Yves Garnier

Perinatal brain injury

Abstract Perinatal brain damage in the mature fetus is usually brought about by severe intrauterine asphyxia following an acute reduction of the uterine or umbilical circulation. The areas most heavily affected are the parasagittal region of the cerebral cortex and the basal ganglia. The fetus reacts to a severe lack of oxygen with activation of the sympathetic-adrenergic nervous system and a redistribution of cardiac output in favor of the central organs (brain, heart and adrenals). If the asphyxic insult persists, the fetus is unable to maintain circulatory centralization, and the cardiac output and extent of cerebral perfusion fall. Owing to the acute reduction in oxygen supply, oxidative phosphorylation in the brain comes to a standstill. The Na+/K+ pump at the cell membrane has no more energy to maintain the ionic gradients. In the absence of a membrane potential, large amounts of calcium ions flow through the voltage-dependent ion channels, down an extreme extra-/intracellular concentration gradient, into the cell. Current research suggests that the excessive increase in levels of intracellular calcium, so-called calcium overload, leads to cell damage through the activation of proteases, lipases and endonucleases. During ischemia, besides the influx of calcium ions into the cells via voltage-dependent calcium channels, more calcium enters the cells through glutamate-regulated ion channels. Glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles during ischemia following anoxic cell depolarization. The acute lack of cellular energy arising during ischemia induces almost complete inhibition of cerebral protein biosynthesis. Once the ischemic period is over, protein biosynthesis returns to preischemic levels in non-vulnerable regions of the brain, while in more vulnerable areas it remains inhibited. The inhibition of protein synthesis, therefore, appears to be an early indicator of subsequent neuronal cell death. A second wave of neuronal cell damage occurs during the reperfusion phase. This cell damage is thought to be caused by the postischemic release of oxygen radicals, synthesis of nitric oxide (NO), inflammatory reactions and an imbalance between the excitatory and inhibitory neurotransmitter systems. Part of the secondary neuronal cell damage may be caused by induction of a kind of cellular suicide programme known as apoptosis. Interestingly, there is increasing evidence from recent clinical studies that perinatal brain damage is closely associated with ascending intrauterine infection before or during birth. However, a major part of this damage is likely to be of hypoxic-ischemic nature due to LPS-induced effects on fetal cerebral circulation. Knowledge of these pathophysiological mechanisms has enabled scientists to develop new therapeutic strategies with successful results in animal experiments. The potential of such therapies is discussed here, particularly the promising effects of intravenous administration of magnesium or postischemic induction of cerebral hypothermia.

Perinatal brain damage: underlying mechanisms and neuroprotective strategies.

Children who suffer from perinatal brain injury often deal with the dramatic consequences of this misfortune for the rest of their lives. Despite the severe clinical and socioeconomic significance, no effective clinical strategies have yet been developed to counteract this condition. As shown in recent studies, perinatal brain injury is usually brought about by cerebral ischemia, cerebral hemorrhage, or an ascending intrauterine infection. This review focuses on the pathophysiologic pathways activated by these insults and describes neuroprotective strategies that can be derived from these mechanisms. Fetal cerebral ischemia causes an acute breakdown of neuronal membrane potential followed by the release of excitatory amino acids such as glutamate and aspartate. Glutamate binds to postsynaptically located glutamate receptors that regulate calcium channels. The resulting calcium influx activates proteases, lipases, and endonucleases, which in turn destroy the cellular skeleton. A second wave of neuronal cell damage occurs during the reperfusion phase. This cell damage is thought to be caused by the postischemic release of oxygen radicals, synthesis of nitric oxide, inflammatory reactions, and an imbalance between the excitatory and inhibitory neurotransmitter systems. Furthermore, secondary neuronal cell damage may be brought about in part by induction of a cellular suicide program known as apoptosis. Recent studies have shown that inflammatory reactions not only aggravate secondary neuronal damage after cerebral ischemia, but may also injure the immature brain directly. This damage may be mediated by cardiovascular effects of endotoxins leading to cerebral hypoperfusion and by activation of apoptotic pathways in oligodendrocyte progenitors through the release of proinflammatory cytokines. Periventricular or intraventricular hemorrhage (PIVH) is a typical lesion of the immature brain. The inability of preterm fetuses to redistribute cardiac output in favor of the central organs and their lack of cerebral autoregulation may cause significant fluctuations in cerebral blood flow when oxygen is in short supply. Disruption of the thin-walled blood vessels in the germinal matrix with subsequent cerebral hemorrhage is often the inevitable result and is at times associated with cerebral hemorrhagic infarction. Knowledge of these pathophysiologic mechanisms has enabled scientists do develop new therapeutic strategies, which have been shown to be neuroprotective in animal experiments. The potential of such therapies is discussed here, particularly the promising effects of postischemic induction of cerebral hypothermia, the application of the calcium-antagonist flunarizine, and the administration of magnesium.

