrent risk for premature delivery. We examined the effects of single and multiple glucocorticoid courses on Cx-32, Cx-36, and Cx-43 protein expression in fetal cerebral cortex, cerebellum, and spinal cord, and differences in connexin expression among brain regions under basal conditions. In the single course groups, the ewes received dexamethasone (6 mg) or placebo as four intramuscular injections every 12 h over 48 h. In the multiple course groups, the ewes received the same treatment, once a week for five weeks starting at 76–78 days of gestation. Connexins were measured by Western immunoblot on brain samples from 105–108 day gestation fetuses. A single dexamethasone course was associated with increases (P

The objective of the present study was to examine the effect of antenatal or postnatal treatment with corticosteroids on the NMDA receptor, one of the mediators of both normal brain development and hypoxic-ischemic injury, by determining the characteristics of the receptor MK-801 binding site in untreated and corticosteroid-treated fetal and newborn lambs. (3)H-MK-801 binding was performed in cerebral cortical cell membranes from fetal sheep at 88, 120, and 136 d gestation (term = 150 d), and from 5-d-old lambs and adult ewes. Animals were randomized to receive dexamethasone [fetuses: 6 mg, i.m. every 12 hr for four doses to mother; lambs: 0.01 mg/kg (low dose) or 0.25 mg/kg (high dose) every 12 hr for four doses] or placebo. During development, B(max) (apparent number of receptors) increased, reaching a maximum in 5-d-old lambs (p < 0.05) and decreasing in the adult brain. K(d) (dissociation constant) did not change, suggesting that receptor affinity was not altered during maturation. Dexamethasone treatment had no effect on MK-801 binding in the fetus or adult, but in lambs was associated with a significant decrease in B(max) from 2.17 +/- 0.18 pmol/mg protein in placebo-treated animals to 1.65 +/- 0.8 and 1.62 +/- 0.07 pmol/mg protein in low-dose and high-dose animals, respectively. Affinity for (3)H-MK-801 decreased 20% after dexamethasone treatment in lambs only (p < 0.05). Thus, dexamethasone treatment appears to modify the NMDA receptor only during a specific period of brain development.

We have been studying the ontogeny of the blood-brain barrier function in ovine fetuses and lambs. During these studies, we have found that the duration of ventilation also influences blood-brain barrier permeability in premature lambs. Chronically instrumented hysterotomy-delivered surfactant-treated premature lambs were studied at 90% or 137 days of gestation ( n = 9). Blood-brain barrier function was quantified with the blood-to-brain transfer constant Kito α-aminoisobutyric acid. Linear regression analysis was used to compare the Kivalues in the brain regions, as the dependent variable, to the duration of ventilation, as the independent variable. There were direct correlations ( P < 0.05) between the Kivalues and the duration of ventilation [306 min (mean), 162–474 min (range)] in the cerebral cortex, cerebellum, medulla, caudate nucleus, hippocampus, superior colliculus, inferior colliculus, thalamus, pons, cervical spinal cord, and choroid plexus, but not in the pituitary gland. Ventilatory pressures and rates were established before the onset of the permeability studies. Calculated mean airway pressures [14 cmH2O (mean), 7–20 cmH2O (range)] from similarly studied premature lambs did not correlate with the duration of positive-pressure ventilation. We conclude that increases in the duration of positive-pressure ventilation predispose premature lambs to increases in regional blood-brain barrier permeability. These alterations in barrier function occur over relatively short time intervals (minutes to hours). In our study, these changes in permeability are most likely not attributable to changes in mean airway pressure.