Endotoxemia severely affects circulation during normoxia and asphyxia in immature fetal sheep.

Objective: The purpose of the present study was to determine whether endotoxins (lipopolysaccharides, LPS) affect the fetal cardiovascular system in a way likely to cause brain damage. Methods: Thirteen fetal sheep were chronically instrumented at a mean gestational age of 107 ± 1 days. After control measurements of organ blood flow (microsphere method), blood gases, and acid base balance were obtained, seven of 13 fetuses received LPS (53 ± 3 μg/kg fetal weight) intravenously. Sixty minutes later, asphyxia was induced by occlusion of the maternal aorta for 2 minutes. Measurements of organ blood flows were made at -60, -1, +2, +4, +30, and +60 minutes. Results: Unlike in the control group, after LPS infusion there was a significant decrease in arterial oxygen saturation (-46%; P <.001) and pH (P <.001). In LPS-treated fetuses the portion of combined ventricular output directed to the placenta decreased significantly (-76%; P <.001), whereas output to the fetal body (+60%; P <.001), heart (+167%; P <.05), and adrenals (+229%; P <.01) increased. Furthermore, during asphyxia circulatory centralization was impaired considerably in LPS-treated fetuses, and there was clear evidence of circulatory decentralization. This decentralization caused a severe decrease in cerebral oxygen delivery by 70%. Wihin 30 minutes after induction of asphyxia five of seven LPS-treated featuses died, whereas all control fetuses recovered completely. Conclusions: Endotoxemia severely impaired fetal cardiovascular control during normoxia and asphyxia, resulting in a considerable decrease in cerebral oxygen delivery. These effects might have important effects in the development of fetal brain damage associated with intrauterine infection.

Perinatal brain damage—from pathophysiology to prevention

Children undergoing perinatal brain injury often suffer from the dramatic consequences of this misfortune for the rest of their lives. Despite the severe clinical and socio-economic significance, no effective clinical strategies have yet been developed to counteract this condition. This review describes the pathophysiological mechanisms that are implicated in perinatal brain injury. These include the acute breakdown of neuronal membrane potential followed by the release of excitatory amino acids such as glutamate and aspartate. Glutamate binds to postsynaptically located glutamate receptors that regulate calcium channels. The resulting calcium influx activates proteases, lipases and endonucleases which in turn destroy the cellular skeleton. The acute lack of cellular energy during ischemia induces almost complete inhibition of cerebral protein biosynthesis. Once the ischemic period is over, protein biosynthesis returns to preischemic levels in non-vulnerable regions of the brain, while in more vulnerable areas it remains inhibited. A second wave of neuronal cell damage occurs during the reperfusion phase induced by the postischemic release of oxygen radicals, synthesis of nitric oxide (NO), inflammatory reactions and an imbalance between the excitatory and inhibitory neurotransmitter systems. Clinical studies have shown that intrauterine infection increases the risk of periventricular white matter damage especially in the immature fetus. This damage may be mediated by cardiovascular effects of endotoxins leading to cerebral hypoperfusion and by activation of apoptotic pathways in oligodendrocyte progenitors through the release of pro-inflammatory cytokines. Knowledge of these pathophysiological mechanisms has enabled scientists to develop new therapeutic strategies which have been shown to be neuroprotective in animal experiments. The potential of such therapies is discussed here, particularly the promising effects of postischemic induction of mild cerebral hypothermia, the application of the calcium-antagonist flunarizine and the administration of magnesium.

Preterm birth and inflammation-The role of genetic polymorphisms.

Spontaneous preterm labour and preterm births are still the leading cause of perinatal morbidity and mortality in the developed world. Previous efforts to prevent preterm birth have been hampered by a poor understanding of the underlying pathophysiology, inadequate diagnostic tools and generally ineffective therapies. Clinical, epidemiological and experimental studies indicate that genito-urinary tract infections play a critical role in the pathogenesis of preterm birth. Moreover, intrauterine infection increases perinatal mortality and morbidity, such as cerebral palsy and chronic lung disease, significantly. It has recently been suggested that gene-environment interactions play a significant role in determining the risk of preterm birth. Polymorphisms of certain critical genes may be responsible for a harmful inflammatory response in those who possess them. Accordingly, polymorphisms that increase the magnitude or the duration of the inflammatory response were associated with an increased risk of preterm birth. In contrast polymorphisms that decrease the inflammatory response were associated with a lower risk of preterm birth. This article will review the current understanding of pathogenetic pathways in the aetiology of preterm birth.

Intravenous lipopolysaccharide-induced pulmonary maturation and structural changes in fetal sheep

BACKGROUND Antenatal pulmonary inflammation is associated with reduced risk for respiratory distress syndrome but with an increased risk for bronchopulmonary dysplasia (BPD) with impaired alveogenesis. OBJECTIVE We hypothesized that fetal systemic inflammation induced by intravenous (IV) lipopolysaccharide (LPS) would affect lung development in utero. STUDY DESIGN Twenty-one fetal sheep were instrumented (107 days gestational age). Control fetuses received saline (N = 12) and 9 in the study group received 100 ng of LPS IV 3 days after surgery. Animals were assessed for lung maturation and structure after 3 (N = 5) and 7 (N = 4) days. RESULTS Interleukin-6 concentration increased in the bronchoalveolar lavage more than 40-fold 3 days after LPS IV. Processing of pro-surfactant protein (SP)-B to mature SP-B and increased SP-B concentrations were shown 7 days after LPS IV. Deposition of elastin fibers at sites of septation was disturbed within 3 days after LPS IV. CONCLUSION Lung maturation and disturbed lung structure occurred after short-term exposure to fetal inflammation and suggests new targeted therapies for BPD.

Increased Maternal/Fetal Blood S100B Levels Following Systemic Endotoxin Administration and Periventricular White Matter Injury in Preterm Fetal Sheep

Objective. Intrauterine infection is suggested to cause perinatal brain white matter injury. In the current study, we evaluated whether S100B, a brain damage marker, may be also assessed in maternal bloodstream after white matter injury induced by fetal intravenous application of lypopolisaccharide (LPS) endotoxin. Methods. Fourteen fetal sheeps were chronically catheterized at a mean gestational age of 107 days. Three days after surgery, fetuses (n = 7) received 500 ng of LPS or 2 mL 0.9% saline (n = 7) intravenously (IV). Lypopolisaccharide and placebo groups were monitored by continuous hemodynamic data recordings and at 6 predetermined time points (control value; 3, 6, 24, 48, and 72 hours after LPS/placebo administration) blood was drawn for laboratory parameters and S100B assessment. Brain damage was evaluated by light microscopy after Klüver-Barrera staining. Selected areas of the periventricular white matter were also examined by electron microscopy. Results. White matter injury was detected in all LPS-treated fetuses, whereas no abnormalities were seen in control animals or in LPS-treated mothers. Maternal and fetal S100B protein levels were significantly higher in the LPS group than in the control group at all monitoring time points (P < .001). The highest fetal-maternal S100B levels were observed at 3-hour time-point (P < .001). Conclusions. We found that S100B protein is increased in the maternal district in presence of fetal periventricular brain white matter injury induced by endotoxin. The present data offer additional support for S100B assessment in the maternal circulation in pregnancies complicated by intrauterine infection at risk of white matter injury.

Low dose flunarizine protects the fetal brain from ischemic injury in sheep

Flunarizine, a calcium channel blocker, reduced cerebral damage caused by hypoxic-ischemic insults in neonatal rats and in fetal sheep near term. However, the high dose regimen used in these studies produced cardiovascular side effects that might have counteracted the neuroprotective properties of flunarizine. Therefore, the neuroprotective effect was tested in a low dose protocol (1 mg/kg estimated body weight). Twelve fetal sheep near term were instrumented chronically. Six fetuses were pretreated with 1 mg of flunarizine per kg of estimated body weight 1 h before ischemia, whereas the remainder (n = 6) received solvent. Cerebral ischemia was induced by occluding both carotid arteries for 30 min. To exclude the possibility that the neuroprotective effects of flunarizine were caused by cerebrovascular alterations we measured cerebral blood flow by injecting radiolabeled microspheres before (-1 h), during (3 min and 27 min) and after (40 min, 3 h, and 72 h) cerebral ischemia. At the end of the experiment (72 h) the ewe was given a lethal dose of sodium pentobarbitone and saturated potassium chloride i.v., and the fetal brain was perfused with formalin. Neuronal cell damage was assessed in various brain structures by light microscopy after cresyl violet/fuchsin staining using a scoring system: 1, 0-5% damage; 2, 5-50% damage; 3, 50-95% damage; 4, 95-99% damage; and 5, 100% damage. In 10 other fetal sheep effects of low dose flunarizine on circulatory centralization caused by acute aphyxia could be excluded. In the treated group neuronal cell damage was reduced significantly in many cerebral areas to varying degrees(range for control group, 1.03-2.14versus range for treated group, 1.00-1.13; p < 0.05 to p < 0.001, respectively). There were only minor differences in blood flow to the various brain structures between groups. We conclude that pretreatment with low dose flunarizine protects the brain of fetal sheep near term from ischemic injury. This neuroprotective effect is not mediated by changes in cerebral blood flow. We further conclude that low dose flunarizine may be clinically useful as a treatment providing fetal neuroprotection, particularly because the fetal cardiovascular side effects are minimal.