To compare the efficacy of intravenous and endotracheal epinephrine administration, and intravenous administration above and below the diaphragm, during cardiopulmonary resuscitation in newborn piglets.Prospective, randomized, experimental laboratory protocol.Perinatal cardiovascular research laboratory at a university school of medicine.Forty newborn piglets (Sus domesticus).After cardiac arrest by ventricular fibrillation, cardiopulmonary resuscitation was begun. Radiolabeled epinephrine or placebo (0.9% sodium chloride) was administered into the right atrium, femoral vein, or endotracheal tube. Chest compressions and ventilation were continued for 10 mins.After epinephrine or placebo administration, samples were obtained from the systemic arterial circulation for measurement of radioisotope activity and plasma epinephrine concentrations. Mean carotid arterial blood pressure, right atrial, and inferior vena caval pressures were measured continuously. Epinephrine administration via the right atrium and femoral vein resulted in significant increases in plasma epinephrine concentration, percent of radioisotope recovery, and mean carotid arterial blood pressure, whereas endotracheal epinephrine administration did not. Placebo administered into the femoral vein resulted in a significant increase in percent radioisotope recovery, but not in plasma epinephrine concentration or carotid arterial blood pressure. Endotracheal administration of placebo did not result in significant increases in plasma epinephrine concentration, percent radioisotope recovery, or carotid arterial blood pressure. There were no significant differences between right atrial or inferior vena caval pressures among the groups.During cardiopulmonary resuscitation in newborn piglets, intravenous administration of epinephrine is more efficacious than endotracheal administration. Furthermore, efficacy is similar between femoral venous and right atrial administration.

We tested the hypotheses that, in hypoxic young pigs, reductions in cardiac output restrict systemic oxygen transport to a greater extent than does hypoxia alone and that compensatory responses to this restriction are more effective in higher than in lower priority vasculatures. To study this, 10- to 14-day-old instrumented awake hypoxic (arterial oxygen tension = 39 Torr) pigs were exposed to reduced venous return by inflation of a right atrial balloon-tipped catheter. Blood flow was measured with radionuclide-labeled microspheres, and oxygen metabolism was determined with arterial and venous oxygen contents from appropriate vessels. Hypoxia resulted in a reduction in oxygen tension; increases in cardiac output and perfusion to brain (72% over baseline), heart, adrenal glands, and liver without reductions to other organs except for the spleen; reductions in systemic and intestinal oxygen delivery; and increases in systemic and intestinal oxygen extraction without changes in systemic, cerebral, or intestinal oxygen uptake. During hypoxia, decreasing venous return was associated with increases in arterial lactic acid concentration and central venous pressure; attenuation of the hypoxia-related increase in cardiac output; sustained increases in brain (72% over baseline) and heart perfusion; reductions in lung (bronchial artery), pancreatic, renal, splenic, and intestinal (−50% below baseline) perfusion; decreases in systemic and gastrointestinal oxygen delivery; sustained increases in systemic and intestinal oxygen extraction; and decreases in intestinal oxygen uptake, without changes in cerebral oxygen metabolism. We conclude that when venous return to the heart is reduced in hypoxic young pigs, the hypoxia-related increase in cardiac output was attenuated and the relative reduction in cardiac output was associated with preserved cerebral oxygen uptake and compromised intestinal oxygen uptake. Regional responses to hypoxia combined with relative reductions in cardiac output differ from that of hypoxia alone, with the greatest effects on lower priority organs such as the gastrointestinal tract.

We tested the hypothesis that nitric oxide (NO) modulates postprandial hyperemia in young pigs. To test this hypothesis, we studied five groups of 3-wk-old pigs: group 1: milk fed, N(G)-monomethyl-L-arginine (L-NMMA) and L-arginine (L-Arg) treated (n = 10); group 2: milk fed, placebo treated (n = 8); group 3: water fed, L-NMMA and L-Arg treated (n = 6); group 4: water fed, placebo treated (n = 4); and group 5: fasted sham fed, L-NMMA and L-Arg treated (n = 6). After catheter placement and electromagnetic flow probe instrumentation of the mesenteric artery, systemic blood pressure and mesenteric artery blood flow were measured during preprandial baseline, postprandial, and postprandial intra-arterial L-NMMA- and L-Arg-infused study periods. The same measurements were made in the milk- and water-fed placebo-treated groups except that diluent replaced the L-NMMA and L-Arg infusions. In the milk- and water-fed placebo-treated groups, a significant (analysis of variance, P < 0.05), postprandial hyperemia was observed. The change in blood flow was greater (P < 0.05) in the milk-fed group than in the water-fed group. Inhibition of NO synthesis with L-NMMA diminished (P < 0.05) the hyperemic responses to both water and milk feeding and resulted in a decrease (P < 0.05) in mesenteric artery perfusion in the fasted sham-fed state. We conclude that, in young pigs, NO is a modulator of mesenteric vascular tone in both the postprandial and fasted states.