Neuroprotective effects of magnesium on metabolic disturbances in fetal hippocampal slices after oxygen-glucose deprivation: mediation by nitric oxide system.

Objective: We investigated the effects of magnesium on metabolic disturbances in hippocampal slices prepared from fetal guinea pigs after oxygen-glucose deprivation (OGD). Methods: Metabolic disturbances were assessed by measuring changes in energy metabolism and protein synthesis. In addition we determined cyclic guanosine monophosphate (cGMP) concentrations in the slices after OGD, as a measure of nitric oxide (NO) production, to clarify whether a possible neuprotective effect of magnesium is mediated in part through the NO system. Results: Twelve hours after oxygen-glucose deprivation, adenosine triphosphate (ATP) concentration and protein synthesis in the hippocampal slices were significantly reduced depending on the severity of OGD. A higher magnesium concentration in the incubation medium from 1.3 mM to 3.9 mM 2 hours before OGD significantly improved the recovery of ATP and protein synthesis, whereas treatment after OGD was ineffective. The cGMP concentrations increased dramatically in hippocampal slices 10 minutes after OGD, indicating a significant increase in NO production. When the concentration of magnesium in the artificial cerebrospinal fluid was increased 2 hours before OGD the rise in tissue levels of cGMP was considerably reduced. Again, treatment after OGD had no effect. Conclusion: We conclude that increasing magnesium concentration in the artificial cerebrospinal fluid before OGD alleviated metabolic disturbances in hippocampal slices from mature fetal guinea pigs, whereas treatment after OGD had no effect. This neuroprotective property of magnesium might be mediated in part through the inhibition of NO production shortly after OGD.

Nitric oxide and fetal organ blood flow during normoxia and hypoxemia in endotoxin-treated fetal sheep.

OBJECTIVE: To investigate the role of nitric oxide in the process of circulatory decentralization during fetal hypoxemia. METHODS: Fifteen sheep with singleton pregnancies were chronically instrumented at 107 days of gestation (term is 147 days). Three days later, 8 of the fetuses received nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis. Fifteen minutes after L-NAME administration, all 15 fetuses received lipopolysaccharides (LPS) from a strain of Escherichia coli. The 7 fetuses that received LPS only were used as controls. Sixty minutes after LPS was administered, the maternal aorta was occluded for 2 minutes in all fetuses. Organ blood flow and physiological variables were measured at 75 minutes before the start of occlusion (ie, at the time of L-NAME administration to the experimental group), at 1 minute before the start of occlusion, and at 2, 4, and 30 minutes after the start of occlusion. RESULTS: Arterial pH was lower in the L-NAME group than in the control group at 1 minute before and 2 minutes after occlusion. Mean arterial pressure was higher in the L-NAME group than in the control group at 2 and 4 minutes after occlusion. Cardiac output fell in the L-NAME group and was lower than in the control group; the percentage of cardiac output to the cerebrum in the L-NAME group was 35% lower than that in the control group. Throughout the study, placental blood flow decreased by more than 80% in both groups and remained low. Blood flow to the fetal body decreased by 65% in the L-NAME group and was lower than in the control group. Blood flow to the carcass also decreased in the L-NAME group and was 36% of that in the control group. CONCLUSION: Inhibition of nitric oxide synthesis causes a general vasoconstriction in practically all organs and leads to a reduction in LPS-induced circulatory decentralization. The changes in blood flow distribution in endotoxin-treated fetal sheep seem to be mediated in part by nitric oxide.