We tested the hypothesis that, in newborn piglets, magnesium results in a dose-dependent prostanoid-mediated cerebrovasodilation. Pial arterioles (50-200 microns in diameter) were serially measured, and cortical subarachnoid cerebrospinal fluid (CSF) was collected for radioimmunoassay of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha, hydrolysis product of prostacyclin) and thromboxane B2 (TxB2, metabolite of thromboxane A2) before and after CSF containing 1.2, 2.4, 4.8, and 9.6 mM MgCl2 was suffused over the parietal cortex under a closed cranial window in twelve 2- to 4-day-old piglets. Magnesium suffusion resulted (P < 0.05) in a dose-dependent pial arteriolar vasodilation. The increase in vessel diameter was greater (P < 0.001) with 2.4, 4.8, and 9.6 mM than with 1.2 mM concentration of magnesium. The increase in vessel diameter with 9.6 mM was also greater (P < 0.001) than with the 2.4 mM concentration of magnesium. Magnesium suffusion did not result in changes in cortical CSF prostanoid concentrations. The effect of intravenous indomethacin (5 mg/kg) on cyclooxygenase inhibition in the pial arterioles was confirmed by a 24 +/- 3% decrease in vessel diameter at the baseline (1.2 mM) magnesium concentration. In contrast, cyclooxygenase inhibition with intravenous indomethacin did not attenuate the magnesium-induced cerebrovasodilation. We conclude that in new born piglets magnesium suffusion over the parietal cortex results in a dose-dependent cerebrovasodilation that is most likely not mediated by prostanoids.

To test the hypothesis that administration of indomethacin prophylaxis before 6 hours of life results in a lower incidence of intraventricular hemorrhage (IVH) compared with administration after 6 hours of life, and that the effects of early prophylaxis depend on gestational age (GA) and sex in very low birth weight infants (birth weight1250 g).Very low birth weight infants admitted to our neonatal intensive care unit between 2003 and 2010 who received indomethacin prophylaxis were analyzed retrospectively. Exclusion criteria included unknown time of indomethacin prophylaxis, death at12 hours of life, congenital anomalies, and unavailable head ultrasound report. Infants were dichotomized based on the timing of indomethacin prophylaxis (6 hours or6 hours of life) to compare incidence of IVH all grades and severe (grade 3-4) IVH. Secondary analyses examined the effects of the time of indomethacin prophylaxis initiation by GA and sex on the incidence of IVH.A total of 868 infants (431 males and 437 females) met the criteria for analysis. Indomethacin prophylaxis was given at6 hours of life in 730 infants and at6 hours of life to 168 infants. The 2 groups differed with respect to antenatal steroid exposure, GA, outborn prevalence, and pneumothoraces. After multivariate analysis, there were no between-group differences in all-grade or severe IVH. However, females, but not males, treated at6 hours of life had a lower incidence of severe IVH (P.05), particularly at lower GAs.Prophylactic indomethacin administered before 6 hours of life is not associated with lower incidence of IVH.

OBJECTIVES Fetuses of diabetic women exhibit hypoxemia, elevated catecholamine concentrations at birth, and increased incidence of death. Our previous findings suggested that experimental fetal hyperinsulinemia results in a surge in catecholamines with cardiovascular changes supported by increased beta-adrenergic activity. The present experiments were designed to assess the contribution of alpha-adrenergic stimulation to the hemodynamic changes in the hyperinsulinemic ovine fetus. METHODS Combined ventricular output, regional organ blood flow, vascular resistance, metabolism, and catecholamine concentrations were measured before and during an infusion of insulin and during continued infusion with alpha-adrenergic blockade (phentolamine) in eight chronically catheterized fetal sheep. RESULTS Fetal insulin infusion produced hyperinsulinemic-hypoglycemia, a surge in epinephrine and norepinephrine concentration, and increases in the combined ventricular output (blood flow to the fetus plus placenta) and regional blood flow to the fetus, heart, stomach, gastrointestinal tract, fat, and carcass. In the hyperinsulinemic state, alpha-adrenergic blockade was associated with additional increases in fetal norepinephrine concentration and no major changes in combined ventricular output or blood flow to the body of the fetus, except for decreased blood flow to the stomach and lungs, and a decrease in stroke volume. CONCLUSIONS Because vasodilation characterizes the hyperinsulinemic state, alpha-adrenergic stimulation contributes less to compensatory cardiovascular changes in the hyperinsulinemic fetus than that which we previously have shown for beta-adrenergic stimulation.

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (P

The neonatal gastrointestinal tract is a site of intense anabolic and metabolic activity, as it is responsible for the assimilation of nutritional intake and exhibits accelerated growth shortly after birth. The hypermetabolic state of the gastrointestinal tract requires sufficient blood flow and oxygen delivery to sustain adequate oxygen consumption to meet these metabolic needs. Therefore, an understanding of the mechanisms regulating intestinal vascular perfusion in the normal state and during pathophysiological conditions in the perinatal period is important to elucidate potential contributions to the development of intestinal pathologies in the neonate. The goal of this review is to summarize the available literature on the regulation of intestinal blood flow and oxygenation in the fetus and newborn in normal states and during pathological stress.

Interleukin (IL)-1β and IL-6 have been implicated in brain development, injury progression, and fetal/maternal immune interactions. We examined IL-1β and IL-6 protein expression in cerebral cortex (CC) and white matter (WM) from non-ischemic ovine fetuses at 87-90, 122-127, and 135-137 days of gestation, pregnant ewes at 87-90 and 135-137 days of gestation, and fetuses exposed to 48 or 72h of reperfusion after ischemia. Protein expression was determined by Western immunoblot. In non-ischemic CC, IL-1β was higher (P

BACKGROUND AND OBJECTIVE: Delayed cord clamping (DCC) has been advocated during preterm delivery to improve hemodynamic stability during the early neonatal period. The hemodynamic effects of DCC in premature infants after birth have not been previously examined. Our objective was to compare the hemodynamic differences between premature infants randomized to either DCC or immediate cord clamping (ICC). METHODS: This prospective study was conducted on a subset of infants who were enrolled in a randomized controlled trial to evaluate the effects of DCC versus ICC. Entry criteria included gestational ages of 24 to 316 weeks. Twins and infants of mothers with substance abuse were excluded. Serial Doppler studies were performed at 6 ± 2, 24 ± 4, 48 ± 6, and 108 ± 12 hours of life. Measurements included superior vena cava blood flow, right ventricle output, middle cerebral artery blood flow velocity (BFV), superior mesenteric artery BFV, left ventricle shortening fraction, and presence of a persistent ductus arteriosus. RESULTS: Twenty-five infants were enrolled in the DCC group and 26 in the ICC group. Gestational age, birth weight, and male gender were similar. Admission laboratory and clinical events were also similar. DCC resulted in significantly higher superior vena cava blood flow over the study period, as well as greater right ventricle output and right ventricular stroke volumes at 48 hours. No differences were noted in middle cerebral artery BFV, mean superior mesenteric artery BFV, shortening fraction, or the incidence of a persistent ductus arteriosus. CONCLUSIONS: DCC in premature infants is associated with potentially beneficial hemodynamic changes over the first days of life. * Abbreviations: ANOVA — : analysis of variance BFV — : blood flow velocity DCC — : delayed cord clamping ICC — : immediate cord clamping MCA — : middle cerebral artery PDA — : persistent ductus arteriosus P/IVH — : periventricular or intraventricular hemorrhage RVO — : right ventricle output RV-SV — : right ventricle stroke volume SMA — : superior mesenteric artery SVC — : superior vena cava

Cytokines have gained increasing attention as therapeutic targets in inflammation-related disorders and inflammatory conditions have been investigated in sheep. Monoclonal antibodies (mAbs) specific for the ovine pro-inflammatory cytokines interleukin (IL)-1β and IL-6 could be used to study the effects of blocking pro-inflammatory cytokines in sheep. Ovine-specific IL-1β and IL-6 proteins and mAbs specific for these molecules were produced and the ability of the mAbs to neutralize the proteins was tested in cultures of ovine splenic mononuclear cells. Expression of nuclear factor (NF)-κβ and signal transducer and activator of transcription (STAT)-3 was evaluated by western blotting and densitometric quantification. Treatment with purified IL-1β and IL-6 proteins increased NF-κβ (P

We examined the effects of hyperglycemic hyperosmolality on blood-brain barrier (BBB) permeability during development. We hypothesized that the barrier becomes more resistant to hyperglycemic hyperosmolality during development, and the immature BBB is more resistant to glucose than to mannitol hyperosmolality. We quantified the BBB response to hyperosmolality with the blood-to-brain transfer constant ( Ki) in immature fetuses, premature, and newborn lambs. Ki increased as a function of increases in osmolality. A segmented regression model described the relationship between Ki and osmolality. At lower osmolalities, changes in Ki were minimal but after a threshold, increases were linear. We examined responses of Ki to hyperglycemic hyperosmolality by comparing the thresholds and slopes of the second regression segments. Lower thresholds and steeper slopes indicate greater vulnerability to hyperosmolality. Thresholds increased ( P < 0.05) during development in pons and superior colliculus. Thresholds were higher ( P < 0.05) during glucose than mannitol hyperosmolality in thalamus, superior colliculus, inferior colliculus and medulla of premature lambs, and in cerebrum and cerebellum of newborns. We conclude that BBB permeability increased as a function of changes in glucose osmolality, the barrier becomes more resistant to glucose hyperosmolality in two brain regions during development, and the barrier is more resistant to glucose than to mannitol hyperosmolality in some brain regions of premature and newborn lambs.

A multicenter, randomized, double-blind, controlled trial compared three beractant (Survanta) administration procedures in the treatment of neonatal respiratory distress syndrome. Infants weighingor = 600 gm with respiratory distress syndrome who required assisted ventilation were treated within 8 hours of birth with beractant administered intratracheally. Procedure A required administration in two fractional doses after removal of the infant from the ventilator. Procedure B required administration in two fractional doses through a neonatal suction valve and did not require removal of the infant from the ventilator, and procedure C required administration in four fractional doses during removal from the ventilator. Procedure C is the method used in all previous beractant studies. Of the 299 infants enrolled, 103 were randomly assigned to procedure A, 100 to procedure B, and 96 to procedure C. The results indicate no significant differences among the treatment groups in the clinical outcome measures of fractional inspired oxygen, mean airway pressure, and arterial-alveolar ratio of partial pressure of oxygen at 72 hours of life, or in the incidences of air leaks, pulmonary interstitial emphysema, or death through 72 hours of life. There were no significant differences in the lowest heart rates recorded during administration of doses, but there was less oxygen desaturation during administration of dose 1 with procedure B than with procedure A (p = 0.001), and more reflux of beractant after procedure B than after procedure C (p = 0.007). We conclude that the three procedures are equally effective and can be used to administer beractant safely and effectively. Procedure B has the advantage of allowing administration without interrupting mechanical ventilation.

We tested the hypotheses that in newborn piglets indomethacin (Indo) pretreatment blunts the hyperemic brain blood flow (BF) and alters the cerebral metabolic responses to hypoxia and that these responses are dose dependent. We studied 23 chronically instrumented piglets exposed to graded hypoxia (O2 content: 7.1-0.4 microM O2/ml) after pretreatment with high (5 mg/kg, n = 8)-or low (0.3 mg/kg, n = 6)-dose Indo or placebo (diluent, n = 9). Total and regional brain BF increased significantly with decreasing O2 content values (P < 0.01) in all three groups. However, the rise in the brain BF curves with decreasing O2 content values was significantly (P < 0.05) lower in the high-compared with the low-dose group in all brain regions with the greatest effect in the caudal regions. Furthermore, the BF curves in the placebo-treated animals were similar to the low-dose group. The cerebral metabolic rate of O2 (CMR(O2)) and glucose metabolism were preserved in the three groups over all hypoxic ranges until severe hypoxia (O2 content < or = 1.1 microM O2/ml) was achieved in the high-dose group, when CMR(O2) decreased (P < 0.05), and glucose metabolism increased (P < 0.05). The mean arterial blood pressure in the high-dose group during severe hypoxia was 45 mmHg (P > 0.05). Although coupling of cerebral BF and CMR(O2) was preserved in the three groups, this association was significantly altered with high-dose pretreatment. We conclude that an attenuation in the hypoxia-induced brain perfusion by Indo is dose dependent. Alterations in CMR(O2) and glucose metabolism are observed with high-dose pretreatment during severe hypoxia, and the responses to hypoxia are similar with placebo and low-dose Indo pretreatment.